60-35-5DLFVBJFMPXGRIB-UHFFFAOYSA-NDLFVBJFMPXGRIB-UHFFFAOYSA-N
AcetamideAcetamid
acetamida
Acetic acid amide
Acetimidic acid
Ethanamide
Ethanimidic acid
Methanecarboxamide
NSC 25945
DTXSID7020005103-90-2RZVAJINKPMORJF-UHFFFAOYSA-NRZVAJINKPMORJF-UHFFFAOYSA-N
Acetaminophen4-Acetamidophenol
APAP
Paracetamol
4-hydroxyacetanilide
Acetamide, N-(4-hydroxyphenyl)-
4-(Acetylamino)phenol
4-(N-Acetylamino)phenol
4-Acetaminophenol
4'-Hydroxyacetanilide
Abensanil
Acetagesic
Acetalgin
ACETAMIDE, N-(4-HYDROXYPHENYL)
Acetaminofen
Acetanilide, 4'-hydroxy-
ACETANILIDE, 4-HYDROXY-
Algotropyl
Alvedon
Anaflon
Apamide
Banesin
Ben-u-ron
Bickie-mol
Biocetamol
Cetadol
Citramon P
Claratal
Clixodyne
Dafalgan
Daphalgan
Dial-a-gesic
Disprol
Doliprane
Dolprone
Dymadon
Efferalgan
Endophy
Febrilex
Febrilix
Febro-Gesic
Febrolin
Fepanil
Finimal
Gattaphen T
Gelocatil
Gutte Enteric
Homoolan
Jin Gang
Lestemp
Liquagesic
Lonarid
Lyteca Syrup
Minoset
Momentum
N-(4-Hydroxyphenyl)acetamide
N-Acetyl-4-aminophenol
N-Acetyl-4-hydroxyaniline
N-Acetyl-p-aminophenol
Napafen
Naprinol
Nobedon
NSC 109028
NSC 3991
Ortensan
p-(Acetylamino)phenol
p-Aceaminophenol
Pacemol
p-Acetamidophenol
p-Acetoaminophen
P-ACETYLAMINOPHENOL
Paldesic
panadeine
Panadol
Panadol Actifast
Panadol Extend
Panaleve
Panasorb
Panodil
Paracetamol DC
Paracetamole
Parageniol
Paramol
Paraspen
Parelan
Pasolind N
Perfalgan
Phenaphen
Phendon
p-Hydroxyacetanilide
Prodafalgan
Puerxitong
Pyrinazine
Resfenol
Resprin
Rhodapop NCR
Salzone
Tabalgin
Tachipirina
Tempanal
Tralgon
Tylenol
TylolHot
Valadol
Valgesic
Vermidon
Vick Pyrena
DTXSID2020006968-81-0VGZSUPCWNCWDAN-UHFFFAOYSA-NVGZSUPCWNCWDAN-UHFFFAOYSA-N
AcetohexamideBenzenesulfonamide, 4-acetyl-N-[(cyclohexylamino)carbonyl]-
1-(p-Acetylbenzenesulfonyl)-3-cyclohexylurea
1-[(p-Acetylphenyl)sulfonyl]-3-cyclohexylurea
Acetohexamid
acetohexamida
Dimelin
Dimelor
Dymelor
Gamadiabet
Hypoglicil
Metaglucina
Minoral
N-(p-Acetylphenylsulfonyl)-N'-cyclohexylurea
Ordimel
Tsiklamid
Urea, 1-[(p-acetylphenyl)sulfonyl]-3-cyclohexyl-
DTXSID702000767-66-3HEDRZPFGACZZDS-UHFFFAOYSA-NHEDRZPFGACZZDS-UHFFFAOYSA-N
ChloroformTrichloromethane
Methane, trichloro-
CARBON TRICHLORIDE
Chloroforme
cloroformo
Formyl trichloride
Methane trichloride
Methane,trichloro-
NSC 77361
Trichloroform
UN 1888
DTXSID1020306110-00-9YLQBMQCUIZJEEH-UHFFFAOYSA-NYLQBMQCUIZJEEH-UHFFFAOYSA-N
FuranDivinylene oxide
furanne
Furfuran
Oxacyclopentadiene
Tetrole
UN 2389
DTXSID60206467429-90-5XAGFODPZIPBFFR-UHFFFAOYSA-NAZDRQVAHHNSJOQ-UHFFFAOYSA-N
AluminumAisin Metal Fiber
Al 050P-H24
ALC Fine
Alcan XI 1391
Almi-Paste SSP 303AR
Aloxal 3010
Alpaste 00-0506
Alpaste 0100M
Alpaste 0100MA
Alpaste 0100M-C
Alpaste 0200M
Alpaste 0200T
Alpaste 0230M
Alpaste 0230T
Alpaste 0241M
Alpaste 0300M
Alpaste 0500M
Alpaste 0539X
Alpaste 0620MS
Alpaste 0625TS
Alpaste 0638-70C
Alpaste 0700M
Alpaste 0780M
Alpaste 0900M
Alpaste 100M
Alpaste 100MS
Alpaste 100MSR
Alpaste 1100M
Alpaste 1100MA
Alpaste 1100N
Alpaste 1100NA
Alpaste 1109MA
Alpaste 1109MC
Alpaste 1200M
Alpaste 1200T
Alpaste 1260MS
Alpaste 1500MA
Alpaste 1700NL
Alpaste 1810YL
Alpaste 1830YL
Alpaste 1900M
Alpaste 1900XS
Alpaste 1950M
Alpaste 1950N
Alpaste 210N
Alpaste 2172EA
Alpaste 2173
Alpaste 240T
Alpaste 241M
Alpaste 417
Alpaste 46-046
Alpaste 4-621
Alpaste 4919
Alpaste 50-63
Alpaste 50-635
Alpaste 51-148B
Alpaste 51-231
Alpaste 5205N
Alpaste 5207N
Alpaste 52-509
Alpaste 52-568
Alpaste 5301N
Alpaste 5302N
Alpaste 53-119
Alpaste 5422NS
Alpaste 54-452
Alpaste 54-497
Alpaste 54-542
Alpaste 55-516
Alpaste 55-519
Alpaste 55-574
Alpaste 5620NS
Alpaste 5630NS
Alpaste 5640NS
Alpaste 56-501
Alpaste 5650NS
Alpaste 5653NS
Alpaste 5654NS
Alpaste 5680N
Alpaste 5680NS
Alpaste 60-600
Alpaste 60-760
Alpaste 60-768
Alpaste 62-356
Alpaste 6340NS
Alpaste 6370NS
Alpaste 6390NS
Alpaste 640NS
Alpaste 65-388
Alpaste 66NLB
Alpaste 710N
Alpaste 7130N
Alpaste 7160N
Alpaste 7160NS
Alpaste 725N
Alpaste 740NS
Alpaste 7430NS
Alpaste 7580NS
Alpaste 7620NS
Alpaste 7640NS
Alpaste 7670M
Alpaste 7670NS
Alpaste 7675NS
Alpaste 7679NS
Alpaste 7680N
Alpaste 7680NS
Alpaste 76840NS
Alpaste 7730N
Alpaste 7770N
Alpaste 7830N
Alpaste 8004
Alpaste 8080N
Alpaste 8260NAR
Alpaste 891K
Alpaste 91-0562
Alpaste 92-0592
Alpaste 93-0595
Alpaste 93-0647
Alpaste 94-2315
Alpaste 95-0570
Alpaste 96-0635
Alpaste 96-2104
Alpaste 97-0510
Alpaste 97-0534
Alpaste AW 520B
Alpaste AW 612
Alpaste AW 9800
Alpaste F 795
Alpaste FM 7680K
Alpaste FX 440
Alpaste FX 910
Alpaste FZ 0534
Alpaste FZU 40C
Alpaste G
Alpaste HR 8801
Alpaste HS 2
Alpaste J
Alpaste K 9800
Alpaste MC 666
Alpaste MC 707
Alpaste MF 20
Alpaste MG 01
Alpaste MG 1000
Alpaste MG 1300
Alpaste MG 500
Alpaste MG 600
Alpaste MH 6601
Alpaste MH 8801
Alpaste MH 9901
Alpaste MR 7000
Alpaste MR 9000
Alpaste MS 630
Alpaste N 1700NL
Alpaste NS 7670
Alpaste O 100N
Alpaste O 2130
Alpaste O 300M
Alpaste P 0100
Alpaste P 1950
Alpaste S
Alpaste SAP 110
Alpaste SAP 414P
Alpaste SAP 550N
Alpaste SCR 5070
Alpaste TCR 2020
Alpaste TCR 2060
Alpaste TCR 2070
Alpaste TCR 3010
Alpaste TCR 3030
Alpaste TCR 3040
Alpaste TCR 3130
Alpaste TD 200T
Alpaste UF 500
Alpaste WB 0230
Alpaste WD 500
Alpaste WJP-U 75C
Alpaste WX 0630
Alpaste WX 7830
Alpaste WXA 7640
Alpaste WXM 0630
Alpaste WXM 0650
Alpaste WXM 0660
Alpaste WXM 1415
Alpaste WXM 1440
Alpaste WXM 5422
Alpaste WXM 760b
Alpaste WXM 7640
Alpaste WXM 7675
Alpaste WXM-T 60B
Alpaste WXM-U 75
Alpaste WXM-U 75C
Altop X
Aluchrome Ultrafin Super
Alumat 1600
Alumet H 30
aluminio
Aluminium
Aluminium Flake
Aluminum 27
Aluminum atom
Aluminum element
Aluminum Flake PCF 7620
Aluminum granules
ALUMINUM METAL/GRANULE
ALUMINUM PASTE
ALUMINUM PIGMENT
ALUMINUM TURNINGS
Alumi-paste 640NS
Alumipaste 91-0562
Alumipaste 98-1822T
Alumipaste AW 620
Alumipaste CR 300
Alumipaste GX 180A
Alumipaste GX 201A
Alumipaste HR 7000
Alumipaste HR 850
Alumipaste MG 11
Alumipaste MH 8801
Aquamet NPW 2900
Aquapaste 205-5
Aquasilver LPW
Astroflake 40
Astroflake Black N 020
Astroflake Black N 070
Astroflake LG 40
Astroflake LG 70
Astroflake Silver N 040
Astroshine NJ 1600
Astroshine T 8990
Atomizalumi VA 200
C.I. PIGMENT METAL 1
Chromal IV
Chromal X
Decomet 1001/10
Decomet 2018/10
Decomet High Gloss Al 1002/10
Ecka AS 081
Eckart 9155
Eterna Brite 301-1
Eterna Brite 601-1
Eterna Brite 651-1
Eterna Brite EBP 251PA
Eterna Brite Primier 251PA
Ferro FX 53-038
Friend Color F 500GR-W
Friend Color F 500WT
Friend Color F 700RE-W
Friend Color F 701RE-W
Hi Print 60T
High Print 60T
Hisparkle HS 2
Hydro Paste 8726
Hydrolac WHH 2153
Hydrolan 3560
Hydrolux Reflexal 100
Hydroshine WS 1001
JISA 51010P
Kryal Z
Lansford 243
LE Sheet 800
Leafing Alpaste
LG-H Silver 25
Lunar Al-V 95
Metallux 161
Metallux 2154
Metallux 2192
Metalure
Metalure 55350
Metalure L 55350
Metalure L 59510
Metalure W 2001
Metapor
Metasheen 1800
Metasheen HR 0800
Metasheen KM 100
Metasheen KM 1000
Metasheen Slurry 1807
Metasheen Slurry 1811
Metasheen Slurry KM 100
Metax G
Metax S
Mirror Glow 1000
Mirror Glow 600
Mirrorsheen
Noral Aluminium
Noral Ink Grade Aluminium
Obron 10890
Offset FM 4500
Puratronic
Reflexal 145
Reynolds 400
Reynolds 4-301
Reynolds 4-591
Reynolds 667
SAP 260PW-HS
SAP-FM 4010
SBC 516-20Z
Scotchcal 7755SE
Serumekku
Setanium 50MIS-H8
Siberline ET 2025
Siberline ST 21030E1
Silvar A
Silver VT 522
Silverline SSP 353
Silvex 793-20C
Sparkle Silver 3141ST
Sparkle Silver 3500
Sparkle Silver 3641
Sparkle Silver 5000AR
Sparkle Silver 516AR
Sparkle Silver 5242AR
Sparkle Silver 5245AR
Sparkle Silver 5271AR
Sparkle Silver 5500
Sparkle Silver 5745
Sparkle Silver 7000AR
Sparkle Silver 7005AR
Sparkle Silver 7500
Sparkle Silver 960-25E1
Sparkle Silver E 1745AR
Sparkle Silver L 1526AR
Sparkle Silver Premier 751
Sparkle Silver SS 3130
Sparkle Silver SS 5242AR
Sparkle Silver SS 5588
Sparkle Silver SSP 132AR
Special PCR 507
Splendal 6001BG
Spota Mobil 801
SSP 760-20C
Stapa Aloxal PM 2010
Stapa Aloxal PM 3010
Stapa Aloxal PM 4010
Stapa Hydrolac BG 8n.1
Stapa Hydrolac BGH Chromal X
Stapa Hydrolac PM Chromal VIII
Stapa Hydrolac W 60NL
Stapa Hydrolac WH 16
Stapa Hydrolac WH 66NL
Stapa Hydrolux 2192
Stapa Hydrolux 8154
Stapa IL Hydrolan 2192-55900G
Stapa Metallic R 607
Stapa Metallux 1050
Stapa Metallux 211
Stapa Metallux 212
Stapa Metallux 2196
Stapa Metallux 274
Stapa Mobilux 181
Stapa Offset 3000
Stapa PV 10
Stapa VP 46432G
Starbrite 2100
Super Fine 18000
Super Fine 22000
Supramex 2022
Toyo Aluminum 02-0005
Toyo Aluminum 93-3040
Transmet K 102HE
Tufflake 3645
Tufflake 5843
UN 1396
US Aluminum 809
Valimet H 2
Valimet H 3
White Silver 7080N
White Silver 7130N
DTXSID30402737440-43-9BDOSMKKIYDKNTQ-UHFFFAOYSA-NBDOSMKKIYDKNTQ-UHFFFAOYSA-N
CadmiumCadimium
CADMIUM BLUE
CADMIUM, IN PLATTEN, STANGEN, BROCKEN,KOERNER
DTXSID10239407439-97-6QSHDDOUJBYECFT-UHFFFAOYSA-NQSHDDOUJBYECFT-UHFFFAOYSA-N
MercuryLiquid silver
Mercure
MERCURIC METAL TRIPLE DISTILLED
mercurio
Mercury element
Quecksilber
Quicksilver
UN 2024
UN 2809
DTXSID10241727440-61-1JFALSRSLKYAFGM-UHFFFAOYSA-NJFALSRSLKYAFGM-UHFFFAOYSA-N
UraniumUranium, isotope of mass 238
238U Element
UN 2979 (DOT)
Uranium I
DTXSID10425227440-38-2RQNWIZPPADIBDY-UHFFFAOYSA-NRQNWIZPPADIBDY-UHFFFAOYSA-N
ArsenicAs
Arsenic black
ARSENIC METAL
arsenico
Grey arsenic
UN 1558
DTXSID40238867440-22-4BQCADISMDOOEFD-UHFFFAOYSA-NBQCADISMDOOEFD-UHFFFAOYSA-N
SilverAg Nanopaste NPS-J 90
Ag Sphere 2
Ag-C-GS
Algaedyn
Arctic Silver 3
Argentum
Astroflake 5
Carey Lea silver
Colloidal silver
Dotite XA 208
Du Pont 4943
ECM 100AF4810
Enlight 600
Enlight silver plate 600
Epinall
Finesphere SVND 102
Fordel DC
FP 5369-502
Jelcon SH 1
Jungindai Takasago 300
KS (metal)
LCP 1-19SFS
Metz 3000-1
Nanomelt AGC-A
Nanomelt Ag-XA 301
Nanomelt Ag-XF 301
Nanomelt Ag-XF 301H
Nanopaste NPS-J 90
Perfect Silver
Puff Silver X 1200
RT 1710S-C1
SD (metal)
Shell Silver
Silbest E 20
Silbest F 20
Silbest J 18
Silbest TC 12
Silbest TC 20E
Silbest TC 25A
Silbest TCG 1
Silbest TCG 7
Silcoat AgC 103
Silcoat AgC 2011
Silcoat AgC 209
Silcoat AgC 2190
Silcoat AgC 222
Silcoat AgC 2411
Silcoat AgC 74T
Silcoat AgC-A
Silcoat AgC-AO
Silcoat AgC-B
Silcoat AgC-BO
Silcoat AgC-D
Silcoat AgC-G
Silcoat AgC-GS
Silcoat AgC-L
Silcoat AgC-O
Silcoat GS
Silcoat RF 200
Silflake 135
Silsphere 514
Silver atom
Silver element
Silver Flake 1
Silver Flake 25
Silver Flake 52
Silver Flake 7A
SILVER FLAKES
Silver metal
Silvest TCG 11N
Technic 299
Technic 450
Techno Alpha 175
DTXSID40243057439-96-5PWHULOQIROXLJO-UHFFFAOYSA-NPWHULOQIROXLJO-UHFFFAOYSA-N
ManganeseColloidal manganese
Cutaval
Manganese element
Manganese fulleride
Manganese metal alloy
Manganese-55
manganeso
DTXSID20241697440-02-0PXHVJJICTQNCMI-UHFFFAOYSA-NPXHVJJICTQNCMI-UHFFFAOYSA-N
NickelCarbonyl 255
Carbonyl Ni 123
Carbonyl Ni 283
Carbonyl Nickel 123
Carbonyl Nickel 283
Carbonyl Nickel 287
Cerac N 2003
CNS 10 Micron
Exmet 4 Ni X-4/0
Fibrex P
Incofoam
Nickel element
NICKEL ROUND ANODES
Nicrobraz LM:BNi 2
Ni-Flake 95
Novamet 123
Novamet 4SP
Novamet 4SP10
Novamet 525
Novamet CNS 400
Novamet HCA 1
Novamet NI 255
Raney nickel
Raney nickel 2800
UN 1325
UN 2881
DTXSID20209257440-66-6HCHKCACWOHOZIP-UHFFFAOYSA-NHCHKCACWOHOZIP-UHFFFAOYSA-N
ZincZn
Asarco L 15
C.I. Pigment Black 16
Merrillite
NC-Zinc
Rheinzink
Stapa TE Zinc AT
UF (metal)
UN 1436
Zinc dust
Zinc Dust 3
Zinc Dust 500 mesh
Zinc Dust LS 2
Zinc Dust MCS
Zinc Flakes GTT
ZINC METAL
ZINC MOSSY
ZINC STRIP
ZINC, MOSSY
Zincsalt GTT
DTXSID7035012UBERON:0001986endotheliumUBERON:0001981blood vesselMP:0003674oxidative stressGO:0002526acute inflammatory responseGO:0033484nitric oxide homeostasisGO:0001974blood vessel remodeling1increased7functional change3occurrenceIonizing Radiation<p>Ionizing radiation can vary in energy, dose, charge, and in the spatial distributions of energy transferred to other matter (linear energy transfer per unit length or LET) (ICRU 1970). At the same dose, low and high LET both generate energy deposition events, including many higher energy events (Goodhead and Nikjoo 1989). However, they differ in the spatial distribution and upper range of intensity of energy deposited. Lower LET such as gamma rays sparsely deposit many individual excitations or small clusters of excitations of low energy (Goodhead 1988). In contrast, high LET such as alpha particles have fewer tracks but readily transfer their energy to matter and therefore deposit their energy over a much smaller area (Goodhead 1994). Consequently, alpha and other high LET particles penetrate less deeply into tissue, interactions are densely focused on a narrow track, and individual energy depositions can be large (Goodhead 1988). These different energy deposition patterns can lead to differences in radiation effects including the pattern of DNA damage.</p>
<p>Exposure to ionizing radiation can come from natural and industrial sources. Space and terrestrial radiation includes a range of LET particles, while diagnostic radiation methods such as X-ray imaging, mammography and CT scans use low LET X-rays. Radiation therapy can use an external beam to direct radiation on a focused tissue area, or deposit solid or liquid radioactive materials in the body that release (mostly gamma) radiation internally. External radiotherapy typically uses X-rays but is moving towards higher LET charged particles such as protons and heavy ions (Durante, Orecchia et al. 2017).</p>
2019-05-03T12:36:362019-05-07T12:12:13Acetaminophen2016-11-29T18:42:262016-11-29T18:42:26Chloroform2016-11-29T18:42:272016-11-29T18:42:27furan2020-05-01T14:35:222020-05-01T14:35:22Platinum2022-02-04T14:36:542022-02-04T14:36:54Aluminum2022-02-04T14:42:112022-02-04T14:42:11Cadmium2017-10-25T08:33:122017-10-25T08:33:12Mercury2016-11-29T18:42:192016-11-29T18:42:19Uranium2021-08-05T14:28:502021-08-05T14:28:50Arsenic2021-04-27T00:15:212021-04-27T00:15:21Silver 2022-02-03T11:20:112022-02-03T11:20:11Manganese2022-02-04T14:47:232022-02-04T14:47:23Nickel2022-02-04T14:47:592022-02-04T14:47:59Zinc2022-02-04T15:05:002022-02-04T15:05:00nanoparticles2016-12-21T09:40:062016-12-21T09:40:06Topoisomerase inhibitors2019-05-19T20:21:242019-05-19T20:21:24Radiomimetic compounds2019-05-19T20:21:422019-05-19T20:21:42WCS_9606human10116rat10090mouse6239nematodeWCS_7955zebrafish3702thale-cress3349Scotch pineWCS_35525Daphnia magna3055Chlamydomonas reinhardtiiWCS_6396common brandling wormWCS_4472Lemna minor8030Salmo salarWikiUser_26rodents9606Homo sapiensWikiUser_28VertebratesWCS_9986rabbitWikiUser_25human and other cells in culture9913bovineWikiUser_24Pig9823pigsDeposition of EnergyEnergy DepositionMolecular<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">Deposition of energy refers to events where energetic subatomic particles, nuclei, or electromagnetic radiation deposit energy in the media through which they transverse. The energy may either be sufficient (</span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">e.g. ionizing radiation) or insufficient (e.g. non-ionizing radiation) to ionize atoms or molecules<span style="background-color:white"> (Beir et al.,1999</span>). </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Ionizing radiation can cause the ejection of electrons from atoms and molecules, thereby</span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"> resulting in their ioniz</span></span><span style="font-family:"Times New Roman",serif">ation</span></span> <span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">and the </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">breakage of</span></span> <span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">chemical<span style="font-size:16px"> <span style="font-family:Times New Roman,Times,serif">bonds. </span></span></span></span></span></span></span><span style="font-size:14px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:16px">The excitation of molecules can also occur without ionization. </span>T</span></span><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">hese events are stochastic and unpredictable. </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:16px">Th</span>e energy of these subatomic particles or electromagnetic waves mostly range from 124 KeV to 5.4 MeV and is dependent on the source and type of radiation (Zyla et al., 2020). To be ionizing the incident radiation must have sufficient energy to free electrons from atomic or molecular electron orbitals. The energy deposited can induce direct and indirect ionization events and this</span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"> can be via internal (injections, inhalation, or absorption of radionuclides) or external exposure from radiation fields -- this also applies to non-ionizing radiation. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">Not all electromagnetic radiation is ionizing; as the incident radiation must have sufficient energy to free electrons from the electron orbitals of the atom or molecule. The energy deposited can induce direct and indirect ionization events and can result from internal (injections, inhalation, ingestion) or external exposure. </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">Direct ionization is the principal path where charged particles interact with biological structures such as DNA, proteins or membranes to cause biological damage. Photons, which are electromagnetic waves can also create electrons that can cause direct ionization. </span></span></span></span></span><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">Indirect ionization produces free radicals from other molecules, specifically water, which can then transform to cause damage to critical targets such as DNA.</span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif"> </span></span>Ionization of water, which is a major constituent of tissues and organs, produces free radical and molecular species, which themselves can indirectly damage critical targets such as DNA (Beir et al., 1999; </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Balagamwala et al., 2013) or alter cellular processes. Given the fundamental nature of energy deposition by radioactive/unstable nuclei, nucleons or elementary particles in material, this process is universal to all biological contexts. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">Energy deposition differs with the linear energy transfer (LET) defined as deposition of energy per unit distance (Hall and Giaccia, 2018 UNSCEAR, 2020). High LET radiation refers to energy mostly above 10 keV μm-1 which often produces more complex, dense structural damage than low LET radiation (below 10 keV μm-1). High LET radiation includes heavy ions, alpha particles and high-energy neutrons. Low-LET radiation such as photons (X- and gamma rays), electrons as well as high-energy protons produces sparse ionization events. Low LET radiation travels farther into tissue but deposits smaller amounts of energy, whereas typically high LET particles, do not travel as far but deposits larger amounts of energy into tissue at the same absorbed dose. The biological effect of the deposition of energy can be modulated by varying dose and dose rate of exposure, such as in acute, chronic, or fractionated exposures (Hall and Giaccia, 2018). </span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Non-ionizing radiation is electromagnetic waves that does not have enough energy to break bonds and induce ion formation but it can cause molecules to excite and vibrate faster resulting in ensuing biological effects. </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Examples of non-ionizing radiation include radio waves (wavelength: 100 km-1m), microwaves (wavelength: 1m-1mm), infrared radiation (wavelength: 1mm- 1 um), visible light (wavelengths: 400-700 nm), and ultraviolet radiation of longer wavelengths such as UVB (wavelengths: 315-400nm) and UVA (wavelengths: 280-315 nm). UVC radiation (200-289 nm) is, in contrast to UVB and UVA, considered to be a type of ionizing radiation<span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">. </span></span></span></span></span></span><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">Exposure to non-ionizing radiation occurs either from natural or anthropogenic sources, and include radio waves used for communication (broadcasting and cell phones), microwaves used in cooking food and in radar systems, infrared radiation emitted by warm objects or used in remote controls, thermal imaging and medical treatments. Visible light is the range of electromagnetic radiation and is commonly used in photosynthesis in primary producers. UV radiation has key functions in melanisation (tanning) of a number of species and exhibits key signalling roles in navigation and communication (e.g insects, aquatic invertebrates and fish), locomotory and predatory behavior (e.g. reptiles, birds and crustaceans) and growth and development (e.g. plants). UV radiation is also used in some medical treatments such as skin diseases (e.g. psoriasis, eczema, vitiligo and skin cancers). </span></span></p>
<table border="1" bordercolor="#ccc" cellpadding="5" cellspacing="0" style="border-collapse:collapse">
<tbody>
<tr>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Radiation type</strong></span></span></td>
<td style="background-color:#eeeeee; text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Assay Name</strong></span></span></p>
</td>
<td style="background-color:#eeeeee; text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>References</strong></span></span></p>
</td>
<td style="background-color:#eeeeee; text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Description</strong></span></span></p>
</td>
<td style="background-color:#eeeeee; text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>OECD Approved Assay</strong></span></span></p>
</td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ionizing radiation</span></span></td>
<td style="text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Monte Carlo Simulations (Geant4)</span></span></p>
</td>
<td style="text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Douglass et al., 2013; Douglass et al. 2012; Zyla et al., 2020</span></span></p>
</td>
<td style="text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Monte Carlo simulations are based on a computational algorithm that mathematically models the deposition of energy into materials.</span></span></p>
</td>
<td style="text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></p>
</td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ionizing radiation</span></span></td>
<td style="text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Fluorescent Nuclear Track Detector (FNTD)</span></span></p>
</td>
<td style="text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sawakuchi, 2016; Niklas, 2013; Koaira & Konishi, 2015</span></span></p>
</td>
<td style="text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">FNTDs are biocompatible chips with crystals of aluminium oxide doped with carbon and magnesium; used in conjuction with fluorescent microscopy, these FNTDs allow for the visualization and the linear energy transfer (LET) quantification of tracks produced by the deposition of energy into a material.</span></span></p>
</td>
<td style="text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></p>
</td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ionizing radiation</span></span></td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Tissue equivalent proportional counter (TEPC)</span></span></td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Straume et al, 2015</span></span></td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Measure the LET spectrum and calculate the dose equivalent.</span></span></td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ionizing radiation</span></span></td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">alanine dosimeters/NanoDots</span></span></td>
<td style="text-align:center">
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Lind et al. 2019; Xie et al., 2022</span></span></p>
</td>
<td style="text-align:center"> </td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Non-ionizing radiation</span></span></td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">UV meters or radiameters</span></span></td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Xie et at., 2020</span></span></td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">UVA/UVB (irradiance intensity), UV dosimeters (accumulated irradiance over time), Spectrophoto meter (absorption of UV by a substance or material)</span></span></td>
<td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></td>
</tr>
</tbody>
</table>
<p> </p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Energy can be deposited into any substrate, both living and non-living; it is independent of age, taxa, sex, or life-stage. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Taxonomic applicability: </strong>This MIE is not taxonomically specific. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Life stage applicability: </strong>This MIE is not life stage specific. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Sex applicability: </strong>This MIE is not sex specific. </span></span></p>
LowUnspecificHighAll life stagesModerateModerateModerateHighHighHighModerateHighModerateModerateHighLow<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Balagamwala, E. H. et al. (2013), “Introduction to radiotherapy and standard teletherapy techniques”,<em> Dev Ophthalmol,</em> Vol. 52, Karger, Basel, https://doi.org/10.1159/000351045 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Beir, V. et al. (1999), “The Mechanistic Basis of Radon-Induced Lung Cancer”, in <em>Health Risks of Exposure to Radon: BEIR V</em>I, National Academy Press, Washington, D.C., https://doi.org/10.17226/5499 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Douglass, M. et al. (2013), “Monte Carlo investigation of the increased radiation deposition due to gold nanoparticles using kilovoltage and megavoltage photons in a 3D randomized cell model”<em>, Medical Physics</em>, Vol. 40/7, American Institute of Physics, College Park, https://doi.org/10.1118/1.4808150 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Douglass, M. et al. (2012), “Development of a randomized 3D cell model for Monte Carlo microdosimetry simulations.”, <em>Medical Physics</em>, Vol. 39/6, American Institute of Physics, College Park, https://doi.org/10.1118/1.4719963 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hall, E. J. and Giaccia, A.J. (2018), <em>Radiobiology for the Radiologist</em>, 8th edition, Wolters Kluwer, Philadelphia. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kodaira, S. and Konishi, T. (2015), “Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector.”, <em>Journal of Radiation Research</em>, Vol. 56/2, Oxford University Press, Oxford, https://doi.org/10.1093/jrr/rru091 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Lind, O.C., D.H. Oughton and Salbu B. (2019), "The NMBU FIGARO low dose irradiation facility", <em>International Journal of Radiation Biology</em>, Vol. 95/1, Taylor & Francis, London, https://doi.org/10.1080/09553002.2018.1516906.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sawakuchi, G.O. and Akselrod, M.S. (2016), “Nanoscale measurements of proton tracks using fluorescent nuclear track detectors.”,<em> Medical Physics</em>, Vol. 43/5, American Institute of Physics, College Park, https://doi.org/10.1118/1.4947128 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Straume, T. et al. (2015), “Compact Tissue-equivalent Proportional Counter for Deep Space Human Missions.”,<em> Health physics,</em> Vol. 109/4, Lippincott Williams & Wilkins, Philadelphia, https://doi.org/10.1097/HP.0000000000000334 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Niklas, M. et al. (2013), “Engineering cell-fluorescent ion track hybrid detectors.”, <em>Radiation Oncology</em>, Vol. 8/104, BioMed Central, London, https://doi.org/10.1186/1748-717X-8-141 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">UNSCEAR (2020), <em>Sources, effects and risks of ionizing radiation</em>, United Nations. </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Xie, Li. et al. (2022), "Ultraviolet B Modulates Gamma Radiation-Induced Stress Responses in Lemna Minor at Multiple Levels of Biological Organisation", <em>SSRN</em>, Elsevier, Amsterdam, http://dx.doi.org/10.2139/ssrn.4081705 .</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Zyla, P.A. et al. (2020)<em>, Review of particle physics: Progress of Theoretical and Experimental Physics,</em> 2020 Edition, Oxford University Press, Oxford. </span></span></p>
<p> </p>
<p> </p>
2019-08-22T09:44:232024-03-08T11:49:39Oxidative Stress Oxidative Stress Molecular<p style="text-align:justify">Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell (Pizzino et al., 2017; Sharifi-Rad et al., 2020; Jena et al., 2023). As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al. 2009) and can be used as indicators of the presence of oxidative stress in the cell.</p>
<p style="text-align:justify">In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides (2SH àSS) on neighboring amino acids (Antelmann and Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010).</p>
<p><span style="font-size:16px"><span style="background-color:white"><span style="color:#2f5597">ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase, <span style="background-color:white">catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol</span></span></span><span style="color:#2f5597">, or antioxidant vitamins and minerals including vitamin E, C, carotene, lutein, zeaxanthin, selenium, and zinc (Fletcher, 2010). The enzymes, vitamins and minerals catalyze the conversion of ROS to non-toxic molecules such as water and O<sub>2</sub></span><span style="background-color:white"><span style="color:#2f5597"><span style="background-color:white">. However, these antioxidant systems are not perfect and endogenous metabolic processes and/or exogenous oxidative influences can trigger cumulative oxidative injuries to the mitochondria, causing a decline in their functionality and efficiency, which further promotes cellular oxidative stress (</span></span></span></span><span style="color:#2f5597">Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017)<span style="font-size:16px"><span style="background-color:white"><span style="background-color:white">.</span></span></span></span></p>
<p><span style="color:#27ae60"><span style="font-size:18px"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif"><span style="background-color:white">However, an emerging viewpoint suggests that ROS-induced modifications may not be as detrimental as previously thought, but rather contribute to signaling processes (Foyer et al., 2017). </span></span></span></span></span></span></p>
<p style="text-align:justify">Protection against oxidative stress is relevant for all tissues and organs, although some tissues may be more susceptible. For example, the brain possesses several key physiological features, such as high O2 utilization, high polyunsaturated fatty acids content, presence of autooxidable neurotransmitters, and low antioxidant defenses as compared to other organs, that make it highly susceptible to oxidative stress (Halliwell, 2006; Emerit and al., 2004; Frauenberger et al., 2016).</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Sources of ROS Production</span></span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Direct Sources:</span></span></strong><span style="font-size:12.0pt"><span style="color:#2f5597"> Direct sources involve the deposition of energy onto water molecules, breaking them into active radical species. When ionizing radiation hits water, it breaks it into hydrogen (H*) and hydroxyl (OH*) radicals by destroying its bonds. The hydrogen will create hydroxyperoxyl free radicals (HO<sub>2</sub>*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and more O<sub>2</sub> (Elgazzar and Kazem, 2015). Antioxidant mechanisms are also affected by radiation, with catalase (CAT) and peroxidase (POD) levels rising as a result of exposure (Seen et al. 2018; Ahmad et al. 2021). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Indirect Sources:</span></span></strong><span style="font-size:12.0pt"><span style="color:#2f5597"> An indirect source of ROS is the mitochondria, which is one of the primary producers in eukaryotic cells (Powers et al., 2008). As much as 2% of the electrons that should be going through the electron transport chain in the mitochondria escape, allowing them an opportunity to interact with surrounding structures. Electron-oxygen reactions result in free radical production, including the formation of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O<sub>2</sub>, and inorganic phosphate (P<sub>i</sub>) (Hargreaves et al. 2020; Raimondi et al. 2020; Vargas-Mendoza et al. 2021). The first and third complexes of the transport chain are the most relevant to mammalian ROS production (Raimondi et al., 2020). The mitochondria have its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS are also produced through nicotinamide adenine dinucleotide phosphate oxidase (NOX) stimulation, an event commenced by angiotensin II, a product/effector of the renin-angiotensin system (Nguyen Dinh Cat et al. 2013; Forrester et al. 2018). Other ROS producers include xanthine oxidase, immune cells (macrophage, neutrophils, monocytes, and eosinophils), phospholipase A<sub>2</sub> (PLA<sub>2</sub>), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021).</span></span></span></span></p>
<p><strong>Oxidative Stress. Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage.</strong><span style="color:#27ae60"> Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed</span></p>
<ul>
<li>Detection of ROS by chemiluminescence <span style="font-size:12px">(<span style="font-family:arial,helvetica,sans-serif">https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683)</span></span></li>
<li>Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential.</li>
<li>Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html). </li>
<li>TBARS. Oxidative damage to lipids can be measured by assaying for lipid peroxidation using TBARS (thiobarbituric acid reactive substances) using a commercially available kit. </li>
<li>8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or HPLC, described in Chepelev et al. (Chepelev, et al. 2015).</li>
</ul>
<p><strong>Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include:</strong></p>
<ul>
<li>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus</li>
<li>Western blot for increased Nrf2 protein levels</li>
<li>Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleus</li>
<li>qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences)</li>
<li>Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014)</li>
<li>OECD TG422D describes an ARE-Nrf2 Luciferase test method</li>
<li>In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation</li>
</ul>
<p> </p>
<table border="1" cellpadding="1" cellspacing="1">
<tbody>
<tr>
<td><strong>Assay Type & Measured Content</strong></td>
<td><strong>Description</strong></td>
<td><strong>Dose Range Studied</strong></td>
<td>
<p><strong>Assay Characteristics </strong><strong>(Length / Ease of use/Accuracy)</strong></p>
</td>
</tr>
<tr>
<td>
<p><strong>ROS Formation in the Mitochondria assay</strong> (Shaki et al., 2012)</p>
</td>
<td>“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 μM) in respiration buffer containing (0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 μM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 μM) to mitochondria and was then incubated for 10 min. Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.”</td>
<td>0, 50, 100 and 200 μM of Uranyl Acetate</td>
<td>
<p>Long/ Easy</p>
<p>High accuracy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Mitochondrial Antioxidant Content Assay</strong> Measuring GSH content</p>
(Shaki et al., 2012)</td>
<td>“GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as μg/mg protein.”</td>
<td>
<p>0, 50, 100, or 200 <em>μ</em>M Uranyl Acetate</p>
</td>
<td> </td>
</tr>
<tr>
<td>
<p><strong>H<sub>2</sub>O<sub>2</sub> Production Assay</strong> Measuring H<sub>2</sub>O<sub>2</sub> Production in isolated mitochondria</p>
(Heyno et al., 2008)</td>
<td>“Effect of CdCl<sub>2</sub> and antimycin A (AA) on H<sub>2</sub>O<sub>2</sub> production in isolated mitochondria from potato. H<sub>2</sub>O<sub>2</sub> production was measured as scopoletin oxidation. Mitochondria were incubated for 30 min in the measuring buffer (see the Materials and Methods) containing 0.5 mM succinate as an electron donor and 0.2 µM mesoxalonitrile 3‐chlorophenylhydrazone (CCCP) as an uncoupler, 10 U horseradish peroxidase and 5 µM scopoletin.” (</td>
<td>
<p>0, 10, 30  <em>μ</em>M Cd<sup>2+</sup></p>
2  <em>μ</em>M<br />
antimycin A</td>
<td> </td>
</tr>
<tr>
<td>
<p><strong>Flow Cytometry ROS & Cell Viability</strong></p>
(Kruiderig et al., 1997)</td>
<td>“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At <em>t </em>5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”</td>
<td> </td>
<td>
<p>Strong/easy</p>
medium</td>
</tr>
<tr>
<td>
<p><strong>DCFH-DA Assay</strong> Detection of hydrogen peroxide production (Yuan et al., 2016)</p>
</td>
<td>
<p>Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H<sub>2</sub>O<sub>2 </sub>to form fluorescent production. </p>
</td>
<td>0-400 µM</td>
<td>
<p>Long/ Easy</p>
<p>High accuracy</p>
</td>
</tr>
<tr>
<td>
<p><strong>H2-DCF-DA Assay</strong> Detection of superoxide production (Thiebault et al., 2007)</p>
</td>
<td>This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer.</td>
<td>0–600 µM</td>
<td>
<p>Long/ Easy</p>
<p>High accuracy</p>
</td>
</tr>
<tr>
<td><strong>CM-H2DCFDA Assay</strong></td>
<td>**Come back and explain the flow cytometry determination of oxidative stress from Pan et al. (2009)**</td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>
<p>Direct Methods of Measurement</p>
<table cellspacing="0" class="Table" style="border-collapse:collapse; width:623px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Method of Measurement</span></span></strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">References</span></span></strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Description</span></span></strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">OECD-Approved Assay</span></span></strong></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Chemiluminescence </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Lu, C. et al., 2006; </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">ROS can induce electron transitions in molecules, leading to electronically excited products. When the electrons transition back to ground state, chemiluminescence is emitted and can be measured. Reagents such as uminol and lucigenin are commonly used to amplify the signal. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
<p> </p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Spectrophotometry </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">NO has a short half-life. However, if it has been reduced to nitrite (NO2-), stable azocompounds can be formed via the Griess Reaction, and further measured by spectrophotometry. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Direct or Spin Trapping-Based Electron Paramagnetic Resonance (EPR) Spectroscopy </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The unpaired electrons (free radicals) found in ROS can be detected with EPR, and is known as electron paramagnetic resonance. A variety of spin traps can be used. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Nitroblue Tetrazolium Assay </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The Nitroblue Tetrazolium assay is used to measure O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> levels. O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> reduces nitroblue tetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescence analysis of DHE is used to measure O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> levels. O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> is reduced to O2 as DHE is oxidized to 2-hydroxyethidium, and this reaction can be measured by fluorescence. Similarly, hydrocyans can be oxidized by any ROS, and measured via fluorescence. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Amplex Red Assay </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescence analysis to measure extramitochondrial or extracellular H<sub>2</sub>O<sub>2</sub> levels. In the presence of horseradish peroxidase and H<sub>2</sub>O<sub>2</sub>, Amplex Red is oxidized to resorufin, a fluorescent molecule measurable by plate reader. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Dichlorodihydrofluorescein Diacetate (DCFH-DA) </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">An indirect fluorescence analysis to measure intracellular H<sub>2</sub>O<sub>2</sub> levels. H<sub>2</sub>O<sub>2</sub> interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it to dichlorofluorescein (DCF), a fluorescent product. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">HyPer Probe </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescent measurement of intracellular H<sub>2</sub>O<sub>2</sub> levels. HyPer is a genetically encoded fluorescent sensor that can be used for <em>in vivo</em> and<em> in situ </em>imaging. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Cytochrome c Reduction Assay </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The cytochrome c reduction assay is used to measure O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> levels. O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> is reduced to O2 as ferricytochrome c is oxidized to ferrocytochrome c, and this reaction can be measured by an absorbance increase at 550 nm. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Proton-electron double-resonance imagine (PEDRI)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The redox state of tissue is detected through nuclear magnetic resonance/magnetic resonance imaging, with the use of a nitroxide spin probe or biradical molecule. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Glutathione (GSH) depletion </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Biesemann, N. et al., 2018) </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">A downstream target of the Nrf2 pathway is involved in GSH synthesis. As an indication of oxidation status, GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., </span></span><span style="color:#2f5597"><a href="http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html"><span style="font-size:12.0pt"><span style="color:#2f5597">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</span></span></a></span><span style="font-size:12.0pt"><span style="color:#2f5597">). </span></span></span></span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Thiobarbituric acid reactive substances (TBARS) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit. </span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Protein oxidation (carbonylation)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Azimzadeh et al., 2017; Azimzadeh etal., 2015; Ping et al., 2020)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Can be determined with enzyme-linked immunosorbent assay (ELISA) or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress.</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"><span style="color:#27ae60">Seahorse XFp Analyzer </span></td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"><span style="color:#27ae60">Leung et al. 2018 </span></td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"><span style="color:#27ae60">The Seahorse XFp Analyzer provides information on mitochondrial function, oxidative stress, and metabolic dysfunction of viable cells by measuring respiration (oxygen consumption rate; OCR) and extracellular pH (extracellular acidification rate; ECAR). </span></td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"><span style="color:#27ae60">No </span></td>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Molecular Biology:</span></span></strong><span style="font-size:12.0pt"><span style="color:#2f5597"> Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include: </span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Method of Measurement</span></span></strong> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">References</span></span></strong> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Description</span></span></strong> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">OECD-Approved Assay</span></span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Immunohistochemistry </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Amsen, D., de Visser, K. E., and Town, T., 2009)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus </span></span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Quantitative polymerase chain reaction (qPCR) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Forlenza et al., 2012)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences) </span></span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px">
<p style="text-align:center"><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Jackson, A. F. et al., 2014)</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway</span></span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="color:#27ae60"><strong>Taxonomic applicability: </strong>Occurrence of oxidative stress is not species specific. </span></p>
<p><span style="color:#27ae60"><strong>Life stage applicability:</strong> Occurrence of oxidative stress is not life stage specific. </span></p>
<p><span style="color:#27ae60"><strong>Sex applicability: </strong>Occurrence of oxidative stress is not sex specific. </span></p>
<p><span style="color:#27ae60"><strong>Evidence for perturbation by prototypic stressor:</strong> There is evidence of the increase of oxidative stress following perturbation from a variety of stressors including exposure to ionizing radiation and altered gravity (Bai et al., 2020; Ungvari et al., 2013; Zhang et al., 2009). </span></p>
HighMixedHighAll life stagesHighHigh<p style="margin-left:48px; text-align:left"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Ahmad, S. et al. (2021), “60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression”, <em>Journal Insect Science,</em> Vol. 21/5, Oxford University Press, Oxford, </span><a href="https://doi.org/10.1093/jisesa/ieab080" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/jisesa/ieab080</a></span></span></p>
<p style="margin-left:48px; text-align:left"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, <em>Antioxidants & Redox Signaling</em>, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3400" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1089/ars.2010.3400</a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in <em>Inflammation and Cancer</em>, Humana Press, Totowa, </span></span><a href="https://doi.org/10.1007/978-1-59745-447-6_5" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#1155cc">https://doi.org/10.1007/978-1-59745-447-6_5</span></span></span></a> </span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, <em>Journal of Proteome Research</em>, Vol. 14/2, American Chemical Society, Washington, </span></span></span><a href="https://doi.org/10.1021/pr501141b" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1021/pr501141b</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, <em>International Journal of Radiation Biology</em>, Vol. 93/9, Informa, London, </span></span></span><a href="https://doi.org/10.1080/09553002.2017.1339332" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1080/09553002.2017.1339332</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="color:#27ae60">Azzam, E. I. et al. (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, Cancer Letters, Vol. 327/1-2, Elsevier, Ireland, https://doi.org/10.1016/j.canlet.2011.12.012 </span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, <em>American Journal of Physiology - Cell Physiology</em>, Vol. 318/5, American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/ajpcell.00520.2019." style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/ajpcell.00520.2019.</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, <em>Journal of ocular pharmacology and therapeutics</em>, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, <a href="https://doi.org/10.1089/jop.2000.16.285.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a> </span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”, <em>Scientific Reports, </em>Vol. 8/1,</span></span> <span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Nature Portfolio, London, </span></span><a href="https://doi.org/10.1038/s41598-018-27614-8" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1038/s41598-018-27614-8</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in <em>The Pathophysiologic Basis of Nuclear Medicine</em>, Springer, New York, pp. 540-548</span></span></span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, <em>Ophthalmic Research</em>, Vol. 44, Karger International, Basel, <a href="https://doi.org/10.1159/000316476.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a> </span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Forlenza, M. et al. (2012), “The use of real-time quantitative PCR for the analysis of cytokine mRNA levels” in <em>Cytokine Protocols, </em>Springer, New York, </span></span><a href="https://doi.org/10.1007/978-1-61779-439-1_2" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1007/978-1-61779-439-1_2</span></span></a><strong> </strong></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Forrester, S.J. et al. </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">(2018), “Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology”, <em>Physiological Reviews, </em>Vol. 98/3<strong>,</strong> American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/physrev.00038.201" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/physrev.00038.201</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="color:#27ae60"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Foyer, C. H., A. V. Ruban, and G. Noctor (2017), “Viewing oxidative stress through the lens of oxidative signalling rather than damage”, Biochemical Journal, Vol. 474/6, Portland Press, England, https://doi.org/10.1042/BCJ20160814 </span></span></span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Ganea, E. and J. J. Harding (2006), “Glutathione-related enzymes and the eye”, <em>Current eye research</em>, Vol. 31/1, Informa, London, <a href="https://doi.org/10.1080/02713680500477347.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/02713680500477347.</a> </span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#222222">Griendling, K. K. et al. (2016), “Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent sign</span></span></span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">aling in the cardiovascular system: a scientific statement from the American Heart Association”, <em>Circulation research</em>, Vol. 119/5, Lippincott Williams & Wilkins, Philadelphia, </span></span></span></span><a href="https://doi.org/10.1161/RES.0000000000000110" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1161/RES.0000000000000110</span></span></span></a> </span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#303030">Guo, C.</span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"> et al. (2013), “Oxidative stress, mitochondrial damage and neurodegenerative diseases”, <em>Neural regeneration research</em>, Vol. 8/21, Publishing House of Neural Regeneration Research, China, </span></span><a href="https://doi.org/10.3969/j.issn.1673-5374.2013.21.009" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/<span style="background-color:white">10.3969/j.issn.1673-5374.2013.21.009</span></span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#222222">Hargreaves, M., and L. L. Spriet (2020), “Skeletal muscle energy metabolism during exercise.”, <em>Nature Metabolism</em>, Vol. 2, Nature Portfolio, London, </span></span></span><a href="https://doi.org/10.1038/s42255-020-0251-4" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1038/s42255-020-0251-4</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Hladik, D. and S. Tapio (2016), “Effects of ionizing radiation on the mammalian brain”, <em>Mutation Research/Reviews in Mutation Research</em>, Vol. 770, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.mrrev.2016.08.003" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/j.mrrev.2016.08.003</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Itoh, K., J. Mimura and M. Yamamoto (2010), “Discovery of the negative regulator of Nrf2, Keap1: a historical overview”, <em>Antioxidants & Redox Signaling</em>, Vol. 13/11, Mary Ann Leibert Inc., Larchmont, </span></span><a href="https://doi.org/10.1089/ars.2010.3222" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1089/ars.2010.3222</span></span></a> </span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Jackson, A.F. et al. (2014), “Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan.”, <em>Toxicology and Applied Pharmacology, </em>Vol. 274/11, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.taap.2013.10.019" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/j.taap.2013.10.019</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Jacobsen, N.R. et al. </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">(2008), “Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C<sub>60</sub> fullerenes in the FE1-Muta<sup>TM </sup>Mouse lung epithelial cells”, <em>Environmental and Molecular Mutagenesis,</em> Vol. 49/6, John Wiley & Sons, Inc., Hoboken, </span></span></span><a href="https://doi.org/10.1002/em.20406" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1002/em.20406</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Karimi, N. et al. (2017), “Radioprotective effect of hesperidin on reducing oxidative stress in the lens tissue of rats”, <em>International Journal of Pharmaceutical Investigation</em>, Vol. 7/3, Phcog Net, Bengaluru, <a href="https://doi.org/10.4103/jphi.JPHI_60_17.%E2%80%AF" rel="noreferrer noopener" target="_blank">https://doi.org/10.4103/jphi.JPHI_60_17.</a></span></p>
<p style="margin-left:48px"><span style="color:#27ae60"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Leung, D.T.H., and Chu, S. (2018), “Measurement of Oxidative Stress: Mitochondrial Function Using the Seahorse System” In: Murthi, P., Vaillancourt, C. (eds) Preeclampsia. Methods in Molecular Biology, vol 1710. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7498-6_22 </span></span></span></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Lu, C., G. Song, and J. Lin (2006), “Reactive oxygen species and their chemiluminescence-detection methods”, <em>TrAC Trends in Analytical Chemistry, </em>Vol. 25/10, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.trac.2006.07.007" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/j.trac.2006.07.007</span></span></a></span></span></p>
<p style="margin-left:48px; text-align:left"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH oxidase, and redox signaling in the vasculature”, <em>Antioxidants & redox signaling,</em> Vol. 19/10<strong>,</strong> </span><span style="background-color:white"><span style="color:black">Mary Ann Liebert, Larchmont, </span></span><a href="https://doi.org/10.1089/ars.2012.4641" style="color:#0563c1; text-decoration:underline"><span style="background-color:white">https://doi.org/10.1089/ars.2012.4641</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Ping, Z. et al. (2020), “Oxidative Stress in Radiation-Induced Cardiotoxicity”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2020, Hindawi, </span></span></span><a href="https://doi.org/10.1155/2020/3579143" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1155/2020/3579143</span></span></a></span></span></p>
<p style="margin-left:48px">Pizzino, G. et al. (2017) “Oxidative Stress: Harms and Benefits for Human Health.” Oxidative medicine and cellular longevity, Vol. 2017: 8416763, Hindawi, https://doi.org/10.1155/2017/8416763 </p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Powers, S.K. and M.J. Jackson. (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, <em>Physiological Reviews,</em> Vol. 88/4<strong>,</strong> American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/physrev.00031.2007" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/physrev.00031.2007</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Raimondi, V., F. Ciccarese and V. Ciminale. (2020), “Oncogenic pathways and the electron transport chain: a dangeROS liason”, <em>British Journal of Cancer, </em>Vol. 122/2, Nature Portfolio, London, </span></span></span><a href="https://doi.org/10.1038/s41416-019-0651-y" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1038/s41416-019-0651-y</span></span></a></span></span></p>
<p style="margin-left:48px; text-align:left"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Seen, S. and L. Tong. (2018), “Dry eye disease and oxidative stress”, <em>Acta Ophthalmologica,</em> Vol. 96/4<strong>,</strong> John Wiley & Sons, Inc., Hoboken, </span><a href="https://doi.org/10.1111/aos.13526" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1111/aos.13526</a></span></span></p>
<p style="margin-left:48px; text-align:left"><span style="font-size:1rem">Sharifi-Rad, M. et al. (2020), “Lifestyle, Oxidative Stress, and Antioxidants: Back and Forth in the Pathophysiology of Chronic Diseases.” Frontiers in physiology Vol. 11:694, https://doi.org/10.3389/fphys.2020.00694 </span></p>
<p style="margin-left:48px; text-align:left">Snezhkina, A. V. et al. (2019), “ROS Generation and Antioxidant Defense Systems in Normal and Malignant Cells.” Oxidative medicine and cellular longevity Vol. 2019: 6175804, https://doi.org/10.1155/2019/6175804 </p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, <em>The Journals of Gerontology Series A: Biological Sciences and Medical Sciences</em>, Vol. 68/12, Oxford University Press, Oxford, </span></span></span><a href="https://doi.org/10.1093/gerona/glt057." style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1093/gerona/glt057.</span></span></a> </span></span></p>
<p style="margin-left:48px"> </p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, <em>Life, </em>Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, </span></span></span><a href="https://doi.org/10.3390/life11111269" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.3390/life11111269</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Wang, H. et al. (2019), “Radiation-induced heart disease: a review of classification, mechanism and prevention”, <em>International Journal of Biological Sciences, </em>Vol. 15/10, Ivyspring International Publisher, Sydney, <a href="https://doi.org/10.7150/ijbs.35460" style="color:#0563c1; text-decoration:underline">https://doi.org/10.7150/ijbs.35460</a> </span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, <em>Journal of applied physiology</em>, Vol. 106/1, American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/japplphysiol.01278.2007" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/japplphysiol.01278.2007</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">.</span></span></span></span></span></p>
<p style="margin-left:48px"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Zhao, R. Z. et al. (2019), “Mitochondrial electron transport chain, ROS generation and uncoupling”, <em>International journal of molecular medicine</em>, Vol. 44/1, </span><span style="color:black">Spandidos</span><span style="background-color:white"><span style="color:black"> Publishing Ltd</span></span><span style="color:black">., Athens, </span><a href="https://doi.org/10.3892/ijmm.2019.4188" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3892/ijmm.2019.4188</a></span></span></p>
2017-05-30T13:58:172024-03-08T12:28:08Increased Pro-inflammatory mediatorsIncreased pro-inflammatory mediatorsTissue<p>Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites. They can be divided into exogenous and endogenous mediators.</p>
<p>Exogenous mediators of inflammation are bacterial products or toxins like endotoxin or LPS. Endogenous mediators of inflammation are produced from within the (innate and adaptive) immune system itself, as well as other systems. They can be derived from molecules that are normally present in the plasma in an inactive form, such as peptide fragments of some components of complement, coagulation, and kinin systems. Or they can be released at the site of injury by a number of cell types that either contain them as preformed molecules within storage granules, e.g. histamine, or which can rapidly switch on the machinery required to synthesize the mediators.</p>
<p>Table1: a non-exhaustive list of examples for pro-inflammatory mediators</p>
<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width:253px">
<p><strong>Classes of inflammatory mediators</strong></p>
</td>
<td style="width:361px">
<p><strong>Examples</strong></p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Pro-inflammatory cytokines</p>
</td>
<td style="width:361px">
<p>TNF-a, Interleukins (IL-1, IL-6, IL-8), Interferons (IFN-g), chemokines (CXCL, CCL, GRO-α, MCP-1), GM-CSF</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Prostaglandins</p>
</td>
<td style="width:361px">
<p>PGE2</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Bradykinin</p>
</td>
<td style="width:361px">
<p> </p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Vasoactive amines</p>
</td>
<td style="width:361px">
<p>histamine, serotonin</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Reactive oxygen species (ROS)</p>
</td>
<td style="width:361px">
<p>O<sup>2-</sup>, H<sub>2</sub>O<sub>2</sub></p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Reactive nitrogen species (RNS)</p>
</td>
<td style="width:361px">
<p>NO, iNOS</p>
</td>
</tr>
</tbody>
</table>
<p>The increased production of pro-inflammatory mediators can have negative consequences on the parenchymal cells leading even to cell death, as described for TNF-a or peroxynitrite on neurons (Chao et al., 1995; Brown and Bal-Price, 2003). <span style="color:#2980b9">Along with TNF-α, IL-1β and IL-6 have been shown to exhibit negative consequences on neurogenesis and neuronal precursor cell proliferation when overexpressed. IFN-γ is also associated with neuronal damage, although it is not as extensively studied compared to TNF-α, IL-1β and IL-6.</span> In addition, via a feedback loop, they can act on the reactive resident cells thus maintaining or exacerbating their reactive state; and by modifying elements of their signalling pathways, they can favour the M1 phenotypic polarization and the chronicity of the inflammatory process (Taetzsch et al., 2015).</p>
<p>Basically, this event occurs equally in various tissues and does not require tissue-specific descriptions. Nevertheless, there are some specificities such as the release of glutamate by brain reactive glial cells (Brown and Bal-Price, 2003; Vesce et al., 2007).The differences may rather reside in the type of insult favouring the increased expression and/or release of a specific class of inflammatory mediators, as well the time after the insult reflecting different stages of the inflammatory process. For these reasons, the analyses of the changes of a battery of inflammatory mediators rather than of a single one is a more adequate measurement of this KE.</p>
<p style="margin-right:13px; text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="font-size:7pt"><span style="font-size:11.0pt">Regulatory examples using the KE</span></span></strong></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:7pt"><span style="font-size:11.0pt">CD54 and CD 86 as well as IL-8 expression is used to assess skin sensitization potential (OECD TG 442E). IL-2 expression is used to assess immunotoxicity (and will become an OECD test guideline); for the latter see also doi: 10.1007/s00204-018-2199-7. </span></span></span></p>
<p> </p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>When activated, resident macrophages (Kupffer cells) release inflammatory mediators including cytokines, chemokines, lysosomal, and proteolytic enzymes and are a main source of TGF-β1 - the most potent pro-fibrogenic cytokine. Following the role of TGF-β is described in more detail.</p>
<p>Transforming growth factor β (TGF-β) is a pleiotropic cytokine with potent regulatory and</p>
<p>inflammatory activity [Sanjabi et al., 2009; Li and Flavell, 2008a;2008b]. The multi-faceted effects of TGF-β on numerous immune functions are cellular and environmental context dependent [Li et al., 2006]. TGF-β binds to TGF-β receptor II (TGF-βRII) triggering the kinase activity of the cytoplasmic domain that in turn activates TGF-βRI. The activated receptor complex leads to nuclear translocation of Smad molecules,</p>
<p>and transcription of target genes [Li et al., 2006a]. The role of TGF-β as an immune modulator of T cell activity is best exemplified by the similarities between TGF-β1 knockout and T cell specific</p>
<p>TGF-β receptor II knockout mice [Li et al., 2006b; Marie et al., 2006;Shull et al., 1992]. The animals in both of these models develop severe multi-organ autoimmunity and succumb to death within a few weeks after birth [Li et al., 2006b; Marie et al., 2006; Shull et al., 1992]. In addition, in mice where TGF-β signaling is blocked specifically in T cells, the development of natural killer T (NKT) cells, natural regulatory T (nTreg) cells, and CD8+ T cells was shown to be dependent on TGF-β signaling in the thymus [Li et al., 2006b; Marie et al., 2006].</p>
<p>TGF-β plays a major role under inflammatory conditions. TGF-β in the presence of IL-6 drives the differentiation of T helper 17 (Th17) cells, which can promote further inflammation and augment autoimmune conditions [Korn et al., 2009]. TGF-β orchestrates the differentiation of both Treg and Th17 cells in a concentration-dependent manner [Korn et al., 2008]. In addition, TGF-β in combination with IL-4, promotes the differentiation of IL-9- and IL-10-producing T cells, which lack</p>
<p>suppressive function and also promote tissue inflammation [Dardalhon et al., 2008; Veldhoen et al., 2008]. The biological effects of TGF-β under inflammatory conditions on effector and memory CD8+ T cells are much less understood. In a recent study, it was shown that TGF-β has a drastically opposing role on naïve compared to antigen-experienced/memory CD8+ T cells [Filippi et al., 2008]. When cultured <em>in vitro</em>, TGF-β suppressed naïve CD8+ T cell activation and IFN-γ production, whereas TGF-β enhanced survival of memory CD8+ T cells and increased the production of IL-17 and IFN-γ [Filippi et al., 2008]. TGF-β also plays an important role in suppressing the cells of the innate immune system.</p>
<p>The transforming growth factor beta (TGF-β) family of cytokines are ubiquitous, multifunctional, and essential to survival. They play important roles in growth and development, inflammation and repair, and host immunity. The mammalian TGF-β isoforms (TGF-β1, β2 and β3) are secreted as latent precursors and have multiple cell surface receptors of which at least two mediate signal transduction. Autocrine and paracrine effects of TGF-βs can be modified by extracellular matrix, neighbouring cells and other cytokines. The vital role of the TGF-β family is illustrated by the fact that approximately 50% of TGF-1 gene knockout mice die in utero and the remainder succumb to uncontrolled inflammation after birth. The role of TGF-β in homeostatic and pathogenic processes suggests numerous applications in the diagnosis and treatment of various diseases characterised by inflammation and fibrosis. [Clark and Coker, 1998; Santibañez et al., 2011; Pohlers et al., 2009] Abnormal TGF-β regulation and function are implicated in a growing number of fibrotic and inflammatory pathologies, including pulmonary fibrosis, liver cirrhosis, glomerulonephritis and diabetic nephropathy, congestive heart failure, rheumatoid arthritis, Marfan syndrome, hypertrophic scars, systemic sclerosis, myocarditis, and Crohn’s disease. [Gordon and Globe,2008] TGF-β1 is a polypeptide member of the TGF-β superfamily of cytokines. TGF-β is synthesized as a non-active pro-form, forms a complex with two latent associated proteins latency-associated protein (LAP) and latent TGF- β binding protein (LTBP) and undergoes protolithic cleavage by the endopeptidase furin to generate the mature TGF-β dimer. Among the TGF-βs, six distinct isoforms have been discovered although only the TGF-β1, TGF-β2 and TGF-β3 isoforms are expressed in mammals, and their human genes are located on chromosomes 19q13, 1q41 and 14q24, respectively. Out of the three TGF-β isoforms (β1, β2 and β3) only TGF-β1 was linked to fibrogenesis and is the most potent fibrogenic factor for hepatic stellate cells. [Roberts, 1998; Govinden and Bhoola, 2003]. During fibrogenesis, tissue and blood levels of active TGF-β are elevated and overexpression of TGF-β1 in transgenic mice can induce fibrosis. Additionally, experimental fibrosis can be inhibited by anti-TGF-β treatments with neutralizing antibodies or soluble TGF-β receptors [Qi et al.; 1999; Shek and Benyon , 2004; De Gouville et al., 2005; Chen et al., 2009]. TGF-β1 induces its own mRNA to sustain high levels in local sites of injury. The effects of TGF-β1 are classically mediated by intracellular signalling via Smad proteins. Smads 2 and 3 are stimulatory whereas Smad 7 is inhibitory. [Parsons et al., 2013; Friedman, 2008; Kubiczkova et al., 2012] Smad1/5/8, MAP kinase (mitogen-activated protein) and PI3 kinase are further signalling pathways in different cell types for TGF-β1 effects.</p>
<p>TGF-β is found in all tissues, but is particularly abundant in bone, lung, kidney and placental tissue. TGF-β is produced by many, but not all parenchymal cell types, and is also produced or released by infiltrating cells such as lymphocytes, monocytes/macrophages, and platelets. Following wounding or inflammation, all these cells are potential sources of TGF-β. In general, the release and activation of TGF-β stimulates the production of various extracellular matrix proteins and inhibits the degradation of these matrix proteins. [Branton and Kopp, 1999]</p>
<p>TGF-β 1 is produced by every leukocyte lineage, including lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these immune cells. [Letterio and Roberts; 1998]</p>
<p>In the liver TGF-β1 is released by activated Kupffer cells, liver sinusoidal endothelial cells, and platelets; in the further course of events also activated hepatic stellate cells express TGF-β1. Hepatocytes do not produce TGF-β1 but are implicated in intracellular activation of latent TGF-β1. [Roth et al., 1998; Kisseleva and Brenner, 2007; Kisseleva and Brenner, 2008; Poli, 2000; Liu et al., 2006]</p>
<p>TGF-β1 is the most established mediator and regulator of epithelial-mesenchymal-transition (EMT) which further contributes to the production of extracellular matrix. It has been shown that TGF-β1 mediates EMT by inducing snail-1 transcription factor and tyrosine phosphorylation of Smad2/3 with subsequent recruitment of Smad4. [Kolios et al., 2006; Bataller and Brenner, 2005; Guo and Friedman,2007; Brenner,2009; Kaimori et al., 2007; Gressner et al., 2002; Kershenobich Stalnikowitz and Weisssbrod, 2003; Li et al., 2008; Matsuoka and Tsukamoto, 1990; Kisseleva and Brenner, 2008; Poli, 200; Parsons et al., 2007; Friedman 2008; Liu et al., 2006]</p>
<p>TGF-β1 induces apoptosis and angiogenesis in vitro and in vivo through the activation of vascular endothelial growth factor (VEGF) High levels of VEGF and TGF-β1 are present in many tumors. Crosstalk between the signalling pathways activated by these growth factors controls endothelial cell apoptosis and angiogenesis. [Clark and Coker; 1998]</p>
<p> </p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:11pt">T<span style="font-size:14px">he specific type of measurement(s) might vary with tissue, environment and context and will need to be described for different tissue contexts as used within different AOP descriptions</span></span><span style="font-size:14px">.</span></span></p>
<p><span style="font-size:14px">In general, quantification of inflammatory markers can be done by:</span></p>
<ul>
<li><span style="font-size:14px">qRT-PCR (mRNA expression)</span></li>
<li><span style="font-size:14px">ELISA</span></li>
<li><span style="font-size:14px">Immunocytochemistry</span></li>
<li><span style="font-size:14px">Immunoblotting</span></li>
</ul>
<p><span style="font-size:14px">For descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</span><br />
</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>There are several assays for TGB-β1 measurement available.</p>
<p>e.g. Human TGF-β1 ELISA Kit. The Human TGF-β 1 ELISA (Enzyme –Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human TGF-β1 in serum, plasma, cell culture supernatants, and urine. This assay employs an antibody specific for human TGF-β1 coated on a 96-well plate. Standards and samples are pipetted into the wells and TGF-β1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human TGF-β1 antibody is added. After washing away unbound biotinylated antibody, HRP- conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and colour develops in proportion to the amount of TGF-β1 bound. The StopSolution changes the colour from blue to yellow, and the intensity of the colour is measured at 450 nm [Mazzieri et al., 2000]</p>
<p><span style="color:#2980b9">Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="color:#2980b9">Assay </span></p>
</td>
<td>
<p><span style="color:#2980b9">Reference </span></p>
</td>
<td>
<p><span style="color:#2980b9">Description </span></p>
</td>
<td>
<p><span style="color:#2980b9">OECD Approved Assay </span></p>
</td>
</tr>
<tr>
<td>
<ul>
<li>
<p><span style="color:#2980b9">RT-qPCR </span></p>
</li>
<li>
<p><span style="color:#2980b9">Q-PCR </span></p>
</li>
</ul>
</td>
<td>
<p><span style="color:#2980b9">(Veremeyko et al., 2012; Alwine et al, 1977; Forlenza et al., 2012) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Measures mRNA expression of cytokines, chemokines and inflammatory markers </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunoblotting (western blotting) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Lee et al., 2008) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Uses antibodies specific to proteins of interest, can used to detect presence of pro-inflammatory mediators in samples of cell or tissue lysate </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Whole blood stimulation assay </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Thurm & Halsey, 2005) </span></p>
</td>
<td>
<p><span style="color:#2980b9"> Detects inflammatory cytokines in blood </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Imaging tests </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Rollins & Miskolci, 2014) </span></p>
</td>
<td>
<p><span style="color:#2980b9">A qualitative technique using a cytokine specific antibodies and fluorophores can be used to visualize expression patterns, subcellular location of the target and protein-protein interactions. </span></p>
<p><span style="color:#2980b9">Common examples include double immunofluorescence confocal microscopy or other molecular imaging modalities. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Flow-cytometry </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Karanikas et al., 2000) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Detects the intracellular cytokines with stimulation. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunoassays (ex. enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISpot), radioimmunoassay) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009; Engvall & Perlmann, 1972; Ji & Forsthuber, 2016; Goldsmith, 1975) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Plate based assay technique using antibodies to detect presence of a protein in a liquid sample. </span></p>
<p><span style="color:#2980b9">Can be used to identify presence of an inflammatory cytokine of interest especially when in low concentrations. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Inflammatory cytokine arrays </span></p>
<p> </p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009) </span></p>
<p> </p>
</td>
<td>
<p><span style="color:#2980b9">Similar to the ELISA, except using a membrane-based rather than plate-based approach. Can be used to measure multiple cytokine targets concurrently. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunohistochemistry (IHC) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009; Coons et al., 1942) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Immobilized tissue or cell cultures are stained using antibodies for specificity of ligands of interest. Versions of the assays can be used to visualize localization of inflammatory cytokines. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Human [Santibañez et al., 2011]</p>
<p>Rat [Luckey and Petersen, 2001]</p>
<p>Mouse [Nan et al., 2013]</p>
<p><strong>BRAIN:</strong></p>
<p><span style="font-size:14px">Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</span></p>
<p> </p>
<p><span style="color:#2980b9"><strong>Taxonomic applicability</strong>: The inflammatory response and increase of the pro-inflammatory mediators has been observed across species from simple invertebrates such as Daphnia to higher order vertebrates (Weavers & Martin, 2020).</span></p>
<p><span style="color:#2980b9"><strong>Life stage applicability</strong>: This key event is not life stage specific (Kalm et al., 2013; Veeraraghan et al., 2011; Hladik & Tapio, 2016). </span></p>
<p><span style="color:#2980b9"><strong>Sex applicability</strong>: Most studies conducted were on male models, although sex-dependent differences in pro-inflammatory markers have been previously reported (Cekanaviciute et al., 2018; Parihar et al., 2020). </span></p>
<p><span style="color:#2980b9"><strong>Evidence for perturbation by a prototypic stressor</strong>: There is evidence of the increase of pro-inflammatory mediators following perturbation from a variety of stressors including exposure to ionizing radiation. (Abdel-Magied et al., 2019; Cho et al., 2017; Gaber et al., 2003; Ismail et al., 2016; Kim et al. 2002; Lee et al., 2010; Parihar et al., 2018).</span></p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesNot SpecifiedNot Specified<p> <span style="color:windowtext">Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355</span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004 Jan;88(1):181-93. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Hamadi N, Sheikh A, Madjid N, Lubbad L, Amir N, Shehab SA, Khelifi-Touhami F, Adem A: Increased pro-inflammatory cytokines, glial activation and oxidative stress in the hippocampus after short-term bilateral adrenalectomy. BMC Neurosci 2016, <strong>17:</strong>61.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Taetzsch T, Levesque S, McGraw C, Brookins S, Luqa R, Bonini MG, Mason RP, Oh U, Block ML (2015) Redox regulation of NF-kappaB p50 and M1 polarization in microglia. Glia 5, <strong>63:</strong>423-440.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Vesce S, Rossi D, Brambilla L, Volterra A (2007) Glutamate release from astrocytes in physiological conditions and in neurodegenerative disorders characterized by neuroinflammation. Int Rev Neurobiol. 82 :57-71.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext"> <strong>LIVER:</strong></span></span></p>
<ul style="list-style-type:circle">
<li><span style="font-size:14px">Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</span></li>
<li><span style="font-size:14px">Branton, M.H. and J.B. Kopp (1999), TGF-beta and fibrosis, Microbes Infect, vol. 1, no. 15, pp. 1349-1365.</span></li>
<li><span style="font-size:14px">Brenner, D.A. (2009), Molecular Pathogenesis of Liver Fibrosis, Trans Am Clin Climatol Assoc, vol. 120, pp. 361–368.</span></li>
<li><span style="font-size:14px">Cheng, K., N.Yang and R.I. Mahato (2009), TGF-beta1 gene silencing for treating liver fibrosis, Mol Pharm, vol. 6, no. 3, pp. 772–779.</span></li>
<li><span style="font-size:14px">Clark, D.A. and R.Coker (1998), Transforming growth factor-beta (TGF-beta), Int J Biochem Cell Biol, vol. 30, no. 3, pp. 293-298.</span></li>
<li><span style="font-size:14px">Dardalhon V, Awasthi A, Kwon H, Galileos G, Gao W, Sobel RA, Mitsdoerffer M, Strom TB,</span></li>
<li><span style="font-size:14px">De Gouville, A.C. et al. (2005), Inhibition of TGF-beta signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis, Br J Pharmacol, vol. 145, no. 2, pp. 166–177.</span></li>
<li><span style="font-size:14px">Filippi CM, Juedes AE, Oldham JE, Ling E, Togher L, Peng Y, Flavell RA, von Herrath MG, Transforming growth factor-beta suppresses the activation of CD8+ T-cells when naive but promotes their survival and function once antigen experienced: a two-faced impact on autoimmunity. Diabetes 2008;57:2684–2692.</span></li>
<li><span style="font-size:14px">Friedman, S.L. (2008), Mechanisms of Hepatic Fibrogenesis, Gastroenterology, vol. 134, no. 6, pp. 1655–1669.</span></li>
<li><span style="font-size:14px">Gordon, K.J. and G.C. Blobe (2008), Role of transforming growth factor-β superfamily signalling pathways in human disease, Biochim Biophys Acta, vol. 1782, no. 4, pp. 197–228.</span></li>
<li><span style="font-size:14px">Govinden, R. and K.D. Bhoola (2003), Genealogy, expression, and cellular function of transforming growth factor-β, Pharmacol. Ther, vol. 98, no. 2, pp. 257–265.</span></li>
<li><span style="font-size:14px">Gressner, A.M. et al. (2002), Roles of TGF-β in hepatic fibrosis. Front Biosci, vol. 7, pp. 793-807.</span></li>
<li><span style="font-size:14px">Guo, J. and S.L. Friedman (2007), Hepatic fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.</span></li>
<li><span style="font-size:14px">Kaimori, A. et al. (2007), Transforming growth factor-beta1 induces an epithelial-to-mesenchymal transition state in mouse hepatocytes in vitro, J Biol Chem, vol. 282, no. 30, pp. 22089-22101.</span></li>
<li><span style="font-size:14px">Kershenobich Stalnikowitz, D. and A.B. Weisssbrod (2003), Liver Fibrosis and Inflammation. A Review, Annals of Hepatology, vol. 2, no. 4, pp.159-163.</span></li>
<li><span style="font-size:14px">Kisseleva T and Brenner DA, (2008), Mechanisms of Fibrogenesis, Exp Biol Med, vol. 233, no. 2, pp. 109-122.</span></li>
<li><span style="font-size:14px">Kisseleva, T. and Brenner, D.A. (2007), Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis, Journal of Gastroenterology and Hepatology, vol. 22, Suppl. 1; pp. S73–S78.</span></li>
<li><span style="font-size:14px">Kolios, G., V. Valatas and E. Kouroumalis (2006), Role of Kupffer cells in the pathogenesis of liver disease, World J.Gastroenterol, vol. 12, no. 46, pp. 7413-7420.</span></li>
<li>Korn T, Mitsdoerffer M, Croxford AL, Awasthi A, Dardalhon VA, Galileos G, Vollmar P, Stritesky GL, Kaplan MH, Waisman A, Kuchroo VK, Oukka M., IL-6 controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells into Foxp3+ regulatory T cells, Proceedings of the National Academy of Sciences Nov 2008, 105 (47) 18460-18465; DOI: 10.1073/pnas.0809850105</li>
<li><span style="font-size:14px">Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol. 2009</span></li>
<li><span style="font-size:14px">Kubiczkova, L. et al, (2012), TGF-β - an excellent servant but a bad master, J Transl Med, vol. 10, p. 183.</span></li>
<li><span style="font-size:14px">Letterio, J.J. and A.B. Roberts (1998), Regulation of immune responses by TGF-beta, Annu Rev Immunol, vol.16, pp. 137-161.</span></li>
<li><span style="font-size:14px">Li MO, Flavell RA. Contextual regulation of inflammation: a duet by transforming growth factor-beta and interleukin-10. Immunity 2008a;28:468–476.</span></li>
<li><span style="font-size:14px">Li MO, Flavell RA. TGF-beta: a master of all T cell trades. Cell 2008b;134:392–404.</span></li>
<li><span style="font-size:14px">Li MO, Sanjabi S, Flavell RA. Transforming growth factor-beta controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms. Immunity 2006b;25:455–471.</span></li>
<li><span style="font-size:14px">Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol 2006a;24:99–146.</span></li>
<li><span style="font-size:14px">Li, Jing-Ting et al. (2008), Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies, J Gastroenterol, vol. 43, no. 6, pp. 419–428.</span></li>
<li><span style="font-size:14px">Liu, Xingjun et al. (2006), Therapeutic strategies against TGF-beta signaling pathway in hepatic fibrosis. Liver Int, vol.26, no.1, pp. 8-22.</span></li>
<li><span style="font-size:14px">Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.</span></li>
<li><span style="font-size:14px">Marie JC, Liggitt D, Rudensky AY. Cellular mechanisms of fatal early-onset autoimmunity in mice with the T cell-specific targeting of transforming growth factor-beta receptor. Immunity 2006;25:441–454.</span></li>
<li><span style="font-size:14px">Matsuoka, M. and H. Tsukamoto, (1990), Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor beta: implication for a pathogenetic role in alcoholic liver fibrogenesis, Hepatology, vol. 11, no. 4, pp. 599-605.</span></li>
<li><span style="font-size:14px">Mazzieri, R .et al. (2000), Measurements of Active TGF-β Generated by Culture Cells, Methods in Molecular Biology, vol. 142, pp. 13-27.</span></li>
<li><span style="font-size:14px">Nan, Y.M. et al. (2013), Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol mediated liver fibrosis in mice, Lipids Health Dis, vol. 12, p.11.</span></li>
<li><span style="font-size:14px">Parsons, C.J., M.Takashima and R.A. Rippe (2007), Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, vol. 22, Suppl.1, pp. S79-S84.</span></li>
<li><span style="font-size:14px">Pohlers , D. et al. (2009), TGF-β and fibrosis in different organs – molecular pathway imprints, Biochim. Biophys. Acta, vol. 1792, no. 8, pp.746–756.</span></li>
<li><span style="font-size:14px">Poli, G. (2000), Pathogenesis of liver fibrosis: role of oxidative stress, Mol Aspects Med, vol. 21, no. 3, pp. 49 – 98.</span></li>
<li><span style="font-size:14px">Qi Z et al.(1999),Blockade of type beta transforming growth factor signalling prevents liver fibrosis and dysfunction in the rat, Proc Natl Acad Sci USA, vol. 96, no. 5, pp. 2345-2349.</span></li>
<li><span style="font-size:14px">Roberts, A.B. (1998), Molecular and cell biology of TGF-β, Miner Electrolyte Metab, vol. 24, no. 2-3, pp. 111-119.</span></li>
<li><span style="font-size:14px">Roth, S., K. Michel and A.M. Gressner (1998), (Latent) transforming growth factor beta in liver parenchymal cells, its injury-dependent release, and paracrine effects on rat HSCs, Hepatology, vol. 27, no. 4, pp. 1003-1012.</span></li>
<li><span style="font-size:14px">Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA. Anti- and Pro-inflammatory Roles of TGF-β, IL-10, and IL-22 In Immunity and Autoimmunity. Current opinion in pharmacology. 2009;9(4):447-453.</span></li>
<li><span style="font-size:14px">Santibañez J.F., M. Quintanilla and C. Bernabeu (2011), TGF-β/TGF-β receptor system and its role in physiological and pathological conditions, Clin Sci (Lond), vol. 121, no. 6, pp. 233-251.</span></li>
<li><span style="font-size:14px">Shek, F.W. and R.C. Benyon (2004), How can transforming growth factor beta be targeted usefully to combat liver fibrosis? Eur J Gastroenterol Hepatol, vol. 16, no. 2, pp.123-126.</span></li>
<li><span style="font-size:14px">Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, </span> <span style="font-family:times new roman,serif; font-size:10.5pt">Calvin D, et al.</span> Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature. 1992 Oct 22;359(6397):693-9.</li>
<li><span style="font-size:14px">Veldhoen M, Uyttenhove C, van Snick J, Helmby H, Westendorf A, Buer J, Martin B, Wilhelm C, Stockinger B. Transforming growth factor-beta 'reprograms' the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nat Immunol 2008;9:1341–1346.</span></li>
</ul>
<p><span style="font-size:14px"><span style="color:windowtext"> </span></span> </p>
<p><span style="font-size:14px"><span style="color:#2980b9">Abdel-Magied, N., S. M., Shedid and Ahmed, A. G. (2019), “Mitigating effect of biotin against irradiation-induced cerebral cortical and hippocampal damage in the rat brain tissue”, Environmental Science and Pollution Research, Vol. 26/13, Springer, London, </span><a href="https://doi.org/10.1007/S11356-019-04806-X" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1007/S11356-019-04806-X</span></a><span style="color:#2980b9">. </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Alwine, J. C., D. J. Kemp and G. R. Stark (1977), “Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 74/12, United States National Academy of Sciences, Washington, D.C., </span><a href="https://doi.org/10.1073/pnas.74.12.5350" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1073/pnas.74.12.5350</span></a><span style="color:#2980b9"> </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, </span><a href="https://doi.org/10.1007/978-1-59745-447-6_5" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1007/978-1-59745-447-6_5</span></a></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Cekanaviciute, E., S. Rosi and S. Costes. (2018), "Central Nervous System Responses to Simulated Galactic Cosmic Rays", International Journal of Molecular Sciences, Vol. 19/11, Multidisciplinary Digital Publishing Institute (MDPI) AG, Basel, https://doi.org/10.3390/ijms19113669. </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Cho, H. J. et al. (2017), “Role of NADPH Oxidase in Radiation-induced Pro-oxidative and Pro-inflammatory Pathways in Mouse Brain”, International Journal of Radiation Biology, Vol. 93/11, Informa, London, </span><a href="https://doi.org/10.1080/09553002.2017.1377360" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1080/09553002.2017.1377360</span></a><span style="color:#2980b9">. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Coons, A. H. et al. (1942), “The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluorescent Antibody”, The Journal of Immunology, Vol. 45/3, American Association of Immunologists, Minneapolis, pp. 159-169 </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Engvall, E., and P. Perlmann (1972), “Enzyme-Linked Immunosorbent Assay, Elisa”, The Journal of Immunology, Vol. 109/1, American Association of Immunologists, Minneapolis, pp. 129-135 </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Fan, L. W. and Y. Pang. (2017), "Dysregulation of neurogenesis by neuroinflammation: Key differences in neurodevelopmental and neurological disorders", Neural Regeneration Research, Vol. 12/3, Wolters Kluwer, Alphen aan den Rijn, </span><a href="https://doi.org/10.4103/1673-5374.202926" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.4103/1673-5374.202926</span></a><span style="color:#2980b9">. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Forlenza, M. et al. (2012), “The use of real-time quantitative PCR for the analysis of cytokine mRNA levels” in Cytokine Protocols, Springer, New York, https://doi.org/10.1007/978-1-61779-439-1_2 </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Gaber, M. W. et al. (2003), “Differences in ICAM-1 and TNF-alpha expression between large single fraction and fractionated irradiation in mouse brain”, International Journal of Radiation Biology, Vol. 79/5, Informa, London, </span><a href="https://doi.org/10.1080/0955300031000114738" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1080/0955300031000114738</span></a><span style="color:#2980b9">. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Goldsmith, S. J. (1975), "Radioimmunoassay: Review of basic principles", Seminars in Nuclear Medicine, Vol. 5/2, https://doi.org/10.1016/S0001-2998(75)80028-6. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Hladik, D. and S. Tapio. (2016), "Effects of ionizing radiation on the mammalian brain", Mutation Research/Reviews in Mutation Research, Vol. 770, Elsevier B. b., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.08.003. </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Ismail, A. F. M., A.A.M. Salem and M.M.T. Eassawy (2016), “Modulation of gamma-irradiation and carbon tetrachloride induced oxidative stress in the brain of female rats by flaxseed oil”, Journal of Photochemistry and Photobiology B: Biology, Vol. 161, Elsevier, Amsterdam, </span><a href="https://doi.org/10.1016/J.JPHOTOBIOL.2016.04.031" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1016/J.JPHOTOBIOL.2016.04.031</span></a><span style="color:#2980b9">. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Ji, N. and T. G. Forsthuber. (2014), "ELISPOT Techniques" (pp. 63–71), https://doi.org/10.1007/7651_2014_111. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Kalm, M., K. Roughton and K. Blomgren. (2013), "Lipopolysaccharide sensitized male and female juvenile brains to ionizing radiation", Cell Death & Disease, Vol. 4/12, Nature Publishing Group, Berlin, https://doi.org/10.1038/cddis.2013.482. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Karanikas, V. et al. (2000), “Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy”, Clinical Cancer Research, Vol. 6/3, American Association for Cancer Research, Philadelphia, pp. 829–837 </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Kim, S. H. et al. (2002), “Expression of TNF-alpha and TGF-beta 1 in the rat brain after a single high-dose irradiation”, Journal of Korean Medical Science, Vol. 17/2, Korean Medical Association, Seoul, </span><a href="https://doi.org/10.3346/JKMS.2002.17.2.242" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.3346/JKMS.2002.17.2.242</span></a><span style="color:#2980b9">. </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Lee, J. W. et al. (2008), “Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation”, Journal of Neuroinflammation, Vol. 5/1, BioMed Central, London, </span><a href="https://doi.org/10.1186/1742-2094-5-37" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1186/1742-2094-5-37</span></a><span style="color:#2980b9"> </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Lee, W. H. et al. (2010), “Irradiation induces regionally specific alterations in pro-inflammatory environments in rat brain”, International Journal of Radiation Biology, Vol. 86/2, Informa, London, https://doi.org/10.3109/09553000903419346. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Parihar, V. K. et al. (2018), “Persistent nature of alterations in cognition and neuronal circuit excitability after exposure to simulated cosmic radiation in mice”, Experimental Neurology, Vol. 305, Elsevier, Amsterdam, https://doi.org/10.1016/J.EXPNEUROL.2018.03.009. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Parihar, V. K. et al. (2020), "Sex-Specific Cognitive Deficits Following Space Radiation Exposure", Frontiers in Behavioral Neuroscience, Vol. 14, https://doi.org/10.3389/fnbeh.2020.535885. </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Rollins, J. and V. Miskolci (2014), “Immunofluorescence and subsequent confocal microscopy of intracellular TNF in human neutrophils” in Cytokines Bioassays, Springer, London, </span><a href="https://doi.org/10.1007/978-1-4939-0928-5_24" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1007/978-1-4939-0928-5_24</span></a></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Thurm, C. W. and J. F. Halsey (2005), “Measurement of Cytokine Production Using Whole Blood”, in Current Protocols in Immunology, John Wiley & Sons, Inc., Hoboken, </span><a href="https://doi.org/10.1002/0471142735.im0718bs66" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1002/0471142735.im0718bs66</span></a><span style="color:#2980b9"> </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Veeraraghavan, J. et al. (2011), "Low-dose γ-radiation-induced oxidative stress response in mouse brain and gut: Regulation by NFκB–MnSOD cross-signaling", Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol. 718/1–2, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrgentox.2010.10.006. </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Veremeyko, T. et al. (2012), “Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation”, Journal of Visualized Experiments: JoVE, Vol. 65, MyJove Corporation, Cambridge, </span><a href="https://doi.org/10.3791/4097" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.3791/4097</span></a></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Weavers, H. and P. Martin (2020), “The cell biology of inflammation: From common traits to remarkable immunological adaptations”, Journal of Cell Biology, Vol. 219, Rockefeller University Press, New York, </span><a href="https://doi.org/10.1083/jcb.202004003" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1083/jcb.202004003</span></a><span style="color:#2980b9"> </span></span></p>
