Increase, Reactive oxygen species

60-35-5

DLFVBJFMPXGRIB-UHFFFAOYSA-N

Acetamide

Acetamid acetamida Acetic acid amide Acetimidic acid Ethanamide Ethanimidic acid Methanecarboxamide NSC 25945

DTXSID7020005

103-90-2

RZVAJINKPMORJF-UHFFFAOYSA-N

Acetaminophen

4-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

DTXSID2020006

968-81-0

VGZSUPCWNCWDAN-UHFFFAOYSA-N

Acetohexamide

Benzenesulfonamide, 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-

DTXSID7020007

67-66-3

HEDRZPFGACZZDS-UHFFFAOYSA-N

Chloroform

Trichloromethane Methane, trichloro- CARBON TRICHLORIDE Chloroforme cloroformo Formyl trichloride Methane trichloride Methane,trichloro- NSC 77361 Trichloroform UN 1888

DTXSID1020306

110-00-9

YLQBMQCUIZJEEH-UHFFFAOYSA-N

Furan

Divinylene oxide furanne Furfuran Oxacyclopentadiene Tetrole UN 2389

DTXSID6020646

7429-90-5

XAGFODPZIPBFFR-UHFFFAOYSA-N

AZDRQVAHHNSJOQ-UHFFFAOYSA-N

Aluminum

Aisin 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

DTXSID3040273

7440-43-9

BDOSMKKIYDKNTQ-UHFFFAOYSA-N

Cadmium

Cadimium CADMIUM BLUE CADMIUM, IN PLATTEN, STANGEN, BROCKEN,KOERNER

DTXSID1023940

7439-97-6

QSHDDOUJBYECFT-UHFFFAOYSA-N

Mercury

Liquid silver Mercure MERCURIC METAL TRIPLE DISTILLED mercurio Mercury element Quecksilber Quicksilver UN 2024 UN 2809

DTXSID1024172

7440-61-1

JFALSRSLKYAFGM-UHFFFAOYSA-N

Uranium

Uranium, isotope of mass 238 238U Element UN 2979 (DOT) Uranium I

DTXSID1042522

7440-38-2

RQNWIZPPADIBDY-UHFFFAOYSA-N

Arsenic

As Arsenic black ARSENIC METAL arsenico Grey arsenic UN 1558

DTXSID4023886

7440-22-4

BQCADISMDOOEFD-UHFFFAOYSA-N

Silver

Ag 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

DTXSID4024305

7439-96-5

PWHULOQIROXLJO-UHFFFAOYSA-N

Manganese

Colloidal manganese Cutaval Manganese element Manganese fulleride Manganese metal alloy Manganese-55 manganeso

DTXSID2024169

7440-02-0

PXHVJJICTQNCMI-UHFFFAOYSA-N

Nickel

Carbonyl 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

DTXSID2020925

7440-66-6

HCHKCACWOHOZIP-UHFFFAOYSA-N

Zinc

Zn 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

DTXSID7035012

CHEBI:26523

CHEBI reactive oxygen species

CHEBI:39026

CHEBI low-density lipoprotein

GO:1903409

GO reactive oxygen species biosynthetic process

MP:0003674

MP oxidative stress

GO:0072577

GO endothelial cell apoptotic process

GO:0006954

GO inflammatory response

GO:0042116

GO macrophage activation

GO:0006898

GO receptor-mediated endocytosis

WIKI increased

WIKI decreased

Reactive oxygen species

2017-06-16T08:32:10

2017-08-15T10:43:27

Acetaminophen

2016-11-29T18:42:26

Chloroform

2016-11-29T18:42:27

furan

2020-05-01T14:35:22

Platinum

2022-02-04T14:36:54

Aluminum

2022-02-04T14:42:11

Cadmium

2017-10-25T08:33:12

Mercury

2016-11-29T18:42:19

Uranium

2021-08-05T14:28:50

Arsenic

2021-04-27T00:15:21

Silver

2022-02-03T11:20:11

Manganese

2022-02-04T14:47:23

Nickel

2022-02-04T14:47:59

Zinc

2022-02-04T15:05:00

nanoparticles

2016-12-21T09:40:06

WikiUser_28

Vertebrates

WCS_9606

common toxicological species human

WikiUser_25

Wikiuser: Cyauk human and other cells in culture

10090

NCBI mouse

WCS_35525

common ecological species crustaceans

WCS_4472

common ecological species Lemna minor

WCS_7955

common ecological species zebrafish

WikiUser_26

ApacheUser rodents

9606

NCBI Homo sapiens

10116

NCBI rat

39108

NCBI Murinae gen. sp.