2017-11-28T09:00:542023-03-21T15:50:49Altered Signaling PathwaysAltered SignalingMolecular<p><span style="font-family:Times New Roman,Times,serif">Cells receive, process, and transmit signals to respond to their environment via signaling pathways. </span><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="font-family:Times New Roman,Times,serif">Signaling</span> pathways are groups of molecules that work together in a cell to control physiological and metabolic processe<span style="font-family:Times New Roman,Times,serif">s.</span><span style="color:black"><span style="font-family:Times New Roman,Times,serif"> </span></span></span></span></span></span></span><span style="font-family:Times New Roman,Times,serif">Kinases, for example, are important signaling molecules that can phosphorylate other proteins (Svoboda & Reenstra, 2002).</span><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black"><span style="font-family:Times New Roman,Times,serif"> Initiation of signaling pathways is an important co</span>mponent of </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">cellular homeostasis including normal cell development and the response to cellular damage from exposure to external stressors (</span></span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Esbenshade & Duzic, 2006</span></span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">). </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Signaling pathways are themselves activated by signals and the same signal can lead to different responses depending on the tissue type </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">(Hamada, et al. 2011; Svoboda & Reenstra, 2002</span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">)</span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">. </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Examples of signals include the activation of receptors to activate transcriptional targets, induction of receptor-ligand interactions and the initiation of cell-cell contact, or cell-extracellular matrix contact (Hunter, 2000). </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Many signalling pathways are crucial to intercellular communication via membrane receptors that transduce signals into the cell, while others are activated in an intracellular manner (Svoboda & Reenstra, 2002). Altered signalling (i.e., increase/decrease) can lead to different physiological outcomes, meaning that the </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">directionality of the signaling response, determines the end outcome. For example, increase of the PI3K/Akt/mTOR pathway, which under physiological conditions is responsible for regulating the cell cycle, can lead to increased proliferation and decreased apoptosis. However, a decrease expression of this pathway can lead to an increase in apoptosis and decreased proliferation (<span style="background-color:white">Porta et al., 2014;</span> Venkatesulu et al., 2018).</span></span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Method of Measurement</span></span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Reference</span></span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Description</span></span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">OECD Approved Assay</span></span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Kinase assays </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Svoboda & Reenstra, 2002) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Block kinase with inhibitors to monitor the activity of a kinase of interest. </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Cell behaviour assays </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Svoboda & Reenstra, 2002) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Signal transduction events of cells are monitored. Cells are exposed to varying levels of signaling proteins and the resulting actions of a cell are observed (changes in structure, cell shape, matrix binding etc.). </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Ratiometric or single-wavelength dyes </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Svoboda & Reenstra, 2002) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Detects alterations in signal-transduction activities via monitoring changes in detectable wavelengths. </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Fluorescence microscopy/spectroscopy </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Oksvold et al., 2002) </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Measures cell localization, protein interactions, signal propagation, amplification, and integration in the cell in real-time, or upon stimulation. </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Yes</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Green Fluorescent Protein (GFP) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Zaccolo and Pozzan, 2000) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">GFP assays act as fluorescent reporters but also as a marker of intracellular signalling events i.e. second messengers Ca2+ and cAMP, or for pH in different various cell compartments </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Fluorescence Resonance Energy Transfer (</span></span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">FRET) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Bunt and Wouters, 2017) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Assay helps illuminate the interactions between biological molecules </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Fluorescence recovery after photobleaching (</span></span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">FRAP) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Svoboda & Reenstra, 2002) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Determines mobility and diffusion of small molecules. </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Immunoprecipitation </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Svoboda & Reenstra, 2002) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Involves isolating and concentrating a particular protein from mixed samples to detect changes in signalling molecule activity. </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Chromatin immunoprecipitation approved for analyzing histone modifications</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Immunohistochemistry </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Kurien et al., 2011; Svoboda & Reenstra, 2002) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Northern, western and southern blotting techniques can be used to visualize signal transduction events. For example, antibodies with recognition epitopes can be used to locate active configurations or phosphorylated proteins within a cell or cell lysate. </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Veremeyko et al., 2012; Alwine et al, 1977)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Measures mRNA expression of the gene of interest. </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Enzyme-linked immunosorbent assay (ELISA)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Amsen et al., 2009; Engvall & Perlmann, 1972)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Plate-based assay technique using antibodies to detect presence of a protein in a liquid sample. Can be used to identify presence of a protein of interest especially when in low concentrations</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Taxonomic applicability: </span></span></strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Altered signaling is applicable to all animals as cell signaling occurs among animal cells. This includes vertebrates such as humans, mice and rats (Nair et al., 2019). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Life stage applicability: </span></span></strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">This key event is not life stage specific. </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Sex applicability: </span></span></strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">This key event is not sex specific. </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Evidence for perturbation by a stressor: </span></span></strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Multiple studies show that signaling pathways can be disrupted by many types of stressors including ionizing radiation and altered gravity (Cheng et al., 2020; Coleman et al., 2021; Su et al., 2020; </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Yentrapalli</span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"> et al., 2013). </span></span></span></span></p>
LowUnspecificModerateAll life stagesModerateModerateModerate<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Alwine, J. C., D. J. Kemp and G. R. Stark (1977), “Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes”, <em>Proceedings of the National Academy of Sciences of the United States of America</em>, Vol. 74/12, United States National Academy of Sciences, Washington, D.C., </span></span></span><a href="https://doi.org/10.1073/pnas.74.12.5350" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1073/pnas.74.12.5350</span></span></a></span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in <em>Inflammation and Cancer</em>, Humana Press, Totowa, </span></span></span><a href="https://doi.org/10.1007/978-1-59745-447-6_5" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1007/978-1-59745-447-6_5</span></span></a></span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Bunt, G., and F. S. Wouters (2017), “FRET from single to multiplexed signaling events”, <em>Biophysical reviews</em>, Vol. 9, Springer, London, </span></span></span></span><a href="https://doi.org/10.1007/s12551-017-0252-z" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1007/s12551-017-0252-z</span></span></span></a></span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Cheng, Y. P. et al. (2017), “Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats”,<em> Pflugers Archiv European Journal of Physiology</em>, Vol. 469, Springer, New York, </span></span></span></span><a href="https://doi.org/10.1007/s00424-017-1969-z" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1007/s00424-017-1969-z</span></span></span></a></span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Coleman, M. A. et al. (2015), “Low-dose radiation affects cardiac physiology: gene networks and molecular signaling in cardiomyocytes”, <em>American Journal of Physiology - Heart and Circulatory Physiology</em>, Vol. 309/11, American Physiological Society, Rockville, </span></span></span></span><a href="https://doi.org/10.1152/ajpheart.00050.2015" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/ajpheart.00050.2015</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Engvall, E., and P. Perlmann (1972), “Enzyme-Linked Immunosorbent Assay, Elisa”, <em>The Journal of Immunology</em>, Vol. 109/1, American Association of Immunologists, Minneapolis, pp. 129-135</span></span></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Esbenshade, T. A., and E. Duzic (2006), “Overview of signal transduction”, <em>Current Protocols in Pharmacology</em>, Vol. 31/1, John Wiley & Sons, Ltd., Hoboken, </span></span></span></span><a href="https://doi.org/10.1002/0471141755.ph0201s31" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1002/0471141755.ph0201s31</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Hamada, N. et al. (2011), “Signaling pathways underpinning the manifestations of ionizing radiation-induced bystander effects”, <em>Current Molecular Pharmacology</em>, Vol. 4/2, Bentham Science Publishers, Sharjah UAE, <a href="https://doi.org/10.2174/1874467211104020079" style="color:#0563c1; text-decoration:underline">https://doi.org/10.2174/1874467211104020079</a></span></span></span></span></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Hunter, T. (2000), “Signaling - 2000 and beyond”, <em>Cell</em>, Vol. 100/1, Cell Press, Cambridge, </span></span></span></span><a href="https://doi.org/10.1016/s0092-8674(00)81688-8" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/s0092-8674(00)81688-8</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Kurien, B. T. et al. (2011), “An overview of Western blotting for determining antibody specificities for immunohistochemistry”, in</span></span></span></span> <em><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Signal Transduction Immunohistochemistry Methods and Protocols</span></span></span></span></em><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">, Springer, London, </span></span></span></span><a href="https://doi.org/10.1007/978-1-61779-024-9_3" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1007/978-1-61779-024-9_3</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Nair, A. et al. (2019), “Conceptual Evolution of Cell Signaling”, <em>International journal of molecular sciences</em>, Vol. 20/13, Multidisciplinary Digital Publishing Institute, Basel, </span></span></span></span><a href="https://doi.org/10.3390/ijms20133292" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.3390/ijms20133292</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Oksvold, M. P. et al. (2002), “Fluorescent histochemical techniques for analysis of intracellular signaling”, <em>The Journal of Histochemistry and Cytochemistry</em>, Vol. 50/3, SAGE Publications, Thousand Oaks, </span></span></span></span><a href="https://doi.org/10.1177/002215540205000301" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1177/002215540205000301</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Porta, C., C. Paglino and A. Mosca (2014), “Targeting PI3K/Akt/mTOR Signaling in Cancer”, <em>Frontiers in Oncology</em>, Vol. 4, Frontiers Media SA, Lausanne, </span></span></span></span><a href="https://doi.org/10.3389/fonc.2014.00064" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.3389/fonc.2014.00064</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Su, Y. T. et al. (2020), “Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats”, <em>Life Sciences</em>, Vol. 243, Elsevier, Amsterdam, </span></span></span></span><a href="https://doi.org/10.1016/j.lfs.2019.117253" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/j.lfs.2019.117253</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Svoboda, K. K. and W. R. Reenstra (2002), “Approaches to studying cellular signaling: a primer for morphologists”, <em>The Anatomical record</em>, Vol. 269/2, John Wiley & Sons, Ltd., Hoboken, </span></span></span></span><a href="https://doi.org/10.1002/ar.10074" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1002/ar.10074</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Venkatesulu, B. P. et al. </span></span></span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">(2018), “Radiation-Induced Endothelial Vascular Injury</span></span></span></span>: <span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">A Review of Possible Mechanisms”, <em>JACC: Basic to Translational Science</em>, Vol. 3/4, Elsevier, Amsterdam, </span></span></span></span><a href="https://doi.org/10.1016/j.jacbts.2018.01.014" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/j.jacbts.2018.01.014</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Veremeyko, T. et al. (2012), “Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation”, <em>Journal of Visualized Experiments: JoVE</em>, Vol. 65, MyJove Corporation, Cambridge, </span></span></span><a href="https://doi.org/10.3791/4097" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.3791/4097</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Yentrapalli, R. et al. (2013), “The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation”, <em>PloS one</em>, Vol. 8/8, PLOS, San Francisco, </span></span></span></span><a href="https://doi.org/10.1371/journal.pone.0070024" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1371/journal.pone.0070024</span></span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Zaccolo, M. and T. Pozzan (2000), “Imaging signal transduction in living cells with GFP-based probes”, <em>IUBMB life</em>, Vol. 49/5, John Wiley & Sons, Ltd., Hoboken, https://doi.org/10.1080/152165400410218</span></span></span></span></span></span></p>
2022-09-28T11:42:432024-02-13T07:31:37Altered, Nitric Oxide LevelsAltered, Nitric Oxide LevelsCellular<p><span style="font-family:Times New Roman,Times,serif">Nitric oxide (NO) is a diffusible molecule produced by many cell types, including endothelial cells, and is responsible for vasodilation (Schulz, Gori & Münzel, 2011; Soloviev & Kizub, 2019). The source of endogenous NO is L-arginine (Burov et al., 2022). Production of NO in the body can occur through nitric oxide synthase (NOS), an enzyme that degrades L-arginine in the presence of oxygen and nicotinamide adenine dinucleotide phosphate (NADPH) (Luiking, Engelen & Deutz, 2010). Tetrahydrobiopterin (BH4) is an important cofactor of NOS, allowing the enzymatic production of NO. A non-enzymatic method to produce NO includes the reduction of nitrite (Luiking, Engelen & Deutz, 2010). NO is constitutively produced by endothelial nitric oxide synthase (eNOS) and neuronal NOS (nNOS), and can be increased by inducible NOS (iNOS) (Powers & Jackson, 2008). Changes in the expression or activity of NOS enzymes can cause changes in NO levels. For example, iNOS is mainly regulated through transcription and its upregulation can result in increased production of NO (Farah, Michel & Balligand, 2018). Also, eNOS can be regulated by Ca2+ concentrations and blood flow shear stress through phosphorylation at Ser1177 (activating) and Thr495 (inhibiting) (Förstermann, 2010).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Without measuring NO levels directly, NOS levels can be used as a proxy to measure NO production. eNOS and iNOS are common points for assessing NO levels indirectly. Decreased NOS protein expression often corresponds to a decrease in NO. However, it is important to note that NOS levels do not perfectly correlate with NO levels. Increased NOS can also decrease NO if paired with a simultaneous increase in ROS, which, through oxidizing the enzyme’s cofactor BH4, causes NOS uncoupling (Forstermann, 2010; Zhang et al., 2009). Uncoupled NOS produces additional ROS that react with NO and reduce its overall abundance. Therefore, in this case, higher levels of NOS correlate to increased quantity of uncoupled NOS and a subsequent drop in NO bioavailability (Soloviev & Kizub, 2019). </span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Assay </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Reference </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Description </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">OECD Approved Assay </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Western blotting/immunoblotting </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">(Hong et al., 2013; Baker et al., 2009; Yan et al., 2020; Zhang et al., 2009; Zhang et al., 2008; Shi et al., 2012; Azimzadeh et al., 2017; Azimzadeh et al., 2015) </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Western blotting/immunoblotting is used to determine levels of inducible and endothelial NOS (NO synthesizing enzyme) in its phosphorylated and unphosphorylated forms, as well as nitrotyrosine (an indicator of NO). NOS and nitrotyrosine are detected by antibodies of each protein, visualized using chemiluminescence, and quantified using densitometry. </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Nitric oxide/nitrate/nitrate (NOx) assay kit (Griess assay) </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">(Azimzadeh et al., 2017; Adbel-Magied & Shedid, 2019; Yan et al., 2020; Cervelli et al., 2017; Siamwala et al., 2010) </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Levels of nitrite/nitrate (NOx) are determined using the NO assay kit. Nitrate reductase is used to convert nitrate into nitrite and the Griess reagent is then used to quantify levels of nitrite. </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Immunohistochemical staining </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">(Fuji et al., 2016) </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Uses an antibody to detect and measure levels of eNOS. </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Immunofluorescence </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">(Hamada et al., 2019) </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Uses fluorescent dye-labeled eNOS antibodies to visualize and determine eNOS levels. </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-family:Times New Roman,Times,serif">ELISA kit </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">(Hasan et al., 2020; Azimzadeh et al., 2015) </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Used to determine levels of NO and iNOS in serum by immobilizing the target antigen and binding it to associated antibodies linked to reporter enzymes. The activity of the reporter enzymes is then measured to determine levels of NO and iNOS. </span></p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p><span style="font-family:Times New Roman,Times,serif">4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) fluorescent probe </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">(Soucy et al., 2011; Soucy et al., 2010) </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Used to detect low concentrations of NO by reacting with it to become a fluorescent benzotriazole that can then be visualized and measured. </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Taxonomic applicability: </span></span></strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Altered nitric oxide is applicable to vertebrates only, as endothelial NO synthase (eNOS) is required for the formation of NO from the amino acid, L-arginine, and only vertebrates have a true endothelial lining (Yano et al., 2007).</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Life stage applicability: </span></span></strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">This key event is not life stage specific.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Sex applicability:</span></span></strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"> This key event is not sex specific (Soucy et al., 2011; Takeda et al., 2003).</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Evidence for perturbation by a stressor: </span></span></strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Current literature provides ample evidence of external stressors, including ionizing radiation exposure and altered gravity, inducing significant changes to levels of nitric oxide, nitrate, and NO synthase (Soucy et al., 2011; Zhang et al., 2009).</span></span></span></span></p>
HighMaleLowFemaleModerateUnspecificModerateAdultModerateNot Otherwise SpecifiedHighHighModerateModerate<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Abdel-Magied, N.<span style="background-color:white"><span style="color:black"> and S. M. </span></span></span><span style="font-size:12.0pt">Shedid </span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">(2019), “</span></span></span><span style="font-size:12.0pt">Impact of zinc oxide nanoparticles on thioredoxin-interacting protein and asymmetric dimethylarginine as biochemical indicators of cardiovascular disorders in gamma-irradiated rats”</span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">, </span></span></span><em><span style="font-size:12.0pt">Environmental Toxicology</span></em><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">, Vol. 35/4, John Wiley & Sons, Inc., Hoboken, </span></span></span> <a href="https://doi.org/10.1002/tox.22879" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1002/tox.22879</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Lo<span style="color:black">ng-Term Radiation-Induced Vascular Dysfunction”, <em>Journal of Proteome Research</em>, Vol. 14/2, American Chemical Society, Washington, </span></span><a href="https://doi.org/10.1021/pr501141b" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1021/pr501141b</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:black">Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, <em>International Journal of Radiation Biology</em>, Vol. 93/9, Informa, London, </span></span><a href="https://doi.org/10.1080/09553002.2017.1339332" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1080/09553002.2017.1339332</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Baker, J. E. et al. (2009), “10 Gy total body irradiation increases risk of coronary sclerosis, degeneration of heart structure and function in a rat model”, <em>International Journal of Radiation Biology</em>, Vol. 85/12, Informa, London, </span><a href="https://doi.org/10.3109/09553000903264473" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.3109/09553000903264473</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Burov, O. N. et al. (2022), “Mechanisms of nitric oxide generation in living systems”, <em>Nitric Oxide</em>, Vol. 118, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.niox.2021.10.003" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.niox.2021.10.003</a>.</span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Cervelli, T. et al. (2017), “A new natural antioxidant mixture protects against oxidative and DNA damage in endothelial cell exposed to low-dose irradiation”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2017, Hindawi, London, </span></span></span><a href="https://doi.org/10.1155/2017/9085947" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">https://doi.org/10.1155/2017/9085947</span></span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:#212529">Farah, C., L. Y. M. Michel and J.-L. Balligand. (2018), "Nitric oxide signalling in cardiovascular health and disease", <em>Nature Reviews Cardiology</em>, Vol. 15/5, Springer Nature, London, </span></span><a href="https://doi.org/10.1038/nrcardio.2017.224" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1038/nrcardio.2017.224</span></a><span style="font-size:12.0pt"><span style="color:#212529">.</span></span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:#212529">Förstermann, U. (2010), "Nitric oxide and oxidative stress in vascular disease", <em>Pflügers Archiv - European Journal of Physiology</em>, Vol. 459, Springer Nature, London, </span></span><a href="https://doi.org/10.1007/S00424-010-0808-2" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1007/S00424-010-0808-2</span></a><span style="font-size:12.0pt"><span style="color:#212529">.</span></span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">Fuji, S. et al. (2016), “Association between endothelial function and micro-vascular remodeling measured by synchrotron radiation pulmonary micro-angiography in pulmonary arterial hypertension”, </span></span></span><em><span style="font-size:12.0pt"><span style="color:#212529">General Thoracic and Cardiovascular Surgery</span></span></em><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">, Vol. 64/10, Springer, London, </span></span></span><a href="https://doi.org/10.1007/s11748-016-0684-6" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1007/s11748-016-0684-6</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Hamada, N. et al. (2020), “Ionizing Irradiation Induces Vascular Damage in the Aorta of Wild-Type Mice”, <em>Cancers</em>, Vol. 12/10, Multidisciplinary Digital Publishing Institute, Basel, </span><a href="https://doi.org/10.3390/CANCERS12103030" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.3390/CANCERS12103030</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Hamada, N. et al. (2022), “Temporal Changes in Sparing and Enhancing Dose Protraction Effects of Ionizing Irradiation for Aortic Damage in Wild-Type Mice”, <em>Cancers</em>, Vol. 14/14, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/cancers1414331</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:black">Hasan, H. F., R. R. Radwan and S. M. Galal (2020), “Bradykinin‐potentiating factor isolated from Leiurus quinquestriatus scorpion venom alleviates cardiomyopathy in irradiated rats via remodelling of the RAAS pathway”, <em>Clinical and Experimental Pharmacology and Physiology</em>, Vol. 47/2, Wiley-Blackwell, Hoboken, </span></span><a href="https://doi.org/10.1111/1440-1681.13202" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1111/1440-1681.13202</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Hong, C. W. et al. (2013), “Involvement of inducible nitric oxide synthase in radiation-induced vascular endothelial damage”, <em>Journal of Radiation Research</em>, Vol. 54/6, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/JRR/RRT066" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/JRR/RRT066</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Powers, S. K. and M. J. Jackson. (2008), "Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production", <em>Physiological Reviews</em>, Vol. 88/4, The American Physiological Society, Rockville, </span><a href="https://doi.org/10.1152/physrev.00031.2007" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1152/physrev.00031.2007</span></a><span style="font-size:12.0pt">.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Schulz, E., T. Gori and T. Münzel. (2011), "Oxidative stress and endothelial dysfunction in hypertension", <em>Hypertension Research</em>, Vol. 34/6, Nature Portfolio, London, </span><a href="https://doi.org/10.1038/hr.2011.39" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1038/hr.2011.39</span></a><span style="font-size:12.0pt">.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:black">Shi, F. et al. (2012), “Effects of Simulated Microgravity on Human Umbilical Vein Endothelial Cell Angiogenesis and Role of the PI3K-Akt-eNOS Signal Pathway”, <em>PLoS ONE</em>, Vol. 7/7, PLOS, San Francisco, </span></span><a href="https://doi.org/10.1371/journal.pone.0040365" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1371/journal.pone.0040365</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Siamwala, J. H. et al. (2010), “Simulated microgravity perturbs actin polymerization to promote nitric oxide-associated migration in human immortalized Eahy926 cells”, <em>Protoplasma</em>, Vol. 242/1, Springer, London, </span><a href="https://doi.org/10.1007/S00709-010-0114-Z" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/S00709-010-0114-Z</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soloviev, A. I. and I. V. Kizub. (2019), "Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction", <em>Biochemical Pharmacology</em>, Vol. 159, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/J.BCP.2018.11.019" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/J.BCP.2018.11.019</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, <em>Journal of Applied Physiology</em>, Vol. 108/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.00946.2009" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/japplphysiol.00946.2009</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soucy, K. G. et al. (2011), “HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, <em>Radiation Research</em>, Vol. 176/4, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RR2598.1" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1667/RR2598.1</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Yan, T., et al. (2020), “Ionizing radiation induces BH4 deficiency by downregulating GTP-cyclohydrolase 1, a novel target for preventing and treating radiation enteritis”, <em>Biochemical Pharmacology</em>, Vol. 180, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/J.BCP.2020.114102" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/J.BCP.2020.114102</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:#212121">Takeda, I., et al. (2013), “Possible Role of Nitric Oxide in Radiation-Induced Salivary Gland Dysfunction”, <em>Radiation Research</em>, Vol. 159/4, BioOne, </span></span><a href="https://doi.org/10.1667/0033-7587(2003)159%5b0465:PRONOI%5d2.0.CO;2" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1667/0033-7587(2003)159[0465:PRONOI]2.0.CO;2</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Yano, K., et al. (2007), “Phenotypic heterogeneity is an evolutionarily conserved feature of the endothelium”, <em>Blood</em>, Vol. 109/2, American Society of Hematology, Washington, D.C., </span></span></span><a href="https://doi.org/10.1182/blood-2006-05-026401" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1182/blood-2006-05-026401</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Yao, L. et al. (2010), "The role of RhoA/Rho kinase pathway in endothelial dysfunction", <em>Journal of Cardiovascular Disease Research</em>, Vol. 1/4, Elsevier, Amsterdam, </span><a href="https://doi.org/10.4103/0975-3583.74258" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.4103/0975-3583.74258</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, <em>Journal of Applied Physiology</em>, Vol. 106, American Physiological Society, Rockville, </span><a href="https://doi.org/10.1152/japplphysiol.01278.2007" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1152/japplphysiol.01278.2007</span></a></span></span></p>
2022-09-28T12:00:332023-04-13T09:00:04Increase, Endothelial DysfunctionIncrease, Endothelial DysfunctionTissue<p><span style="font-family:Times New Roman,Times,serif">The endothelium is the innermost lining of blood vessels consisting of a single layer of endothelial cells. As the layer separating blood and vessel walls, the endothelium controls the flow of molecules, fluid, and circulating blood cells between the two. However, the specific functions and even the structure of endothelial cells vary greatly depending on the organ (Ricard et al., 2021). Dysfunction to the vascular endothelium can age arteries and is the result of increased proliferation and apoptotic behaviour of cells including an increased response to endothelial constrictors. It is also represented by an imbalance between vasodilators and vasoconstrictors which are produced by the endothelium. The dysfunction can encompass vasospasm, thrombosis, penetration of immune cells (i.e macrophage) and an increase in cyclooxygenase. These processes can activate the endothelium and a prolonged state of activation is problematic and is referred to as endothelial dysfunction (Sitia et al., 2010; Deanfield et al., 2005; Konukoglu & Uzun, 2017; Korpela & Liu, 2014). Other factors leading to endothelial dysfunction are loss in endothelial function leading to cell senescence and a low proliferative capacity of endothelial progenitor cells.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Endothelial cell senescence </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Assay </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Reference </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Description </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">OECD Approved Assay </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Senescence-associated beta-galactosidase staining (SA-beta-gal) </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">(Farhat et al., 2008; González-Gualda et al., 2021; Hooten et al., 2017) </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Can be used to measure senescence-associated β-galactosidase activity, a marker for senescent cells. </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
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<td>
<p><span style="font-family:Times New Roman,Times,serif">Bromodeoxyuridine (BrdU) detected with staining incorporation </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">(González-Gualda et al., 2021) </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Reduced BrdU incorporation can indicate a lack of DNA synthesis. </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Immunohistochemistry to detect senescence markers. </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">(González-Gualda et al., 2021) </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Markers include Ki67 and Lamin B1. Reduced Ki67 can indicate reduced proliferation. Reduced Lamin B1 indicates impaired structural integrity of the nucleus. </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Cell morphology and size measured with light microscopy or flow cytometry. </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">(González-Gualda et al., 2021) </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Senescent cells exhibit an enlarged and flattened morphology. </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
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</tbody>
</table>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Cell death: </span></p>
<p><span style="font-family:Times New Roman,Times,serif">See the <a href="https://aopwiki.org/events/1825" rel="noreferrer noopener" target="_blank">increase, cell death KE</a> for methods to measure endothelial cell death. </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Impaired vasomotion</span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Assay </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Reference </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Description </span></p>
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<td>
<p><span style="font-family:Times New Roman,Times,serif">OECD Approved Assay </span></p>
</td>
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<td>
<p><span style="font-family:Times New Roman,Times,serif">Concentration-response curves to vasodilators/vasoconstrictors </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">(Deanfield et al., 2005; Verma et al., 2003) </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">Measurement of endothelial relaxation/contraction of blood vessels can give insight into endothelial dysfunction. This can be induced by endothelium-independent stimuli to stimulate vasodilation or vasoconstriction. A decreased stimuli response can be indicative of endothelial dysfunction. </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
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<td>
<p><span style="font-family:Times New Roman,Times,serif">Detection of contractile factors (eg. endothelin) using enzyme-linked immunosorbent assay (ELISA). </span></p>
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<p><span style="font-family:Times New Roman,Times,serif">(Abdel-Sayed et al., 2003) </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">Endothelin is an endothelium-derived vasoconstrictor. </span></p>
</td>
<td>
<p><span style="font-family:Times New Roman,Times,serif">No </span></p>
</td>
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</tbody>
</table>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><strong><span style="font-size:12.0pt">Taxonomic applicability: </span></strong><span style="font-size:12.0pt">Endothelial dysfunction is applicable to vertebrates as only vertebrates have a true endothelial lining (Yano et al., 2007). </span></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><strong><span style="font-size:12.0pt">Life stage applicability: </span></strong></span>Although endothelial dysfunction may occur due to aging (Hererra et al., 2010), this key event can occur at any life stage (Chang et al., 2017; Lee et al., 2020).</span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><strong><span style="font-size:12.0pt">Sex applicability: </span></strong><span style="font-size:12.0pt">This key event is not sex specific (Hughson et al., 2018; Lee et al., 2020). </span></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><strong><span style="font-size:12.0pt">Evidence for perturbation by a stressor: </span></strong><span style="font-size:12.0pt">Multiple studies show that endothelial dysfunction can be triggered by many types of stressors including ionizing radiation and altered gravity (Cheng et al., 2017; Soucy et al., 2011; Su et al., 2020; Yentrapalli et al., 2013). </span></span></span></p>
UBERON:0001986endotheliumModerateUnspecificModerateAll life stagesModerateModerateModerate<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Abdel-Sayed, S. et al. (2003), “Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects”, <em>American Journal of Hypertension</em>, Vol. 16/7, Oxford University Press, Oxford, <a href="http://doi.org/10.1016/S0895-7061(03)00903-8">https://doi.org/10.1016/S0895-7061(03)00903-8</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Chang, P. Y. et al. (2017), “MSC-derived cytokines repair radiation-induced intra-villi microvascular injury”, <em>Oncotarget</em>, Vol. 8/50, Impact Journals, Orchard Park, </span></span></span><a href="https://doi.org/10.18632/oncotarget.21236" style="color:#0563c1; text-decoration:underline">https://doi.org/10.18632/oncotarget.21236</a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Cheng, Y. P. et al. (2017), “Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats”, <em>Pflugers Archiv European Journal of Physiology</em>, Vol. 469, Springer, New York, <a href="http://doi.org/10.1007/s00424-017-1969-z">https://doi.org/10.1007/s00424-017-1969-z</a></span></span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Deanfield, J. et al. (2005), “Endothelial function and dysfunction”, <em>Journal of hypertension</em>, Vol. 23/1, Lippincott Williams & Wilkins, Philadelphia, </span></span></span><a href="https://doi.org/10.1097/00004872-200501000-00004" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1097/00004872-200501000-00004</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Farhat, N. et al. (2008), “Stress-induced senescence predominates in endothelial cells isolated from atherosclerotic chronic smokers”, <em>Canadian Journal of Physiology and Pharmacology</em>, Vol. 86/11, Canadian Science Publishing, Ottawa, </span></span></span><a href="https://doi.org/10.1139/Y08-082" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1139/Y08-082</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:1rem">González-Gualda, E. et al. (2021), “A guide to assessing cellular senescence in vitro and in vivo”, <em>The FEBS Journal</em>, Vol. 288, FEBS press, <a href="http://doi.org/10.1111/febs.15570">https://doi.org/10.1111/febs.15570</a> </span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Herrera, M. D. et al. (2010), “Endothelial dysfunction and aging: An update”, <em>Ageing Research Reviews</em>, Vol 9/2, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.arr.2009.07.002" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.arr.2009.07.002</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Hooten, N. N. and M. K. Evans (2017), “Techniques to Induce and Quantify Cellular Senescence”, <em>Journal of Visualized Experiments: JoVE</em>, Vol. 123, MyJove Corporation, Cambridge, </span></span></span><a href="https://doi.org/10.3791/55533" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.3791/55533</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Hughson, R. L., A. Helm and M. Durante (2018), “Heart in space: effect of the extraterrestrial environment on the cardiovascular system”, <em>Nature Reviews Cardiology</em>, Vol. 15/3, Nature Portfolio, London, </span></span></span><a href="https://doi.org/10.1038/nrcardio.2017.157" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1038/nrcardio.2017.157</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Konukoglu, D., and H. Uzun (2017), “Endothelial Dysfunction and Hypertension”, in <em>Hypertension: from basic research to clinical practice</em>, Springer, London, </span></span></span><a href="https://doi.org/10.1007/5584_2016_90" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1007/5584_2016_90</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Korpela, E., and S. K. Liu (2014), “Endothelial perturbations and therapeutic strategies in normal tissue radiation damage”, <em>Radiation Oncology, </em>Vol. 9, BioMed Central, London, </span></span></span><a href="https://doi.org/10.1186/s13014-014-0266-7" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1186/s13014-014-0266-7</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">Lee, S. et al. (2020), “Arterial structure and function during and after long-duration spaceflight”, <em>Journal of Applied Physiology, </em>Vol. 129/1, American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/japplphysiol.00550.2019" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1152/japplphysiol.00550.2019</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:"Times New Roman",Times,serif; font-size:1rem">Ricard, N. et al. (2021), “The quiescent endothelium: signalling pathways regulating organ-specific endothelial normalcy”, Nature reviews cardiology, Vol. 18/8, Springer Nature, <a href="http://doi.org/10.1038/s41569-021-00517-4 ">https://doi.org/10.1038/s41569-021-00517-4 </a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Sitia, S. et al. (2010), “From endothelial dysfunction to atherosclerosis”, Autoimmunity Reviews, Vol. 9/12, Elsevier, Amsterdam, <a href="http://doi.org/10.1016/j.autrev.2010.07.016">https://doi.org/10.1016/j.autrev.2010.07.016</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Soucy, K. G. et al. (2011), “HZE <sup>56</sup>Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, <em>Radiation Research</em>, Vol. 176/4, Radiation Research Society, Bozeman, </span></span></span><a href="https://doi.org/10.1667/RR2598.1" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1667/RR2598.1</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Su, Y. T. et al. (2020), “Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats”, <em>Life Sciences</em>, Vol. 243, Elsevier, Amsterdam, <a href="http://doi.org/10.1016/j.lfs.2019.117253">https://doi.org/10.1016/j.lfs.2019.117253</a></span></span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Verma, S., M. R. Buchanan and T. J. Anderson (2003), “Endothelial function testing as a biomarker of vascular disease”, <em>Circulation</em>, Vol. 108/17, Lippincott Williams & Wilkins, Philadelphia, </span></span></span><a href="https://doi.org/10.1161/01.CIR.0000089191.72957.ED" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1161/01.CIR.0000089191.72957.ED</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Yano, K. et al. (2007), “Phenotypic heterogeneity is an evolutionarily conserved feature of the endothelium”, <em>Blood</em>, Vol. 109/2, American Society of Hematology, Washington, D.C., </span></span></span><a href="https://doi.org/10.1182/blood-2006-05-026401" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1182/blood-2006-05-026401</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212121">Yentrapalli, R. et al. (2013), “The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation”, <em>PloS one</em>, Vol. 8/8, PLOS, San Francisco, </span></span></span><span style="font-size:11.0pt"><a href="https://doi.org/10.1371/journal.pone.0070024" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1371/journal.pone.0070024</span></span></a></span></span></p>
2022-09-28T12:39:532023-03-21T12:17:07Occurrence, Vascular Remodeling Occurrence, Vascular Remodeling Organ<p><span style="font-family:Times New Roman,Times,serif">The vascular wall is composed of endothelial, smooth muscle and fibroblast cell interactions (Gibbons & Dzau, 1994; Renna, Heras & Miatello, 2013). The vasculature is capable of detecting changes in its surroundings and maintaining homeostasis (Gibbons & Dzau, 1994; Renna, Heras & Miatello, 2013). The functionality of blood vessels is highly dependent on their structure, with changes in arterial morphology being associated with downstream impacts (Gibbons & Dzau, 1994). Vascular remodeling is a term for many histological changes, including increased vascular stiffness, wall shear stress, intima-media thickening (IMT), increased intima-media section area, and increased vessel diameter (Herity et al., 1999). As blood vessels stiffen, this impacts systolic and diastolic pressure and pulse which can be indicators of vascular remodeling. Cellular level changes characterized by processes of growth, death, migration and production or degradation of the extracellular matrix (ECM) result in inflammation (increase in VCAM, ICAM, cytokines, chemokines) and calcification (changes in ratios of collagen and elastin) (Gibbons & Dzau, 1994). Initial tissue injury and resulting remodeling can also lead to turbulent blood flow causing further structural changes like increased vessel fibrosis. Increased vascular remodelling is often associated with a build-up of plaque in the arteries (known as atherosclerosis) due to impaired healing, which forces the vessel walls to attempt to remodel to maintain blood flow (Sylvester et al., 2018).</span></p>
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<tbody>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Assay</span></span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Reference</span></span></strong></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:19px; vertical-align:top; width:226px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Description</span></span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">OECD Approved Assay</span></span></strong></span></span></p>
</td>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:top; width:188px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Pulse wave velocity (PWV)</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011)</span></span></span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Used to measure blood vessel stiffness. Calculated using measurements from a Doppler probe and electrocardiogram (ECG).</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:top; width:128px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">NIS-Elements image analysis software (Nikon)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Soucy et al., 2011)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Used to measure intraluminal perimeter (which in turn is used to calculate circular luminal diameter) and vessel wall thickness.</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Hematoxylin-eosin (HE) staining</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Shen et al., 2018; Su et al., 2020; Delp et al., 2000, Cheng et al., 2017, Yu et al., 2011)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Used to measure aortic wall thickness, intima-media wall thickness (IMT), wall shear stress, outer media perimeter, and media cross section area (CSA).</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Wire myography</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Tarasova et al., 2020)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Blood vessels are mounted in wire myograph systems and the relaxed inner diameter is estimated from the passive length-tension relationship between each artery.</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Verhoeff-van Gieson staining</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Sofronova et al., 2015)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Measures elastin-collagen content in blood vessels, with Verhoeff stain highlighting elastin and van Gieson highlighting collagen. The higher the ratio of elastin to collagen, the greater the distensibility of the vessel. A higher collagen ratio is associated with increased vascular stiffness.</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Sonography</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(Lee et al., 2020; Sarkozy et al., 2019; Sridharan et al., 2020)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Uses ultrasound waves to measure IMT and intima-media area, both of which are markers of vascular structure and are used to calculate vascular stiffness.</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">No</span></span></span></span></p>
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<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><strong><span style="font-size:12.0pt">Taxonomic applicability: </span></strong><span style="font-size:12.0pt">Vascular remodelling is applicable to all species with a closed circulatory system where blood is transported throughout the body via blood vessels with corresponding vessel walls (Renna,</span><span style="font-size:12.0pt"> Heras & Miatello, 2013). Closed circulatory systems are present in most vertebrates and some invertebrates.</span></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><strong><span style="font-size:12.0pt">Life stage applicability: </span></strong><span style="font-size:12.0pt">This key event is not life stage specific. </span></span>However, advancing age is a risk factor for vascular remodeling (Harvey, Montezano & Touyz, 2015).</span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><strong><span style="font-size:12.0pt">Sex applicability:</span></strong><span style="font-size:12.0pt"> This key event is not sex specific. </span></span>However, men are shown to develop vascular remodeling younger than women (Kessler et al., 2019).</span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><strong><span style="font-size:12.0pt">Evidence for perturbation by a stressor: </span></strong><span style="font-size:12.0pt">Current literature provides ample evidence of vascular remodelling being induced by stressors including ionizing radiation exposure and altered gravity (Shen et al. 2018; Su et al., 2020; Delp et al., 2000, Cheng et al., 2017, Yu et al., 2011; Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011). </span></span></span></p>
UBERON:0001981blood vesselModerateMaleLowFemaleModerateUnspecificModerateAdultModerateNot Otherwise SpecifiedModerateModerateModerate<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Cheng, Y. P. et al. (2017), "Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats", <em>Pflugers Archiv European Journal of Physiology</em>, Vol. 469, Springer Nature, London, </span><a href="https://doi.org/10.1007/s00424-017-1969-z" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1007/s00424-017-1969-z</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Delp, M.D. et al. (2000), “Structural and functional remodeling of skeletal muscle microvasculature is induced by simulated microgravity”, <em>American Journal of Physiology - Heart and Circulatory Physiology</em>, Vol. 278, American Physiological Society, Rockville, </span><a href="https://doi.org/10.1152/ajpheart.2000.278.6.h1866" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1152/ajpheart.2000.278.6.h1866</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Gibbons, G. H., and V. J. Dzau (1994), “The Emerging Concept of Vascular Remodeling”, <em>New England Journal of Medicine</em>, Vol. 330/20, Massachusetts Medical Society, Waltham, <a href="https://doi.org/10.1056/NEJM199405193302008" rel="noreferrer noopener" target="_blank">https://doi.org/10.1056/NEJM199405193302008</a></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Harvey, A., A. C. Montezano, & R. M. Touyz. (2015), “Vascular biology of ageing-Implications in hypertension”, <em>Journal of molecular and cellular cardiology</em>, Vol. 83, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.yjmcc.2015.04.011" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.yjmcc.2015.04.011</a></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Herity, N.A. et al. (1999), “Review: Clinical Aspects of Vascular Remodeling”, <em>Journal of Cardiovascular Electrophysiology</em>, Vol.10/7, Wiley, <a href="http://doi.org/10.1111/j.1540-8167.1999.tb01273.x">https://doi.org/10.1111/j.1540-8167.1999.tb01273.x</a></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Kessler, E. L. et al. (2019), “Sex-specific influence on cardiac structural remodeling and therapy in cardiovascular disease”, <em>Biology of Sex Differences</em>, Vol. 10, Springer Nature, <a href="http://doi.org/10.1186/s13293-019-0223-0">https://doi.org/10.1186/s13293-019-0223-0</a></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Lee, S. M. C. et al. (2020), “Arterial structure and function during and after long-duration spaceflight”, <em>Journal of Applied Physiology</em>, Vol. 129, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00550.2019</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Patel, S. (2020), "The effects of microgravity and space radiation on cardiovascular health: From low-Earth orbit and beyond", <em>IJC Heart and Vasculature</em>, Vol. 30, Elsevier, Amsterdam, </span><a href="https://doi.org/10.1016/j.ijcha.2020.100595" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1016/j.ijcha.2020.100595</span></a><span style="font-size:12.0pt">.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Renna, N. F., N. Heras, R. M. Miatello (2013), “Pathophysiology of Vascular Remodeling in Hypertension”, <em>International Journal of Hypertension, </em>Vol. 2013, Hindawi, London, </span><a href="http://doi.org/10.1155/2013/808353" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">http://doi.org/10.1155/2013/808353</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Sárközy, M. et al. (2019), "Selective heart irradiation induces cardiac overexpression of the pro-hypertrophic miR-212", <em>Frontiers in Oncology</em>, Vol. 9, Frontiers Media S.A., Lausanne, </span><a href="https://doi.org/10.3389/FONC.2019.00598/FULL" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.3389/FONC.2019.00598/FULL</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Shen, Y. et al. (2018), “Transplantation of bone marrow mesenchymal stem cells prevents radiation-induced artery injury by suppressing oxidative stress and inflammation”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2018, Hindawi, London, </span><a href="https://doi.org/10.1155/2018/5942916" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1155/2018/5942916</span></a><span style="font-size:12.0pt">.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Sofronova, S. I. et al. (2015), “Spaceflight on the Bion-M1 biosatellite alters cerebral artery vasomotor and mechanical properties in mice”, <em>Journal of Applied Physiology</em>, Vol. 118/7, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00976.2014.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Soucy, K. G. et al. (2011), “HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, <em>Radiation Research</em>, Vol. 176/4, Radiation Research Society, Bozeman, https://doi.org/10.1667/RR2598.1. </span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, <em>Journal of Applied Physiology</em>, Vol. 108/5, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00946.2009. </span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Soucy, K. G. et al. (2007), “Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta”, <em>Radiation and Environmental Biophysics</em>, Vol. 46, Springer, New York, https://doi.org/10.1007/s00411-006-0090-z.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Sridharan, V. et al. (2020), "Effects of single-dose protons or oxygen ions on function and structure of the cardiovascular system in male Long Evans rats", <em>Life Sciences in Space Research</em>, Vol. 26, Elsevier, Amsterdam, https://doi.org/10.1016/j.lssr.2020.04.002.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Su, Y. T. et al. (2020), "Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats", <em>Life Sciences</em>, Vol. 243, Elsevier, Amsterdam, https://doi.org/10.1016/j.lfs.2019.117253.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Sylvester, C. B. et al. (2018), "Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer", <em>Frontiers in Cardiovascular Medicine</em>, Vol. 5, Frontiers Media SA, Lausanne, https://doi.org/10.3389/fcvm.2018.00005.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Tarasova, O. S. et al. (2020), "Simulated Microgravity Induces Regionally Distinct Neurovascular and Structural Remodeling of Skeletal Muscle and Cutaneous Arteries in the Rat", <em>Frontiers in Physiology</em>, Vol. 1, Frontiers Media SA, Lausanne, https://doi.org/10.3389/fphys.2020.00675.</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Yu, T. et al. (2011), "Iron-ion radiation accelerates atherosclerosis in apolipoprotein E-Deficient mice", <em>Radiation Research</em>, Vol. 175/6, Radiation Research Society, Bozeman, https://doi.org/10.1667/RR2482.1.</span></span></span></p>
2022-10-17T16:49:012023-03-21T12:24:23Increase, DNA strand breaksIncrease, DNA strand breaksMolecular<p>DNA strand breaks are a type of damage resulting from the hydrolysis of phosphodiester groups in the backbone of DNA molecules (Gates, 2009) and can occur on a single strand (single strand breaks; SSBs) or both strands (double strand breaks; DSBs) (Cannan and Pederson, 2016; Tamanoi and Yoshikawa, 2022; Tripathy et al., 2021). SSBs arise when the phosphate backbone connecting adjacent nucleotides in DNA is broken on one strand. DSBs are generated when both strands are simultaneously broken at sites that are sufficiently close to one another that base-pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. As a consequence, the two DNA ends generated by a DSB can physically dissociate from one another, becoming difficult to repair and increasing the chance of inappropriate recombination with other sites in the genome (Jackson, 2002). SSBs can also turn into DSBs if the replication fork stalls at the lesion leading to fork collapse. It is also worth noting that there are error-prone and error-free forms of DSB repair (Jackson, 2002), and that the SSB repair pathway are distinct form the DSB repair pathways.</p>
<p>Strand breaks are intermediates in various biological events, including DNA repair (e.g., base excision repair), V(D)J recombination in developing lymphoid cells and chromatin remodeling in both somatic cells and germ cells. The spectrum of damage can be intricate, resulting in complex lesions, leading to mutations, and clustered damage defined as two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns. These lesions are more difficult to repair and have been studied in many types of models (Barbieri et al., 2019 and Asaithamby et al., 2011)<span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">. </span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">DSBs and complex lesions are of particular concern, as they are considered the most lethal and deleterious type of DNA lesion. If misrepaired or left unrepaired, DSBs may drive the cell towards genomic instability, apoptosis or tumorigenesis (Beir, 1999).</span></span></p>
<p> </p>
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Please refer to the table below for details regarding these and other methodologies for detecting DNA DSBs.</span></span></p>
<p style="text-align:center"> </p>
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<p style="text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Assay Name</span></span></strong></p>
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<p style="text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">References</span></span></strong></p>
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<p style="text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Description</span></span></strong></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">OECD </span></span></strong><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Approved Assay</span></span></strong></p>
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<p style="margin-left:10px; margin-right:9px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Comet Assay (Single Cell Gel Eletrophoresis - Alkaline)</span></span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Collins, 2004; Olive </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">and Banath, 2006; Platel et al., 2011; Nikolova et al., 2017</span></span></p>
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<p style="margin-left:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect SSBs or DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at an alkaline pH (pH >13); DNA fragments are forced to move, forming a "comet"-like </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">appearance</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">Yes (No. 489)</span></p>
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<p style="margin-left:11px; margin-right:10px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Comet Assay (Single Cell Gel Eltrophoresis - Neutral)</span></span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Collins, 2014; Olive </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">and Banath, 2006; Anderson and Laubenthal, 2013; Nikolova et al., 2017</span></span></p>
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<p style="margin-left:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at a neutral pH; DNA fragments, which are not denatured at the neutral pH, are forced to move, forming a "comet"-</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">like appearance</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:1px; margin-right:-1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX Foci Quantification - Flow </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Cytometry</span></span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Rothkamm and Horn, 2009; Bryce et al., </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">2016</span></span></p>
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<p style="margin-left:26px; margin-right:-1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Measurement of <span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX immunostaining in cells by flow cytometry, normalized to total levels of H2AX</span></span></p>
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<p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></p>
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<p style="margin-left:12px; margin-right:11px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX Foci Quantification - </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Western Blot</span></span></p>
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<p style="margin-left:9px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Burma et al., 2001; Revet et al., 2011</span></span></p>
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<p style="margin-left:14px; margin-right:9px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Measurement of <span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX immunostaining in cells by Western blotting, normalized to total levels of H2AX</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:12px; margin-right:11px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX Foci Quantification - Microscopy</span></span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Redon et al., 2010; Mah et al., 2010; Garcia-Canton et al., </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">2013</span></span></p>
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<p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Quantification of <span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX immunostaining by counting <span style="font-family:"MS UI Gothic",sans-serif">γ</span>- H2AX foci visualized with a microscope</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:12px; margin-right:11px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX Foci Detection -</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif"> </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">ELISA and flow cytometry</span></span></p>
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<p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Ji et al., 2017; </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Bryce et al., 2016</span></span></p>
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<p style="margin-right:-1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Detection of <span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX in cells by ELISA, normalized to total levels of H2AX; γH2AX foci detection can be high-throughput and automated using flow cytometry-based immunodetection.</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">Pulsed Field Gel Electrophoresis (PFGE)</span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Ager et al., 1990; Gardiner et al., 1985; Herschleb et al., 2007; Kawashima et </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">al., 2017</span></span></p>
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<p style="margin-left:9px; margin-right:8px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect DSBs, cells are embedded and lysed in agarose, and the released DNA undergoes gel electrophoresis in which the direction of the voltage is periodically alternated; Large DNA fragments are thus </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">able to be separated by size</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">The TUNEL </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">(Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) Assay</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">Loo, 2011</span></p>
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<p style="margin-left:5px; margin-right:4px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect strand breaks, dUTPs added to the 3’OH end of a strand break by the DNA polymerase terminal deoxynucleotidyl transferase (TdT) are tagged with a fluorescent dye or a reporter enzyme to allow visualization (We note that this method is typically used to measure apoptosis)</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:7px; margin-right:6px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><em>In Vitro </em>DNA Cleavage Assays using </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Topoisomerase</span></span></p>
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<p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Nitiss, 2012</span></span></p>
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<p style="margin-left:15px; margin-right:15px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Cleavage of DNA can be achieved using purified topoisomerase; DNA strand breaks can then be separated and quantified using gel electrophoresis</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">PCR assay </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Figueroa‑González & Pérez‑Plasencia, 2017 </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">Assay of strand breaks through the observation of DNA amplification prevention. Breaks block Taq polymerase, reducing the number of DNA templates, preventing amplification </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
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<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">Sucrose density gradient centrifuge </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Raschke et al. 2009 </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">Division of DNA pieces by density, increased fractionation leads to lower density pieces, with the use of a sucrose cushion </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
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<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">Alkaline Elution Assay </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Kohn, 1991 </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">Cells lysed with detergent-solution, filtered through membrane to remove all but intact DNA </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
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<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">Unwinding Assay </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Nacci et al. 1992 </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">DNA is stored in alkaline solutions with DNA-specific dye and allowed to unwind following removal from tissue, increased strand damage associated with increased unwinding </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
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<p><span style="font-size:11px"><strong>Taxonomic applicability: </strong>DNA strand breaks are relevant to all species, including vertebrates such as humans, that contain DNA (Cannan & Pederson, 2016). </span></p>
<p><span style="font-size:11px"><strong>Life stage applicability: </strong>This key event is not life stage specific as all life stages display strand breaks. However, there is an increase in baseline levels of DNA strand breaks seen in older individuals though it is unknown whether this change due to increased break induction or a greater retention of breaks due to poor repair (White & Vijg, 2016). </span></p>
<p><span style="font-size:11px"><strong>Sex applicability:</strong> This key event is not sex specific as both sexes display evidence of strand breaks. In some cell types, such as peripheral blood mononuclear cells, males show higher levels of single strand breaks than females (Garm et al., 2012). </span></p>
<p><span style="font-size:11px"><strong>Evidence for perturbation by a stressor: </strong>There are studies demonstrating that increased DNA strand breaks can result from exposure to multiple stressor types including ionizing & non-ionizing radiation, chemical agents, and oxidizing agents (EPRI, 2014; Hamada, 2014; Cencer et al., 2018; Cannan & Pederson, 2016; Yang et al., 1998). </span></p>
HighUnspecificHighAll life stagesNot Specified<p>Ager, D. D. et al. (1990). “Measurement of Radiation- Induced DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis.” Radiat Res. 122(2), 181-7.</p>
<p>Anderson, D. & Laubenthal J. (2013), “Analysis of DNA Damage via Single-Cell Electrophoresis. In: Makovets S, editor. DNA Electrophoresis. Totowa.”, NJ: Humana Press. p 209-218.</p>
<p>Asaithamby, A., B. Hu and D.J. Chen. (2011) Unrepaired clustered DNA lesions induce chromosome breakage in human cells. Proc Natl Acad Sci U S A 108(20): 8293-8298 .</p>
<p>Barbieri, S., G. Babini, J. Morini et a l (2019). . Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures. Scientific Reports 9(1): 14019</p>
<p>Beir, V. et al. (1999), “The Mechanistic Basis of Radon-Induced Lung Cancer”, in Health Risks of Exposure to Radon: BEIR VI, National Academy Press, Washington, D.C., https://doi.org/10.17226/5499 </p>
<p>Bryce, S. et al. (2016), “Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.”, Environ Mol Mutagen. 57:171-189. Doi: 10.1002/em.21996.</p>
<p>Burma, S. et al. (2001), “ATM phosphorylates histone H2AX in response to DNA double-strand breaks.”, J Biol Chem, 276(45): 42462-42467. doi:10.1074/jbc.C100466200</p>
<p>Cannan, W.J. and D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", Journal of Cellular Physiology, Vol.231/1, Wiley, New York, https://doi.org/10.1002/jcp.25048. </p>
<p>Cannan, W.J. and D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", Journal of Cellular Physiology, Vol.231/1, Wiley, New York, https://doi.org/10.1002/jcp.25048. </p>
<p>Cencer, C. et al. (2018), “PARP-1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light”, Photochemistry and Photobiology, Vol.94/1, Wiley-Blackwell, Hoboken, https://doi.org/10.1111/php.12814. </p>
<p>Charlton, E. D. et al. (1989), “Calculation of Initial Yields of Single and Double Stranded Breaks in Cell Nuclei from Electrons, Protons, and Alpha Particles.”, Int. J. Radiat. Biol. 56(1): 1-19. doi: 10.1080/09553008914551141.</p>
<p>Collins, R. A. (2004), “The Comet Assay for DNA Damage and Repair. Molecular Biotechnology.”, Mol Biotechnol. 26(3): 249-61. doi:10.1385/MB:26:3:249</p>
<p>Durdik, M et al. (2015), “Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes.” Cytometry. Part A. 87(12): 1070-8. Doi:10.1002/cyto.a.22731 </p>
<p>EPRI (2014), Epidemiology and mechanistic effects of radiation on the lens of the eye: Review and scientific appraisal of the literature, EPRI, California. </p>
<p>Figueroa‑González, G. and C. Pérez‑Plasencia. (2017), “Strategies for the evaluation of DNA damage and repair mechanisms in cancer”, Oncology Letters, Vol.13/6, Spandidos Publications, Athens, https://doi.org/10.3892/ol.2017.6002. </p>
<p>Garcia-Canton, C. et al. (2013), “Assessment of the in vitro p-H2AX assay by High Content Screening asa novel genotoxicity test.”, Mutat Res. 757:158-166. Doi: 10.1016/j.mrgentox.2013.08.002</p>
<p>Gardiner, K. et al. (1986), “Fractionation of Large Mammalian DNA Restriction Fragments Using Vertical Pulsed-Field Gradient Gel Electrophoresis.”, Somatic Cell and Molecular Genetics. 12(2): 185-95.Doi: 10.1007/bf01560665.</p>
<p>Garm, C. et al. (2012), “Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells”, Aging Cell, Vol.12/1, Blackwell Publishing Ltd, Oxford, https://doi.org/10.1111/acel.12019. </p>
<p>Guo, X. et al. (2018), “Acetylation of 53BP1 dictates the DNA double strand break repair pathway.” Nucleic acids research. 46(2): 689-703. doi:10.1093/nar/gkx1208 </p>
<p>Hamada, N. (2014), “What are the intracellular targets and intratissue target cells for radiation effects?”, Radiation research, Vol. 181/1, The Radiation Research Society, Indianapolis, https://doi.org/10.1667/RR13505.1. </p>
<p>Herschleb, J. et al. (2007), “Pulsed-field gel electrophoresis.”, Nat Protoc. 2(3): 677-684. doi:10.1038/nprot.2007.94</p>
<p>Jackson, S. (2002). “Sensing and repairing DNA double-strand breaks.”, Carcinogenesis. 23:687-696. Doi:10.1093/carcin/23.5.687.</p>
<p>Ji, J. et al. (2017), “Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay.”, PLoS One. 12(2): e0171582. doi:10.1371/journal.pone.0171582</p>
<p>Kawashima, Y.(2017), “Detection of DNA double-strand breaks by pulsed-field gel electrophoresis.”, Genes Cells 22:84-93. Doi: 10.1111/gtc.12457.</p>
<p>Khoury, L. et al. (2013), “Validation of high-throughput genotoxicity assay screening using cH2AX in-cell Western assay on HepG2 cells.”, Environ Mol Mutagen, 54:737-746. Doi: 10.1002/em.21817.</p>
<p>Khoury, L. et al. (2016), “Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening.”, Mutagenesis, 31:83-96. Doi: <a href="https://doi.org/10.1093/mutage/gev058" target="_blank">10.1093/mutage/gev058</a>.</p>
<p>Kohn, K.W. (1991), “Principles and practice of DNA filter elution”, Pharmacology & Therapeutics, Vol.49/1, Elsevier, Amsterdam, https://doi.org/10.1016/0163-7258(91)90022-E. </p>
<p>Loo, DT. (2011), “In Situ Detection of Apoptosis by the TUNEL Assay: An Overview of Techniques. In: Didenko V, editor. DNA Damage Detection In Situ, Ex Vivo, and In Vivo. Totowa.”, NJ: Humana Press. p 3-13.doi: <a href="https://doi.org/10.1007/978-1-60327-409-8_1" target="_blank">10.1007/978-1-60327-409-8_1</a>.</p>
<p>Mah, L. J. et al. (2010), “Quantification of gammaH2AX foci in response to ionising radiation.”, J Vis Exp(38). doi:10.3791/1957.</p>
<p>Nacci, D. et al. (1992), “Application of the DNA alkaline unwinding assay to detect DNA strand breaks in marine bivalves”, Marine Environmental Research, Vol.33/2, Elsevier BV, Amsterdam, https://doi.org/10.1016/0141-1136(92)90134-8. </p>
<p>Nikolova, T., F. et al. (2017), “Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.”, Mutat Res 822:10-18. Doi: <a href="https://doi.org/10.1016/j.mrgentox.2017.07.004" target="_blank">10.1016/j.mrgentox.2017.07.004</a>.</p>
<p>Nitiss, J. L. et al. (2012), “Topoisomerase assays. ”, Curr Protoc Pharmacol. Chapter 3: Unit 3 3.</p>
<p>OECD. (2014). Test No. 489: “In vivo mammalian alkaline comet assay.” OECD Guideline for the Testing of Chemicals, Section 4 .</p>
<p>Olive, P. L., & Banáth, J. P. (2006), “The comet assay: a method to measure DNA damage in individual cells.”, Nature Protocols. 1(1): 23-29. doi:10.1038/nprot.2006.5.</p>
<p>Platel A. et al. (2011), “Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the <em>in vitro </em>modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels.”, Mutat Res 726:151-159. Doi: 10.1016/j.mrgentox.2011.09.003.</p>
<p>Popp, H. D. et al. (2017), “Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks”, Journal of visualized experiments, 129: 56617, doi:10.3791/56617 </p>
<p>Raschke, S., J. Guan and G. Iliakis. (2009), “Application of alkaline sucrose gradient centrifugation in the analysis of DNA replication after DNA damage”, Methods in Molecular Biology, Vol.521, Humana Press, Totowa, https://doi.org/10.1007/978-1-60327-815-7_18. </p>
<p>Redon, C. et al. (2010), “The use of gamma-H2AX as a biodosimeter for total-body radiation exposure in non-human primates.”, PLoS One. 5(11): e15544. doi:10.1371/journal.pone.0015544</p>
<p>Revet, I. et al. (2011), “Functional relevance of the histone γH2Ax in the response to DNA damaging agents.” Proc Natl Acad Sci USA.108:8663-8667. Doi: 10.1073/pnas.1105866108</p>
<p>Rogakou, E.P. et al. (1998), “DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139.” , J Biol Chem, 273:5858-5868. Doi: 10.1074/jbc.273.10.5858</p>
<p>Rothkamm, K. & Horn, S. (2009), “γ-H2AX as protein biomarker for radiation exposure.”, Ann Ist Super Sanità, 45(3): 265-71.</p>
<p>Tamanoi, F., & Yoshikawa, K. (2022), “Overview of DNA damage and double-strand breaks”, The Enzymes, Vol.51, 1–5. https://doi.org/10.1016/bs.enz.2022.08.001 </p>
<p>Tripathy, B. K., Pal, K., Shabrish, S., & Mittra, I. (2021), “A New Perspective on the Origin of DNA Double-Strand Breaks and Its Implications for Ageing” Genes, Vol.12/2, 163. <a href="https://doi.org/10.3390/genes12020163" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/genes12020163</a> </p>
<p>White, R.R. and J. Vijg. (2016), “Do DNA Double-Strand Breaks Drive Aging?”, Molecular Cell, Vol.63, Elsevier, Amsterdam, http://doi.org/10.1016/j.molcel.2016.08.004. </p>
<p>Yang, Y. et al. (1998), “The effect of catalase amplification on immortal lens epithelial cell lines”, Experimental Eye Research, Vol.67/6, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1998.0560. </p>
2019-05-19T16:33:202024-03-08T12:05:51f1940943-69e5-458b-8c03-896b1c61c3f981eb87ac-fb24-4667-bd3a-9be52450f362<p>Energy deposited onto biomolecules stochastically in the form on ionizing and non-ionizing radiation can cause direct and indirect molecular-level damage. As energy is deposited in an aqueous solution, water molecules can undergo radiolysis, breaking bonds to produce reactive oxygen species (ROS) (Ahmadi et al., 2021; Karimi et al., 2017) or directly increase function of enzymes involved in ROS generation (i.e. catalaze). Various species of ROS can be generated with differing degrees of biological effects. For example, singlet oxygen, superoxide, and hydroxyl radical are highly unstable, with short half-lives and react close to where they are produced, while species like H<sub>2</sub>O<sub>2</sub> are much more stable and membrane permeable, meaning they can travel from the site of production, reacting elsewhere as a much weaker oxidant (Spector, 1990). In addition, enzymes involved in reactive oxygen and nitrogen species (RONS) production can be directly upregulated following the deposition of energy (de Jager, Cockrell and Du Plessis, 2017). Although less common than ROS, reactive nitrogen species (RNS) can also be produced by energy deposition resulting in oxidative stress (Cadet et al., 2012; Tangvarasittichai & Tangvarasittichai, 2019), a state in which the amount of ROS and RNS, collectively known as RONS, overwhelms the cell’s antioxidant defense system. This loss in redox homeostasis can lead to oxidative damage to macromolecules including proteins, lipids, and nucleic acids (Schoenfeld et al., 2012; Tangvarasittichai & Tangvarasittichai, 2019; Turner et al., 2002). </p>
<p>Overal weight of evidence: High</p>
<p>A large body of literature supports the linkage between the deposition of energy and oxidative stress. Multiple reviews describe the relationship in the context of ROS production (Marshall, 1985; Balasubramanian, 2000; Jurja et al., 2014), antioxidant depletion (Cabrera et al., 2011; Fletcher, 2010; Ganea & Harding, 2006; Hamada et al., 2014; Spector, 1990; Schoenfeld et al., 2012; Wegener, 1994), and overall oxidative stress (Eaton, 1994, Tangvarasittichai & Tangvarasittichain, 2019). This includes investigations into the mechanism behind the relationship (Ahmadi et al., 2021; Balasubramanian, 2000; Cencer et al., 2018; Eaton, 1994; Fletcher, 2010; Jiang et al., 2006; Jurja et al., 2014; Padgaonkar et al., 2015; Quan et al., 2021; Rong et al., 2019; Slezak et al., 2015; Soloviev & Kizub, 2019; Tian et al., 2017; Tahimic & Globus, 2017; Varma et al., 2011; Venkatesulu et al., 2018; Wang et al., 2019a; Yao et al., 2008; Yao et al., 2009; Zigman et al., 2000). </p>
<p>Water radiolysis is a main source of free radicals. Energy ionizes water and free radicals are produced that combine to create more stable ROS, such as hydrogen peroxide, hydroxide, superoxide, and hydroxyl (Eaton, 1994; Rehman et al., 2016; Tahimic & Globus, 2017; Tian et al., 2017; Varma et al., 2011; Venkatesulu et al., 2018). ROS formation causes ensuing damage to the body, as ~80% of tissues are comprised of water (Wang et al., 2019a). Ionizing radiation (IR) is a source of energy deposition, it can also interact with molecules, such as nitric oxide (NO), to produce less common free radicals, including RNS (Slezak et al., 2015; Tahimic & Globus, 2017; Wang et al., 2019a). Free radicals can diffuse throughout the cell and damage vital cellular components, such as proteins, lipids, and DNA, as well as dysregulate cellular processes, such as cell signalling (Slezak et al., 2015; Tian et al., 2017). </p>
<p>ROS are also commonly produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Deposition of energy can activate NOX and induce expression of its catalytic and cytosolic components, resulting in increased intracellular ROS (Soloviev & Kizub, 2019). Intracellular ROS production can also be initiated through the expression of protein kinase C, which in turn activates NOX through phosphorylation of its cytosolic components (Soloviev & Kizub, 2019). Alternatively, ROS are often formed at the electron transport chain (ETC) of the mitochondria, due to IR-induced electron leakage leading to ionization of the surrounding O<sub>2</sub> to become superoxide (Soloviev & Kizub, 2019). Additionally, energy reaching a cell can be absorbed by an unstable molecule, often NADPH, known as a chromophore, which leads to the production of ROS (Balasubramanian, 2000; Cencer et al., 2018; Jiang et al., 2006; Jurja et al., 2014; Padgaonkar et al., 2015; Yao et al., 2009; Zigman et al., 2000). </p>
<p>Energy deposition can also weaken a cell’s antioxidant defense system through the depletion of certain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT). Antioxidants are consumed during the process of neutralizing ROS, so as energy deposition stimulates the formation of ROS it begins to outpace the rate at which antioxidants are replenished; this results in an increased risk of oxidative stress when their concentrations are low (Belkacémi et al., 2001; Giblin et al., 2002; Ji et al., 2014; Kang et al., 2020; Karimi et al., 2017; Padgaonkar et al., 2015; Rogers et al., 2004; Slezak et al., 2015; Tahimic & Globus, 2017; Wang et al., 2019a; Wegener, 1994; Weinreb & Dovrat, 1996; Zhang et al., 2012; Zigman et al., 1995; Zigman et al., 2000). When the amount of ROS overwhelms the antioxidant defense system, the cell will enter oxidative stress leading to macromolecular and cellular damage (Tangvarasittichai & Tangvarasittichai, 2019). </p>
<p>The relationship between energy deposition and oxidative stress is strongly supported by primary research on the effects of IR on ROS and antioxidant levels (Bai et al., 2020; Cervelli et al., 2017; Hatoum et al., 2006; Huang et al., 2018; Huang et al., 2019; Karam & Radwan, 2019; Kook et al., 2015; Liu et al., 2018; Liu et al., 2019; Mansour, 2013; Philipp et al., 2020; Ramadan et al., 2020; Sharma et al., 2018; Shen et al., 2018; Soltani et al., 2016; Soucy et al., 2010; Soucy et al., 2011; Ungvari et al., 2013; Wang et al., 2016; Wang et al., 2019b; Zhang et al., 2018; Zhang et al., 2020). Of note is that the relationship is demonstrated across studies conducted using various cell types, models and using broad dose-ranges as summarized below. Much evidence is available and described to help discern the quantitative understanding of the relationship, since it is well established. </p>
<p> </p>
<p><strong>Dose Concordance </strong></p>
<p>It is well-accepted that any dose of radiation will deposit energy onto matter. Doses as low as 1 cGy support this relationship (Tseung et al., 2014). Following the deposition of energy, markers of oxidative stress are observed in the form of RONS, a change in levels of antioxidants, and oxidative damage to macromolecules. These effects have been shown across various organs/tissues and cell types as described below. </p>
<p> </p>
<p><u>RONS </u></p>
<p>Cardiovascular tissue: </p>
<p>There is a considerable amount of evidence to support this relationship in cell types and tissues of relevance to the cardiovascular system. Recent studies have shown a linear increase in ROS in human umbilical vein endothelial cells (HUVECs) following 0-5 Gy gamma irradiation (Wang et al., 2019b). HUVECs irradiated with 0.25 Gy X-rays (Cervelli et al., 2017) and 9 Gy 250kV photons (Sharma et al., 2018) show increased ROS. Gamma ray irradiated rats at 5 Gy display increased ROS levels in the aorta (Soucy et al., 2010). A study using cerebromicrovascular endothelial cell (CMVECs) showed a dose-dependent increase in ROS from 0-8 Gy gamma irradiation (Ungvari et al., 2013). Additionally, telomerase-immortalized coronary artery endothelial (TICAE) and telomerase-immortalized microvascular endothelial (TIME) cells irradiated with 0.1 and 5 Gy of X-rays displayed increased ROS production (Ramadan et al., 2020). Gut arterioles of rats showed increased ROS following multiple fractions of 2.5 Gy X-ray rat irradiation (Hatoum et al., 2006). Additionally, rats irradiated with 1 Gy of 56Fe expressed increased ROS levels in the aorta (Soucy et al., 2011). </p>
<p> </p>
<p>Brain tissue: </p>
<p>Markers of oxidative stress have also been consistently observed in brain tissue. Human neural stem cells subjected to 1, 2 or 5 Gy gamma rays showed a dose-dependent increase in RONS production (Acharya et al., 2010). A dose-dependent increase in ROS was observed in rat brains following 1-10 Gy gamma rays (Collins-Underwood et al., 2008). Neural precursor cells exposed to 0-10 Gy of X-irradiation showed increased ROS levels (Giedzinski et al., 2005; Limoli et al., 2004). Mice brain tissue displayed increased ROS following proton irradiation (Baluchamy et al., 2012; Giedzinski et al., 2005). Neural processor cells expressed linearly increased ROS levels following doses of 56Fe (Limoli et al., 2007). A dose-dependent increase in RONS was also observed after exposure to 1-15 cGy 56Fe irradiation in mice neural stem/precursor cell (Tseng et al., 2014). Human neural stem cells exposed to 5-100 cGy of various ions demonstrated a dose-dependent increase in RONs (Baulch et al., 2015). </p>
<p> </p>
<p>Eye tissue: </p>
<p>The eye is also sensitive to the accumulation of free radicals, in a state of antioxidant decline. It has been shown in human lens epithelial cells (HLECs) and HLE-B3 following gamma irradiation of 0.25 and 0.5 Gy that ROS levels are markedly increased (Ahmadi et al., 2021). Exposure to non-ionizing radiation, such as ultraviolet (UV)-B, has also led to increased ROS in HLECs and mice lenses (Ji et al., 2015; Kubo et al., 2010; Rong et al., 2019; Yang et al., 2020) </p>
<p> </p>
<p>Bone tissue: </p>
<p>Rat bone marrow-derived mesenchymal stem cell (bmMSCs) irradiated with 2, 5 and 10 Gy gamma rays and Murine MC3T3-E1 osteoblast cells irradiated with 2, 4, and 8 Gy of X-rays have shown a dose-dependent increase in ROS levels (Bai et al., 2020; Kook et al., 2015). Murine RAW264.7 cells and rat bmMSC irradiated with 2 Gy of gamma rays displayed increased ROS levels (Huang et al., 2019; Huang et al., 2018; hang et al., 2020). Human bone marrow-derived mesenchymal stem cell (hBMMSCs) irradiated with 2 or 8 Gy X-rays showed increased ROS (Liu et al., 2018; Zhang et al., 2018). Similarly, murine MC3T3-E1 osteoblast-like cells irradiated with 6 Gy of X-rays also displayed increased ROS (Wang et al., 2016). Finally, whole-body irradiation of mice with 2 Gy of 31.6 keV/mm LET 12C heavy ions showed increased ROS (Liu et al., 2019) </p>
<p> </p>
<p><u>Antioxidants </u></p>
<p>Blood: </p>
<p>Workers exposed to X-rays at less than 1 mSv/year for an average of 15 years showed around 20% decreased antioxidant activity compared to unexposed controls (Klucinski et al., 2008). Similarly, adults exposed to high background irradiation of 260 mSv/year showed about 50% lower antioxidant activity power compared to controls (Attar, Kondolousy and Khansari, 2007). </p>
<p> </p>
<p>Cardiovascular tissue: </p>
<p>Heart tissue of rats following gamma irradiation of rats at 5 and 6 Gy resulted in a decrease in antioxidant levels (Karam & Radwan, 2019; Mansour, 2013). Similarly, HUVECs (Soltani, 2016) and TICAE cells (Philipp et al., 2020) irradiated at 2 Gy and 0.25-10 Gy gamma rays, respectively, displayed decreased antioxidant levels. Mice exposed to 18 Gy of X-ray irradiation showed decreased antioxidants in the aorta (Shen et al., 2018).</p>
<p> </p>
<p>Brain tissue: </p>
<p>Mice brain tissue following 2, 10 and 50 cGy whole-body gamma irradiation revealed a dose-dependent change in SOD2 activity (Veeraraghan et al., 2011). Mice brain tissue showed decreased glutathione (GSH) and SOD levels following proton irradiation (Baluchamy et al., 2012) </p>
<p> </p>
<p>Eye tissue: </p>
<p>Rats exposed to 15 Gy gamma rays demonstrated decreased antioxidants in the lens tissue (Karimi et al, 2017). Neutron irradiation of rats at 3.6 Sv resulted in a decrease in antioxidants in lens (Chen et al., 2021). A few studies found a dose concordance between UV irradiation and decreased antioxidant levels (Hua et al, 2019; Ji et al, 2015; Zigman et al., 2000; Zigman et al, 1995). HLECs following UVB exposure from 300 J/m2 to 14,400 J/m2 in HLECs showed linear decreases in antioxidant activity (Ji et al., 2015). Similarly, HLEC exposed to 4050, 8100 and 12,150 J/m2 found decreased antioxidant levels (Hua et al., 2019). Following UV irradiation of rabbit and squirrel lens epithelial cells (LECs) showed a linear decrease of antioxidant level, CAT (Zigman et al., 2000; Zigman et al., 1995). Mice exposed to UV irradiation found decreased antioxidant levels in lens (Zhang et al., 2012). Similarly, SOD levels decreased following 0.09 mW/cm 2 UVB exposure of HLECs (Kang et al., 2020). </p>
<p> </p>
<p>Bone tissue: </p>
<p>Rat bmMSCs irradiated with 2, 5 and 10 Gy gamma rays and Murine MC3T3-E1 osteoblast cells irradiated with 2, 4, and 8 Gy of X-rays showed a dose-dependent decrease in antioxidant levels (Bai et al., 2020; Kook et al., 2015). hBMMSCs irradiated with 8 Gy X-rays also showed a decrease in antioxidant, SOD, levels (Liu et al., 2018). </p>
<p> </p>
<p><u>Oxidative Damage </u></p>
<p>Cardiovascular tissue: </p>
<p>HUVECs and rat hearts irradiated by gamma rays at 2 and 6 Gy, respectively, resulted in increased levels of oxidative stress markers, such as malondialdehyde (MDA), and thiobarbituric reactive substances (TBARS) (Mansour, 2013; Soltani, 2016). </p>
<p> </p>
<p>Brain tissue: </p>
<p>Mice brain tissue were shown to have increased lipid peroxidation (LPO) as determined by MDA measurements, following proton irradiation at 1 and 2 Gy (Baluchamy et al., 2012). Neural precursor cells from rat hippocampus exposed to 0, 1, 5 and 10 Gy of X-irradiation resulted in increased lipid peroxidation (Limoli et al., 2004).</p>
<p> </p>
<p>Eye tissue: </p>
<p>Rats exposed to 15 Gy gamma rays demonstrated increased MDA in lens tissue (Karimi et al, 2017). Neutron irradiation of rats at 3.6 Sv resulted in an initial decrease, followed by an increase in MDA in lens (Chen et al., 2021). Following UV irradiation at 300 4050, 8100 and 12,150 J/m2, there was an increase in LPO in in human lens (Chitchumroonchokchai et al, 2004; Hua et al, 2019). Similarly, LPO increased following 0.09 mW/cm 2 UVB exposure of HLECs (Kang et al., 2020). </p>
<p> </p>
<p><strong>Time Concordance </strong></p>
<p>It is well-accepted that deposition of energy into matter results in immediate vibrational changes to molecules or ionization events. Deposition of energy is therefore an upstream event to all follow-on latent events like oxidative stress. </p>
<p> </p>
<p><u>RONS </u></p>
<p>Cardiovascular tissue: </p>
<p>In TICAE and TIME cells, ROS increased at 45 mins after X-ray irradiation (Ramadan et al., 2020). Superoxide and peroxide production were increased 1 day after 2-8 Gy of gamma irradiation in CMVECs (Unvari et al., 2013). </p>
<p> </p>
<p>Bone tissue: </p>
<p>hBMMSCs irradiated with X-rays at 2 Gy showed peak ROS production at 2-8h post-irradiation (Zhang et al., 2018). Murine RAW264.7 cells (can undergo osteoclastogenesis) irradiated with 2 Gy of gamma rays showed increased ROS at 2-8h post-irradiation (Huang et al., 2018). </p>
<p> </p>
<p>Brain tissue: </p>
<p>In human lymphoblast cells exposed to 2 Gy of X-rays, ROS were increased at various times between 13 and 29 days post-irradiation (Rugo and Schiestl, 2004). RONS were increased in human neural stem cells at 12-48h post-irradiation with 2 and 5 Gy of gamma rays (Acharya et al., 2010). ROS levels were increased in rat neural precursor cells at 6-24h after irradiation with 1-10 Gy of protons (Giedzinksi et al., 2005). Both 56Fe (1.3 Gy) and gamma ray (2 Gy) irradiation of mice increased ROS levels after 2 months post-irradiation in the cerebral cortex (Suman et al., 2013). ROS were also increased 12 months after 56Fe irradiation (Suman et al., 2013). RONS increased as early as 12h post-irradiation continuing to 8 weeks with 2-200 cGy doses of 56Fe irradiation of mouse neural stem/precursor cells (Tseng et al., 2014). The same cell type irradiated with 1 and 5 Gy of 56Fe irradiation showed increased ROS at 6h post-irradiation, with the last increase observed 25 days post-irradiation (Limoli et al., 2004). </p>
<p> </p>
<p>Eye tissue: </p>
<p>Mice exposed to 11 Gy of X-rays showed increased ROS at 9 months post-irradiation in lenses (Pendergrass et al., 2010). In human lens cells, ROS were found increased at 1h after 0.25 Gy gamma ray irradiation (Ahmadi et al., 2021), 15 minutes after 30 mJ/cm2 UV radiation (Jiang et al., 2006), 2.5-120 minutes after 0.014 and 0.14 J/cm2 UV radiation (Cencer et al., 2018), and 24h after 30 mJ/cm2 UVB radiation (Yang et al., 2020). </p>
<p> </p>
<p><u>Antioxidants </u></p>
<p>Cardiovascular tissue: </p>
<p>CAT antioxidant enzyme was decreased in mice aortas as early as 3 days post-irradiation, remaining decreased until 84 days after irradiation with 18 Gy of X-rays (Shen et al., 2018). The antioxidant enzymes peroxiredoxin 5 (PRDX5) and SOD were both shown to have the greatest decrease at 24h after 2 Gy gamma irradiation of TICAE cells (Philipp et al., 2020). </p>
<p> </p>
<p>Eye tissue: </p>
<p>Bovine lenses irradiated with 44.8 J/cm2 of UVA radiation showed decreased CAT levels at 48-168h post-irradiation (Weinreb and Dovrat, 1996). UV irradiation of mice at 20.6 kJ/m2 led to decreased GSH at both 1 and 16 months post-irradiation in the lens (Zhang et al., 2012). Bovine lens cells exposed to 10 Gy of X-rays showed decreased levels of the antioxidant GSH at 24 and 120h after exposure (Belkacemi et al., 2001). </p>
<p> </p>
<p><u>Oxidative damage markers </u></p>
<p>Cardiovascular tissue: </p>
<p>Oxidative damage markers 4-hydroxynonemal (4-HNE) and 3-Nitrotyosine (3-NT) were both significantly increased in the aorta of mice at 3 days post-irradiation, remaining increased until 84 days after irradiation with 18 Gy of X-rays (Shen et al., 2018). </p>
<p> </p>
<p><strong>Essentiality </strong></p>
<p>Radiation has been found to induce oxidative stress above background levels. Many studies have shown that lower doses of ionizing radiation resulted in decreased levels in markers of oxidative stress in multiple cell types (Acharya et al., 2010; Ahmadi et al., 2021; Bai et al., 2020; Baluchamy et al., 2012 Chen et al., 2021; Collins-Underwood et al., 2008; Giedzinski et al., 2005; Kook et al., 2015; Kubo et al., 2010; Philipp et al., 2020; Ramadan et al., 2020; Ungvari et al., 2013; Veeraraghan et al., 2011; Wang et al., 2019b; Zigman et al., 2000; Zigman et al., 1995). The essentiality of deposition of energy can be assessed through the removal of deposited energy, a physical stressor that does not require to be metabolized in order to elicit downstream effects on a biological system. Studies that do not deposit energy are observed to have no downstream effects. </p>
<p>There are several uncertainties and inconsistencies in this KER. </p>
<ul>
<li>
<p>Chen et al. (2021) found that radiation can have adaptive responses. The study used three neutron radiation doses, 0.4 and 1.2 Sv, and 3.6 Sv. After 0.4 and 1.2 Sv, the activity of antioxidant enzymes GSH and SOD increased, and the concentration of malondialdehyde, a product of oxidative stress, decreased. After 3.6 Sv, the opposite was true. </p>
</li>
</ul>
<ul>
<li>
<p>While the concentration of most antioxidant enzymes decreases after energy deposition, there is some uncertainty with SOD. Certain papers have found that its concentration decreases with dose (Chen et al., 2021; Hua et al., 2019; Ji et al., 2015; Kang et al., 2020) while others found no difference after irradiation (Rogers et al., 2004; Zigman et al., 1995). Several studies have also found that higher levels of SOD do not increase resistance to UV radiation (Eaton, 1994; Hightower, 1995). </p>
</li>
<li>
<p>At 1-week post-irradiation with 10 Gy of 60Co gamma rays, TICAE cells experienced a significant increase in levels of the antioxidant, PRDX5, contrary to the decrease generally seen in antioxidant levels following radiation exposure (Philipp et al., 2020). </p>
</li>
<li>
<p>Various studies found an increase in antioxidant SOD levels within the brain after radiation exposure (Acharya et al., 2010; Baluchamy et al., 2012; Baulch et al., 2015; Veeraraghan et al., 2011). </p>
</li>
</ul>
<p>The table below provides some representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All data is statistically significant unless otherwise stated. </p>
HighMaleModerateFemaleHighUnspecificHighJuvenileModerateAdultModerateModerateHighLow<p><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">Most evidence is derived from in vitro studies, predominately using rabbit models. Evidence in humans and mice is moderate, while there is considerable available data using rat models. The relationship is applicable in both sexes, however, males are used more often in animal studies. No studies demonstrate the relationship in preadolescent animals, while adolescent animals were used very often, and adults were used occasionally in in vivo studies. </span></span></p>
<p>Acharya, M. M. et al. (2010), “Consequences of ionizing radiation-induced damage in human neural stem cells”, Free radical biology & medicine, Vol. 49/12, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.freeradbiomed.2010.08.021" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.freeradbiomed.2010.08.021</a>. </p>
<p>Ahmadi, M. et al. (2021), “Early responses to low-dose ionizing radiation in cellular lens epithelial models”, Radiation research, Vol. 197/1, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RADE-20-00284.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RADE-20-00284.1</a> </p>
<p>Attar, M., Y. M. Kondolousy, N. Khansari, (2007), “Effect of High Dose Natural Ionizing Radiation on the Immune System of the Exposed Residents of Ramsar Town, Iran”, Iranian Journal of Allergy, Asthma and Immunology, Vol. 6/2, pp. 73-78. </p>
<p>Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, American Journal of Physiology - Cell Physiology, Vol. 318/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/ajpcell.00520.2019." rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/ajpcell.00520.2019.</a> </p>
<p>Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, Journal of ocular pharmacology and therapeutics, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, <a href="https://doi.org/10.1089/jop.2000.16.285." rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a> </p>
<p>Baluchamy, S. et al. (2012), “Reactive oxygen species mediated tissue damage in high energy proton irradiated mouse brain”, Molecular and cellular biochemistry, Vol. 360/1-2, Springer, London, <a href="https://doi.org/10.1007/s11010-011-1056-2." rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s11010-011-1056-2.</a> </p>
<p>Baulch, J. E. et al. (2015), “Persistent oxidative stress in human neural stem cells exposed to low fluences of charged particles Redox biology, Vol. 5, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.redox.2015.03.001." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.redox.2015.03.001.</a> </p>
<p>Belkacémi, Y. et al. (2001), “Lens epithelial cell protection by aminothiol WR-1065 and anetholedithiolethione from ionizing radiation”, International journal of cancer, Vol. 96, John Wiley & Sons, Ltd., Hoboken, <a href="https://doi.org/10.1002/ijc.10346." rel="noreferrer noopener" target="_blank">https://doi.org/10.1002/ijc.10346.</a> </p>
<p>Cabrera M., R. Chihuailaf and F. Wittwer Menge (2011), “Antioxidants and the integrity of ocular tissues”, Veterinary medicine international, Vol. 2011, Hindawi, London, <a href="https://doi.org/10.4061/2011/905153." rel="noreferrer noopener" target="_blank">https://doi.org/10.4061/2011/905153.</a> </p>
<p>Cadet, J. et al. (2012), “Oxidatively generated complex DNA damage: tandem and clustered lesions”, Cancer letters, Vol. 327, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.canlet.2012.04.005." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.canlet.2012.04.005.</a> </p>
<p>Cencer, C. et al. (2018), “PARP-1/PAR activity in cultured human lens epithelial cells exposed to tow levels of UVB light”, Photochemistry and photobiology, Vol. 94, John Wiley & Sons, Ltd., Hoboken, <a href="https://doi.org/10.1111/php.12814." rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/php.12814.</a> </p>
<p>Cervelli, T. et al. (2017), “A New Natural Antioxidant Mixture Protects against Oxidative and DNA Damage in Endothelial Cell Exposed to Low-Dose Irradiation”, Oxidative medicine and cellular longevity, Vol. 2017, Hindawi, London, <a href="https://doi.org/10.1155/2017/9085947." rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2017/9085947.</a> </p>
<p>Chen, Y. et al. (2021), “Effects of neutron radiation on Nrf2-regulated antioxidant defense systems in rat lens”, Experimental and therapeutic medicine, Vol. 21/4, Spandidos Publishing Ltd, Athens, <a href="https://doi.org/10.3892/etm.2021.9765." rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/etm.2021.9765.</a> </p>
<p>Chitchumroonchokchai, C. et al. (2004), “Xanthophylls and α-tocopherol decrease UVB-induced lipid peroxidation and stress signaling in human lens epithelial cells”, The Journal of Nutrition, Vol. 134/12, American Society for Nutritional Sciences, Bethesda, <a href="https://doi.org/10.1093/jn/134.12.3225." rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jn/134.12.3225.</a> </p>
<p>Collins-Underwood, J. R. et al. (2008), “NADPH oxidase mediates radiation-induced oxidative stress in rat brain microvascular endothelial cells”, Free radical biology & medicine, Vol. 45/6, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.freeradbiomed.2008.06.024." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.freeradbiomed.2008.06.024. </a> </p>
<p>de Jager, T.L., Cockrell, A.E., Du Plessis, S.S. (2017), “Ultraviolet Light Induced Generation of Reactive Oxygen Species”, in Ultraviolet Light in Human Health, Diseases and Environment. Advances in Experimental Medicine and Biology, Springer, Cham, https://doi.org/10.1007/978-3-319-56017-5_2</p>
<p>de Freitas, L. F. and Hamblin, M. R. (2016), “Proposed Mechanisms of Photobiomodulation or Low-Level Light Therapy”, IEEE journal of selected topics in quantum electronics : a publication of the IEEE Lasers and Electro-optics Society, Vol. 22/3, IEEE Xplore, <a href="https://doi.org/10.1109/JSTQE.2016.2561201%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1109/JSTQE.2016.2561201</a> </p>
<p>de Jager, T.L., Cockrell, A.E., Du Plessis, S.S et al. (2017), “Ultraviolet Light Induced Generation of Reactive Oxygen Species”, in Ultraviolet Light in Human Health, Diseases and Environment. Advances in Experimental Medicine and Biology, Springer, Cham, https://doi.org/10.1007/978-3-319-56017-5_2 </p>
<p>Demir, E. et al. (2020), “Nigella sativa oil and thymoquinone reduce oxidative stress in the brain tissue of rats exposed to total head irradiation”, International journal of radiation biology, Vol. 96/2, Informa, London, <a href="https://doi.org/10.1080/09553002.2020.1683636." rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2020.1683636.</a> </p>
<p>Eaton, J. W. (1994), “UV-mediated cataractogenesis: A radical perspective”, Documenta ophthalmologica, Vol. 88, Springer, London, <a href="https://doi.org/10.1007/BF01203677." rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/BF01203677.</a> </p>
<p>Fatma, N. et al. (2005), “Impaired homeostasis and phenotypic abnormalities in Prdx6-/- mice lens epithelial cells by reactive oxygen species: Increased expression and activation of TGFβ”, Cell death and differentiation, Vol. 12, Nature Portfolio, London, <a href="https://doi.org/10.1038/sj.cdd.4401597." rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/sj.cdd.4401597.</a> </p>
<p>Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, Ophthalmic Research, Vol. 44, Karger International, Basel, <a href="https://doi.org/10.1159/000316476." rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a> </p>
<p>Ganea, E. and J. J. Harding (2006), “Glutathione-related enzymes and the eye”, Current eye research, Vol. 31/1, Informa, London, <a href="https://doi.org/10.1080/02713680500477347." rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/02713680500477347.</a> </p>
<p>Giblin, F. J. et al. (2002), “UVA light in vivo reaches the nucleus of the guinea pig lens and produces deleterious, oxidative effects”, Experimental eye research, Vol. 75/4, Elsevier, Amsterdam, <a href="https://doi.org/10.1006/exer.2002.2039." rel="noreferrer noopener" target="_blank">https://doi.org/10.1006/exer.2002.2039.</a> </p>
<p>Giedzinski, E. et al. (2005), “Efficient production of reactive oxygen species in neural precursor cells after exposure to 250 MeV protons”, Radiation research, Vol. 164/4, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/rr3369.1." rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/rr3369.1.</a> </p>
<p>Hamada, N. et al. (2014), “Emerging issues in radiogenic cataracts and cardiovascular disease”, Journal of radiation research, Vol. 55/5, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jrr/rru036." rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jrr/rru036.</a> </p>
<p>Hamblin, M. R. (2018), “Mechanisms and Mitochondrial Redox Signaling in Photobiomodulation”, Photochemistry and Photobiology, Vol. 94/2, John Wiley & Sons, Ltd., Hoboken, <a href="https://doi.org/10.1111/php.12864%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/php.12864</a>. </p>
<p>Hatoum, O. A. et al. (2006), “Radiation induces endothelial dysfunction in murine intestinal arterioles via enhanced production of reactive oxygen species”, Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 26/2, Lippincot Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/01.ATV.0000198399.40584.8C/FORMAT/EPUB" rel="noreferrer noopener" target="_blank">https://doi.org/10.1161/01.ATV.0000198399.40584.8C.</a> </p>
<p>Hightower, K. and J. McCready (1992), “Mechanisms involved in cataract development following near-ultraviolet radiation of cultured lenses”, Current eye research, Vol. 11/7, Informa, London, <a href="https://doi.org/10.3109/02713689209000741." rel="noreferrer noopener" target="_blank">https://doi.org/10.3109/02713689209000741.</a> </p>
<p>Hightower, K. R. (1995), “The role of the lens epithelium in development of UV cataract”, Current eye research, Vol. 14/1, Informa, London, <a href="https://doi.org/10.3109/02713689508999916." rel="noreferrer noopener" target="_blank">https://doi.org/10.3109/02713689508999916.</a> </p>
<p>Hua, H. et al. (2019), “Protective effects of lanosterol synthase up-regulation in UV-B-induced oxidative stress”, Frontiers in pharmacology, Vol. 10, Frontiers Media SA, Lausanne, <a href="https://doi.org/10.3389/fphar.2019.00947." rel="noreferrer noopener" target="_blank">https://doi.org/10.3389/fphar.2019.00947.</a> </p>
<p>Huang, L. et al. (2006), “Oxidation-induced changes in human lens epithelial cells 2. Mitochondria and the generation of reactive oxygen species”, Free radical biology & medicine, Vol. 41/6, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.freeradbiomed.2006.05.023" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.freeradbiomed.2006.05.023</a>. </p>
<p>Huang, B. et al. (2019), “Amifostine suppresses the side effects of radiation on BMSCs by promoting cell proliferation and reducing ROS production”, Stem Cells International, Vol. 2019, Hindawi, London, <a href="https://doi.org/10.1155/2019/8749090." rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2019/8749090.</a> </p>
<p>Huang, B. et al. (2018), “Sema3a inhibits the differentiation of raw264.7 cells to osteoclasts under 2gy radiation by reducing inflammation”, PLoS ONE, Vol. 13/7, PLOS, San Francisco, <a href="https://doi.org/10.1371/journal.pone.0200000." rel="noreferrer noopener" target="_blank">https://doi.org/10.1371/journal.pone.0200000.</a> </p>
<p>ICRU (1998), “ICRU report 57: conversion coefficients for use in radiological protection against external radiation”, Journal of the ICRU, Vol. 29/2, SAGE Publishing </p>
<p>Ismail, A. F. and S. M. El-Sonbaty (2016), “Fermentation enhances Ginkgo biloba protective role on gamma-irradiation induced neuroinflammatory gene expression and stress hormones in rat brain”, Journal of photochemistry and photobiology. B, Biology, Vol. 158, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.jphotobiol.2016.02.039." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.jphotobiol.2016.02.039.</a> </p>
<p>Ji, Y. et al. (2015), “The mechanism of UVB irradiation induced-apoptosis in cataract”, Molecular and cellular biochemistry, Vol. 401, Springer, London, <a href="https://doi.org/10.1007/s11010-014-2294-x." rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s11010-014-2294-x.</a> </p>
<p>Jiang, Q. et al. (2006), “UV radiation down-regulates Dsg-2 via Rac/NADPH oxidase-mediated generation of ROS in human lens epithelial cells”, International Journal of Molecular Medicine, Vol. 18/2, Spandidos Publishing Ltd, Athens, https://doi.org/10.3892/ijmm.18.2.381. </p>
<p>Jurja, S. et al. (2014), “Ocular cells and light: harmony or conflict?”, Romanian Journal of Morphology & Embryology, Vol. 55/2, Romanian Academy Publishing House, Bucharest, pp. 257–261. </p>
<p>Kang, L. et al. (2020), “Ganoderic acid A protects lens epithelial cells from UVB irradiation and delays lens opacity”, Chinese journal of natural medicines, Vol. 18/12, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/S1875-5364(20)60037-1." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/S1875-5364(20)60037-1.</a> </p>
<p>Karam, H. M. and R. R. Radwan (2019), “Metformin modulates cardiac endothelial dysfunction, oxidative stress and inflammation in irradiated rats: A new perspective of an antidiabetic drug”, Clinical and Experimental Pharmacology and Physiology, Vol. 46/12, Wiley-Blackwell, Hoboken, <a href="https://doi.org/10.1111/1440-1681.13148." rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/1440-1681.13148.</a> </p>
<p>Karimi, N. et al. (2017), “Radioprotective effect of hesperidin on reducing oxidative stress in the lens tissue of rats”, International Journal of Pharmaceutical Investigation, Vol. 7/3, Phcog Net, Bengaluru, https://doi.org/10.4103/jphi.JPHI_60_17. </p>
<p>Kłuciński, P. et al. (2008), “Erythrocyte antioxidant parameters in workers occupationally exposed to low levels of ionizing radiation”, Annals of Agricultural and Environmental Medicine, Vol. 15/1, pp. 9-12. </p>
<p>Kook, S. H. et al. (2015), “Irradiation inhibits the maturation and mineralization of osteoblasts via the activation of Nrf2/HO-1 pathway”, Molecular and Cellular Biochemistry, Vol. 410/1-2, Springer, London, <a href="https://doi.org/10.1007/s11010-015-2559-z." rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s11010-015-2559-z.</a> </p>
<p>Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, International Journal of Radiation Biology, 1–12. https://doi.org/10.1080/09553002.2022.2110306 </p>
<p>Kubo, E. et al. (2010), “Protein expression profiling of lens epithelial cells from Prdx6-depleted mice and their vulnerability to UV radiation exposure”, American Journal of Physiology, Vol. 298/2, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/ajpcell.00336.2009." rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/ajpcell.00336.2009.</a> </p>
<p>Lee, J. et al. (2004), “Reactive oxygen species, aging, and antioxidative nutraceuticals”, Comprehensive reviews in food science and food safety, Vol. 3/1, Blackwell Publishing Ltd, Oxford, <a href="http://doi.org/10.1111/j.1541-4337.2004.tb00058.x." rel="noreferrer noopener" target="_blank">http://doi.org/10.1111/j.1541-4337.2004.tb00058.x.</a> </p>
<p>Limoli, C. L. et al. (2007), “Redox changes induced in hippocampal precursor cells by heavy ion irradiation”, Radiation and environmental biophysics, Vol. 46/2, Springer, London, <a href="https://doi.org/10.1007/s00411-006-0077-9." rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00411-006-0077-9.</a> </p>
<p>Limoli, C. L. et al. (2004), “Radiation response of neural precursor cells: linking cellular sensitivity to cell cycle checkpoints, apoptosis and oxidative stress”, Radiation research, Vol. 161/1, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/rr3112." rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/rr3112.</a> </p>
<p>Liu, F. et al. (2019), “Transcriptional response of murine bone marrow cells to total-body carbon-ion irradiation”, Mutation Research - Genetic Toxicology and Environmental Mutagenesis, Vol. 839, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.mrgentox.2019.01.014." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.mrgentox.2019.01.014.</a> </p>
<p>Liu, Y. et al. (2018), “Protective effects of α2macroglobulin on human bone marrow mesenchymal stem cells in radiation injury”, Molecular Medicine Reports, Vol. 18/5, Spandidos Publishing Ltd, Athens, <a href="https://doi.org/10.3892/mmr.2018.9449." rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/mmr.2018.9449.</a> </p>
<p>Manda, K. et al. (2007), “Melatonin attenuates radiation-induced learning deficit and brain oxidative stress in mice”, Acta neurobiologiae experimentalis, Vol. 67/1, Nencki Institute of Experimental Biology, Warsaw, pp. 63 –70. </p>
<p>Manda, K., M. Ueno and K. Anzai (2008), “Memory impairment, oxidative damage and apoptosis induced by space radiation: ameliorative potential of alpha-lipoic acid”, Behavioural brain research, Vol. 187/2, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.bbr.2007.09.033." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.bbr.2007.09.033.</a> </p>
<p>Mansour, H. H. (2013), “Protective effect of ginseng against gamma-irradiation-induced oxidative stress and endothelial dysfunction in rats”, EXCLI Journal, Vol. 12, Leibniz Research Centre for Working Environment and Human Factors, Dortmund, pp. 766-777. </p>
<p>Marshall, J. (1985), “Radiation and the ageing eye”, Ophthalmic & physiological optics, Vol. 5, Wiley-Blackwell, Hoboken, <a href="https://doi.org.10.1111/j.1475-1313.1985.tb00666.x." rel="noreferrer noopener" target="_blank">https://doi.org.10.1111/j.1475-1313.1985.tb00666.x. </a> </p>
<p>Padgaonkar, V. A. et al. (2015) “Thioredoxin reductase activity may be more important than GSH level in protecting human lens epithelial cells against UVA light”, Photochemistry and photobiology, Vol. 91/2, Wiley-Blackwell, Hoboken, <a href="https://doi.org/10.1111/php.12404." rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/php.12404. </a> </p>
<p>Pendergrass, W. et al. (2010), “X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts”, Molecular Vision, Vol. 16, Emory University, Atlanta, pp. 1496-513. </p>
<p>Philipp, J. et al. (2020), “Radiation Response of Human Cardiac Endothelial Cells Reveals a Central Role of the cGAS-STING Pathway in the Development of Inflammation”, Proteomes, Vol. 8/4, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/proteomes8040030." rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/proteomes8040030.</a> </p>
<p>Quan, Y. et al. (2021), “Connexin hemichannels regulate redox potential via metabolite exchange and protect lens against cellular oxidative damage”, Redox biology, Vol. 46, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.redox.2021.102102." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.redox.2021.102102.</a> </p>
<p>Ramadan, R. et al. (2020), “Connexin43 Hemichannel Targeting With TAT-Gap19 Alleviates Radiation-Induced Endothelial Cell Damage”, Frontiers in Pharmacology, Vol. 11, Frontiers Media SA, Lausanne, <a href="https://doi.org/10.3389/fphar.2020.00212" rel="noreferrer noopener" target="_blank">https://doi.org/10.3389/fphar.2020.00212</a> </p>
<p>Rehman, M. U. et al. (2016), “Comparison of free radicals formation induced by cold atmospheric plasma, ultrasound, and ionizing radiation”, Archives of biochemistry and biophysics, Vol. 605, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.abb.2016.04.005" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.abb.2016.04.005</a>. </p>
<p>Rogers, C. S. et al. (2004), “The effects of sub-solar levels of UV-A and UV-B on rabbit corneal and lens epithelial cells”, Experimental eye research, Vol. 78, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.exer.2003.12.011" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.exer.2003.12.011</a>. </p>
<p>Rong, X. et al. (2019), “TRIM69 inhibits cataractogenesis by negatively regulating p53”, Redox biology, Vol. 22, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.redox.2019.101157" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.redox.2019.101157</a>. </p>
<p>Rugo, R. E. and R. H. Schiestl (2004), “Increases in oxidative stress in the progeny of X-irradiated cells”, Radiation research, Vol. 162/4, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/rr3238." rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/rr3238.</a> </p>
<p>Santos, A. L., S. Sinha, and A. B. Linder (2018), “The good, the bad, and the ugly of ROS: New insights on aging and aging-related diseases from eukaryotic and prokaryotic model organisms”, Oxidative medicine and cellular longevity, Vol. 2018, Hindawi, London, <a href="https://doi.org/10.1155/2018/1941285" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2018/1941285</a>. </p>
<p>Schoenfeld, M. P. et al. (2012), “A hypothesis on biological protection from space radiation through the use of new therapeutic gases as medical counter measures”, Medical gas research, Vol. 2/8, BioMed Central Ltd, London, <a href="https://doi.org/10.1186/2045-9912-2-8" rel="noreferrer noopener" target="_blank">https://doi.org/10.1186/2045-9912-2-8</a>. </p>
<p>Sharma, U. C. et al. (2018), “Effects of a novel peptide Ac-SDKP in radiation-induced coronary endothelial damage and resting myocardial blood flow”, Cardio-oncology, Vol. 4, BioMed Central Ltd, London, <a href="https://doi.org/10.1186/s40959-018-0034-1." rel="noreferrer noopener" target="_blank">https://doi.org/10.1186/s40959-018-0034-1.</a> </p>
<p>Shen, Y. et al. (2018), “Transplantation of bone marrow mesenchymal stem cells prevents radiation-induced artery injury by suppressing oxidative stress and inflammation”, Oxidative Medicine and Cellular Longevity, Vol. 2018, Hindawi, London, <a href="https://doi.org/10.1155/2018/5942916" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2018/5942916</a>. </p>
<p>Slezak, J. et al. (2017), “Potential markers and metabolic processes involved in the mechanism of radiation-induced heart injury”, Canadian journal of physiology and pharmacology, Vol. 95/10, Canadian Science Publishing, Ottawa, <a href="https://doi.org/10.1139/cjpp-2017-0121." rel="noreferrer noopener" target="_blank">https://doi.org/10.1139/cjpp-2017-0121.</a> </p>
<p>Soloviev, A. I. and I.V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, Biochemical pharmacology, Vol. 159, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.bcp.2018.11.019." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.bcp.2018.11.019. </a> </p>
<p>Soltani, B. (2016), “Nanoformulation of curcumin protects HUVEC endothelial cells against ionizing radiation and suppresses their adhesion to monocytes: potential in prevention of radiation-induced atherosclerosis”, Biotechnology Letters, Vol. 38, Springer, London, <a href="https://doi.org/10.1007/s10529-016-2189-x." rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s10529-016-2189-x.</a> </p>
<p>Soucy, K. G. et al. (2011), “HZE 56Fe-Ion Irradiation Induces Endothelial Dysfunction in Rat Aorta: Role of Xanthine Oxidase”, Radiation Research, Vol. 176/4, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RR2598.1.%E2%80%AF" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RR2598.1. </a> </p>
<p>Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, Journal of Applied Physiology, Vol. 108/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.00946.2009." rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/japplphysiol.00946.2009.</a> </p>
<p>Spector, A. (1990), “Oxidation and aspects of ocular pathology”, The CLAO journal, Vol. 16, Contact Lens Association of Ophthalmologists, Colorado, pp. S8-10. </p>
<p>Stohs, S. (1995), “The role of free radicals in toxicity and disease”, Journal of Basic and Clinical Physiology and Pharmacology, Vol. 6/3-4, Walter de Gruyter GmbH, Berlin, pp. 205-228. </p>
<p>Suman, S. et al. (2013), “Therapeutic and space radiation exposure of mouse brain causes impaired DNA repair response and premature senescence by chronic oxidant production”, Aging, Vol. 5/8, Impact Journals, Orchard Park, <a href="https://doi.org/10.18632/aging.100587." rel="noreferrer noopener" target="_blank">https://doi.org/10.18632/aging.100587. </a> </p>
<p>Tahimic, C. G. T., and R. K. Globus (2017), “Redox signaling and its impact on skeletal and vascular responses to spaceflight”, International Journal of Molecular Sciences, Vol. 18/10, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/ijms18102153." rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/ijms18102153.</a> </p>
<p>Tangvarasittichai, O. and S. Tangvarasittichai (2018), “Oxidative stress, ocular disease and diabetes retinopathy”, Current pharmaceutical design, Vol. 24/40, Bentham Science Publishers, Sharjah, <a href="https://doi.org/10.2174/1381612825666190115121531." rel="noreferrer noopener" target="_blank">https://doi.org/10.2174/1381612825666190115121531.</a> </p>
<p>Taysi, S. et al. (2012), “Zinc administration modulates radiation-induced oxidative injury in lens of rat”, Pharmacognosy Magazine, Vol. 8/2, https://doi.org/10.4103/0973-1296.103646 </p>
<p>Tian, Y. et al. (2017), “The Impact of Oxidative Stress on the Bone System in Response to the Space Special Environment”, International Journal of Molecular Sciences, Vol. 18/10, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/ijms18102132." rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/ijms18102132.</a> </p>
<p>Tseng, B. P. et al. (2014), “Functional consequences of radiation-induced oxidative stress in cultured neural stem cells and the brain exposed to charged particle irradiation”, Antioxidants & redox signaling, Vol. 20/9, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2012.5134." rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2012.5134. </a> </p>
<p>Turner, N. D. et al. (2002), “Opportunities for nutritional amelioration of radiation-induced cellular damage”, Nutrition, Vol. 18/10, Elsevier Inc, New York, <a href="http://doi.org/10.1016/S0899-9007(02)00945-0." rel="noreferrer noopener" target="_blank">http://doi.org/10.1016/S0899-9007(02)00945-0.</a> </p>
<p>Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, Vol. 68/12, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/gerona/glt057." rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/gerona/glt057.</a> </p>
<p>Varma, S. D. et al. (2011), “Role of ultraviolet irradiation and oxidative stress in cataract formation-medical prevention by nutritional antioxidants and metabolic agonists”, Eye & contact lens, Vol. 37/4, Lippincot Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1097/ICL.0b013e31821ec4f2." rel="noreferrer noopener" target="_blank">https://doi.org/10.1097/ICL.0b013e31821ec4f2.</a> </p>
<p>Venkatesulu, B. P. et al. (2018), “Radiation-Induced Endothelial Vascular Injury: A Review of Possible Mechanisms”, JACC: Basic to Translational Science, Vol. 3/4, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.jacbts.2018.01.014." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.jacbts.2018.01.014. </a> </p>
<p>Veeraraghavan, J. et al. (2011), “Low-dose gamma-radiation-induced oxidative stress response in mouse brain and gut: regulation by NFκB-MnSOD cross-signaling”, Mutation research, Vol. 718/1-2, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.mrgentox.2010.10.006." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.mrgentox.2010.10.006.</a> </p>
<p>Wang, C. et al. (2016), “Protective effects of cerium oxide nanoparticles on MC3T3-E1 osteoblastic cells exposed to X-ray irradiation”, Cellular Physiology and Biochemistry, Vol. 38/4, Karger International, Basel, <a href="https://doi.org/10.1159/000443092." rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000443092.</a> </p>
<p>Wang, H. et al. (2019a), “Radiation-induced heart disease: a review of classification, mechanism and prevention”, International Journal of Biological Sciences, Vol. 15/10, Ivyspring International Publisher, Sydney, <a href="https://doi.org/10.7150/ijbs.35460." rel="noreferrer noopener" target="_blank">https://doi.org/10.7150/ijbs.35460.</a> </p>
<p>Wang, H. et al. (2019b), “Gamma Radiation-Induced Disruption of Cellular Junctions in HUVECs Is Mediated through Affecting MAPK/NF-κB Inflammatory Pathways”, Oxidative Medicine and Cellular Longevity, Vol. 2019, Hindawi, London, <a href="https://doi.org/10.1155/2019/1486232." rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2019/1486232.</a> </p>
<p>Wegener, A. R. (1994), “In vivo studies on the effect of UV-radiation on the eye lens in animals”, Documenta ophthalmologica, Vol. 88, Springer, London, <a href="https://doi.org/10.1007/BF01203676" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/BF01203676</a>. </p>
<p>Weinreb O. and A. Dovrat (1996), “Transglutaminase involvement in UV-A damage to the eye lens”, Experimental eye research, Vol. 63/5, Elsevier, London, <a href="https://doi.org/10.1006/exer.1996.0150" rel="noreferrer noopener" target="_blank">https://doi.org/10.1006/exer.1996.0150</a>. </p>
<p>Yang, H. et al. (2020), “Cytoprotective role of humanin in lens epithelial cell oxidative stress-induced injury”, Molecular medicine reports, Vol. 22/2, Spandidos Publishing Ltd, Athens, <a href="https://doi.org/10.3892/mmr.2020.11202" rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/mmr.2020.11202</a>. </p>
<p>Yao, K. et al. (2008), “The flavonoid, fisetin, inhibits UV radiation-induced oxidative stress and the activation of NF-κB and MAPK signaling in human lens epithelial cells”, Molecular Vision, Vol. 14, Emory University, Atlanta, pp. 1865-1871. </p>
<p>Yao, J. et al. (2009), “UVB radiation induces human lens epithelial cell migration via NADPH oxidase-mediated generation of reactive oxygen species and up-regulation of matrix metalloproteinases”, International Journal of Molecular Medicine, Vol. 24/2, Spandidos Publishing Ltd, Athens, <a href="https://doi.org/10.3892/ijmm_00000218" rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/ijmm_00000218</a>. </p>
<p>Yves, C. (2000), "Oxidative stress and Alzheimer disease", The American Journal of Clinical Nutrition, Vol. 71/2, <a href="https://doi.org/10.1093/ajcn/71.2.621s" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/ajcn/71.2.621s</a>. </p>
<p>Zhang, J. et al. (2012), “Ultraviolet radiation-induced cataract in mice: The effect of age and the potential biochemical mechanism”, Investigative ophthalmology & visual science, Vol. 53, Association for Research in Vision and Ophthalmology, Rockville, <a href="https://doi.org/10.1167/iovs.12-10482" rel="noreferrer noopener" target="_blank">https://doi.org/10.1167/iovs.12-10482</a>. </p>
<p>Zhang, L. et al. (2020), “Amifostine inhibited the differentiation of RAW264.7 cells into osteoclasts by reducing the production of ROS under 2 Gy radiation”, Journal of Cellular Biochemistry, Vol. 121/1, John Wiley & Sons, Ltd., Hoboken, <a href="https://doi.org/10.1002/jcb.29247." rel="noreferrer noopener" target="_blank">https://doi.org/10.1002/jcb.29247.</a> </p>
<p>Zhang, L. et al. (2018), “Astragalus polysaccharide inhibits ionizing radiation-induced bystander effects by regulating MAPK/NF-κB signaling pathway in bone mesenchymal stem cells (BMSCs)”, Medical Science Monitor, Vol. 24, International Scientific Information, Inc., Melville, <a href="https://doi.org/10.12659/MSM.909153." rel="noreferrer noopener" target="_blank">https://doi.org/10.12659/MSM.909153.</a> </p>
<p>Zigman, S. et al. (2000), “Effects of intermittent UVA exposure on cultured lens epithelial cells”, Current Eye Research, Vol. 20/2, Informa UK Limited, London, <a href="https://doi.org/10.1076/0271-3683(200002)2021-DFT095." rel="noreferrer noopener" target="_blank">https://doi.org/10.1076/0271-3683(200002)2021-DFT095.</a> </p>
<p>Zigman, S. et al. (1995), “Damage to cultured lens epithelial cells of squirrels and rabbits by UV-A (99.9%) plus UV-B (0.1%) radiation and alpha tocopherol protection”, Molecular and cellular biochemistry, Vol. 143, Springer, London, <a href="https://doi.org/10.1007/BF00925924." rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/BF00925924.</a> </p>
<p> </p>
2022-09-28T12:01:482024-03-08T13:28:15f1940943-69e5-458b-8c03-896b1c61c3f9f703f4b6-e87b-4be7-8ffa-ff2f1a670694<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Direct deposition of ionizing energy refers to imparted energy interacting directly with the DNA double helix and producing randomized damage. This can be in the form of double strand breaks (DSBs), single-strand breaks, base damage, or the crosslinking of DNA to other molecules (Smith et al., 2003; Joiner, 2009; Christensen, 2014; Sage and Shikazono, 2017). Among these, the most detrimental type of DNA damage to a cell is DSBs. They are caused by the breaking of the sugar-phosphate backbone on both strands of the DNA double helix molecule, either directly across from each other or several nucleotides apart (Ward, 1988; Iliakis et al., 2015). This occurs when high-energy subatomic particles interact with the orbital electrons of the DNA causing ionization (where electrons are ejected from atoms) and excitation (where electrons are raised to higher energy levels) (Joiner, 2009). The number of DSBs produced and the complexity of the breaks is highly dependent on the amount of energy deposited on and absorbed by the cell. This can vary as a function of the dose-rate (Brooks et al., 2016) and the radiation quality which is a function of its linear energy transfer (LET) (Sutherland et al., 2000; Nikjoo et al., 2001; Jorge et al., 2012). LET describes the amount of energy that an ionizing particle transfers to media per unit distance (Smith et al., 2003; Okayasu, 2012a; Christensen et al., 2014). High LET radiation, such as alpha particles, heavy ion particles, and neutrons can deposit larger quantities of energy within a single track than low LET radiation, such as γ-rays, X-rays, electrons, and protons (Kadhim et al., 2006; Franken et al., 2012; Frankenberg et al., 1999; Rydberg et al., 2002; Belli et al., 2000; Antonelli et al., 2015). As such, radiation with higher LETs tends to produce more complex, dense structural damage, particularly in the form of clustered damage, in comparison to lower LET radiation (Nikjoo et al., 2001; Terato and Ide, 2005; Hada and Georgakilas, 2008; Okayasu, 2012a; Lorat et al., 2015; Nikitaki et al., 2016). Some data reports that low dose and low LER radiation can lead to complex lesions, which can cause unrepairable DNA damage. However, determining the actual frequency of the complexity of these lesions has proven challenging (Wilkinson et al., 2023). The complexity and yield of clustered DNA damage increases with ionizing density (Ward, 1988; Goodhead, 2006). However, clustered damage can also be induced even by a single radiation track through a cell.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">While the amount of DSBs produced depends on the radiation dose (see dose concordance), it also depends on several other factors. As the LET increases, the complexity of DNA damage increases, decreasing the repair rate, and increasing toxicity (Franken et al., 2012; Antonelli et al., 2015).</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Overall Weight of Evidence for this KER: High</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The biological rationale linking the direct deposition of energy on DNA with an increase in DSB formation is strongly supported by numerous literature reviews that are available on this topic (J .F. Ward, 1988; Lipman, 1988; Hightower, 1995; Terato & Ide, 2005; Goodhead, 2006; Kim & Lee, 2007; Asaithamby et al., 2008; Hada & Georgakilas, 2008; Jeggo, 2009; Clement, 2012; Okayasu, 2012b; Stewart, 2012; M. E. Lomax et al., 2013; EPRI, 2014; Hamada, 2014; Moore et al., 2014; Desouky et al., 2015; Ainsbury, 2016; Foray et al., 2016; Hamada & Sato, 2016; Hamada, 2017a; Sage & Shikazono, 2017; Chadwick, 2017; Wang et al., 2021; Nagane et al., 2021; Sylvester et al., 2018; Baselet et al., 2019). Ionizing radiation can be in the form of high energy particles (such as alpha particles, beta particles, or charged ions) or high energy photons (such as gamma-rays or X-rays). Ionizing radiation can break the DNA within chromosomes both directly and indirectly, as shown through using velocity sedimentation of DNA through neutral and alkaline sucrose gradients. The most direct path entails a collision between a high-energy particle or photon and a strand of DNA.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Additionally, excitation of secondary electrons in the DNA allows for a cascade of ionization events to occur, which can lead to the formation of multiple damage sites (Joiner, 2009). As an example, high-energy electrons will traverse a DNA molecule in a mammalian cell within 10<sup>-18</sup> s and 10<sup>-14</sup> s, resulting in 100,000 ionizing events per 1 Gy dose in a 10 μm cell (Joiner, 2009). The amount of damage can be influenced by factors such as the cell cycle stage and chromatin structure. It has been shown that in more condensed, packed chromatin structures such as those present in intact cells and heterochromatin, it is more difficult for the DNA to be damaged (Radulescu et al., 2006; Agrawala et al., 2008; Falk et al., 2008; Venkatesh et al., 2016). In contrast, DNA damage is more easily induced in lightly-packed chromatin such as euchromatin and nucleoids, (Radulescu et al., 2006; Falk et al., 2008; Venkatesh et al., 2016).</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Of the possible radiation-induced DNA damage types, DSB is considered to be the most harmful to the cell, as there may be severe consequences if this damage is not adequately repaired (Khanna & Jackson, 2001; Smith et al., 2003; Okayasu, 2012a; M. E. Lomax et al., 2013; Rothkamm et al., 2015).</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">A considerable fraction of DSBs can also be formed in cells through indirect mechanisms. In this case, deposited energy can split water molecules near DNA, which can generate a significant quantity of reactive oxygen species in the form of hydroxyl free radicals (Ward, 1988; Wolf, 2008; Desouky et al., 2015; Maier et al., 2016, Cencer et al., 2018; Bains, 2019; Ahmadi et al., 2021). Estimates using models and experimental results suggest that hydroxyl radicals may be present within nanoseconds of energy deposition by radiation (Yamaguchi et al., 2005). These short-lived but highly reactive hydroxyl radicals may react with nearby DNA. This will produce DNA damage, including single-strand breaks and DSBs (Ward, 1988; Sasaki, 1998; Desouky et al., 2015; Maier et al., 2016). DNA breaks are especially likely to be produced if the sugar moiety is damaged, and DSBs occur when two single-strand breaks are in close proximity to each other (Ward, 1988).</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Empirical data strongly supports this KER. The evidence presented below is summarized in table 1. The types of DNA damage produced by ionizing radiation and the associated mechanisms, including the induction of DSBs, are reviewed by Lomax et al. (2013) and documents produced by international radiation governing frameworks (Valentin, 1998; UNSCEAR, 2000). Other reviews also highlight the relationship between the deposition of energy by radiation and DSB induction, and discuss the various methods available to detect these DSBs (Terato & Ide, 2005; Rothkamm et al., 2015; Sage & Shikazono, 2017). A visual representation of the time frames and dose ranges probed by the dedicated studies discussed here is shown in Figures 1 & 2 below.</span></span></p>
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<p><img alt="" src="https://aopwiki.org/system/dragonfly/production/2022/10/12/4zw4nw353c_ke1_mie_dsb_dose_v2.png" style="height:734px; width:1000px" /></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Figure 1: Plot of example studies (y-axis) against equivalent dose (Sv) used to determine the empirical link between direct deposition of energy and DSBs. The z-axis denotes the equivalent dose rate used in each study. The y-axis is ordered from low LET to high LET from top to bottom. </span></span></p>
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<p> </p>
<p><img alt="" src="https://aopwiki.org/system/dragonfly/production/2022/10/12/a85jspx5_ke1_mie_dsb_time_v2.png" style="height:706px; width:1000px" /></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Figure 2: Plot of example studies (y-axis) against time scales used to determine the empirical link between direct deposition of energy and DSBs. The z-axis denotes the equivalent dose rate used in each study. The y-axis is ordered from low LET to high LET from top to bottom. </span></span></p>
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<p><u><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dose Concordance</span></span></u></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is evidence in the literature suggesting a dose concordance between the direct deposition of energy by ionizing radiation and the incidence (Grudzenski et al., 2010) of DNA DSBs. Results from in vitro (Aufderheide et al., 1987; Sidjanin, 1993; Bucolo, 1994; Frankenberg et al., 1999; Rogakou et al., 1999; Belli et al., 2000; Sutherland et al., 2000; Lara et al., 2001; Rydberg et al., 2002; Baumstark-Kham et al., 2003; Rothkamm and Lo, 2003; Long, 2004; Kuhne et al., 2005; Sudprasert et al., 2006; Beels et al., 2009; Grudzenski et al., 2010; Liao, 2011; Franken et al., 2012; Bannik et al., 2013; Shelke & Das, 2015; Antonelli et al., 2015; Markiewicz et al., 2015; Allen, 2018; Dalke, 2018; Bains, 2019; Ahmadi et al., 2021; Sabirzhanov et al., 2020; Ungvari et al., 2013; Rombouts et al., 2013; Baselet et al., 2017</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">), in vivo (Reddy, 1998; Sutherland et al., 2000; Rube et al., 2008; Beels et al., 2009; Grudzenski et al., 2010; Markiewicz et al., 2015; Barnard, 2018; Barnard, 2019; Barnard, 2022; Schmal et al., 2019; Barazzuol et al., 2017; Geisel et al., 2012</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">), ex vivo (Rube et al., 2008; Flegal et al., 2015) and simulation studies (Charlton et al., 1989) suggest that there is a positive, linear, dose-dependent increase in DSBs with increasing deposition of energy across a wide range of radiation types (iron ions, X-rays, ultrasoft X-rays, gamma-rays, photons, UV light, and alpha particles) and radiation doses (1 mGy - 100 Gy) (Aufderheide et al., 1987; Sidjanin, 1993; Frankenberg et al., 1999; Sutherland et al., 2000; de Lara et al., 2001; Baumstark-Khan et al., 2003; Rothkamm & Lo, 2003; Kuhne et al., 2005; Rube et al., 2008; Grudzenski et al., 2010; Bannik et al., 2013; Shelke & Das, 2015; Antonelli et al., 2015; Dalke, 2018; Barazzuol et al., 2017; Ungvari et al., 2013; Rombouts et al., 2013; Baselet et al., 2017; Geisel et al., 2012</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">). DSBs have been predicted to occur at energy deposition levels as low as 75 eV (Charlton et al., 1989). </span></span></p>
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<p><u><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Time Concordance</span></span></u></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is evidence suggesting a time concordance between the direct deposition of energy and the incidence of DSBs. A number of different models and experiments have provided evidence of ionizing radiation-induced foci (IRIF), which can be used to infer DSB formation seconds (Mosconi et al., 2011) or minutes after radiation exposure (Rogakou et al., 1999; Rothkamm and</span></span><span style="font-size:11px"><span style="font-family:Arial,Helvetica,sans-serif"> </span>Löbrich</span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">, 2003; Rube et al., 2008; Beels et al., 2009; Kuefner et al., 2009; Grudzenski et al., 2010; Antonelli et al., 2015; Acharya et al., 2010; Sabirzhanov et al., 2020; Rombouts et al., 2013; Nübel et al., 2006; Baselet et al., 2017; Zhang et al., 2017). </span></span></p>
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<p><u><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Essentiality</span></span></u></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Deposition of energy is essential for DNA strand breaks. They can also be caused through other routes, such as oxidative stress (Cadet et al., 2012), but under normal physiological conditions deposition of energy is necessary. This was tested through many studies using various indicators such as 53BP1 foci/cell, γH2AX foci/cell, DNA migration, and the amount of DNA in tails for the comet assay. Various organisms such as humans, mice, rabbits, guinea pigs, and cattle were used. They showed that without the deposition of energy, there was only a negligible amount of DNA strand breaks (Aufderheide et al., 1987; Sidjanin, 1993; Bucolo, 1994; Reddy, 1998; Rogers, 2004; Bannik et al., 2013; Dalke, 2018; Bains, 2019; Barnard, 2019; Barnard, 2021). </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Uncertainties and inconsistencies in this KER are as follows:</span></span></p>
<ul>
<li><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Studies have shown that dose-rates (Brooks et al., 2016) and radiation quality (Sutherland et al., 2000; Nikjoo et al., 2001; Jorge et al., 2012) are factors that can influence the dose-response relationship. </span></span></li>
<li><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Low-dose radiation has been observed to have beneficial effects and may even invoke protection against spontaneous genomic damage (Feinendegen, 2005; Day et al., 2007; Feinendegen et al., 2007; Shah et al., 2012; Nenoi et al., 2015; Dalke, 2018). This protective effect has been documented in in vivo and in vitro, as reviewed by ICRP (2007) and UNSCEAR (2008) and can vary depending on the cell type, the tissue, the organ, or the entire organism (Brooks et al., 2016).</span></span></li>
<li><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Depositing ionizing energy is a stochastic event; as such this can influence the location, degree and type of DNA damage imparted on a cell. As an example, studies have shown that mitochondrial DNA may also be an important target for genotoxic effects of ionizing radiation (Wu et al., 1999).</span></span></li>
</ul>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Quantitative understanding of this linkage suggests that DSBs can be predicted upon exposure to ionizing radiation. This is dependent on the biological model, the type of radiation and the radiation dose. In general, 1 Gy of radiation is thought to result in 3000 damaged bases (Maier et al., 2016), 1000 single-strand breaks, and 40 DSBs (Ward, 1988; Foray et al., 2016; Maier et al., 2016) . The table below provides representative examples of the calculated DNA damage rates across different model systems, most of which are examining DNA DSBs.</span></span></p>
<p> </p>
<p> </p>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dose Concordance </span></span></strong></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The following tables provide representative examples of the relationship, unless otherwise indicated, all data is significantly significant. </span></span></p>
<table border="1">
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<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Reference </span></span></strong></p>
</td>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Experiment Description </span></span></strong></p>
</td>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Result </span></span></strong></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ward, 1988 </span></span></p>
</td>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Cells containing approximately 6 pg of DNA were exposed to 1 Gy. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Under the assumption of 6 pg of DNA per cell. 60 eV of energy deposited per event over a total of 1 Gy. Deoxyribose (2.3 pg/cell): 14,000 eV deposited, 235 events. Bases (2.4 pg/cell): 14.7 keV deposited, 245 events. Phosphate (1.2 pg/cell): 7,300 eV deposited, 120 events. Bound water (3.1 pg/cell): 19 keV deposited, 315 events. Inner hydration shell (4.2 pg/cell): 25,000 eV deposited 415 events. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Charlton, 1989 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In-silico. A computer simulation/model was used to test various types of radiation with doses from 0 to 400 eV (energy deposited) on the amount of DNA damage produced. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Simulated dose-concordance prediction of increase in number of DSBs/54 nucleotide pairs as direct deposition of energy increases in the range 75-400 eV. In the range 100 - 150 eV: 0.38 DSBs/54 nucleotide pairs and at 400 eV: ~0.80 DSBs per 64 nucleotide pairs. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sutherland, 2000 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human cells were exposed to <sup>137</sup>Cs γ-rays (0 – 100 Gy, 0.16 – 1.6 Gy/min). The frequency of DSBs was determined using gel electrophoresis. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Using isolated bacteriophage T7 DNA and 0-100 Gy of γ radiations, observed a response of 2.4 DSBs per megabase pair per Gy. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rogakou et al., 1999 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Normal human fibroblasts (IMR90) and human breast cancer cells (MCF7 were exposed to 0.6 and 2 Gy <sup>137</sup>Cs γ-rays delivered at 15.7 Gy/min. The number of DSBs were determined by immunoblotting for γ-H2AX. </span></span></p>
<p> </p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Radiation doses of 0.6 Gy & 2 Gy to normal human fibroblasts (IMR90) and MCF7 cells resulted in 10.1 & 12.2 DSBs per nucleus on average (0.6 Gy), respectively; increasing to 24 & 27.1 DSBs per nucleus (2 Gy). </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kuhne et al., 2005 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary human skin fibroblasts (HSF2) were exposed to 0 – 70 Gy <sup>60</sup>Co γ-rays (0.33 Gy/min), X-rays (29 kVp, 1.13 Gy/min), and CKX-rays (0.14 Gy/min). The number of DSBs were determined with pulsed-field gel electrophoresis. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">γ-ray and X-ray irradiation of primary human skin fibroblasts (HSF2) at 0 - 70 Gy. γ-rays: (6.1 ± 0.2) x 10-9 DSBs per base pair per Gy, X-rays: (7.0 ± 0.2) x 10-9 DSBs per base pair per Gy. CKX -rays: (12.1 ± 1.9) x 10-9 DSBs per base pair per Gy. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rothkamm, 2003 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary human fibroblast cell lines MRC-5 (lung), HSF1 and HSF2 (skin), and180BR (deficient in DNA ligase IV) were exposed to 1 mGy – 100 Gy X-rays (90 kV). Low doses were delivered at 6 – 60 mGy/min and high doses were delivered at 2 Gy/min. The number of DSBs were determined with pulsed-field gel electrophoresis. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">X-ray irradiation of primary human fibroblasts (MRC-5) in the range 1 mGy - 100 Gy, 35 DSBs per cell per Gy. </span></span></p>
</td>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Grudzenski et al, 2010 </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary human fibroblasts (HSF1) and C57BL/6NCrl adult mice were exposed to X-rays (2.5 – 200 mGy, 70 mGy/min), and photons (10 mGy – 1 Gy, 2 Gy/min (100 mGy and 1 Gy), and 0.35 Gy/min (10 mGy)). γ-H2AX immunofluorescence was observed to determine DSBs. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">X-rays irradiating primary human fibroblasts (HSF1) in the range 2.5 - 100 mGy yielded a response of 21 foci per Gy. When irradiating adult C57BL/6NCrl mice with photons a response of 0.07 foci per cell at 10 mGy was found. At 100 mGy the response was 0.6 foci per cell and finally, at 1 Gy; 8 foci per cell. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">de Lara, 2001 </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Chinese hamster cells (V79-4) were exposed to 0 – 20 Gy of<sup> 60</sup>Co γ-rays (2 Gy/min), and ultrasoft X-rays (0.7 – 35 Gy/min): carbon-K shell (0.28 keV), copper L-shell (0.96 keV), aluminum K-shell (1.49 keV), and titanium K-shell (4.55 keV). The number of DSBs were determined with pulsed-field gel electrophoresis. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">V79-4 cells irradiated with γ-rays and ultrasoft X-rays (carbon K-shell, copper L-shell, aluminium K-shell and titanum K-shell) in the range 0 - 20 Gy. Response (DSBs per Gy per cell): γ-rays: 41, carbon K-shell: 112, copper L-shell: 94, aluminum K-shell: 77, titanium K-shell: 56. </span></span></p>
</td>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rübe et al., 2008 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Brain, lung, heart and small intestine tissue from adult SCID, A-T, BALB/c and C57BL/6NCrl mice; Whole blood and isolated lymphocytes from BALB/c and C57BL/6NCrl mice were exposed to 0.1 – 2 Gy of photons (whole body irradiation, 6 MV, 2 Gy/min) and X-rays (whole body irradiation, 90 kV, 2 Gy/min). γ-H2AX foci were determined with immunochemistry to measure DSBs. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Linear dose-dependent increase in DSBs in the brain, small intestine, lung and heart of C57BL/6CNrl mice after whole-body irradiation with 0.1 - 1.0 Gy of radiation. 0.8 foci per cell (0.1 Gy) and 8 foci per cell (1 Gy). </span></span></p>
</td>
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<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Antonelli et al., 2015 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary human foreskin fibroblasts (AG01522) were exposed to 0 – 1 Gy of <sup>136</sup>Cs γ-rays (1 Gy/min), protons (0.84 MeV, 28.5 keV/um), carbon ions (58 MeV/u, 39.4 keV/um), and alpha particles (americium-241, 0.75 MeV/u, 0.08 Gy/min, 125.2 keV/um). γ-H2AX foci were determined with immunochemistry to measure DSBs. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Linear dose-dependent increase in the number of DSBs from 0 - 1 Gy for γ-rays and alpha particles as follows: γ-rays: 24.1 foci per Gy per cell nucleus, alpha particles: 8.8 foci per Gy per cell nucleus. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barnard et al., 2019 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. 10-week-old female C57BL/6 mice were whole-body exposed to 0.5, 1, and 2 Gy of 60Co γ-rays at 0.3, 0.063, and 0.014 Gy/min. p53 binding protein 1 (53BP1) foci were determined via immunofluorescence. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Central LECs showed a linear increase in mean 53BP1 foci/cell with the maximum dose and dose-rate displaying a 78x increase compared to control. Peripheral LECs and lower dose rates displayed similar results, with slightly fewer foci. </span></span><span style="font-size:11px">Although an increase in dose-response was observed, an inverse-dose rate response was reported, with higher 53BP1 foci persisting at lower dose rates.</span></p>
</td>
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<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ahmadi et al., 2021 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human LEC cells were exposed to 137Cs γ-rays at doses of 0, 0.1, 0.25, and 0.5 Gy and dose rates of 0.065 and 0.3 Gy/min. DNA strand breaks were measured using the comet assay. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Human LECs showed a gradual increase in the tail from the comet assay with the maximum dose and dose-rate displaying a 3.7x increase compared to control. Lower dose-rates followed a similar pattern with a lower amount of strand breaks. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada et al., 2006 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary normal human diploid fibroblast (HE49) cells were exposed to 0.1, 0.5, and 4 Gy X-rays at 240 kV with a dose rate of 0.5 Gy/min. The number of H2AX foci/cell, which represented DNA strand breaks, was determined 6 – 7 minutes after irradiation through fluorescence microscopy. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cells displayed a linear increase in the number of H2AX foci/cell, with the maximum dose displaying a 125x increase compared to control. </span></span></p>
<p> </p>
</td>
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<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dubrova & Plumb, 2002</span></span></td>
<td> </td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At 1 Gy observe 70 DSBs, 1000 single-strange breaks and 2000 damaged DNA bases per cell per Gy.</span></span></p>
</td>
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<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sabirzhanov et al., 2020</span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Rat cortical neurons were exposed to 2, 8 or 32 Gy of X rays (320 kV) at a dose rate of 1.25 Gy/min. Western blot was used to measure γ-H2AX, p-ataxia telangiectasia mutated (ATM) and p- ATM/RAD3-related (ATR) levels. </span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In rat cortical neurons, p-ATM increased at 2, 8, and 32 Gy, with a 15-fold increase at 8 and 32 Gy. γ-H2AX levels increased at 8 and 32 Gy. </span></span></td>
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<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Geisel et al., 2012 </span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Patients with suspected coronary artery disease receiving X-rays from computed tomography or conventional coronary angiography had levels of DSBs assessed in blood lymphocytes by γ-H2AX fluorescence. </span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There was a correlation between effective dose (in mSv) and DSBs. For both conventional coronary angiography and computed tomography, a dose of 10 mSv produced about 2-fold more DNA DSBs than a dose of 5 mSv. </span></span></td>
</tr>
<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ungvari et al., 2013 </span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Rat cerebromicrovascular endothelial cells and hippocampal neurons were irradiated with 2-10 Gy of <sup>137</sup>Cs gamma rays. DNA strand breaks were assessed with the comet assay. </span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DNA damage increased at all doses (2-10 Gy). In the control, less than 5% of DNA was in the tail, while by 6 Gy, 35% of the DNA was in the tail in cerebromicrovascular endothelial cells and 25% was in the tail in neurons. </span></span></td>
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<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rombouts et al., 2013 </span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. EA.hy926 cells and human umbilical vein endothelial cells were irradiated with various doses of X-rays (0.25 Gy/min). γ-H2AX foci were assessed with immunofluorescence. </span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">More γ-H2AX foci were observed at higher doses in both cell types. In human umbilical vein endothelial cells, few foci/nucleus were observed at 0.05 Gy, with about 23 at 2 Gy. In EA.hy926 cells, few foci/nucleus were observed at 0.05 Gy, with about 37 at 2 Gy. </span></span></td>
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<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Baselet et al., 2017 </span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human telomerase-immortalized coronary artery endothelial cells were irradiated with various doses of X-rays (0.5 Gy/min). Immunocytochemical staining was performed for γ-H2AX and 53BP1 foci. </span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Doses of 0.05 and 0.1 Gy did not increase the number of γ-H2AX foci, but 0.5 Gy increased foci number by 5-fold and 2 Gy by 15-fold. A dose of 0.05 Gy did not increase the number of 53BP1 foci, but 0.1 Gy, 0.5 Gy and 2 Gy increased levels by 3-fold, 7-fold and 8-fold, respectively.</span></span> </td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Time Concordance </span></span></strong></p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Reference </span></span></strong></p>
</td>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Experiment Description </span></span></strong></p>
</td>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Result </span></span></strong></p>
</td>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rogakou et al., 1999 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Normal human fibroblasts (IMR90), human breast cancer cells (MCF7), human astrocytoma cells (SF268), Indian muntjac Muntiacus muntjak normal skin fibroblasts, Xenopus laevisA6 normal kidney cells, Drosophila melanogaster epithelial cells, and Saccharomyces cerevisiae were exposed to 0.6, 2, 20, 22, 100, and 200 Gy 137Cs γ-rays. Doses below 20 Gy were delivered at 15.7 Gy/min and other doses were delivered in 1 minute. DNA breaks were visualized using γ-H2AX antibodies and microscopy. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DSBs were present at 3 min and persisted from 15 - 60 min. </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada & Woloschak, 2017 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. human LECs were exposed to 0.025 Gy X-rays at 0.42 – 0.45 Gy/min. 53BP1 foci were measured via indirect immunofluorescence. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In cells immediately exposed to 0.025 Gy, the level of 53BP1 foci/cell increased to 3.3x relative to control 0.5 h post-irradiation. </span></span></p>
</td>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada et al., 2006 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary normal human diploid fibroblast (HE49) cells were exposed to 0.1, 0.5, and 4 Gy (deposition of energy) at 240 kV with a dose rate of 0.5 Gy/min. The number of H2AX foci/cell, which represented DNA strand breaks, was determined through fluorescence microscopy. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In cells immediately exposed to 0.5 Gy, 11% of cells had 18 foci six min post-irradiation, compared to 90% of controls having 0 foci. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Acharya et al., 2010 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human neural stem cells were exposed to 1, 2 and 5 Gy of γ-rays at a dose rate of 2.2 Gy/min. The levels of γ-H2AX phosphorylation post irradiation were assessed by immunocytochemistry, fluorescence-activated cell sorting (FACS) analysis and γ-H2AX foci enumeration. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The number of cells positive for nuclear γ-H2AX foci peaked at 20 min post-irradiation. After 1h, this level quickly declined. </span></span></p>
</td>
</tr>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Schmal et al., 2019 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Juvenile and adult C57BL/6 mice were exposed to whole body 6-MV photons at 2 Gy/min. Irradiations were done in 5x, 10x, 15x and 20x fractions of 0.1 Gy. Double staining for NeuN and 53BP1 was used to quantify DNA damage foci and the possible accumulation in the hippocampal dentate gyrus. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">To assess possible accumulation of persisting 53BP1-foci during fractionated radiation, juvenile and adult mice were examined 72 h after exposure to 5×, 10×, 15×, or 20× fractions of 0.1 Gy, compared to controls. The number of persisting 53BP1-foci increased significantly in both juvenile and adult mice during fractionated irradiation (maximum at 1 m post-IR). </span></span></p>
</td>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dong et al., 2015 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. C57BL/6J mice were exposed to 2 Gy of X-rays at 2 Gy/min using a 6 MV source. γ-H2AX foci were assessed with immunofluorescence in the brain. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At 0.5 h, about 14 γ-H2AX foci/cell were present. This decreased linearly to about 2 foci/cell at 24 h, with no foci/cell from 48 h to 6 weeks. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barazzuol et al., 2017 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. C57BL/6 mice were exposed to 0.1 or 2 Gy of X-rays (250 kV) at a rate of 0.5 Gy/min. 53BP1 foci were quantified with immunofluorescence in neural stem cells and neuron progenitors in the lateral ventricle. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At both 0.5 and 6 h post-irradiation, increased 53BP1 foci were observed, with the highest level at 0.5 h. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sabirzhanov et al., 2020 </span></span></p>
<p> </p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Rat cortical neurons were exposed to 2, 8 or 32 Gy of X rays (320 kV) at a dose rate of 1.25 Gy/min. Western blot was used to measure γ-H2AX, p-ATM and p-ATR levels. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In rat cortical neurons, γ-H2AX, p-ATM and p-ATR all increased at 30 minutes post-irradiation, with a sustained increase until 6 h. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Zhang et al., 2017 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. HT22 hippocampal neuronal cellsT were irradiated with X-rays (320 kVp) at 8 or 12 Gy at a dose rate of 4 Gy/min. The comet assay was preformed to assess the DNA double strand breaks in HT22 cells. Western blot was used to measure γ-H2AX and p-ATM. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At 8 Gy, the comet assay showed an increased tail moment at both 30 minutes and 24 h post-irradiation. At 12 Gy, p-ATM was increased over 4-fold at both 30 minutes and 1 h post-irradiation. γ-H2AX was increased over 3-fold at 30 minutes post-irradiation and almost 2-fold at 1 and 24 h. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Geisel et al., 2012 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Patients with suspected coronary artery disease receiving X-rays from computed tomography or conventional coronary angiography had levels of DSBs assessed in blood lymphocytes by γ-H2AX fluorescence. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DSBs were increased at 1 h post-irradiation and returned to pre-irradiation levels by 24 h. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Park et al., 2022 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human aortic endothelial cells were irradiated with 137Cs gamma rays at 4 Gy (3.5 Gy/min). γ-H2AX was measured with western blot. p-ATM and 53BP1 were determined with immunofluorescence. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">γ-H2AX, p-ATM, and 53BP1 were shown increased at 1 h post-irradiation and slightly decreased for the rest of the 6 h but remained elevated above the control. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kim et al., 2014 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human umbilical vein endothelial cells were irradiated with 4 Gy of 137Cs gamma rays. γ-H2AX levels were determined with immunofluorescence. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">γ-H2AX foci greatly increased at 1 and 6 h post-irradiation, with the greatest increase at 1 h. </span></span></p>
</td>
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<tr>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dong et al., 2014 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human umbilical vein endothelial cells were irradiated with 2 Gy of 137Cs gamma rays. γ-H2AX levels were determined with immunofluorescence. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">γ-H2AX foci increased 8-fold at 3 h, 7-fold at 6 h, and 2-fold at 12 and 24 h post-irradiation. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rombouts et al., 2013 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. EA.hy926 cells and human umbilical vein endothelial cells were irradiated with X-rays (0.25 Gy/min). γ-H2AX foci were assessed with immunofluorescence. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The greatest increase in γ-H2AX foci was observed 30 minutes post-irradiation, while levels were still slightly elevated at 24 h. </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Nübel et al., 2006 </span></span></p>
</td>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human umbilical vein endothelial cells were irradiated with gamma rays at 20 Gy. DNA strand breaks were assessed with the comet assay and western blot for γ-H2AX. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The olive tail moment increased 5-fold immediately after irradiation and returned to control levels by 4 h. A large increase in γ-H2AX was observed at 0.5 h post-irradiation, with lower levels at 4 h but still above the control. </span></span></p>
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</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Baselet et al., 2017 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human telomerase-immortalized coronary artery endothelial cells were irradiated with various doses of X-rays (0.5 Gy/min). Immunocytochemical staining was performed for γ-H2AX and 53BP1 foci. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Increased γ-H2AX and 53BP1 foci were observed at 0.5 h post-irradiation, remaining elevated at 4 h but returning to control levels at 24 h. </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Gionchiglia et al., 2021 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Male CD1 and B6/129 mice were irradiated with X-rays at 10 Gy. Brain sections were single or double-stained with antibodies against γ-H2AX and p53BP1. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In the forebrain, cerebral cortex, hippocampus and subventricular zone (SVZ)/ rostral migratory stream (RMS)/ olfactory bulb (OB), γH2AX and p53BP1 positive cells increased at both 15 and 30 minutes post-irradiation, with the greatest increase at 30 minutes. </span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
HighUnspecificHighAll life stagesHighHighHighLowLowLow<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">This KER is plausible in all life stages, sexes, and organisms with DNA. The majority of the evidence is from In vivo adult mice and human In vitro models that do not specify the sex. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Agrawala, P.K. et al. (2008), "Induction and repairability of DNA damage caused by ultrasoft X-rays: Role of core events.", Int. J. Radiat. Biol., 84(12):1093–1103. doi:10.1080/09553000802478083.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ahmadi, M. et al. (2021), “Early responses to low-dose ionizing radiation in cellular lens epithelial models”, Radiation research, Vol.197/1, <em>Radiation Research Society</em>, United States, https://doi.org/10.1667/RADE-20-00284.1 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ainsbury, E. A. et al. (2016), “Ionizing radiation induced cataracts: Recent biological and mechanistic developments and perspectives for future research”, <em>Mutation research. Reviews in mutation research</em>, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.07.010 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Alexander, J. L. and Orr-Weaver, T. L. (2016), “Replication fork instability and the consequences of fork collisions from replication”, <em>Genes & Development</em>, Vol. 30/20, Cold Spring Harbor Laboratory Press, https://doi.org/ 10.1101/gad.288142.116 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Allen, C. H. et al. (2018), “Raman micro-spectroscopy analysis of human lens epithelial cells exposed to a low-dose-range of ionizing radiation”, <em>Physics in medicine & biology</em>, Vol. 63/2, IOP Publishing, Bristol, https://doi.org/10.1088/1361-6560/aaa176 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Antonelli, A.F. et al. (2015), "Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death", Radiat. Res. 183(4):417-31, doi:10.1667/RR13855.1.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Acharya, M. et al. (2010), “Consequences of ionizing radiation-induced damage in human neural stem cells”, Free Radical Biology and Medicine. 49(12):1846-1855, doi:10.1016/j.freeradbiomed.2010.08.021. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Asaithamby, A. et al. (2008), "Repair of HZE-Particle-Induced DNA Double-Strand Breaks in Normal Human Fibroblasts.", Radiat Res. 169(4):437–446. doi:10.1667/RR1165.1.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Aufderheide, E. et al. (1987), “Heavy ion effects on cellular DNA: Strand break induction and repair in cultured diploid lens epithelial cells”, <em>International journal of radiation biology and related studies in physics, chemistry and medicin</em>e, Vol. 51/5, Taylor & Francis, London, https://doi.org/10.1080/09553008714551071 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Bannik, K. et al. (2013), “Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes?”, <em>Radiation and environmental biophysics</em>, Vol. 52/2, Springer-Verlag, Berlin/Heidelberg, https://doi.org/10.1007/s00411-012-0451-8 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Bains, S. K. et al. (2019), “Effects of ionizing radiation on telomere length and telomerase activity in cultured human lens epithelium cells”, I<em>nternational journal of radiation biology</em>, Vol. 95/1, Taylor & Francis, Abingdon, https://doi.org/10.1080/09553002.2018.1466066 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barazzuol, L et al. (2017), “A coordinated DNA damage response promotes adult quiescent neural stem cell activation. PLOS Biology, 15(5). <a href="https://doi.org/10.1371/journal.pbio.2001264" rel="noreferrer noopener" target="_blank">https://doi.org/10.1371/journal.pbio.2001264</a> </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barnard, S. G. R. et al. (2018), “Dotting the eyes: mouse strain dependency of the lens epithelium to low dose radiation-induced DNA damage”, <em>International journal of radiation biology</em>, Vol. 94/12, Taylor & Francis, Abingdon, https://doi.org/10.1080/09553002.2018.1532609 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barnard, S. G. R. et al. (2019), “Inverse dose-rate effect of ionising radiation on residual 53BP1 foci in the eye lens”, <em>Scientific Reports</em>, Vol. 9/1, Nature Publishing Group, England, https://doi.org/10.1038/s41598-019-46893-3 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barnard, S. G. R. et al. (2022), “Radiation-induced DNA damage and repair in lens epithelial cells of both Ptch1 (+/-) and Ercc2 (+/-) mutated mice”, <em>Radiation Research</em>, Vol. 197/1, Radiation Research Society, United States, https://doi.org/10.1667/RADE-20-00264.1 </span></span></p>
<p> </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Baselet, B. et al. (2019), “Pathological effects of ionizing radiation: endothelial activation and dysfunction”, Cellular and molecular life sciences, Vol. 76/4, Springer Nature, https://doi.org/10.1007/s00018-018-2956-z </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Baselet, B. et al. (2017), “Functional Gene Analysis Reveals Cell Cycle Changes and Inflammation in Endothelial Cells Irradiated with a Single X-ray Dose”, Frontiers in pharmacology, Vol. 8, Frontiers, https://doi.org/10.3389/fphar.2017.00213 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Baumstark-Khan, C., J. Heilmann, and H. Rink (2003), ‘Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation”, <em>Advances in space research</em>, Vol. 31/6, Elsevier Ltd, England, https://doi.org/10.1016/S0273-1177(03)00095-4 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Beels, L. et al. (2009), "g-H2AX Foci as a Biomarker for Patient X-Ray Exposure in Pediatric Cardiac Catheterization", Are We Underestimating Radiation Risks?":1903–1909. doi:10.1161/CIRCULATIONAHA.109.880385.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Belli M, Cherunbini R, Vecchia MD, Dini V, Moschini G, Signoretti C, Simon G, Tabocchini MA, Tiveron P. 2000. DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study. Int J Radiat Biol. 76(8):1095-1104.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Brooks, A.L., D.G. Hoel & R.J. Preston (2016), "The role of dose rate in radiation cancer risk: evaluating the effect of dose rate at the molecular, cellular and tissue levels using key events in critical pathways following exposure to low LET radiation.", Int. J. Radiat. Biol. 92(8):405–426. doi:10.1080/09553002.2016.1186301.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Bucolo, C. et al. (1994), “The effect of ganglioside on oxidation-induced permeability changes in lens and in epithelial cells of lens and retina”, <em>Experimental eye research,</em> Vol. 58/6, Elsevier Ltd, London, https://doi.org/10.1006/exer.1994.1067 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cabrera et al. (2011), “Antioxidants and the integrity of ocular tissues”, in Veterinary medicine international, SAGE-Hindawi Access to Research, United States. DOI: 10.4061/2011/905153 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cadet, J. et al. (2012), “Oxidatively generated complex DNA damage: tandem and clustered lesions”, Cancer letters, Vol. 327/1, Elsevier Ireland Ltd, Ireland. https://doi.org/10.1016/j.canlet.2012.04.005 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cannan, W.J. & D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", J. Cell Physiol. 231(1):3–14. doi:10.1002/jcp.25048. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cencer, C. S. et al. (2018), “PARP-1/PAR activity in cultured human lens epithelial cells exposed to two levels of UVB light”, Photochemistry and photobiology, Vol. 94/1, Wiley, Hoboken, https://doi.org/10.1111/php.12814 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Chadwick, K.H., (2017), Towards a new dose and dose-rate effectiveness factor (DDREF)? Some comments., J Radiol Prot., 37:422-433. doi: 10.1088/1361-6498/aa6722. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Charlton, D.E., H. Nikjoo & J.L. Humm (1989), "Calculation of initial yields of single- and double-strand breaks in cell nuclei from electrons, protons and alpha particles.", Int. J. Rad. Biol., 53(3):353-365, DOI: 10.1080/09553008814552501 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Christensen, D.M. (2014), "Management of Ionizing Radiation Injuries and Illnesses, Part 3: Radiobiology and Health Effects of Ionizing Radiation.", 114(7):556–565. doi:10.7556/jaoa.2014.109. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Citrin, D.E. & J.B. Mitchel (2014), "Public Access NIH Public Access.", 71(2):233–236. doi:10.1038/mp.2011.182.doi. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dalke, C. et al. (2018), “Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk”, Radiation and environmental biophysics, Vol. 57/2, Springer Berlin Heidelberg, Berling/Heidelberg, https://doi.org/10.1007/s00411-017-0728-z </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Day, T.K. et al. (2007), "Adaptive Response for Chromosomal Inversions in pKZ1 Mouse Prostate Induced by Low Doses of X Radiation Delivered after a High Dose.", Radiat Res. 167(6):682–692. doi:10.1667/rr0764.1. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">De Angelis, P. M. et al. (2006), “Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery”, Molecular Cancer, Vol. 5/20, BioMed Central, https://doi.org/10.1186/1476-4598-5-20 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DeGraff, W. G. et al. (1992), “Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage”, Free Radical Biology and Medicine, Vol. 13/5, Elsevier, https://doi.org/10.1016/0891-5849(92)90142-4 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Desouky, O., N. Ding & G. Zhou (2015), "ScienceDirect Targeted and non-targeted effects of ionizing radiation.", J. Radiat. Res. Appl. Sci. 8(2):247–254. doi:10.1016/j.jrras.2015.03.003. </span></span></p>
<p> </p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dong et al. (2015), “Relationship between irradiation-induced neuro-inflammatory environments and impaired cognitive function in the developing brain of Mice. International Journal of Radiation Biology, 91(3):224–239. <a href="https://doi.org/10.3109/09553002.2014.988895" rel="noreferrer noopener" target="_blank">https://doi.org/10.3109/09553002.2014.988895</a> </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dong, X. et al. (2014), “NEMO modulates radiation-induced endothelial senescence of human umbilical veins through NF-κB signal pathway”, Radiation Research, Vol. 183/1, BioOne, https://doi.org/10.1667/RR13682.1 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dubrova, Y.E. & M.A. Plumb (2002), "Ionising radiation and mutation induction at mouse minisatellite loci The story of the two generations", Mutat. Res. 499(2):143–150. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">EPRI (2014), Epidemiology and mechanistic effects of radiation on the lens of the eye: Review and scientific appraisal of the literature, EPRI, California </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Falk, M., E. Lukášová & S. Kozubek (2008), "Chromatin structure influences the sensitivity of DNA to γ-radiation.", Biochim. Biophys. Acta. - Mol. Cell. Res. 1783(12):2398–2414. doi:10.1016/j.bbamcr.2008.07.010. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Feinendegen, L.E. (2005), "UKRC 2004 debate Evidence for beneficial low level radiation effects and radiation hormesis. Radiology.", 78:3–7. doi:10.1259/bjr/63353075. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Feinendegen, L.E., M. Pollycove & R.D. Neumann (2007), "Whole-body responses to low-level radiation exposure: New concepts in mammalian radiobiology.", Exp. Hematol. 35(4 SUPPL.):37–46. doi:10.1016/j.exphem.2007.01.011. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Flegal, M. et al. (2015), "Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and γ-Radiation.", (July):1–9. doi:10.3791/52912. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Foray, N., M. Bourguignon and N. Hamada (2016), “Individual response to ionizing radiation”, Mutation research. Reviews in mutation research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.09.001 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Franken NAP, Hovingh S, Cate RT, Krawczyk P, Stap J, Hoebe R, Aten J, Barendsen GW. 2012. Relative biological effectiveness of high linear energy transfer alpha-particles for the induction of DNA-double-strand breaks, chromosome aberrations and reproductive cell death in SW-1573 lung tumour cells. Oncol reports. 27:769-774. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Frankenberg D, Brede HJ, Schrewe UJ, Steinmetz C, Frankenberg-Scwager M, Kasten G, Pralle E. 1999. Induction of DNA Double-Strand Breaks by 1H and 4He Ions in Primary Human Skin Fibroblasts in the LET range of 8 to 124 keV/µm. Radiat Res. 151:540-549. </span></span></p>