9823

NCBI pigs

Increase, Reactive oxygen species

Increase, ROS

Cellular

Biological State: increased reactive oxygen species (ROS) Biological compartment: an entire cell -- may be cytosolic, may also enter organelles. Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017).  However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015).  <div> Reactive oxygen species (ROS) refers to the chemical species superoxide, hydrogen peroxide, and their secondary reactive products. In the biological context, ROS are signaling molecules with important roles in cell energy metabolism, cell proliferation, and fate. Therefore, balancing ROS levels at the cellular and tissue level is an important part of many biological processes. Disbalance, mainly an increase in ROS levels, can cause cell dysfunction and irreversible cell damage. ROS are produced from both exogenous stressors and normal endogenous cellular processes, such as the mitochondrial electron transport chain (ETC). Inhibition of the ETC can result in the accumulation of ROS. Exposure to chemicals, heavy metal ions, or ionizing radiation can also result in increased production of ROS. Chemicals and heavy metal ions can deplete cellular antioxidants reducing the cell’s ability to control cellular ROS and resulting in the accumulation of ROS. Cellular antioxidants include glutathione (GSH), protein sulfhydryl groups, superoxide dismutase (SOD). ROS are radicals, ions, or molecules that have a single unpaired electron in their outermost shell of electrons, which can be categorized into two groups: free oxygen radicals and non-radical ROS [Liou et al., 2010]. <Free oxygen radicals> <div> <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px"> superoxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px"> O2·- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> hydroxyl radical </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ·OH </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> nitric oxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> NO· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> organic radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> R· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> peroxyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ROO· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> alkoxyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RO· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> thiyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RS· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> sulfonyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ROS· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> thiyl peroxyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RSOO· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> disulfides </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RSSR </td> </tr> </tbody> </table> </div> <Non-radical ROS> <div> <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px"> hydrogen peroxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px"> H2O2 </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> singlet oxygen </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> 1O2 </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ozone/trioxygen </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> O3 </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> organic hydroperoxides </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ROOH </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> hypochlorite </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ClO- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> peroxynitrite </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ONOO- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> nitrosoperoxycarbonate anion </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> O=NOOCO2- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> nitrocarbonate anion </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> O2NOCO2- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> dinitrogen dioxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> N2O2 </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> nitronium </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> NO2+ </td> </tr> <tr> <td colspan="2" style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:580px"> highly reactive lipid- or carbohydrate-derived carbonyl compounds </td> </tr> </tbody> </table> </div> Potential sources of ROS include NADPH oxidase, xanthine oxidase, mitochondria, nitric oxide synthase, cytochrome P450, lipoxygenase/cyclooxygenase, and monoamine oxidase [Granger et al., 2015]. ROS are generated through NADPH oxidases consisting of p47phox and p67phox. ROS are generated through xanthine oxidase activation in sepsis [Ramos et al., 2018]. Arsenic produces ROS [Zhang et al., 2011]. Mitochondria-targeted paraquat and metformin mediate ROS production [Chowdhury et al., 2020]. ROS are generated by bleomycin [Lu et al., 2010]. Radiation induces dose-dependent ROS production [Ji et al., 2019]. ROS are generated in the course of cellular respiration, metabolism, cell signaling, and inflammation [Dickinson and Chang 2011; Egea et al. 2017]. Hydrogen peroxide is also made by the endoplasmic reticulum in the course of protein folding. Nitric oxide (NO) is produced at the highest levels by nitric oxide synthase in endothelial cells and phagocytes. NO production is one of the main mechanisms by which phagocytes kill bacteria [Wang et al., 2017]. The other species are produced by reactions with superoxide or peroxide, or by other free radicals or enzymes. ROS activity is principally local. Most ROS have short half-lives, ranging from nano- to milliseconds, so diffusion is limited, while reactive nitrogen species (RNS) nitric oxide or peroxynitrite can survive long enough to diffuse across membranes [Calcerrada et al. 2011]. Consequently, local concentrations of ROS are much higher than average cellular concentrations, and signaling is typically controlled by colocalization with redox buffers [Dickinson and Chang 2011; Egea et al. 2017]. Although their existence is limited temporally and spatially, ROS interact with other ROS or with other nearby molecules to produce more ROS and participate in a feedback loop to amplify the ROS signal, which can increase RNS. Both ROS and RNS also move into neighboring cells, and ROS can increase intracellular ROS signaling in neighboring cells [Egea et al. 2017]. In the primary event, photoreactive chemicals are excited by the absorption of photon energy.  The energy of the photoactivated chemicals transfer to oxygen and then generates the reactive oxygen species (ROS), including superoxide (O2−) via type I reaction and singlet oxygen (1O2) via type II reaction, as principal intermediate species in phototoxic reaction (Foote, 1991, Onoue et al. , 2009). </div>

Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies. Yuan, Yan, et al., (2013) described ROS monitoring by using H2-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H2-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm. Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013). Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS. On the basis of the pathogenesis of drug-induced phototoxicity, a reactive oxygen species (ROS) assay was proposed to evaluate the phototoxic risk of chemicals. The ROS assay can monitor generation of ROS, such as singlet oxygen and superoxide, from photoirradiated chemicals, and the ROS data can be used to evaluate the photoreactivity of chemicals (Onoue et al. , 2014, Onoue et al. , 2013, Onoue and Tsuda, 2006).  The ROS assay is a recommended approach by guidelines to evaluate the phototoxic risk of chemicals (ICH, 2014, PCPC, 2014). <div> <Direct detection> Many fluorescent compounds can be used to detect ROS, some of which are specific, and others are less specific. ・ROS can be detected by fluorescent probes such as p-methoxy-phenol derivative [Ashoka et al., 2020]. ・Chemiluminescence analysis can detect the superoxide, where some probes have a wider range for detecting hydroxyl radical, hydrogen peroxide, and peroxynitrite [Fuloria et al., 2021]. ・ROS in the blood can be detected using superparamagnetic iron oxide nanoparticles (SPION)-based biosensor [Lee et al., 2020]. ・Hydrogen peroxide (H2O2) can be detected with a colorimetric probe, which reacts with H2O2 in a 1:1 stoichiometry to produce a bright pink colored product, followed by the detection with a standard colorimetric microplate reader with a filter in the 540-570 nm range. ・The levels of ROS can be quantified using multiple-step amperometry using a stainless steel counter electrode and non-leak Ag|AgCl reference node [Flaherty et al., 2017]. ・Singlet oxygen can be measured by monitoring the bleaching of p-nitrosodimethylaniline at 440 nm using a spectrophotometer with imidazole as a selective acceptor of singlet oxygen [Onoue et al., 2014]. <Indirect Detection> Alternative methods involve the detection of redox-dependent changes to cellular constituents such as proteins, DNA, lipids, or glutathione [Dickinson and Chang 2011; Wang et al. 2013; Griendling et al. 2016]. However, these methods cannot generally distinguish between the oxidative species behind the changes and cannot provide good resolution for the kinetics of oxidative activity. </div>

ROS is a normal constituent found in all organisms, lifestages, and sexes.

UBERON:0000062

UBERON organ

CL:0000000

CL cell

High Unspecific High Mixed

High

All life stages

High Moderate Moderate Moderate High High High

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Ozcan, Ayla, and Metin Ogun. 2015. “Biochemistry of Reactive Oxygen and Nitrogen Species.” In Basic Principles and Clinical Significance of Oxidative Stress, edited by Sivakumar Joghi Thatha Gowder. Rijeka: IntechOpen. Parrish, A. R. 2010. “2.27 - Hypoxia/Ischemia Signaling.” In Comprehensive Toxicology (Second Edition), edited by Charlene A. McQueen, 529–42. Oxford: Elsevier. PCPC. PCPC 2014 safety evaluation guidelines; Chapter 7: Evaluation of Photoirritation and Photoallergy potential. Personal Care Products Council; 2014. Ramos, M. F. P., et al. (2018). "Xanthine oxidase inhibitors and sepsis." Int J Immunopathol Pharmacol. 32:2058738418772210. doi:10.1177/2058738418772210 Ravanat, J. L., et al. (2014). "Radiation-mediated formation of complex damage to DNA: a chemical aspect overview." Br J Radiol 87(1035): 20130715. Schutzendubel, A. and Polle, A. (2002). "Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization." 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2025-06-12T01:27:08

Increase, Oxidative Stress

Molecular

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.  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.  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 on neighboring amino acids (Antelmann & Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010).  ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol, 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 O2. 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 (Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017).   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).    Sources of ROS Production  Direct Sources: 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 (HO2*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H2O2) and more O2 (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).   Indirect Sources: 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 (H2O2) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O2, and inorganic phosphate (Pi) (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 has its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS is 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 A2 (PLA2), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021).