<p> </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Geisel, D. et al. (2012), “DNA double-strand breaks as potential indicators for the biological effects of ionising radiation exposure from cardiac CT and conventional coronary angiography: a randomised, controlled study”, European Radiology, Vol. 22/8, Springer Nature, <a href="https://doi.org/10.1007/s00330-012-2426-1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00330-012-2426-1</a> </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Gionchiglia, N. et al. (2021), “Association of Caspase 3 Activation and H2AX γ Phosphorylation in the Aging Brain: Studies on Untreated and Irradiated Mice,” Biomedicines. 9(9):1166. doi: 10.3390/biomedicines9091166. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Goodhead, D.T. (2006), "Energy deposition stochastics and track structure: What about the target?", Radiat. Prot. Dosimetry. 122(1–4):3–15. doi:10.1093/rpd/ncl498. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Grudzenski, S. et al. (2010), "Inducible response required for repair of low-dose radiation damage in human fibroblasts.", Proc. Natl. Acad. Sci. USA. 107(32): 14205-14210, doi:10.1073/pnas.1002213107. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hada, M. & A.G. Georgakilas (2008), "Formation of Clustered DNA Damage after High-LET Irradiation: A Review.", J. Radiat. Res., 49(3):203–210. doi:10.1269/jrr.07123. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada, N. et al. (2006), “Histone H2AX phosphorylation in normal human cells irradiated with focused ultrasoft X rays: evidence for chromatin movement during repair”, Radiation Research, Vol. 166/1, Radiation Research Society, United States, https://doi.org/10.1667/RR3577.1 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada, N. (2014), “What are the intracellular targets and intratissue target cells for radiation effects?”, Radiation research, Vol. 181/1, The Radiation Research Society, Lawrence, https://doi.org/10.1667/RR13505.1 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada, N. and T. Sato (2016), “Cataractogenesis following high-LET radiation exposure”, Mutation Research. Reviews in mutation research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.08.005 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada, N. (2017a), “Ionizing radiation sensitivity of the ocular lens and its dose rate dependence”, International journal of radiation biology, Vol. 93/10, Taylor & Francis, Abingdon, https://doi.org/10.1080/09553002.2016.1266407 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada, N. and G. E. Woloschak (2017), “Ionizing radiation response of primary normal human lens epithelial cells”, PloS ONE, Vol. 12/7, Public Library Science, San Francisco, https://doi.org/10.1371/journal.pone.0181530 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Havas, M. (2017), “When theory and observation collide: Can non-ionizing radiation cause cancer?”, Environmental pollution, Vol. 221, Elsevier Ltd, England. https://doi.org/10.1016/j.envpol.2016.10.018 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hightower, K. R. (1995), “The role of the lens epithelium in development of UV cataract”, Current eye research, Vol. 14/1, Informal UK Ltd, Philadelphia, https://doi.org/10.3109/02713689508999916 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Iliakis, G., T. Murmann & A. Soni (2015), "Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations.", Mutat. Res. - Genet. Toxicol. Environ. Mutagen. 793:166–175. doi:10.1016/j.mrgentox.2015.07.001. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Joiner, M. (2009), "Basic Clinical Radiobiology", Edited by. [1] P.J. Sadler, Next-Generation Met Anticancer Complexes Multitargeting via Redox Modul Inorg Chem 52 21.:375. doi:10.1201/b13224. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Jorge, S.-G. et al. (2012), "Evidence of DNA double strand breaks formation in Escherichia coli bacteria exposed to alpha particles of different LET assessed by the SOS response.", Appl. Radiat. Isot. 71(SUPPL.):66–70. doi:10.1016/j.apradiso.2012.05.007. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kadhim, M.A., M.A. Hill & S.R. Moore, (2006), "Genomic instability and the role of radiation quality.", Radiat. Prot. Dosimetry. 122(1–4):221–227. doi:10.1093/rpd/ncl445. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Khanna, K.K. & S.P. Jackson (2001), "DNA double-strand breaks: signaling, repair and the cancer connection.", Nature Genetics. 27(3):247-54. doi:10.1038/85798. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kim, K. S. et al. (2014), “Characterization of DNA damage-induced cellular senescence by ionizing radiation in endothelial cells”, International Journal of Radiation Biology, Vol. 90/1, Informa, London, https://doi.org/10.3109/09553002.2014.859763 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kim, J. N. and B. M. Lee (2007), “Risk factors, health risks, and risk management for aircraft personnel and frequent flyers”, Journal of toxicology and environmental health. Part B, Critical reviews, Vol. 10/3, Taylor & Francis Group, Philadelphia, https://doi.org/10.1080/10937400600882103 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kleiman, N. J., R. Wang and A. Spector (1990), “Ultraviolet light induced DNA damage and repair in bovine lens epithelial cells”, Current eye research, Vol. 9/12, Informa UK Ltd, Oxford, https://doi.org/10.3109/02713689009003475 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, International Journal of Radiation Biology, 1–12. https://doi.org/10.1080/09553002.2022.2110306 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kuefner, M.A. et al. (2009), "DNA Double-Strand Breaks and Their Repair in Blood Lymphocytes of Patients Undergoing Angiographic Procedures.", Investigative radiology. 44(8):440-6. doi:10.1097/RLI.0b013e3181a654a5. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kuefner, M.A. et al. (2015), "Chemoprevention of Radiation-Induced DNA Double-Strand Breaks with Antioxidants.", Curr Radiol Rep (2015) 3: 81. https://doi.org/10.1007/s40134-014-0081-9 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kuhne, M., G. Urban & M. Lo, (2005), "DNA Double-Strand Break Misrejoining after Exposure of Primary Human Fibroblasts to CK Characteristic X Rays, 29 kVp X Rays and Co γ-Rays.", Radiation Research. 164(5):669-676. doi:10.1667/RR3461.1. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">de Lara, C.M. et al. (2001), "Dependence of the Yield of DNA Double-Strand Breaks in Chinese Hamster V79-4 Cells on the Photon Energy of Ultrasoft X Rays.", Radiation Research. 155(3):440-8. doi:10.1667/0033-7587(2001)155[0440:DOTYOD]2.0.CO;2. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Liao, J. et al. (2011), “Anti-UVC irradiation and metal chelation properties of 6-benzoyl-5,7-dihydroxy-4-phenyl-chromen-2-one: An implications for anti-cataract agent”, International journal of molecular sciences, Vol. 12/10, MDPI, Basel. https://doi.org/10.3390/ijms12107059 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Lipman, R. M., B. J. Tripathi, R. C. Tripathi (1998), “Cataracts induced by microwave and ionizing radiation”, Survey of ophthalmology, Vol. 33/3, Elsevier Inc, United States, https://doi.org/10.1016/0039-6257(88)90088-4 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Lomax, M.E., L.K. Folkes & P.O. Neill (2013). "Biological Consequences of Radiation-induced DNA Damage: Relevance to Radiotherapy", Statement of Search Strategies Used and Sources of Information Why Radiation Damage is More Effective than Endogenous Damage at Killing Cells Ionising Radiation-induced Do. 25:578–585. doi:10.1016/j.clon.2013.06.007. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Long, A. C., C. M. H. Colitz, and J. A. Bomser (2001), “Apoptotic and necrotic mechanisms of stress-induced human lens epithelial cell death”, Experimental biology and medicine, SAGE Publications, London, https://doi.org/10.1177/153537020422901012 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Lorat, Y. et al. (2015), "Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy – The heavy burden to repair.", DNA Repair (Amst). 28:93–106. doi:10.1016/j.dnarep.2015.01.007. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Maier, P. et al. (2016), "Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization.", Int. J. Mol. Sci., 14;17(1), pii:E102, doi:10.3390/ijms17010102. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Markiewicz, E. et al. (2015), “Nonlinear ionizing radiation-induced changes in eye lens cell proliferation, cyclin D1 expression and lens shape”, Open biology, Vol. 5/4, Royal society, London, https://doi.org/10.1098/rsob.150011 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Milligan, J. R. et al. (1995), « DNA repair by thiols in air shows two radicals make a double-strand break”, Radiation Research, Vol 143/3, pp. 273-280 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Moore, S., F.K.T. Stanley & A.A. Goodarzi (2014), "The repair of environmentally relevant DNA double strand breaks caused by high linear energy transfer irradiation – No simple task.", DNA repair (Amst), 17:64–73. doi: 10.1016/j.dnarep.2014.01.014. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Mosconi, M., U. Giesen & F. Langner (2011), "53BP1 and MDC1 foci formation in HT-1080 cells for low- and high-LET microbeam irradiations.", Radiat. Envrion. Biophys. 50(3):345–352. doi:10.1007/s00411-011-0366-9. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular disease”, Journal of Radiation Research, Vol. 62/4, https://doi.org/10.1093/jrr/rrab032 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Nenoi, M., B. Wang & G. Vares (2015), "In vivo radioadaptive response: A review of studies relevant to radiation-induced cancer risk.", Hum. Exp. Toxicol. 34(3):272–283. doi:10.1177/0960327114537537. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Nikitaki, Z. et al. (2016), "Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET).", Free Radiac. Res. 50(sup1):S64-S78, doi:10.1080/10715762.2016.1232484. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Nikjoo, H. et al. (2001), "Computational approach for determining the spectrum of DNA damage induced by ionizing radiation.", Radiat. Res. 156(5 Pt 2):577–83.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Nübel, T. et al. (2006), “Lovastatin protects human endothelial cells from killing by ionizing radiation without impairing induction and repair of DNA double-strand breaks”, Clinical Cancer Research, Vol. 12/3, American Association for Cancer Research, https://doi.org/10.1158/1078-0432.CCR-05-1903 </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Ojima, M., N. Ban, and M. Kai (2008), “DNA double-strand breaks induced by very low X-ray doses are largely due to bystander effects”, Radiation Research, Vol. 170/3, Radiation Research Society, United States, https://doi.org/10.1667/RR1255.1 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Okayasu, R. (2012a), "Repair of DNA damage induced by accelerated heavy ions-A mini review.", Int. J. Cancer. 130(5):991–1000. doi:10.1002/ijc.26445. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Okayasu, R. (2012b), "Heavy ions — a mini review.", 1000:991–1000. doi:10.1002/ijc.26445. </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Park, J. W. et al. (2022), “Metformin alleviates ionizing radiation-induced senescence by restoring BARD1-mediated DNA repair in human aortic endothelial cells”, Experimental Gerontology, Vol. 160, Elsevier, Amsterdam, https://doi.org/10.1016/j.exger.2022.111706 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Parris, C.N. et al. (2015), "Enhanced γ-H2AX DNA damage foci detection using multimagnification and extended depth of field in imaging flow cytometry.", Cytom. Part A. 87(8):717–723. doi:10.1002/cyto.a.22697. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Radulescu I., K. Elmroth & B. Stenerlöw (2006), "Chromatin Organization Contributes to Non-randomly Distributed Double-Strand Breaks after Exposure to High-LET Radiation.", Radiat. Res. 161(1):1–8. doi:10.1667/rr3094. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rastogi, R. P. et al. (2010), “Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair”, Journal of nucleic acids, Hindawi Ltd, United States. https://doi.org/10.4061/2010/592980 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Reddy, V. N. et al. (1998), “The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium”, Investigative ophthalmology & visual science, Vol. 39/2, Arvo, pp. 344-350 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rogakou, E.P. et al. (1999), "Megabase Chromatin Domains Involved in DNA Double-Strand Breaks In Vivo.", J. Cell Biol, 146(5):905-16. doi: 10.1083/jcb.146.5.905. </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Rogers, C. S. et al. (2004), “The effects of sub-solar levels of UV-A and UV-B on rabbit corneal and lens epithelial cells”, Experimental eye research, Vol. 78/5, Elsevier Ltd, London, https://doi.org/10.1016/j.exer.2003.12.011</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Rombouts, C. et al. (2013), “Differential response to acute low dose radiation in primary and immortalized endothelial cells”, International Journal of Radiation Biology, Vol. 89/10, Informa, London, https://doi.org/10.3109/09553002.2013.806831 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rothkamm, K. et al. (2015), "Review DNA Damage Foci: Meaning and Significance.", Environ. Mol. Mutagen., 56(6):491-504, doi: 10.1002/em.21944. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rothkamm, K. & M. Lo (2003), "Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses.", PNAS, 100(9):5057-62. doi:10.1073/pnas.0830918100. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rübe, C.E. et al. (2008), "Cancer Therapy: Preclinical DNA Double-Strand Break Repair of Blood Lymphocytes and Normal Tissues Analysed in a Preclinical Mouse Model: Implications for Radiosensitivity Testing.", Clin. Cancer Res., 14(20):6546–6556. doi:10.1158/1078-0432.CCR-07-5147. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Russo, A. et al. (2015), "Review Article Genomic Instability: Crossing Pathways at the Origin of Structural and Numerical Chromosome Changes.", Envrion. Mol. Mutagen. 56(7):563-580. doi:10.1002/em. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rydberg B, Heilbronn L, Holley WR, Lobrich M, Zeitlin C et al. 2002. Spatial Distribution and Yield of DNA Double-Strand Breaks Induced by 3-7 MeV Helium Ions in Human Fibroblasts. Radiat Res. 158(1):32-42.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Sabirzhanov, et al. (2020), “Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.”, Int J Mol Sci. 21(15):5239. doi: 10.3390/ijms21155239.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sage, E. & N. Shikazono (2017), "Free Radical Biology and Medicine Radiation-induced clustered DNA lesions: Repair and mutagenesis.", Free Radic. Biol. Med. 107(December 2016):125–135. doi:10.1016/j.freeradbiomed.2016.12.008. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sasaki, H. et al. (1998), “TEMPOL protects against lens DNA strand breaks and cataract in the x-rayed rabbit”, Investigative ophthalmology & visual sciences, Vol. 39/3, Arvo, Rockville, pp. 544-552</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Schmal, Z. et al. (2019), “DNA damage accumulation during fractionated low-dose radiation compromises hippocampal neurogenesis”, Radiotherapy and Oncology. 137:45-54. doi:10.1016/j.radonc.2019.04.021.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sears, C. R. and J. J. Turchi (2012), “Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways”, Journal of biological chemistry, Vol 287/29, The American Society for Biochemistry and Molecular Biology, Inc, USA, https://doi.org/ 10.1074/jbc.M112.344911 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Shah, D.J., R.K. Sachs & D.J. Wilson (2012), "Radiation-induced cancer: A modern view." Br. J. Radiol. 85(1020):1166–1173. doi:10.1259/bjr/25026140. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Shelke, S. & B. Das (2015), "Dose response and adaptive response of non- homologous end joining repair genes and proteins in resting human peripheral blood mononuclear cells exposed to γ radiation.", (December 2014):365–379. doi:10.1093/mutage/geu081. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sidjanin, D., S. Zigman and J. Reddan (1993), “DNA damage and repair in rabbitlens epithelial cells following UVA radiation”, Current eye research, Vol. 12/9, Informa UK Ltd, Oxford, https://doi.org/10.3109/02713689309020382 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Smith, J. et al. (2003), "Impact of DNA ligase IV on the delity of end joining in human cells.", Nucleic Acids Research. 31(8):2157-2167.doi:10.1093/nar/gkg317. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Smith, T.A. et al. (2017), "Radioprotective agents to prevent cellular damage due to ionizing radiation." Journal of Translational Medicine.15(1).doi:10.1186/s12967-017-1338-x. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Stewart, F. A. et al. (2012), “ICRP publication 118: ICRP statement on tissue reactions and early and late effects of radiation in normal tissues and organs – threshold doses for tissue reactions in a radiation protection context”, Annals of the ICRP, Vol, 41/1-2, Elsevier Ltd, London, https://doi.org/10.1016/j.icrp.2012.02.001 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sudprasert, W., P. Navasumrit & M. Ruchirawat (2006), "Effects of low-dose γ radiation on DNA damage, chromosomal aberration and expression of repair genes in human blood cells.", Int. J. Hyg. Envrion. Health, 209:503–511. doi:10.1016/j.ijheh.2006.06.004. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sutherland, B.M. et al. (2000), "Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation.", J. of Rad. Res. 43 Suppl(S):S149-52. doi: 10.1269/jrr.43.S149</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sylvester, C. B. et al. (2018), “Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer”, Frontiers in cardiovascular medicine, Vol. 5, Frontiers, https://doi.org/10.3389/fcvm.2018.00005 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Terato, H. & H. Ide (2005), "Clustered DNA damage induced by heavy ion particles.", Biol. Sci. Sp. 18(4):206–215. doi:10.2187/bss.18.206.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ungvari, Z. et al. (2013), “Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosensitivity”, The journals of gerontology. Series A, Biological sciences and medical sciences, Vol. 68/12, Oxford University Press, Oxford, https://doi.org/10.1093/gerona/glt057 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Valentin, J.D.J (1998), "Chapter 1. Ann ICRP.", 28(4):5–7. doi:10.1016/S0146-6453(00)00002-6. http://www.ncbi.nlm.nih.gov/pubmed/10882804. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Venkatesh, P. et al. (2016), "Effect of chromatin structure on the extent and distribution of DNA double strand breaks produced by ionizing radiation; comparative study of hESC and differentiated cells lines.", Int J. Mol. Sci. 17(1). doi:10.3390/ijms17010058. </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Wang, et al. (2021), “Ionizing Radiation-Induced Brain Cell Aging and the Potential Underlying Molecular Mechanisms.”, Cells. 10(12):3570. doi: 10.3390/cells10123570 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ward, J. F. (1988), "DNA Damage Produced by Ionizing Radiation in Mammalian Cells: Identities, Mechanisms of Formation, and Reparability.", Prog. Nucleic Acid Res. Mol. Biol. 35(C):95–125. doi:10.1016/S0079-6603(08)60611-X. </span></span></p>
<p>Wilkinson, Beth et al. (2023) “The Cellular Response to Complex DNA Damage Induced by Ionising Radiation.” Int J Mol Sci. 24(5): 4920. doi:10.3390/ijms24054920 </p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Wolf, N. et al. (2008), “Radiation cataracts: Mechanisms involved in their long delayed occurrence but then rapid progression”, Molecular vision, Vol. 14/34-35, Molecular Vision, Atlanta, pp. 274-285 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Wu, L.J. et al. (1999), "Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells.", Proc. Natl. Acad. Sci. 96(9):4959–4964. doi:10.1073/pnas.96.9.4959. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Yamaguchi, H. et al. (2005), "Estimation of Yields of OH Radicals in Water Irradiated by Ionizing Radiation.", J. of Rad. Res. 46(3):333-41. doi: 10.1269/jrr.46.333.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Zhang, L. et al. (2017), “The inhibitory effect of minocycline on radiation-induced neuronal apoptosis via AMPKα1 signaling-mediated autophagy.”, Sci Rep.7(1):16373. doi: 10.1038/s41598-017-16693-8. </span></span></p>
2019-08-26T12:00:062024-03-08T12:44:2681eb87ac-fb24-4667-bd3a-9be52450f362f703f4b6-e87b-4be7-8ffa-ff2f1a670694<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Oxidative stress is an event that involves both a reduction in free radical scavengers and enzymes, and an increase in free radicals (Brennan et al., 2012). Oxidative stress needs to be maintained within an organism to avoid an excess of damage to biological structures, such as DNA. A redox homeostasis between the radicals and the scavengers is necessary. Between reactive oxygen species (ROS) and reactive nitrogen species (RNS), collectively known as RONS, ROS is particularly significant to oxidative damage and disease states. Radicals such as singlet oxygen and hydroxyl radical are highly unstable and will react with molecules near their generation point, while radicals such as H<sub>2</sub>O<sub>2</sub> are more stable and membrane permeable, meaning they can travel further to find electrons (Spector, 1990). Since DNA is mainly found in nucleus, ROS needs to reach the nucleus to induce breaks. Hydroxyl radicals, in addition to being highly reactive, are capable of causing DNA damage leading to single stranded breaks (SSBs), double stranded breaks (DSBs) and complex lesions (Cadet and Davies, 2017; Halliwell et al., 2021; Engwa et al., 2020; Wilkinson et al., 2023). The regulation of these radicals is achieved by the antioxidant defense response (ADR), which includes enzymatic and non-enzymatic processes. The ADR is recruited to manage RONS levels, with antioxidants such as superoxide dismutase (SOD) functioning as the first line of defense, this includes the three isotypes copper-zinc SOD (CuZn-SOD), manganese SOD (Mn SOD) and extracellular SOD (EC SOD) (Engwa et al., 2020). These antioxidants act as scavengers to oxidants, reacting with them before reaching other structures within the cell such as DNA strands (Cabrera et al., 2011; Engwa et al., 2020). The backbone of DNA can fragment upon sustained exposure to ROS (Uwineza et al., 2019; Cannan et al., 2016). Due to low oxidation potentials, adenine and guanine are the DNA bases more prone to oxidation, with oxidation potentials (normal hydrogen electrode) at pH 7 of 1.3 eV and 1.42 eV compared to the 1.6 eV and 1.7 eV of cytosine and thymine (Fong, 2016; Halliwell et al., 2021; Poetsch, 2020). In fact, certain radicals even target guanine in a selective fashion, including carbonate anion radical (CO<sub>3</sub><sup>•-</sup>) and singlet oxygen (<sup>1</sup>O<sub>2</sub>) (Halliwell et al., 2021).</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Overall Weight of Evidence: Moderate </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">The biological plausibility of the relationship between increased oxidative stress leading to increased DNA damage (e.g. SSBs, DSBs, complex lesions, abasic sites, and oxidized bases) (Cadet et al., 2012; Cadet and Davies, 2017</span></span>)<span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"> is highly supported by the literature. Evidence was collected from studies conducted using in vitro lens epithelial cell models and derived from humans, bovine and germ line cells (Spector, 1990; Stohs, 1995; Aitken et al., 2001; Spector, 1995). As this evidence is derived from studies using a human cell model it limits the ability to compare between different taxonomies (Ahmadi et al., 2022; Cencer et al., 2018; Liu et al., 2013; Meng et al., 2021; Smith et al., 2015; Zhou et al., 2016). Other evidence comes from human-derived and rodent models of neuronal and endothelial cells (Cervelli et al., 2014; El-Missiry et al., 2018; Huang et al., 2021; Sakai et al., 2017; Ungvari et al., 2013; Zhang et al., 2017). </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">ROS that are generated specifically as a result of radiation are highly localized, increasing the likelihood of clustered regions of damage. Naturally generated ROS are more widespread and as a result less capable of generating clusters of damage. ROS will act on DNA bases to oxidize or delete them from the sequence, which create nicks on the strand (Cannan et al., 2016). This damage can occur to any DNA base but bases such as guanine and adenine are most vulnerable due to their low oxidation potentials (Fong, 2016). The mechanism through which the strand break occurs is a result of base excision repair (BER) happening at multiple sites that are too close together, resulting in the spontaneous conversion to DSBs prior to completion of repair. Indirect SSB formation could also occur during the repair process by the BER pathway and thus unrepaired SSB could stall the replication mechanism and may lead to a potential DSB (Cadet and Davies, 2017).</span></span> <span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">ROS damage to bases clustered together means that multiple sites of BER are happening very close together and while the strand may be able to support the damaged area for one repair, concurrent repairs make surrounding areas more fragile and the strand breaks at the nick sites are under added strain (Cannan et al., 2016). Endogenous damage to DNA as a result of radicals appears over time and mainly as isolated lesions, a pattern understood to be due to the diffusion of the radicals resulting in homogenous distribution patterns. This differs from the specific situations where radiation acts as the stressor to increase oxidative stress, as the radiation track will be highly localized and form radicals within that hit space. This leads to non-homologous lesions and clustered damage to the DNA (Ward et al., 1985).</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">This relationship is well supported through empirical evidence from studies using stressors such as H<sub>2</sub>O<sub>2</sub>, photons, γ- and X-ray, which cause an increase in markers of oxidative stress such as ROS-generating enzymes (lactate dehydrogenase, LDH), and a decrease in free radical scavengers, resulting in DNA strand fragmentation. These studies include both in vivo and in vitro human lens epithelial cells (LECs), mouse, rat and rabbit models, including neuronal cells lines and endothelial cells (Ahmadi et al., 2022; Cencer et al., 2018; Cervelli et al., 2014; El-Missiry et al., 2018; Huang et al., 2021; Liu et al., 2013; Meng et al., 2021; Spector et al., 1997; Ungvari et al., 2013; Zhang et al., 2017; Zhou et al., 2016; Sakai et al., 2017). </span></span></p>
<p> </p>
<p><strong><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Dose/Incidence Concordance </span></span></strong></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">There is high evidence to support a dose concordance between oxidative stress and DNA strand breaks. One in vitro study demonstrated that when ROS levels in LECs are 10% above control following 0.5 Gy gamma ray exposure, DNA strand breaks increased 15-20% above control (Ahmadi et al., 2021). Another study with ultraviolet (UV)B radiation demonstrated higher ROS levels after exposure to 0.14 J/cm<sup>2</sup> on in vitro LECs as compared to a lower dose exposure (0.014 J/cm<sup>2</sup>) for the same time. This corresponded to DNA strand break levels also increasing following high dose rate exposure, but not with the low dose exposure (Cencer et al., 2018). </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">A 30 µM of H<sub>2</sub>O<sub>2</sub> treatment of in vitro LECs is associated with a 1.4x increase in lactate dehydrogenase (LDH) and 55% more DNA strand breaks (Liu et al., 2013; Smith et al., 2015). Following exposure of in vitro LECs to 50 µM H<sub>2</sub>O<sub>2</sub>, increased ROS levels, 4x for LDH, and decreased antioxidant levels, 2x control for GSH-Px and SOD, are associated with a 3x increase in γ-H2AX, a marker of DNA strand breaks (Meng et al., 2021). SOD and GSH decreased by 2-fold following 100 µM H<sub>2</sub>O<sub>2</sub> exposure on LECs with an in vitro model (Zhou et al., 2016). At 125 µM H<sub>2</sub>O<sub>2</sub> intact DNA can be reduced to near 1% of pre-treatment levels for in vitro LECs (Spector et al., 1997). Following 400 µM H<sub>2</sub>O<sub>2</sub> LDH increased to 1200% of control in neuroblastoma cells (Feng et al., 2016) and DNA strand breaks increased to over 150% of control in in vitro LECs (Li et al., 1998). </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Exposure of in vitro mouse hippocampal neuronal cells (HT22 cell line) to 10 Gy of X-irradiation resulted in a 5x increase in ROS generation and 3x increase in γ-H2AX (Huang et al., 2021). Another study exposed the same cell line to 8 and 12 Gy of X-irradiation and found a ~2x increase in ROS at 8 Gy and a 4.4x and 3.2x increase in phosphorylation of ataxia telangiectasia mutated (ATM) and γ-H2AX, respectively, 30 minutes after 12 Gy (Zhang et al., 2017). A separate study exposed adult male rats to 4 Gy of γ-irradiation and found 2x increase in 4-hydroxy-2-nonenal (4-HNE) (lipid peroxidation marker) and 3x increase in protein carbonylation. Glutathione reductase decreased by approximately 5x, whereas glutathione and glutathione peroxidase levels decreased by approximately 3x each. Tail DNA %, tail length and tail moment (DNA strand break parameters) increased by approximately 2x, 3x and 6x, respectively (El-Missiry et al., 2018). </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Endothelial cells exposed to irradiation also demonstrated the relation between oxidative stress and DNA strand breaks. Rat cerebromicrovascular endothelial cells (CMVECs) exposed to 8 Gy <sup>137</sup>Cs gamma rays showed increased cellular peroxide production and mitochondrial oxidative stress. Tail DNA content indicating DNA damage was also increased from 0 to 45% (Ungvari et al., 2013). Human umbilical vein endothelial cells (HUVECs) were irradiated with single (0.125, 0.25, 0.5 Gy), or fractionated (2 × 0.125 Gy, 2 × 0.250 Gy) doses of X-rays. Intracellular ROS production increased in a dose-dependent manner following 0.125, 0.25, 0.5 Gy, and γ-H2AX foci positive cells were observed at all doses (Cervelli et al., 2014). Human aortic endothelial cells (HAECs) exposed to 100µM H<sub>2</sub>O<sub>2</sub> showed 3.7-fold increase in intracellular ROS and a 3.4- and 4.7-fold increase in γ-H2AX and p-ATM, respectively (Sakai et al., 2017).</span></span></p>
<p> </p>
<p><strong><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Time Concordance </span></span></strong></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">There is low evidence to support a time concordance between oxidative stress to strand breaks on DNA. Non-protein-thiol levels, an antioxidant, in in vitro LECs decreased to near zero by 30 min post-exposure to 300 µM H<sub>2</sub>O<sub>2</sub>, before recovering to 70% of control by 120 min. At 60 min post-exposure to 125 µM H<sub>2</sub>O<sub>2</sub> there was a start to a divergence from control level DNA fragmentation, one that increased logarithmically, with the treated group having a 14~18% reduction in intact DNA by 9 h post-exposure (Yang et al., 1998). Time response information is difficult to monitor for DNA strand breaks because repair will occur, reducing the number of breaks over time. At 0 minutes post in vitro exposure to 40 µM H<sub>2</sub>O<sub>2</sub> LECs had ~145% of control level DNA strand breaks but that number dropped to ~105% by 30 minutes post-exposure (Li et al., 1998). </span></span></p>
<p> </p>
<p><strong><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Essentiality </span></span></strong></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Oxidative stress has been found to increase levels of DNA strand breaks above background levels (Li et al., 1998; Liu et al., 2013; Cencer et al., 2018; Ahmadi et al., 2022; El-Missiry et al., 2018; Huang et al., 2021; Cervelli et al., 2017; Sakai et al., 2017). It has been shown that inhibition of oxidative stress leads to a reduction in DNA strand breaks. Sulforaphane (SFN) is an isothiocyanate, which provides chemical protection against ROS by activating the release of enzymatic scavengers. When SFN was added to in vitro LECs exposed to 30 µM H<sub>2</sub>O<sub>2</sub>, LDH decreased to near unexposed cell levels from the 1.4x control level without SFN. This LDH drop was associated with reducing the levels of DNA strand breaks induced by oxidative stress almost 3-fold as compared to cells without SFN (Liu et al., 2013). In another study, intact DNA levels were returned to control when treated with µPx-11 (peroxidase that breaks down H<sub>2</sub>O<sub>2</sub>), following exposure to 125 µM H<sub>2</sub>O<sub>2</sub>. This was a near 100% recovery compared to the drop seen in LECs that did not contain µPx-11 (Spector et al., 1997). </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Within the brain of Wistar rats, epigallocatechin-3-gallate (EGCG) ameliorated radiation-induced increases in lipid peroxidation and protein carbonylation, as well as decreases in glutathione (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) and reverted the levels back to those similar to controls. DNA strand break parameters also returned to those similar to controls after treatment with EGCG (El-Missiry et al., 2018). Similar effects were also shown in another study using treatment mesenchymal stem cell-conditioned medium in mouse hippocampal cells exposed to 10 Gy of X-irradiation (Huang et al., 2021). </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">HUVECs pretreated with the antioxidant mixture RiduROS blunted ROS generation in a concentration-dependent manner by 65% ± 5.6% and 98% ± 2%, at 0.1 and 1 μg/mL, respectively, compared with cells irradiated without pretreatment. Low-dose irradiation also increased DSB-induced γ-H2AX foci compared with control cells and 24 h of RiduROS pretreatment reduced the γ-H2AX foci number by 41% (Cervelli et al., 2017). Additionally, HAECs treated with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) found significantly reduced intracellular ROS at 100µM, as well as reduced γ-H2AX foci formation by 47% and 48% following EPA and DHA treatment respectively. (Sakai et al., 2017). </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The following tables provide representative examples of the relationship, unless otherwise indicated, all data is significantly significant. </span></span></p>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dose Concordance </span></span></strong></p>
<table border="1">
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<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Reference </span></span></strong></p>
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<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Experiment Description </span></span></strong></p>
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<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Result </span></span></strong></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cencer et al., 2018 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, human LECs exposed to UVB and tested for 120 min post exposure with fluorescent probes to detect ROS production and mitochondrial superoxide, and tetramethylrhodamine-dUTP (TMR) red assay to detect strand breaks. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Both ROS and DNA strand breaks were increased by both 0.014 J/cm<sup>2</sup> and 0.14 J/cm<sup>2</sup> UVB radiation. At 0.014 J/cm<sup>2</sup>, cellular ROS increased a maximum of 15 fluorescence units above the control at 5 minutes post-UVB, while DNA strand breaks increased about 115 fluorescence units above the control at this time. At 0.14 J/cm<sup>2</sup>, cellular ROS increased a maximum of about 35 fluorescence units above the control at 90 minutes post-UVB, while mitochondrial superoxide increased about 30 fluorescence units above the control and DNA strand breaks increased about 125 fluorescence units above the control at this time. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> </span></span><br />
</p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ahmadi et al., 2021 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, human LECs exposed to 0.065-0.3 Gy/min gamma radiation, with dihydroethdium (DHE) fluorescent probes to measure ROS levels and comet assay to measure strand breaks. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Human LECs exposed in vitro to 0.1 - 0.5 Gy gamma rays showed a gradual increase in ROS levels and a corresponding gradual increase in DNA in the tail from the comet assay (indicative of increased DNA strand breaks) with the maximum dose displaying a 10% increase in ROS levels and a 17% increase in DNA strand damage. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Li et al., 1998 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, bovine LECs were exposed to 40 and 400 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> with an alkaline unwinding assay to determine strand break levels. </span></span></p>
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<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Immediately after LECs were exposed to 40 µM and 400 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">, there were ~145% and ~150% DNA strand breaks compared to the unexposed control level, respectively. The amounts of DNA strand breaks in cells exposed to both concentrations were reduced to ~105% of the unexposed control level after 30 mins. After 400 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">, oxidative stress as measured by LDH was 1200% of control in neuroblastoma cells. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Spector et al., 1997 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, rat LECs exposed to 100 and 125 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> with alkaline elution assay to determine single strand break level. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Exposure to 125 µM of </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> to lens epithelial cells resulted in reduction of intact DNA to near 1% by 9 hr post-exposure. Exposure to 100 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> reduced SOD and GSH levels by 2-fold. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">El-Missiry et al., 2018 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo, albino Wistar rats were exposed to 4 Gy of γ radiation (<sup>137</sup>Cs source) at 0.695 rad/s. Kits were used to measure 4-HNE (secondary product of lipid peroxidation) and protein </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">carbonyl group levels as markers of oxidative stress. Antioxidants including GSH, GPx </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">and GR were also assessed. The comet assay was used to analyze DNA strand breaks by visualizing DNA tail %, tail length and tail moment. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">4-HNE and protein carbonyl levels increased by approximately 2- and 3-fold after radiation exposure. GSH and GPx levels decreased by approximately 3-fold each, whereas GR levels decreased by approximately 5-fold. Tail DNA %, tail length and tail moment increased by approximately 2-, 3- and 6-fold after exposure to 4 Gy. </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ungvari et al., 2013 </span></span></p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. CMVECs and rat hippocampal neurons were irradiated with 2-8 Gy <sup>137</sup>Cs gamma rays. 5(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) staining, and flow cytometry were used to measure ROS production. DNA damage was quantified by measuring the tail DNA content (as a percentage of total DNA) using the Comet Assay-IV software. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Day 1 post-irradiation showed increased cellular peroxide production and increased mitochondrial oxidative stress in CMVECs in a dose-dependent manner, increasing a maximum of ~3-fold at 8 Gy. Tail DNA content also increased in a dose-dependent manner with an approximate increase from 0 to 45% at 8 Gy. </span></span></p>
<p> </p>
</td>
</tr>
<tr>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Huang et al., 2021 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, HT22 cells (mouse hippocampal neuronal cell line) were exposed to 10 Gy of X-irradiation at 6 Gy/min. ROS levels were measured using H2-DCFDA staining and fluorescence microscope analysis, whereas western blotting was used to detect γ-H2AX. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At 10 Gy, intracellular ROS generation increased by 5-fold and γ-H2AX increased by 3-fold. </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Zhang et al., 2017 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. HT22 cells were exposed to 8 and 12 Gy X-rays. Relative intracellular ROS levels were determined by DCFDA. p-ATM, γ-H2AX were measured with Western blot. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Following 8 Gy irradiation, intracellular ROS levels increased ~1.8-fold. Phosphorylation of ATM and γ-H2AX were increased 4.4-fold and 3.2-fold, respectively, 30 minutes after 12 Gy. </span></span></p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cervelli et al., 2014 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. HUVECs were irradiated with single doses (0.125, 0.25, 0.5 Gy), or fractionated doses (2 × 0.125 Gy, 2 × 0.250 Gy) of X-rays. Intracellular ROS generation was measured with a fluorescent dye, C-DCFDA, using a spectrofluorometer. Immunofluorescence microscopy was used to measure γ-H2AX foci. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Intracellular ROS production was significantly increased in a dose-dependent manner (1.6-, 2- and 2.8-fold at 0.125, 0.25, 0.5 Gy, respectively). When HUVECs were exposed to fractionated doses, no increase in ROS generation was observed, compared with respective single doses. 24h post-irradiation the percentage of foci-positive cells exposed to 0.125 Gy, 2 × 0.125 Gy, 0.250 Gy, 2 × 0.250 Gy and 0.5 Gy, was 1.68, 1.48, 3.53, 2.59, 8.74-fold over the control, respectively. </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sakai et al., 2017 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. HAECs were exposed to 100uM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">. Intracellular ROS was measured by CM-H2DCFDA. DNA DSBs were detected by immunofluorescent analysis with γ-H2AX as a marker. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Intracellular ROS increased by ~3.7-fold </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">p-ATM increased by ~4.7-fold. γ-H2AX increased by ~3.4-fold. </span></span></p>
<p> </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Incidence Concordance </span></span></strong></p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Reference </span></span></strong></p>
</td>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Experiment Description </span></span></strong></p>
</td>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Result </span></span></strong></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Meng et al., 2021 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, human LECs exposed to 50 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> with DCFH-DA fluorescent probe to detect ROS levels and immunofluorescence and western blot assay to detect γ-H2AX. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">50 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub> </span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">exposure to lens epithelial cells increased oxidative stress, with ROS measured by LDH, by 4-fold and decreased the level of antioxidants by 2-fold as measured by SOD and GSH-PX. This resulted in 3-fold increase in γ-H2AX. </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Smith et al., 2015 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, human LECs exposed to 30 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> with alkaline comet assay to determine amount of strand breaks. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Treatment of lens epithelial cells to 30 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> induced DNA strand breaks by 55% at 0.5 hr after exposure and increased the level of LDH by ~1.4 fold at 24 hr post-exposure. </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Liu et al. 2013 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, human LECs exposed to 30 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> with alkaline comet assay determination of strand breaks. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">LDH increased by ~1.4 fold at 24 hr post-exposure, with a 5x increase from control levels in DNA strand breaks. </span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Time Concordance </span></span></strong></p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Reference </span></span></strong></p>
</td>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Experiment Description </span></span></strong></p>
</td>
<td>
<p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Result </span></span></strong></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Yang et al., 1998 </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, rabbit LECs exposed to </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> with TCA addition and thiol assay to determine non-protein thiol (NP-SH) level and alkaline elusion assay to determine strand breaks. </span></span></p>
</td>
<td>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"> In rabbit LECs exposed in vitro to 125 µM </span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">H<sub>2</sub>O<sub>2</sub></span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">, non-protein thiol levels decreased to <5% control (indicates oxidative stress) 30 min post-irradiation, and % DNA retained using alkaline elution decreased by 1.6 log (indicates increased DNA fragmentation) within the next 8.5 h. </span></span></p>
</td>
</tr>
</tbody>
</table>
LowUnspecificLowMaleLowAdultLowNot Otherwise SpecifiedLowLowLowLowLow<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">This KER is plausible in all life stages, sexes, and organisms with DNA. The evidence is from human, rodent, rabbit and bovine in vitro studies that do not specify the sex, as well as an adult rat in vivo study. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ahmadi, M. et al. (2021), “Early Responses to Low-Dose Ionizing Radiation in Cellular Lens Epithelial Models”, Radiation Research, Vol.197, Radiation Research Society, Indianapolis, https://doi.org/10.1667/RADE-20-00284.1. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Aitken, R.J. and C. Krausz. (2001), “Oxidative stress, DNA damage and the Y chromosome”, Reproduction, Vol.122/2001, Bioscientifica, Bristol, https://doi.org/10.1530/rep.0.1220497. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Annesley, S.J. and P.R. Fisher. (2019), “Mitochondria in Health and Disease”, Cells, Vol.8/7, MDPI, Basel, https://doi.org/10.3390/cells8070680. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Brennan, L., R. McGreal and M. Kantorow. (2012), “Oxidative stress defense and repair systems of the ocular lens”, Frontiers in Bioscience – Elite, Vol.4/E(1), Frontiers in Bioscience, Singapore, https://doi.org/10.2741/365. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Britton, S. et al. (2020), “ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks”, Nucleic Acids Research, Vol.48/17, Oxford University Press, Oxford, https://doi.org/10.1093/nar/gkaa723. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cabrera, M. and R. Chihuailaf. (2011), “Antioxidants and the integrity of ocular tissues”, Veterinary Medicine International, Vol.2011, Hindawi Limited, London, https://doi.org/10.4061/2011/905153. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cannan, W. and D. Pederson. (2016), “Mechanisms and consequences of double-strand DNA break formation in chromatin”, Journal of Cell Physiology, Vol.231/1, Wiley, Hoboken, https://doi.org/10.1002/jcp.25048. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cencer, C. et al. (2018), “PARP-1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light”, Photochemistry and Photobiology, Vol.94/1, Wiley-Blackwell, Hoboken, https://doi.org/10.1111/php.12814. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cervelli, T.et al. (2014), “Effects of single and fractionated low-dose irradiation on vascular endothelial cells”, Atherosclerosis, Vol.235/2, Elsevier, Amsterdam, https://doi.org/10.1016/j.atherosclerosis.2014.05.932. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cervelli, T. et al. (2017), “A New Natural Antioxidant Mixture Protects against Oxidative and DNA Damage in Endothelial Cell Exposed to Low-Dose Irradiation”, Oxidative medicine and cellular longevity, Vol. 2017, Hindawi, London, <a href="https://doi.org/10.1155/2017/9085947." rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2017/9085947.</a> </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Climent, M. et al. (2020), “MicroRNA and ROS Crosstalk in Cardiac and Pulmonary Diseases”, International Journal of Molecular Science, Vol.21/12, MDPI, Basel, https://doi.org/10.3390/ijms21124370. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dahm-Daphie, J., C. Sass, and W. Alberti. (2000), “Comparison of biological effects of DNA damage induced by ionizing radiation and hydrogen peroxide in CHO cells”, International Journal Radiation Biology, Vol.76/1, Informa, London, https://doi.org/10.1080/095530000139023. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">El-Missiry, M. A. et al. (2018), "Neuroprotective effect of epigallocatechin-3-gallate (EGCG) on radiation-induced damage and apoptosis in the rat hippocampus", International Journal of Radiation Biology, Vol. 94/9, https://doi.org/10.1080/09553002.2018.1492755. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Engwa, G.A., F.N. Nweke and B.N. Nkeh-Chungag. (2020), “Free Radicals, Oxidative Stress-Related Diseases and Antioxidant Supplementation”, Alternative therapies in health and medicine, Vol.28/1, InnoVision Health Media, Eagan, pp.114-128. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Feng, C. et al. (2016), “Lycopene protects human SH-SY5Y neuroblastoma cells against hydrogen peroxide-induced death via inhibition of oxidative stress and mitochondria-associated apoptotic pathways”, Molecular Medicine Reports, Vol.13/5, Spandidos Publications, Athens, https://doi.org/10.3892/mmr.2016.5056. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Fong, C.W. (2016), “Platinum anti-cancer drugs: Free radical mechanism of Pt-DNA adduct formation and anti-neoplastic effect”, Free Radical Biology and Medicine, Vol.95/June 2016, Elsevier, Amsterdam, https://doi.org/10.1016/j.freeradbiomed.2016.03.006. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Halliwell, B. et al. (2021), “Hydroxyl radical is a significant player in oxidative DNA damage in vivo”, Chemical Society Reviews, Vol.50, Royal Society of Chemistry, London, https://doi.org/10.1039/d1cs00044f. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Huang, Y. et al. (2021), "Mesenchymal Stem Cell-Conditioned Medium Protects Hippocampal Neurons From Radiation Damage by Suppressing Oxidative Stress and Apoptosis", Dose-Response, Vol. 19/1, https://doi.org/10.1177/1559325820984944. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kay, J. et al. (2019), “Inflammation-induced DNA damage, mutations and cancer”, DNA Repair, Vol.83, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.dnarep.2019.102673.%E2%80%AF" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.dnarep.2019.102673. </a> </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Jeggo, P.A., V. Geuting and M. Löbrich. (2011), “The role of homologous recombination in radiation-induced double-strand break repair”, Radiotherapy and Oncology, Vol.101/1, Elsevier, Amsterdam, https://doi.org/10.1016/j.radonc.2011.06.019. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kurutas E. B. (2016), “The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state”, Nutrition journal, Vol.15/1, Biomed Central, London, https://doi.org/10.1186/s12937-016-0186-5. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kruk, J., K. Kubasik-Kladna and H. Aboul-Enein. (2016), “The Role Oxidative Stress in the Pathogenesis of Eye Diseases: Current Status and a Dual Role of Physical Activity”, Mini-Review in Medicinal Chemistry, Vol.16/3, Bentham Science Publishers, Sharjah, https://doi.org/10.2174/1389557516666151120114605. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Li, Y. et al. (1998), “Response of lens epithelial cells to hydrogen peroxide stress and the protective effect of caloric restriction”, Experimental Cell Research, Vol.239/2, Elsevier, Amsterdam, https://doi.org/10.1006/excr.1997.3870. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Liu, H. et al. (2013), “Sulforaphane can protect lens cells against oxidative stress: Implications for cataract prevention”, Investigative Ophthalmology and Visual Science, Vol.54/8, Association for Research in Vision and Ophthalmology, Rockville, https://doi.org/10.1167/iovs.13-11664. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Meng, K. and C. Fang. (2021), “Knockdown of Tripartite motif-containing 22 (TRIM22) relieved the apoptosis of lens epithelial cells by suppressing the expression of TNF receptor-associated factor 6 (TRAF6)”, Bioengineered, Vol.12/1, Taylor & Francis, Oxfordshire, https://doi.org/10.1080/21655979.2021.1980645. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Nishida, M. et al. (2005), “Ga12/13- and Reactive Oxygen Species-dependent Activation of c-Jun NH2-terminal Kinase and p38 Mitogen-activated Protein Kinase by Angiotensin Receptor Stimulation in Rat Neonatal Cardiomyocytes”, Journal of Biological Chemistry, Vol.280/18, American Society for Biochemistry and Molecular Biology, Rockville, https://doi.org/10.1074/jbc.M409710200. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Poetsch, A.R. (2020), “The genomics of oxidative DNA damage, repair, and resulting mutagenesis”, Computational and Structural Biotechnology Journal, Vol.18, Elsevier, Amsterdam, https://doi.org/10.1016/j.csbj.2019.12.013. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Quinlan, R.A., and P.J. Hogg. (2018), “γ-Crystallin redox–detox in the lens”, Journal of Biological Chemistry, Vol.293/46, American Society for Biochemistry and Molecular Biology, Rockville, https://doi.org/10.1074/jbc.H118.006240. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sakai, C. et al. (2017), “Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA damage in vascular endothelial cells”, PloS one, Vol.12/11, https://doi.org/10.1371/journal.pone.0187934. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Scully, R. and A. Xie. (2013), “Double strand break repair functions of histone H2AX”, Mutation Research, Vol.750/1-2, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.mrfmmm.2013.07.007.%E2%80%AF" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.mrfmmm.2013.07.007. </a> </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Smith, A. et al. (2015), “Ku80 counters oxidative stress-induced DNA damage and cataract formation in the human lens”, Investigative Ophthalmology and Visual Science, Vol.56/13, Association for Research in Vision and Ophthalmology, Rockville, https://doi.org/10.1167/iovs.15-18309. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Spector, A. et al. (1997), “Microperoxidases catalytically degrade reactive oxygen species and may be anti-cataract agents”, Experimental Eye Research, Vol.65/4, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1997.0336. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Spector, A. et al. (1996), “Variation in cellular glutathione peroxidase activity in lens epithelial cells, transgenics and knockouts does not significantly change the response to H2O2 stress”, Experimental Eye Research, Vol.62/5, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1996.0063. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Spector, A. (1995), “Oxidative stress‐induced cataract: mechanism of action”, The FASEB Journal, Vol.9/12, Federation of American Societies for Experimental Biology, Bethesda, https://doi.org/10.1096/fasebj.9.12.7672510. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Spector, A. (1990), “Oxidation and Aspects of Ocular Pathology”, CLAO Journal, Vol.16/1, Lippincott, Williams and Wilkins Ltd, Philadelphia, S8-S10. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Stohs, S. (1995), “The role of free radicals in toxicity and disease”, Journal of Basic Clinical Physiology and Pharmacology, Vol.6/3-4, Walter de Gruyter GmbH, Berlin, https://doi.org/10.1515/jbcpp.1995.6.3-4.205. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sweeney, M.H.J. and R.J.W. Truscott. (1998), “An Impediment to Glutathione Diffusion in Older Normal Human Lenses: a Possible Precondition for Nuclear Cataract”, Experimental Eye Research, Vol.67, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1998.0549. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Taylor, A. and K. J. A. Davies (1987), “Protein oxidation and loss of protease activity may lead to cataract formation in the aged lens”, Free Radical Biology & Medicine, Vol. 3, Pergamon Journals Ltd, United States of America, pp. 371-377 </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ungvari, Z. et al. (2013), "Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosens", The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, Vol. 68/12, https://doi.org/10.1093/gerona/glt057. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Uwineza, A. et al. (2019), “Cataractogenic load – A concept to study the contribution of ionizing radiation to accelerated aging in the eye lens”, Mutation Research - Reviews in Mutation Research, Vol.779, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.mrrev.2019.02.004.%E2%80%AF" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.mrrev.2019.02.004. </a> </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ward, J.F., W.F. Blakely & E.I. Joner. (1985), “Mammalian Cells Are Not Killed by DNA Single-Strand Breaks Caused by Hydroxyl Radicals from Hydrogen Peroxide”, Radiation Research, Vol.103/3, Radiation Research Society, Indianapolis, hptts://doi.org/10.2307/3576760. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Wu, H. et al. (2021), “Lactate dehydrogenases amplify reactive oxygen species in cancer cells in response to oxidative stimuli”, Signal Transduction and Targeted Therapy, Vol.6/1, Nature Portfolio, Berlin, https://doi.org/10.1038/s41392-021-00595-3. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Yang, Y. et al. (1998), “The effect of catalase amplification on immortal lens epithelial cell lines”, Experimental Eye Research, Vol.67/6, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1998.0560. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Yuan, J., R. Adamski and J. Chen. (2010), “Focus on histone variant H2AX: To be or not to be”, FEBS Letters, Vol.584/17, Wiley, Hoboken, https://doi.org/10.1016/j.febslet.2010.05.021. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Zhang, L. et al. (2017), "The inhibitory effect of minocycline on radiation-induced neuronal apoptosis via AMPKα1 signaling-mediated autophagy", Scientific Reports, Vol. 7/1, https://doi.org/10.1038/s41598-017-16693-8. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Zhou, Y. et al. (2016), “Protective Effect of Rutin Against H2O2-Induced Oxidative Stress and Apoptosis in Human Lens Epithelial Cells”, Current Eye Research, Vol.41/7, Taylor & Francis, Oxfordshire, <a href="https://doi.org/10.3109/02713683.2015.1082186.%E2%80%AF" rel="noreferrer noopener" target="_blank">https://doi.org/10.3109/02713683.2015.1082186. </a> </span></span></p>
<p> </p>
2022-12-16T16:30:262024-03-08T14:44:49f703f4b6-e87b-4be7-8ffa-ff2f1a670694908b38ae-23f7-446a-bb32-46656376b24b<p>DNA strand breaks can lead to altered signaling of various pathways through the DNA damage response. DNA strand breaks, which are a form of DNA damage, can induce ataxia telangiectasia mutated (ATM) and ATM/RAD3-related (ATR), two phosphoinositide 3-kinase (PI3K)-related serine/threonine kinases (PIKKs) (Abner and McKinnon, 2004; Lee and McKinnon, 2007; Nagane et al., 2021; Sylvester et al., 2018; Thadathil et al., 2019; Wang et al., 2020; Wang et al., 2017). Following DNA strand breaks, DNA damage response cellular signaling can phosphorylate downstream proteins and activate several transcription factors and pathways (Wang et al., 2017). Spontaneous DNA strand breaks from endogenous sources will induce signaling as a normal response to facilitate DNA repair. However, excessive DNA damage induced by a stressor will result in increased activation of these pathways and subsequent harmful downstream effects. Signaling pathways induced by DNA strand breaks include p53/p21 (Abner and McKinnon, 2004; Baselet et al., 2018; Lee and McKinnon, 2007; Nagane et al., 2021; Sylvester et al., 2018; Thadathil et al., 2019; Wang et al., 2020; Wang et al., 2017), caspase (Abner and McKinnon, 2004; Baselet et al., 2019; Wang et al., 2020; Wang et al., 2016) and mitogen-activated protein kinase (MAPK) family pathways (Ghahremani et al., 2002; Nagane et al., 2021). </p>
<p>Overall weight of evidence: Moderate</p>
<p>There is strong evidence supporting the link between DNA strand breaks leading to altered signaling pathways. Single strand breaks (SSBs) or double strand breaks (DSBs) in DNA from both endogenous and exogenous sources can induce the DNA damage response, which can result in the induction of various signaling pathways (Baselet et al., 2019). DNA strand breaks are well known to lead to the activation of ATM and ATR as part of the normal DNA damage response (Abner and McKinnon, 2004; Baselet et al., 2019; Lee and McKinnon, 2007; Nagane et al., 2021; Sylvester et al., 2018; Thadathil et al., 2019; Wang et al., 2020; Wang et al., 2017; Wang et al., 2016). While ATM tends to be recruited to DSBs, ATR is recruited by many types of DNA damage including both DSBs and SSBs (Maréchal and Zou, 2013; Wang et al., 2017). Following a DNA DSB, the Mre11-Rad50-Nbs1 (MRN) complex senses and directly binds to the DNA ends at the site of the break, which subsequently activates ATM (Lee and McKinnon, 2007; Maréchal and Zou, 2013). Following a DNA SSB, resection of the damaged strand by apurinic/apyrimidinic endonuclease (APE)1/APE2 is followed by coating the single-stranded DNA with replication protein A (RPA), where the recruitment of the ATR-ATR interacting protein (ATRIP) complex and the activation of ATR occurs (Caldecott, 2022; Maréchal and Zou, 2013). </p>
<p>ATM and ATR can phosphorylate over 700 proteins (Nagane et al., 2021), and phosphorylation of key signaling proteins by ATM/ATR will alter signaling in their respective pathways. High levels of DNA strand breaks induced by exogenous stressors will enhance ATM/ATR activation and subsequently further activate downstream signaling, leading to downstream consequences. The extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK subfamily pathways can be phosphorylated and activated by ATM/ATR (Ghahremani et al., 2002; Nagane et al., 2021). Additionally, ATM/ATR can phosphorylate p53 on serine 15 to enhance the stability of p53, leading to activation of the p53 pathway and changes in the transcriptional activity of p53 (Abner and McKinnon, 2004; Baselet et al., 2019; Lee and McKinnon, 2007; Nagane et al., 2021; Sylvester et al., 2018; Thadathil et al., 2019; Wang et al., 2020; Wang et al., 2017; Wang et al., 2016). The apoptosis pathway downstream of p53 can also be activated by DNA strand breaks (Abner and McKinnon, 2004; Baselet et al., 2019; Lee and McKinnon, 2007; Thadathil et al., 2019; Wang et al., 2020). </p>
<p>Evidence for this relationship was collected from studies using in vivo mouse and rat models as well as in vitro mouse-derived, rat-derived and human-derived cell models. The stressors used to support this relationship include <sup>137</sup>Cs gamma rays and X rays. Markers of DNA strand breaks in this KER include p53 binding protein 1 (53BP1), phosphorylation of H2AX (γ-H2AX), phosphorylation of ATR (p-ATR) and phosphorylation of ATM (p-ATM). Altered signaling was measured mostly by the protein expression of the p53/p21 and apoptosis pathways. </p>
<p> </p>
<p><strong>Dose Concordance </strong></p>
<p>A few studies have indicated a dose concordance between the increase in DNA strand breaks and altered signaling pathways. X-ray irradiation of rat cortical neurons showed increased DNA damage markers, γ-H2AX, p-ATM and p-ATR and increased levels of signaling proteins, including p21, p-p53 and cleaved caspase 3 at both doses of 8 and 32 Gy (Sabirzhanov et al., 2020). <sup>137</sup>Cs gamma irradiated cerebromicrovascular endothelial cells (CMVECs) and rat hippocampal neurons showed increased DNA strand breaks, measured by comet assay, at 2-10 Gy, and increased caspase 3/7 activity at 2, 4 and 6 Gy (Ungvari et al., 2013). </p>
<p> </p>
<p><strong>Time Concordance </strong></p>
<p>Many studies demonstrate that DNA strand breaks occur before altered signaling in a time course. Although both KEs can occur quickly, Gionchiglia et al. (2021) showed in mice that γ-H2AX and p53BP1 foci were increased as early as 15 minutes after 10 Gy of X-ray irradiation while cleaved caspase 3 did not increase until 30 minutes after irradiation. In HT22 hippocampal neurons irradiated with 12 Gy of X-rays, γ-H2AX and p-ATM were increased at 30 minutes post-irradiation while p53 was increased after 1 h and caspase 3 was increased after 48 h (Zhang et al., 2017). Similarly, rat cortical neurons irradiated with 8 Gy of X-rays showed increased p-ATM, γ-H2AX and p-ATR after 30 minutes, while p-p53, p21 and cleaved caspase 3 did not increase until 3 or 6 h post-irradiation (Sabirzhanov et al., 2020). Multiple studies using human- and rat-derived endothelial cells irradiated with 4 Gy of <sup>137</sup>Cs gamma rays show increased DNA strand breaks at 1 h post-irradiation, with altered signaling to p53 and p21 at 6 h and to caspase 3/7 at 18 h post-irradiation (Kim et al., 2014; Park et al., 2022; Ungvari et al., 2013). In a longer-term study irradiating human lung microvascular endothelial cells (HMVEC-L) with 15 Gy of X-rays, increased DNA strand breaks were observed at 14 days post-irradiation, while altered signaling in the p53 pathway was observed at 21 days post-irradiation (Lafargue et al., 2017). </p>
<p> </p>
<p><strong>Incidence concordance </strong></p>
<p>A few studies have demonstrated an incidence concordance between DNA strand breaks and altered signaling at equivalent doses. Following X-ray irradiation of mice, DNA damage markers, γ-H2AX and p53BP1, increased by 10, 15 and 5-fold in different region of the brain, while cleaved caspase 3 signaling molecule increased by 1.4 and 2.6-fold (Gionchiglia et al., 2021). Gamma ray irradiation of Wistar rats showed a 6-fold increase in DNA damage marker compared to a 0.2-fold decrease in (B-cell lymphoma 2) Bcl-2 and a 2- to 4-fold increase in signaling proteins p53, Bcl-2-associated protein X (Bax) and caspase 3/8/9 (El-Missiry et al., 2018). </p>
<p> </p>
<p><strong>Essentiality </strong></p>
<p>Some studies show that preventing an increase in DNA strand breaks will restore signaling. Treatment with mesenchymal stem cell-conditioned medium (MSC-CM) reduced γ-H2AX, decreased the levels of p53, Bax, cleaved caspase 3 and increased the levels of Bcl-2 in HT22 cells irradiated with 10 Gy of X-rays (Huang et al., 2021). The inhibition of microRNA (miR)-711 decreased levels of DNA damage markers, p-ATM, p-ATR and γ-H2AX, and decreased signaling molecules including p-p53, p21 and cleaved caspase 3 (Sabirzhanov et al., 2020).</p>
<p>None identified</p>
<p>The tables below provide some representative examples of quantitative linkages between the two key events. All data that is represented is statistically significant unless otherwise indicated.</p>
ModerateMaleLowFemaleLowJuvenileModerateAdultLowModerateModerate<p>Evidence for this relationship is predominantly from studies using rat- and mouse-derived cells, with some in vivo evidence in mice and rats. There is in vivo evidence in male animals, but no in vivo studies specify the use of female animals. In vivo evidence is from adult models.</p>
<p>Abner, C. W. and P. J. McKinnon. (2004), "The DNA double-strand break response in the nervous system", <em>DNA Repair</em>, Vol. 3/8–9, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.dnarep.2004.03.009" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.dnarep.2004.03.009</a>. </p>
<p>Baselet, B. et al. (2019), "Pathological effects of ionizing radiation: endothelial activation and dysfunction", <em>Cellular and Molecular Life Sciences</em>, Vol. 76/4, Springer Nature, <a href="https://doi.org/10.1007/s00018-018-2956-z" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00018-018-2956-z</a>. </p>
<p>Caldecott, K. W. (2022), “DNA single-strand break repair and human genetic disease”, <em>Trends in Cell Biology</em>, 32(9), Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.tcb.2022.04.010" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.tcb.2022.04.010</a> </p>
<p>El-Missiry, M. A. et al. (2018), "Neuroprotective effect of epigallocatechin-3-gallate (EGCG) on radiation-induced damage and apoptosis in the rat hippocampus", <em>International Journal of Radiation Biology</em>, Vol. 94/9, Informa, London, <a href="https://doi.org/10.1080/09553002.2018.1492755" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2018.1492755</a>. </p>
<p>Ghahremani, H. et al. (2002), “Interaction of the c-Jun/JNK Pathway and Cyclin-dependent Kinases in Death of Embryonic Cortical Neurons Evoked by DNA Damage”, <em>Journal of Biological Chemistry</em>, Vol. 277/38, Elsevier, Amsterdam, https://doi.org/10.1074/jbc.M204362200 </p>
<p>Gionchiglia, N. et al. (2021), "Association of Caspase 3 Activation and H2AX γ Phosphorylation in the Aging Brain: Studies on Untreated and Irradiated Mice", <em>Biomedicines</em>, Vol. 9/9, MDPI, Basel, <a href="https://doi.org/10.3390/biomedicines9091166" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/biomedicines9091166</a>. </p>
<p>Huang, Y. et al. (2021), "Mesenchymal Stem Cell-Conditioned Medium Protects Hippocampal Neurons From Radiation Damage by Suppressing Oxidative Stress and Apoptosis", <em>Dose-Response</em>, Vol. 19/1, SAGE publications, <a href="https://doi.org/10.1177/1559325820984944" rel="noreferrer noopener" target="_blank">https://doi.org/10.1177/1559325820984944</a>. </p>
<p>Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. <a href="http://doi.org/10.1080/09553002.2022.2110306">https://doi.org/10.1080/09553002.2022.2110306</a></p>
<p>Kim, K. S. et al. (2014), "Characterization of DNA damage-induced cellular senescence by ionizing radiation in endothelial cells", <em>International Journal of Radiation</em> Biology, Vol. 90/1, Informa, London, <a href="https://doi.org/10.3109/09553002.2014.859763" rel="noreferrer noopener" target="_blank">https://doi.org/10.3109/09553002.2014.859763</a>. </p>
<p>Lafargue, A. et al. (2017), "Ionizing radiation induces long-term senescence in endothelial cells through mitochondrial respiratory complex II dysfunction and superoxide generation", <em>Free Radical Biology and Medicine</em>, Vol. 108, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.freeradbiomed.2017.04.019" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.freeradbiomed.2017.04.019</a>. </p>
<p>Lee, Y. and P. J. McKinnon. (2007), "Responding to DNA double strand breaks in the nervous system", <em>Neuroscience</em>, Vol. 145/4, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.neuroscience.2006.07.026" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.neuroscience.2006.07.026</a>. </p>
<p>Maréchal, A. and L. Zou. “DNA damage sensing by the ATM and ATR kinases”, <em>Cold Spring Harbor Perspectives in Biology</em>, 5(9), Cold Spring Harbor Laboratory Press, <a href="https://doi.org/10.1101/cshperspect.a012716" rel="noreferrer noopener" target="_blank">https://doi.org/10.1101/cshperspect.a012716</a> </p>
<p>Nagane, M. et al. (2021), "DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases", <em>Journal of Radiation Researc</em>h, Vol. 62/4, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jrr/rrab032" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jrr/rrab032</a>. </p>
<p>Park, J.-W. et al. (2022), "Metformin alleviates ionizing radiation-induced senescence by restoring BARD1-mediated DNA repair in human aortic endothelial cells", <em>Experimental Gerontology</em>, Vol. 160, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.exger.2022.111706" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.exger.2022.111706</a>. </p>
<p>Sabirzhanov, B. et al. (2020), "Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways", <em>International Journal of Molecular Sciences</em>, Vol. 21/15, MDPI, Basel, <a href="https://doi.org/10.3390/ijms21155239" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/ijms21155239</a>. </p>
<p>Sylvester, C. B. et al. (2018), "Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer", <em>Frontiers in Cardiovascular Medicine</em>, Vol. 5, Fronteirs, <a href="https://doi.org/10.3389/fcvm.2018.00005" rel="noreferrer noopener" target="_blank">https://doi.org/10.3389/fcvm.2018.00005</a>. </p>
<p>Thadathil, N. et al. (2019), "DNA double-strand breaks: a potential therapeutic target for neurodegenerative diseases", <em>Chromosome Research</em>, Vol. 27/4, Springer Nature, <a href="https://doi.org/10.1007/s10577-019-09617-x" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s10577-019-09617-x</a>. </p>
<p>Ungvari, Z. et al. (2013), "Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity", <em>The journals of gerontology. Series A, Biological sciences and medical sciences</em>, Vol. 68/12, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/gerona/glt057" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/gerona/glt057</a>. </p>
<p>Wang, Q. et al. (2020), "Radioprotective Effect of Flavonoids on Ionizing Radiation-Induced Brain Damage", <em>Molecules</em>, Vol. 25/23, MDPI, Basel, <a href="https://doi.org/10.3390/molecules25235719" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/molecules25235719</a>. </p>
<p>Wang, H. et al. (2017), "Chronic oxidative damage together with genome repair deficiency in the neurons is a double whammy for neurodegeneration: Is damage response signaling a potential therapeutic target?", <em>Mechanisms of Ageing and Development</em>, Vol. 161, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.mad.2016.09.005" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.mad.2016.09.005</a>. </p>
<p>Wang, Y., M. Boerma and D. Zhou. (2016), "Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases", <em>Radiation Research</em>, Vol. 186/2, BioOne, <a href="https://doi.org/10.1667/RR14445.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RR14445.1</a>. </p>
<p>Zhang, L. et al. (2017), "The inhibitory effect of minocycline on radiation-induced neuronal apoptosis via AMPKα1 signaling-mediated autophagy", <em>Scientific Reports</em>, Vol. 7/1, Springer Nature, <a href="https://doi.org/10.1038/s41598-017-16693-8" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41598-017-16693-8</a>. </p>
2023-03-10T15:07:202023-03-21T13:09:3481eb87ac-fb24-4667-bd3a-9be52450f362908b38ae-23f7-446a-bb32-46656376b24b<p><span style="font-family:Times New Roman,Times,serif">Oxidative stress occurs when the production of free radicals exceeds the capacity of cellular antioxidant defenses (Cabrera & Chihuailaf, 2011). Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are both free radicals that can contribute to oxidative stress (Ping et al., 2020); however, ROS are more commonly studied than RNS (Nagane et al., 2021). ROS can mediate oxidative damage to biomacromolecules as they react with DNA, proteins and lipids, resulting in functional changes to these molecules (Ping et al., 2020). For example, ROS acting on lipids creates lipid peroxidation (Cabrera & Chihuailaf, 2011). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Many signaling pathways control and maintain physiological balance within a living organism, and these can be impacted by oxidative stress. Excessive reactive oxygen and nitrogen species (RONS) during oxidative stress can modify biological molecules and directly cause DNA damage, which can lead to altered signal transduction pathways (Hughson, Helm & Durante, 2018; Lehtinen & Bonni, 2006; Nagane et al., 2021; Ping et al., 2020; Ramadan et al., 2021; Schmidt-Ullrich et al., 2000; Soloviev & Kizub, 2019; Wang, Boerma & Zhou, 2016; Venkatesulu et al., 2018; Zhang et al., 2016). Different cell types can express distinct cellular pathways that can have varied response to an increase in oxidative stress. For example, oxidative stress in endothelial cells has been shown to inhibit the insulin-like growth factor 1 receptor (IGF-1R) and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway and to activate the mitogen-activated protein kinase (MAPK) pathway, which can then have downstream detrimental effects (Ping et al., 2020). The MAPK family pathway is also activated in the central nervous system (CNS) in response to oxidative stress through calcium-induced phosphorylation of several kinases. These include phosphoinositide 3-kinase (PI3K), protein kinase A (PKA) and protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII) (Lehtinen & Bonni, 2006; Li et al., 2013; Ramalingam & Kim, 2012). Oxidative stress in bone cells can lead to increased expression of the receptor activator of nuclear factor kappa B ligand (RANKL) and Nrf2 activation (Tahimic & Globus, 2017; Tian et al., 2017). Following activation, Nrf2 then interferes with the activation of runt-related transcription factor 2 (Runx2), and depending on the level of oxidative stress, this may result in altered bone cell function (Kook et al., 2015).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: High</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Many reviews describe the role of oxidative stress in altered signaling. The mechanisms through which oxidative stress can contribute to changes in various signaling pathways are well-described. For example, oxidative stress can directly alter signaling pathways through protein oxidation (Ping et al., 2020; Schmidt-Ullrich et al., 2000; Valerie et al., 2007). Oxidation of cysteine and methionine residues, which are particularly sensitive to oxidation, can cause conformational change, protein expansion, and degradation, leading to changes in the protein levels of signaling pathways (Ping et al., 2020). Furthermore, oxidation of key residues in signaling proteins can alter their function, resulting in altered signaling. For example, oxidation of methionine 281 and 282 in the Ca<sup>2+</sup>/calmodulin binding domain of Ca<sup>2+</sup>/calmodulin-dependent protein kinase II (CaMKII) leads to constitutive activation of its kinase activity and subsequent downstream alterations in signaling pathways (Li et al., 2013; Ping et al., 2020). Similarly, during oxidative stress, tyrosine phosphatases can be inhibited by oxidation of a catalytic cysteine residue, resulting in increased phosphorylation of proteins in various signaling pathways (Schmidt-Ullrich et al., 2000; Valerie et al., 2007). Particularly relevant to this are the MAPK pathways. The extracellular signal-regulated kinase (ERK) pathway is activated by upstream tyrosine kinases and relies on tyrosine phosphatases for deactivation (Lehtinen & Bonni, 2006; Valerie et al., 2007). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Furthermore, oxidative stress can indirectly influence signaling pathways through oxidative DNA damage which can lead to mutations or changes in the gene expression of proteins in signaling pathways (Ping et al., 2020; Schmidt-Ullrich et al., 2000; Valerie et al., 2007). DNA damage surveillance proteins like ataxia telangiectasia mutated (ATM) kinase and ATM/Rad3-related (ATR) protein kinase phosphorylate over 700 proteins, leading to changes in downstream signaling (Nagane et al., 2021; Schmidt-Ullrich et al., 2000; Valerie et al., 2007). For example, ATM, activated by oxidative DNA damage, phosphorylates many proteins in the ERK, p38, and Jun N-terminal kinase (JNK) MAPK pathways, leading to various downstream effects (Nagane et al., 2021; Schmidt-Ullrich et al., 2000). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">The response of oxidative stress on signaling pathways has been studied extensively in various diseases. Herein presented are examples relevant to a few cell types related to vascular disease, impaired learning and memory, and bone loss. Many other pathways are plausible but available research has highlighted these to be critical to disease. </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Endothelial cells: . Antioxidant enzymes and the glutathione redox buffer control the redox state of vascular tissues. However, the dysregulation of signaling pathways can occur in the endothelium when oxidative stress is favored (Soloviev & Kizub, 2019). Oxidative stress can activate the acidic sphingomyelinase (ASMase)/ceramide pathway, the MAPK pathways, the p53/p21 pathway, and the signaling proteins p16 and p21, as well as inhibit the PI3K/Akt pathway (Hughson, Helm & Durante, 2018; Nagane et al., 2021; Ping et al., 2020; Ramadan et al., 2021; Soloviev & Kizub, 2019; Wang, Boerma & Zhou, 2016). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Bone cells: Oxidative stress can induce signaling changes in the Wnt/β-catenin pathway, the RANK/RANKL pathway, the Nrf2/HO-1 pathway, and the MAPK pathways (Domazetovic et al., 2017; Manolagas & Almeida, 2007; Tian et al., 2017). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Brain cells: Oxidative stress can induce alterations to various pathways such as the PI3K/Akt pathway, cAMP response element-binding protein (CREB) pathway, the p53/p21 pathway, as well as the MAPK family pathways, including JNK, ERK and p38 (Lehtinen & Bonni, 2006; Ramalingam & Kim, 2012). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Additionally, the electron transport chain in the mitochondria is an important source of ROS, which can damage mitochondria by inducing mutations in mitochondrial DNA. These mutations lead to mitochondrial dysfunction due to alterations in cellular respiration mechanisms that perpetuates oxidative stress and can then induce the release of signaling molecules related to apoptosis from the mitochondria. Pro-apoptotic markers (Bax, Bak and Bad) and anti-apoptotic markers (Bcl-2 and Bcl-xL) can regulate the caspase pathway that ultimately mediate apoptosis (Annunziato et al., 2003; Wang & Michaelis, 2010; Wu et al., 2019). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">The mechanisms of oxidative stress leading to altered signaling may be different for each pathway. For example, although both the PI3K/Akt and MAPK pathways can be regulated by insulin-like growth factor (IGF)-1, ROS results in selective inhibition of the IGF-1R/PI3K/Akt pathway by inhibiting the IGF-1 receptor (IGF-1R) activation of IRS1 (Ping et al., 2020). Additionally, ROS-induced MAPK activation can occur through Ras-dependent signaling. Firstly, oxygen radicals mediate the phosphorylation of upstream epidermal growth factor receptors (EGFRs) on tyrosine residues, resulting in increased binding of growth factor receptor-bound protein 2 (Grb2) and subsequent activation of Ras signaling (Lehtinen & Bonni, 2006). Direct inhibition of MAPK phosphatases with hydroxyl radicals also activates this pathway (Li et al., 2013). In another mechanism, ROS competitively inhibit the Wnt/β-catenin pathway through the activation of forkhead box O (FoxO), which are involved in the antioxidant response and require binding of β-catenin for transcriptional activity (Tian et al., 2017).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Evidence for this relationship was collected from studies using <em>in vivo</em> mouse, rat, and pig models, as well as <em>in vitro</em> mouse-derived, rat-derived, bovine-derived, and human-derived models. The stressors used to support this relationship include gamma rays, X rays, microgravity, hydrogen peroxide, chronic cold stress, heavy ion radiation, simulated ischemic stroke and growth differentiation factor (GDF) 15 overexpression. These stressors were shown to increase levels of oxidative stress and induce changes within relevant signaling pathways (Azimzadeh et al., 2021; Azimzadeh et al., 2015; Fan et al., 2017; Xu et al., 2019; Suman et al., 2013; Limoli et al., 2004; Tian et al., 2020; Hladik et al., 2020; Diao et al., 2018; Hasan, Radwan & Galal, 2019; Xin et al., 2015; El-Missiry et al., 2018; Kenchegowda et al., 2018; Kook et al., 2015; Sun et al., 2013; Yoo, Han & Kim, 2016, Zhao et al., 2013; Bai et al., 2020; Chen et al., 2009; Carvour et al., 2008; Wortel et al., 2019; Azimzadeh et al., 2017; Park et al., 2016; Sakata et al., 2015; Ruffels et al., 2004; Crossthwaite et al., 2002). </span></p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Incidence concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">A few studies demonstrate greater changes to oxidative stress than to altered signaling. Human umbilical vein endothelial cells (HUVECs) irradiated with 10 Gy of X-rays showed a 20-fold increase in ROS and a 0.5-fold decrease in the ratio of p-Akt/Akt (Sakata et al., 2015). Microgravity exposure to preosteoblast cells showed a 0.24-fold decrease to the antioxidant Cu/Zn-superoxide dismutase (SOD) and a 0.36-fold decrease to p-Akt (Yoo, Han & Kim, 2016). It was also shown in rats that MDA levels increased by 1.5-fold while angiotensin and aldosterone increased by 1.4-fold after 6 Gy of gamma rays (Hasan, Radwan & Galal, 2020). Bai et al. (2020) demonstrated with multiple endpoints that ROS levels increased, and antioxidant enzyme levels decreased more than signaling pathways were altered. </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Dose Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Many studies demonstrate dose concordance for this relationship, at the same doses. Low-dose (0.5 Gy) X-ray irradiation of human coronary artery endothelial cells (HCAECs) show increased protein carbonylation with decreased glutathione S-transferase omega-1 (GSTO1) antioxidant levels and a simultaneous alteration of signaling proteins Rho GDP-dissociation inhibitor (RhoGDI), p16, and p21 (Azimzadeh et al., 2017). A dose of about 2 Gy of gamma rays showed decreased antioxidants as well as decreased protein levels and activation of the PI3K/Akt pathway in pig cardiac tissue (Kenchegowda et al., 2018). Similarly, gamma irradiation at 6 Gy resulted in reduced levels of the antioxidant glutathione (GSH) and increased levels of the lipid peroxidation marker MDA as well as an increase in the renin angiotensin aldosterone system (RAAS) measured in rat heart tissue and blood serum, respectively (Hasan, Radwan & Galal, 2020). HUVECs irradiated with 10 Gy of X-rays demonstrated increased ROS while p-Akt decreased and p-ERK1/2 increased (Sakata et al., 2015). Gamma radiation at 15 Gy led to both increased ROS as well as attenuated p38 MAPK and Nrf2 signaling pathways in murine cardiac tissue (Fan et al., 2017). In contrast, 16 Gy X-ray exposure led to decreased levels of the antioxidant SOD, increased MDA as well as increased MAPK signaling in murine heart tissue (Azimzadeh et al., 2021). After simulated microgravity, changes to signaling pathways, increased ROS and MDA, and decreased antioxidants were found both in <em>in vitro</em> mouse-derived bone cells and in <em>in vivo</em> rat femurs. Increased ROS levels and decreased antioxidants were found with changes in the RANK/RANKL pathway, Wnt/β-catenin pathway, Runx2, PI3K/Akt pathway, and MAPK pathways (Diao et al., 2018; Sun et al., 2013; Xin et al., 2015; Yoo, Han & Kim, 2016). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">A few studies also find that oxidative stress often occurs at lower doses than altered signaling pathways. Bai et al. (2020) measured oxidative stress, shown by increased ROS and decreased antioxidant expression, at 2, 5, and 10 Gy of gamma rays. The authors also found Runx2 increased at the same doses, but the p53/p21 pathway was only significantly altered at 5 and 10 Gy (Bai et al., 2020). At similar doses, X-ray irradiated mouse osteoblast-like cell line MC3T3-E1 cells showed increased ROS and decreased antioxidants both 4 and 8 Gy (Kook et al., 2015). While HO-1 also increased at both 4 and 8 Gy, Nrf2 and Runx2 were measured altered at 8 Gy (Kook et al., 2015). In another study, X-ray irradiation at 16 Gy resulted in decreased SOD and increased MDA and protein carbonylation, which were associated with decreased PI3K/Akt pathway activity and protein levels, decreased ERK activity and protein levels, increased p38 activity, and increased p16 and p21 protein levels in heart tissue (Azimzadeh et al., 2015). Azimzadeh et al. (2015) also showed that at 8 Gy oxidative stress was still observed, but fewer signaling molecule levels and activity were altered at this. Particularly, no changes to MAPK pathways were observed. </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Within the rat hippocampus, El-Missiry et al. (2018) demonstrated that exposure to 4 Gy of X-irradiation results in increased 4-HNE (oxidative stress marker) levels, reduced antioxidant activity and an increase in p53 expression. In the cerebral cortex of mice, Suman et al. (2013) reported that 1.6 Gy of <sup>56</sup>Fe and 2 Gy of gamma rays increased ROS levels, consequently increased p21 and p53 levels. Limoli et al. (2004) also reported increased ROS levels in mice and rat neural precursor cells after exposure to X-irradiation (1-10 Gy), accompanied by increased expression of p21 and p53. Hladik et al. (2020) exposed female mice to 0.063, 0.125 or 0.5 Gy of gamma-radiation, which resulted in increases of protein carbonylation, as well as increased phosphorylation of CREB, ERK1/2 and p38. Radiation-induced changes in apoptotic markers were also reported. More specifically, there was a significant rise in pro-apoptotic markers Bax and caspase 3, with significant reduction in anti-apoptotic marker Bcl-xL (Hladik et al., 2020). Furthermore, middle cerebral artery occlusion (MCAO) surgery known to simulate ischemic stroke in C57BL/6J mice was shown to increase ROS levels, as well as the phosphorylation of ERK1/2, p38 and JNK (Tian et al., 2020). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Other studies that have used hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) to induce oxidative stress within cell cultures, have also observed alterations in signaling pathways. Zhao et al. (2013) exposed mouse hippocampal-derived HT22 cells to varying concentrations of H<sub>2</sub>O<sub>2</sub> and found a dose-dependent increase in ROS production from 250-1000 μM. Additionally, treating the cells to H<sub>2</sub>O<sub>2</sub> resulted in a concentration-dependent increase of ERK1/2, JNK1/2 and p38 phosphorylation. Ruffels et al. (2004) incubated human neuroblastoma cells (SH-SY5Y) to varying concentrations of H<sub>2</sub>O<sub>2</sub> that ranged from 0.5-1.25 mM and found a dose-dependent increase in JNK1/2, ERK1/2 and Akt phosphorylation. Another study exposed SH-SY5Y and rat pheochromocytoma (PC12) cells to 0.05-2 mM H<sub>2</sub>O<sub>2</sub> and found a dose-dependent increase in ROS from 0-1 mM in SH-SY5Y cells, and from 0-2 mM in PC12 cells with a concentration-dependent increase in ERK1/2, p38 and JNK phosphorylation (Chen et al., 2009). Furthermore, Crossthwaite et al. (2002) incubated neuronal cultures from 15- to 16-day-old Swiss mice to 100, 300 and 1000 μm H<sub>2</sub>O<sub>2</sub> and showed increased levels of ROS. A corresponding increase in ERK1/2 and Akt activation was observed at 100-300 μm, and for JNK1/2 the observation was observed at 1000 μm. Carvour et al. (2008) treated N27 cells (rat dopaminergic cell line) to 3-30 μM H<sub>2</sub>O<sub>2</sub> and measured increased ROS levels, as well as increased apoptotic signaling molecules caspase 3 and proapoptotic kinase protein kinase C-δ (PKCδ) cleavage. </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Time Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Limited evidence shows that oxidative stress leads to altered signaling pathways in a time concordant manner. When irradiated with X-rays, HCAECs, BAECs and MCT3T3-E1 osteoblast-like cells show increase in ROS or levels of protein carbonylation, or a decrease in the levels of superoxide dismutase (SOD), catalase (CAT), GSTO1 or GSH at earlier timepoints than alterations in the signaling molecules p16, p21, Ceramide, Runx2, and HO-1 (Azimzadeh et al., 2017; Kook et al., 2015; Wortel et al., 2019). As the key events are both molecular-level changes, both can occur quickly after irradiation. Wortel et al. (2019) found that increased hydrogen peroxide levels could be observed <em>in vitro</em> as early as 2 minutes post-irradiation, while ASMase activity and ceramide levels were only increased 5 minutes post-irradiation. </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">When exposed to H<sub>2</sub>O<sub>2</sub>, PC12 cells show an increase production of ROS with a corresponding increase in phosphorylation of MAPK proteins in a time-dependent fashion. An increase in ERK1/2, JNK and p38 phosphorylation was observed within 5-15 minutes and sustained for over 2 hours (Chen et al., 2009). When exposed to cold stress for 1, 2 and 3 weeks, MDA levels increased in a time-dependent manner from 1-3 weeks within the brain tissue isolated from C57BL/6 mice. The expressions of JNK, ERK and p38 phosphorylation levels were all also significantly upregulated in chronic cold-stressed groups for all time-points (Xu et al., 2019). After gamma irradiation (2 Gy), ROS increased 2 months post-irradiation, while increased p21 and decreased Bcl-2 were only observed at 12 months (Suman et al., 2013). However, other signaling molecules were increased at both times. </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Essentiality </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Several studies have investigated the essentiality of the relationship, where the blocking or attenuation of the upstream KE causes a change in frequency of the downstream KE. The increase in oxidative stress can be modulated by certain drugs, antioxidants and media. L-carnitine injections decreased ROS and increased p-p38/p38 and p-Nrf2/Nrf2 signaling (Fan et al., 2017). Fenofibrate was found to return levels of SOD, phosphorylated MAPK signaling proteins and increase Nrf2 levels (Azimzadeh et al., 2021). Antioxidants (N-acetyl cysteine, curcumin) were shown to restore or reduce ROS levels closer to control levels following radiation or microgravity exposure, respectively. Signaling proteins in the Nrf2/HO-1 pathway and the RANKL/osteoprotegerin (OPG) ratio were decreased and brought closer to control levels (Kook et al., 2015; Xin et al., 2015). Hydrogen rich medium showed reduced ROS with restoration of OPG and RANKL signaling levels to controls (Sun et al., 2013). Polyphenol S3 (60 mg/kg/d) treatment was found to reverse the effect of microgravity on CAT, SOD and MDA, returning the levels to near control values. Meanwhile, Runx2 mRNA levels and β-catenin/β-actin levels increased following treatment (Diao et al., 2018). Sildenafil is another drug that was found to reduce ROS generation by inhibiting O<sub>2</sub><sup>-</sup> production and intracellular peroxynitrite levels in bovine aortic endothelial cells (BAECs) after gamma irradiation. As well, ASMase activity and ceramide levels were inhibited by sildenafil (Wortel et al., 2019). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Within brain cells, several antioxidants have been found to attenuate oxidative stress-induced alterations in signaling pathways. These antioxidants include Melandrii Herba extract, N-acetyl-L-cysteine (NAC), gallocatechin gallate/epigallocatechin-3-gallate, Cornus officinalis (CC) and fermented CC (FCC), L-165041, fucoxanthin, and edaravone. These antioxidants were shown to reduce ROS and subsequently decrease phosphorylation of MAPKs such as ERK1/2, JNK1/2 and p38 after exposure to radiation, H<sub>2</sub>O<sub>2</sub> or LPS (Lee et al., 2017; Deng et al., 2012; Park et al., 2021; Tian et al., 2020; Schnegg et al., 2012; Zhao et al., 2017; Zhao et al., 2013; El-Missiry et al., 2018). Another documented modulator is mesenchymal stem-cell conditioned medium (MSC-CM), which was able to alleviate oxidative stress in HT22 cells and restore levels of p53 (Huang et al., 2021).</span></p>
<ul>
<li><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">MAPK pathways can exhibit varied responses after exposure to oxidative stress. The expected response is an increase in the activity of the ERK, JNK, and p38 pathways as protein phosphatases, involved in the inactivation of MAPK pathways, are deactivated by oxidative stress (<span style="background-color:white">Valerie et al., 2007). Although some studies observe this (</span>Azimzadeh et al., 2021; Sakata et al., 2015<span style="background-color:white">), others show a decrease (Fan et al., 2017; </span>Yoo, Han & Kim, 2016<span style="background-color:white">) or varying changes (</span>Azimzadeh et al., 2015<span style="background-color:white">) in the MAPK pathways.</span></span></span></li>
</ul>
<p><span style="font-family:Times New Roman,Times,serif">The tables below provide representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All data that is represented is statistically significant unless otherwise indicated. </span></p>
HighMaleLowFemaleLowUnspecificModerateAdultModerateJuvenileLowHighHighModerate<p><span style="font-family:Times New Roman,Times,serif">Based on the prioritized studies presented here, the evidence of taxonomic applicability is low for humans despite there being strong plausibility as the evidence only includes <em>in vitro</em> human cell-derived models. The taxonomic applicability for mice and rats is considered high as there is much available data using <em>in vivo</em> rodent models that demonstrate the concordance of the relationship. The taxonomic applicability was determined to be moderate for pigs as only one <em>in vivo</em> study provided meaningful support to the relationship. In terms of sex applicability, all <em>in vivo</em> studies that indicated the sex of the animals used male animals, therefore, the evidence for males is high and females is considered to be low for this KER. The majority of studies used adolescent animals, with a few using adult animals. Preadolescent animals were not used to support the KER; however, the relationship in preadolescent animals is still plausible.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Annunziato, L. (2003), "Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions", <em>Toxicology Letters</em>, Vol. 139/2–3, <a href="https://doi.org/10.1016/S0378-4274(02)00427-7" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/S0378-4274(02)00427-7</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Arfin, S. et al. (2021), “Oxidative Stress in Cancer Cell Metabolism”, <em>Antioxidants 2021</em>, Vol. 10/5, MDPI, Basel, <a href="https://doi.org/10.3390/ANTIOX10050642" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/ANTIOX10050642</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Azimzadeh, O. et al. (2021), "Activation of pparα by fenofibrate attenuates the effect of local heart high dose irradiation on the mouse cardiac proteome", <em>Biomedicines</em>, Vol. 9/12, MDPI, Basel, <a href="https://doi.org/10.3390/biomedicines9121845" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/biomedicines9121845</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Azimzadeh, O. et al. (2017), "Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways", <em>International Journal of Radiation Biology</em>, Vol. 93/9, Informa, London, <a href="https://doi.org/10.1080/09553002.2017.1339332" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2017.1339332</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Azimzadeh, O. et al. (2015), "Integrative proteomics and targeted transcriptomics analyses in cardiac endothelial cells unravel mechanisms of long-term radiation-induced vascular dysfunction", <em>Journal of Proteome Research</em>, Vol. 14/2, American Chemical Society, Washington, <a href="https://doi.org/10.1021/pr501141b" rel="noreferrer noopener" target="_blank">https://doi.org/10.1021/pr501141b</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Bai, J. et al. (2020), "Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling", <em>American Journal of Physiology - Cell Physiology</em>, Vol. 318/5, American Physiological Society, <a href="https://doi.org/10.1152/ajpcell.00520.2019" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/ajpcell.00520.2019</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Boyce, B. F. and L. Xing. (2007), "The RANKL/RANK/OPG pathway", <em>Current Osteoporosis Reports</em>, Vol. 5/3,<a href="https://doi.org/10.1007/s11914-007-0024-y" rel="noreferrer noopener" target="_blank"> </a><a href="https://doi.org/10.1007/s11914-007-0024-y" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s11914-007-0024-y</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Cabrera, M. P. and R. H. Chihuailaf. (2011), "Antioxidants and the Integrity of Ocular Tissues", <em>Veterinary Medicine International</em>, Vol. 2011, Hindawi, London, <a href="https://doi.org/10.4061/2011/905153" rel="noreferrer noopener" target="_blank">https://doi.org/10.4061/2011/905153</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Carvour, M. et al. (2008), "Chronic Low-Dose Oxidative Stress Induces Caspase-3-Dependent PKCδ Proteolytic Activation and Apoptosis in a Cell Culture Model of Dopaminergic Neurodegeneration", <em>Annals of the New York Academy of Sciences</em>, Vol. 1139/1, <a href="https://doi.org/10.1196/annals.1432.020" rel="noreferrer noopener" target="_blank">https://doi.org/10.1196/annals.1432.020</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Chen, L. et al. (2009), "Hydrogen peroxide-induced neuronal apoptosis is associated with inhibition of protein phosphatase 2A and 5, leading to activation of MAPK pathway", <em>The International Journal of Biochemistry & Cell Biology</em>, Vol. 41/6, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.biocel.2008.10.029" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.biocel.2008.10.029</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Crossthwaite, A. J., S. Hasan and R. J. Williams. (2002), "Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca2+ and PI3-kinase", <em>Journal of Neurochemistry</em>, Vol. 80/1, John Wiley & Sons, Hoboken, <a href="https://doi.org/10.1046/j.0022-3042.2001.00637.x" rel="noreferrer noopener" target="_blank">https://doi.org/10.1046/j.0022-3042.2001.00637.x</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Deng, Z. et al. (2012), "Radiation-Induced c-Jun Activation Depends on MEK1-ERK1/2 Signaling Pathway in Microglial Cells", (I. Ulasov, Ed.) <em>PLoS ONE</em>, Vol. 7/5, <a href="https://doi.org/10.1371/journal.pone.0036739" rel="noreferrer noopener" target="_blank">https://doi.org/10.1371/journal.pone.0036739</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Diao, Y. et al. (2018), "Polyphenols (S3) Isolated from Cone Scales of Pinus koraiensis Alleviate Decreased Bone Formation in Rat under Simulated Microgravity", <em>Scientific Reports</em>, Vol. 8/1, Nature, <a href="https://doi.org/10.1038/s41598-018-30992-8" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41598-018-30992-8</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Domazetovic, V. et al. (2017), "Oxidative stress in bone remodeling: role of antioxidants", <em>Clinical cases in mineral and bone metabolism</em>, Vol. 14/2, pp. 209-216 </span></p>
<p><span style="font-family:Times New Roman,Times,serif">El-Missiry, M. A. et al. (2018), "Neuroprotective effect of epigallocatechin-3-gallate (EGCG) on radiation-induced damage and apoptosis in the rat hippocampus", <em>International Journal of Radiation Biology</em>, Vol. 94/9, <a href="https://doi.org/10.1080/09553002.2018.1492755" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2018.1492755</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Fan, Z. et al. (2017), "L-carnitine preserves cardiac function by activating p38 MAPK/Nrf2 signalling in hearts exposed to irradiation", <em>European Journal of Pharmacology</em>, Vol. 804, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.ejphar.2017.04.003" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.ejphar.2017.04.003</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Hasan, H. F., R. R. Radwan and S. M. Galal. (2020), "Bradykinin‐potentiating factor isolated from Leiurus quinquestriatus scorpion venom alleviates cardiomyopathy in irradiated rats via remodelling of the RAAS pathway", <em>Clinical and Experimental Pharmacology and Physiology</em>, Vol. 47/2, Wiley, <a href="https://doi.org/10.1111/1440-1681.13202" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/1440-1681.13202</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Hladik, D. et al. (2020), "CREB Signaling Mediates Dose-Dependent Radiation Response in the Murine Hippocampus Two Years after Total Body Exposure", <em>Journal of Proteome Research</em>, Vol. 19/1, <a href="https://doi.org/10.1021/acs.jproteome.9b00552" rel="noreferrer noopener" target="_blank">https://doi.org/10.1021/acs.jproteome.9b00552</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Huang, Y. et al. (2021), "Mesenchymal Stem Cell-Conditioned Medium Protects Hippocampal Neurons From Radiation Damage by Suppressing Oxidative Stress and Apoptosis", <em>Dose-Response</em>, Vol. 19/1, <a href="https://doi.org/10.1177/1559325820984944" rel="noreferrer noopener" target="_blank">https://doi.org/10.1177/1559325820984944</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Hughson, R. L., A. Helm and M. Durante. (2018), "Heart in space: Effect of the extraterrestrial environment on the cardiovascular system", <em>Nature Reviews Cardiology</em>, Vol. 15/3, Nature, <a href="https://doi.org/10.1038/nrcardio.2017.157" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/nrcardio.2017.157</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Kenchegowda, D. et al. (2018), "Selective Insulin-like Growth Factor Resistance Associated with Heart Hemorrhages and Poor Prognosis in a Novel Preclinical Model of the Hematopoietic Acute Radiation Syndrome", <em>Radiation Research</em>, Vol. 190/2, BioOne, <a href="https://doi.org/10.1667/RR14993.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RR14993.1</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Kook, S. H. et al. (2015), "Irradiation inhibits the maturation and mineralization of osteoblasts via the activation of Nrf2/HO-1 pathway", <em>Molecular and Cellular Biochemistry</em>, Vol. 410/1–2, Nature, <a href="https://doi.org/10.1007/s11010-015-2559-z" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s11010-015-2559-z</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. https://doi.org/10.1080/09553002.2022.2110306</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Lee, K., A. Lee and I. Choi. (2017), "Melandrii Herba Extract Attenuates H2O2-Induced Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells and Scopolamine-Induced Memory Impairment in Mice", <em>Molecules</em>, Vol. 22/10, MDPI, Basel, <a href="https://doi.org/10.3390/molecules22101646" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/molecules22101646</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Lehtinen, M. and A. Bonni. (2006), "Modeling Oxidative Stress in the Central Nervous System", <em>Current Molecular Medicine</em>, Vol. 6/8, <a href="https://doi.org/10.2174/156652406779010786" rel="noreferrer noopener" target="_blank">https://doi.org/10.2174/156652406779010786</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Li, J. et al. (2013), "Oxidative Stress and Neurodegenerative Disorders", <em>International Journal of Molecular Sciences</em>, Vol. 14/12, <a href="https://doi.org/10.3390/ijms141224438" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/ijms141224438</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Limoli, C. L. et al. (2004), "Radiation Response of Neural Precursor Cells: Linking Cellular Sensitivity to Cell Cycle Checkpoints, Apoptosis and Oxidative Stress", <em>Radiation Research</em>, Vol. 161/1, <a href="https://doi.org/10.1667/RR3112" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RR3112</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Manolagas, S. C. and M. Almeida. (2007), "Gone with the Wnts: β-Catenin, T-Cell Factor, Forkhead Box O, and Oxidative Stress in Age-Dependent Diseases of Bone, Lipid, and Glucose Metabolism", <em>Molecular Endocrinology</em>, Vol. 21/11, Oxford University Press, Oxford, <a href="https://doi.org/10.1210/me.2007-0259" rel="noreferrer noopener" target="_blank">https://doi.org/10.1210/me.2007-0259</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Essers, M. A. et al. (2004), “FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK”. <em>The EMBO journal</em>, Vol. 23/24, EMBO, <a href="https://doi.org/10.1038/sj.emboj.7600476" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/sj.emboj.7600476</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Nagane, M. et al. (2021), "DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases", <em>Journal of Radiation Research</em>, Vol. 62/4, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jrr/rrab032" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jrr/rrab032</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Park, H. et al. (2016), "GDF15 contributes to radiation-induced senescence through the ROS-mediated p16 pathway in human endothelial cells", <em>Oncotarget</em>, Vol. 7/9, <a href="https://doi.org/10.18632/oncotarget.7457" rel="noreferrer noopener" target="_blank">https://doi.org/10.18632/oncotarget.7457</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Park, D. H. et al. (2021), "Neuroprotective Effect of Gallocatechin Gallate on Glutamate-Induced Oxidative Stress in Hippocampal HT22 Cells", <em>Molecules</em>, Vol. 26/5, MDPI, Basel, <a href="https://doi.org/10.3390/molecules26051387" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/molecules26051387</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Ping, Z. et al. (2020), "Oxidative Stress in Radiation-Induced Cardiotoxicity",<em> Oxidative Medicine and Cellular Longevity</em>, Vol. 2020, Hindawi, London, <a href="https://doi.org/10.1155/2020/3579143" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2020/3579143</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Ramadan, R. et al. (2021), "The role of connexin proteins and their channels in radiation-induced atherosclerosis", <em>Cellular and Molecular Life Sciences</em>, Vol. 78, Nature, <a href="https://doi.org/10.1007/s00018-020-03716-3" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00018-020-03716-3</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Ramalingam, M. and S.-J. Kim. (2012), "Reactive oxygen/nitrogen species and their functional correlations in neurodegenerative diseases", <em>Journal of Neural Transmission</em>, Vol. 119/8, Springer Nature, Berlin, <a href="https://doi.org/10.1007/s00702-011-0758-7" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00702-011-0758-7</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Ruffels, J., M. Griffin and J. M. Dickenson. (2004), "Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells: role of ERK1/2 in H2O2-induced cell death", <em>European Journal of Pharmacology</em>, Vol. 483/2–3, Elsevier, Amsterdam <a href="https://doi.org/10.1016/j.ejphar.2003.10.032" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.ejphar.2003.10.032</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Sakata, K. et al. (2015), "Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells", <em>Vascular Pharmacology</em>, Vol. 70, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.vph.2015.03.016" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.vph.2015.03.016</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Schmidt-Ullrich, R. K. et al. (2000), "Signal transduction and cellular radiation responses.", <em>Radiation research</em>, Vol. 153/3, BioOne, <a href="https://doi.org/10.1667/0033-7587(2000)153%5B0245:stacrr%5D2.0.co;2" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/0033-7587(2000)153[0245:stacrr]2.0.co;2</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Schnegg, C. I. et al. (2012), "PPARδ prevents radiation-induced proinflammatory responses in microglia via transrepression of NF-κB and inhibition of the PKCα/MEK1/2/ERK1/2/AP-1 pathway", <em>Free Radical Biology and Medicine</em>, Vol. 52/9, <a href="https://doi.org/10.1016/j.freeradbiomed.2012.02.032" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.freeradbiomed.2012.02.032</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Soloviev, A. I. and I. V. Kizub. (2019), "Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction", <em>Biochemical Pharmacology</em>, Vol. 159, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.bcp.2018.11.019" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.bcp.2018.11.019</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Suman, S. et al. (2013), "Therapeutic and space radiation exposure of mouse brain causes impaired DNA repair response and premature senescence by chronic oxidant production", <em>Aging</em>, Vol. 5/8, <a href="https://doi.org/10.18632/aging.100587" rel="noreferrer noopener" target="_blank">https://doi.org/10.18632/aging.100587</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Sun, Y. et al. (2013), "Treatment of hydrogen molecule abates oxidative stress and alleviates bone loss induced by modeled microgravity in rats", <em>Osteoporosis International</em>, Vol. 24/3, Nature, <a href="https://doi.org/10.1007/s00198-012-2028-4" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00198-012-2028-4</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Tahimic, C. G. T. and R. K. Globus. (2017), “Redox Signaling and Its Impact on Skeletal and Vascular Responses to Spaceflight”, <em>International Journal of Molecular Sciences</em>, Vol. 18/10, MDPI, Basel, <a href="https://doi.org/10.3390/IJMS18102153" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/IJMS18102153</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Tian, Y. et al. (2017), "The impact of oxidative stress on the bone system in response to the space special environment", <em>International Journal of Molecular Sciences</em>, Vol. 18/10, MDPI, Basel, <a href="https://doi.org/10.3390/ijms18102132" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/ijms18102132</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Tian, W. et al. (2019), "Neuroprotective Effects of Cornus officinalis on Stress-Induced Hippocampal Deficits in Rats and H2O2-Induced Neurotoxicity in SH-SY5Y Neuroblastoma Cells", <em>Antioxidants</em>, Vol. 9/1, MDPI, Basel, <a href="https://doi.org/10.3390/antiox9010027" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/antiox9010027</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Tian, R. et al. (2020), "miR-137 prevents inflammatory response, oxidative stress, neuronal injury and cognitive impairment via blockade of Src-mediated MAPK signaling pathway in ischemic stroke", <em>Aging</em>, Vol. 12/11, <a href="https://doi.org/10.18632/aging.103301" rel="noreferrer noopener" target="_blank">https://doi.org/10.18632/aging.103301</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Valerie, K. et al. (2007), "Radiation-induced cell signaling: inside-out and outside-in", <em>Molecular Cancer Therapeutics</em>, Vol. 6/3, American Association for Cancer Research, <a href="https://doi.org/10.1158/1535-7163.MCT-06-0596" rel="noreferrer noopener" target="_blank">https://doi.org/10.1158/1535-7163.MCT-06-0596</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Venkatesulu, B. P. et al. (2018), “Radiation-Induced Endothelial Vascular Injury: A Review of Possible Mechanisms”, <em>JACC: Basic to translational science</em>, Vol. 3/4, Elsevier, Amsterdam,<a href="https://doi.org/10.1016/j.jacbts.2018.01.014." rel="noreferrer noopener" target="_blank"> </a><a href="https://doi.org/10.1016/j.jacbts.2018.01.014." rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.jacbts.2018.01.014.</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Wang. (2010), "Selective neuronal vulnerability to oxidative stress in the brain", <em>Frontiers in Aging Neuroscience</em>, https://doi.org/10.3389/fnagi.2010.00012. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Wang, Y., M. Boerma and D. Zhou. (2016), "Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases", <em>Radiation Research</em>, Vol. 186/2, BioOne, <a href="https://doi.org/10.1667/RR14445.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RR14445.1</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Wortel, R. C. et al. (2019), "Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress", <em>The Journal of Sexual Medicine</em>, Vol. 16/11, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.jsxm.2019.08.015" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.jsxm.2019.08.015</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Wu, Y., M. Chen and J. Jiang. (2019), "Mitochondrial dysfunction in neurodegenerative diseases and drug targets via apoptotic signaling", <em>Mitochondrion</em>, Vol. 49, https://doi.org/10.1016/j.mito.2019.07.003. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Xin, M. et al. (2015), "Attenuation of hind-limb suspension-induced bone loss by curcumin is associated with reduced oxidative stress and increased vitamin D receptor expression", <em>Osteoporosis International</em>, Vol. 26/11, Nature, <a href="https://doi.org/10.1007/s00198-015-3153-7" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00198-015-3153-7</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Xu, B. et al. (2019), "Oxidation Stress-Mediated MAPK Signaling Pathway Activation Induces Neuronal Loss in the CA1 and CA3 Regions of the Hippocampus of Mice Following Chronic Cold Exposure", <em>Brain Sciences</em>, Vol. 9/10, MDPI, Basel, <a href="https://doi.org/10.3390/brainsci9100273" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/brainsci9100273</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Yoo, Y. M., T. Y. Han and H. S. Kim. (2016), "Melatonin suppresses autophagy induced by clinostat in preosteoblast MC3T3-E1 cells", <em>International Journal of Molecular Sciences</em>, Vol. 17/4, MDPI, Basel, <a href="https://doi.org/10.3390/ijms17040526" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/ijms17040526</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Zhao, Z.-Y. et al. (2013), "Edaravone Protects HT22 Neurons from H 2 O 2 -induced Apoptosis by Inhibiting the MAPK Signaling Pathway",<em> CNS Neuroscience & Therapeutics</em>, Vol. 19/3, John Wiley & Sons, Hoboken, <a href="https://doi.org/10.1111/cns.12044" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/cns.12044</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Zhao, D. et al. (2017), "Anti-Neuroinflammatory Effects of Fucoxanthin via Inhibition of Akt/NF-κB and MAPKs/AP-1 Pathways and Activation of PKA/CREB Pathway in Lipopolysaccharide-Activated BV-2 Microglial Cells", <em>Neurochemical Research</em>, Vol. 42/2, Springer Nature, Berlin, <a href="https://doi.org/10.1007/s11064-016-2123-6" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s11064-016-2123-6</a>. </span></p>
2022-09-28T12:40:292024-02-13T16:53:53f1940943-69e5-458b-8c03-896b1c61c3f9383161af-8fa6-4cd9-81d4-8c7c472b341d<p><span style="font-family:Times New Roman,Times,serif">Deposition of energy from irradiation can affect nitric oxide (NO), a diffusible signaling molecule responsible for vasodilation (Dong et al., 2020; Mitchell et al., 2019; Soloviev & Kizub, 2019; Wang, Boerma & Zhou, 2016). NO activity is regulated by nitric oxide synthase (NOS) enzymes, which can be affected by NOS protein concentrations and cofactors tetrahydobiopterin (BH4), nicotinamide adenine dinucleotide phosphate (NADPH) and Ca<sup>2+</sup> (Luiking, Engelen & Deutz, 2010). The deposition of energy can alter certain pathways involving NO and therefore indirectly alter NO levels. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway, the RhoA/Rho kinase (ROCK) pathway, the renin-angiotensin-aldosterone system (RAAS), and the acidic sphingomyelinase/ceramide pathway can influence NO levels (Hemmings & Restuccia 2012; Millatt, Abdel-Rahman & Siragy, 1999; Nagane et al., 2021; Soloviev & Kizub, 2019; Yao et al., 2010). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Deposition of energy can alter NO levels through radiolysis and the direct formation of reactive oxygen species (ROS) (Azzam, Jay-Gerin & Pain, 2013). An increase in ROS and reactive nitrogen species (RNS) can influence NO levels; however, the involvement of RNS in NO production has not been strongly demonstrated in literature (Nagane et al., 2021). ROS acts as a modulator for the relationship between energy deposition leading to altered NO levels. Following ionizing radiation (IR) exposure, there are various enzymes and immune cells involved with indirectly increasing ROS, thereby influencing NO levels (Powers & Jackson, 2008; Soloviev & Kizub, 2019; Vargas-Mendoza et al., 2021). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: High</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The biological plausibility surrounding the connection between deposition of energy and altered NO levels is well-supported by reviews in the literature and mechanistic understanding. NO is a diffusible molecule produced by endothelial cells (Soloviev & Kizub, 2019; Wang, Boerma & Zhou, 2016). The enzyme NOS produces NO and can be used as a proxy to measure NO levels. eNOS and iNOS are common endpoints for assessing NO levels. Changes in eNOS phosphorylation (p-eNOS) can also indicate NO levels, with phosphorylation at serine 1177 increasing eNOS activity and phosphorylation at threonine 495 decreasing eNOS activity (Nagane et al., 2021). The deposition of energy from IR can indirectly lead to changes in NO levels through various pathways (Nagane et al., 2021; Soloviev & Kizub, 2019). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">NO levels can be altered by deposition of energy through ROS. ROS can be produced directly through the radiolysis of water or indirectly through the mitochondrial electron transport chain (ETC) and various enzymes and immune cells (Azzam, Jay-Gerin & Pain, 2013; Powers & Jackson, 2008; Soloviev & Kizub, 2019; Vargas-Mendoza et al., 2021). NO bioavailability is reduced through the molecule’s reaction with ROS that produces peroxynitrite, or oxidation of the NOS cofactor BH4. This uncouples NOS causing it to produce ROS instead of NO, further driving down NO bioavailability (Forrester et al., 2019; Soloviev & Kizub, 2019). NO can also increase as a result of deposition of energy through activation of iNOS during oxidative stress (Nathan & Xie, 1994). However, this additional NO reacts with ROS to form peroxynitrite (Nagane et al., 2021; Soloviev & Kizub, 2019). The reaction of ROS with NO produces the RNS peroxynitrite, which can also further oxidize BH4 and uncouple NOS, resulting in further NO reduction (Soloviev & Kizub, 2019). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Deposition of energy can also alter NO levels by activating signaling pathways involved in NO regulation. Under normal conditions, the PI3K/Akt pathway can activate NOS through phosphorylation (Hemmings & Restuccia 2012; Nagane et al., 2021). The RhoA/ROCK pathway prevents both the expression and phosphorylation of NOS (Yao et al., 2010). Furthermore, the RAAS pathway can increase the production of ROS through NADPH oxidase (NOX), which causes decreased NO (Nguyen Dinh Cat et al., 2013). Additionally, the RAAS pathway can activate NOS as a countermeasure for vasoconstriction (Millatt, Abdel-Rahman & Siragy, 1999). Lastly, the acidic sphingomyelinase/ceramide pathway can also activate NOX, resulting in lower NO levels (Soloviev & Kizub, 2019). Deposition of energy from IR can change these pathways through oxidative stress and changes in protein expression, which results in altered NO levels (Schmidt-Ullrich et al., 2000).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The empirical evidence supporting this KER was collected from research using both <em>in vivo</em> and <em>in vitro</em> models. The use of X-rays and gamma rays as stressors with doses ranging from 0.05 Gy to 60 Gy demonstrates the relationship between deposition of energy and altered NO levels. The dose, dose rate, and radiation type will all influence the level of energy deposition and therefore of NO production, but insufficient evidence exists to quantify the relationship between these factors and NO production. Human coronary artery endothelial cells (HCAECs) (Azimzadeh et al. 2017), human aortic endothelial cells (HAoECs) (Azimzadeh et al., 2021), human umbilical vein endothelial cells (HUVECs) (Hong et al. 2013; Sakata et al., 2015; Sonveaux et al., 2003), bovine aortic endothelial cells (BAECs) (Hirakawa et al., 2002; Sonveaux et al., 2003), tumor models (Sonveaux et al., 2003), cardiac microvascular endothelial cells and serum (Azimzadeh et al. 2015), as well as <em>in vivo</em> rat pulmonary arterioles (Fuji et al., 2016), Wistar albino rat heart tissue and serum (Abdel-Mageid & Shedid, 2019), B6J mice aortic endothelium (Hamada et al., 2020; Hamada et al., 2021), WAG/RijCmcr rat heart tissue (Baker et al., 2009), and serum from the abdominal inferior vena cava (Ohta et al., 2007) were the models used in these studies. Both direct NO measures, such as ELISA assay, and indirect NO measures, such as eNOS, iNOS, citrulline (NOS product) and NOx (nitrite and nitrate, NO metabolites) levels and activity, were used to determine changes in NO levels following energy deposition. </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Dose Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Evidence shows concordant changes in NO levels and deposition of energy. The study by Dias et al. (2018) showed varying changes to eNOS after chronic gamma irradiation, with a general trend of decreasing eNOS expression with increasing energy deposition (i.e., at 0.5, 1, and 2 Gy). Similarly, HUVECs exposed to gamma irradiation showed slightly lower eNOS levels at 12.5 Gy than at 10 Gy (Sadhukhan et al., 2020). X-ray irradiation of mouse cardiac microvascular endothelial cells showed 0.6 and 0.2-fold decreases in p-eNOS at 8 and 16 Gy, respectively (Azimzadeh et al., 2015). X-ray irradiation of mouse blood serum showed NO decreased 0.3-fold and 0.2-fold following 8 Gy and 16 Gy X-ray irradiation, respectively (Azimzadeh et al., 2015). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">In contrast, BAECs and HUVECs irradiated with X-rays showed a trend of increasing eNOS expression and activation from 2 to 20 Gy (Sonveaux et al., 2003). Using similar doses of X-rays also showed that NOx increased dose-dependently from 1 to 20 Gy and reached a maximum increase of 10-fold (Sakata et al., 2015). Other high X-ray doses from 19.6 to 31.5 Gy on mice showed about a 2-fold increase in serum nitrate concentration, which had a general increasing trend at larger doses (Ohta et al., 2017). In BAECs, eNOS expression did not significantly change from 1 to 60 Gy of X-rays, while iNOS expression increased to 6% of GAPDH expression after 1 Gy, 26% after 2 Gy and remained around 40% of GAPDH expression from 10 to 60 Gy (Hirakawa et al., 2002). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Many studies do not examine this relationship at multiple doses. After 0.5 Gy X-ray irradiation of HCAECs, p-eNOS and NO were both decreased (Azimzadeh et al., 2017). HUVECs irradiated with 4 Gy X-rays showed increases in iNOS and nitrotyrosine (peroxynitrite biomarker) levels (Hong et al., 2013). eNOS levels decreased in mouse aortic endothelial cells when exposed to 5 Gy gamma and X-ray irradiation at various acute and chronic regimens (Hamada et al., 2020; Hamada et al., 2021; Hamada et al., 2021). Rat cardiac and serum NOx increased as a response to 8 Gy gamma irradiation (Abdel-Mageid & Shedid, 2019). Total body irradiation (TBI) of rats with 10 Gy X-rays resulted in a decrease in eNOS, iNOS and NOx levels (Baker et al., 2009). Both cardiac eNOS and serum NO decreased to 73% and 63%, respectively, following 16 Gy X-ray irradiation (Azimzadeh et al., 2021). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Time Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is evidence that a change in NO levels occurs after energy deposition (i.e., irradiation) occurred. HUVECs irradiated with 4 Gy X-rays displayed an increase in nitrotyrosine (peroxynitrite biomarker) at 6 hours post-irradiation (Hong et al., 2013). They also showed increased iNOS after 1.5, 3, and 6 hours (Hong et al., 2013). Following a 26 Gy X-ray irradiation of mice, serum nitrate concentration showed a maximum increase after 3h, followed by a return to pre-irradiation levels at 12 h and 24 h post-irradiation (Ohta et al., 2007). As well, in HUVECs, 10 and 12 Gy gamma rays led to decreased eNOS levels after 4 and 24 h (Sadhukhan et al., 2020). BAECs and HUVECs irradiated with 6 Gy X-rays did not exhibit significant changes in eNOS 1-6 h post-irradiation, but eNOS increased after 12, 24, and 48 hours (Sonveaux et al., 2003) </span></p>
<p><span style="font-family:Times New Roman,Times,serif">HUVECs irradiated with 10 Gy X-rays also had no change in eNOS levels over 72 h, while p-eNOS (Ser1177) increased consistently over 72 h, p-eNOS (Thr495) decreased after 6 h and returned to initial levels after 72 h, iNOS did not change other than a non-significant increased after 24 h, citrulline increased after 6 h and remained the same for 72 h and NOx increased over 72 h (Sakata et al., 2015). After 2 Gy X-ray irradiation of BAECs, eNOS expression did not significantly change from 0-120 h, iNOS expression increased to a maximum 6 h post-irradiation and NO increased to a maximum 12 h (Hirakawa et al., 2002). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Following 0.5 Gy X-ray irradiation of HCAECs, NO was not significantly lower after 1 day, while the 7 and 14 day time-points showed significant decreases (Azimzadeh et al., 2017). In human aortic endothelial cells, acute gamma radiation consistently showed decreased eNOS at each time-point up to 16 days after irradiation, while chronic irradiation decreased eNOS levels 1 and 4 days post-irradiation (Dias et al., 2018). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Finally, eNOS expression was lower 1, 3, and 6 months post-irradiation of mice aortic endothelial cells with 5 Gy gamma rays (Hamada et al., 2020). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Essentiality </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Altered NO levels can occur due to a deposition of energy in the form of ionizing radiation. Since deposited energy initiates events immediately, the removal of deposited energy also supports the essentiality of the key event. Studies that do not deposit energy are observed to have no downstream effects. Deposition of energy cannot be blocked by chemicals, but radiation could be shielded. Currently, no studies show the effect of shielding on this relationship. </span></p>
<ul>
<li><span style="font-family:Times New Roman,Times,serif">Due to the high reactivity of NO, it can be difficult to directly measure it (Luiking, Engelen & Deutz, 2010). The inconsistencies in NO levels may be attributed to the challenges in measuring NO, such as its availability in cell (Azimzadeh et al., 2017; Hirakawa et al., 2002; Hong et al., 2013; Sakata et al., 2015; Sonveaux et al., 2003), serum (Abdel-Magied & Shedid,. 2019; Azimzadeh et al. 2015; Ohta et al., 2007) or tissue (Abdel-Magied & Shedid, 2019; Baker et al., 2009; Fuji et al., 2016; Hamada et al., 2020), its homeostasis with ROS, and its relationship to nitrosylation as accumulated damage and whether p-eNOS/eNOS is being measured.</span></li>
</ul>
<p><span style="font-family:Times New Roman,Times,serif">Examples of quantitative understanding of the relationship are provided. All data that is represented is statistically significant unless otherwise indicated.</span></p>
ModerateMaleLowFemaleLowUnspecificModerateAdultModerateJuvenileLowHighModerate<p><span style="font-family:Times New Roman,Times,serif">The evidence for the taxonomic applicability to humans is low as evidence comes from <em>in vitro</em> human cell-derived models. The relationship has been shown <em>in vivo</em> in mice and rats, with considerable evidence in mice. The relationship has been shown<em> in vivo</em> in males and is likely in females. Most <em>in vivo</em> studies indicate adult or adolescent animal models used. In addition, the relationship is also likely in preadolescent animals.</span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Abdel-Magied, N. and S. M. Shedid (2019), “Impact of zinc oxide nanoparticles on thioredoxin-interacting protein and asymmetric dimethylarginine as biochemical indicators of cardiovascular disorders in gamma-irradiated rats”, <em>Environmental Toxicology</em>, Vol. 35, John Wiley & Sons, Ltd., Hoboken, <a href="https://doi.org/10.1002/tox.22879" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1002/tox.22879</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Azimzadeh, O. et al. (2021), “Activation of PPARα by feno fibrate attenuates the effect of local heart high dose irradiation on the mouse cardiac proteome”, <em>Biomedicines</em>, Vol. 9/12, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/biomedicines9121845" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/biomedicines9121845</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, <em>International Journal of Radiation Biology</em>, Vol. 93/9, Informa, London, <a href="https://doi.org/10.1080/09553002.2017.1339332" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1080/09553002.2017.1339332</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Azimzadeh, O. et al. (2015), “Integrative proteomics and targeted transcriptomics analyses in cardiac endothelial cells unravel mechanisms of long-term radiation-induced vascular dysfunction”, <em>Journal of Proteome Research</em>, Vol. 14/2, American Chemical Society, Washington, <a href="https://doi.org/10.1021/pr501141b" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1021/pr501141b</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Azzam, E. I., J. P. Jay-Gerin and D. Pain (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, <em>Cancer Letters</em>, Vol. 327, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.canlet.2011.12.012" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.canlet.2011.12.012</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Baker, J. E. et al. (2009), “10 Gy total body irradiation increases risk of coronary sclerosis, degeneration of heart structure and function in a rat model”, <em>International Journal of Radiation Biology</em>, Vol. 85/12, Informa, London, <a href="https://doi.org/10.3109/09553000903264473" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3109/09553000903264473</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Dias, J. et al. (2018), “Gamma Low- Dose-Rate Ionizing Radiation Stimulates Adaptive Functional and Molecular Response in Human Aortic Endothelial Cells in a Threshold-, Dose-, and Dose Rate–Dependent Manner”, <em>Dose-Response</em>, Vol. 16/1, SAGE Publications, Thousand Oaks, <a href="https://doi.org/10.1177/1559325818755238" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1177/1559325818755238</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Dong, S. et al. (2020), “Oxidative stress: A critical hint in ionizing radiation induced pyroptosis”, <em>Radiation Medicine and Protection</em>, Vol. 1/4, National Institute of Radiological Protection, <a href="https://doi.org/10.1016/j.radmp.2020.10.001" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.radmp.2020.10.001</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Forrester, S. J. et al. (2018), “Reactive Oxygen Species in Metabolic and Inflammatory Signaling”, <em>Circulation Research</em>, Vol. 122/6, Lippincot Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/CIRCRESAHA.117.311401" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/CIRCRESAHA.117.311401</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Fuji, S. et al. (2016), “Association between endothelial function and micro-vascular remodeling measured by synchrotron radiation pulmonary micro-angiography in pulmonary arterial hypertension”, <em>General Thoracic and Cardiovascular Surgery</em>, Vol. 64, Springer, New York, <a href="https://doi.org/10.1007/s11748-016-0684-6" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/s11748-016-0684-6</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Hamada, N. et al. (2022), “Temporal Changes in Sparing and Enhancing Dose Protraction Effects of Ionizing Irradiation for Aortic Damage in Wild-Type Mice”, Cancers, Vol. 14/14, MDPI, Basel, <a href="https://doi.org/10.3390/cancers14143319" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/cancers14143319</a></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Hamada, N. et al. (2021), “Vascular damage in the aorta of wild-type mice exposed to ionizing radiation: Sparing and enhancing effects of dose protraction”, <em>Cancers</em>, Vol.13/21, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/cancers13215344" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/cancers13215344</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Hamada, N. et al. (2020), “Ionizing Irradiation Induces Vascular Damage in the Aorta of Wild-Type Mice”, <em>Cancers</em>, Vol. 12/10, </span>Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/CANCERS12103030" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/CANCERS12103030</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Hemmings, B. A. and D. F. Restuccia (2012). “PI3K-PKB/Akt Pathway”, <em>Cold Spring Harbor Perspectives in Biology</em>, Vol. 4/9, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, <a href="https://doi.org/10.1101/CSHPERSPECT.A011189" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1101/CSHPERSPECT.A011189</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Hirakawa, M. et al. (2002), “Tumor Cell Apoptosis by Irradiation-induced Nitric Oxide Production in Vascular Endothelium”, <em>Cancer Research,</em> Vol. 62/5, American Association for Cancer Research, Philadelphia, pp. 1450–1457.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Hong, C. W. et al. (2013), “Involvement of inducible nitric oxide synthase in radiation-induced vascular endothelial damage”, <em>Journal of Radiation Research</em>, Vol. 54/6, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/JRR/RRT066" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/JRR/RRT066</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Ježek, P. and L. Hlavatá (2005), “Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism”, <em>The International Journal of Biochemistry & Cell Biology</em>, Vol. 37/12, Elsevier, Amsterdam, https://doi.org/10.1016/j.biocel.2005.05.013. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. https://doi.org/10.1080/09553002.2022.2110306</span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Luiking, Y. C., M. P. Engelen and N. E. Deutz (2010), “Regulation of nitric oxide production in health and disease”, <em>Current Opinion in Clinical Nutrition and Metabolic Care</em>, Vol. 13/1, Lippincott Williams and Wilkins Ltd, Philadelphia, <a href="https://doi.org/10.1097/MCO.0b013e328332f99d" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1097/MCO.0b013e328332f99d</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Millatt, L. J., E. M. Abdel-Rahman and H. M. Siragy (1999), “Angiotensin II and nitric oxide: a question of balance”, <em>Regulatory Peptides</em>, Vol. 81/1-3, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/S0167-0115(99)00027-0" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/S0167-0115(99)00027-0</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Mitchell, A. et al. (2019), “Cardiovascular effects of space radiation: implications for future human deep space exploration”, <em>European Journal of Preventive Cardiology</em> Vol. 26/16, SAGE Publishing, Thousand Oaks, <a href="https://doi.org/10.1177/2047487319831497" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1177/2047487319831497</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases”, <em>Journal of Radiation Research</em>, Vol. 62/4, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/JRR/RRAB032" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/JRR/RRAB032</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Nathan, C. and Q. W. Xie (1994), “Regulation of biosynthesis of nitric oxide”, <em>Journal of Biological Chemistry</em>, Vol. 269/19, American Society for Biochemistry and Molecular Biology, Rockville, <a href="https://doi.org/10.1016/S0021-9258(17)36703-0" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/S0021-9258(17)36703-0</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH Oxidase, and Redox Signaling in the Vasculature”, <em>Antioxidants & Redox Signaling</em>, Vol. 19/10, Mary Ann Liebert, Inc., Larchmont, <a href="https://doi.org/10.1089/ARS.2012.4641" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1089/ARS.2012.4641</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Ohta, S. et al. (2007), “The Role of Nitric Oxide in Radiation Damage”, <em>Biological and Pharmaceutical Bulletin</em>, Vol. 30/6<em>, </em>Pharmaceutical Society of Japan, Tokyo, <a href="https://doi.org/10.1248/bpb.30.1102" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1248/bpb.30.1102</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Powers, S. K. and M. J. Jackson (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, <em>Physiological Reviews</em>, Vol. 88/4, The American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00031.2007" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/physrev.00031.2007</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Sadhukhan, R. et al. (2020), “Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose”, <em>Scientific Reports</em>, Vol. 10/1, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41598-020-64672-3" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/s41598-020-64672-3</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Sakata, K. et al. (2015), “Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells”, <em>Vascular Pharmacology</em>, Vol. 70, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.vph.2015.03.016" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.vph.2015.03.016</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Schmidt-Ullrich, R. K. et al. (2000), “Signal Transduction and Cellular Radiation Responses”, <em>Radiation Research</em>, Vol. 153, R<span style="color:black">adiation Research</span><span style="background-color:white"><span style="color:black"> Society, Bozeman, </span></span><a href="https://doi.org/10.1667/0033-7587(2000)153%5b0245:STACRR%5d2.0.CO;2" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1667/0033-7587(2000)153[0245:STACRR]2.0.CO;2</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soloviev, A. I. and I.V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, <em>Biochemical pharmacology</em>, Vol. 159, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.bcp.2018.11.019" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.bcp.2018.11.019</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Sonveaux, P. et al. (2003), “Irradiation-induced Angiogenesis through the Up-Regulation of the Nitric Oxide Pathway: Implications for Tumor Radiotherapy”, <em>Cancer Research</em>, Vol. 63/5, American Association for Cancer Research, Philadelphia, pp. 1012–1019.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, <em>Life</em>, Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/life11111269" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/life11111269</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Wang, Y., M. Boerma and D. Zhou (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, <em>Radiation research</em>, Vol. 186/2, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RR14445.1" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1667/RR14445.1</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Yao, L. et al. (2010), “The role of RhoA/Rho kinase pathway in endothelial dysfunction”, <em>Journal of Cardiovascular Disease Research</em>, Vol. 1/4, Elsevier, Amsterdam, <a href="https://doi.org/10.4103/0975-3583.74258" style="color:#0563c1; text-decoration:underline">https://doi.org/10.4103/0975-3583.74258</a>.</span></span></p>
2022-09-28T12:44:422023-03-21T11:06:40f1940943-69e5-458b-8c03-896b1c61c3f942ee9daa-42e3-49b5-9aa0-9dc2b7d09c20<p><span style="font-family:Times New Roman,Times,serif">Energy deposition can lead to ionization events that can directly interact with molecules within the cell and can subsequently lead to biological changes such as the formation of free radicals and the initiation of DNA damage repair mechanisms. Different radiation types have different physical properties and as a result the biological effects on cells may differ. Dose and dose rate of the deposited energy also play a role as these factors affect the amount and rate of energy deposited (Donaubauer et al., 2020). Repeated or prolonged exposure to radiation can exhaust the protective effect of the endothelium and lead to endothelial dysfunction (Baselet et al., 2019). Consequently, cells within the vascular endothelium may lose their integrity and become senescent or apoptotic via alterations to signaling pathways related to cell survival, leading to dysregulation of vasodilation and eventual endothelial dysfunction (Deanfield et al., 2007; Bonetti et al., 2003). Activation of the endothelium, consisting of inflammation, proliferation, thrombosis and low nitric oxide, occurs as a normal response to pathological conditions and oxidative stress from deposited energy (Krüger-Genge et al., 2019).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: Moderate</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The biological plausibility surrounding the connection between deposition of energy leading to endothelial dysfunction is well-supported by reviews in the literature and mechanistic understanding. The impact on endothelial dysfunction from deposited energy onto cells may vary with the radiation source and associated parameters of dose, dose rate, and type, which can influence the amount of energy absorbed, among other factors such as tissue type. </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Radiation types such as gamma rays, X-rays, and charged particles at doses ranging from 0.05-18 Gy and dose rates as low as 2.4 mGy/h induce endothelial dysfunction through an increase in cellular markers of apoptosis and cellular senescence in human cell and animal models as well as diminished relaxation response of vessels in animal models (Yentrepalli et al., 2013a; Yentrepalli et al., 2013b; Soucy et al., 2011; On et al., 2001; Hatoum et al., 2006; Soucy et al., 2010; Soloviev et al., 2003; Baselet et al., 2017; Shen et al., 2017). Following irradiation, endothelial cells may lose their integrity and become senescent or apoptotic via alterations to signaling pathways related to cell survival, leading to endothelial dysfunction (Deanfield et al., 2007; Bonetti et al., 2003). Senescent endothelial cells show changes in cell morphology, cell-cycle arrest, and increased senescence-associated β-galactosidase (SA-β-gal) staining. They also have a pro-inflammatory secretory phenotype, which further contributes to negative effects. These changes lead to endothelial dysfunction, which results in dysregulation of vasodilation (Wang et al., 2016; Hughson et al., 2018; Ramadan et al., 2021). Prolonged chronic inflammation following irradiation causes an ineffective healing process, further worsened by a decrease in endothelium-dependent relaxation. This leads to endothelial dysfunction, making the vasculature more vulnerable to damage from non-laminar flow (Sylvester et al., 2018). Since the endothelium is largely responsible for controlling fluid flow, dysfunctions in the endothelium can lead to fluid imbalance, blood pressure changes, and blood clot formation (Konukoglu & Uzun, 2017; Korpela & Liu, 2014; Verma et al., 2003). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">The empirical evidence supporting this KER is gathered from research utilizing both <em>in vivo</em> and <em>in vitro</em> models. Many <em>in vitro</em> studies have examined this relationship using human endothelial cell cultures, such as telomerase-immortalized coronary artery endothelial cells (TICAE) and human umbilical vein endothelial cells (HUVECs) (Baselet et al., 2017; Yentrapalli et al., 2013b). <em>In vivo</em> studies analyzed changes in murine aorta, white rabbit thoracic aorta, and rat aorta and microvessels (Hatoum et al., 2006; Shen et al., 2018; Soloviev et al., 2003; Soucy et al., 2010; Soucy et al., 2011). The evidence includes use of gamma, X-ray, and heavy ion radiation in the dose range of 0.05-18 Gy. SA-β-gal, a marker for cellular senescence, and therefore endothelial dysfunction, relaxation in response to acetylcholine (ACh) and apoptosis were examined in these studies (Baselet et al., 2017; Hatoum et al., 2006; Shen et al., 2018; Soloviev et al., 2003; Soucy et al., 2010; Soucy et al., 2011; Yentrapalli et al., 2013). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Dose Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Studies using <em>in vitro</em> and <em>in vivo</em> models have shown that deposition of energy as produced by acute and chronic doses of ionizing radiation administered from 0.05-18 Gy, with dose rates ranging from 2.4 mGy/h to 2.43 Gy/min, cause endothelial dysfunction as indicated by cellular markers such as cellular senescence and apoptosis as well as decreased maximum relaxation response of vessels in response to ACh. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">For example, chronic gamma irradiation of human endothelial cells at a dose rate as low as 2.4 mGy/h led to an increase of 2-fold in SA-β-gal staining (Yentrapalli et al., 2013a). A similar study showed that 4.1 mGy/h chronic gamma irradiation caused no significant changes after 0.69 Gy, but there was a 2-fold increase in SA-β-gal at 2.07 Gy and a 3-fold increase after 4.13 Gy (Yentrapalli et al., 2013b). Another study also looking at SA-β-gal found a 1.7-fold maximum increase in SA-β-gal after irradiating human endothelial cells with 0.05-2 Gy X-rays (Baselet et al., 2017). A study measuring radiation-induced apoptosis in mouse aorta found a 5-fold increase in the number of apoptotic cells after 18 Gy X-ray irradiation (Shen et al., 2018). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Many studies have measured endothelial dysfunction through the relaxation response of vessels in response to ACh. After rabbit thoracic aortas were irradiated with 6 Gy gamma rays, there was a 0.5-fold decrease in maximum relaxation response to ACh, with a linear decrease in relaxation from 0, 1, 2, and 4 Gy gamma rays (Soloviev et al., 2003). A study that irradiated rat aorta with 0.5 and 1 Gy <sup>56</sup>Fe ions found a 0.8-fold decrease in maximum relaxation response to ACh (Soucy et al., 2011). Microvessels from rat intestines irradiated with 2250 cGy of fractionated X-rays showed an ACh-induced maximum dilation of 3%, while controls showed a maximum dilation of 87%. A significant decrease was seen after only three doses of 250 cGy (Hatoum et al., 2006). Gamma ray irradiation of rat aorta at 5 Gy showed a 0.6-fold decrease in relaxation response to ACh (Soucy et al., 2010). Similar results were found in a study using 10 Gy gamma rays on rat aortas, which showed a 9% maximum relaxation response to ACh compared to the 48% maximum relaxation in the control group (On et al., 2001). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Time Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is moderate evidence to suggest a time concordance between the deposition of energy and endothelial dysfunction. A chronic gamma irradiation study at a dose rate of 2.4 mGy/h examined human endothelial cells <em>in vitro</em> after 1, 6, 10 and 12 weeks and showed an increase in SA-β-gal as early as 10 weeks, with levels remaining significantly increased at 12 weeks (Yentrapalli et al., 2013a). A study by the same group also looked at the effects of 4.1 mGy/h gamma rays on human endothelial cells <em>in vitro</em>, but at 1, 3 and 6 weeks of chronic irradiation, revealing an increase in cellular senescence, indicated by increased SA-β-gal, as soon as 3 weeks, with levels remaining significantly increased at 6 weeks (Yentrepalli et al., 2013b). After mouse aorta were irradiated with 18 Gy X-rays, the number of apoptotic cells were determined over 84 days. The number of apoptotic cells was significantly higher than the controls after 3, 7, 14, 28, and 84 days, but was highest (5-fold higher than control) after 7 days followed by a linear decrease (Shen et al., 2018). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Similarly, rabbit thoracic aortas irradiated with 6 Gy gamma rays and exposed to ACh showed a 0.5-fold decrease in maximum relaxation after both 9 and 30 days (Soloviev et al., 2003). When rats were irradiated with 1 Gy <sup>56</sup>Fe ions, ACh-induced relaxation in the aorta decreased 0.25-fold compared to controls 4 months after irradiation, and went from a 67.5% relaxation response in the control group to 59% in the irradiated group after 6 months (although this change was not statistically significant). Relaxation response to ACh remained non-significant at 8 months post-irradiation (Soucy et al., 2011). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Essentiality </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Endothelial dysfunction can be triggered in response to an injury or a stressor. Therefore, with a reduction in stressor severity, there should be less endothelial dysfunction. As deposition of energy is a physical stressor, it cannot be blocked/decreased using chemicals; however, it can be shielded, though currently no available data used shielding of radiation and measured the impact on endothelial dysfunction. Since deposited energy initiates events immediately, the removal of deposited energy also supports the essentiality of the key event. Studies that do not deposit energy are observed to have no downstream effects.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Much evidence for this relationship comes from high dose studies (>2 Gy); further work is needed at varying doses and dose rates to better understand the relationship. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Examples of quantitative understanding of the relationship are shown in the table below. All data represented is statistically significant unless otherwise indicated. </span></p>
ModerateMaleModerateFemaleLowUnspecificModerateAdultLowJuvenileModerateNot Otherwise SpecifiedLowModerateModerateModerate<p><span style="font-family:Times New Roman,Times,serif">The evidence for the taxonomic applicability to humans is low as the majority of the evidence is from <em>in vitro</em> human-derived cells. The relationship is supported by both sexes of mouse, rat, and rabbit models. The <em>in vivo</em> studies were mostly undertaken in adolescent or adult rats and mice. In addition, the relationship is likely at any life stage.</span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Baselet, B. et al. (2019), “Pathological effects of ionizing radiation: endothelial activation and dysfunction”, Cellular and Molecular Life Science, Vol. 76, Springer, New York, https://doi.org/10.1007/s00018-018-2956-z.</span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Baselet, B. et al. (2017), “Functional Gene Analysis Reveals Cell Cycle Changes and Inflammation in Endothelial Cells Irradiated with a Single X-ray Dose”, <em>Frontiers in pharmacology</em>, Vol. 8, Frontiers Media SA, Lausanne, </span><a href="https://doi.org/10.3389/fphar.2017.00213" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.3389/fphar.2017.00213</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Boerma, M. et al. (2015), “Space radiation and cardiovascular disease risk”, <em>World Journal of Cardiology</em>, Vol. 7/12, Baishideng Publishing Group, Pleasanton, </span><a href="https://doi.org/10.4330/wjc.v7.i12.882" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.4330/wjc.v7.i12.882</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Bonetti, P. O., L. O. Lerman and A. Lerman (2003), “Endothelial dysfunction: a marker of atherosclerotic risk”, <em>Arteriosclerosis, thrombosis, and vascular biology</em>, Vol. 23/2, Lippincott Williams & Wilkins, Philadelphia, </span><a href="https://doi.org/10.1161/01.atv.0000051384.43104.fc " style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1161/01.atv.0000051384.43104.fc</span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Deanfield, J. E., J. P. Halcox and T. J. Rabelink (2007), “Endothelial Function and Dysfunction”, <em>Circulation</em>, Vol. 115/10, Lippincott Williams & Wilkins, Philadelphia, </span><a href="https://doi.org/10.1161/CIRCULATIONAHA.106.652859" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1161/CIRCULATIONAHA.106.652859</span></a><span style="font-size:12.0pt">. </span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">Donaubauer, A. J. et al. (2020), “The Influence of Radiation on Bone and Bone Cells-Differential Effects on Osteoclasts and Osteoblasts”, <em>International journal of molecular sciences</em>, Vol. 21/7, Multidisciplinary Digital Publishing Institute, Basel, </span></span></span><a href="https://doi.org/10.3390/ijms21176377" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.3390/ijms21176377</span></a><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">. </span></span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:#212121">Finch, W., K. Shamsa, M. S. Lee (2014), “Cardiovascular complications of radiation exposure”, <em>Reviews in Cardiovascular Medicine</em>, Vol. 15/3, IMR Press, </span></span><a href="https://doi.org/10.3909/ricm0689" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.3909/ricm0689</span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">Hatoum, O. A. et al. (2006), “Radiation Induces Endothelial Dysfunction in Murine Intestinal Arterioles via Enhanced Production of Reactive Oxygen Species”, <em>Arteriosclerosis, Thrombosis, and Vascular Biology</em>, Vol. 26/2, Lippincott Williams & Wilkins, Philadelphia, </span></span></span><a href="https://doi.org/10.1161/01.ATV.0000198399.40584.8c" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1161/01.ATV.0000198399.40584.8c</span></span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">Hughson, R.L., A. Helm and M. Durante (2018), “Heart in space: Effect of the extraterrestrial environment on the cardiovascular system”, <em>Nature Reviews Cardiology</em>, Vol. 15/3, Nature Portfolio, London, </span></span></span><a href="https://doi.org/10.1038/nrcardio.2017.157" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1038/nrcardio.2017.157</span></a><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">. </span></span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="background-color:white"><span style="color:black">Konukoglu, D., and H. Uzun (2017), “Endothelial Dysfunction and Hypertension”, <em>Advances in experimental medicine and biology</em>, Vol. 956, Springer, New York, </span></span><a href="https://doi.org/10.1007/5584_2016_90" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/5584_2016_90</a><span style="background-color:white"><span style="color:black">. </span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Korpela, E., and S. K. Liu (2014), “Endothelial perturbations and therapeutic strategies in normal tissue radiation damage”, <em>Radiation oncology, </em>Vol. 9, BioMed Central, London, </span><a href="https://doi.org/10.1186/s13014-014-0266-7" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1186/s13014-014-0266-7</a>. </span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. https://doi.org/10.1080/09553002.2022.2110306</span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">Krüger-Genge, A. et al. (2019), “Vascular Endothelial Cell Biology: An Update”, <em>International Journal of Molecular Sciences</em>, Vol. 20/18, Multidisciplinary Digital Publishing Institute, Basel, </span></span></span><a href="https://doi.org/10.3390/IJMS20184411" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.3390/IJMS20184411</span></a><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">. </span></span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="background-color:white"><span style="color:black">On, Y. K. et al. (2001), “Vitamin C prevents radiation-induced endothelium-dependent vasomotor dysfunction and de-endothelialization by inhibiting oxidative damage in the rat”, <em>Clinical and experimental pharmacology & physiology</em>, Vol. 28/10, Wiley-Blackwell, Hoboken, </span></span><a href="https://doi.org/10.1046/j.1440-1681.2001.03528.x" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1046/j.1440-1681.2001.03528.x</a><span style="background-color:white"><span style="color:black">. </span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="background-color:white"><span style="color:black">Ramadan, R. et al. (2021), “The role of connexin proteins and their channels in radiation-induced atherosclerosis”, <em>Cellular and molecular life sciences: CMLS</em>, Vol. 78/7, Springer, New York, </span></span><a href="https://doi.org/10.1007/s00018-020-03716-3" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/s00018-020-03716-3</a>. </span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="background-color:white"><span style="color:black">Shen, Y. et al. (2018), “Transplantation of bone marrow mesenchymal stem cells prevents radiation-induced artery injury by suppressing oxidative stress and inflammation”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2018, Hindawi, London, </span></span><a href="https://doi.org/10.1155/2018/5942916" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1155/2018/5942916</a><span style="background-color:white"><span style="color:black">. </span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Soloviev, A. I. et al. (2003), “Mechanisms of endothelial dysfunction after ionized radiation: selective impairment of the nitric oxide component of endothelium-dependent vasodilation”, <em>British journal of pharmacology</em>, Vol. 138/5, Wiley, </span><a href="https://doi.org/10.1038/sj.bjp.0705079" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1038/sj.bjp.0705079</span></a></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Soucy, K. G. et al. (2011), “HZE 56Fe-Ion Irradiation Induces Endothelial Dysfunction in Rat Aorta: Role of Xanthine Oxidase”, <em>Radiation Research</em>, Vol. 176/4, Radiation Research Society, Bozeman, </span><a href="https://doi.org/10.1667/RR2598.1" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1667/RR2598.1</span></a><span style="font-size:12.0pt">. </span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, <em>Journal of Applied Physiology</em>, Vol. 108/5, American Physiological Society, Rockville, </span><a href="https://doi.org/10.1152/japplphysiol.00946.2009" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1152/japplphysiol.00946.2009</span></a><span style="font-size:12.0pt">. </span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Sylvester, C. B. et al. (2018), “Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer”, <em>Frontiers in Cardiovascular Medicine</em>, Vol. 5, Frontiers Media SA, Lausanne, </span><a href="https://doi.org/10.3389/fcvm.2018.00005" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.3389/fcvm.2018.00005</span></a><span style="font-size:12.0pt">.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">Verma, S., M. R. Buchanan and T. J. Anderson (2003), “Endothelial function testing as a biomarker of vascular disease”, <em>Circulation</em>, Vol. 108/17, Lippincott Williams & Wilkins, Philadelphia, </span></span></span><a href="https://doi.org/10.1161/01.CIR.0000089191.72957.ED" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1161/01.CIR.0000089191.72957.ED</span></a><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">.</span></span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">Wang, Y., M. Boerma and D. Zhou (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, <em>Radiation research</em>, Vol. 186/2, Radiation Research Society, Bozeman, </span></span></span><a href="https://doi.org/10.1667/RR14445.1" style="color:#0563c1; text-decoration:underline" target="_blank"><span style="font-size:12.0pt"><span style="background-color:white">https://doi.org/10.1667/RR14445.1</span></span></a><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">. </span></span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Yentrapalli, R. et al. (2013a), “The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation”, <em>PloS one</em>, Vol. 8/8, PLOS, San Francisco, </span><a href="https://doi.org/10.1371/journal.pone.0070024" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1371/journal.pone.0070024</span></a><span style="font-size:12.0pt">. </span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Yentrapalli, R. et al. (2013b), “Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low-dose rate gamma radiation”, <em>Proteomics</em>, Vol. 13/7, John Wiley & Sons, Ltd., Hoboken, </span><a href="https://doi.org/10.1002/pmic.201200463" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt">https://doi.org/10.1002/pmic.201200463</span></a><span style="font-size:12.0pt">. </span></span></span></p>
2022-09-28T12:44:532023-03-21T10:37:2681eb87ac-fb24-4667-bd3a-9be52450f362f487d2a3-b9c7-4777-958c-8ebecaa04071<p><span style="font-family:Times New Roman,Times,serif">The increase in reactive oxygen species (ROS) and reactive nitrogen species (RNS) during a state of oxidative stress can stimulate an increase in pro-inflammatory mediators. Reactive oxygen and nitrogen species (RONS) cause cellular damage, which leads to the production of pro-inflammatory mediators (Slezak et al., 2015; Sylvester et al., 2018; Wang et al., 2019a). In addition, ROS can act as second messenger signalling molecules in activating pro-inflammatory transcription factor nuclear factor kappa B (NF-κB), resulting in increased production of pro-inflammatory cytokines and adhesion factors (Ping et al., 2020; Slezak et al., 2017; Slezak et al., 2015; Sylvester et al., 2018; Venkatesulu et al., 2018; Wang et al., 2019a). The inflammatory state induced by RONS will further increase RONS, leading to a cycle of chronic inflammation and oxidative stress (Venkatesulu et al., 2018; Wang et al., 2019a).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: Moderate</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The biological plausibility of the linkage between oxidative stress and pro-inflammatory mediators is strongly supported by review papers on the subject (Ping et al., 2020; Ramadan et al., 2021; Slezak et al., 2017; Slezak et al., 2015; Sylvester et al., 2018; Venkatesulu et al., 2018; Wang et al., 2019a). Pro-inflammatory mediators are released in instances of cell damage to recruit macrophages, monocytes, and other scavengers to ingest and degrade dead and damaged cells. As a major pathway of cell damage, oxidative stress causes upregulation of pro-inflammatory mediators including NF-κB, transforming growth factor-β (TGF-β), tumour necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) (Ping et al., 2020; Slezak et al., 2017; Slezak et al., 2015; Sylvester et al., 2018; Venkatesulu et al., 2018; Wang et al., 2019a). Oxidative stress also stimulates a rise in pro-inflammatory adhesion factors, such as E-selectin, intercellular adhesion molecule-1 (ICAM1), and vascular cell adhesion molecule-1 (VCAM1), which facilitate inflammation by assisting the entrance of inflammatory cells into tissues and recruiting macrophages (Ping et al., 2020; Slezak et al., 2017; Slezak et al., 2015; Sylvester et al., 2018; Venkatesulu et al., 2018; Wang et al., 2019a). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Once antioxidant levels become exhausted in a state of oxidative stress, ROS are present in higher concentrations and can therefore act more effectively as second messenger signalling molecules in activating pro-inflammatory transcription factors, such as NF-κB, and stimulating production of pro-inflammatory cytokines, such as IL-1, IL-6 and TNF-α (Ping et al., 2020; Sylvester et al., 2018; Wang et al., 2019a). NF-κB is normally kept in an inactive state through formation of a complex with the IkB family of inhibitor proteins but is activated by oxidative stress through nuclear translocation of the complex to the promoter areas of inflammation regulatory genes (Ping et al., 2020; Slezak et al., 2017). The macrophages that are recruited in the resulting inflammatory response can also produce ROS and activate the pro-inflammatory mediator, TGF-β, forming a positive feedback loop (Venkatesulu et al., 2018). Another positive feedback loop is formed by ROS and NF-κB, as ROS activates NF-κB, resulting in the expression of the genes cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LPO), which are responsible for ROS production (Ping et al., 2020). In addition, NF-κB is also involved in the production of the pro-inflammatory adhesion factors ICAM and VCAM (Ping et al., 2020; Slezak et al., 2017; Slezak et al., 2015). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Oxidative stress may also result in oxidation of low-density lipoproteins, allowing the lipoproteins to be ingested by macrophages. This could initiate the atherosclerotic process (plaque build-up in the arteries) and subsequently lead to lipid cells secreting pro-inflammatory cytokines, such as IL-1β and TGF-β (Ramadan et al., 2021; Slezak et al., 2017; Sylvester et al., 2018; Ping et al., 2020; Venkatesulu et al., 2018). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">The empirical evidence relevant to this KER provides moderate support for the linkage between increases in oxidative stress and increases in pro-inflammatory mediators. The evidence to support this relationship comes from studies examining the effects of ionizing radiation, such as <sup>137</sup>Cs gamma-rays, on the cardiovascular system. There is moderate evidence for a dose- and time-dependent relationship between oxidative stress and pro-inflammatory mediators (Abdel-Magied & Shedid, 2019; Chen et al., 2019; Cho et al., 2017; Ismail et al., 2015; Ismail et al., 2016; Karam et al., 2019; Philipp et al., 2020; Wang et al., 2019a). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Dose Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Current literature on the dose-dependent relationship between oxidative stress and increases in pro-inflammatory mediators is moderate. Chen et al. (2019) exposed male Sprague Dawley rats to simulated microgravity for 7 and 21 days and measured levels of the oxidative stress marker H<sub>2</sub>O<sub>2</sub> along with the pro-inflammatory mediators, IL-6, interferon-gamma (IFN-γ), and TNF-α. Their finding provided evidence of dose concordance between oxidative stress and pro-inflammatory mediators, as the study observed more significant changes to H2O2 levels at 7 days than 21 days, while IL-6, IFN-γ, and TNF-α levels were more significantly affected at day 21 than day 7 (Chen et al., 2019). Philipp et al. (2020) irradiated human telomerase-immortalized coronary artery endothelial cells (TICAE cells) with 0.25, 0.5, 2, and 10 Gy of 137Cs gamma rays. They found that superoxide dismutase (SOD) decreased consistently at 2 Gy, while pro-inflammatory mediators showed consistent increases at 2 or 10 Gy, but not at lower doses. Other studies using gamma rays show levels of oxidative stress markers increase with subsequent increases to pro-inflammatory mediators following exposure to high doses (>2 Gy), with some markers being significantly affected at low doses (<2 Gy) (Abdel-Magied & Shedid, 2019; Cho et al., 2017; Ismail et al., 2016; Ismail et al., 2015; Karam et al., 2019). However, not all studies demonstrated dose concordance. Wang et al. (2019b) irradiated human endothelial cells with various doses of gamma rays. They found pro-inflammatory mediators significantly increased at lower doses (0.2-5Gy) than oxidative stress (5 Gy only), although this could be due to the sensitivity of the assays. </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Time Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Current literature on the time-dependent relationship between oxidative stress and increases in pro-inflammatory mediators is low. Cho et al. (2017) irradiated male C57BL/6 mice and measured levels of superoxide and the pro-inflammatory mediators TNF-α and monocyte chemoattractant protein (MCP-1), at 4, 8, and 24 hours post-irradiation. Oxidative stress and pro-inflammatory mediators demonstrated a time concordant relationship, as ROS levels were significantly increased at 4 hours post-irradiation, while both pro-inflammatory mediators did not significantly change until 8 hours (Cho et al., 2017). Philipp et al. (2020) irradiated telomerase immortalized human coronary artery endothelial cells and measured levels of the antioxidant, SOD1, and the pro-inflammatory adhesion factor, ICAM1, at 4 hours, 24 hours, 48 hours, and 1 week post-irradiation. Neither SOD1 nor ICAM1 followed consistent changes over time across all doses. However, the earliest decrease in SOD1 and the earliest increase in ICAM1 were both found at 4 hours (Philipp et al., 2020). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Incidence concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Few studies demonstrated incidence concordance between oxidative stress and increased pro-inflammatory mediators. In human TICAE cells irradiated with 2 Gy of gamma rays, SOD1 was decreased 0.5-fold at 24h post-irradiation, while ICAM1 was increased 1.1-fold at this dose and time (Philipp et al., 2020). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Essentiality </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Studies that treated models with countermeasures to suppress the increase in oxidative stress caused by ionizing radiation exposure found that subsequent increases in pro-inflammatory mediators were also significantly attenuated (Abdel-Magied & Shedid, 2019; Karam et al., 2019). Blocking ionizing radiation effect on oxidative stress (upstream KE) and analyzing the subsequent effect on pro-inflammatory mediators (downstream KE) provided evidence for essentiality between the KEs. </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Treatment of irradiated albino rat heart tissue with the antidiabetic drug, metformin (50 mg/kg daily for 2 weeks), provided evidence for its efficacy as an antioxidant and anti-inflammatory drug. Metformin treatment following irradiation resulted in a recovery of SOD and catalase (CAT) activity to 90% and 44%, respectively, compared to irradiated groups. In addition to attenuating ionizing radiation effect on oxidative stress, metformin mitigated increases to NF-κB, TNF-α, and IL-6 levels, as well as reduced elevated E-selectin, ICAM, and VCAM levels by 0.5-, 0.55-, and 0.6-fold, respectively (Karam et al., 2019). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Irradiated Wistar albino rats were treated with the food and drug additive ZnO-NP (10 mg/kg daily for 2 weeks), which has antioxidant effects. Treatment with ZnO-NPs attenuated all IR-induced changes to oxidative and inflammatory markers. Compared to the irradiated group, ZnO-NP treatment resulted in restoration of CAT, SOD, glutathione (GSH), and glutathione peroxidase (GPx) levels by ~104%, ~73%, ~91%, and ~73%, respectively. Elevated levels of ICAM, TNF-α, IL-18, and C-reactive protein (CRP) were reduced by ~44%, ~46%, ~45%, and ~42%, respectively, compared to irradiated groups (Abdel-Magied & Shedid, 2019). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Irradiated Wistar rats were treated with flaxseed oil (FSO) (500 mg/kg-bw daily for 7 days), which has a uniquely high content of the antioxidant lignans. Treatment with FSO significantly reduced IR-induced changes to both antioxidants and pro-inflammatory mediators. IR-induced reductions to the activity of the antioxidants SOD, CAT, and GSH-Px were significantly alleviated back to levels statistically similar to the controls. Reduced levels of pro-inflammatory mediators, TNF-α, IL-1β, and IL-6, back to control levels was also observed (Ismail et al., 2016).</span></p>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Times New Roman",serif">Chen et al. (2019) found that levels of the pro-inflammatory mediators, <span style="color:black">IL-6, IFN-γ</span>, and <span style="color:black">TNF-α</span>, decreased following 7 and 21 days of microgravity exposure, contrary to the trend generally observed following ionizing radiation exposure (Chen et al., 2019).</span></span></span></li>
</ul>
<p><span style="font-family:Times New Roman,Times,serif">Several examples of studies that provide quantitative understanding of the relationship are summarized. All data represented is statistically significant unless otherwise indicated.</span></p>
HighMaleLowFemaleLowUnspecificLowJuvenileLowAdultModerateNot Otherwise SpecifiedLowHighModerate<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">Most evidence defining the relationship is derived from mice or rat models. A low number of <em>in-vitro</em> human studies were available. Males have been studied more often than females. The age of the models remained unspecified in several studies, while a few studies reported evidence from adult and adolescent models.</span></span></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Abdel-Magied, N. and S. M. Shedid (2019), “Impact of zinc oxide nanoparticles on thioredoxin-interacting protein and asymmetric dimethylarginine as biochemical indicators of cardiovascular disorders in gamma-irradiated rats”, <em>Environmental Toxicology</em>, Vol. 35, John Wiley & Sons, Ltd., Hoboken, <a href="https://doi.org/10.1002/tox.22879" rel="noreferrer noopener" target="_blank">https://doi.org/10.1002/tox.22879</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Chen, B. et al. (2019), “The Impacts of Simulated Microgravity on Rat Brain Depended on Durations and Regions”, <em>Biomedical and Environmental Sciences</em>, Vol. 32/7, Elsevier, Amsterdam, <a href="https://doi.org/10.3967/bes2019.067" rel="noreferrer noopener" target="_blank">https://doi.org/10.3967/bes2019.067</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Cho, H. J. et al. (2017), “Role of NADPH oxidase in radiation-induced pro-oxidative and pro-inflammatory pathways in mouse brain”, <em>International Journal of Radiation Biology</em>, Vol. 93/11, Informa, London, <a href="https://doi.org/10.1080/09553002.2017.1377360" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2017.1377360</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Ismail, A. F. M., F.S.M. Moawed and M. A. Mohamed (2015), “Protective mechanism of grape seed oil on carbon tetrachloride-induced brain damage in γ-irradiated rats”, <em>Journal of Photochemistry and Photobiology B: Biology</em>, Vol. 153, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.jphotobiol.2015.10.005" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.jphotobiol.2015.10.005</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Ismail, A. F. M., A. A. M. Salem and M. M. T. Eassawy (2016), “Modulation of gamma-irradiation and carbon tetrachloride induced oxidative stress in the brain of female rats by flaxseed oil”, <em>Journal of Photochemistry and Photobiology B: Biology</em>, Vol. 161, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.jphotobiol.2016.04.031" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.jphotobiol.2016.04.031</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Karam, H. M. and R. R. Radwan (2019), “Metformin modulates cardiac endothelial dysfunction, oxidative stress and inflammation in irradiated rats: A new perspective of an antidiabetic drug”, <em>Clinical and Experimental Pharmacology and Physiology</em>, Vol. 46/12, Wiley-Blackwell, Hoboken, <a href="https://doi.org/10.1111/1440-1681.13148" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/1440-1681.13148</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. <a href="http://doi.org/10.1080/09553002.2022.2110306">https://doi.org/10.1080/09553002.2022.2110306</a></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Philipp, J. et al. (2020), “Radiation Response of Human Cardiac Endothelial Cells Reveals a Central Role of the cGAS-STING Pathway in the Development of Inflammation”, <em>Proteomes</em>, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/proteomes8040030" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/proteomes8040030</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Ping, Z. et al. (2020), “Review Article Oxidative Stress in Radiation-Induced Cardiotoxicity”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2020, Hindawi, London, <a href="https://doi.org/10.1155/2020/3579143" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2020/3579143</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Ramadan, R. et al. (2021), “The role of connexin proteins and their channels in radiation-induced atherosclerosis”, <em>Cellular and molecular life sciences: CMLS</em>, Vol. 78/7, Springer, London, <a href="https://doi.org/10.1007/s00018-020-03716-3" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00018-020-03716-3</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Slezak, J. et al. (2017), “Potential markers and metabolic processes involved in the mechanism of radiation-induced heart injury”, <em>Canadian Journal of Physiology and Pharmacology</em>, Vol. 95/10, Canadian Science Publishing, Ottawa, <a href="https://doi.org/10.1139/cjpp-2017-0121" rel="noreferrer noopener" target="_blank">https://doi.org/10.1139/cjpp-2017-0121</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Slezak, J. et al. (2015), “Mechanisms of cardiac radiation injury and potential preventive approaches”, <em>Canadian Journal of Physiology and Pharmacology</em>, Vol. 93/9, Canadian Science Publishing, Ottawa, <a href="https://doi.org/10.1139/CJPP-2015-0006" rel="noreferrer noopener" target="_blank">https://doi.org/10.1139/CJPP-2015-0006</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Sylvester, C. B. et al. (2018), “Radiation-induced Cardiovascular Disease: Mechanisms and importance of Linear energy Transfer”, <em>Frontiers in Cardiovascular Medicine</em>, Vol. 5, Frontiers Media SA, Lausanne, <a href="https://doi.org/10.3389/fcvm.2018.00005" rel="noreferrer noopener" target="_blank">https://doi.org/10.3389/fcvm.2018.00005</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Venkatesulu, B. P. et al. (2018), “Radiation-Induced Endothelial Vascular Injury: A Review of Possible Mechanisms”, <em>JACC: Basic to translational science</em>, Vol. 3/4, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.jacbts.2018.01.014" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.jacbts.2018.01.014</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Wang, H. et al. (2019a), “Radiation-induced heart disease: a review of classification, mechanism and prevention”, <em>International Journal of Biological Sciences</em>, Vol. 15/10, Ivyspring International Publisher, Sydney, <a href="https://doi.org/10.7150/ijbs.35460" rel="noreferrer noopener" target="_blank">https://doi.org/10.7150/ijbs.35460</a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Wang, H. et al. (2019b), “Gamma Radiation-Induced Disruption of Cellular Junctions in HUVECs Is Mediated through Affecting MAPK/NF-κB Inflammatory Pathways”, <em>Oxidative medicine and cellular longevity</em>, Vol. 2019, Hindawi, London, <a href="https://doi.org/10.1155/2019/1486232" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2019/1486232</a>. </span></p>