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. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed  <ul> <li>Detection of ROS by chemiluminescence (https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683) </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>    Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include:  <ul> <li>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus 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 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> </ul> In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation. <table border="1"> <tbody> <tr> <td> Assay Type & Measured Content  </td> <td> Description  </td> <td> Dose Range Studied  </td> <td> Assay Characteristics (Length/Ease of use/Accuracy)  </td> </tr> <tr> <td> ROS  Formation in the Mitochondria assay (Shaki et al., 2012)  </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, 10mM 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>  Long/ Easy High accuracy    </td> </tr> <tr> <td> Mitochondrial Antioxidant Content Assay Measuring GSH content (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> 0, 50,  100, or  200 µM  Uranyl Acetate  </td> <td>   </td> </tr> <tr> <td> H2O2 Production Assay Measuring H2O2 Production in isolated mitochondria (Heyno et al., 2008)    </td> <td> “Effect of CdCl2 and antimycin A (AA) on H2O2 production in isolated mitochondria from potato. H2O2 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> 0, 10, 30  µM Cd2+     2 µM antimycin A  </td> <td>   </td> </tr> <tr> <td> Flow Cytometry ROS & Cell Viability (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 t 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)”“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 t 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>           Strong/easy medium  </td> </tr> <tr> <td> DCFH-DA  Assay Detection of hydrogen peroxide production (Yuan et al.,  2016)  </td> <td> 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 H2O2 to form fluorescent production.    </td> <td> 0-400  µM  </td> <td> Long/ Easy High accuracy  </td> </tr> <tr> <td> H2-DCF-DAAssay Detection of superoxide production (Thiebault etal., 2007)    </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> Long/ Easy High accuracy  </td> </tr> <tr> <td> CM-H2DCFDA  Assay (Eruslanov  & Kusmartsev, 2009)  </td> <td> The dye (CM-H2DCFDA) diffuses into the cell and is cleaved by esterases, the thiol reactive chlormethyl group reacts with intracellular glutathione which can be detected using flow cytometry.  </td> <td>   </td> <td> Long/Easy/ High Accuracy  </td> </tr> </tbody> </table>   <table border="1"> <tbody> <tr> <td> Method of Measurement   </td> <td> References   </td> <td> Description   </td> <td colspan="2"> OECD-Approved Assay  </td> </tr> <tr> <td> Chemiluminescence   </td> <td> (Lu, C. et al., 2006;   Griendling, K. K., et al., 2016)  </td> <td> 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 luminol and lucigenin are commonly used to amplify the signal.   </td> <td colspan="2"> No    </td> </tr> <tr> <td> Spectrophotometry   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> 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.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Direct or Spin Trapping-Based electron paramagnetic resonance (EPR) Spectroscopy   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> 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.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Nitroblue Tetrazolium Assay   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> The Nitroblue Tetrazolium assay is used to measure O2.− levels. O2.− reduces nitroblue tetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> Fluorescence analysis of DHE is used to measure O2.− levels.  O2.− 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.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Amplex Red Assay   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> Fluorescence analysis to measure extramitochondrial or extracellular H2O2 levels. In the presence of horseradish peroxidase and H2O2, Amplex Red is oxidized to resorufin, a fluorescent molecule measurable by plate reader.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Dichlorodihydrofluorescein Diacetate (DCFH-DA)   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> An indirect fluorescence analysis to measure intracellular H2O2 levels.  H2O2 interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it to dichlorofluorescein (DCF), a fluorescent product.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> HyPer Probe   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> Fluorescent measurement of intracellular H2O2 levels. HyPer is a genetically encoded fluorescent sensor that can be used for in vivo and in situ imaging.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Cytochrome c Reduction Assay   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> The cytochrome c reduction assay is used to measure O2.− levels. O O2.− 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.   </td> <td colspan="2"> No  </td> </tr> <tr> <td> Proton-electron double-resonance imaging (PEDRI)   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> 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.   </td> <td colspan="2"> No              </td> </tr> <tr> <td> Glutathione (GSH) depletion   </td> <td> (Biesemann, N. et al., 2018)   </td> <td> 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., <a href="http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html" rel="noreferrer noopener" target="_blank">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</a>).    </td> <td colspan="2"> No  </td> </tr> <tr> <td> Thiobarbituric acid reactive substances (TBARS)   </td> <td> (Griendling, K. K., et al., 2016)  </td> <td> Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit.    </td> <td colspan="2"> No  </td> </tr> <tr> <td> Protein oxidation (carbonylation)  </td> <td> (Azimzadeh et al., 2017; Azimzadeh et al., 2015; Ping et al., 2020)  </td> <td> Can be determined with ELISA or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress.  </td> <td colspan="2"> No  </td> </tr> <tr> <td> Seahorse XFp Analyzer  </td> <td> Leung et al. 2018  </td> <td> 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).  </td> <td> No  </td> </tr> </tbody> </table>   Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include:   <table border="1"> <tbody> <tr> <td> Method of Measurement   </td> <td> References   </td> <td> Description   </td> <td> OECD-Approved Assay  </td> </tr> <tr> <td> Immunohistochemistry   </td> <td> (Amsen, D., de Visser, K. E., and Town, T., 2009)  </td> <td> Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus    </td> <td> No  </td> </tr> <tr> <td> qPCR   </td> <td> (Forlenza et al., 2012)  </td> <td> 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)   </td> <td> No  </td> </tr> <tr> <td> Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis  </td> <td> (Jackson, A. F. et al., 2014)  </td> <td> 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  </td> <td> No  </td> </tr> </tbody> </table>