2022-09-28T12:41:202023-03-21T14:44:43f1940943-69e5-458b-8c03-896b1c61c3f9ce63fcef-8305-4862-ad5a-db8fc07c2ae8<p><span style="font-family:Times New Roman,Times,serif">Deposition of energy can trigger vascular remodeling through many pathways (Tapio, 2016) including changes to vessel structure and blood flow (Patel, 2020; Sylvester et al., 2018). Pro-inflammatory mediators can be increased, which can result in a low level of inflammation causing intimal thickening (Sylvester et al., 2018). Deposition of energy can generate reactive oxygen species (ROS) and highly reactive radicals sparsely from low- linear energy transfer (LET) radiation and densely from high-LET radiation, which can cause endothelial dysfunction and subsequent vascular remodeling (Boerma et al., 2015; Hughson, Helm & Durante, 2017; Slezak et al., 2017; Soloviev & Kizub, 2019; Sylvester et al., 2018). Increased production of ROS changes the bioavailability of nitric oxide (NO), a diffusible molecule responsible for vasodilation, which leads to inhibited vasomotion and cellular senescence as components of endothelial dysfunction (Patel, 2020; Soloviev & Kizub, 2019). Changes in the expression or activity of proteins in many signaling pathways can lead to endothelial dysfunction (Schmidt-Ullrich et al., 2000; Tapio, 2016). In addition, the increased pro-inflammatory mediators can lead to endothelial dysfunction and therefore, vascular remodeling (Tapio, 2016). Another possible vascular remodeling change is age accelerated atherosclerosis (EPRI, 2020; Hamada et al., 2014). Studies using varying LET, delivered at acute and chronic dose-rates, have shown remodeling of the vasculature (reviewed in Tapio, 2016).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: High</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The biological plausibility suggesting that deposition of energy leads to vascular remodeling is well-supported by reviews and mechanistic studies published in the literature. Vascular remodeling may occur due to aging and diet (Zieman, Melenovsky & Kass, 2005). However, the deposition of energy from ionizing radiation (IR) can accelerate vascular remodeling in the form of accelerated atherosclerosis (Boerma et al., 2015; Boerma et al., 2016; EPRI, 2020; Hamada et al., 2014; Hughson, Helm & Durante, 2017; Mitchell et al., 2019; Sylvester et al., 2018), which can be demonstrated by arterial thickening or the amount of oxidized low-density lipoprotein (oxLDL) (Poznyak et al., 2021). Remodeling normally allows adaptation to long-term hemodynamic changes but can also contribute to vascular diseases (Gibbons & Dzau, 1994). Various short-term post-spaceflight studies have shown vascular remodeling after deposition of energy from space IR (Patel, 2020). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif"> Under physiological conditions, the body maintains a balance of ROS and NO levels. IR generates ROS that can react with NO and reduce its bioavailability, causing endothelial dysfunction and vascular stiffness (Patel, 2020). Similarly, signaling pathways can cause vascular remodeling through endothelial dysfunction and altered NO (Tapio, 2016). Increased ROS or altered signaling can cause a prolonged inflammatory response; this has been observed in animal models exposed to high-LET radiation (Hughson, Helm & Durante, 2017; Sylvester et al., 2018; Tapio, 2016). The low level of inflammation results in intimal thickening and inhibits tissue and vessel recovery (Sylvester et al., 2018). Microvascular injury and inflammation may cause angiogenesis, which prevents vascular resistance (Slezak et al., 2017). However, depending on the source, radiation may have different effects on angiogenesis (Grabham & Sharma, 2013). An increase in pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), secreted as a consequence of photon irradiation can promote angiogenesis. Exposure to low-LET protons and high-LET heavy ion radiation can disturb angiogenesis due to decreased VEGF secretion and tubule formation (Grabham & Sharma, 2013; Sylvester et al., 2018). Matrix metalloproteinases (MPPs) are involved in remodeling of the extracellular matrix (ECM) and can affect various pathological processes after irradiation (Slezak et al., 2017). Following radiation, existing collagen in the heart may be remodeled, which indicates ECM remodeling (Shen et al., 2018; Sridharan et al., 2020; Zieman, Melenovsky & Kass, 2005). Thus, many mechanisms exist via which the deposition of energy can lead to vascular remodeling; these are generally well understood and described in the literature, leading to a strong weight of evidence for the biological plausibility of this KER. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">There is moderate empirical evidence supporting the connection between deposition of energy leading to vascular remodeling. The evidence was gathered from studies using <em>in vivo</em> rat and mouse models, as well as <em>in vitro</em> models of human vessels and <em>ex vivo</em> human vessel biopsies (Grabham et al., 2011; Hamada et al., 2020; Hamada et al., 2021; Hamada et al., 2022; Russel et al., 2009; Sarkozy et al., 2019; Shen et al., 2018; Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011; Sridharan et al., 2020; Yu et al., 2011). The models used stressors such as iron ions (<sup>56</sup>Fe) (Soucy et al., 2011; Yu et al., 2011), gamma rays (Hamada et al., 2020; Hamada et al., 2021; Hamada et al., 2022; Soucy et al., 2007; Soucy et al., 2010), X-rays (Hamada et al., 2021; Hamada et al., 2022; Shen et al., 2018), electrons (Sarkozy et al., 2019), protons and oxygen ions (<sup>16</sup>O) (Sridharan et al., 2020). Vascular stiffness measured by pulse wave velocity (PWV) and vascular composition (Hamada et al., 2020; Hamada et al., 2021; Hamada et al., 2022; Shen et al., 2018; Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011; Sridharan et al., 2020), vessel thickness/diameter measured directly (Sarkozy et al., 2019; Shen et al., 2018; Soucy et al., 2011; Sridharan et al., 2020; Yu et al., 2011) and other morphological changes (Hamada et al., 2020; Hamada et al., 2021; Hamada et al., 2022) were used as endpoints for vascular remodeling.</span></p>
<p> </p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Dose Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">PWV is often measured to determine vascular stiffness and therefore remodeling, where PWV is quadratically proportional to Young's modulus (measure of a material’s stiffness) (Pereira, Correia & Cardoso, 2015; Soucy et al., 2011). The PWV of irradiated rats increased with an increase in radiation dose. Across several studies, 0.5, 1, 1.6, and 5 Gy doses were tested, with responses of 1.1 to 1.2-fold changes to PWV (Soucy et al., 2007, 2010, 2011). PWV is also quadratically proportional to the vessel diameter and inversely quadratically proportional to the vessel wall thickness. However, Soucy et al. (2011) found no significant change in the aortic wall thickness:lumen diameter ratio, indicating the change in PWV was due to changes in vessel composition and not geometric remodeling. Collagen composition in the membrane was also measured to determine vascular composition and therefore remodeling, with increased collagen indicating stiffness (Zieman, Melenovsky & Kass, 2005). Collagen accumulation increased 1.4-fold after 18 Gy X-ray irradiation (Shen et al., 2018) and 1.5-fold after various regiments of 5 Gy X-rays (Hamada et al., 2021; Hamada et al., 2022) in mice. Following 12 months of 16O exposure, the tissue content of collagen type III peptide increased 2.3-fold and 2-fold at 0.05 Gy and 0.25 Gy, respectively (Sridharan et al, 2020). VE-cadherin, a marker for adherens junctions, decreased 0.2- to 0.3-fold in mice after various regimens of 5 Gy gamma and X-rays, with a high decrease in the acute and fractionated doses and no change after chronic gamma rays (Hamada et al., 2022; Hamada et al., 2021; Hamada et al., 2020). oxLDL increased in mice after X-ray irradiation at both 8 and 16 Gy; however, the increase was not greater at 16 Gy relative to 8 Gy (Azimzadeh et al., 2015). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Vessel thickness was also measured in various studies. Aortic thickness increased 1.4-fold after 18 Gy X-ray irradiation of mice (Shen et al., 2018). Iron ion irradiation on apolipoprotein E (apoE)-deficient mice, a model for atherosclerosis, at both 2 and 5 Gy showed a 1.4-fold increase in carotid artery intima thickness compared to sham-irradiated apoE-deficient mice (Yu et al., 2011). Markers of vascular remodeling, anterior and inferior wall thicknesses in systole (AWTs and IWTs) and diastole (AWTd and IWTd) were measured after a high dose of 50 Gy electrons. AWTs increased 1.3-fold, IWTs increased 1.1-fold, AWTd increased 1.5-fold and IWTd increased 1.2-fold (Sarkozy et al., 2019). In turn, patients having undergone radiation treatment for head and neck or breast cancer with radiation doses totaling 66 Gy and 49 Gy, respectively, observed increased intima-media ratios (IMR) of 1.5- and 1.4-fold, respectively (Russel et al., 2009). Mice that did not show endothelial detachments before irradiation showed high frequencies of detachments after acute doses of 5 Gy gamma and X-rays (Hamada et al., 2020; Hamada et al., 2021). Vascular permeability was also significantly increased after 5 Gy gamma rays in mice (Hamada et al., 2020). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Radiation type can affect the nature of vascular remodeling following exposure. A study using 3D vessel models of endothelial cells in a gel matrix showed that high-LET iron-ions reduces vessel length in both mature and developing vessels after only 0.8 Gy. In contrast, low-LET protons only inhibited growth in developing vessels at 0.8 Gy and required a dose of 3.2 Gy to affect mature vessels. Gamma radiation required a dose of 0.8 Gy to inhibit vessel growth and 6.4 Gy for any significant breakdown of mature vessels (Grabham et al., 2011).</span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Time Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">In rats irradiated with 5 Gy gamma rays, PWV as a measure of arterial stiffness increased from 3.9 m/s before irradiation to around 4.5 m/s after 1 day, 1 week and 2 weeks (Soucy et al., 2007). Under the same conditions, PWV increased from 4.1 m/s before irradiation to 4.6 m/s after 1 day, 4.9 m/s after 1 week, and 4.8 after 2 weeks in a separate study by the same authors (Soucy et al., 2010). Using 1 Gy iron ions instead, PWV increased by 0.5 m/s both 4 months and 8 months post-irradiation (Soucy et al., 2011). Mice irradiated with 18 Gy X-rays showed significantly increased aortic thickness 1.4-fold after 7 days and 1.3-fold after 14 and 28 days, while collagen accumulation increased 1.4-fold after 14, 28 and 84 days (Shen et al., 2018). ApoE-deficient mice that were irradiated with both 2 and 5 Gy iron ions showed a 1.4-fold increase in carotid artery intima thickness after 13 weeks compared to controls (Yu et al., 2011). Mice irradiated with 8 and 16 Gy of X-rays showed increased oxLDL levels at 16 weeks post-irradiation (Azimzadeh et al., 2015). Ventricular posterior wall thickness decreased at 3- and 5-months post-irradiation, and PWV increased at 12 months post-irradiation with <sup>16</sup>O irradiation (Sridharan et al., 2020). The results suggest that <sup>16</sup>O irradiation may lead to long-term vascular dysfunction in rats similar to iron ions in the studies by Soucy et al. (2011) and Yu et al. (2011). Collagen type III increased 2.4-fold after 12 months of 0.5 Gy proton irradiation (Sridharan et al., 2020). CD2, CD4 and CD8 markers for the T-protein lymphocytes that are thought to be involved with promoting hypertension and microvascular remodeling increased in rat hearts 6 months after a 0.1 Gy dose of 16O, and 12 months after a single 0.5 Gy dose of <sup>16</sup>O (Sridharan et al., 2020). Mice irradiated with 5 Gy gamma rays showed increased endothelial detachments, increased vascular permeability and VE-cadherin expression at 1, 3 and 6 months after irradiation (Hamada et al., 2020; Hamada et al., 2021). VE-cadherin was also increased 12 months after irradiation, but endothelial detachments were no longer present (Hamada et al., 2022). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Essentiality </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif"> Vascular remodeling such as arterial stiffness occurs naturally with aging, but deposition of energy can accelerate this process (Zieman, Melenovsky & Kass, 2005). Since deposited energy initiates events immediately, the removal of deposited energy also supports the essentiality of the key event. Studies that do not deposit energy are observed to have no downstream effects.</span></p>
<ul>
<li>
<p><span style="font-family:Times New Roman,Times,serif">Not all results show the expected dose-response. For example, total collagen and collagen type III peptide levels studied in Sridharan et al. (2020) did not consistently increase with increasing dose. Similarly, oxLDL levels were higher at the 8 Gy dose compared to the 16 Gy dose (Azimzadeh et al., 2015). </span></p>
</li>
<li>
<p><span style="font-family:Times New Roman,Times,serif">Yu et al. (2011) showed that intimal thickness increased at 13 weeks after iron ion irradiation of apoE-deficient mice. At 40 weeks post-irradiation, intimal thickness remained at similar levels, but the level was no longer statistically significant because the sham-irradiated group showed higher intimal thickness. </span></p>
</li>
</ul>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.</span></span></span></span></p>
HighMaleHighFemaleModerateJuvenileHighAdultHighModerateHigh<p><span style="font-family:Times New Roman,Times,serif">The relationship has been shown <em>in vivo</em> in mice and rats and <em>ex vivo</em> in human models. Majority of studies used males. Evidence came from either adult or adolescent animals. However, the relationship is plausible at any life stage.</span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="background-color:white"><span style="color:#212121">Andreassi, M. G. et al. (2015), “Subclinical Carotid Atherosclerosis and Early Vascular Aging From Long-Term Low-Dose Ionizing Radiation Exposure: A Genetic, Telomere, and Vascular Ultrasound Study in Cardiac Catheterization Laboratory Staff”, <em>JACC: Cardiovascular Interventions</em>, Vol. 8/4, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.jcin.2014.12.233" style="color:#0563c1; text-decoration:underline"><span style="background-color:white">https://doi.org/10.1016/j.jcin.2014.12.233</span></a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="background-color:white"><span style="color:#212121">Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, <em>Journal of Proteome Research</em>, Vol. 14/2, American Chemical Society, Washington, </span></span><a href="https://doi.org/10.1021/pr501141b" style="color:#0563c1; text-decoration:underline"><span style="background-color:white">https://doi.org/10.1021/pr501141b</span></a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="background-color:white"><span style="color:#212121">Berk, B. C. and V. A. Korshunov (2006), “Genetic determinants of vascular remodelling”, <em>The Candian Journal of Cardiology</em>, Vol. 22, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/s0828-282x(06)70980-1" style="color:#0563c1; text-decoration:underline"><span style="background-color:white">https://doi.org/10.1016/s0828-282x(06)70980-1</span></a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="background-color:white">Boerma, M. et al. (2016), “Effects of ionizing radiation on the heart”,<em> Mutation Research/Reviews in Mutation Research</em>, Vol. 770, Elsevier, Amsterdam, </span><a href="https://doi.org/10.1016/j.mrrev.2016.07.003" style="color:#0563c1; text-decoration:underline"><span style="background-color:white">https://doi.org/10.1016/j.mrrev.2016.07.003</span></a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="background-color:white">Boerma, M. et al. (2015), “Space radiation and cardiovascular disease risk”, <em>World Journal of Cardiology</em>, Vol. 7/12, Baishideng Publishing Group, Pleasanton, </span><a href="https://doi.org/10.4330/wjc.v7.i12.882" style="color:#0563c1; text-decoration:underline"><span style="background-color:white">https://doi.org/10.4330/wjc.v7.i12.882</span></a> </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Dorresteijn, L. D. A. et al. (2005), “Increased carotid wall thickening after radiotherapy on the neck”, <em>European Journal of Cancer</em>, Vol. 41/7, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.ejca.2005.01.020" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.ejca.2005.01.020</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">EPRI. (2020). <em>Cardiovascular Risks from Low Dose Radiation Exposure: Review and Scientific Appraisal of the Literature</em></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Gianicolo, M. E. et al. (2010), “Effects of external irradiation of the neck region on intima media thickness of the common carotid artery”, <em>Cardiovascular Ultrasound</em>, Vol. 8, Nature, <a href="https://doi.org/10.1186/1476-7120-8-8" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1186/1476-7120-8-8</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Gibbons, G. H., and V. J. Dzau (1994), “The Emerging Concept of Vascular Remodeling”, <em>New England Journal of Medicine</em>, Vol. 330/20, Massachusetts Medical Society, Waltham, <a href="https://doi.org/10.1056/NEJM199405193302008." style="color:#0563c1; text-decoration:underline">https://doi.org/10.1056/NEJM199405193302008.</a> </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Grabham, P. et al. (2011), “Effects of ionizing radiation on three-dimensional human vessel models: differential effects according to radiation quality and cellular development”, <em>Radiation Research, </em>Vol. 175, BioOne, <a href="https://doi.org/10.1667/RR2289.1" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1667/RR2289.1</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Grabham, P. and P. Sharma (2013), “The effects of radiation on angiogenesis”, <em>Vascular Cell</em>, Vol. 5/1, Publiverse Online S.R.L., Bucharest, <a href="https://doi.org/10.1186/2045-824X-5-19." style="color:#0563c1; text-decoration:underline">https://doi.org/10.1186/2045-824X-5-19</a>.</span></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Hamada, N. et al. (2022), “Temporal Changes in Sparing and Enhancing Dose Protraction Effects of Ionizing Irradiation for Aortic Damage in Wild-Type Mice”, Cancers, Vol. 14/14, MDPI, Basel, https://doi.org/10.3390/cancers14143319</span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Hamada, N. et al. (2021), “Vascular damage in the aorta of wild-type mice exposed to ionizing radiation: Sparing and enhancing effects of dose protraction”, <em>Cancers</em>, Vol.13/21, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/cancers13215344" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/cancers13215344</a>.</span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Hamada, N. et al. (2020), “Ionizing Irradiation Induces Vascular Damage in the Aorta of Wild-Type Mice”, <em>Cancers</em>, Vol. 12/10, </span>Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/CANCERS12103030." style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/CANCERS12103030.</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:#212121">Hamada, N. et al. (2014), “Emerging issues in radiogenic cataracts and cardiovascular disease”, <em>Journal of radiation research</em>, Vol. 55/5, Oxford University Press. </span><a href="https://doi.org/10.1093/jrr/rru036" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/jrr/rru036</a> </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Harvey, A., A. C. Montezano and R. M. Touyz (2015), “Vascular biology of ageing—Implications in hypertension”, <em>Journal of Molecular and Cellular Cardiology</em>, Vol. 83, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.yjmcc.2015.04.011" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.yjmcc.2015.04.011</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Hughson, R.L., A. Helm and M. Durante (2017), “Heart in space: Effect of the extraterrestrial environment on the cardiovascular system”, <em>Nature Reviews Cardiology</em>, Vol. 15/3, Nature Portfolio, London, <a href="https://doi.org/10.1038/nrcardio.2017.157." style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/nrcardio.2017.157.</a> </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Kessler, E. L. et al. (2019), “Sex-specific influence on cardiac structural remodeling and therapy in cardiovascular disease”, <em>Biology of Sex Differences</em>, Vol. 10/7, BioMed Central, London, <a href="https://doi.org/10.1186/s13293-019-0223-0" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1186/s13293-019-0223-0</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">King, L. J. et al. (1999), “Asymptomatic carotid arterial disease in young patients following neck radiation therapy for Hodgkin lymphoma”, <em>Radiology</em>, Vol. 213/1, Radiological Society of North America, <a href="https://doi.org/10.1148/radiology.213.1.r99oc07167" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1148/radiology.213.1.r99oc07167</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. https://doi.org/10.1080/09553002.2022.2110306</span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Mitchell, A. et al. (2019), “Cardiovascular effects of space radiation: implications for future human deep space exploration”, <em>European Journal of Preventive Cardiology</em>, Vol. 26/16, SAGE Publishing, Thousand Oaks, <a href="https://doi.org/10.1177/2047487319831497" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1177/2047487319831497</a> </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">North, B. J. and D. A. Sinclair (2012), “The Intersection Between Aging and Cardiovascular Disease”, <em>Circulation Research</em>, Vol. 110/8, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/CIRCRESAHA.111.246876" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/CIRCRESAHA.111.246876</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Patel, S. (2020), “The effects of microgravity and space radiation on cardiovascular health: From low-Earth orbit and beyond”, <em>IJC Heart and Vasculature, </em>Vol. 30, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.ijcha.2020.100595" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.ijcha.2020.100595</a>. </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Pereira, T., C. Correia and J. Cardoso (2015), “Novel Methods for Pulse Wave Velocity Measurement”, <em>Journal of Medical and Biological Engineering</em>, Vol. 35/5,<em> </em>Springer, New York, <a href="https://doi.org/10.1007/s40846-015-0086-8" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/s40846-015-0086-8</a>. </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Poznyak, A. V. et al. (2021), “Overview of OxLDL and Its Impact on Cardiovascular Health: Focus on Atherosclerosis”, <em>Frontiers in Pharmacology</em>, Vol. 11, Frontiers, <a href="https://doi.org/10.3389/fphar.2020.613780" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fphar.2020.613780</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Russel et al., (2009), “Novel insights into pathological changes in muscular arteries of radiotherapy patients”, <em>Radiotherapy and Oncology</em>, Vol. 92, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.radonc.2009.05.021" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.radonc.2009.05.021</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Sárközy, M. et al. (2019), “Selective heart irradiation induces cardiac overexpression of the pro-hypertrophic miR-212”, <em>Frontiers in Oncology, </em>Vol. 9, Frontiers Media SA, Lausanne, <a href="https://doi.org/10.3389/fonc.2019.00598" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fonc.2019.00598</a> </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Shen, Y. et al. (2018), “Transplantation of bone marrow mesenchymal stem cells prevents radiation-induced artery injury by suppressing oxidative stress and inflammation”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2018, Hindawi, London, <a href="https://doi.org/10.1155/2018/5942916" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1155/2018/5942916</a>. </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Schmidt-Ullrich, R. K. et al. (2000), “Signal Transduction and Cellular Radiation Responses”, <em>Radiation Research</em>, Vol. 153, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/0033-7587(2000)153%5b0245:STACRR%5d2.0.CO;2" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1667/0033-7587(2000)153[0245:STACRR]2.0.CO;2</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Slezak, J. et al. (2017), “Potential markers and metabolic processes involved in the mechanism of radiation-induced heart injury”, <em>Canadian journal of physiology and pharmacology</em>, Vol. 95/10, Canadian Science Publishing, Ottawa, <a href="https://doi.org/10.1139/cjpp-2017-0121" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1139/cjpp-2017-0121</a> </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soloviev, A. I. and I.V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, <em>Biochemical pharmacology</em>, Vol. 159, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.bcp.2018.11.019" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.bcp.2018.11.019</a> </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soucy, K. G. et al. (2011), “HZE 56Fe-Ion Irradiation Induces Endothelial Dysfunction in Rat Aorta: Role of Xanthine Oxidase”, <em>Radiation Research</em>, Vol. 176/4, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RR2598.1" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1667/RR2598.1</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, <em>Journal of Applied Physiology</em>, Vol. 108/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.00946.2009" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/japplphysiol.00946.2009</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soucy, K. G. et al. (2007), “Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta”, <em>Radiation and Environmental Biophysics</em>, Vol. 46/2, Springer, New York, <a href="https://doi.org/10.1007/s00411-006-0090-z" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/s00411-006-0090-z</a>. </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Sridharan, V. et al. (2020), “Effects of single-dose protons or oxygen ions on function and structure of the cardiovascular system in male Long Evans rats”, <em>Life Sciences in Space Research</em>, Vol. 26, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.lssr.2020.04.002" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.lssr.2020.04.002</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Sylvester, C. B. et al. (2018), “Radiation-induced Cardiovascular Disease: Mechanisms and importance of Linear energy Transfer”, <em>Frontiers in Cardiovascular Medicine</em>, Vol. 5, Frontiers Media SA, Lausanne, <a href="https://doi.org/10.3389/fcvm.2018.00005" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fcvm.2018.00005</a>. </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Tapio, S. (2016), “Pathology and biology of radiation-induced cardiac disease”, <em>Journal of Radiation Research</em>, Vol. 57/5, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jrr/rrw064" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/jrr/rrw064</a>. </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Ungvari, Z. et al. (2018), “Mechanisms of Vascular Aging”, <em>Circulation Research</em>, Vol. 123/7, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/CIRCRESAHA.118.311378" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/CIRCRESAHA.118.31137</a></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Winham, S. J., M. de Andrade and V. M. Miller (2015), “Genetics of cardiovascular disease: Importance of sex and ethnicity”, <em>Atherosclerosis, </em>Vol. 241/1, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.atherosclerosis.2015.03.021" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.atherosclerosis.2015.03.021</a> </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Yu, T. et al. (2011), “Iron-ion radiation accelerates atherosclerosis in apolipoprotein E-Deficient mice”, <em>Radiation Research</em>, Vol. 175/6, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RR2482.1." style="color:#0563c1; text-decoration:underline">https://doi.org/10.1667/RR2482.1</a>. </span></span></p>
<p><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Zieman, S. J., V. Melenovsky and D. A. Kass (2005), “Mechanisms, Pathophysiology, and Therapy of Arterial Stiffness”, <em>Arteriosclerosis, Thrombosis, and Vascular Biology</em>, Vol. 25/5, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/01.ATV.0000160548.78317.29" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/01.ATV.0000160548.78317.29</a> </span></span></p>
2022-10-17T16:54:292023-03-21T10:32:53908b38ae-23f7-446a-bb32-46656376b24b383161af-8fa6-4cd9-81d4-8c7c472b341d<p style="text-align:justify"><span style="font-family:Times New Roman,Times,serif">Multiple signaling pathways can regulate nitric oxide (NO) levels. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can activate nitric oxide synthase (NOS), an enzyme that produces NO, through phosphorylation (Hemmings & Restuccia 2012; Nagane et al., 2021). The RhoA/Rho kinase (ROCK) pathway inhibits both the expression and phosphorylation of NOS (Yao et al., 2010). Furthermore, the renin-angiotensin-aldosterone system (RAAS) can both inhibit NOS to reduce vasodilation and activate NOS as a countermeasure for vasoconstriction (Millatt, Abdel-Rahman & Siragy, 1999). The extracellular signal-regulated protein kinase 5 (ERK5)/kruppel-like factor 2 (KLF2) pathway can increase transcription of endothelial NOS (eNOS), which results in increased NO levels (Paudel, Fusi & Schmidt, 2021). Lastly, the acidic sphingomyelinase/ceramide pathway can activate NADPH oxidase (NOX) production of reactive oxygen species (ROS) that react with NO, resulting in lower NO levels (Soloviev & Kizub, 2019). Alterations in these pathways will result in altered NO levels. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: Moderate</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The biological plausibility surrounding the connection between altered signaling pathways and altered NO levels is well-supported by the literature. Studies have shown that altered signaling pathways lead to altered NO levels (Azimzadeh et al., 2015; Azimzadeh et al., 2017; Hasan, Radwan & Galal, 2019; Shi et al., 2012; Siamwala et al., 2010). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Multiple signaling pathways can influence NO levels. Under normal physiological conditions, the PI3K/Akt pathway is regulated by various growth factors and other signaling molecules to modulate eNOS phosphorylation and therefore NO production (Hemmings & Restuccia, 2012). Phosphorylation of eNOS can affect its function. Phosphorylation at Ser1177 activates the enzyme, while phosphorylation at Thr495 acts in reverse and decreases enzymatic activity instead (Forstermann, 2010; Nagane et al., 2021). In endothelial cells, Thr495 is constitutively phosphorylated by kinases like protein kinase C (PKC) (Forstermann, 2010). Following Akt activation, eNOS is phosphorylated on Ser1177 to activate the enzyme and thus NO production is upregulated (Karar & Maity, 2011). Although peroxisome proliferator-activated receptor α (PPARα) can increase eNOS directly through transcription, it can also increase vascular endothelial growth factor (VEGF) (Du, Wagner & Wagner, 2020), which can activate the PI3K pathway to activate eNOS (Hicklin & Ellis, 2005). Alternatively, activation of the RhoA/ROCK pathway leads to a decrease in NO bioavailability (Yao et al., 2010). The RhoA/ROCK pathway causes eNOS mRNA destabilization and prevents Ser1177 eNOS phosphorylation by Akt (Yao et al., 2010). However, phosphorylation of RhoGDI, a regulator in the RhoA/ROCK pathway, causes increased affinity to GTP-RhoA, sequestering the active form of RhoA and preventing eNOS inhibition (Dovas & Couchman, 2005). The RAAS pathway results in the production of angiotensin II (AngII) which can cause various downstream effects on vascular homeostasis, including inducing vasoconstriction (Millatt, Abdel-Rahman & Siragy, 1999). AngII can downregulate eNOS Ser1177 phosphorylation to prevent vasodilation (Ding et al., 2020; Millatt, Abdel-Rahman & Siragy, 1999), or activate eNOS as a corrective measure (Millatt, Abdel-Rahman & Siragy, 1999). As a result, depending on the mechanism, NO levels can either increase or decrease after AngII stimulation. The ERK5/KLF2 pathway results in activation of the KLF2 transcription factor, which increases transcription of eNOS (Paudel, Fusi & Schmidt, 2021).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The empirical evidence to support this KER is provided by <em>in vivo</em> mouse and rat models and <em>in vitro</em> models of human coronary artery endothelial cell (HCAECs), human umbilical vein endothelial cells (HUVECs), and EA.hy96 cells (a hybrid of HUVECs and A549 cells). The effects of altered signaling pathways on the levels of NO as well as inducible NOS (iNOS) and eNOS have been investigated. These studies examined levels of signaling molecules in insulin-dependent PI3K/Akt pathway such as IGFR1, PPARα, PI3K, Akt, and the phosphorylated versions of each (Azimzadeh et al., 2015; Azimzadeh et al., 2021; Shi et al., 2012), RAAS pathway indicator, AngII (Hasan, Radwan & Galal, 2019), phosphorylated Rho GDP-dissociation inhibitor (p-RhoGDI) in the RhoA/ROCK pathway (Azimzadeh et al., 2017), ERK5 and KLF2 (Sadhukhan et al., 2020), and the effect they have on NO. The studies used stressors such as X-rays (Azimzadeh et al., 2015; Azimzadeh et al., 2017; Azimzadeh et al., 2021), gamma rays (Hasan, Radwan & Galal, 2019; Sadhukhan et al., 2020) and altered gravity (Shi et al., 2012; Siamwala et al., 2010). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Dose Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is moderate evidence to demonstrate dose concordance between altered signaling pathways leading to altered NO levels. Activated RhoA reduces the activity and abundance of eNOS (Yao et al., 2010). HCAECs irradiated with 0.5 Gy demonstrated a 0.7-fold decrease in p-RhoGDI, a 0.6-fold decrease in p-eNOS and a 0.8-fold decrease in cellular NO, 7 days after exposure (Azimzadeh et al., 2017). The decrease in p-RhoGDI, involved in the RhoA/ROCK pathway, was correlated with a decrease in p-eNOS and NO (Azimzadeh et al., 2017). HSP90, a protein that binds to and activates eNOS (Karar & Maity, 2011), decreased 0.8-fold following 0.5 Gy X-ray irradiation (Azimzadeh et al., 2017). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">High doses (>2 Gy) also show dose concordance between altered signaling pathways and altered NO levels. X-ray irradiation of BAECs and HUVECs caused a 5.6-fold increase in p-Akt/Akt after 6 Gy (Sonveaux et al., 2003). eNOS increased 3-fold after 6 Gy, while p-eNOS increased 1.2-fold after 2 Gy and 1.7-fold after 6 Gy (Sonveaux et al., 2003). Although the p-Akt/Akt ratio was not significantly increased after 2 Gy, p-Akt alone had increased, indicating changes in the PI3K/Akt pathway had occurred at 2 Gy. Therefore, a decrease in PI3K/Akt pathway proteins, such as p-IGFR1, p-Akt and p-PI3K are correlated with decreases in p-eNOS and NO (Azimzadeh et al., 2015), while an increase in p-Akt correlates with increases in eNOS and p-eNOS (Shi et al., 2012; Sonveaux et al., 2003). In rat blood serum, 6 Gy gamma ray irradiation led to a 1.4-fold increase in AngII and aldosterone levels and a 3.3-fold increase in iNOS expression indicating that a change in the RAAS pathway can lead to altered NO levels (Hasan, Radwan & Galal, 2019). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Mice that received high dose (8 or 16 Gy) X-ray irradiation on the heart showed alterations in the levels of signalling proteins involved in the PI3K/Akt pathway, including a 0.4 and 0.3-fold decrease of p-IGFR1, a 0.5-fold decrease of PI3K, a 0.2 and 0.1-fold decrease of p-Akt and a 0.6 and 0.2-fold decrease of p-eNOS at 8 Gy and 16 Gy, respectively (Azimzadeh et al., 2015). Concurrently to changes to PI3K/Akt pathway signaling molecules, the level of serum NO decreased 0.3-fold and 0.3-fold following 8 Gy and 16 Gy X-ray irradiation, respectively (Azimzadeh et al., 2015). As well, following X-ray irradiation, the ERK/MAPK pathway decreased 0.5-fold at 16 Gy and the p38/MAPK pathway increased 1.3-fold at 16 Gy (Azimzadeh et al., 2015). X-ray irradiation in mice at 16 Gy also inhibited PPARα resulting in decreased activity and phosphorylation of eNOS through the PI3K/Akt pathway and decreased NO levels (Azimzadeh et al., 2021). This study also showed an increase in p-ERK, and p-p38 at 16 Gy (Azimzadeh et al., 2021). After irradiation of HUVECs with 10 or 12 Gy, p-ERK5, KLF2 and eNOS were all found to decrease after fractionated doses, while KLF2 and eNOS both decreased after acute doses (Sadhukhan et al., 2020). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Microgravity in HUVEC-C was simulated by a clinostat using rotation at a slow speed to negate centrifugal force. Following clinorotation, levels of eNOS and p-eNOS expression were significantly increased by 5.2-fold and 5.5-fold, respectively. As well, p-Akt increased by 2.9-fold after exposure to simulated microgravity in HUVEC-C (Shi et al., 2012). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Time Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is some evidence to demonstrate time concordance between altered signaling pathways leading to altered NO levels. After 0.5 Gy X-ray of HCAECs, p-Rho-GDI significantly decreased after 1 day, while NO was not significantly lower after 1 day (Azimzadeh et al., 2017). After 24 h, p-Akt was increased, while eNOS was increased 24 and 48 h after X-ray irradiation of BAECs and HUVECs (Sonveaux et al., 2003). After 7 days, p-Rho-GDI decreased further and NO showed a significant decrease. HUVECs irradiated with 10 or 12 Gy acute or fractionated X-rays showed decreased levels of p-ERK5, KLF2 and eNOS at 4 h, and decreased KLF2 and eNOS at 24 h after irradiation (Sadhukhan et al., 2020). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Incidence Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">The evidence of incidence concordance for this relationship is moderate, as a few studies demonstrated incidence concordance. In mice hearts irradiated with X-rays, p-Akt, the direct upstream activator of eNOS, decreased 0.2-fold after 8 Gy and 0.1-fold after 16 Gy, while NO decreased 0.3-fold after 8 Gy and 0.2-fold after 16 Gy (Azimzadeh et al., 2015). Similarly, after 0.5 Gy X-ray irradiation of HCAECs, p-RhoGDI decreased 0.7-fold while NO decreased 0.8-fold (Azimzadeh et al., 2017). X-ray irradiation of BAECs and HUVECs at 6 Gy resulted in a 5.6-fold increase in the ratio of p-Akt/Akt and a 3-fold increase to p-eNOS (Sonveaux et al., 2003). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Essentiality </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Several studies have investigated the essentiality of various signalling pathways in altering NO levels. Under normal conditions, the phosphorylation of eNOS by the PI3K/Akt pathway activates NOS, resulting in NO production. LY294002 treatment, a PI3K inhibitor, led to significant decreases in eNOS and p-eNOS levels in HUVEC-C samples (Shi et al., 2012). After exposure to simulated microgravity by clinorotation, LY294002 treatment led to decreased p-Akt levels to below control levels (Shi et al., 2012). The inhibition of the PI3K/Akt pathway with wortmanin, another PI3K inhibitor, also led to a 0.3-fold decrease in NO production (Siamwala et al., 2010). Irradiation inhibited PPARα and eNOS, while treatment with fenofibrate, a PPARα activator, kept both p-PPARα and NO at control levels (Azimzadeh et al., 2021). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Bradykinin-potentiating factor (BPF) is implicated in muscle contraction, inflammatory responses and angiotensin-converting enzyme (ACE) inhibition. ACE in endothelial cells converts AngI to AngII. Irradiation at 6 Gy led to increased iNOS, AngII, and aldosterone, while the subsequent treatment with BPF decreased all endpoints measured following irradiation, including the levels of iNOS, AngII, and aldosterone. The recovery of iNOS serum levels in 6 Gy gamma irradiated rats following BPF treatment indicates that the signaling pathways play a role in NO levels (Hasan, Radwan & Galal, 2019). In addition, bradykinin signaling through bradykinin receptor 2 (B2R) activates eNOS (Ancion et al., 2019). Nitrite concentration was found to increase after both microgravity and treatment with bradykinin (Siamwala et al., 2010). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Various inhibitors of the mevalonate pathway were used to recover KLF2 levels after irradiation, which resulted in increased KLF2 and eNOS levels (Sadhukhan et al., 2020). </span></p>
<ul>
<li><span style="font-family:Times New Roman,Times,serif">Due to the high reactivity of NO, it can be difficult to obtain its direct measures (Luiking, Engelen & Deutz, 2010). The inconsistencies in NO levels may be attributed to the challenges in measuring NO. Directionality of NO changes cannot be compared between studies due to a variety of experimental conditions like stressor type, dose, dose rate, model and time course of the experiment.</span></li>
</ul>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.</span></span></span></span></p>
LowMaleLowFemaleModerateUnspecificLowAdultModerateNot Otherwise SpecifiedLowModerateModerate<p><span style="font-family:Times New Roman,Times,serif">The majority of the evidence for this KER is from rat and mouse models. Most evidence regarding sex and lifestage is unspecified with a small amount of evidence from adult models.</span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Ancion, A. et al. (2019), “A Review of the Role of Bradykinin and Nitric Oxide in the Cardioprotective Action of Angiotensin-Converting Enzyme Inhibitors: Focus on Perindopril”, <em>Cardiology and Therapy</em>, Vol. 8/2, Springer, London, </span><a href="https://doi.org/10.1007/S40119-019-00150-W" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/S40119-019-00150-W</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Azimzadeh, O. et al.</span> (2021), “Activation of pparα by fenofibrate attenuates the effect of local heart high dose irradiation on the mouse cardiac proteome”, <em>Biomedicines</em>, Vol. 9/12, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/biomedicines9121845" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/biomedicines9121845</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, <em>International Journal of Radiation Biology</em>, Vol. 93/9, Informa, London, </span><a href="https://doi.org/10.1080/09553002.2017.1339332" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1080/09553002.2017.1339332</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, <em>Journal of Proteome Research</em>, Vol. 14/2, American Chemical Society, Washington, </span><a href="https://doi.org/10.1021/pr501141b" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1021/pr501141b</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Ding, J. et al. (2020), “Angiotensin II Decreases Endothelial Nitric Oxide Synthase Phosphorylation <em>via</em> AT1R Nox/ROS/PP2A Pathway”, <em>Frontiers in physiology</em>, Vol.11, Frontiers Media SA, Lausanne, </span><a href="https://doi.org/10.3389/fphys.2020.566410" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fphys.2020.566410</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Dovas, A. and J. R. Couchman (2005), “RhoGDI: multiple functions in the regulation of Rho family GTPase activities”, <em>Biochemical Journal</em>, Vol. 390, Portland Press, London, </span><a href="https://doi.org/10.1042/BJ20050104" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1042/BJ20050104</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Du, S., N. Wagner and K. D. Wagner (2020), “The Emerging Role of PPAR Beta/Delta in Tumor Angiogenesis”, <em>PPAR Research</em>, Vol. 2020, Hindawi, London, <a href="https://doi.org/10.1155/2020/3608315" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1155/2020/3608315</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Förstermann, U. (2010), “Nitric oxide and oxidative stress in vascular disease”, <em>Pflugers Archiv : European journal of physiology</em>, Vol. 459/6, Springer, London, </span><a href="https://doi.org/10.1007/s00424-010-0808-2" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/s00424-010-0808-2</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Hasan, H. F., R. R. Radwan and S. M. Galal (2019), “Bradykinin‐potentiating factor isolated from Leiurus quinquestriatus scorpion venom alleviates cardiomyopathy in irradiated rats via remodelling of the RAAS pathway”, <em>Clinical and Experimental Pharmacology and Physiology</em>, Vol. 47/2, Wiley-Blackwell, Hoboken, </span><a href="https://doi.org/10.1111/1440-1681.13202" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1111/1440-1681.13202</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Hemmings, B. A. and D. F. Restuccia (2012), “PI3K-PKB/Akt Pathway”, <em>Cold Spring Harbor Perspectives in Biology</em>, Vol. 4/9, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, </span><a href="https://doi.org/10.1101/CSHPERSPECT.A011189" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1101/CSHPERSPECT.A011189</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Hicklin, D. J. and L. M. Ellis (2005), “Role of the Vascular Endothelial Growth Factor Pathway in Tumor Growth and Angiogenesis”, <em>Journal of Clinical Oncology</em>, Vol. 23/5, American Society of Clinical Oncology, Virginia, <a href="https://doi.org/10.1200/JCO.2005.06.081" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1200/JCO.2005.06.081</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Karar, J. and A. Maity (2011), “PI3K/AKT/mTOR Pathway in Angiogenesis”, <em>Frontiers in Molecular Neuroscience</em>, Vol. 4, Frontiers Media SA, Lausanne, </span><a href="https://doi.org/10.3389/FNMOL.2011.00051" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/FNMOL.2011.00051</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:1rem">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, </span><em style="font-size:1rem">International Journal of Radiation Biolog</em><span style="font-size:1rem">y, Vol. 98/12. <a href="http://doi.org/10.1080/09553002.2022.2110306">https://doi.org/10.1080/09553002.2022.2110306</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Luiking, Y. C., M. P. Engelen and N. E. Deutz (2010), “Regulation of nitric oxide production in health and disease”, <em>Current Opinion in Clinical Nutrition and Metabolic Care</em>, Vol. 13/1, Lippincott Williams and Wilkins Ltd., Philadelphia, </span><a href="https://doi.org/10.1097/MCO.0b013e328332f99d" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1097/MCO.0b013e328332f99d</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Millatt, L. J., E. M. Abdel-Rahman and H. M. Siragy (1999), “Angiotensin II and nitric oxide: a question of balance”, <em>Regulatory Peptides</em>, Vol. 81/1-3, Elsevier, Amsterdam, </span><a href="https://doi.org/10.1016/S0167-0115(99)00027-0" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/S0167-0115(99)00027-0</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases”, <em>Journal of Radiation Research</em>, Vol. 62/4, Oxford University Press, Oxford, </span><a href="https://doi.org/10.1093/JRR/RRAB032" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/JRR/RRAB032</a> </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Paudel, R., L. Fusi and M. Schmidt (2021), “The MEK5/ERK5 Pathway in Health and Disease”, <em>International Journal of Molecular Sciences</em>, Vol. 22/14, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/ijms22147594" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/ijms22147594</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Sadhukhan, R. et al. (2020), “Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose”, <em>Scientific Reports</em>, Vol. 10/1, Springer, London, <a href="https://doi.org/10.1038/s41598-020-64672-3" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/s41598-020-64672-3</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Shi, F. et al. (2012), “Effects of Simulated Microgravity on Human Umbilical Vein Endothelial Cell Angiogenesis and Role of the PI3K-Akt-eNOS Signal Pathway”, <em>PLoS ONE</em>, Vol. 7/7, PLOS, San Francisco, </span><a href="https://doi.org/10.1371/journal.pone.0040365" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1371/journal.pone.0040365</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Siamwala, J. H. et al. (2010), “Simulated microgravity perturbs actin polymerization to promote nitric oxide-associated migration in human immortalized Eahy926 cells”, <em>Protoplasma</em>, Vol. 242/1, Springer, London, </span><a href="https://doi.org/10.1007/S00709-010-0114-Z" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/S00709-010-0114-Z</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Soloviev, A. I. and I.V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, <em>Biochemical pharmacology</em>, Vol. 159, Elsevier, Amsterdam, </span><a href="https://doi.org/10.1016/j.bcp.2018.11.019" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.bcp.2018.11.019</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Sonveaux, P. et al. (2003), “Irradiation-induced Angiogenesis through the Up-Regulation of the Nitric Oxide Pathway: Implications for Tumor Radiotherapy”, <em>Cancer Research</em>, Vol. 63/5, American Association for Cancer Research, Philadelphia, </span><a href="https://aacrjournals.org/cancerres/article/63/5/1012/511021/Irradiation-induced-Angiogenesis-through-the-Up" style="color:#0563c1; text-decoration:underline">https://aacrjournals.org/cancerres/article/63/5/1012/511021/Irradiation-induced-Angiogenesis-through-the-Up</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"><span style="color:black">Yao, L. et al. (2010), “The role of RhoA/Rho kinase pathway in endothelial dysfunction”, <em>Journal of Cardiovascular Disease Research</em>, Vol. 1/4, Elsevier, Amsterdam, </span><a href="https://doi.org/10.4103/0975-3583.74258" style="color:#0563c1; text-decoration:underline">https://doi.org/10.4103/0975-3583.74258</a></span></span></p>
2022-09-28T12:41:392023-03-21T11:44:5581eb87ac-fb24-4667-bd3a-9be52450f362383161af-8fa6-4cd9-81d4-8c7c472b341d<p><span style="font-family:Times New Roman,Times,serif">The increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during oxidative stress can lead to altered nitric oxide (NO) levels, specifically a reduction in its bioavailability. </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Although RNS can also interfere with NO levels, most studies focus on ROS and not RNS (Nagane et al., 2021). Oxidative stress influences the production and activity of endothelial nitric oxide synthase (eNOS), thereby altering and reducing NO levels and its bioavailability. eNOS, otherwise known as NOS3, is an enzyme that catalyzes NO production from the amino acid L-arginine in vascular endothelial cells. NO mediates vascular tone and blood flow via the activation of soluble guanylate cyclase (sGC) within the vascular smooth muscle (Chen, Pittman and & Popel, 2008). A form of ROS known as superoxide anion (O<sub>2</sub><sup>-</sup>) causes increased NO degradation (Incalza et al., 2018), converting NO into the RNS peroxynitrite. In addition, ROS can uncouple eNOS by oxidation of the enzyme’s cofactor, BH4 (Matsubara et al., 2015; Forstermann, 2010). Uncoupled eNOS produces ROS instead of NO, which can further convert existing NO into peroxynitrite (Forstermann, 2010). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: Moderate</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The biological rationale for the relationship between increased oxidative stress and altered NO levels is well-supported by the literature, validated by many studies presented in this area of research. The biological mechanism of this relationship is well-known and widely accepted. The reaction between NO and free radicals leads to the creation of peroxynitrite, which results in reduced NO bioavailability (Incalza et al., 2018; Mitchell et al., 2019; Nagane et al., 2021; Soloviev & Kizub, 2019; Wang, Boerma & Zhou, 2016). In addition, in vascular tissues, increased levels of O2- or peroxynitrite can oxidize BH4 and lead to uncoupled eNOS (Forstermann, 2010; Matsubara et al., 2015; Soloviev & Kizub, 2019). When uncoupled, eNOS transfers electrons to O2- rather than L-arginine, causing O2- production instead of NO production, further reducing NO bioavailability. Although much of the biological plausibility indicates that NO decreases with oxidative stress, ROS have been associated with increased NO as well (Nagane et al., 2021; Soloviev & Kizub, 2019). This is likely due to the complexity of NO regulation in signaling pathways, upregulation of inducible nitric oxide synthase (iNOS), as well as variations in stressors, doses, dose rates, models, duration of study and diseases present in studies (Nagane et al., 2021).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The empirical data for this KER somewhat supports the relationship of increased oxidative stress eventuating in altered NO levels. The evidence was gathered from both <em>in vivo</em> and <em>in vitro</em> models. Gamma rays (Abdel-Magied & Shedid, 2020; Hasan, Radwan & Galal, 2019; Soucy et al., 2010), X-rays (Cervelli et al., 2017; Yan et al., 2020), altered gravity (Zhang et al., 2009) and heavy ions (Soucy et al., 2011) were used as stressors with dose levels ranging from 0.25 to 10 Gy. Direct and indirect measures of NO, including iNOS, eNOS and nitrite levels, were used as endpoints to investigate the effect of increased oxidative stress on NO levels. Evidence from radiation sources that induce oxidative stress in human umbilical vein endothelial cells (HUVECs) or rat aorta have demonstrated a change in NO levels (Abdel-Magied & Shedid, 2020; Cervelli et al., 2017; Hasan, Radwan & Galal, 2019; Soucy et al., 2010; Soucy et al., 2011; Yan et al., 2020; Zhang et al., 2009). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif"><strong>Dose Concordance</strong></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Moderate evidence is available within current literature that demonstrates dose-concordance between increased oxidative stress and altered NO levels. Elevations in the production of ROS have been reported with exposure to altered gravity and different doses (0.25 Gy, 1 Gy, 4 Gy, 5 Gy, and 10 Gy) of radiation in HUVECs and rat aorta/tissue models, and provide supporting evidence that an increase in oxidative stress can lead to subsequent alterations in NO levels (Abdel-Magied & Shedeed, 2020; Cervelli et al., 2017; Hasan, Radwan & Galal, 2019; Sakata et al., 2015; Soucy et al., 2010; Soucy et al., 2011; Yan et al., 2020; Zhang et al., 2009). For example, a study involving HUVEC X-ray irradiation at 0.25 Gy found increases in both ROS and nitrite/nitrate levels (Cervelli et al., 2017). As well, a study involving 1 Gy of iron ions demonstrated elevations in ROS production with accompanying reductions in NO levels using the DAF-FM DA fluorescent probe (Soucy et al., 2011). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">At high doses (>2 Gy), dose concordance between increased oxidative stress and altered nitric oxide can be observed. For example, 5 Gy gamma irradiation leads to increased ROS production with decreased NO levels measured using the DAF-FM DA fluorescent probe (Soucy et al., 2010) Gamma irradiation of rats with 6 Gy led to changes in oxidative stress indices that showed an increase in ROS, while iNOS levels were also increased (Hasan, Radwan & Galal, 2019). Rat cardiac tissue exposed to 8 Gy gamma irradiation also showed an increase in ROS through oxidative stress indices and an increase in nitrite/nitrate content (Abdel-Magied & Shedid, 2020). A study investigating HUVECs and rat aorta/tissue responses to 10 Gy and 4 Gy X-ray radiation, respectively, showed reduced eNOS dimerization and nitrite levels with elevations in ROS production in both models (Yan et al., 2020). Also in HUVECs, 10 Gy X-rays resulted in increased ROS, increased p-eNOS (Ser1177), decreased p-eNOS (Thr495), increased citrulline and increased NOx (nitrite and nitrate, NO proxies) (Sakata et al., 2015). NOx was also significantly increased after 20 Gy (Sakata et al., 2015). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Simulated microgravity showed increased eNOS and iNOS in rats, while superoxide levels, although not quantitatively shown, increased following altered gravity (Zhang et al., 2009). Although, the authors state that the increase in NOS would likely cause decreased NO production because of NOS uncoupling and peroxynitrite production (Zhang et al., 2009). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif"><strong>Time Concordance </strong></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Very few studies demonstrate time concordance of this relationship. In HUVECs irradiated with 0.25 Gy of X-rays, ROS levels increased after 45 minutes, while nitrite and nitrate levels increased after 24 hours. In rats exposed to 6 Gy of gamma rays, both oxidative stress indices and iNOS increased 4 weeks post-irradiation (Hasan, Radwan & Galal, 2019). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif"><strong>Incidence concordance </strong></span></p>
<p><span style="font-family:Times New Roman,Times,serif">There is moderate evidence of incidence concordance for this relationship. Yan et al. (2020) showed both <em>in vivo</em> in a rat model irradiated with 4 Gy of X-rays and <em>in vitro</em> in HUVECs irradiated with 10 Gy of X-rays that superoxide levels increased more than NO and eNOS were decreased following irradiation. Rats irradiated with 1 Gy of <sup>56</sup>Fe ions showed a 1.8-fold increase in ROS and just a 0.8-fold decrease in NO levels (Soucy et al., 2011). Similarly, gamma irradiation of rats at 5 Gy resulted in a 1.7-fold increase in ROS and a 0.7-fold decrease in NO levels (Soucy et al., 2010). In HUVECs irradiated with 10 Gy of X-rays, ROS increased 15.5-fold while NOx levels increased just 10-fold (Sakata et al., 2015).</span></p>
<p> </p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif"><strong>Essentiality </strong></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Studies scrutinizing the usefulness of antioxidants in inhibiting oxidative stress show a moderately supported relationship between elevated oxidative stress and altered NO levels. Antioxidants have been implicated in the activation of the NOS family of enzymes by preventing BH4 oxidation, thereby increasing NO bioavailability (Kojsova et al., 2006). One study observed that treatment with bradykinin potentiating factor (BPF), which can affect eNOS regulation through the renin aldosterone angiotensin system, contributed to elevations in the antioxidant glutathione (GSH) and a marker of antioxidant potential, ferric reducing antioxidant power (FRAP) (Hasan, Radwan & Galal, 2019). BPF contributed to reductions in malondialdehyde (MDA), a biomarker for oxidative stress, while also increasing iNOS levels (Hasan, Radwan & Galal, 2019). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Another study demonstrated that a composition of antioxidants (resveratrol, extramel, seleno-L-methionine, Curcuma longa, reduced glutathione, and vitamin C (RiduROS) inhibited increases in ROS while restoring NO levels (Cervelli et al., 2017). Furthermore, rat mesenteric arteries and HUVECs treated with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of Gch1 that is important for BH4 synthesis, lowered both the dimer:monomer eNOS ratio and the nitrite (measure of NO) concentration, and increased superoxide following irradiation (Yan et al., 2020). The notable elevation in O2- demonstrates that inhibiting Gch1 and limiting BH4 activity can prevent eNOS activity and elevate ROS Levels (Yan et al., 2020). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">In addition, two studies demonstrated that treatment with oxypurinol (OXP), a xanthine oxidase (XO) inhibitor where XO is responsible for generating cardiac ROS, led to reductions in ROS well under the control level whereas NO increased to control levels demonstrating that oxidative damage contributes to a decrease in NO bioavailability (Soucy et al., 2010; Soucy et al., 2011). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Zinc oxide nanoparticles (ZnO-NPs) can act as antioxidants; Abdel-Magied & Shedid (2020) found that low concentrations (10 mg/kg ZnO-NPs) decreased oxidation and NO levels in rats after gamma irradiation. Angiotensin II type 1 (AT1) receptors are able to regulate NOS levels. Treatment with losartan, an AT1 receptor antagonist, led to a decrease in O2- , iNOS and eNOS levels, demonstrating that the increase in NOS and ROS can be prevented by blocking AT1 receptors (Zhang et al., 2009). Biotin was found to increase GSH content, superoxide dismutase (SOD), catalase (CAT) activity and return NOx levels to near control values following irradiation in hippocampus (Abdel-Magied & Shedid, 2020).</span></p>
<ul>
<li><span style="font-family:Times New Roman,Times,serif">The directionality of changes to NO is inconsistent between studies, as some studies show increased NO levels and other studies show decreased NO levels. Improved methods are needed to assess NO levels directly, which may facilitate an understanding of the relationship (Cervelli et al., 2017, Hasan, Radwan & Galal, 2019). This, along with variation in experimental conditions, can account for the inconsistencies in NO changes between studies.</span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.</span></span></span></span></span></p>
ModerateMaleLowFemaleLowUnspecificModerateAdultLowJuvenileLowHigh<p><span style="font-family:Times New Roman,Times,serif">The evidence for the taxonomic applicability to humans is low as evidence comes from <em>in vitro</em> human cell-derived models. Many studies use <em>in vivo</em> rat models, predominately males. Occasionally, animal age is not specified in studies; most studies indicate the animals are adult or adolescent. In addition, the relationship is also plausible in preadolescent animals.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Abdel-Magied, N. and S. M. Shedid (2020), “Impact of zinc oxide nanoparticles on thioredoxin-interacting protein and asymmetric dimethylarginine as biochemical indicators of cardiovascular disorders in gamma-irradiated rats”, <em>Environmental Toxicology</em>, Vol. 35, Wiley, <a href="http://doi.org/10.1002/tox.22879">https://doi.org/10.1002/tox.22879</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Cervelli, T. et al. (2017), “A new natural antioxidant mixture protects against oxidative and DNA damage in endothelial cell exposed to low-dose irradiation”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2017, Hindawi, London, <a href="https://doi.org/10.1155/2017/9085947" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2017/9085947</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Chen, K., R. N. Pittman and A. S. Popel (2008), “Nitric oxide in the vasculature: where does it come from and where does it go? A quantitative perspective”, <em>Antioxidants & redox signaling</em>, Vol. 10/7, Mary Ann Liebert, Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2007.1959" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2007.1959</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Förstermann, U. (2010), “Nitric oxide and oxidative stress in vascular disease”, <em>Pflugers Archiv : European journal of physiology</em>, Vol. 459/6, Springer, Berlin, <a href="https://doi.org/10.1007/s00424-010-0808-2" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00424-010-0808-2</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Hasan, H. F., R. R. Radwan and S. M. Galal (2019), “Bradykinin‐potentiating factor isolated from Leiurus quinquestriatus scorpion venom alleviates cardiomyopathy in irradiated rats via remodelling of the RAAS pathway”, <em>Clinical and Experimental Pharmacology and Physiology</em>, Vol. 47/2, Wiley-Blackwell, Hoboken, <a href="https://doi.org/10.1111/1440-1681.13202" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/1440-1681.13202</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Incalza, M. A. et al. (2018), “Oxidative stress and reactive oxygen species in endothelial dysfunction associated with cardiovascular and metabolic diseases”, <em>Vascular pharmacology</em>, Vol. 100, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.vph.2017.05.005" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.vph.2017.05.005</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Kojsová, S. et al. (2006), “The effect of different antioxidants on nitric oxide production in hypertensive rats”, <em>Physiological research</em>, Vol. 55, Czech Academy of Sciences, Prague, <a href="https://doi.org/10.33549/physiolres.930000.55.S1.3." rel="noreferrer noopener" target="_blank">https://doi.org/10.33549/physiolres.930000.55.S1.3. </a> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. <a href="http://doi.org/10.1080/09553002.2022.2110306">https://doi.org/10.1080/09553002.2022.2110306</a></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Matsubara, K. et al. (2015), “Nitric oxide and reactive oxygen species in the pathogenesis of preeclampsia”, <em>International journal of molecular sciences</em>, Vol. 16/3, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/ijms16034600" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/ijms16034600</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Mitchell, A. et al. (2019), “Cardiovascular effects of space radiation: implications for future human deep space exploration”, <em>European Journal of Preventive Cardiology</em>, Vol. 26/16, SAGE Publishing, Thousand Oaks, <a href="https://doi.org/10.1177/2047487319831497" rel="noreferrer noopener" target="_blank">https://doi.org/10.1177/2047487319831497</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases”, <em>Journal of Radiation Research</em>, Vol. 62/4, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/JRR/RRAB032" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/JRR/RRAB032</a>. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Sakata, K. et al. (2015). “Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells”, <em>Vascular Pharmacology</em>, Vol. 70, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.vph.2015.03.016" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.vph.2015.03.016</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Soloviev, A. I. and I.V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, <em>Biochemical pharmacology</em>, Vol. 159, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.bcp.2018.11.019" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.bcp.2018.11.019</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Soucy, K. G. et al. (2011), “HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, <em>Radiation Research</em>, Vol. 176/4, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RR2598.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RR2598.1</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, <em>Journal of Applied Physiology</em>, Vol. 108/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.00946.2009" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/japplphysiol.00946.2009</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Wang, Y., M. Boerma and D. Zhou (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, <em>Radiation research</em>, Vol. 186/2, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RR14445.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RR14445.1</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Yan, T. et al. (2020), “Ionizing radiation induces BH4 deficiency by downregulating GTP-cyclohydrolase 1, a novel target for preventing and treating radiation enteritis”, <em>Biochemical Pharmacology</em>, Vol. 180, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.bcp.2020.114102" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.bcp.2020.114102</a>.</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, <em>Journal of applied physiology</em>, Vol. 106/1, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.01278.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/japplphysiol.01278.2007</a>. </span></p>
2022-09-28T12:41:552023-03-21T11:35:5681eb87ac-fb24-4667-bd3a-9be52450f36242ee9daa-42e3-49b5-9aa0-9dc2b7d09c20<p><span style="font-family:Times New Roman,Times,serif">Oxidative stress describes the imbalances in reactive oxygen and reactive nitrogen species (RONS) radical formation as well as antioxidants and reactive oxygen species (ROS) scavengers (Beckhauser et al., 2016; Elahi et al., 2009; Ray et al., 2012). Oxidative stress can lead to endothelial dysfunction. </span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="font-family:Times New Roman,Times,serif">Within the cardiovascular system, every vessel is lined with a single layer of endothelial cells (Augustin et al., 1994; Fishman, 1982). This endothelial layer plays a crucial role in the regulation of vascular homeostasis through controlling various factors such as vascular permeability, vasomotion, and immune response (Baran et al., 2021; Bonett</span>i et al., 2003; Hughson et al., 2018; Slezak et al., 2017; Sylvester et al., 2018). Of the vascular wall components, the endothelium is also the most vulnerable to damage from ROS (Soloviev & Kizub, 2018). Endothelial cells normally exist in a quiescent state characterized by high nitric oxide (NO) bioavailability (Carmeliet & Jain, 2011); however, cells can become activated as part of a normal host-defence response following tissue injury or oxidative stress (Deanfield et al., 2007; Krüger-Genge et al., 2019). Sustained activation leads to the pathological state of endothelial dysfunction which is defined by decreased NO bioavailability, increased vessel permeability, altered vasomotion, and a pro-thrombotic and inflammatory environment (Baran et al., 2021; Bonetti et al., 2003; Deanfield et al., 2007; Schiffrin, 2008).</span></span></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Shifting redox balance towards oxidation is known to indirectly lead to endothelial dysfunction through various mechanisms (Hughson et al., 2018; Ramadan et al., 2020; Soloviev & Kizub, 2018). There are several ways through which imbalanced ROS can affect endothelium function, including decreasing NO bioavailability through direct scavenging, which forms the RNS peroxynitrite (ONOO<sup>-</sup>) (Hatoum et al., 2006; Li et al., 2002; Schiffrin, 2008; Soloviev & Kizub, 2018; Venkatesulu et al., 2018), as well as impeding NO production and diffusion (Hatoum et al., 2006; Li et al., 2002; Schiffrin, 2008; Soloviev & Kizub, 2018; Venkatesulu et al., 2018; Schiffrin, 2008; Soloviev & Kizub, 2018). Additionally, elevated ROS contribute to introducing a pro-inflammatory and pro-thrombotic milieu characteristic of dysfunction (Hughson et al., 2018; Schiffrin, 2008; Slezak et al., 2017; Tapio, 2016; Venkatesulu et al., 2018). It is also linked to decreased vasomotion (Schiffrin, 2008; Soloviev & Kizub, 2018; Venkatesulu et al., 2018) and finally the onset of endothelial cell apoptosis and premature senescence (Borghini et al., 2013; Hughson et al., 2018; Tapio, 2016; Wang et al., 2016).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: Moderate</span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Mechanisms for oxidative stress leading to endothelial dysfunction are outlined in various reviews on the topic (Hughson et al., 2018; Nagane et al., 2021; Slezak et al., 2017; Soloviev & Kizub, 2018; Venkatesulu et al., 2018; Wang et al., 2016).</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Elevated ROS can indirectly lead to endothelial dysfunction by causing an imbalance of NO, specifically the decrease in NO bioavailability. Firstly, ROS can react with NO directly; if quenching outpaces NO production, it will cause reduced NO bioavailability underlying endothelial dysfunction (Hatoum et al., 2006; Li et al., 2002; Soloviev & Kizub, 2018). In particular, the superoxide anion (</span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">•</span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">O<sup>-</sup><sub>2</sub>) reacts with NO to form peroxynitrite, both reducing available NO and further accelerating NO degradation (Li et al., 2002; Soloviev & Kizub, 2018). In addition, superoxide and peroxynitrite can uncouple eNOS which produces more ROS instead of NO (Soloviev & Kizub, 2018). Peroxynitrite can cause cellular senescence as a part of endothelial dysfunction (Nagane et al., 2021). eNOS downregulation and subsequent drop in NO levels are caused in part by increased endothelin-1 (ET-1), a vasoconstrictor with enhanced secretion during an oxidative stress state (Marasciulo, </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Montagnani & Potenza,</span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"> 2006; Ramadan et al., 2020). ROS is also involved in perturbing NO diffusion from the endothelial cells (Soloviev & Kizub, 2018). Overall, the decreased NO bioavailability causes reduced vasodilation and endothelial dysfunction (Soloviev & Kizub, 2018).</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Oxidative stress also affects endothelial function through inhibition of endothelium-dependent vasodilation (Soloviev & Kizub, 2018; Venkatesulu et al., 2018). ROS in both endothelial cells and surrounding vascular smooth muscle cells (VSMCs) act as second messengers to many cellular pathways that mediate VSMC contractility and endothelial permeability and function, causing disruption to these endothelial functions (Hughson et al., 2018; Li et al., 2002; Ramadan et al., 2020; Soloviev & Kizub, 2018; Ungvari et al., 2013; Venkatesulu et al., 2018). Specifically, impaired endothelium-dependent vasomotion following radiation (Venkatesulu et al., 2018) was suggested to be due to the loss of PGF2α inhibition and therefore, vasoconstriction (Li et al., 2002). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Oxidative stress is also involved in inducing the pro-thrombotic and inflammatory environment of endothelial dysfunction. In the case of radiation induced endothelial injury, radiation type, fraction size used, and endothelial cell model used all influence the resulting downstream endpoints (Venkatesulu et al., 2018). Possible changes to the endothelial milieu include alterations of cell adhesion molecule levels, creation of pro-thrombotic environment, endothelial cell apoptosis and inflammation (Hughson et al., 2018; Nagane et al., 2021; Slezak et al., 2017; Tapio, 2016; Venkatesulu et al., 2018). When induced by oxidative stress, nuclear factor kappa B (NF-кB) can target genes involved with the upregulation of prothrombotic markers associated with endothelial dysfunction (Slezak et al., 2017). Free radicals produced by macrophages have also been shown to stimulate TGF-β, thus accelerating the creation of a profibrotic milieu (Venkatesulu et al., 2018). ROS can also oxidize low-density lipoproteins (LDL) resulting in structural complications as oxidized LDL accumulates in blood circulation due to decreased cell uptake (Nagane et al., 2021; Slezak et al., 2017). Furthermore, endothelial cells can undergo morphological changes following oxidative injury, as the cells become enlarged, and form fibrin networks, showing increased levels of activated platelets and leukocytes with membrane protrusions and pseudopodial extensions, which are all indicative of an inflammatory and pro-thrombotic state (Li et al., 2002). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Furthermore, ROS can induce premature endothelial cell senescence, which in turn contributes to overall endothelial dysfunction (Hughson et al., 2018; Nagane et al., 2021; Tapio, 2016). In contrast to replicative senescence attributed to telomere dysfunction, oxidative stress is one of several injuries causing stress-induced premature senescence (Nagane et al., 2021). This is thought to occur through oxidative stress causing the induction of the p53/p21 pathway which regulates cell senescence (Borghini et al., 2013; Wang et al., 2016). Once senescent, the endothelial cells contribute to dysfunction in multiple ways. Firstly, senescence can stimulate a pro-inflammatory response and trigger apoptosis through decreased cell repair (Nagane et al., 2021; Ramadan et al., 2020). Additionally, senescent cells themselves are sources of ROS, furthering both genomic instability causing additional senescence in neighbouring cells and endothelial dysfunction itself (Tapio, 2016; Wang et al., 2016). Senescent cells also lack proper endothelial cell function, contributing to changing the environment to a dysfunctional one (Hughson et al., 2018; Tapio, 2016). This lack of function includes a decrease in NO production, increased monocyte adhesion, and loss of cell barrier integrity paired with increased levels of ET-1 (Hughson et al., 2018; Nagane et al., 2021; Tapio, 2016). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Finally, oxidative stress has been shown to lead to mitochondrial dysfunction and dysregulation, which is thought to play an important role in the development of endothelial dysfunction (Borghini et al., 2013; Hughson et al., 2018; Nagane et al., 2021; Slezak et al., 2017). </span></span></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Empirical evidence provides a moderate level of support to this KER. Examples of this evidence are summarized here and further in attached tables.</span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="font-family:Times New Roman,Times,serif">The evidence to support the relationship between oxidative stress leading to endothelial dysfuncti</span>on was gathered from studies using <em>in vitro </em>and <em>in vivo</em> rat and mice models (Delp et al., 2016; Hatoum et al., 2006; Shen et al., 2018; Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011; Ungvari et al., 2013), <em>in vivo</em> pig models (Li et al., 2002) and human <em>in vitro</em> cells (Ramadan et al., 2020). Various stressors were applied, including X-rays, hindlimb unloading (HU), heavy ions (<sup>56</sup>Fe ions and <sup>32</sup>P) and gamma rays with a dose range of 0.1 to 22.5 Gy. To determine the effect of oxidative stress on endothelial dysfunction, various assays and end-points were measured. Senescence-associated β-galactosidase (</span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">SA ꞵ-gal), </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">insulin-like growth factor-binding protein-7</span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"> (IGFBP-7) and growth differentiation factor 15 (GDF-15) as senescence markers, caspase 3/7 activity as an apoptosis marker, endothelin-1 levels for the ratio of apoptotic to normal cells, <span style="background-color:white"><span style="color:#212121">4-hydroxynonenal</span></span> (4-HNE) and 3-nitrotyrosine (3-NT) as aortic oxidative damage markers, superoxide production, vascular tension, and ROS detection via xanthine oxidase (XO) activity, and fluorescent dyes, such as dihydroethidium fluorescence were all used as end-point measures. </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Dose Concordance</span></span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Dose concordance between the two key events is supported by numerous studies. Hatoum et al. (2006) explored the effect of 3 to 9 cumulative X-ray doses of 0.25 Gy on murine intestinal arterioles. The study found that superoxide and hydrogen peroxide generation was increased, while at the same doses vasodilation in response to acetylcholine (ACh) was decreased (Hatoum et al., 2006). Another study using variou<span style="font-family:Times New Roman,Times,serif">s doses </span></span></span></span></span><span style="font-family:Times New Roman,Times,serif">on cerebral microvascular endothelial cells</span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="font-family:Times New Roman,Times,serif"> found a</span> significant change in cellular peroxide and mitochondrial oxidative stress following the 4 Gy dose, while SA β-gal showed the first large increase at 4 Gy (Ungvari et al., 2013).</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Ramadan et al. (2020) used X-ray irradiation in multiple types of human endothelial cells has shown that ROS production is significantly higher in both the 0.1 Gy and 5 Gy compared to the control. These changes were correlated to endothelial dysfunction as SA β-gal activity and endothelial apoptosis were also affected, showing a response of greater magnitude following the 5 Gy dose compared to 0.1 Gy (Ramadan et al., 2020). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Exposure of mice to 18 Gy X-rays led to a ~1.8-fold and ~2.2-fold decrease in 4-HNE and 3-NT respectively, both being markers of aortic oxidative damage. Simultaneously, the 18 Gy dose caused a ~5-fold increase in aortic apoptosis, a marker of endothelial dysfunction (Shen et al., 2018). Two studies (Soucy et al., 2007; Soucy et al., 2010) found that varying doses (0.5 and 5 Gy) of gamma radiation led to significant increases in ROS levels in rat aorta. Subsequently, endothelial function was affected with a 0.5 Gy dose resulting in a ~30% decrease in ACh-induced vasodilation response (Soucy et al., 2007), and a 5 Gy dose leading to a ~13-15% decrease in ACh-induced vasodilation (Soucy et al., 2010).</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Iron ion irradiation resulted in oxidative stress and endothelial dysfunction both occurring after 1 Gy (Soucy et al., 2011). Similarly, Delp et al. (2016) showed total body <sup>56</sup>Fe irradiation at 1 Gy led to a ~2-fold increase in XO activity and a ~10% decrease in ACh response in mice (Delp et al., 2016). Delp et al. (2016) also explored the effects of HU on mice and found no significant changes to XO activity or ACh response following 2-week HU, while HU in combination with 1 Gy <sup>56</sup>Fe radiation led to a ~2.2-fold increase in XO activity and the same ~10% decrease in ACh response. Li et al. (2002) showed ~3.5-fold increase in superoxide anion production and a ~25-80% decrease in vasoconstriction and vasodilation response to various vasomotive substances following 20 Gy <sup>32</sup>P radiation in pig coronary arteries.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Time Concordance</span></span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">There is some evidence of time concordance between oxidative stress and endothelial dysfunction. Ramadan et al. (2020) used human endothelial cells and showed ROS production first increased 45 minutes after 5 Gy X-ray exposure before returning to baseline levels after 2 hours. Apoptosis markers Annexin V and Caspase 3/7 were first increased after 4 hours. Cellular senescence evaluated with the SA-β-gal activity was first measured only after 7 days (Ramadan et al., 2020). ROS production and dilation response to ACh after irradiation were measured at various times as cumulative doses were given, which showed increased ROS and decreased dilation of rat intestinal microvessels both after 5 days of cumulative 0.25 Gy X-ray doses (Hatoum et al., 2006). Work using 4-HNE and 3-NT as biomarkers of oxidative stress in mice aorta following 18 Gy 6MV X-ray at 3, 7, 14, 28, and 84 days after irradiation showed both markers to be significantly elevated starting after 3 days. Exposure also led to significantly increased apoptosis, indicating endothelial dysfunction after 3 days (Shen et al., 2018).</span></span></span></span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Incidence Concordance</span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is moderate support in current literature for an incidence concordance relationship between oxidative stress and endothelial dysfunction. Three of the primary research studies used to support this AOP demonstrated an average change to endpoints of oxidative stress that was greater or equal to that of endothelial dysfunction (Soucy et al., 2011; Soucy et al., 2010; Soucy et al., 2007).</span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Essentiality </span></span></strong> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Essentiality in the relationship was demonstrated in the following studies. Studies by Soucy et al. (2007, 2010, 2011) explored the relationship between radiation exposure, ROS levels and endothelial function, all focusing on the role of XO. Soucy et al. (2007) incubated aortic rings from irradiated rats in the XO inhibitor oxypurinol (Oxp) and saw this treatment result in recovery of ACh vasodilation response. Soucy et al. (2010) showed that administration of allopurinol (a superoxide scavenger) following irradiation led to significantly decreased ROS levels. Additionally, the latter two studies showed that XO inhibition by dietary administration of Oxp significantly decreased XO activity and ROS levels while simultaneously recovering ACh response (Soucy et al., 2010, 2011). Similar results were observed when treatment with manganese tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), a SOD mimetic, returned peroxide and superoxide levels and significantly improved ACh response irradiated rats (Hatoum et al., 2006). Dietary treatment with Tempol, a water-soluble SOD-mimetic likewise increased vasomotion and decreased superoxide levels (Hatoum et al., 2006). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Human bone marrow mesenchymal stem cells (hBMSCs) have also been studied for their ability to prevent radiation-induced aortic injury. Both high and low doses of hBMSCs were shown to increase catalase and heme oxygenase 1 (HO-1) antioxidant activity, and decrease levels of the aortic oxidative damage markers 4-HNE and 3-NT. Subsequently, this treatment also significantly decreased levels of apoptosis in the aorta (Shen et al., 2018). Finally, blocking Connexin43 hemichannels using TAT-Gap19 peptide also significantly reduced oxidative stress and resultant cell senescence and death, suggesting the role of intracellular communication in mediating radiation response (Ramadan et al., 2020).</span></span></span></span></p>
<p><span style="font-family:Times New Roman,Times,serif">Studies have also shown that transgenic mice overexpressing superoxide dismutase (SOD) have a twofold reduction in aortic lesions following X-ray exposure compared to control (Hughson et al., 2018). Overexpression of SOD also mitigates atherosclerotic plaque formation, further outlining the relationship between oxidative stress and the pathological environment of endothelial dysfunction (Tapio, 2016). </span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Work by Ramadan et al. (2020) explored the use of TAT-Gap19 to block endothelial intracellular communication in order to modulate radiation response of intercellular connexin proteins. Overall, TAT-Gap19 was shown to reduce ROS production and subsequent senescence (SA β-gal activity) and apoptosis (Annexin V and Caspase 3/7) markers. However, treatment with TAT-Gap19 led to an increase in SA β-gal in non-irradiated control at the 9-day point. Additionally, the 0.1 Gy irradiated group showed persistent SA β-gal activity at all time points studied, </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">while the 5 Gy<span style="color:black"> group demonstrated an unexpected decrease before day 14. </span></span></span></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.</span></span></span></span></span></p>
HighMaleLowFemaleLowUnspecificModerateAdultLowNot Otherwise SpecifiedLowModerateHighLow<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">The evidence is derived from rat <em>in vivo </em>and <em>in vitro </em>models. Mice cell-derived studies were also available but less <em>in-vivo </em>evidence was available from this species. There was a low number of studies containing human or pig models to support this KER. Males have been studied more often than females. There are a few studies with unspecified lifestage of models, while the studies with a defined age typically used adult models. </span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">References </span></span></strong></span></span></p>
<p style="margin-left:30px"><span style="font-family:Calibri,sans-serif; font-size:12pt"><span style="font-family:"Times New Roman",serif">Augustin, H. G., D. H. Kozian and R. C. Johnson (1994), “Differentiation of endothelial cells: Analysis of the constitutive and activated endothelial cell phenotypes”, <em>BioEssays</em>, Vol. 16/12, Wiley, Hoboken, </span></span><a href="https://doi.org/10.1002/bies.950161208" style="font-family: Calibri, sans-serif; font-size: 11pt; color: rgb(5, 99, 193); text-decoration: underline;">https://doi.org/10.1002/bies.950161208</a><span style="font-family:Times New Roman,serif">.</span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Baran, R. et al. (2021), “The Cardiovascular System in Space: Focus on In Vivo and In Vitro Studies”, <em>Biomedicines</em>, Vol. 10/1, Multidisciplinary Digital Publishing Institute, Basel, </span></span><a href="https://doi.org/10.3390/biomedicines10010059" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/biomedicines10010059</a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Beckhauser, T. F., J. Francis-Oliveira and R. De Pasquale (2016), “Reactive Oxygen Species: Physiological and Physiopathological Effects on Synaptic Plasticity”, <em>Journal of Experimental Neuroscience</em>, Vol. 10, SAGE Publishing, Thousand Oaks, </span></span><a href="https://doi.org/10.4137/JEN.S39887" style="color:#0563c1; text-decoration:underline">https://doi.org/10.4137/JEN.S39887</a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Bonetti, P. O., L. O. Lerman and A. Lerman (2003), “Endothelial Dysfunction: a marker of atherosclerotic risk”, <em>Arteriosclerosis, Thrombosis, and Vascular Biology</em>, Vol. 23/2, Lippincott Williams & Wilkins, Philadelphia, </span></span><a href="https://doi.org/10.1161/01.ATV.0000051384.43104.FC" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1161/01.ATV.</span></span></a><a name="_Int_6pXfOe1l"><span style="color:#0563c1"><u><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">0000051384.43104.FC</span></span></u></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Borghini, A. et al. (2013), “Ionizing radiation and atherosclerosis: Current knowledge and future challenges”, <em>Atherosclerosis</em>, Vol. 230/1, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.atherosclerosis.2013.06.010" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/j.atherosclerosis.2013.06.010</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Carmeliet, P., and R. K. Jain. (2011), “Molecular mechanisms and clinical applications of angiogenesis”, <em>Nature</em>, Vol. 473/7347, <span style="color:black">Nature Portfolio, London, </span></span></span><a href="https://doi.org/10.1038/nature10144" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1038/nature10144</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Deanfield, J. E., J. P. Halcox and T. J. Rabelink (2007), “Endothelial Function and Dysfunction”, <em>Circulation</em>, Vol. 115/10, Lippincott Williams & Wilkins, Philadelphia, </span></span><a href="https://doi.org/10.1161/CIRCULATIONAHA.106.652859" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1161/CIRCULATIONAHA.106.652859</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Delp, M. D. et al. (2016), “Apollo Lunar Astronauts Show Higher Cardiovascular Disease Mortality: Possible Deep Space Radiation Effects on the Vascular Endothelium”, <em>Scientific Reports</em>, Vol. 316/23, Nature Portfolio, London, </span></span><a href="https://doi.org/10.1038/SREP29901" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1038/SREP29901</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Elahi, M. M., Y. X. Kong and B. M. Matata (2009), “Oxidative Stress as a Mediator of Cardiovascular Disease”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2/5, Hindawi, London, </span></span><a href="https://doi.org/10.4161/oxim.2.5.9441" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.4161/oxim.2.5.9441</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Fishman, A. P. (1982), “ENDOTHELIUM: A DISTRIBUTED ORGAN OF DIVERSE CAPABILITIES”, <em>Annals of the New York Academy of Sciences</em>, Vol. 401/1, Wiley-Blackwell, Hoboken, </span></span><a href="https://doi.org/10.1111/j.1749-6632.1982.tb25702.x" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1111/j.1749-6632.1982.tb25702.x</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Hatoum, O. A. et al. </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(2006), “Radiation Induces Endothelial Dysfunction in Murine Intestinal Arterioles via Enhanced Production of Reactive Oxygen Species”, <em>Arteriosclerosis, Thrombosis, and Vascular Biology</em>, Vol. 26/2, Lippincott Williams & Wilkins, Philadelphia, </span></span><a href="https://doi.org/10.1161/01.ATV.0000198399.40584.8c" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1161/01.ATV.0000198399.40584.8c</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-family:Calibri,sans-serif; font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Hughson, R.L., A. Helm and M. Durante (2018), “Heart in space: Effect of the extraterrestrial environment on the cardiovascular system”, <em>Nature Reviews Cardiology</em>, Vol. 15/3, Nature Portfolio, London, </span></span></span><a href="https://doi.org/10.1038/nrcardio.2017.157" style="font-family: Calibri, sans-serif; font-size: 11pt; color: rgb(5, 99, 193); text-decoration: underline;"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1038/nrcardio.2017.157</span></span></a><span style="color:#000000; font-family:Times New Roman,serif">.</span></p>