Taxonomic applicability: Occurrence of oxidative stress is not species specific.   Life stage applicability: Occurrence of oxidative stress is not life stage specific.  Sex applicability: Occurrence of oxidative stress is not sex specific.  Evidence for perturbation by prototypic stressor: 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).

High Mixed

High

All life stages

High High

Ahmad, S. et al. (2021), “60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression”, Journal Insect Science, Vol. 21/5, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jisesa/ieab080" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jisesa/ieab080</a>  Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, Antioxidants & Redox Signaling, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3400" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2010.3400</a>  Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, <a href="https://doi.org/10.1007/978-1-59745-447-6_5" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/978-1-59745-447-6_5</a>   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, <a href="https://doi.org/10.1021/pr501141b" rel="noreferrer noopener" target="_blank">https://doi.org/10.1021/pr501141b</a>  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, <a href="https://doi.org/10.1080/09553002.2017.1339332" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2017.1339332</a>  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  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>  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.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a>    Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”, Scientific Reports, Vol. 8/1, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41598-018-27614-8" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41598-018-27614-8</a>.   Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in The Pathophysiologic Basis of Nuclear Medicine, Springer, New York, pp. 540-548  Eruslanov, E., & Kusmartsev, S. (2010). Identification of ROS using oxidized DCFDA and flow-cytometry. Methods in molecular biology ,N.J.,  Vol. 594,  https://doi.org/10.1007/978-1-60761-411-1_4  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.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a>   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   Forrester, S.J. et al. (2018), “Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology”, Physiological Reviews, Vol. 98/3, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00038.201" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/physrev.00038.201</a>  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  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.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/02713680500477347.</a>   Griendling, K. K. et al. (2016), “Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association”, Circulation research, Vol. 119/5, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/RES.0000000000000110" rel="noreferrer noopener" target="_blank">https://doi.org/10.1161/RES.0000000000000110</a>   Guo, C. et al. (2013), “Oxidative stress, mitochondrial damage and neurodegenerative diseases”, Neural regeneration research, Vol. 8/21, Publishing House of Neural Regeneration Research, China, <a href="https://doi.org/10.3969/j.issn.1673-5374.2013.21.009" rel="noreferrer noopener" target="_blank">https://doi.org/10.3969/j.issn.1673-5374.2013.21.009</a>  Hargreaves, M., and L. L. Spriet (2020), “Skeletal muscle energy metabolism during exercise.”, Nature Metabolism, Vol. 2, Nature Portfolio, London, <a href="https://doi.org/10.1038/s42255-020-0251-4" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s42255-020-0251-4</a>  Hladik, D. and S. Tapio (2016), “Effects of ionizing radiation on the mammalian brain”, Mutation Research/Reviews in Mutation Research, Vol. 770, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.mrrev.2016.08.003" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.mrrev.2016.08.003</a>  Itoh, K., J. Mimura and M. Yamamoto (2010), “Discovery of the negative regulator of Nrf2, Keap1: a historical overview”, Antioxidants & Redox Signaling, Vol. 13/11, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3222" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2010.3222</a>   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.”, Toxicology and Applied Pharmacology, Vol. 274/11, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.taap.2013.10.019" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.taap.2013.10.019</a>  Jacobsen, N.R. et al. (2008), “Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C60 fullerenes in the FE1-MutaTM Mouse lung epithelial cells”, Environmental and Molecular Mutagenesis, Vol. 49/6, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1002/em.20406" rel="noreferrer noopener" target="_blank">https://doi.org/10.1002/em.20406</a>  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, <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>  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  Lu, C., G. Song, and J. Lin (2006), “Reactive oxygen species and their chemiluminescence-detection methods”, TrAC Trends in Analytical Chemistry, Vol. 25/10, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.trac.2006.07.007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.trac.2006.07.007</a>  Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH oxidase, and redox signaling in the vasculature”, Antioxidants & redox signaling, Vol. 19/10, Mary Ann Liebert, Larchmont, <a href="https://doi.org/10.1089/ars.2012.4641" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2012.4641</a>  Ping, Z. et al. (2020), “Oxidative Stress in Radiation-Induced Cardiotoxicity”, Oxidative Medicine and Cellular Longevity, Vol. 2020, Hindawi, <a href="https://doi.org/10.1155/2020/3579143" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2020/3579143</a>  Powers, S.K. and M.J. Jackson. (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, Physiological Reviews, Vol. 88/4, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00031.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/physrev.00031.2007</a>  Raimondi, V., F. Ciccarese and V. Ciminale. (2020), “Oncogenic pathways and the electron transport chain: a dangeROS liason”, British Journal of Cancer, Vol. 122/2, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41416-019-0651-y" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41416-019-0651-y</a>  Seen, S. and L. Tong. (2018), “Dry eye disease and oxidative stress”, Acta Ophthalmologica, Vol. 96/4, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1111/aos.13526" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/aos.13526</a>  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>     Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, Life, Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/life11111269" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/life11111269</a>  Wang, H. et al. (2019), “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>   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”, Journal of applied physiology, 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>.  Zhao, R. Z. et al. (2019), “Mitochondrial electron transport chain, ROS generation and uncoupling”, International journal of molecular medicine, Vol. 44/1, Spandidos Publishing Ltd., Athens, <a href="https://doi.org/10.3892/ijmm.2019.4188" rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/ijmm.2019.4188</a>  AOPWiki 2017-05-30T13:58:17

2026-02-11T07:05:27

Increase, Endothelial Dysfunction

Tissue

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.

Endothelial cell senescence  <table border="1"> <tbody> <tr> <td> Assay  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> Senescence-associated beta-galactosidase staining (SA-beta-gal)  </td> <td> (Farhat et al., 2008; González-Gualda et al., 2021; Hooten et al., 2017)  </td> <td> Can be used to measure senescence-associated β-galactosidase activity, a marker for senescent cells.  </td> <td> No  </td> </tr> <tr> <td> Bromodeoxyuridine (BrdU) detected with staining incorporation  </td> <td> (González-Gualda et al., 2021)  </td> <td> Reduced BrdU incorporation can indicate a lack of DNA synthesis.  </td> <td> No  </td> </tr> <tr> <td> Immunohistochemistry to detect senescence markers.  </td> <td> (González-Gualda et al., 2021)  </td> <td> Markers include Ki67 and Lamin B1. Reduced Ki67 can indicate reduced proliferation. Reduced Lamin B1 indicates impaired structural integrity of the nucleus.  </td> <td> No  </td> </tr> <tr> <td> Cell morphology and size measured with light microscopy or flow cytometry.  </td> <td> (González-Gualda et al., 2021)  </td> <td> Senescent cells exhibit an enlarged and flattened morphology.  </td> <td> No  </td> </tr> </tbody> </table>   Cell death:  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.    Impaired vasomotion <table border="1"> <tbody> <tr> <td> Assay  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> Concentration-response curves to vasodilators/vasoconstrictors  </td> <td> (Deanfield et al., 2005; Verma et al., 2003)  </td> <td> 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.   </td> <td> No  </td> </tr> <tr> <td> Detection of contractile factors (eg. endothelin) using enzyme-linked immunosorbent assay (ELISA).  </td> <td> (Abdel-Sayed et al., 2003)  </td> <td> Endothelin is an endothelium-derived vasoconstrictor.  </td> <td> No  </td> </tr> </tbody> </table> Endothelial barrier  <table border="1"> <tbody> <tr> <td> Assay  </td> <td> Reference  </td> <td> Description  </td> <td> OECD Approved Assay  </td> </tr> <tr> <td> Permeability assays  </td> <td> (Kabacik & Raj, 2017)  </td> <td> Measurement of endothelial permeability using fluorescent dyes or stains to detect the various sized macromolecules that cross the barrier.  </td> <td> No  </td> </tr> <tr> <td> Electric Cell Substrate Impedance Sensing (ECIS)  </td> <td> (Young, 2012; Young & Smilenov, 2011)  </td> <td> Measurement of endothelial barrier changes and monolayer resistance using a range of frequencies.  </td> <td> No  </td> </tr> </tbody> </table>

Taxonomic applicability: Endothelial dysfunction is applicable to vertebrates as only vertebrates have a true endothelial lining (Yano et al., 2007). Life stage applicability: 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). Sex applicability: This key event is not sex specific (Hughson et al., 2018; Lee et al., 2020). Evidence for perturbation by a stressor: 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).