<p style="margin-left:30px"><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. https://doi.org/10.1080/09553002.2022.2110306</span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Krüger-Genge, A. et al. </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(2019), “Vascular Endothelial Cell Biology: An Update”, <em>International Journal of Molecular Sciences</em>, Vol. 20/18, Multidisciplinary Digital Publishing Institute, Basel<span style="color:black">, </span></span></span><a href="https://doi.org/10.3390/IJMS20184411" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.3390/IJMS20184411</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Li, J. et al. (2002), “Endovascular irradiation impairs vascular functional responses in noninjured pig coronary arteries”, <em>Cardiovascular Radiation Medicine</em>, Vol. 3/3–4, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/S1522-1865(03)00096-9" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/S1522-1865(03)00096-9</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Marasciulo, F., M. Montagnani and M. Potenza </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(2006), “Endothelin-1: The Yin and Yang on Vascular Function”, <em>Current Medicinal Chemistry</em>, Vol. 13/14, Bentham Science Publishers, Sharjah, </span></span><a href="https://doi.org/10.2174/092986706777441968" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.2174/092986706777441968</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases”, <em>Journal of Radiation Research</em>, Vol. 62/4, Oxford University Press, Oxford, </span></span><a href="https://doi.org/10.1093/JRR/RRAB032" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1093/JRR/RRAB032</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Ramadan, R. et al. (2020), “Connexin43 Hemichannel Targeting With TAT-Gap19 Alleviates Radiation-Induced Endothelial Cell Damage”, <em>Frontiers in Pharmacology</em>, Vol. 11, Frontiers Media SA, Lausanne, </span></span><a href="https://doi.org/10.3389/fphar.2020.00212" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.3389/fphar.2020.00212</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Ray, P. D., B. W. Huang and Y. Tsuji (2012), “Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling”, <em>Cellular Signalling</em>, Vol. 24/5, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.cellsig.2012.01.008" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/j.cellsig.2012.01.008</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Schiffrin, E. L. (2008), “Oxidative Stress, Nitric Oxide Synthase, and Superoxide Dismutase”, <em>Hypertension</em>, Vol. 51/1, Lippincott Williams & Wilkins, Philadelphia, </span></span><a href="https://doi.org/10.1161/HYPERTENSIONAHA.107.103226" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1161/HYPERTENSIONAHA.107.103226</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Shen, Y. et al. (2018), “Transplantation of bone marrow mesenchymal stem cells prevents radiation-induced artery injury by suppressing oxidative stress and inflammation”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2018, Hindawi, London, </span></span><a href="https://doi.org/10.1155/2018/5942916" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1155/2018/5942916</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Slezak, J. et al. (2017), “Potential markers and metabolic processes involved in the mechanism of radiation-induced heart injury”, <em>Canadian Journal of Physiology and Pharmacology</em>, Vol. 95/10, Canadian Science Publishing, Ottawa, </span></span><a href="https://doi.org/10.1139/cjpp-2017-0121" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1139/cjpp-2017-0121</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">.</span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Soloviev, A. I. and I.V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, <em>Biochemical pharmacology</em>, Vol. 159, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.bcp.2018.11.019" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/j.bcp.2018.11.019</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Soucy, K. G. et al. </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(2011), “HZE 56Fe-Ion Irradiation Induces Endothelial Dysfunction in Rat Aorta: Role of Xanthine Oxidase”, <em>Radiation Research</em>, Vol. 176/4, Radiation Research Society, Bozeman, </span></span><a href="https://doi.org/10.1667/RR2598.1" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1667/RR2598.1</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">.</span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Soucy, K. G. et al. </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, <em>Journal of Applied Physiology</em>, Vol. 108/5, American Physiological Society, Rockville, </span></span><a href="https://doi.org/10.1152/japplphysiol.00946.2009" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/japplphysiol.00946.2009</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">.</span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Soucy, K. G. et al. </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(2007), “Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta”, <em>Radiation and Environmental Biophysics</em>, Vol. 46/2, Springer, New York, </span></span><a href="https://doi.org/10.1007/s00411-006-0090-z" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1007/s00411-006-0090-z</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Sylvester, C. B. et al. </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(2018), “Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer”, <em>Frontiers in Cardiovascular Medicine</em>, Vol. 5, Frontiers Media SA, Lausanne, </span></span><a href="https://doi.org/10.3389/fcvm.2018.00005" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.3389/fcvm.2018.00005</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Tapio, S. (2016), “Pathology and biology of radiation-induced cardiac disease”, <em>Journal of Radiation Research</em>, Vol. 57/5, Oxford University Press, Oxford, </span></span><a href="https://doi.org/10.1093/jrr/rrw064" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1093/jrr/rrw064</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, <em>The Journals of Gerontology Series A: Biological Sciences and Medical Sciences</em>, Vol. 68/12, Oxford University Press, Oxford, </span></span><a href="https://doi.org/10.1093/gerona/glt057" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1093/gerona/glt057</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Venkatesulu, B. P. et al. </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">(2018), “Radiation-Induced Endothelial Vascular Injury”, <em>JACC: Basic to Translational Science</em>, Vol. 3/4, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.jacbts.2018.01.014" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1016/j.jacbts.2018.01.014</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:30px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Wang, Y., M. Boerma and D. Zhou (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, <em>Radiation research</em>, Vol. 186/2, Radiation Research Society, Bozeman, </span></span><a href="https://doi.org/10.1667/RR14445.1" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1667/RR14445.1</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">.</span></span></span></span></p>
2022-09-28T12:43:322023-03-21T09:23:09908b38ae-23f7-446a-bb32-46656376b24b42ee9daa-42e3-49b5-9aa0-9dc2b7d09c20<p><span style="font-family:Times New Roman,Times,serif">Altered signaling pathways can disrupt cellular homeostasis and induce endothelial dysfunction, characterized by a prolonged state of endothelial activation (Deanfield et al., 2007). Signaling pathways involved in triggering endothelial dysfunction include the p53-p21 pathway, the Akt/phosphtidylinositol-3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway, the RhoA-Rho-kinase pathway, and the acid sphingomyelinase (ASM)/ceramide (Cer) pathway (Venkatsulu et al., 2018; Soloviev et al., 2019; Wang et al., 2016). Activation of the signaling molecule p53 by phosphorylation enhances its stability, leading to cell cycle arrest and premature senescence in endothelial cells and can alternatively lead to a caspase cascade resulting in cellular apoptosis. Activation of the sphingomyelinase ceramide pathway can also contribute to endothelial apoptosis through production of ceramide that activates mitogen-activated protein kinase (MAPK) and extracellular-signal-regulated kinase (ERK). Signaling molecules MAPK and ERK can also be activated as a direct response to a stressor and prompt a cascade of events resulting in endothelial cell apoptosis. Impairment of the Akt/PI3K/mTOR pathway can lead to apoptosis by preventing cell survival signaling and can also lead to downregulation of Rho cytoskeletal proteins for senescence of endothelial cells (Venkatesulu et al., 2018; Soloviev et al., 2019; Nagane et al., 2021; Ramadan et al., 2021; Hughson et al., 2018).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: Moderate</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The biological plausibility of the connection between altered signaling pathways leading to an increase in endothelial dysfunction is well-supported by literature and the mechanisms are generally understood. Multiple signaling pathways influence endothelial function. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Modulation of the Akt/PI3K/mTOR pathway downregulates downstream Rho cytoskeletal proteins, which leads to partial-to-full senescence of endothelial cells, resulting in increased vascular permeability and endothelial dysfunction (Venkatsulu et al., 2018). Modulation of the Akt/PI3K/mTOR pathway also acts upstream of the p53-p21 pathway to mediate endothelial cell senescence (Wang et al., 2016). Unlike in other cell types, in endothelial cells the p53-p21 pathway is more important than the p16-Rb pathway for induction of cell senescence (Wang et al., 2016). Senescent endothelial cells show changes in cell morphology, cell-cycle arrest, and increased senescence-associated β-galactosidase (SA-β-gal) staining. These changes lead to endothelial dysfunction, which results in dysregulation of vasodilation (Wang et al., 2016; Hughson et al., 2018; Ramadan et al., 2021). Phosphorylation of p53 is another important moderator of apoptosis in endothelial cells, as well as the ASM/Cer pathway, where the production of ceramide mediates endothelial apoptosis through sphingomyelinase, activating MAPK and ERK, which prompt a cascade of events culminating in endothelial cell apoptosis, another cellular marker for endothelial dysfunction (Venkatsulu et al., 2018; Soloviev et al., 2019).</span></p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Empirical Evidence </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">The empirical evidence supporting this KER was gathered from research utilizing both <em>in vivo </em>and <em>in vitro</em> models. Many in vitro studies have examined the relationship using endothelial cell cultures. Endothelial dysfunction due to altered signaling can be measured by premature endothelial cell senescence, apoptosis and impaired contractile response (Korpela & Liu, 2014). Levels of signaling molecules in pathways such as the Akt/PI3K/mTOR, RhoA-Rho-kinase, and ASM/Cer pathways, and the effect they have on endothelial dysfunction as characterized by endothelial cellular senescence, apoptosis and contractile response, was examined in these studies (Chang et al., 2017; Cheng et al., 2017; Su et al., 2020; Summers et al., 2008; Yentrapalli et al., 2013a; Yentrapalli et al., 2013b). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Dose Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Studies have used <em>in vitro</em> models with acute and chronic doses of radiation administered from 0.23 to 10 Gy to show that the dose at which significant alterations in signaling pathways occurs is concordant with doses at which endothelial dysfunction occurs. Alterations in various signaling pathways, including decreases in the Akt/PI3K/mTOR pathway and increases in the p53-p21 pathway, both occurred at 2.4 and 4 Gy; endothelial cell senescence was found only at 4 Gy (Yentrapalli et al., 2013a). Another study using 4.1 mGy/h chronic gamma irradiation found significant changes in the p53-p21 and ERK signaling pathways after both 2.1 and 4.1 Gy (Yentrapalli et al., 2013b). These changes were associated with an increase in endothelial dysfunction at both doses as well, with only a slight increase in cell senescence after 2.1 Gy (Yentrapalli et al., 2013b). Endothelial cells exposed to a single dose of 10 Gy of X-rays showed an increase in Akt and p-Akt that was correlated with an increase in endothelial dysfunction indicated by a 5-fold increase in apoptosis (Chang et al., 2017). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Various studies also showed that altered signaling and endothelial dysfunction can occur at the same stressor severity during hindlimb unloading (HU). Rats exposed to microgravity for 20 days showed both decreased RhoA signaling molecule and impaired vasodilation (Summers et al., 2008). Two studies observing the effects of microgravity for 4 weeks in rats found that a decrease in the ASM/Cer pathway resulted in better endothelial function, while an increase in the pathway resulted in endothelial dysfunction (Cheng et al., 2017; Su et al., 2020). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif"><strong>Time Concordance</strong> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">There is limited evidence to suggest a time concordance between altered signaling pathways and endothelial dysfunction. In a study using gamma irradiation in vitro, the p53-p21 and ERK signaling pathways were altered after 3 weeks of chronic gamma irradiation, while cell senescence was increased only slightly after 3 weeks, and increased more after 6 weeks (Yentrapalli et al., 2013b). In a similar study, alterations in various signaling pathways including decreases in the Akt/PI3K/mTOR pathway were found as early as 1 week during chronic gamma irradiation (Yentrapalli et al., 2013a). The earliest significant increase in cellular senescence occurred after 10 weeks (Yentrapalli et al., 2013a). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Incidence Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is limited support in current literature for an incidence concordance relationship between altered signaling and endothelial dysfunction. One out of the 6 primary research studies used to support this KER demonstrated an average change to endpoints of altered signaling that was greater or equal to that of endothelial dysfunction (Cheng et al., 2017; Yentrapalli et al., 2013b). After 6 weeks of gamma irradiation of HUVECs, altered signaling marker, p21, was increased by 3.5-fold, compared to the 3-fold increase in SA-β-gal staining, a marker for endothelial cell senescence (Yentrapalli et al., 2013b). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Essentiality </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Changes in endothelial signaling pathways can trigger endothelial dysfunction. Therefore, in the absence of altered signaling endothelial dysfunction is not expected. Through the use of signaling molecule inhibitors such as Y27632 and dpm, altered signaling can be suppressed which results in reduced endothelial dysfunction (Venkatsulu et al., 2018; Soloviev et al., 2019; Wang et al., 2016). A study observing the effects of Y27632, a Rho kinase inhibitor, found that when rat abdominal aortas were incubated with Y27632 the contractile response, which was hindered by HU, was returned to control levels (Summers et al., 2008). Similar results have been shown by other groups that have looked at endothelial cells incubated in mesenchymal stem cell conditioned media (MSC-CM), which is thought to exhibit therapeutic potential for microvascular injury through angiogenic cytokines. MSC-CM prevented an increase in cleaved capsase-3 and increased both Akt and p-Akt, which was associated with a substantial decrease in apoptosis (Chang et al., 2017). It has also been found that enhancing the ASM/Cer pathway with C6-ceramide increases downstream caspase-3 and endothelial dysfunction, measured by apoptosis, while ASM inhibitors dpm and DOX partially decreased both caspase-3 and apoptosis (Su et al., 2020; Cheng et al., 2017).</span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Much of the evidence for this relationship comes from <em>in vitr</em>o<em> </em>studies; further work is needed to determine the certainty of the relationship at the tissue level.</span></span></span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.</span></span></span></span></span></p>
ModerateMaleLowFemaleLowUnspecificModerateAdultLowJuvenileLowModerate<p><span style="font-family:Times New Roman,Times,serif">Evidence for this KER is supported through <em>in vivo</em> rat and <em>in vitro</em> human studies. The in vivo studies were conducted in male animals, although the relationship is still plausible in females. The in vivo studies were undertaken in adolescent and adult rats. </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Chang, P. Y. et al. (2017), “MSC-derived cytokines repair radiation-induced intra-villi microvascular injury”,<em> Oncotarget</em>, Vol. 8/50, Impact Journals, Orchard Park, <a href="https://doi.org/10.18632/oncotarget.21236" style="color:#0563c1; text-decoration:underline">https://doi.org/10.18632/oncotarget.21236.</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Cheng, Y. P. et al. (2017), “Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats”, <em>Pflugers Archiv European Journal of Physiology</em>, Vol. 469, Springer, New York, <a href="https://doi.org/10.1007/s00424-017-1969-z" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/s00424-017-1969-z</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="color:black">Deanfield, J.E., J. P. Halcox and T. J. Rabelink (2007), “Endothelial function and dysfunction: Testing and clinical relevance”, <em>Circulation</em>, Vol. 115/10, Lippincott Williams & Wilkins, Philadelphia, </span><a href="https://doi.org/10.1161/CIRCULATIONAHA.106.652859" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/CIRCULATIONAHA.106.652859</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Hughson, R.L., A. Helm and M. Durante (2018), “Heart in space: Effect of the extraterrestrial environment on the cardiovascular system”, <em>Nature Reviews Cardiology</em>, Vol. 15/3, Nature Portfolio, London, </span></span><a href="https://doi.org/10.1038/nrcardio.2017.157" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1038/nrcardio.2017.157</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Korpela, E., and S. K. Liu (2014), “Endothelial perturbations and therapeutic strategies in normal tissue radiation damage”, <em>Radiation oncology, </em>Vol. 9, BioMed Central, London, </span></span><a href="https://doi.org/10.1186/s13014-014-0266-7" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1186/s13014-014-0266-7</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. https://doi.org/10.1080/09553002.2022.2110306</span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases”, <em>Journal of Radiation Research</em>, Vol. 62/4, Oxford University Press, Oxford, </span></span><a href="https://doi.org/10.1093/JRR/RRAB032" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/JRR/RRAB032</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Ramadan, R. et al. (2021), “The role of connexin proteins and their channels in radiation-induced atherosclerosis”, <em>Cellular and molecular life sciences: CMLS</em>, Vol. 78/7, Springer, New York, </span></span><a href="https://doi.org/10.1007/s00018-020-03716-3" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1007/s00018-020-03716-3</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Soloviev, A. I. and I. V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, <em>Biochemical pharmacology</em>, Vol. 159, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.bcp.2018.11.019" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1016/j.bcp.2018.11.019</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Su, Y. T. et al. (2020), “Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats”, <em>Life sciences</em>, Vol. 243, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.lfs.2019.117253" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.lfs.2019.117253</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Summers, S. M., S. V. Nguyen, and R. E. Purdy (2008), “Hindlimb unweighting induces changes in the RhoA-Rho-kinase pathway of the rat abdominal aorta”, <em>Vascular pharmacology</em>, Vol. 48/4-6, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.vph.2008.03.006" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.vph.2008.03.006</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Venkatesulu, B. P. et al. (2018), “Radiation-Induced Endothelial Vascular Injury: A Review of Possible Mechanisms”, <em>JACC: Basic to translational science</em>, Vol. 3/4, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.jacbts.2018.01.014" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1016/j.jacbts.2018.01.014</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Wang, Y., M. Boerma and D. Zhou (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, <em>Radiation research</em>, Vol. 186/2, Radiation Research Society, Bozeman, </span></span><a href="https://doi.org/10.1667/RR14445.1" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1667/RR14445.1</a><span style="background-color:white"><span style="color:black">. </span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Yentrapalli, R. et al. (2013a), “The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation”, <em>PloS one</em>, Vol. 8/8, PLOS, San Francisco, </span></span><a href="https://doi.org/10.1371/journal.pone.0070024" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1371/journal.pone.0070024</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Yentrapalli, R. et al. (2013b), “Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low-dose rate gamma radiation”, <em>Proteomics</em>, Vol. 13/7, John Wiley & Sons, Ltd., Hoboken, </span></span><a href="https://doi.org/10.1002/pmic.201200463" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1002/pmic.201200463</a></span></p>
2022-09-28T12:42:182023-03-21T09:20:47f487d2a3-b9c7-4777-958c-8ebecaa0407142ee9daa-42e3-49b5-9aa0-9dc2b7d09c20<p><span style="font-family:Times New Roman,Times,serif">An increase in pro-inflammatory mediators including the cytokines tumor necrosis factor-α (TNF-α), interleukin 1 beta and 6 (IL-1β, IL-6), chemokines monocyte chemoattractant protein 1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) can lead to inflammatory response which can disrupt cellular homeostasis and if persistent can lead to eventual endothelial dysfunction (Venkatesulu et al., 2018; Korpela & Liu, 2014). Normally, an inflammatory response provides a protective effect to the endothelium but if prolonged (over months) it can exhaust this protective inflammatory effect, as a result, endothelial cells may become senescent or apoptotic, leading to endothelial dysfunction (Deanfield et al., 2007; Bonetti et al., 2003, Wang et al., 2016; Hughson et al., 2018; Ramadan et al., 2021).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: Moderate</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The biological plausibility connecting increased pro-inflammatory mediators to increased endothelial dysfunction is well-supported by literature (Bonetti et al., 2003; Deanfield et al., 2007; Hughson et al., 2018; Ramadan et al., 2021; Wang et al., 2019; Wang et al., 2016), and has been demonstrated in animal studies and human cell models (Shen et al., 2018; Chang et al., 2017; Baselet et al., 2017; Ramadan et al., 2020; Ungvari et al., 2013). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Inflammation can initially provide a protective effect to the endothelium, but chronic inflammation can exhaust this protective inflammatory effect resulting in loss of endothelial integrity and resident cells becoming senescent or apoptotic, leading to endothelial dysfunction (Deanfield et al., 2007; Bonetti et al., 2003). Senescent endothelial cells show changes in cell morphology, cell-cycle arrest, and increased senescence-associated β-galactosidase (SA-β-gal) staining. These changes lead to endothelial dysfunction, which also leads to dysregulation of vasodilation (Wang et al., 2016; Hughson et al., 2018; Ramadan et al., 2021). The inflammatory response is regulated by a balance between pro-inflammatory and anti-inflammatory mediators, and specific cytokine profiles are dependent on parameters of the stressor/exposure/insult (Wang et al., 2019). The pro-inflammatory cytokines TNF-α and IL-1 play a critical role by triggering a cytokine cascade, which initiates an inflammatory response to promote healing and restore tissue function. TNF-α is able to induce apoptotic cell death, which is implicated in endothelial dysfunction. Nuclear factor kappa B (NF-кB) is also activated, which targets multiple genes coding for vascular cell adhesion proteins (VCAM), intercellular adhesion molecule (ICAM), and IL-1, as well as prothrombotic markers (Slezak et al., 2017). NF-кB mediates a pro-survival and pro-inflammatory state. Inflammation persisting for months leads to prolonged chronic inflammation, which causes an ineffective healing process that is further worsened by a decrease in endothelium-dependent relaxation. This causes endothelial dysfunction, making vasculature more vulnerable to damage from non-laminar flow (Sylvester et al., 2018). Senescent cells also have a pro-inflammatory secretory phenotype, which further contributes to negative effects on the endothelium. Increased pro-inflammatory mediators may be due to increased expression but may also be attributed to increased permeability of the endothelium as seen after irradiation in animal models, which results in increased transmigration of inflammatory cells into the endothelium and can lead to eventual dysfunction (Hughson et al., 2018).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The empirical evidence supporting this KER is gathered from research utilizing both <em>in vivo</em> and <em>in vitro</em> models. Many <em>in vitro</em> studies have examined the relationship using endothelial cell cultures. Levels of pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, IL-8, MCP-1 and ICAM-1, and the effect they have on endothelial dysfunction, as characterized by endothelial cellular senescence and apoptosis, have been examined in these studies. The evidence is derived from stressors of gamma and X-ray radiation in the range of 0.05-18 Gy (Shen et al., 2018; Baselet et al., 2017; Ramadan et al., 2020; Ungvari et al., 2013; Chang et al., 2017). </span></p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Dose Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is moderate evidence to demonstrate dose concordance between an increase in pro-inflammatory mediators and endothelial dysfunction. Most studies do not show statistically significant effects across all doses; however, biological trends across multiple doses have been included to support this relationship. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Studies examining a range of doses from 0.05 Gy to 18 Gy using predominantly X-rays as the source of stressor support the relationship between increased pro-inflammatory mediators and endothelial dysfunction. An <em>in vitro</em> study using X-ray irradiation on human endothelial cells showed an increase in pro-inflammatory cytokines IL-6 and CCL2 at a dose as low as 0.05 Gy. Though the increase was not statistically significant, there was a biological trend showing significant increases at higher doses. All doses demonstrated a correlation to endothelial dysfunction (Baselet et al., 2017). Another study using human endothelial cells exposed to X-rays showed similar results with increases in pro-inflammatory mediators, including IL-6, VCAM-1, and IL-8, as well as SA-β-gal activity at 5 Gy (Ramadan et al., 2020). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">A gamma ray study that exposed rat endothelial cells to a 6 Gy dose showed an increase in pro-inflammatory mediators IL-6, IL-1α, IL-1β, and MCP-1 associated with an increase in endothelial cell senescence (Ungvari et al., 2013). Another single dose X-ray study at 10 Gy also revealed increases in pro-inflammatory mediators with a 1.2-fold increase in IL-1α, and a 6-fold increase in IL-6 and TNF-α. This was associated with an increase in endothelial dysfunction, indicated by a 5-fold increase in apoptotic cells (Chang et al., 2017). A study using X-rays on mouse aortas found that there was a 2-fold increase in the pro-inflammatory mediators TNF-α and ICAM-1 after 18 Gy, and a 5-fold increase in endothelial apoptosis, which is a defined marker for endothelial dysfunction (Shen et al., 2018). </span></p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Time Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is limited evidence to suggest a time concordance between increased pro-inflammatory mediators and endothelial dysfunction. An <em>in vitro</em> study using 5 Gy of X-rays found that pro-inflammatory mediators, including IL-6, VCAM-1, TNF-α, ICAM-1, IL-1β and MCP-1, increased as soon as 1 day post-irradiation. SA-β-gal, a marker for cellular senescence, showed the first increase 7 days post-irradiation (Ramadan et al., 2020). A study using 18 Gy of X-rays examined mouse aortas after 3-84 days post-irradiation and found an increase in both pro-inflammatory mediators and endothelial dysfunction as early as 3 days post-irradiation (Shen et al., 2018). </span></p>
<p><span style="font-family:Times New Roman,Times,serif"><strong>Incidence concordance</strong> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Few studies demonstrated incidence concordance. In an <em>in vitro</em> study using human endothelial cells irradiated with X-rays, incidence concordance was demonstrated at both 0.5 and 2 Gy as pro-inflammatory mediators IL-6 and CCL2 were increased 2-fold or greater while SA-β-gal increased a maximum of 1.5-fold (Baselet et al., 2017). Similarly, 5 Gy of X-rays resulted in increases to multiple pro-inflammatory mediators (IL-1β, IL-6, IL-8, MCP-1, and VCAM) between 1.5- and 4-fold, while SA-β-gal activity increased 1.5-fold in human endothelial cells (Ramadan et al., 2020). </span></p>
<p><span style="font-family:Times New Roman,Times,serif"><strong>Essentiality</strong> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">An increase in inflammation can trigger endothelial dysfunction. Therefore, in the absence of an increase in pro-inflammatory mediators endothelial dysfunction is not expected. Through the use of certain treatments, such as TAT-Gap19 and mesenchymal stem cells, the increase in pro-inflammatory mediators can be greatly supressed, but not fully blocked, which results in reduced but not completely prevented endothelial dysfunction such as apoptosis and cellular senescence (Venkatsulu et al., 2018; Soloviev et al., 2019; Wang et al., 2016). These treatments demonstrate the essentiality of the relationship and are described below; however, the available empirical data supporting essentiality for this KER is limited. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">A study observing the effects of TAT-Gap19, a connexin43 hemichannel blocker, found the increase in pro-inflammatory mediators seen following the stressor was largely, though not fully in all mediators, prevented. This was also associated with a decrease in SA-β-gal, a marker of endothelial cell senescence and dysfunction, compared to the irradiated group (Ramadan et al., 2020). Similar results have been shown by other groups that have examined human endothelial cells incubated in mesenchymal stem cell conditioned media (MSC-CM), which is thought to exhibit therapeutic potential for microvascular injury through angiogenic cytokines. This study revealed a significant but not complete prevention of pro-inflammatory mediators IL-1α, IL-6 and TNF-α. The same pattern was seen in endothelial dysfunction, where apoptosis was significantly prevented but was still slightly above control levels (Chang et al., 2017).</span></p>
<ul>
<li>
<p><span style="font-family:Times New Roman,Times,serif">Much of the evidence for this relationship comes from <em>in vitro</em> studies; further work is needed to determine the certainty of the relationship at the tissue level. </span></p>
</li>
<li>
<p><span style="font-family:Times New Roman,Times,serif">Although studies often measure pro-inflammatory mediators at a few specific time points, chronic inflammation is what contributes to endothelial dysfunction. More human studies should examine the temporal concordance of this relationship to identify whether the inflammation is chronic. </span></p>
</li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.</span></span></span></span></span></p>
ModerateMaleLowFemaleLowUnspecificLowAdultModerateJuvenileLowModerateLow<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">The majority of the evidence is derived from <em>in vitro</em> studies, and a single <em>in vivo</em> study in male pre-adolescent mice. </span></span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Baselet, B. et al. (2017), “Functional Gene Analysis Reveals Cell Cycle Changes and Inflammation in Endothelial Cells Irradiated with a Single X-ray Dose”, <em>Frontiers in pharmacology</em>, Vol. 8, Frontiers Media SA, Lausanne, <a href="https://doi.org/10.3389/fphar.2017.00213" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fphar.2017.00213</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Bonetti, P. O., L. O. Lerman and A. Lerman (2003), “Endothelial dysfunction: a marker of atherosclerotic risk”, <em>Arteriosclerosis, thrombosis, and vascular biology</em>, Vol. 23/2, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/01.atv.0000051384.43104.fc" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/01.atv.0000051384.43104.fc</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Chang, P. Y. et al. (2017), “MSC-derived cytokines repair radiation-induced intra-villi microvascular injury”, <em>Oncotarget</em>, Vol. 8/50, Impact Journals, Buffalo, <a href="https://doi.org/10.18632/oncotarget.21236" style="color:#0563c1; text-decoration:underline">https://doi.org/10.18632/oncotarget.21236</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Deanfield, J. E., J. P. Halcox and T. J. Rabelink. (2007), “Endothelial Function and Dysfunction”, <em>Circulation</em>, Vol. 115/10, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/CIRCULATIONAHA.106.652859" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/CIRCULATIONAHA.106.652859</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Hughson, R. L., A. Helm and M. Durante. (2018), “Heart in space: Effect of the extraterrestrial environment on the cardiovascular system”, <em>Nature Reviews Cardiology</em>, Vol. 15/3, Nature Portfolio, London, <a href="https://doi.org/10.1038/nrcardio.2017.157" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/nrcardio.2017.157</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. https://doi.org/10.1080/09553002.2022.2110306</span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Ramadan, R. et al. (2021), “The role of connexin proteins and their channels in radiation-induced atherosclerosis”, <em>Cellular and molecular life sciences: CMLS</em>, Vol. 78/7, Springer, New York, <a href="https://doi.org/10.1007/s00018-020-03716-3" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1007/s00018-020-03716-3</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Ramadan, R. et al. (2020), “Connexin43 Hemichannel Targeting With TAT-Gap19 Alleviates Radiation-Induced Endothelial Cell Damage”, <em>Frontiers in pharmacology</em>, Vol. 11, Frontiers Media SA, Lausanne, <a href="https://doi.org/10.3389/fphar.2020.00212" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fphar.2020.00212</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Shen, Y. et al. (2018), “Transplantation of Bone Marrow Mesenchymal Stem Cells Prevents Radiation-Induced Artery Injury by Suppressing Oxidative Stress and Inflammation”, <em>Oxidative medicine and cellular longevity</em>, Vol. 2018, Hindawi, London, <a href="https://doi.org/10.1155/2018/5942916" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1155/2018/5942916</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Slezak, J. et al. (2017), “Potential markers and metabolic processes involved in the mechanism of radiation-induced heart injury”, <em>Canadian journal of physiology and pharmacology</em>, Vol. 95/10, Canadian Science Publishing, Ottawa, </span></span><a href="https://doi.org/10.1139/cjpp-2017-0121" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1139/cjpp-2017-0121</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Sylvester, C. B. et al. (2018), “Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer”, <em>Frontiers in Cardiovascular Medicine</em>, Vol. 5, Frontiers Media SA, Lausanne, <a href="https://doi.org/10.3389/fcvm.2018.00005" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fcvm.2018.00005</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Soloviev, A. I. and I.V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, <em>Biochemical pharmacology</em>, Vol. 159, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.bcp.2018.11.019" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.bcp.2018.11.019</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, <em>The Journals of Gerontology Series A: Biological Sciences and Medical Sciences</em>, Vol. 68/12, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/gerona/glt057" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/gerona/glt057</a> </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Venkatesulu, B. P. et al. (2018), “Radiation-Induced Endothelial Vascular Injury: A Review of Possible Mechanisms”, <em>JACC: Basic to translational science</em>, Vol. 3/4, Elsevier, Amsterdam, </span></span><a href="https://doi.org/10.1016/j.jacbts.2018.01.014" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1016/j.jacbts.2018.01.014</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Wang, H. et al. (2019), “Radiation-induced heart disease: a review of classification, mechanism and prevention”, <em>International Journal of Biological Sciences, </em>Vol. 15/10, Ivyspring International Publisher, Sydney, </span></span><a href="https://doi.org/10.7150/ijbs.35460" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.7150/ijbs.35460</a></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="background-color:white"><span style="color:black">Wang, Y., M. Boerma and D. Zhou (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, <em>Radiation research</em>, Vol. 186/2, Radiation Research Society, Bozeman, </span></span><a href="https://doi.org/10.1667/RR14445.1" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1667/RR14445.1</a><span style="background-color:white"><span style="color:black">. </span></span></span></p>
2022-09-28T12:43:542023-03-21T10:39:0742ee9daa-42e3-49b5-9aa0-9dc2b7d09c20ce63fcef-8305-4862-ad5a-db8fc07c2ae8<p><span style="font-family:Times New Roman,Times,serif">Proper endothelial activation is a key step in the growth of new vessels through the process of angiogenesis, a process also affected by a dysfunctional endothelium (Rajashekhar et al., 2006). Regional responses to stressors are also possible, with mechanical stressors differentially affecting pressure in vessels above (superior to) and below (inferior to) the heart (Hargens & Watenpaugh, 1996; Zhang, 2001). The endothelial layer is responsive to these variations in mechanical stresses and can adapt through altering the balance between hypertrophic and hypotrophic remodeling in smooth vessel cells lining vasculature (Baeyens et al., 2016) in part through altering the progression of the acid sphingomyelinase (ASM)/ceramide (Cer) pathway (Cheng et al., 2017).</span></p>
<p><span style="font-family:Times New Roman,Times,serif">Overall weight of evidence: Moderate</span></p>
<p><span style="font-family:Times New Roman,Times,serif">The relationship between endothelial function and vascular remodeling is well supported through a number of in-depth reviews about the mechanisms behind the connection. In a functional endothelial layer, the endothelial cells both contribute and react to the high levels of bioavailable nitric oxide (NO). The result is a vasomotive balance primed for vasodilation and an elevated ratio of antioxidant to pro-oxidant species (Deanfield et al., 2007). Endothelial cells can become activated through various signals, including vascular endothelial growth factor (VEGF), that subsequently induce angiogenesis (Carmeliet & Jain, 2011). Lastly, the endothelial cells form tight junctions and together with pericytes form a basement membrane in tight control of vessel and cell permeability (Carmeliet & Jain, 2011). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Initial endothelial tissue injury can lead to premature cellular senescence resulting in endothelial dysfunction (Hughson et al., 2018). Cell death in the vessels can lead to overall cell loss and reduced vascular density. Although recovery in the form of revascularization following the decrease in vascular density occurs, it is complicated in cases of continuous exposure as it negatively impacts the angiogenic process (Hughson et al., 2018). Stressors such as radiation are thought to disturb angiogenesis through decreasing VEGF secretion causing a decrease in tubule formation (Sylvester et al., 2018). Following the initial tissue injury, the body’s ability to heal is also compromised, in part due to the prolonged state of oxidative stress and simultaneous decrease in endothelium-dependent vasorelaxation leading to increase in non-laminar blood flow. To compensate for the damage caused by this turbulent flow, there is intima-media thickening and potential for eventual atherosclerosis (Bonetti et al., 2003; Hughson et al., 2018; Slezak et al., 2017; Sylvester et al., 2018). Continued injury also leaves the vessels vulnerable to maladaptive repair and ensuing fibrosis (Hsu et al., 2019). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">When activated within healthy limits, endothelial cells loosen at their junction and the presence of VEGF induces increased vessel permeability allowing for the vessels to expand and undergo angiogenesis (Carmeliet & Jain, 2011). However, prolonged increase in permeability is also a marker of dysfunction, where increases in adhesion proteins, and elevated levels of cell senescence accompany this change (Demontis et al., 2017; Hughson et al., 2018). Disruption of endothelial integrity can also lead to cell detachment from the basement membrane; with cardiovascular deconditioning following bedrest leading to significantly elevated levels of circulating endothelial cells in microcirculation (Zhang, 2013). In addition to vessel permeability, there are changes to permeability of endothelial cells themselves, which is shown to increase and not recover following removal of the stressor (Baran et al., 2021). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">The elevated bioavailability of NO associated with proper endothelial function correlates with a decrease in prothrombotic factors, while a dysfunction in the endothelium creates a pro-thrombotic environment (Krüger-Genge et al., 2019). This is also true in the case of certain kinds of vascular damage, where a dysfunctional endothelial layer following stressor exposure has been shown to lead to lymphocyte adhesion and thrombus formation. This pro-thrombotic environment causes vessel occlusion and a decrease in capillary and vascular density, which in turn results in increased vascular resistance requiring further vascular remodeling as compensation (Slezak et al., 2017). Cell senescence following damage also induces monocyte adhesion and can further contribute to creating a pro-atherosclerotic environment (Hughson et al., 2018). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">There is also evidence that the relationship between endothelial function and vascular remodeling is regionally affected following the exposure to localized varied mechanical stressors such as microgravity. Under microgravity conditions (and in simulated microgravity models such as hindlimb unloading (HU)), there is a cephalic shift in blood and fluid resulting in a change of transmural pressure, increasing pressure in vessels above the heart and decreasing in those below (Hargens & Watenpaugh, 1996; Zhang, 2001). The heart continues to pump as usual; however, above the heart the arterial flow is no longer pushing against gravity resulting in increased arterial vascular pressure, while venous return is simultaneously slowed without gravitational assistance. This results in changes such distended veins and arteries in the upper body, increased carotid intima-media thickness and vascular stiffness (Garrett-Bakelman et al., 2019). In the lower limb, the opposite is true as arterial perfusion is decreased and venous return is increased resulting in muscle atrophy. Blood pressure and related fluid shear stress act as important mechanical input for the mechanosensing endothelial cells, which translate these forces into biochemical signals that guide vascular remodeling through affecting the balance between vascular smooth muscle cell proliferation and apoptosis (Baeyens et al., 2016). Above the heart, remodeling presents as hypertrophy and increase in vasoreactivity, while below the heart there is hypotrophy and decrease in myogenic tone and vasoreactivity (Zhang, 2013). This trend has been observed by various reviews and studies; a review examining studies using HU rats summarized that the models studied showed both a decrease in response to drugs inducing vasodilation and constriction and a subsequent increased stiffness in the aorta and carotid arteries (Platts et al., 2014). In humans, bedrest study participants showed both a decrease in endothelium-dependent vasodilation and an increase in circulating endothelial cells – both markers of endothelial dysfunction. Additional bedrest studies also show a decrease in vessel diameter and intimal-medial thickness in arteries below the heart while those above the heart remain unaffected (Zhang, 2013). Ultrasound measurements of cosmonauts having travelled aboard Mir and Salyut-7 showed that after spaceflight, blood supply to the brain remained stable while below the heart vascular tone and arterial resistance was severely compromised (Zhang, 2013). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Research comparing the changes in vascular structure and vasodilation response between the various muscle resistance arteries following HU also showed changes to regionally vary between the vessels studied (Delp et al., 2000; Zhang, 2013). Additionally, HU models showed differences in the arterial response to vasoconstrictors and changes to artery diameter in cutaneous versus skeletal muscle arteries (Tarasova et al., 2020). The ASM and Cer pathway has been investigated for its role in remodeling. The work of Cheng et al. (2017) and Su et al. (2020) both found that a decrease in ASM activity and subsequent Cer production was linked to a decrease in apoptosis levels and a resulting thickening of vessel structure (Cheng et al., 2017; Su et al., 2020). It is important to note that some of the vascular changes following microgravity are protective adaptations that serve to safeguard the cardiovascular system in altered gravity conditions. Under continued microgravity conditions, these changes maintain their protective purpose and are thought not to contribute to adverse outcome progression. The problem arises upon return to earth when the vessels that have adapted to microgravity blood distribution are faced with earth conditions and issues like a decreased orthostatic tolerance surface. </span></p>
<p><span style="font-family:Times New Roman,Times,serif"><strong>Dose Concordance</strong> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">There is some evidence in the literature supporting dose concordance between endothelial dysfunction and vascular remodeling. For example, gamma irradiation at 0.5 Gy led to a 9% decrease in endothelium-dependent vasodilation and no significant changes to vascular stiffness, while a 1 Gy dose led to 13% decrease in vasodilation corresponding to a 16% increase in vascular stiffness (Soucy et al., 2011). A dose of 5 Gy gamma rays significantly attenuated endothelium-dependent vasodilation, while simultaneously increasing vascular stiffness compared to a non-irradiated control (Soucy et al., 2010). Work exploring apoptosis as a measure of endothelial dysfunction demonstrated that an 18 Gy dose of X-rays increased the number of cells with apoptotic DNA fragmentation ~4.5-fold and increased aortic thickness ~1.5 fold compared to control (Shen et al., 2018). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">Studies in rat models of HU by Su et al. (2020), Delp et al. (2000), and Cheng et al. (2017) all demonstrate the regional effects of changes in pressure on resulting vascular adaptation and remodeling. Su et al. (2020) compared the effects of 4-week unloading on the cerebral versus mesenteric artery, showing the balance moving towards cell proliferation above the heart (cerebral artery) with a decrease in apoptosis and increase in intima-media thickness and cross-sectional area. Meanwhile, below the heart (mesenteric artery) apoptosis increased and intima-media thickness and cross-sectional area decreased (Su et al., 2020). This agrees with a similar study that found apoptosis decreased and intima-media thickness of the carotid artery increased following HU (Cheng et al., 2017). Delp et al. (2000) showed that regional adaptations to changes in pressure following HU are also affected by how this change in pressure manifests. Vascular structure changes and endothelium-dependent vasodilation were observed in the gastrocnemius and soleus primary arterioles. Both vessels are in the hindlimbs of mice and therefore are subject to a decrease in pressure following cephalic shift in fluid in 2-week HU. In the gastrocnemius muscle, which saw a decrease in transmural pressure, the decrease in vessel cross sectional area (CSA) was due to muscle atrophy shown by a drop in media thickness but no change in outer media perimeter. In contrast, the soleus muscle experienced a drop in wall shear stress and saw a drop in vessel perimeter with no change in media thickness. Simultaneously, arterioles in the gastrocnemius muscle saw no change in acetylcholine (ACh) response, while those in soleus muscles saw a 50% decrease following the unloading and recovery to control levels at the 4-week time point (Delp et al., 2000). </span></p>
<p><span style="font-family:Times New Roman,Times,serif"> </span></p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Time Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is limited evidence supporting the time concordance of endothelial dysfunction and vascular remodeling. Aortic relaxation response to ACh in Sprague-Dawley rats was found to decrease 20-30% 2 weeks after gamma irradiation with an increase in vascular stiffness measured by pulse-wave velocity (PWV) from around 3.9 m/s to 4.9 m/s (Soucy et al., 2010; Soucy et al., 2007). Shen et al. (2018) demonstrated that endothelial dysfunction assessed via apoptosis became significant 3 days after 18 Gy X-rays in a mouse model, then had a linear decrease which tapered off by day 84. Aortic wall thickness, in turn, showed no significant increase on day 3 post-irradiation, only reaching maximal increase on day 7 before also decreasing linearly to day 84 (Shen et al., 2018). At 4 and 8 months post- <sup>56</sup>Fe-ion irradiation, Wistar rats had 13% and 16% decreased endothelial relaxation response respectively. As well, PWV increased from 4.03 m/s to 4.45 m/s at 4 months and 4.53 m/s to 5.06 m/s at 8 months post-irradiation (Soucy et al., 2011). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif"><strong>Incidence concordance</strong> </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Incidence concordance is moderate in this KER, as multiple studies demonstrate greater changes to endothelial dysfunction than to vascular remodeling. Three studies by Soucy et al. (2011, 2010, 2007) in rats demonstrate greater changes to vasodilation than to vascular stiffness after 5 Gy of gamma rays or 1 Gy of iron ions. In addition, 18 Gy X-ray irradiation of mice showed a 4.5-fold increase in apoptosis and a 1.4-fold increase in aortic thickness (Shen et al., 2018). Following 1 or 4 weeks of HU, greater changes to apoptosis were observed compared to changes in vascular remodeling in the cerebral and small mesenteric arteries of rats (Su et al., 2020). </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Essentiality </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Human bone marrow-derived mesenchymal stem cells (hBMSCs) can prevent endothelial dysfunction and vascular remodeling via their antioxidant and anti-inflammatory properties. While a radiation dose of 18 Gy X-rays in a mouse model increased both the amount of apoptosis in the aorta (indicating endothelial dysfunction) and aortic wall thickness, treatment with hBMSCs reversed these changes. TUNEL positive cells decreased but remained elevated above the control, while aortic wall thickness returned to control levels (Shen et al., 2018). </span></p>
<p> </p>
<p><span style="font-family:Times New Roman,Times,serif">In work exploring the role of the ASM/Cer pathway, a decrease in the ASM activity and resulting Cer production corresponded to a decrease in apoptosis and increase in cell-proliferation in rat models of simulated microgravity (Cheng et al., 2017; Su et al., 2020). Incubation with permeable Cer (C6-Cer) returned apoptosis to control levels. Treatment with the ASM inhibitor desipramine (dpm) led to an overall decrease in apoptosis in all arteries tested, while treatment with doxepin hydrochloride (DOX) led to significant increases in cell proliferation and subsequent intima medial thickness (IMT) and cross-sectional areas (CSA) (Su et al., 2020). </span></p>
<ul>
<li>
<p><span style="font-family:Times New Roman,Times,serif">Lower doses (0.5 Gy and 1.6 Gy) did not show changes in vasomotion compared to control, but vascular stiffness increased at these doses (Soucy et al., 2007). </span></p>
</li>
<li>
<p><span style="font-family:Times New Roman,Times,serif">Tarasova et al. (2020) showed differences in the vascular remodeling and vasoconstriction responses between skeletal and cutaneous arteries. While the two groups demonstrated differences, all vessels followed different trends showing no clear relationship between KEs. </span></p>
</li>
<li>
<p><span style="font-family:Times New Roman,Times,serif">Studies exploring vasoreactivity, vascular structure and vessel stiffness endpoints in humans (Lee et al., 2020) and mice (Sofronova et al., 2015) flown in space, found changes in these endpoints to be inconsistent and/or changes were not statistically significant. </span></p>
</li>
<li>
<p><span style="font-family:Times New Roman,Times,serif">C6-Cer incubation in cerebral arteries showed increased apoptosis with HU in the study by Cheng et al. (2017); however, in Su et al. (2020), there was a slight decrease in apoptosis, measured by TUNEL. </span></p>
</li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.</span></span></span></span></span></p>
HighMaleModerateFemaleModerateAdultLowJuvenileModerateNot Otherwise SpecifiedModerateModerateHigh<p><span style="font-family:Times New Roman,Times,serif">There is a substantial amount of evidence for this KER from <em>in vivo</em> rodent models and from human studies. The sex applicability is high for males and moderate for females as many studies were done only using male animals. Most studies indicated that the animals used were adult. </span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Baeyens, N. et al. (2016), “Endothelial fluid shear stress sensing in vascular health and disease”, <em>The Journal of Clinical Investigation</em>, Vol. 126/3, American Society for Clinical Investigation,</span> <span style="font-size:12.0pt">Ann Arbor, https://doi.org/10.1172/JCI83083.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Baran, R. et al. (2022), “The Cardiovascular System in Space: Focus on In Vivo and In Vitro Studies”, <em>Biomedicines</em>, Vol. 10/1, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/BIOMEDICINES10010059.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Bonetti, P.O., L. O. Lerman and A. Lerman (2003), “Endothelial dysfunction: A marker of atherosclerotic risk”, <em>Arteriosclerosis, Thrombosis, and Vascular Biology</em>, Vol. 23/2, Lippincott Williams & Wilkins, Philadelphia, https://doi.org/10.1161/01.ATV.0000051384.43104.FC.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Carmeliet, P. and R. K. Jain. (2011), “Molecular mechanisms and clinical applications of angiogenesis”, <em>Nature</em>, Vol.473, Nature Portfolio, London, https://doi.org/10.1038/nature10144.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Cheng, Y. P. et al. (2017), “Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats”, <em>Pflugers Archiv European Journal of Physiology</em>, Vol. 469, Springer, New York, https://doi.org/10.1007/s00424-017-1969-z.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Deanfield, J.E., J. P. Halcox and T. J. Rabelink (2007), “Endothelial function and dysfunction: Testing and clinical relevance”, <em>Circulation</em>, Vol. 115/10, Lippincott Williams & Wilkins, Philadelphia, https://doi.org/10.1161/CIRCULATIONAHA.106.652859.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Delp, M.D. et al. (2000), “Structural and functional remodeling of skeletal muscle microvasculature is induced by simulated microgravity”, <em>American Journal of Physiology - Heart and Circulatory Physiology</em>, Vol. 278, American Physiological Society, Rockville, https://doi.org/10.1152/ajpheart.2000.278.6.h1866.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Demontis, G.C. et al. (2017), “Human Pathophysiological Adaptations to the Space Environment”, <em>Frontiers in Physiology</em>, Vol. 8, Frontiers Media SA, Lausanne, https://doi.org/10.3389/fphys.2017.00547.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Garrett-Bakelman, F. E. et al. (2019) “The NASA Twins Study: A multidimensional analysis of year-long human spaceflight”, <em>Science</em>, Vol. 364/6436, American Association for the Advancement of Science, Washington, D.C., https://doi.org/10.1126/science.aau8650</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Hargens, A.R. and D. E. Watenpaugh (1996), “Cardiovascular adaptation to spaceflight”, <em>Medicine & Science in Sports & Exercise</em>, Vol. 28/8, Lippincott Williams & Wilkins, Piladelphia, https://doi.org/10.1097/00005768-199608000-00007.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Hsu, T., H. H. Nguyen-Tran and M. Trojanowska (2019), “Active roles of dysfunctional vascular endothelium in fibrosis and cancer”, <em>Journal of Biomedical Science</em>, Vol. 26/1, BioMed Central, London, https://doi.org/10.1186/S12929-019-0580-3.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Hughson, R.L., A. Helm and M. Durante. (2018), “Heart in space: Effect of the extraterrestrial environment on the cardiovascular system”, <em>Nature Reviews Cardiology</em>, Vol. 15, Nature Portfolio, London, https://doi.org/10.1038/nrcardio.2017.157</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, <em>International Journal of Radiation Biolog</em>y, Vol. 98/12. https://doi.org/10.1080/09553002.2022.2110306</span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Krüger-Genge, A. et al. (2019), “Vascular Endothelial Cell Biology: An Update”, <em>International Journal of Molecular Sciences</em>, Vol. 20/18, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/ijms20184411.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Lee, S. M. C. et al. (2020), “Arterial structure and function during and after long-duration spaceflight”, <em>Journal of Applied Physiology</em>, Vol. 129, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00550.2019.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Platts, S.H. et al. (2014), “Effects of sex and gender on adaptation to space: Cardiovascular alterations”, <em>Journal of Women’s Health</em>, Vol. 23/11, Mary Ann Liebert, Larchmont, https://doi.org/10.1089/jwh.2014.4912.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Rajashekhar, G. et al. (2006), “Continuous Endothelial Cell Activation Increases Angiogenesis: Evidence for the Direct Role of Endothelium Linking Angiogenesis and Inflammation”, <em>Journal of Vascular Research</em>, Vol. 43/2, Karger Publishers, Berlin, https://doi.org/10.1159/000090949.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Shen, Y. et al. (2018), “Transplantation of bone marrow mesenchymal stem cells prevents radiation-induced artery injury by suppressing oxidative stress and inflammation”, <em>Oxidative Medicine and Cellular Longevity</em>, Vol. 2018, Hindawi, London, https://doi.org/10.1155/2018/5942916.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Slezak, J. et al. (2017), “Potential markers and metabolic processes involved in the mechanism of Radiation-Induced heart injury”, <em>Canadian Journal of Physiology and Pharmacology</em>, Vol. 95/10, Canadian Science Publishing, Ottawa, https://doi.org/10.1139/cjpp-2017-0121.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Sofronova, S. I. et al. (2015), “Spaceflight on the Bion-M1 biosatellite alters cerebral artery vasomotor and mechanical properties in mice”, <em>Journal of Applied Physiology</em>, Vol. 118/7, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00976.2014.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Soucy, K. G. et al. (2011), “HZE <sup>56</sup>Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, <em>Radiation Research</em>, Vol. 176/4, Radiation Research Society, Bozeman, https://doi.org/10.1667/RR2598.1.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, <em>Journal of Applied Physiology</em>, Vol. 108/5, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00946.2009.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Soucy, K. G. et al. (2007), “Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta”, <em>Radiation and Environmental Biophysics</em>, Vol. 46, Springer, New York, https://doi.org/10.1007/s00411-006-0090-z.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Su, Y. T. et al. (2020), “Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats”, <em>Life Sciences</em>, Vol. 243, Elsevier, Amsterdam, https://doi.org/10.1016/j.lfs.2019.117253.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Sylvester, C. B. et al. (2018), “Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer”, <em>Frontiers in Cardiovascular Medicine</em>, Vol. 5/5, Frontiers Media SA, Lausanne, https://doi.org/10.3389/fcvm.2018.00005.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Tarasova, O. S. et al. (2020), “Simulated Microgravity Induces Regionally Distinct Neurovascular and Structural Remodeling of Skeletal Muscle and Cutaneous Arteries in the Rat”, <em>Frontiers in Physiology</em>, Vol. 11, Frontiers Media SA, Lausanne, https://doi.org/10.3389/fphys.2020.00675.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Zhang, L. F. (2013), “Region-specific vascular remodeling and its prevention by artificial gravity in weightless environment”, <em>European Journal of Applied Physiology</em>, Vol. 113, American Physiological Society, Rockville, https://doi.org/10.1007/s00421-013-2597-8.</span></span></span></p>
<p style="margin-left:32px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:11pt"><span style="font-size:12.0pt">Zhang, L. F. (2001), “Vascular adaptation to microgravity: What have we learned?”, <em>Journal of Applied Physiology</em>, Vol. 91/6, American Physiological Society, Rockville, https://doi.org/10.1152/jappl.2001.91.6.2415.</span></span></span></p>
2022-10-17T16:54:172023-03-21T09:43:16383161af-8fa6-4cd9-81d4-8c7c472b341d42ee9daa-42e3-49b5-9aa0-9dc2b7d09c20<p><span style="font-family:Times New Roman,Times,serif">Altered nitric oxide (NO) levels can lead to endothelial dysfunction (Soloviev & Kizub, 2019). In a functional endothelium, NO is bioavailable and is involved in preventing inflammation, proliferation and thrombosis (Deanfield, Halcox & Rabelink, 2007; Kruger-Genge et al., 2019). An increase in reactive oxygen species (ROS) along with increased NO can drive cellular senescence in endothelial cells (ECs) and catalyze endothelial dysfunction (Nagane et al., 2021; Wang, Boerma & Zhou, 2016). Another driver of endothelial dysfunction is reduced vasomotion. In a functional state, the endothelium requires a balance of vasoconstrictors and vasodilators (like NO); an interruption of this balance can lead to dysfunction (Deanfield, Halcox & Rabelink, 2007; Marti et al., 2012; Nagane et al., 2021; Schulz, Gori & Münzel, 2011; Soloviev & Kizub, 2019). Decreased NO due to direct reactions with ROS or uncoupling of NOS enzymes will lead to a reduced ability of smooth muscle cells (SMCs) to relax (Soloviev & Kizub, 2019).</span></p>
<p>Overall weight of evidence: Moderate</p>
<p><span style="font-family:Times New Roman,Times,serif">The biological plausibility surrounding the connection between altered NO levels leading to endothelial dysfunction is well-supported by literature. NO is synthesized from L-arginine and oxygen with the aid of enzymes and cofactors (Nagane et al., 2021). NO regulates ECs by binding to soluble guanylyl cyclase (sGC) to create cGMP and activate protein kinase G (PKG), leading to activation of Ca<sup>2+</sup>-dependent vasodilation and smooth muscle relaxation (Nagane et al., 2021; Soloviev & Kizub 2019). NOS isoforms, such as inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) that synthesize NO are indirect measures of NO. Lower NO reduces the ability of SMCs relaxation and dilates the blood vessel leading to an inability to control vasodilation, a component of endothelial dysfunction (Soloviev & Kizub 2019). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Increased expression of NOS enzymes can result in reduced NO levels in the case of insufficient L-arginine substrate or BH4 cofactor leading to ROS production instead of NO (Zhang et al., 2009). ROS can decrease NO bioavailability by uncoupling/downregulating eNOS or converting NO to peroxynitrite (Mitchel et al 2019; Schulz, Gori & Münzel, 2011; Soloviev & Kizub 2019; Wang, Boerma & Zhou, 2016). A further decrease in NO occurs as peroxynitrite oxidizes BH4 to BH2 and induces eNOS to produce ROS, continuing the uncoupling of more NOS enzymes (Hong et al., 2013; Soloviev & Kizub, 2019; Zhang et al., 2009). A reduction in NO bioavailability due to ROS is an important mediator of endothelial dysfunction (Schulz, Gori & Münzel, 2011). </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Another component of endothelial dysfunction influenced by NO levels is cellular senescence (Nagane et al., 2021). iNOS expression increases following an increase in oxidative stress (Nathan & Xie, 1994). In the presence of oxidative stress, NO is converted to peroxynitrite, which is a reactive nitrogen species (RNS) that can modify proteins and lead to cellular senescence (Hong et al., 2013; Nagane et al., 2021; Soloviev & Kizub, 2019). Although NO can increase at first and cause cell senescence, senescent ECs show downregulation and/or uncoupling of eNOS that contributes to a decrease of NO in the endothelium (Wang, Boerma & Zhou, 2016). These changes in senescent ECs lead to endothelial dysfunction. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">The empirical evidence to support this KER was gathered from research that utilized <em>in vivo</em> rat and rabbit models (Soucy et al., 2010; Soucy et al., 2011; Hong et al., 2013; Yan et al., 2020; Zhang et al., 2009), <em>ex vivo</em> rabbit models and <em>in vitro</em> HUVEC models (Hong et al., 2013). Stressors used to alter NO levels and increase endothelial dysfunction include <sup>56</sup>Fe ions (Soucy et al., 2011), X-rays (Hong et al., 2013; Yan et al., 2020), gamma rays (Soucy et al., 2010) and altered gravity by hindlimb unweighting (HU) (Zhang et al., 2009). Irradiation dose levels ranged from 0.5 Gy to 16 Gy (Hong et al., 2013; Soucy et al., 2010; Soucy et al., 2011; Yan et al., 2020). Studies used various endpoints to measure NO levels while endothelial function was consistently determined through relaxation response to acetylcholine (ACh). Methods to measure NO included DAF-FM DA fluorescent probe (Soucy et al., 2010; Soucy et al., 2011), Griess reagent NO assay kit (Yan et al., 2020), eNOS dimer to monomer ratio (Yan et al., 2020), and NOS protein and mRNA levels (Hong et al., 2013; Zhang et al., 2009).</span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Dose Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is moderate evidence to demonstrate dose concordance between a decrease in nitric oxide and endothelial dysfunction. <sup>56</sup>Fe ion irradiation in rat aorta showed a decrease in both NO levels and endothelial relaxation at 1 Gy. However, endothelial relaxation did not significantly decrease at a lower dose of 0.5 Gy (Soucy et al., 2011). After 5 Gy gamma irradiation, NO production and endothelial relaxation both decreased 0.7-fold in rat aorta (Soucy et al., 2010). Increased NOS levels can cause further NO decreases due to uncoupling and production of more peroxynitrite, which is the result of NO reacting with ROS and can therefore, be used to determine NO levels (Hong et al., 2013; Zhang et al., 2009). In rat mesenteric arteries, NO levels decreased 0.6-fold after 4 Gy X-ray irradiation, while endothelial relaxation decreased 0.1-fold compared to non-irradiated arteries (Yan et al., 2020). X-ray irradiated human umbilical vein endothelial cells (HUVECs) showed significantly increased iNOS and peroxynitrite after 4 Gy, and X-ray irradiated rabbit carotid arteries had decreased relaxation after 8 and 16 Gy (Hong et al., 2013). Hong et al (2013) also showed that endothelial relaxation was lower after 16 Gy than 8 Gy ex vivo. </span></p>
<p><span style="font-family:Times New Roman,Times,serif">Altered gravity resulted in an increase of eNOS and increase of iNOS in carotid arteries (Zhang et al., 2009). Increases in eNOS and iNOS corresponded to a decrease in carotid artery relaxation from 64% to 33% at the same level of HU (Zhang et al., 2009). These studies showed increases in NOS isoforms and corresponding decreases in endothelial function, such as increased vasoconstriction and impaired vasodilation following altered gravity. </span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Time Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Evidence of time concordance between altered NO levels and endothelial dysfunction is limited from the studies cited. HUVECs irradiated with 4 Gy X-rays displayed an increase in nitrotyrosine (peroxynitrite biomarker indicating reduced NO) after 6 hours post-irradiation (Hong et al., 2013). Rabbit carotid arteries <em>in vivo</em> and <em>ex vivo</em> irradiated with 8 or 16 Gy X-rays showed decreased ACh-induced endothelial relaxation only after 20 hours post-irradiation (Hong et al., 2013).</span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Incidence Concordance </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">There is limited support in current literature for an incidence concordance relationship between altered NO and endothelial dysfunction. A primary research study that supports this AOP demonstrated an average change to endpoints of altered NO that was greater or equal to that of endothelial dysfunction (Zhang et al., 2009).</span></p>
<p> </p>
<p><strong><span style="font-family:Times New Roman,Times,serif">Essentiality </span></strong></p>
<p><span style="font-family:Times New Roman,Times,serif">Many studies show the essentiality of decreased NO levels in endothelial dysfunction. After stressors like irradiation, xanthine oxidase (XO) can produce cardiac ROS that can react with NO and decrease its concentration or oxidize BH4 and uncouple eNOS. Oxypurinol (Oxp), an inhibitor of XO, has been shown to reverse these effects and reduce ROS levels, restore NO levels, and increase endothelial relaxation following irradiation (Soucy et al., 2010; Soucy et al., 2011). L-nitroarginine (L-NA, general NOS inhibitor) and aminoguanidine (AG, specific iNOS inhibitor) together were able to reduce relaxation of rabbit carotid arteries, suggesting that reduced NO levels are a key cause to endothelial dysfunction (Hong et al., 2013). In addition, L-NA and AG were able to reduce iNOS and nitrotyrosine (peroxynitrite biomarker) levels after irradiation (Hong et al., 2013). Gch1 is an enzyme involved in the synthesis of BH4, a cofactor for eNOS coupling. DAHP, a Gch1 inhibitor, caused the ratio of coupled-to-uncoupled eNOS to decrease and endothelial relaxation to also decrease, showing how coupled eNOS is necessary for endothelial function (Yan et al., 2020). Angiotensin II (AngII) type 1 (AT1) receptor activation can activate NOS. HU rats treated with losartan, an AT1 receptor antagonist, show reduced NOS levels and increased endothelial relaxation (Zhang et al., 2009).</span></p>
<ul>
<li>
<p><span style="font-family:Times New Roman,Times,serif">Directionality of NO changes cannot be compared between studies due to a variety of experimental conditions like stressor type, dose, dose rate, model and time course of the experiment.</span></p>
</li>
<li>
<p><span style="font-family:Times New Roman,Times,serif">Irradiating in vivo rabbit carotid arteries with X-rays showed that endothelial dysfunction was higher after 8 Gy than 16 Gy (Hong et al., 2013). This was inconsistent with the ex vivo model, where endothelial dysfunction was highest after 16 Gy (Hong et al., 2013). Endothelial dysfunction was shown through a relaxation response to ACh.</span></p>
</li>
</ul>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.</span></span></span></span></p>
ModerateMaleLowFemaleLowUnspecificLowAdultModerateNot Otherwise SpecifiedLowModerateLow<p><span style="font-family:Times New Roman,Times,serif">The majority of the evidence is derived from <em>in vivo</em> rat models. A limited number of studies were in human and rabbit models. The relationship has been more commonly shown <em>in vivo</em> in male animals, specifically in adult male rodents.</span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Deanfield, J.E., J. P. Halcox and T. J. Rabelink (2007), “Endothelial function and dysfunction: Testing and clinical relevance”, <em>Circulation</em>, Vol. 115/10, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/CIRCULATIONAHA.106.652859" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1161/CIRCULATIONAHA.106.652859</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Hong, C. W. et al. (2013), “Involvement of inducible nitric oxide synthase in radiation-induced vascular endothelial damage”, <em>Journal of Radiation Research</em>, Vol. 54/6, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/JRR/RRT066" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/JRR/RRT066</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:"Times New Roman",Times,serif; font-size:1rem">Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, </span><em style="font-family:"Times New Roman",Times,serif; font-size:1rem">International Journal of Radiation Biolog</em><span style="font-family:"Times New Roman",Times,serif; font-size:1rem">y, Vol. 98/12. <a href="http://doi.org/10.1080/09553002.2022.2110306">https://doi.org/10.1080/09553002.2022.2110306</a></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Krüger-Genge, A. et al. (2019), “Vascular Endothelial Cell Biology: An Update”, <em>International Journal of Molecular Sciences</em>, Vol. 20/18, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/ijms20184411" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3390/ijms20184411</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Marti, C. N. et al. (2012), “Endothelial dysfunction, arterial stiffness, and heart failure”, <em>Journal of the American College of Cardiology</em>, Vol. 60/16, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/J.JACC.2011.11.082" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/J.JACC.2011.11.082</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Mitchell, A. et al. (2019), “Cardiovascular effects of space radiation: implications for future human deep space exploration”, <em>European Journal of Preventive Cardiology</em>, Vol. 26/16, SAGE Publishing, Thousand Oaks, <a href="https://doi.org/10.1177/2047487319831497." style="color:#0563c1; text-decoration:underline">https://doi.org/10.1177/2047487319831497.</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases”, <em>Journal of Radiation Research</em>, Vol. 62/4, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/JRR/RRAB032" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/JRR/RRAB032</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Nathan, C. and Q. W. Xie (1994), “Regulation of biosynthesis of nitric oxide”, <em>Journal of Biological Chemistry</em>, Vol. 269/19, American Society for Biochemistry and Molecular Biology, Rockville, https://doi.org/10.1016/S0021-9258(17)36703-0 </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt"> Schulz, E., T. Gori and T. Münzel (2011), “Oxidative stress and endothelial dysfunction in hypertension”, <em>Hypertension Research</em>, Vol. 34/6, Nature Portfolio, London, <a href="https://doi.org/10.1038/hr.2011.39" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1038/hr.2011.39</a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soloviev, A. I. and I.V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, <em>Biochemical pharmacology</em>, Vol. 159, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.bcp.2018.11.019" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.bcp.2018.11.019</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soucy, K. G. et al. (2011), “HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, <em>Radiation Research</em>, Vol. 176/4, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RR2598.1" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1667/RR2598.1</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, <em>Journal of Applied Physiology</em>, Vol. 108/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.00946.2009" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/japplphysiol.00946.2009</a>.</span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Wang, Y., M. Boerma and D. Zhou. (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, <em>Radiation research</em>, Vol. 186/2, Radiation Research Society, Bozeman, <a href="https://doi.org/10.1667/RR14445.1" style="color:#0563c1; text-decoration:underline" target="_blank">https://doi.org/10.1667/RR14445.1</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Yan, T., et al. (2020), “Ionizing radiation induces BH4 deficiency by downregulating GTP-cyclohydrolase 1, a novel target for preventing and treating radiation enteritis”, <em>Biochemical Pharmacology</em>, Vol. 180, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/J.BCP.2020.114102" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/J.BCP.2020.114102</a>. </span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:12pt">Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, <em>Journal of Applied Physiology</em>, Vol. 106/1, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.01278.2007" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1152/japplphysiol.01278.2007</a>. </span></span></p>
2022-10-20T15:07:532023-03-21T11:58:08Deposition of energy leads to vascular remodelingDeposition of energy leads to vascular remodeling<p>Tatiana Kozbenko<sup>1,2</sup>, Nadine Adam, Veronica Grybas<sup>1</sup>, Benjamin Smith<sup>1</sup>, Dalya Alomar<sup>1</sup>, Robyn Hocking<sup>1</sup>, Janna Abdelaziz<sup>3</sup>, Amanda Pace<sup>3</sup>, Carole Yauk<sup>2</sup>, Ruth Wilkins<sup>1</sup>, Vinita Chauhan<sup>1</sup></p>
<p>(1) Health Canada, Ottawa, Ontario, K1A 0K9, Canada </p>
<p>(2) University of Ottawa, Ottawa, Ontario K1N 6N5, Canada </p>
<p>(3) Carelton University, Ottawa, Ontario K1S 5B6, Canada </p>
<p><strong>Consultants</strong></p>
<p>Marjan Boerma<sup>1</sup>, Omid Azimzadeh<sup>2</sup>, Steve Blattnig<sup>3</sup>, Nobuyuki Hamada<sup>4</sup></p>
<p>(1) University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA</p>
<p>(2) Federal Office for Radiation Protection (BfS), Section Radiation Biology, 85764 Neuherberg, Germany</p>
<p>(3) NASA Langley Research Center Hampton, VA 23681, USA</p>
<p>(4) Biology and Environmental Chemistry Division, Sustainable System Research Laboratory, Central Research Institute of Electric Power Industry (CRIEPI), Tokyo, Japan</p>
Open for citation & comment<p>The present qualitative AOP (AOP#470) summarizes the evidence for a progression beginning with the deposition of energy to vascular remodeling. The pathway is initiated by ionization/excitation events from the deposition of energy (MIE: Event #1686) leading to an enviorment of reactive oxygen species (ROS), if this occurs at a rate that outpaces the antioxidant defense system, oxidative stress ensues (KE: Event #1392). Deposition of energy can concurrently induce DNA strand breaks (KE: Event #1635) either directly or through damage from ROS. Excessive ROS damages cellular compartments, thereby altering signaling pathways (KE: Event #2066), and increasing levels of pro-inflammatory mediators (KE: Event #1493). Within the vascular wall, activation of certain signaling molecules can alter nitric oxide (NO) levels (KE: Event #2067). All the upstream KEs of the pathway then converge to cause endothelial dysfunction (KE: Event #2068). Modified levels of NO can alter the blood flow within the endothelium resulting in subsequent compensatory vascular remodeling (AO: Event #2069). Vascular remodeling is an important precursor for many diverse cardiovascular pathologies and serves as an important marker for cardiovascular disease. Vascular remodeling includes many structural changes such as increased vessel stiffness, vessel wall thickening, and decreased capillary density. Studies informing this AOP include clinical follow-up studies of radiotherapy patients, epidemiological cohort studies of atomic bomb survivors and nuclear plant workers as well as biological studies using mouse and rat models. Knowledge gaps in the weight of evidence include inconsistencies in NO evaluation, and relatively few studies exploring chronic and low dose exposures including the lack of studies focusing on female biology. </p>
<p>Cardiovascular disease (CVD) includes any health condition affecting the heart and blood vessels. CVD is one of the leading causes of death worldwide, accounting for millions of deaths yearly and is surpassed in some countries, by only cancer (Bray et al., 2021; Tsao et al., 2022). This class of diseases includes congenital defects, as well as CVDs that can develop throughout life such as peripheral artery disease, atherosclerosis, coronary artery disease and myocardial infarction. While the progression to a CVD outcome is slow, many CVDs are often preceded by much earlier changes to vascular structure. Vascular remodeling entails various structural changes of existing vasculature arising from cell death, cell migration and changes to the endothelial cell membrane. It is important to note that changes to vascular structure are not inherently detrimental, and the cardiovascular system undergoes continuous adaptation to protect vascular health (Pries et al., 2001; Santamaría et al., 2020; Zakrzewicz et al., 2002). However, certain remodeling can also serve as an important marker and risk factor for future adverse cardiovascular events (Cohn et al., 2004; Van Varik et al., 2012). Changes to vascular structure can be triggered through perturbations such oxidative stress, inflammation, and alterations to cellular signaling pathways. Adverse remodeling of the vasculature encompasses structural and functional changes to vessel wall intima-media, elevated stiffness, and decreased lumen diameter which are all predictive of the development of and mortality and morbidity from CVD (Heald et al., 2006; Hodis et al., 1998; Polak et al., 2011; Zieman et al., 2005). </p>
<p>The risk of CVD increases with several factors such as age and available evidence suggests that environmental factors such as radiation can also contribute to increased risk (Belzile-Dugas & Eisenberg, 2021; Boerma et al., 2016; Francula-Zaninovic & Nola, 2018; Wang et al., 2019). The deposition of energy from radiation is a stochastic event, with adverse effects emerging years or decades after the exposure (Boerma et al., 2016; Dörr, 2015; EPRI, 2020; Menezes et al., 2018). The effects of high-dose radiation on the cardiovascular system have been well-characterized while the effects of low-dose exposure are more contended. However, growing evidence suggests that lower doses than previously thought are linked to cardiovascular outcomes (Boerma et al., 2016; EPRI, 2020; Little et al., 2021; UNSCEAR, 2008). Much of the high-dose data is from follow up studies in radiotherapy patient cohorts who have elevated risk for adverse cardiovascular events (Zou et al., 2019). In addition to clinical exposure scenarios, epidemiological studies of occupational exposures and Japanese atomic bomb survivors provide supporting evidence. Cohort studies of atomic bomb survivors show CVD risk can be modulated by factors such as age at exposure and estimated dose received (Ozasa et al., 2012; Preston et al., 2003; Shimizu et al., 2010; Takahashi et al., 2017). Long-term follow up of individuals exposed in the Chernobyl disaster also identified statistically significant elevation in CVD risk (Ivanov et al., 2006; Kashcheev et al., 2017). Occupational exposure studies have also been conducted in various countries in an effort to understand the relationship between low-dose chronic exposure and cardiovascular health of nuclear workers (Azizova et al., 2018; Gillies et al., 2017; Zielinski et al., 2009). Occupational exposure studies suggest positive associations between received dose and excessive relative risk of circulatory diseases (Zielinski et al., 2009), CVD mortality (Gillies et al., 2017) and occurrence of ischemic and cerebrovascular disease (Azizova et al., 2018). </p>
<p>Beyond earth, space travel presents an additional radiation exposure scenario. With future missions planned beyond low Earth orbit and the protective shield of the magnetosphere, understanding the unique challenges of space radiation is crucial for protection of travelers. In space, radiation is present in the form of high linear energy transfer (LET) particles and high mass, high energy ions (HZE) which indiscriminately impacts the whole body at a low fluence rate (Baker et al., 2011; Durante & Cucinotta, 2008; Norbury et al., 2016). While the present AOP includes an MIE focused on deposition of energy following radiation exposure, it is important to note that the space exposome contains multiple stressors to which space travelers will be exposed simultaneously. Particularly important, in the case of the cardiovascular system, is microgravity. The cardiovascular system is gravity sensitive, with the endothelial layer being responsive to changes in shear stress and blood pressure (Hughson et al., 2018; Maier et al., 2015; Versari et al., 2013). Variation to the pressure gradient throughout the body can also trigger regional adaptations to vascular structure (L. F. Zhang, 2013). While determining a mechanism for an MIE of microgravity has proven challenging, microgravity exposure has been shown to contribute to KEs in the pathway and therefore, evidence from microgravity studies has been included in the weight of evidence (WOE).</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">It is well documented that ionizing radiation( (eg. X-rays, gamma, photons, alpha, beta, neutrons, heavy ions) leads to energy deposition on the atoms and molecules of the substrate. Many studies, have demonstrated that the type of radiation and distance from source has an impact on the pattern of energy deposition (Alloni, et al. 2014). High linear energy transfer (LET) radiation has been associated with higher-energy deposits (Liamsuwan et al., 2014) that are more densely-packed and cause more complex effects within the particle track (Hada and Georgakilas, 2008; Okayasu, 2012ab; Lorat et al., 2015; Nikitaki et al., 2016) in comparison to low LET radiation. Parameters such as mean lineal energy, dose mean lineal energy, frequency mean specific energy and dose mean specific energy can impact track structure of the traversed energy into a medium (Friedland et al., 2017)</span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">. The detection of energy deposition by ionizing radiation can be demonstrated with the use of fluorescent nuclear track detectors (FNTDs). FNTDs used in conjunction with fluorescent microscopy, are able to visualize radiation tracks produced by ionizing radiation (Niklas et al., 2013; Kodaira et al., 2015; Sawakuchi and Akselrod, 2016). In addition, these FNTD chips can quantify the LET of primary and secondary radiation tracks up to 0.47 keV/um (Sawakuchi and Akselrod, 2016). This co-visualization of the radiation tracks and the cell markers enable the mapping of the radiation trajectory to specific cellular compartments, and the identification of accrued damage (Niklas et al., 2013; Kodaira et al., 2015). There are no known chemical initiators or prototypes that can mimic the MIE.</span></span></p>
adjacentHighHighadjacentHighHighadjacentModerateHighadjacentModerateHighadjacentLowHighadjacentModerateModerateadjacentLowModerateadjacentLowModerateadjacentLowModerateadjacentLowModerateadjacentLowModerateadjacentLowModeratenon-adjacentLowHighnon-adjacentLowModeratenon-adjacentLowHighnon-adjacentLowModerate<p>The essentiality of the MIE to a downstream KE is supported by a non-irradiated control. The comparison of irradiated and non-irradiated groups has shown that the effects of downstream events are enhanced or accelerated by the deposition of energy. </p>
<p>The essentiality of other KEs can be determined by the impact of the manipulation of the upstream KE on the resulting downstream effects. For example, the essentiality of oxidative stress is frequently assessed through antioxidant treatments, which can decrease oxidative stress markers through decreased ROS production or strengthened antioxidant defense activity. SOD administration decreased free radicals, superoxide and peroxide, and improved endothelium-dependent vasodilation, a downstream KE, which had been previously decreased due to radiation exposure (Hatoum et al., 2006). Additionally, oxypurinol treatment inhibited xanthine oxidase (XO) enzyme, which limited the enzyme’s contribution to cardiac ROS and improved endothelium-dependent vasodilation and the recovery of vascular stiffness to control levels (Soucy et al., 2007, 2010, 2011). </p>
<p>The essentiality of DNA strand breaks was not assessed often. One study used mesenchymal stem cell conditioned media (MSC-CM) to reduce the level of ROS-mediated DNA double-stranded breaks and found decreases in signaling molecules including p53, Bax and cleaved caspase 3 (Huang et al., 2021). </p>
<p>The essentiality for altered signaling pathways KE was evaluated by studies using pathway inhibitors or conditioned media. Signaling pathways were shown to be suppressed by inhibitors such as ROCK inhibitor Y27632 and acid sphingomyelinase (ASM) inhibitor desipramine (dpm), which have demonstrated decreased apoptosis and recovered endothelium-dependent vasodilation (Soloviev & Kizub, 2019; Venkatesulu et al., 2018; Wang et al., 2016). Incubation of endothelial cells in MSC-CM was shown to increase cell signaling components, Akt and p-Akt, and decrease apoptosis (Chang et al., 2017). PI3K inhibitors, such as LY294002 and wortmannin, and angiotensin-converting enzyme inhibitor bradykinin-potentiating factor (BPF) were studied for their impact on NO levels. The increase in p-Akt and subsequently eNOS, p-eNOS and NO levels were reversed following PI3K inhibition (Shi et al., 2012; Siamwala et al., 2010). AngII and iNOS levels were returned to control following BPF treatment of irradiated groups (Hasan et al., 2020). Further studies are required for a better understanding of the changes in NO levels and endothelial dysfunction due to altered signaling pathways. Overall, the flexibility of signaling pathways makes it difficult to assess essentiality. </p>
<p>The essentiality for pro-inflammatory mediators was assessed by studies that suppress their expression. The decrease in pro-inflammatory mediators was observed following the use of TAT-Gap19 to block connexin43 hemichannels. This decrease was associated with a decrease in radiation-induced endothelial cell senescence (Ramadan et al., 2020). Additionally, MSC-CM incubation resulted in decreased pro-inflammatory cytokines, IL-1α, IL-6 and TNF-α and decreased endothelial apoptosis (Chang et al., 2017). </p>
<p>Changes in vascular remodeling were evaluated through vascular structure, among other endpoints. Following hindlimb unloading, ASM inhibition in the small mesenteric artery was found to reverse the changes in apoptosis and intima-media thickness (IMT) (Su et al., 2020). Comparisons between irradiated and sham or non-irradiated control groups of various studies using animal and human models have demonstrated differences in vascular structures (Hamada et al., 2020, 2021; Sárközy et al., 2019; Shen et al., 2018; Sridharan et al., 2020; Yu et al., 2011).</p>
<p><strong>Essentiality of the key events </strong></p>
<table border="1">
<tbody>
<tr>
<td>
<p> </p>
</td>
<td>
<p>Defining Question </p>
</td>
<td>
<p>High </p>
</td>
<td>
<p>Moderate </p>
</td>
<td>
<p>Low </p>
</td>
</tr>
<tr>
<td>
<p>Support for Essentiality of KEs </p>
</td>
<td>
<p>Are downstream KEs and/or the AO prevented if an upstream KE is blocked? </p>
</td>
<td>
<p>Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs </p>
</td>
<td>
<p>Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE </p>
</td>
<td>
<p>No or contradictory experimental evidence of the essentiality of any of the KEs </p>
</td>
</tr>
<tr>
<td>
<p>MIE: KE #1689 Deposition of energy </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: High </p>
<p>This event is difficult to test for essentiality as deposition of energy is a physical stressor and cannot be blocked/decreased using chemicals. However, studies show that control or sham-irradiated groups do not show the occurrence of downstream KEs. </p>
</td>
</tr>
<tr>
<td>
<p>KE #1392 Oxidative stress </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: High </p>
<p>Essentiality was well supported within the literature. Antioxidant treatments led to recovery of antioxidant enzyme activity, decreases in DNA strand breaks, and decreases in pro-inflammatory mediators, while ZNO-NP also restored NO levels. Oxypurinol (Oxp) treatment was found to aid in the acetylcholine (ACh) vasodilation response and restore NO levels as it decreased xanthine oxidase (XO) activity and reactive oxygen species (ROS). </p>
</td>
</tr>
<tr>
<td>
<p>KE #1635 </p>
<p>Increase, DNA strand breaks </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Low </p>
<p>Few studies use countermeasures to reduce the number of DNA strand breaks in cells. A few studies show that reducing DNA strand breaks induced by radiation restores signaling pathways and reduces endothelial dysfunction. </p>
</td>
</tr>
<tr>
<td>
<p>KE #1493 Increase, pro-inflammatory mediators </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Low </p>
<p>Essentiality of this event can be determined with countermeasures that limit the increase of pro-inflammatory mediators. Limited research does show essentiality, evidenced by a decrease in apoptosis of endothelial cells following treatment with MSC-CM, which contains angiogenic cytokines that have therapeutic potential for microvascular injury, and a decrease in endothelial cell senescence following treatment with TAT-Gap19, a connexin hemichannel blocker. </p>
</td>
</tr>
<tr>
<td>
<p>KE #2066 </p>
<p>Altered signaling pathways </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Moderate </p>
<p>Essentiality of this relationship can be determined with the use of signaling molecule inhibitors. Signaling molecule inhibitors reduced downstream changes in eNOS, NO, p-Akt, angiotensin II (AngII) and aldosterone following stressors such as irradiation and altered gravity. Inhibitors also prevented impaired contractile response and decreased apoptosis in the arterial endothelium. </p>
</td>
</tr>
<tr>
<td>
<p>KE #2067 Altered, NO levels </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Moderate </p>
<p>The evidence for essentiality of this KE can be determined by using countermeasures that limit changes in NO levels, such as Oxp, L-NA (NOS inhibitor), AG (iNOS inhibitor), DAHP (Gch1 inhibitor) and losartan (AT1 receptor antagonist). Use of these countermeasures reduced NOS levels and decreased the ratio of couple-to-uncoupled eNOS. Endothelial relaxation increased after Oxp and losartan treatment after microgravity exposure, while relaxation decreased in the presence of DAHP, L-NA and AG. When treated with these countermeasures following radiation or microgravity, changes to NO were limited or restored and as a result, endothelial dysfunction was limited. </p>
</td>
</tr>
<tr>
<td>
<p>KE #2068 </p>
<p>Increase, endothelial dysfunction </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Moderate </p>
<p>The essentiality of endothelial dysfunction leading to vascular remodeling is moderately supported within literature. Oxp treatment, an XO inhibitor, restored vasodilator response and reduced vascular stiffness following irradiation. Both dpm and DOX decreased apoptosis and reduced Caspase-3 protein expression. Ceramide treatment following microgravity was found to return proliferation to control levels and increase apoptosis.</p>
</td>
</tr>
</tbody>
</table>
HighUnspecificHighAll life stagesHighHighHighLow<p><strong>Summary of evidence (KE & KER Relationships and evidence) </strong></p>
<p>The AOP is supported by high biological plausibility and moderate empirical evidence. Research, primarily from laboratory studies, has supported dose- and temporal-concordance for each KER. </p>
<p><strong>Biological Plausibility </strong></p>
<p>Described below is the well-established understanding of the mechanisms underlying this AOP with supporting literature. More detailed examples of the empirical data can be found in the individual entries for each KER. </p>
<p>It is well accepted that when energy is deposited in the cell from ionizing radiation (IR), direct damage to cellular structures can occur (Desouky et al., 2015). When traveling through a cell, IR can induce the radiolysis of water forming reactive oxygen species (ROS). Deposition of energy can also induce feedback loops of ROS production where structures and molecules damaged by ROS including the mitochondria and NADPH oxidase (NOX) further produce ROS (Mittal et al., 2014; Soloviev & Kizub, 2019). Additionally, deposited energy can directly upregulate enzymes involved in ROS and reactive nitrogen species (RNS) (collectively RONS) production (de Jager, Cockrell and Du Plessis, 2017). If reactive nitrogen species RONS production outpaces the antioxidant defense, a state of oxidative stress occurs (Fletcher et al., 2010; Slezak et al., 2017; Tahimic & Globus, 2017; Wang et al., 2019). Damage to macromolecules can occur due to oxidative stress, including strand breaks to DNA, oxidation of amino acid residues in proteins and peroxidation of lipids (Ping et al., 2020). Lipid peroxidation can induce further damage to cellular structures as a chain reaction is created by the lipid peroxidation radicals, attacking other lipids, proteins and nucleic acids (Ping et al., 2020). Consequently, oxidative stress can directly lead to multiple downstream KEs including altered signaling pathways, increased DNA strand breaks, increased pro-inflammatory mediators and altered nitric oxide (NO) levels. </p>
<p>DNA strand breaks in endothelial cells can be induced either directly through energy deposition or indirectly through oxidative stress. DNA strand breaks can recruit and activate the protein kinases ataxia telangiectasia mutated (ATM) and ATM/RAD3-related (ATR) (Nagane et al., 2021). Downstream signaling pathways involved in cell death and senescence like the p53/p21 pathway can be activated by ATM/ATR. Furthermore, DNA strand breaks induced by radiation directly or through oxidative stress can cause mutations or changes in transcription of proteins in signaling pathways (Ping et al., 2020; Schmidt-Ullrich et al., 2000). Therefore, DNA strand breaks will induce death and senescence of endothelial cells through altered signaling, resulting in endothelial dysfunction. </p>
<p>Oxidative stress can also induce altered signaling pathways. The effects of oxidative stress on signaling pathways occur through protein oxidation of signaling components (Ping et al., 2020; Schmidt-Ullrich et al., 2000; Valerie et al., 2007). Oxidation of cysteine and methionine residues, which are particularly sensitive to oxidation, can result in structural and functional detriments to the protein (Ping et al., 2020). RONS can influence various pathways including the Akt/PI3K/mTOR pathway, where impaired cell survival signaling can induce cellular senescence (Hassan et al., 2013; Ping et al., 2020). Additionally, inhibition of tyrosine phosphatases by ROS can increase the phosphorylation of mitogen-activated protein kinase (MAPK) pathways, resulting in various downstream effects (Schmidt-Ullrich et al., 2000; Valerie et al., 2007). A phosphorylated p53 induced by oxidative DNA damage can also activate MAPK signaling and initiates a cascade ending in apoptosis (Ashcroft et al., 1999; Gen, 2004). Through affecting cell signaling pathways, damage caused by elevated RONS affects cells beyond those that have been directly irradiated (Ramadan et al., 2021). </p>
<p>Excessive RONS produced by IR disrupt cellular balance and can increase pro-inflammatory mediators (Lumniczky et al., 2021; Schaue et al., 2015). Similar to activation of the immune system by damage from a pathogen, activation by oxidative stress promotes many repair mechanisms, some of which involve rapid release of pro-inflammatory cytokines (Stanojković et al., 2020). The cytokines released vary based on tissue type and radiation parameters (Di Maggio et al., 2015), but tumor necrosis factor (TNF)-α and interleukin (IL)-1 can trigger a cytokine cascade that initiates an inflammatory response (Slezak et al., 2017; Srinivasan et al., 2017). A prolonged state of inflammation in endothelial cells can lead to endothelial dysfunction (Baran et al., 2021). </p>
<p>Both oxidative stress and altered signaling can directly result in altered NO levels. NO is synthesized from L-arginine by the three nitric oxide synthase (NOS) enzymes, endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). ROS can directly reduce NO levels by reacting with NO to produce the RNS peroxynitrite (Deanfield et al., 2007). Furthermore, the cofactor of NOS enzymes, tetrahydrobiopterin (BH4), can be oxidized by RONS leading to inhibition of NOS dimerization, also called NOS uncoupling (Deanfield et al., 2007). Uncoupled NOS will produce superoxide instead of NO, leading to a positive feedback loop of ROS production and reduced NO (Förstermann, 2010; Förstermann & Münzel, 2006; Mitchell et al., 2019; Nagane et al., 2021; Soloviev & Kizub, 2019). </p>
<p>Modulation of NO through altered signaling pathways occurs through changing the activity of NOS enzymes. Phosphorylation of eNOS at Ser1177 will activate the enzyme while phosphorylation at Thr495 inhibits it (Förstermann, 2010; Nagane et al., 2021). Protein kinase B (Akt), part of the phosphoinositide 3-kinase (PI3K)/Akt pathway, can activate eNOS through phosphorylation at Ser1177 to increase NO production (Karar & Maity, 2011). In contrast, activation of the RhoA/Rho kinase (ROCK) pathway will inhibit NO production by destabilizing eNOS mRNA and preventing Ser1177 phosphorylation by Akt (Yao et al., 2010). Angiotensin II (AngII), the end product of the renin-angiotensin-aldosterone system (RAAS), is involved in both downregulating Ser1177 phosphorylation to prevent NO creation (Ding et al., 2020) and activating eNOS as a corrective measure (Millatt et al., 1999). Alterations to these pathways due to IR will result in changes in NO levels. </p>
<p>Each of the components of the pathway described above converge at endothelial dysfunction. Endothelial cells lining the blood vessels throughout the body are an important component for maintaining vascular homeostasis (Bonetti et al., 2003; Deanfield et al., 2007). Endothelial cells are quiescent with high levels of NO most of the time (Carmeliet & Jain, 2011). Endothelial dysfunction can occur due to prolonged activation of the endothelium, characterized by the prolonged lack of bioavailable NO, lack of endothelium-dependent vasodilation and chronic pro-thrombotic and inflammatory state (Baran et al., 2021; Bonetti et al., 2003; Deanfield et al., 2007; Krüger-Genge et al., 2019). A prolonged reduction in NO will decrease vasodilation, increase leukocyte adhesion and increase fibrous plaque formation contributing to the pro-thrombotic dysfunctional environment (Schiffrin, 2008; Senoner & Dichtl, 2019; Venkatesulu et al., 2018). Furthermore, signaling in pathways like p53/p21 or PI3K/Akt/mammalian target of rapamycin (mTOR) can induce apoptosis or premature senescence of endothelial cells as part of endothelial dysfunction due to DNA damage or oxidative stress (Borghini et al., 2013; Hughson et al., 2018; Schiffrin, 2008; Senoner & Dichtl, 2019; Soloviev & Kizub, 2019). Senescent cells have decreased levels of NO production and a pro-inflammatory secretory phenotype, which feed back to further promote endothelial dysfunction (Ungvari et al., 2013; Wang et al., 2016). </p>
<p>Endothelial dysfunction subsequently leads to vascular remodeling, which encompasses multiple structural changes to the vasculature. Chronic inflammation combined with impaired healing and lack of endothelium-dependent vasodilation during endothelial dysfunction increases vulnerability to damage from non-laminar flow and maladaptive repair (Sylvester et al., 2018). As compensation, vessel walls can thicken and atherosclerotic risk can increase (Hughson et al., 2018; Slezak et al., 2017; Sylvester et al., 2018). In cases of maladaptive repair of vessels, vascular remodeling can be exhibited through an increase in fibrosis (Hsu et al., 2019). The pro-thrombotic environment with increased lymphocyte adhesion induced by endothelial cell senescence can increase the likelihood of vessel occlusion, decreasing vascular density such that the corresponding increase in vascular resistance will induce remodeling as a compensatory measure (Slezak et al., 2017). Thus, increased leukocyte adhesion during endothelial dysfunction occurs early in the development of atherosclerosis (Senoner & Dichtl, 2019). Increased arterial stiffness can also occur in response to endothelial dysfunction (Boerma et al., 2015, 2016; Patel et al., 2020), with increased collagen and smooth muscle content paired with decreased elastin and degradation of the extracellular matrix (Zieman et al., 2005). The changes to the vascular structure in response to the deposition of energy are similar to a form of accelerated age-related atherosclerosis (Boerma et al., 2016; Sylvester et al., 2018; Vernice et al., 2020). </p>
<p><strong>Temporal, Dose, and Incidence Concordance </strong></p>
<p>Evidence for time, dose, and incidence concordance in this AOP is moderate. It has been repeatedly shown using many study designs and systems that deposition of energy occurs immediately following irradiation, and downstream events occur at a later timepoint. Endpoints indicating oxidative stress have been observed within minutes following irradiation (Wortel et al., 2019). Studies show that oxidative stress, increased DNA strand breaks, increased pro-inflammatory mediators, and altered signaling may occur over a similar time period; however, alteration in signaling pathways, increased DNA strand breaks, and increased pro-inflammatory mediators can be observed following oxidative stress (Ramadan et al., 2020; Baselet et al., 2017; Sakata et al., 2015; Yang et al., 1998). Increases in NO levels occur in hours to weeks after irradiation (Azimzadeh et al., 2017; Sonveaux et al., 2003; Sakata et al., 2015). Then, from weeks to months following irradiation both endothelial dysfunction and vascular remodeling occur, though concordance between these events is difficult to determine, possibly due to inter-study differences in experimental design and markers (Yentrepalli et al., 2017; Soucy et al., 2007; Yu et al., 2011; Shen et al., 2018). </p>
<p>Overall, the majority of studies demonstrate that upstream KEs occur at the same or lower doses and earlier or the same time as downstream KEs. For example, endothelial cells show a dose-dependent increase in oxidative stress to X-ray irradiation at 0.1 and 5 Gy, while 0.1 Gy induced few changes in pro-inflammatory mediators with significant increases only observed at 5 Gy (Ramadan et al., 2020). Some studies also show that the upstream and downstream KEs can be observed at the same doses of radiation. For example, X-ray irradiation of mice resulted in oxidative stress, altered signaling and reduced NO levels at both 8 and 16 Gy (Azimzadeh et al., 2015). Dose concordance is not consistent across studies, but this may be due to differences in models, timepoints, and radiation types used. </p>
<p>A limited number of studies support incidence concordance. In these, the upstream KE demonstrates a greater change than the downstream KE following exposure to a stressor. For example, mice exposed to 18 Gy of X-rays showed a roughly 2-fold increases in both oxidative stress and pro-inflammatory markers. A 1.3-fold increase in markers for endothelial dysfunction was observed (Shen et al., 2018).</p>
<p><strong>Uncertainties and inconsistencies </strong></p>
<p>The collection of WOE identifies several important uncertainties in the literature. These include lack of quantitative understanding, low-dose or chronic-exposure studies, data from female models and consistency in measurement of NO levels. </p>
<p>The WOE contained data from a wide variety of interdisciplinary fields; consequently, experimental design was equally varied. Overall, studies did not use consistent doses, radiation types, time-points, or evaluation of endpoints. Since dose and type of radiation can affect biological responses, quantitative understanding of relationships could not be determined and was low overall. Additionally, most studies used single or select doses, with limited studies exploring relatively low doses (<0.5 Gy (EPRI, 2020)). Harmonized experiments evaluating changes to adjacent endpoints across a wide range of doses or time-points with consistency of radiation type would greatly benefit quantitative understanding for this AOP. </p>
<p>Similarly, the WOE is lacking in evidence using female models. Sex is an important modulating factor in cardiovascular changes and studies suggest vascular remodeling responses of astronauts can vary by sex (Hughson et al., 2016). The consequence of the general bias in clinical research (Rios et al., 2020; Yakerson, 2019) from which the current WOE draws, is the very large knowledge gaps in mechanistic data for the female body. Filling these knowledge gaps at all levels of biological organization will be an important step in solidifying the AOP. </p>
<p>Evaluation of NO levels was inconsistent between studies. According to the biological plausibility, deposition of energy and subsequent oxidative stress would lead to a decrease in NO that then contributes to impaired vascular relaxation as part of endothelial dysfunction. However, primary research concludes that NO can either increase (Abdel-Magied & Shedid, 2020; Hirakawa et al., 2002; Sakata et al., 2015; Sonveaux et al., 2003) or decrease (Baker et al., 2009; Fuji et al., 2016) following irradiation. Proxy measures used to detect NO, like NOS enzyme activities or nitrite/nitrate levels, may not directly correspond to changes in NO levels. Further standardization in NO measurement and interpretation could refine this KE to become the depletion of NO. </p>
<p>The empirical evidence supports that this AOP is relevant to human (Hong et al., 2013; Siamwala et al., 2010; Jiang et al., 2020; Lee, et al., 2020; Ramadan et al., 2020), rat (Hatoum et al., 2006; Soucy et al., 2010; Hong et al., 2013; Abdel-Magied & Shedid, 2019: Hasan et al., 2020), mouse (Yu et al., 2011; Coleman et al., 2015; Sofronova et al., 2015; Shen et al., 2018; Hamada et al., 2020), and rabbit (Soloviev et al., 2003, Hong et al., 2013) models. Biological plausibility suggests that events in this AOP are not sex specific; however, more studies used male models. Similarly, while biological plausibility suggests the pathway is not age-specific, most studies used adult models. </p>
<p>The essentiality of the MIE to a downstream KE is supported by a non-irradiated control. The comparison of irradiated and non-irradiated groups has shown that the effects of downstream events are enhanced or accelerated by the deposition of energy. </p>
<p>The essentiality of other KEs can be determined by the impact of the manipulation of the upstream KE on the resulting downstream effects. For example, the essentiality of oxidative stress is frequently assessed through antioxidant treatments, which can decrease oxidative stress markers through decreased ROS production or strengthened antioxidant defense activity. SOD administration decreased free radicals, superoxide and peroxide, and improved endothelium-dependent vasodilation, a downstream KE, which had been previously decreased due to radiation exposure (Hatoum et al., 2006). Additionally, oxypurinol treatment inhibited xanthine oxidase (XO) enzyme, which limited the enzyme’s contribution to cardiac ROS and improved endothelium-dependent vasodilation and the recovery of vascular stiffness to control levels (Soucy et al., 2007, 2010, 2011). </p>
<p>The essentiality of DNA strand breaks was not assessed often. One study used mesenchymal stem cell conditioned media (MSC-CM) to reduce the level of ROS-mediated DNA double-stranded breaks and found decreases in signaling molecules including p53, Bax and cleaved caspase 3 (Huang et al., 2021). </p>
<p>The essentiality for altered signaling pathways KE was evaluated by studies using pathway inhibitors or conditioned media. Signaling pathways were shown to be suppressed by inhibitors such as ROCK inhibitor Y27632 and acid sphingomyelinase (ASM) inhibitor desipramine (dpm), which have demonstrated decreased apoptosis and recovered endothelium-dependent vasodilation (Soloviev & Kizub, 2019; Venkatesulu et al., 2018; Wang et al., 2016). Incubation of endothelial cells in MSC-CM was shown to increase cell signaling components, Akt and p-Akt, and decrease apoptosis (Chang et al., 2017). PI3K inhibitors, such as LY294002 and wortmannin, and angiotensin-converting enzyme inhibitor bradykinin-potentiating factor (BPF) were studied for their impact on NO levels. The increase in p-Akt and subsequently eNOS, p-eNOS and NO levels were reversed following PI3K inhibition (Shi et al., 2012; Siamwala et al., 2010). AngII and iNOS levels were returned to control following BPF treatment of irradiated groups (Hasan et al., 2020). Further studies are required for a better understanding of the changes in NO levels and endothelial dysfunction due to altered signaling pathways. Overall, the flexibility of signaling pathways makes it difficult to assess essentiality. </p>
<p>The essentiality for pro-inflammatory mediators was assessed by studies that suppress their expression. The decrease in pro-inflammatory mediators was observed following the use of TAT-Gap19 to block connexin43 hemichannels. This decrease was associated with a decrease in radiation-induced endothelial cell senescence (Ramadan et al., 2020). Additionally, MSC-CM incubation resulted in decreased pro-inflammatory cytokines, IL-1α, IL-6 and TNF-α and decreased endothelial apoptosis (Chang et al., 2017). </p>
<p>Changes in vascular remodeling were evaluated through vascular structure, among other endpoints. Following hindlimb unloading, ASM inhibition in the small mesenteric artery was found to reverse the changes in apoptosis and intima-media thickness (IMT) (Su et al., 2020). Comparisons between irradiated and sham or non-irradiated control groups of various studies using animal and human models have demonstrated differences in vascular structures (Hamada et al., 2020, 2021; Sárközy et al., 2019; Shen et al., 2018; Sridharan et al., 2020; Yu et al., 2011).</p>
<p><strong>Essentiality of the key events </strong></p>
<table border="1">
<tbody>
<tr>
<td>
<p> </p>
</td>
<td>
<p>Defining Question </p>
</td>
<td>
<p>High </p>
</td>
<td>
<p>Moderate </p>
</td>
<td>
<p>Low </p>
</td>
</tr>
<tr>
<td>
<p>Support for Essentiality of KEs </p>
</td>
<td>
<p>Are downstream KEs and/or the AO prevented if an upstream KE is blocked? </p>
</td>
<td>
<p>Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs </p>
</td>
<td>
<p>Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE </p>
</td>
<td>
<p>No or contradictory experimental evidence of the essentiality of any of the KEs </p>
</td>
</tr>
<tr>
<td>
<p>MIE: KE #1689 Deposition of energy </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: High </p>
<p>This event is difficult to test for essentiality as deposition of energy is a physical stressor and cannot be blocked/decreased using chemicals. However, studies show that control or sham-irradiated groups do not show the occurrence of downstream KEs. </p>
</td>
</tr>
<tr>
<td>
<p>KE #1392 Oxidative stress </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: High </p>
<p>Essentiality was well supported within the literature. Antioxidant treatments led to recovery of antioxidant enzyme activity, decreases in DNA strand breaks, and decreases in pro-inflammatory mediators, while ZNO-NP also restored NO levels. Oxypurinol (Oxp) treatment was found to aid in the acetylcholine (ACh) vasodilation response and restore NO levels as it decreased xanthine oxidase (XO) activity and reactive oxygen species (ROS). </p>
</td>
</tr>
<tr>
<td>
<p>KE #1635 </p>
<p>Increase, DNA strand breaks </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Low </p>
<p>Few studies use countermeasures to reduce the number of DNA strand breaks in cells. A few studies show that reducing DNA strand breaks induced by radiation restores signaling pathways and reduces endothelial dysfunction. </p>
</td>
</tr>
<tr>
<td>
<p>KE #1493 Increase, pro-inflammatory mediators </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Low </p>
<p>Essentiality of this event can be determined with countermeasures that limit the increase of pro-inflammatory mediators. Limited research does show essentiality, evidenced by a decrease in apoptosis of endothelial cells following treatment with MSC-CM, which contains angiogenic cytokines that have therapeutic potential for microvascular injury, and a decrease in endothelial cell senescence following treatment with TAT-Gap19, a connexin hemichannel blocker. </p>
</td>
</tr>
<tr>
<td>
<p>KE #2066 </p>
<p>Altered signaling pathways </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Moderate </p>
<p>Essentiality of this relationship can be determined with the use of signaling molecule inhibitors. Signaling molecule inhibitors reduced downstream changes in eNOS, NO, p-Akt, angiotensin II (AngII) and aldosterone following stressors such as irradiation and altered gravity. Inhibitors also prevented impaired contractile response and decreased apoptosis in the arterial endothelium. </p>
</td>
</tr>
<tr>
<td>
<p>KE #2067 Altered, NO levels </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Moderate </p>
<p>The evidence for essentiality of this KE can be determined by using countermeasures that limit changes in NO levels, such as Oxp, L-NA (NOS inhibitor), AG (iNOS inhibitor), DAHP (Gch1 inhibitor) and losartan (AT1 receptor antagonist). Use of these countermeasures reduced NOS levels and decreased the ratio of couple-to-uncoupled eNOS. Endothelial relaxation increased after Oxp and losartan treatment after microgravity exposure, while relaxation decreased in the presence of DAHP, L-NA and AG. When treated with these countermeasures following radiation or microgravity, changes to NO were limited or restored and as a result, endothelial dysfunction was limited. </p>
</td>
</tr>
<tr>
<td>
<p>KE #2068 </p>
<p>Increase, endothelial dysfunction </p>
</td>
<td colspan="4">
<p>Evidence for Essentiality of KE: Moderate </p>
<p>The essentiality of endothelial dysfunction leading to vascular remodeling is moderately supported within literature. Oxp treatment, an XO inhibitor, restored vasodilator response and reduced vascular stiffness following irradiation. Both dpm and DOX decreased apoptosis and reduced Caspase-3 protein expression. Ceramide treatment following microgravity was found to return proliferation to control levels and increase apoptosis.</p>
</td>
</tr>
</tbody>
</table>
<table border="1">
<tbody>
<tr>
<td>
<p> </p>
</td>
<td>
<p>Defining Question </p>
</td>
<td>
<p>High </p>
</td>
<td>
<p>Moderate </p>
</td>
<td>
<p>Low </p>
</td>
</tr>
<tr>
<td>
<p>Review of Biological Plausibility for the KER </p>
<p> </p>
</td>
<td>
<p>Is there a mechanistic (structural or functional) relationship between the upstream KE and downstream KE consistent with established biological knowledge </p>
</td>
<td>
<p>The relationship is well understood based on extensive previous documentation and has an established mechanistic basis and broad acceptance </p>
</td>
<td>
<p>The KER is plausible based on an analogy to accepted biological relationships but scientific understanding is not completely established </p>
</td>
<td>
<p>There is empirical support for a statistical association between KEs but structural or functional relationship between them is not understood </p>
</td>
</tr>
<tr>
<td>
<p>Deposition of energy (MIE: KE #1686) leads to oxidative stress (KE #1392) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Deposition of energy onto the water and biological components of a cell creates ROS, and as ROS production outpaces the cell’s antioxidant defense system, oxidative stress is induced. Both ROS production and subsequent oxidative stress have been extensively studied and the mechanisms are well described in numerous review articles across many biological systems. </p>
</td>
</tr>
<tr>
<td>
<p>Deposition of energy (MIE: KE #1686) leads to increase, DNA strand breaks (KE #1635) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>The deposition of energy onto the DNA molecule will directly cause single- or double-strand breaks in the DNA. Deposited energy can induce chemical modifications to the phosphodiester backbone of both strands of the DNA, possibly resulting in breaks in one or both strands. </p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress (KE #1392) leads to increase, DNA strand breaks (KE #1635) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Increased ROS during oxidative stress can result in the oxidation of bases on the DNA strand, triggering base excision repair, which removes the oxidized bases. When multiple bases in close proximity are removed, the repair efforts cause strain which can lead to strand breaks. </p>
</td>
</tr>
<tr>
<td>
<p>Increase, DNA strand breaks (KE #1635) leads to altered signaling pathways (KE #2066) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Strand breaks induce the recruitment of the kinases ataxia-telangiectasia mutated (ATM) and ATM/RAD3-related (ATR). ATM and ATR can subsequently phosphorylate multiple downstream signaling molecules. High levels of DNA strand breaks can increase the recruitment of ATM and ATR, leading to greater activation of pathways like the p53/p21 pathway and subsequently greater downstream effects. </p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress (KE #1392) leads to increase, pro-inflammatory mediators (KE #1493) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Excess ROS during oxidative stress damages cellular structures and thus activates the immune system and repair mechanisms, many of which involve release of pro-inflammatory mediators. Cells involved with host-defense can themselves also produce ROS, further exacerbating the state of oxidative stress. The biological plausibility of the linkage between oxidative stress and increases in pro-inflammatory mediators is highly supported in literature. </p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress (KE #1392) leads to altered signaling pathways (KE #2066) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Oxidative stress can alter signaling pathways both directly and indirectly. Directly, oxidative stress conditions can lead to oxidation of amino acid residues. This can cause conformational changes, protein expansion, and protein degradation, leading to changes in the activity and level of signaling proteins. Oxidation of key functional amino acids can also alter the activity of signaling proteins, resulting in downstream alterations in signaling pathways. Indirectly, oxidative stress can damage DNA causing changes in the expression of signaling proteins as well as the activation of DNA damage response signaling. The mechanisms of this relationship are widely accepted. </p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress (KE #1392) leads to increase, endothelial dysfunction (KE #2068) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>ROS can interact with NO, taking a vasodilator crucial for endothelial function and turning it into peroxynitrite, a RNS that further contributes to oxidative stress. Furthermore, cellular senescence, inhibition of vasodilation, induced inflammatory environments and cellular apoptosis are all part of endothelial dysfunction that can be indirectly caused by oxidative stress. </p>
</td>
</tr>
<tr>
<td>
<p>Increase, pro-inflammatory mediators (KE #1493) leads to increase, </p>
<p>endothelial dysfunction (KE #2068) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Inflammation provides a protective effect to the endothelium but prolonged or repeated exposure to a stressor can exhaust this, leading to senescence or apoptosis in endothelial cells and subsequent leading to endothelial dysfunction. This endothelial dysfunction can also manifest as a dysregulation of vasodilation. Prolonged inflammation is a widely accepted component in the development of endothelial dysfunction. </p>
</td>
</tr>
<tr>
<td>
<p>Altered signaling pathways (KE #2066) leads to increase, endothelial dysfunction (KE #2068) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Signaling pathways including the PI3K/Akt/mTOR, RhoA-Rho-kinase, ASM/cer pathway, and the p53-p21 pathway have downstream effects on endothelial apoptosis, premature endothelial cell senescence and cytoskeletal proteins to impair contraction, indicators of endothelial dysfunction. </p>
</td>
</tr>
<tr>
<td>
<p>Increase, endothelial dysfunction (KE #2068) leads to occurrence, vascular remodeling (AO: KE #2069) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Key components of endothelial dysfunction include deficiency in bioavailable NO, impaired vasodilation, inflamed endothelium and prothrombotic environment. These events can ultimately lead to vascular remodeling to compensate for decreased capillary and vascular density and increased vascular resistance. Regional pressure changes in vessels due to microgravity can also result in regional changes to vascular structure. </p>
</td>
</tr>
<tr>
<td>
<p>Deposition of energy (MIE: KE #1686) leads to increase, endothelial dysfunction (KE #2068) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Irradiation can cause cellular and tissue level markers of endothelial dysfunction. Following prolonged exposure to radiation, the protective effect of the endothelium can become exhausted and lead to endothelial dysfunction. Consequently, endothelial cells may lose their integrity and become senescent or apoptotic via alterations to signaling pathways, leading to endothelial dysfunction evidenced by dysregulation of vasodilation. Endothelial dysfunction is commonly considered a hallmark for the development of various cardiovascular pathologies. </p>
</td>
</tr>
<tr>
<td>
<p>Deposition of energy (MIE: KE #1686) leads to occurrence, vascular remodeling (AO: KE #2069) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Radiation can accelerate the natural processes of vascular remodeling related to aging. An increase in ROS, produced by IR, can reduce NO bioavailability, leading to endothelial dysfunction and vascular stiffness. In addition, the low level of inflammation during early stages of radiation leads to inhibition of tissue and vessel recovery, and later results in intimal thickening and vascular remodeling. Changes in vessel composition, such as collagen content, may also occur from energy deposition and affect vascular remodeling. </p>
</td>
</tr>
<tr>
<td>
<p>Deposition of energy (MIE: KE#1686) leads to altered, NO levels (KE #2067) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>NO is produced by NOS enzymes or by the reduction of nitrite to NO. Deposition of energy can interfere with this process in several ways. Radiolysis of water forms ROS that interacts with NO to produce peroxynitrite which reduces NO bioavailability. ROS can also cause NOS uncoupling, which can reduce NO levels. In contrast, NO can also increase as a result of IR through activation of iNOS during oxidative stress. IR can also influence various signaling pathways that control NO levels, causing radiation to indirectly affect NO levels. </p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress (KE #1392) leads to altered, NO levels (KE #2067) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>It is thought that excessive ROS production can lead to altered NO bioavailability both through direct interaction and indirectly through decreasing its production. Elevated O2- can interact with NO converting it to peroxynitrite leading to decreased bioavailability. ROS can also oxidize the eNOS cofactor BH4, causing eNOS uncoupling inhibiting NO production. Electron leakage in uncoupled eNOS produces additional ROS, exacerbating the state of oxidative stress. </p>
</td>
</tr>
<tr>
<td>
<p>Altered signaling pathways (KE #2066) leads to altered, NO levels (KE #2067) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Various pathways are well known to influence NO levels. Some well-studied examples include the PI3K/Akt pathway, the RhoA/ROCK pathway, the RAAS pathway and the acidic sphingomyelinase/ceramide pathway. The PI3K/Akt, RhoA/ROCK and RAAS pathways and their components are involved in the phosphorylation of various eNOS residues affecting the enzymes activation. The activation or deactivation of eNOS affects the levels of NO production. In contrast, the acidic sphingomyelinase/ceramide pathway can activate NADPH oxidase (NOX), leading to the production of ROS that goes on to scavenge NO decreasing its bioavailability. </p>
</td>
</tr>
<tr>
<td>
<p>Altered, NO levels (KE #2067) leads to increase, endothelial dysfunction (KE #2068) </p>
</td>
<td colspan="4">
<p>Evidence for Biological Plausibility of KER: High </p>
<p>Lack of bioavailable NO is considered one of the key drivers of endothelial dysfunction. Under normal conditions NO binds with soluble guanylyl cyclase (sGC) creating cGMP and cAMP to activate cellular kinase cascades and Ca2+-dependent vasodilation through smooth-muscle relaxation. Lack of bioavailable NO interrupts this process, reducing the relaxation of smooth muscle cells and dilation of the blood vessels. In contrast, an increase in NO combined with simultaneous excessive ROS can drive cellular senescence through increased peroxynitrite formation. Prolonged impaired vasodilation and elevated premature endothelial cell senescence are important characteristics of endothelial dysfunction. </p>
</td>
</tr>
</tbody>
</table>
<p>Despite biological plausibility and empirical evidence demonstrating the qualitative linkages within the AOP, quantitative understanding is low. As described above, the lack of quantitative understanding of the KERs is due to the diversity in experimental design, including doses tested and radiation types used. The evidence is primarily from laboratory studies that show dose and time response relationships for KEs; however, the strength of the response can vary with factors such as dose-rate, type of radiation, and cell type. Particularly relevant are the relative lack of low-dose studies and exposure scenarios relevant to space radiation. Future work could use the present qualitative AOP to guide experimental design and strengthen quantitative understanding. Standardized studies simultaneously measuring endpoints across several KEs, and across a range of doses and timepoints would be beneficial in filling important gaps in the quantitative understanding.</p>
<table border="1">
<tbody>
<tr>
<td>
<p>Deposition of energy (MIE: KE #1686) leads oxidative stress (KE #1392) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: High </p>
<p>There is a large amount of evidence supporting how much of a change in the deposition of energy is needed to produce a change in the level of oxidative stress. Several different endpoints representing oxidative stress have been used, including changes in the levels or activity of catalase, GSH, superoxide dismutase, GSH-Px, MDA, and ROS. Measurements have also been made over a large range of doses and dose rates, and changes to oxidative stress levels have been shown to depend on the nature, dose and dose rate of energy deposition. </p>
</td>
</tr>
<tr>
<td>
<p>Deposition of energy (MIE: KE #1686) leads to increase, DNA strand breaks (KE #1635) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: High </p>
<p>Studies examining energy deposition leading to strand breaks suggest a positive, linear relationship between these two events. The exact number of strand breaks is difficult to predict from the deposition of energy. The relationship depends on the biological model, the type of radiation, and the dose. </p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress (KE #1392) leads to increase, DNA strand breaks (KE #1635) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Moderate </p>
<p>There is a considerable amount of evidence showing increased DNA strand breaks following exposure to oxidative stress. However, no model has emerged that predicts the number of DNA strand breaks following oxidative stress. Measurements of oxidative stress vary across studies. </p>
</td>
</tr>
<tr>
<td>
<p>Increase, DNA strand breaks (KE #1635) leads to altered signaling pathways (KE #2066) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Moderate </p>
<p>There is much evidence showing changes in the expression or activity of signaling pathways following increased DNA strand breaks. However, no model has been developed to accurately predict the changes to signaling pathways due to increased DNA strand breaks. Furthermore, the changes to signaling pathways are very context- and cell type-dependent. </p>
</td>
</tr>
<tr>
<td>Oxidative Stress (KE #1392) leads to altered signaling pathways (KE #2066)</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>The quantitative understanding of oxidative stress leading to altered signaling pathways is low as a precise quantitative relationship between the key events is difficult to determine due to differences in experimental design. The exact changes to signaling pathways due to oxidative stress will depend on the cell type and species.</p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress (KE #1392) leads to increase, endothelial dysfunction (KE #2068) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Although studies quantitatively measure both oxidative stress and endothelial dysfunction following a stressor, it is difficult to compare results and identify a quantitative relationship as studies use different models, stressors, doses and time scales. In addition, many factors and pathways can contribute to endothelial dysfunction. Thus, no model has been established to predict the extent of changes in endothelial dysfunction after oxidative stress. </p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress (KE #1392) leads to increase, pro-inflammatory mediators (KE #1493) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Moderate </p>
<p>Current primary research shows that an increase in oxidative stress will be followed by a more significant increase in pro-inflammatory mediators. A quantitative association between the two KEs is difficult to determine, as multiple positive feedback mechanisms exist between oxidative stress and inflammation</p>
</td>
</tr>
<tr>
<td>
<p>Increase, pro-inflammatory mediators (KE #1493) leads to increase, endothelial dysfunction (KE #2068) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Although studies reveal increases in markers for endothelial dysfunction in response to increased pro-inflammatory mediators, no quantitative understanding has been established to predict the changes in endothelial dysfunction markers. There are various pro-inflammatory mediators that may contribute to various markers of endothelial dysfunction such as apoptosis and cellular senescence. Studies investigate changes in the levels of different pro-inflammatory mediators and different measures of endothelial dysfunction; therefore, it is difficult to compare the results and identify trends. </p>
</td>
</tr>
<tr>
<td>
<p>Altered signaling pathways (KE #2066) leads to increase, endothelial dysfunction (KE #2068) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Although studies show increases in markers for endothelial dysfunction in response to altered signaling pathways, no quantitative understanding has been established to predict the changes in endothelial dysfunction markers. There are various signaling pathways that may contribute to endothelial dysfunction, including the Akt/PI3K/mTOR pathway, the RhoA-Rho-kinase pathway, and the ASM/cer pathway. Studies investigate changes to the levels of different signaling pathway molecules; therefore, it is difficult to compare the results and identify trends. </p>
</td>
</tr>
<tr>
<td>
<p>Increase, endothelial dysfunction (KE #2068) leads to occurrence, vascular remodeling (AO: KE #2069) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Vascular remodeling is consistently shown with endothelial dysfunction. However, it is difficult to compare results and identify a quantitative relationship as various models, stressors, doses and endpoint measures were used. Thus, no model has been established to accurately predict the changes in vascular remodeling. </p>
</td>
</tr>
<tr>
<td>
<p>Deposition of energy (MIE: KE #1686) leads to increase, endothelial dysfunction (KE #2068) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Studies revealed consistent increases in levels of indicators of endothelial dysfunction such as apoptosis, premature endothelial cell senescence and diminished relaxation response. There is consistent evidence that shows that as the dose increases, the maximum relaxation response decreases. However, more studies are required to quantify this association to show how this relates to levels of cellular markers of apoptosis and senescence. </p>
</td>
</tr>
<tr>
<td>
<p>Deposition of energy (MIE: KE #1686) leads to occurrence, vascular remodeling (AO: KE #2069) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Deposition of energy from IR is consistently demonstrated to drive vascular remodeling. However, it is difficult to compare results and quantify relationships as each study uses different models, stressors, doses and time scales. In addition, many factors and pathways contribute to the components of vascular remodeling. Thus, no model has been established to predict the changes in vascular remodeling after deposition of energy. </p>
</td>
</tr>
<tr>
<td>
<p>Deposition of energy (MIE: KE #1686) leads to altered, NO levels (KE #2067) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Altered nitric oxide levels occur consistently with deposition of energy. However, it is difficult to compare results and determine a quantitative relationship as each study uses different models, stressors, doses and endpoint measures of NO. As well, cancerous cells and normal cells can show different production of NO. Thus, no model has been established to predict the changes in nitric oxide levels at a given dose of IR. </p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress (KE #1392) leads to altered, NO levels (KE #2067) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Alterations in NO levels cannot be predicted from relevant measures of oxidative stress changes, such as increased ROS production and antioxidant enzyme activity. Nevertheless, a general decrease in NO is observed following ROS production. </p>
</td>
</tr>
<tr>
<td>
<p>Altered signaling pathways (KE #2066) leads to altered, NO levels (KE #2067) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Altered NO, iNOS and eNOS levels occur in response to altered signaling pathways; however, a model has not been established to predict the changes in NO levels. Different models, stressors, time scales, doses and dose rates make trends difficult to identify. The studies investigated the levels of different altered signaling pathway molecules and their effects on NO levels, making it difficult to compare and identify quantitative relationships across the results. </p>
</td>
</tr>
<tr>
<td>
<p>Altered, NO levels (KE #2067) leads to increase, endothelial dysfunction (KE #2068) </p>
</td>
<td>
<p>Evidence for Quantitative Understanding of KER: Low </p>
<p>Increased vascular tension occurs consistently with decreased NO levels. Although many studies quantitatively measure a change in endothelial function after changes in NO levels, no model has been established. Each study cited used different models, stressors, time scales, doses and dose rates, which makes it difficult to determine if response levels are consistent between studies.</p>
</td>
</tr>
</tbody>
</table>
<p>The present AOP serves as a platform to promote broader collaborative efforts to understand non-cancer health risks from radiation exposures. It will be a foundational AOP of regulatory interest to researchers seeking areas of knowledge gaps to prioritize research in understanding mechanisms of CVD. The AOP is also relevant to space agencies and clinicians working to improve the guidance on health risks from long-term spaceflight and radiotherapy treatments, respectively. The WOE for this AOP may also inform parameters in biologically based risk models and can serve to develop countermeasures to protect the cardiovascular systems of future space travelers on deep space missions. The present qualitative AOP can be used to guide the design of experiments that will provide quantitative understanding for the KERs to support risk-model development and inform additional guidelines for radiation protection; additionally, the identified research gaps could help prioritize research needs for funding strategies.</p>
Not Specified<p>Abdel-Magied, N., & Shedid, S. M. (2020). Impact of zinc oxide nanoparticles on thioredoxin-interacting protein and asymmetric dimethylarginine as biochemical indicators of cardiovascular disorders in gamma-irradiated rats. Environmental Toxicology, 35(4), 430–442. https://doi.org/10.1002/tox.22879 </p>
<p>Ashcroft, M., Kubbutat, M. H. G., & Vousden, K. H. (1999). Regulation of p53 Function and Stability by Phosphorylation. Molecular and Cellular Biology, 19(3), 1751. https://doi.org/10.1128/MCB.19.3.1751 </p>
<p>Azimzadeh, O., Subramanian, V., Sievert, W., Merl-Pham, J., Oleksenko, K., Rosemann, M., Multhoff, G., Atkinson, M. J., & Tapio, S. (2021). Activation of pparα by fenofibrate attenuates the effect of local heart high dose irradiation on the mouse cardiac proteome. Biomedicines, 9(12). https://doi.org/10.3390/biomedicines9121845 </p>
<p>Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, https://doi.org/10.1080/09553002.2017.1339332. </p>
<p>Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, https://doi.org/10.1021/pr501141b. </p>
<p>Azizova, T. V., Batistatou, E., Grigorieva, E. S., McNamee, R., Wakeford, R., Liu, H., De Vocht, F., & Agius, R. M. (2018). An Assessment of Radiation-Associated Risks of Mortality from Circulatory Disease in the Cohorts of Mayak and Sellafield Nuclear Workers. Radiation Research, 189(4), 371–388. https://doi.org/10.1667/RR14468.1 </p>
<p>Baeyens, N., Bandyopadhyay, C., Coon, B. G., Yun, S., & Schwartz, M. A. (2016). Endothelial fluid shear stress sensing in vascular health and disease. The Journal of Clinical Investigation, 126(3), 821–828. https://doi.org/10.1172/JCI83083 </p>
<p>Baker, J. E., Fish, B. L., Su, J., Haworth, S. T., Strande, J. L., Komorowski, R. A., Migrino, R. Q., Doppalapudi, A., Harmann, L., Allen Li, X., Hopewell, J. W., & Moulder, J. E. (2009). 10 Gy total body irradiation increases risk of coronary sclerosis, degeneration of heart structure and function in a rat model. International Journal of Radiation Biology, 85(12), 1089–1100. https://doi.org/10.3109/09553000903264473 </p>
<p>Baker, J. E., Moulder, J. E., & Hopewell, J. W. (2011). Radiation as a Risk Factor for Cardiovascular Disease. Antioxidant and Redox Signaling, 15(7). https://doi.org/10.1089/ars.2010.3742 </p>
<p>Balasubramanian, D. (2000). Ultraviolet radiation and cataract. Journal of Ocular Pharmacology and Therapeutics, 16(3), 285–297. https://doi.org/10.1089/jop.2000.16.285 </p>
<p>Baran, R., Marchal, S., Campos, S. G., Rehnberg, E., Tabury, K., Baselet, B., Wehland, M., Grimm, D., & Baatout, S. (2021). The Cardiovascular System in Space: Focus on In Vivo and In Vitro Studies. Biomedicines 2022, Vol. 10, Page 59, 10(1), 59. https://doi.org/10.3390/BIOMEDICINES10010059 </p>
<p>Baselet, B., Belmans, N., Coninx, E., Lowe, D., Janssen, A., Michaux, A., Tabury, K., Raj, K., Quintens, R., Benotmane, M. A., Baatout, S., Sonveaux, P., & Aerts, A. (2017). Functional gene analysis reveals cell cycle changes and inflammation in endothelial cells irradiated with a single X-ray dose. Frontiers in Pharmacology, 8. https://doi.org/10.3389/fphar.2017.00213 </p>
<p>Belzile-Dugas, E., & Eisenberg, M. J. (2021). Radiation‐Induced Cardiovascular Disease: Review of an Underrecognized Pathology. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 10(18), 21686. https://doi.org/10.1161/JAHA.121.021686 </p>
<p>Berk, B. C., & Korshunov, V. A. (2006). Genetic determinants of vascular remodelling. Canadian Journal of Cardiology, 22(SUPPL. B). https://doi.org/10.1016/s0828-282x(06)70980-1 </p>
<p>Boerma, M., Nelson, G. A., Sridharan, V., Mao, X.-W., Koturbash, I., & Hauer-Jensen, M. (2015). Space radiation and cardiovascular disease risk. World Journal of Cardiology, 7(12), 882. https://doi.org/10.4330/wjc.v7.i12.882 </p>
<p>Boerma, M., Sridharan, V., Mao, X.-W., Nelson, G. A., Cheema, A. K., Koturbash, I., Singh, S. P., Tackett, A. J., & Hauer-Jensen, M. (2016). Effects of Ionizing Radiation on the Heart. Mutation Research - Reviews in Mutation Research, 770, 319–327. https://doi.org/10.1016/j.mrrev.2016.07.003 </p>
<p>Bonetti, P. O., Lerman, L. O., & Lerman, A. (2003). Endothelial dysfunction: A marker of atherosclerotic risk. Arteriosclerosis, Thrombosis, and Vascular Biology, 23(2), 168–175. https://doi.org/10.1161/01.ATV.0000051384.43104.FC </p>
<p>Borghini, A., Luca Gianicolo, E. A., Picano, E., & Andreassi, M. G. (2013). Ionizing radiation and atherosclerosis: Current knowledge and future challenges. Atherosclerosis, 230(1), 40–47. https://doi.org/10.1016/j.atherosclerosis.2013.06.010 </p>
<p>Bray, F., Laversanne, M., Weiderpass, E., & Soerjomataram, I. (2021). The ever-increasing importance of cancer as a leading cause of premature death worldwide. Cancer, 127(16), 3029–3030. https://doi.org/10.1002/cncr.33587 </p>
<p>Carmeliet, P., & Jain, R. K. (2011). Molecular mechanisms and clinical applications of angiogenesis. https://doi.org/10.1038/nature10144 </p>
<p>Cervelli, T., Panetta, D., Navarra, T., Gadhiri, S., Salvadori, P., Galli, A., Caramella, D., Basta, G., Picano, E., & Del Turco, S. (2017). A New Natural Antioxidant Mixture Protects against Oxidative and DNA Damage in Endothelial Cell Exposed to Low-Dose Irradiation. Oxidative Medicine and Cellular Longevity, 2017. https://doi.org/10.1155/2017/9085947 </p>
<p>Chang, P. Y., Zhang, B. Y., Cui, S., Qu, C., Shao, L. H., Xu, T. K., Qu, Y. Q., Dong, L. H., & Wang, J. (2017). MSC-derived cytokines repair radiation-induced intra-villi microvascular injury. Oncotarget, 8(50). https://doi.org/10.18632/oncotarget.21236 </p>
<p>Chauhan, V., Hamada, N., Monceau, V., Ebrahimian, T., Adam, N., Wilkins, R. C., Sebastian, S., Patel, Z. S., Huff, J. L., Simonetto, C., Iwasaki, T., Kaiser, J. C., Salomaa, S., Moertl, S., & Azimzadeh, O. (2021). Expert consultation is vital for adverse outcome pathway development: a case example of cardiovascular effects of ionizing radiation. International Journal of Radiation Biology, 97(11). https://doi.org/10.1080/09553002.2021.1969466 </p>
<p>Cheng, Y. P., Zhang, H. J., Su, Y. T., Meng, X. X., Xie, X. P., Chang, Y. M., & Bao, J. X. (2017). Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats. Pflugers Archiv European Journal of Physiology, 469(5–6). https://doi.org/10.1007/s00424-017-1969-z </p>
<p>Chiuve, S. E., McCullough, M. L., Sacks, F. M., & Rimm, E. B. (2006). Healthy lifestyle factors in the primary prevention of coronary heart disease among men: Benefits among users and nonusers of lipid-lowering and antihypertensive medications. Circulation, 114(2), 160–167. https://doi.org/10.1161/CIRCULATIONAHA.106.621417 </p>
<p>Chowdhury, R., Shah, D., & Payal, A. (2017). Healthy worker effect phenomenon: Revisited with emphasis on statistical methods-A review. Indian Journal of Occupational and Environmental Medicine, 21(1), 2–8. https://doi.org/10.4103/ijoem.IJOEM_53_16 </p>
<p>Cohn, J. N., Quyyumi, A. A., Hollenberg, N. K., & Jamerson, K. A. (2004). Surrogate markers for cardiovascular disease: Functional markers. Circulation, 109(25). </p>
<p>de Jager, T. L., A. E. Cockrell, S. S. Du Plessis (2017), “Ultraviolet Light Induced Generation of Reactive Oxygen Species”, in: Ultraviolet Light in Human Health, Diseases and Environment, vol 996. Springer Cham, https://doi.org/10.1007/978-3-319-56017-5_2.</p>
<p>Deanfield, J. E., Halcox, J. P., & Rabelink, T. J. (2007). Endothelial function and dysfunction: Testing and clinical relevance. Circulation, 115(10), 1285–1295. https://doi.org/10.1161/CIRCULATIONAHA.106.652859 </p>
<p>Desouky, O., Ding, N., & Zhou, G. (2015). Targeted and non-targeted effects of ionizing radiation. Journal of Radiation Research and Applied Sciences, 8(2), 247–254. https://doi.org/10.1016/J.JRRAS.2015.03.003 </p>
<p>Di Maggio, F. M., Minafra, L., Forte, G. I., Cammarata, F. P., Lio, D., Messa, C., Gilardi, M. C., & Bravatà, V. (2015). Portrait of inflammatory response to ionizing radiation treatment. In Journal of Inflammation (Vol. 12, Issue 1, pp. 1–11). BioMed Central Ltd. https://doi.org/10.1186/s12950-015-0058-3 </p>
<p>Ding, J., Yu, M., Jiang, J., Luo, Y., Zhang, Q., Wang, S., Yang, F., Wang, A., Wang, L., Zhuang, M., Wu, S., Zhang, Q., Xia, Y., & Lu, D. (2020). Angiotensin II Decreases Endothelial Nitric Oxide Synthase Phosphorylation via AT1R Nox/ROS/PP2A Pathway. Frontiers in Physiology, 11, 1245. https://doi.org/10.3389/fphys.2020.566410 </p>
<p>Dörr, W. (2015). Radiobiology of tissue reactions. Annals of the ICRP, 44, 58–68. https://doi.org/10.1177/0146645314560686 </p>
<p>Durante, M., & Cucinotta, F. A. (2008). Heavy ion carcinogenesis and human space exploration. Nature Reviews Cancer, 8(6), 465–472. https://doi.org/10.1038/nrc2391 </p>
<p>Eaton, J. W. (1994). UV-mediated cataractogenesis: A radical perspective. Documenta Ophthalmologica, 88(3–4), 233–242. https://doi.org/10.1007/BF01203677 </p>
<p>EPRI. (2020). Cardiovascular Risks from Low Dose Radiation Exposure: Review and Scientific Appraisal of the Literature. </p>
<p>Fletcher, A. E. (2010). Free radicals, antioxidants and eye diseases: Evidence from epidemiological studies on cataract and age-related macular degeneration. Ophthalmic Research, 44(3), 191–198. https://doi.org/10.1159/000316476 </p>
<p>Forrester, S. J., Kikuchi, D. S., Hernandes, M. S., Xu, Q., & Griendling, K. K. (2018). Reactive oxygen species in metabolic and inflammatory signaling. Circulation Research, 122(6), 877–902. https://doi.org/10.1161/CIRCRESAHA.117.311401 </p>
<p>Förstermann, U. (2010). Nitric oxide and oxidative stress in vascular disease. Pflugers Archiv European Journal of Physiology, 459(6), 923–939. https://doi.org/10.1007/s00424-010-0808-2 </p>
<p>Förstermann, U., & Münzel, T. (2006). Endothelial Nitric Oxide Synthase in Vascular Disease. Circulation, 113(13), 1708–1714. https://doi.org/10.1161/CIRCULATIONAHA.105.602532 </p>
<p>Francula-Zaninovic, S., & Nola, I. A. (2018). Management of Measurable Variable Cardiovascular Disease’ Risk Factors. Current Cardiology Reviews, 14(3), 153–163. https://doi.org/10.2174/1573403x14666180222102312 </p>
<p>Fuji, S., Matsushita, S., Hyodo, K., Osaka, M., Sakamoto, H., Tanioka, K., Miyakawa, K., Kubota, M., Hiramatsu, Y., & Tokunaga, C. (2016). Association between endothelial function and micro-vascular remodeling measured by synchrotron radiation pulmonary micro-angiography in pulmonary arterial hypertension. General Thoracic and Cardiovascular Surgery, 64(10), 597–603. https://doi.org/10.1007/s11748-016-0684-6 </p>
<p>Ganea, E., & Harding, J. J. (2006). Glutathione-related enzymes and the eye. In Current Eye Research (Vol. 31, Issue 1, pp. 1–11). Taylor & Francis. https://doi.org/10.1080/02713680500477347 </p>
<p>Gen, S. W. (2004). The functional interactions between the p53 and MAPK signaling pathways. Cancer Biology and Therapy, 3(2), 156–161. https://doi.org/10.4161/cbt.3.2.614 </p>
<p>Gillies, M., Richardson, D. B., Cardis, E., Daniels, R. D., O’Hagan, J. A., Haylock, R., Laurier, D., Leuraud, K., Moissonnier, M., Schubauer-Berigan, M. K., Thierry-Chef, I., & Kesminiene, A. (2017). Mortality from circulatory diseases and other non-cancer outcomes among nuclear workers in France, the United Kingdom and the United States (inworks). Radiation Research, 188(3), 276–290. https://doi.org/10.1667/RR14608.1 </p>
<p>Grabham, P., & Sharma, P. (2013). The effects of radiation on angiogenesis. Vascular Cell, 5(1), 19. https://doi.org/10.1186/2045-824X-5-19 </p>
<p>Hamada, N., Kawano, K. I., Nomura, T., Furukawa, K., Yusoff, F. M., Maruhashi, T., Maeda, M., Nakashima, A., & Higashi, Y. (2021). Vascular damage in the aorta of wild-type mice exposed to ionizing radiation: Sparing and enhancing effects of dose protraction. Cancers, 13(21). https://doi.org/10.3390/cancers13215344 </p>
<p>Hamada, N., Kawano, K. I., Yusoff, F. M., Furukawa, K., Nakashima, A., Maeda, M., Yasuda, H., Maruhashi, T., & Higashi, Y. (2020). Ionizing irradiation induces vascular damage in the aorta of wild-type mice. Cancers, 12(10), 1–11. https://doi.org/10.3390/cancers12103030 </p>
<p>Hasan, H. F., Radwan, R. R., & Galal, S. M. (2020). Bradykinin-potentiating factor isolated from Leiurus quinquestriatus scorpion venom alleviates cardiomyopathy in irradiated rats via remodelling of the RAAS pathway. Clinical and Experimental Pharmacology and Physiology, 47(2), 263–273. https://doi.org/10.1111/1440-1681.13202 </p>
<p>Hassan, B., Akcakanat, A., Holder, A. M., & Meric-Bernstam, F. (2013). Targeting the PI3-Kinase/Akt/mTOR Signaling Pathway. Surgical Oncology Clinics of North America, 22(4), 641–664. https://doi.org/10.1016/j.soc.2013.06.008 </p>
<p>Hatoum, O. A., Otterson, M. F., Kopelman, D., Miura, H., Sukhotnik, I., Larsen, B. T., Selle, R. M., Moulder, J. E., & Gutterman, D. D. (2006). Radiation induces endothelial dysfunction in murine intestinal arterioles via enhanced production of reactive oxygen species. Arteriosclerosis, Thrombosis, and Vascular Biology, 26(2), 287–294. https://doi.org/10.1161/01.ATV.0000198399.40584.8c </p>
<p>Heald, C. L., Fowkes, F. G. R., Murray, G. D., & Price, J. F. (2006). Risk of mortality and cardiovascular disease associated with the ankle-brachial index: Systematic review. Atherosclerosis, 189(1), 61–69. https://doi.org/10.1016/J.ATHEROSCLEROSIS.2006.03.011 </p>
<p>Hemmings, B. A., & Restuccia, D. F. (2012). PI3K-PKB/Akt pathway. Cold Spring Harbor Perspectives in Biology, 4(9). https://doi.org/10.1101/cshperspect.a011189 </p>
<p>Hirakawa, M., Oike, M., Masuda, K., & Ito, Y. (2002). Tumor cell apoptosis by irradiation-induced nitric oxide production in vascular endothelium. Cancer Research, 62(5), 1450–1457. </p>
<p>Hladik, D., & Tapio, S. (2016). Effects of ionizing radiation on the mammalian brain. Mutation Research - Reviews in Mutation Research, 770, 219–230. https://doi.org/10.1016/j.mrrev.2016.08.003 </p>
<p>Hodis, H. N., Mack, W. J., LaBree, L., Selzer, R. H., Liu, C. R., Liu, C. H., & Azen, S. P. (1998). The role of carotid arterial intima - Media thickness in predicting clinical coronary events. Annals of Internal Medicine, 128(4), 262–269. https://doi.org/10.7326/0003-4819-128-4-199802150-00002 </p>
<p>Hong, C. W., Kim, Y. M., Pyo, H., Lee, J. H., Kim, S., Lee, S., & Noh, J. M. (2013). Involvement of inducible nitric oxide synthase in radiation-Induced vascular endothelial damage. Journal of Radiation Research, 54(6), 1036–1042. https://doi.org/10.1093/jrr/rrt066 </p>
<p>Hsu, T., Nguyen-Tran, H. H., & Trojanowska, M. (2019). Active roles of dysfunctional vascular endothelium in fibrosis and cancer. Journal of Biomedical Science, 26(1), 1–12. https://doi.org/10.1186/s12929-019-0580-3 </p>
<p>Huang, Y. et al. (2021), “Mesenchymal Stem Cell-Conditioned Medium Protects Hippocampal Neurons From Radiation Damage by Suppressing Oxidative Stress and Apoptosis”, Dose-response, Vol. 19/1, Sage Publishing, https://doi.org/10.1177/1559325820984944 </p>
<p>Hughson, R. L., Helm, A., & Durante, M. (2018). Heart in space: Effect of the extraterrestrial environment on the cardiovascular system. In Nature Reviews Cardiology (Vol. 15, Issue 3). https://doi.org/10.1038/nrcardio.2017.157 </p>
<p>Hughson, R. L., Robertson, A. D., Arbeille, P., Shoemaker, J. K., Rush, J. W. E., Fraser, K. S., & Greaves, D. K. (2016). Increased postflight carotid artery stiffness and inflight insulin resistance resulting from 6-mo spaceflight in male and female astronauts. American Journal of Physiology - Heart and Circulatory Physiology, 310(5), H628–H638. https://doi.org/10.1152/ajpheart.00802.2015 </p>
<p>Ismail, A. F. M., & El-Sonbaty, S. M. (2016). Fermentation enhances Ginkgo biloba protective role on gamma-irradiation induced neuroinflammatory gene expression and stress hormones in rat brain. Journal of Photochemistry and Photobiology B: Biology, 158, 154–163. https://doi.org/10.1016/j.jphotobiol.2016.02.039 </p>
<p>Ivanov, V. K., Maksioutov, M. A., Chekin, S. Y., Petrov, A. V., Biryukov, A. P., Kruglova, Z. G., Matyash, V. A., Tsyb, A. F., Manton, K. G., & Kravchenko, J. S. (2006). The risk of radiation-induced cerebrovascular disease in chernobyl emergency workers. Health Physics, 90(3), 199–207. https://doi.org/10.1097/01.HP.0000175835.31663.EA </p>
<p>Karam, H. M., & Radwan, R. R. (2019). Metformin modulates cardiac endothelial dysfunction, oxidative stress and inflammation in irradiated rats: A new perspective of an antidiabetic drug. Clinical and Experimental Pharmacology and Physiology, 46(12), 1124–1132. https://doi.org/10.1111/1440-1681.13148 </p>
<p>Karar, J., & Maity, A. (2011). PI3K/AKT/mTOR Pathway in Angiogenesis. Frontiers in Molecular Neuroscience, 4, 51. https://doi.org/10.3389/fnmol.2011.00051 </p>
<p>Karimi, N., Monfared, A., Haddadi, G., Soleymani, A., Mohammadi, E., Hajian-Tilaki, K., & Borzoueisileh, S. (2017). Radioprotective effect of hesperidin on reducing oxidative stress in the lens tissue of rats. International Journal of Pharmaceutical Investigation, 7(3), 149. https://doi.org/10.4103/jphi.jphi_60_17 </p>
<p>Kashcheev, V. V., Chekin, S. Y., Karpenko, S. V., Maksioutov, M. A., Menyaylo, A. N., Tumanov, K. A., Kochergina, E. V., Kashcheeva, P. V., Gorsky, A. I., Shchukina, N. V., Lovachev, S. S., Vlasov, O. K., & Ivanov, V. K. (2017). Radiation Risk of Cardiovascular Diseases in the Cohort of Russian Emergency Workers of the Chernobyl Accident. Health Physics, 113(1), 23–29. https://doi.org/10.1097/HP.0000000000000670 </p>
<p>Kessler, E. L., Rivaud, M. R., Vos, M. A., & Van Veen, T. A. B. (2019). Sex-specific influence on cardiac structural remodeling and therapy in cardiovascular disease. Biology of Sex Differences, 10(1). https://doi.org/10.1186/s13293-019-0223-0 </p>
<p>Konukoglu, D., & Uzun, H. (2016). Endothelial dysfunction and hypertension. In Advances in Experimental Medicine and Biology (Vol. 956, pp. 511–540). Adv Exp Med Biol. https://doi.org/10.1007/5584_2016_90 </p>
<p>Korpela, E., & Liu, S. K. (2014). Endothelial perturbations and therapeutic strategies in normal tissue radiation damage. Radiation Oncology, 9(1). https://doi.org/10.1186/s13014-014-0266-7 </p>
<p>Kozbenko, T., Adam, N., Lai, V., Sandhu, S., Kuan, J., Flores, D., Appleby, M., Parker, H., Hocking, R., Tsaioun, K., Yauk, C., Wilkins, R., & Chauhan, V. (2022). Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example. International Journal of Radiation Biology, 1–12. https://doi.org/10.1080/09553002.2022.2110306 </p>
<p>Krüger-Genge, A., Blocki, A., Franke, R.-P., & Jung, F. (2019). Vascular Endothelial Cell Biology: An Update. International Journal of Molecular Sciences, 20(18), 4411. https://doi.org/10.3390/ijms20184411 </p>
<p>Lee, M. S., Finch, W., & Mahmud, E. (2013). Cardiovascular complications of radiotherapy. American Journal of Cardiology, 112(10), 1688–1696. https://doi.org/10.1016/j.amjcard.2013.07.031 </p>
<p>Li, J., De Leon, H., Ebato, B., Cui, J., Todd, J., Chronos, N. A. F., & Robinson, K. A. (2002). Endovascular irradiation impairs vascular functional responses in noninjured pig coronary arteries. Cardiovascular Radiation Medicine, 152–162. https://doi.org/10.1016/S1522-1865(03)00096-9 </p>
<p>Little, M. P., Azizova, T. V., & Hamada, N. (2021). Low- and moderate-dose non-cancer effects of ionizing radiation in directly exposed individuals, especially circulatory and ocular diseases: a review of the epidemiology. International Journal of Radiation Biology, 97(6), 782–803. https://doi.org/10.1080/09553002.2021.1876955 </p>
<p>Luiking, Y. C., Engelen, M. P. K. J., & Deutz, N. E. P. (2010). Regulation of nitric oxide production in health and disease. Current Opinion in Clinical Nutrition and Metabolic Care, 13(1), 97–104. https://doi.org/10.1097/MCO.0B013E328332F99D </p>
<p>Lumniczky, K., Impens, N., Armengol, G., Candéias, S., Georgakilas, A. G., Hornhardt, S., Martin, O. A., Rödel, F., & Schaue, D. (2021). Low dose ionizing radiation effects on the immune system. Environment International, 149, 106212. https://doi.org/10.1016/j.envint.2020.106212 </p>
<p>Maier, J. A. M., Cialdai, F., Monici, M., & Morbidelli, L. (2015). The impact of microgravity and hypergravity on endothelial cells. BioMed Research International, 2015. https://doi.org/10.1155/2015/434803 </p>
<p>Matsubara, K., Higaki, T., Matsubara, Y., & Nawa, A. (2015). Nitric oxide and reactive oxygen species in the pathogenesis of preeclampsia. International Journal of Molecular Sciences, 16(3), 4600–4614. https://doi.org/10.3390/ijms16034600 </p>
<p>Menezes, K. M., Wang, H., Hada, M., & Saganti, P. B. (2018). Radiation Matters of the Heart: A Mini Review. Frontiers in Cardiovascular Medicine, 5, 83. https://doi.org/10.3389/FCVM.2018.00083 </p>
<p>Millatt, L. J., Abdel-Rahman, E. M., & Siragy, H. M. (1999). Angiotensin II and nitric oxide: A question of balance. Regulatory Peptides, 81(1–3), 1–10. https://doi.org/10.1016/S0167-0115(99)00027-0 </p>
<p>Mitchell, A., Pimenta, D., Gill, J., Ahmad, H., & Bogle, R. (2019). Cardiovascular effects of space radiation: implications for future human deep space exploration. European Journal of Preventive Cardiology, 26(16), 1707–1714. https://doi.org/10.1177/2047487319831497 </p>
<p>Mittal, M., Siddiqui, M. R., Tran, K., Reddy, S. P., & Malik, A. B. (2014). Reactive oxygen species in inflammation and tissue injury. Antioxidants and Redox Signaling, 20(7), 1126–1167. https://doi.org/10.1089/ars.2012.5149 </p>
<p>Mosca, L., Barrett-Connor, E., & Kass Wenger, N. (2011). Sex/gender differences in cardiovascular disease prevention: What a difference a decade makes. Circulation, 124(19), 2145–2154. https://doi.org/10.1161/CIRCULATIONAHA.110.968792 </p>
<p>Mozaffarian, D., Wilson, P. W. F., & Kannel, W. B. (2008). Beyond Established and Novel Risk Factors. Circulation, 117(23), 3031–3038. https://doi.org/10.1161/CIRCULATIONAHA.107.738732 </p>
<p>Nagane, M., Yasui, H., Kuppusamy, P., Yamashita, T., & Inanami, O. (2021). DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases. Journal of Radiation Research, 62(4), 564–573. https://doi.org/10.1093/jrr/rrab032 </p>
<p>Nathan, C., & Xie, Q. wen. (1994). Nitric oxide synthases: Roles, tolls, and controls. Cell, 78(6), 915–918. https://doi.org/10.1016/0092-8674(94)90266-6 </p>
<p>Norbury, J. W., Schimmerling, W., Slaba, T. C., Azzam, E. I., Badavi, F. F., Baiocco, G., Benton, E., Bindi, V., Blakely, E. A., Blattnig, S. R., Boothman, D. A., Borak, T. B., Britten, R. A., Curtis, S., Dingfelder, M., Durante, M., Dynan, W. S., Eisch, A. J., Robin Elgart, S., … Zeitlin, C. J. (2016). Galactic cosmic ray simulation at the NASA Space Radiation Laboratory. Life Sciences in Space Research, 8, 38–51. https://doi.org/10.1016/j.lssr.2016.02.001 </p>
<p>North, B. J., & Sinclair, D. A. (2012). The intersection between aging and cardiovascular disease. Circulation Research, 110(8), 1097–1108. https://doi.org/10.1161/CIRCRESAHA.111.246876 </p>
<p>Ozasa, K., Shimizu, Y., Suyama, A., Kasagi, F., Soda, M., Grant, E. J., Sakata, R., Sugiyama, H., & Kodama, K. (2012). Studies of the mortality of atomic bomb survivors, report 14, 1950-2003: An overview of cancer and noncancer diseases. In Radiation Research (Vol. 177, Issue 3). https://doi.org/10.1667/RR2629.1 </p>
<p>Padgaonkar, V. A., Leverenz, V. R., Bhat, A. V., Pelliccia, S. E., & Giblin, F. J. (2015). Thioredoxin reductase activity may be more important than GSH level in protecting human lens epithelial cells against UVA light. Photochemistry and Photobiology, 91(2), 387–396. https://doi.org/10.1111/php.12404 </p>
<p>Patel, Z. S., Brunstetter, T. J., Tarver, W. J., Whitmire, A. M., Zwart, S. R., Smith, S. M., & Huff, J. L. (2020). Red risks for a journey to the red planet: The highest priority human health risks for a mission to Mars. Npj Microgravity, 6(1), 1–13. https://doi.org/10.1038/s41526-020-00124-6 </p>
<p>Ping, Z., Peng, Y., Lang, H., Xinyong, C., Zhiyi, Z., Xiaocheng, W., Hong, Z., & Liang, S. (2020). Oxidative Stress in Radiation-Induced Cardiotoxicity. Oxidative Medicine and Cellular Longevity, 2020. https://doi.org/10.1155/2020/3579143 </p>
<p>Polak, J. F., Pencina, M. J., Pencina, K. M., O’Donnell, C. J., Wolf, P. A., & Ralph B. D’Agostino, S. (2011). Carotid-Wall Intima–Media Thickness and Cardiovascular Events. The New England Journal of Medicine, 365(3), 213. https://doi.org/10.1056/NEJMOA1012592 </p>
<p>Preston, D. L., Shimizu, Y., Pierce, D. A., Suyama, A., & Mabuchi, K. (2003). Studies of Mortality of Atomic Bomb Survivors. Report 13: Solid Cancer and Noncancer Disease Mortality: 1950–1997. Https://Doi.Org/10.1667/RR3049, 160(4), 381–407. https://doi.org/10.1667/RR3049 </p>
<p>Pries, A. R., Reglin, B., & Secomb, T. W. (2001). Structural adaptation of microvascular networks: Functional roles of adaptive responses. American Journal of Physiology - Heart and Circulatory Physiology, 281(3). https://doi.org/10.1152/ajpheart.2001.281.3.h1015 </p>
<p>Ramadan, R., Baatout, S., Aerts, A., & Leybaert, L. (2021). The role of connexin proteins and their channels in radiation-induced atherosclerosis. Cellular and Molecular Life Sciences, 78(7), 3087–3103. https://doi.org/10.1007/s00018-020-03716-3 </p>
<p>Ramadan, R., Vromans, E., Anang, D. C., Goetschalckx, I., Hoorelbeke, D., Decrock, E., Baatout, S., Leybaert, L., & Aerts, A. (2020). Connexin43 Hemichannel Targeting With TAT-Gap19 Alleviates Radiation-Induced Endothelial Cell Damage. Frontiers in Pharmacology, 11. https://doi.org/10.3389/fphar.2020.00212 </p>
<p>Rehman, M. U., Jawaid, P., Uchiyama, H., & Kondo, T. (2016). Comparison of free radicals formation induced by cold atmospheric plasma, ultrasound, and ionizing radiation. Archives of Biochemistry and Biophysics, 605, 19–25. https://doi.org/10.1016/j.abb.2016.04.005 </p>
<p>Rios, A., Joshi, R., & Shin, H. (2020). Quantifying 60 Years of Gender Bias in Biomedical Research with Word Embeddings. 1–13. https://doi.org/10.18653/v1/2020.bionlp-1.1 </p>
<p>Rodgers, J. L., Jones, J., Bolleddu, S. I., Vanthenapalli, S., Rodgers, L. E., Shah, K., Karia, K., & Panguluri, S. K. (2019). Cardiovascular risks associated with gender and aging. Journal of Cardiovascular Development and Disease, 6(2). https://doi.org/10.3390/jcdd6020019 </p>
<p>Sadhukhan, R., Leung, J. W. C., Garg, S., Krager, K. J., Savenka, A. V., Basnakian, A. G., & Pathak, R. (2020). Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose. Scientific Reports, 10(1), 1–13. https://doi.org/10.1038/s41598-020-64672-3 </p>
<p>Sakata, K., Kondo, T., Mizuno, N., Shoji, M., Yasui, H., Yamamori, T., Inanami, O., Yokoo, H., Yoshimura, N., & Hattori, Y. (2015). Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells. Vascular Pharmacology, 70, 55–65. https://doi.org/10.1016/j.vph.2015.03.016 </p>
<p>Santamaría, R., González-Álvarez, M., Delgado, R., Esteban, S., & Arroyo, A. G. (2020). Remodeling of the Microvasculature: May the Blood Flow Be With You. Frontiers in Physiology, 11, 1256. https://doi.org/10.3389/FPHYS.2020.586852/XML/NLM </p>
<p>Sárközy, M., Gáspár, R., Zvara, A., Kiscsatári, L., Varga, Z., Kővári, B., Kovács, M. G., Szűcs, G., Fabian, G., Diószegi, P., Cserni, G., Puskás, L. G., Thum, T., Kahán, Z., Csont, T., & Batkai, S. (2019). Selective heart irradiation induces cardiac overexpression of the pro-hypertrophic miR-212. Frontiers in Oncology, 9(JUN), 598. https://doi.org/10.3389/fonc.2019.00598 </p>
<p>Schaue, D., Micewicz, E. D., Ratikan, J. A., Xie, M. W., Cheng, G., & McBride, W. H. (2015). Radiation and Inflammation. In Seminars in Radiation Oncology (Vol. 25, Issue 1, pp. 4–10). W.B. Saunders. https://doi.org/10.1016/j.semradonc.2014.07.007 </p>
<p>Schiffrin, E. L. (2008). Oxidative stress, nitric oxide synthase, and superoxide dismutase: A matter of imbalance underlies endothelial dysfunction in the human coronary circulation. Hypertension, 51(1), 31–32. https://doi.org/10.1161/HYPERTENSIONAHA.107.103226 </p>
<p>Schmidt-Ullrich, R. K., Dent, P., Grant, S., Mikkelsen, R. B., & Valerie, K. (2000). Signal transduction and cellular radiation responses. Radiation Research, 153(3), 245–257. https://doi.org/10.1667/0033-7587(2000)153[0245:STACRR]2.0.CO;2 </p>
<p>Schultz, W. M., Kelli, H. M., Lisko, J. C., Varghese, T., Shen, J., Sandesara, P., Quyyumi, A. A., Taylor, H. A., Gulati, M., Harold, J. G., Mieres, J. H., Ferdinand, K. C., Mensah, G. A., & Sperling, L. S. (2018). Socioeconomic Status and Cardiovascular Outcomes: Challenges and Interventions. Circulation, 137(20). https://doi.org/10.1161/circulationaha.117.029652 </p>
<p>Senoner, T., & Dichtl, W. (2019). Oxidative stress in cardiovascular diseases: Still a therapeutic target? Nutrients, 11(9). https://doi.org/10.3390/nu11092090 </p>
<p>Shen, Y., Jiang, X., Meng, L., Xia, C., Zhang, L., & Xin, Y. (2018). Transplantation of bone marrow mesenchymal stem cells prevents radiation-induced artery injury by suppressing oxidative stress and inflammation. Oxidative Medicine and Cellular Longevity, 2018. https://doi.org/10.1155/2018/5942916 </p>
<p>Shi, F., Wang, Y. C., Zhao, T. Z., Zhang, S., Du, T. Y., Yang, C. Bin, Li, Y. H., & Sun, X. Q. (2012). Effects of simulated microgravity on human umbilical vein endothelial cell angiogenesis and role of the PI3K-Akt-eNOS signal pathway. PLoS ONE, 7(7). https://doi.org/10.1371/journal.pone.0040365 </p>
<p>Shimizu, Y., Kodama, K., Nishi, N., Kasagi, F., Suyama, A., Soda, M., Grant, E. J., Sugiyama, H., Sakata, R., Moriwaki, H., Hayashi, M., Konda, M., & Shore, R. E. (2010). Radiation exposure and circulatory disease risk: Hiroshima and Nagasaki atomic bomb survivor data, 1950-2003. The BMJ, 340(7739), 193. https://doi.org/10.1136/BMJ.B5349 </p>
<p>Siamwala, J. H., Reddy, S. H., Majumder, S., Kolluru, G. K., Muley, A., Sinha, S., & Chatterjee, S. (2010). Simulated microgravity perturbs actin polymerization to promote nitric oxide-associated migration in human immortalized Eahy926 cells. Protoplasma, 242(1), 3–12. https://doi.org/10.1007/s00709-010-0114-z </p>
<p>Slezak, J., Kura, B., Babal, P., Barancik, M., Ferko, M., Frimmel, K., Kalocayova, B., Kukreja, R. C., Lazou, A., Mezesova, L., Okruhlicova, L., Ravingerova, T., Singal, P. K., Bacova, B. S., Viczenczova, C., Vrbjar, N., & Tribulova, N. (2017). Potential markers and metabolic processes involved in the mechanism of Radiation-Induced heart injury. Canadian Journal of Physiology and Pharmacology, 95(10), 1190–1203. https://doi.org/10.1139/cjpp-2017-0121 </p>
<p>Soloviev, A. I., & Kizub, I. V. (2019). Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction. Biochemical Pharmacology, 159, 121–139. https://doi.org/10.1016/j.bcp.2018.11.019 </p>
<p>Sonveaux, P., Brouet, A., Havaux, X., Grégoire, V., Dessy, C., Balligand, J. L., & Feron, O. (2003). Irradiation-induced angiogenesis through the up-regulation of the nitric oxide pathway: Implications for tumor radiotherapy. Cancer Research, 63(5), 1012–1019. https://doi.org/10.1016/s0167-8140(03)80572-8 </p>
<p>Soucy, K. G., Lim, H. K., Attarzadeh, D. O., Santhanam, L., Kim, J. H., Bhunia, A. K., Sevinc, B., Ryoo, S., Vazquez, M. E., Nyhan, D., Shoukas, A. A., & Berkowitz, D. E. (2010). Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta. Journal of Applied Physiology, 108(5), 1250–1258. https://doi.org/10.1152/japplphysiol.00946.2009 </p>
<p>Soucy, K. G., Lim, H. K., Benjo, A., Santhanam, L., Ryoo, S., Shoukas, A. A., Vazquez, M. E., & Berkowitz, D. E. (2007). Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta. Radiation and Environmental Biophysics, 46(2), 179–186. https://doi.org/10.1007/s00411-006-0090-z </p>
<p>Soucy, K. G., Lim, H. K., Kim, J. H., Oh, Y., Attarzadeh, D. O., Sevinc, B., Kuo, M. M., Shoukas, A. A., Vazquez, M. E., & Berkowitz, D. E. (2011). HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase. Radiation Research, 176(4), 474–485. https://doi.org/10.1667/RR2598.1 </p>
<p>Sridharan, V., Seawright, J. W., Landes, R. D., Cao, M., Singh, P., Davis, C. M., Mao, X. W., Singh, S. P., Zhang, X., Nelson, G. A., & Boerma, M. (2020). Effects of single-dose protons or oxygen ions on function and structure of the cardiovascular system in male Long Evans rats. Life Sciences in Space Research, 26, 62–68. https://doi.org/10.1016/j.lssr.2020.04.002 </p>
<p>Srinivasan, L., Harris, M. C., & Kilpatrick, L. E. (2017). Cytokines and Inflammatory Response in the Fetus and Neonate. In Fetal and Neonatal Physiology, 2-Volume Set (pp. 1241–1254). https://doi.org/10.1016/B978-0-323-35214-7.00128-1 </p>
<p>Stanojković, T. P., Matić, I. Z., Petrović, N., Stanković, V., Kopčalić, K., Besu, I., Đorđić Crnogorac, M., Mališić, E., Mirjačić-Martinović, K., Vuletić, A., Bukumirić, Z., Žižak, Ž., Veldwijk, M., Herskind, C., & Nikitović, M. (2020). Evaluation of cytokine expression and circulating immune cell subsets as potential parameters of acute radiation toxicity in prostate cancer patients. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-75812-0 </p>
<p>Su, Y. T., Cheng, Y. P., Zhang, X., Xie, X. P., Chang, Y. M., & Bao, J. X. (2020). Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats. Life Sciences, 243. https://doi.org/10.1016/j.lfs.2019.117253 </p>
<p>Summers, S. M., Nguyen, S. V., & Purdy, R. E. (2008). Hindlimb unweighting induces changes in the RhoA-Rho-kinase pathway of the rat abdominal aorta. Vascular Pharmacology, 48(4–6), 208–214. https://doi.org/10.1016/j.vph.2008.03.006 </p>
<p>Sylvester, C. B., Abe, J. I., Patel, Z. S., & Grande-Allen, K. J. (2018). Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer. Frontiers in Cardiovascular Medicine, 5, 1. https://doi.org/10.3389/FCVM.2018.00005 </p>
<p>Tahimic, C. G. T., & Globus, R. K. (2017). Redox signaling and its impact on skeletal and vascular responses to spaceflight. International Journal of Molecular Sciences, 18(10). https://doi.org/10.3390/ijms18102153 </p>
<p>Takahashi, I., Shimizu, Y., Grant, E. J., Cologne, J., Ozasa, K., & Kodama, K. (2017). Heart disease mortality in the life span study, 1950-2008. In Radiation Research (Vol. 187, Issue 3). https://doi.org/10.1667/RR14347.1 </p>
<p>Tapio, S. (2016). Pathology and biology of radiation-induced cardiac disease. Journal of Radiation Research, 57(5), 439–448. https://doi.org/10.1093/jrr/rrw064 </p>
<p>Tsao, C. W., Aday, A. W., Almarzooq, Z. I., Alonso, A., Beaton, A. Z., Bittencourt, M. S., Boehme, A. K., Buxton, A. E., Carson, A. P., Commodore-Mensah, Y., Elkind, M. S. V., Evenson, K. R., Eze-Nliam, C., Ferguson, J. F., Generoso, G., Ho, J. E., Kalani, R., Khan, S. S., Kissela, B. M., … Martin, S. S. (2022). Heart Disease and Stroke Statistics-2022 Update: A Report From the American Heart Association. Circulation, 145(8), e153–e639. https://doi.org/10.1161/CIR.0000000000001052/FORMAT/EPUB </p>
<p>Ungvari, Z., Podlutsky, A., Sosnowska, D., Tucsek, Z., Toth, P., Deak, F., Gautam, T., Csiszar, A., & Sonntag, W. E. (2013). Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: Role of increased dna damage and decreased dna repair capacity in microvascular radiosens. Journals of Gerontology - Series A Biological Sciences and Medical Sciences, 68(12 A), 1443–1457. https://doi.org/10.1093/gerona/glt057 </p>
<p>UNSCEAR. (2008). UNSCEAR 2006 report. Annex B. Epidemiological evaluation of cardiovascular disease and other non_cancer diseases following radiation exposure. </p>
<p>Valerie, K., Yacoub, A., Hagan, M. P., Curiel, D. T., Fisher, P. B., Grant, S., & Dent, P. (2007). Radiation-induced cell signaling: Inside-out and outside-in. Molecular Cancer Therapeutics, 6(3), 789–801. https://doi.org/10.1158/1535-7163.MCT-06-0596 </p>
<p>Van Varik, B. J., Rennenberg, R. J. M. W., Reutelingsperger, C. P., Kroon, A. A., De Leeuw, P. W., & Schurgers, L. J. (2012). Mechanisms of arterial remodeling: Lessons from genetic diseases. Frontiers in Genetics, 3(DEC), 290. https://doi.org/10.3389/FGENE.2012.00290/BIBTEX </p>
<p>Varma, S. D., Kovtun, S., & Hegde, K. R. (2011). Role of ultraviolet irradiation and oxidative stress in cataract formation-medical prevention by nutritional antioxidants and metabolic agonists. Eye and Contact Lens, 37(4), 233–245. https://doi.org/10.1097/ICL.0b013e31821ec4f2 </p>
<p>Venkatesulu, B. P., Mahadevan, L. S., Aliru, M. L., Yang, X., Bodd, M. H., Singh, P. K., Yusuf, S. W., Abe, J. ichi, & Krishnan, S. (2018). Radiation-Induced Endothelial Vascular Injury: A Review of Possible Mechanisms. JACC: Basic to Translational Science, 3(4), 563–572. https://doi.org/10.1016/j.jacbts.2018.01.014 </p>
<p>Verma, S., Buchanan, M. R., & Anderson, T. J. (2003). Endothelial Function Testing as a Biomarker of Vascular Disease. Circulation, 108(17), 2054–2059. https://doi.org/10.1161/01.CIR.0000089191.72957.ED </p>
<p>Vernice, N. A., Meydan, C., Afshinnekoo, E., & Mason, C. E. (2020). Long-term spaceflight and the cardiovascular system. Precision Clinical Medicine, 3(4), 284–291. https://doi.org/10.1093/PCMEDI/PBAA022 </p>
<p>Versari, S., Longinotti, G., Barenghi, L., Maier, J. A. M., & Bradamante, S. (2013). The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment. FASEB Journal, 27(11), 4466–4475. https://doi.org/10.1096/fj.13-229195 </p>
<p>Wang, H., Wei, J., Zheng, Q., Meng, L., Xin, Y., Yin, X., & Jiang, X. (2019). Radiation-induced heart disease: a review of classification, mechanism and prevention. International Journal of Biological Sciences, 15(10), 2128. https://doi.org/10.7150/IJBS.35460 </p>
<p>Wang, Y., Boerma, M., & Zhou, D. (2016). Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases. Radiation Research, 186(2), 153–161. https://doi.org/10.1667/RR14445.1 </p>
<p>Winham, S. J., de Andrade, M., & Miller, V. M. (2014). Genetics of cardiovascular disease: Importance of sex and ethnicity. Atherosclerosis, 241(1), 219–228. https://doi.org/10.1016/j.atherosclerosis.2015.03.021 </p>
<p>Yakerson, A. (2019). Women in clinical trials: A review of policy development and health equity in the Canadian context. International Journal for Equity in Health, 18(1), 56. https://doi.org/10.1186/s12939-019-0954-x </p>
<p>Yan, T., Zhang, T., Mu, W., Qi, Y., Guo, S., Hu, N., Zhao, W., Zhang, S., Wang, Q., Shi, L., & Liu, L. (2020). Ionizing radiation induces BH4 deficiency by downregulating GTP-cyclohydrolase 1, a novel target for preventing and treating radiation enteritis. Biochemical Pharmacology, 180, 114102. https://doi.org/10.1016/j.bcp.2020.114102 </p>
<p>Yang, Y. et al. (1998), “The Effect of Catalase Amplification on Immortal Lens Epithelial Cell Lines”, Experimental Eye Research, Vol. 67/6, Elsevier, Amsterdam https://doi.org/10.1006/exer.1998.0560 </p>
<p>Yang, E. H., Marmagkiolis, K., Balanescu, D. V., Hakeem, A., Donisan, T., Finch, W., Virmani, R., Herrman, J., Cilingiroglu, M., Grines, C. L., Toutouzas, K., & Iliescu, C. (2021). Radiation-Induced Vascular Disease—A State-of-the-Art Review. Frontiers in Cardiovascular Medicine, 8, 223. https://doi.org/10.3389/FCVM.2021.652761/XML/NLM </p>
<p>Yao, L., Romero, M. J., Toque, H. A., Yang, G., Caldwell, R. B., & Caldwell, R. W. (2010). The role of RhoA/Rho kinase pathway in endothelial dysfunction. Journal of Cardiovascular Disease Research, 1(4), 165–170. https://doi.org/10.4103/0975-3583.74258 </p>
<p>Yentrapalli, R., Azimzadeh, O., Barjaktarovic, Z., Sarioglu, H., Wojcik, A., Harms-Ringdahl, M., Atkinson, M. J., Haghdoost, S., & Tapio, S. (2013). Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low-dose rate gamma radiation. Proteomics, 13(7), 1096–1107. https://doi.org/10.1002/pmic.201200463 </p>
<p>Yentrapalli, R., Azimzadeh, O., Sriharshan, A., Malinowsky, K., Merl, J., Wojcik, A., Harms-Ringdahl, M., Atkinson, M. J., Becker, K. F., Haghdoost, S., & Tapio, S. (2013). The PI3K/Akt/mTOR Pathway Is Implicated in the Premature Senescence of Primary Human Endothelial Cells Exposed to Chronic Radiation. PLoS ONE, 8(8). https://doi.org/10.1371/journal.pone.0070024 </p>
<p>Yu, T., Parks, B. W., Yu, S., Srivastava, R., Gupta, K., Wu, X., Khaled, S., Chang, P. Y., Kabarowski, J. H., & Kucik, D. F. (2011). Iron-ion radiation accelerates atherosclerosis in apolipoprotein E-Deficient mice. Radiation Research, 175(6), 766–773. https://doi.org/10.1667/RR2482.1 </p>
<p>Zakrzewicz, A., Secomb, T. W., & Pries, A. R. (2002). Angioadaptation: Keeping the vascular system in shape. News in Physiological Sciences, 17(5). https://doi.org/10.1152/nips.01395.2001 </p>
<p>Zhang, L. F. (2013). Region-specific vascular remodeling and its prevention by artificial gravity in weightless environment. European Journal of Applied Physiology, 113(12), 2873–2895. https://doi.org/10.1007/S00421-013-2597-8 </p>
<p>Zhang, R., Bai, Y. G., Lin, L. J., Bao, J. X., Zhang, Y. Y., Tang, H., Cheng, J. H., Jia, G. L., Ren, X. L., & Jin, M. (2009). Blockade of at 1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats. Journal of Applied Physiology, 106(1), 251–258. https://doi.org/10.1152/japplphysiol.01278.2007 </p>
<p>Zielinski, J. M., Ashmore, P. J., Band, P. R., Jiang, H., Shilnikova, N. S., Tait, V. K., & Krewski, D. (2009). Low dose ionizing radiation exposure and cardiovascular disease mortality: Cohort study based on Canadian national dose registry of radiation workers. International Journal of Occupational Medicine and Environmental Health, 22(1), 27–33. https://doi.org/10.2478/v10001-009-0001-z </p>
<p>Zieman, S. J., Melenovsky, V., & Kass, D. A. (2005). Mechanisms, Pathophysiology, and Therapy of Arterial Stiffness. Arteriosclerosis, Thrombosis, and Vascular Biology, 25(5), 932–943. https://doi.org/10.1161/01.ATV.0000160548.78317.29 </p>
<p>Zigman, S., McDaniel, T., Schultz, J., & Reddan, J. (2000). Effects of intermittent UVA exposure on cultured lens epithelial cells. Current Eye Research, 20(2), 95–100. https://doi.org/10.1076/0271-3683(200002)2021-DFT095 </p>
<p>Zou, B., Schuster, J. P., Niu, K., Huang, Q., Rühle, A., & Huber, P. E. (2019). Radiotherapy-induced heart disease: a review of the literature. Precision Clinical Medicine, 2(4), 270–282. https://doi.org/10.1093/pcmedi/pbz025</p>
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