UBERON:0001986

UBERON endothelium

Moderate Unspecific

Moderate

All life stages

Moderate Moderate Moderate

Abdel-Sayed, S. et al. (2003), “Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects”, American Journal of Hypertension, Vol. 16/7, Oxford University Press, Oxford, https://doi.org/10.1016/S0895-7061(03)00903-8  Chang, P. Y. et al. (2017), “MSC-derived cytokines repair radiation-induced intra-villi microvascular injury”, Oncotarget, Vol. 8/50, Impact Journals, Orchard Park, <a href="https://doi.org/10.18632/oncotarget.21236" rel="noreferrer noopener" target="_blank">https://doi.org/10.18632/oncotarget.21236</a>  Cheng, Y. P. et al. (2017), “Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats”, Pflugers Archiv European Journal of Physiology, Vol. 469, Springer, New York, https://doi.org/10.1007/s00424-017-1969-z  Deanfield, J. et al. (2005), “Endothelial function and dysfunction”, Journal of hypertension, Vol. 23/1, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1097/00004872-200501000-00004" rel="noreferrer noopener" target="_blank">https://doi.org/10.1097/00004872-200501000-00004</a>  Farhat, N. et al. (2008), “Stress-induced senescence predominates in endothelial cells isolated from atherosclerotic chronic smokers”, Canadian Journal of Physiology and Pharmacology, Vol. 86/11, Canadian Science Publishing, Ottawa, <a href="https://doi.org/10.1139/Y08-082" rel="noreferrer noopener" target="_blank">https://doi.org/10.1139/Y08-082</a>  González-Gualda, E. et al. (2021), “A guide to assessing cellular senescence in vitro and in vivo”, The FEBS Journal, Vol. 288, FEBS press, https://doi.org/10.1111/febs.15570  Herrera, M. D. et al. (2010), “Endothelial dysfunction and aging: An update”, Ageing Research Reviews, 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>  Hooten, N. N. and M. K. Evans (2017), “Techniques to Induce and Quantify Cellular Senescence”, Journal of Visualized Experiments: JoVE, Vol. 123, MyJove Corporation, Cambridge,  <a href="https://doi.org/10.3791/55533" rel="noreferrer noopener" target="_blank">https://doi.org/10.3791/55533</a>  Hughson, R. L., A. Helm and M. Durante (2018), “Heart in space: effect of the extraterrestrial environment on the cardiovascular system”, Nature Reviews Cardiology, Vol. 15/3, Nature Portfolio, London, <a href="https://doi.org/10.1038/nrcardio.2017.157" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/nrcardio.2017.157</a>  Kabacik, S. and K. Raj (2017), “Ionising radiation increases permeability of endothelium through ADAM10-mediated cleavage of VE-cadherin. Oncotarget, Vol. 8/47, Impact Journals, New York, https://doi.org/10.18632/oncotarget.18282       Konukoglu, D., and H. Uzun (2017), “Endothelial Dysfunction and Hypertension”, in Hypertension: from basic research to clinical practice, Springer, London, <a href="https://doi.org/10.1007/5584_2016_90" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/5584_2016_90</a>  Korpela, E., and S. K. Liu (2014), “Endothelial perturbations and therapeutic strategies in normal tissue radiation damage”, Radiation Oncology, Vol. 9, BioMed Central, London, <a href="https://doi.org/10.1186/s13014-014-0266-7" rel="noreferrer noopener" target="_blank">https://doi.org/10.1186/s13014-014-0266-7</a>  Lee, S. et al.  (2020), “Arterial structure and function during and after long-duration spaceflight”, Journal of Applied Physiology, Vol. 129/1, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.00550.2019" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/japplphysiol.00550.2019</a>  Ricard, N. et al. (2021), “The quiescent endothelium: signalling pathways regulating organ-specific endothelial normalcy”, Nature reviews cardiology, Vol. 18/8, Springer Nature, https://doi.org/10.1038/s41569-021-00517-4  Sitia, S. et al. (2010), “From endothelial dysfunction to atherosclerosis”, Autoimmunity Reviews, Vol. 9/12, Elsevier, Amsterdam, https://doi.org/10.1016/j.autrev.2010.07.016.  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" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RR2598.1</a>  Su, Y. T. et al. (2020), “Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats”, Life Sciences, Vol. 243, Elsevier, Amsterdam, https://doi.org/10.1016/j.lfs.2019.117253  Verma, S., M. R. Buchanan and T. J. Anderson (2003), “Endothelial function testing as a biomarker of vascular disease”, Circulation, Vol. 108/17, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/01.CIR.0000089191.72957.ED" rel="noreferrer noopener" target="_blank">https://doi.org/10.1161/01.CIR.0000089191.72957.ED</a>  Yano, K. et al. (2007), “Phenotypic heterogeneity is an evolutionarily conserved feature of the endothelium”, Blood, Vol. 109/2, American Society of Hematology, Washington, D.C., <a href="https://doi.org/10.1182/blood-2006-05-026401" rel="noreferrer noopener" target="_blank">https://doi.org/10.1182/blood-2006-05-026401</a>  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”, PloS one, Vol. 8/8, PLOS, San Francisco, <a href="https://doi.org/10.1371/journal.pone.0070024" rel="noreferrer noopener" target="_blank">https://doi.org/10.1371/journal.pone.0070024</a>  Young, E. F. (2012), “Transient impedance changes in venous endothelial monolayers as a biological radiation dosimetry response”, Journal of Electrical Bioimpedance, Vol. 3/1, Sciendo, Warsaw, <a href="https://doi.org/10.1667/rr2665.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/rr2665.1</a>    Young, E. F. and L. B. Smilenov (2011), “Impedance-based surveillance of transient permeability changes in coronary endothelial monolayers after exposure to ionizing radiation”, Radiation research, Vol. 176/4, BioOne, Washington, <a href="https://doi.org/10.1667/rr2665.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/rr2665.1</a>  AOPWiki 2022-09-28T12:39:53

2024-08-26T10:35:52

Infiltration, Inflammatory cells

Tissue

TNF-induced cytokines and chemokines, such as IL-6, IL-8, GMCSF, CXCL1, and RANTES, can instigate and amplify immune responses through triggering the production of acute phase proteins and the recruitment of neutrophils, macrophages, and basophils to the site of inflammation, and by triggering increased production of monocytes/macrophages from bone marrow<a href="#cite_note-Cullen2013-1">[1]</a>. Monocytes are the precursors of macrophages and dendritic cells and circulate in the blood for 1-3 days. Upon secretion of chemokines such as CCL2 which is also referred to as monocyte chemoattractant protein 1 (MCP1), they can migrate towards affected tissue. This was nicely demonstrated when depletion of MCP-1 in supernatants of Fas-stimulated cells was sufficient to block almost all THP-1 monocyte chemotaxis. Using an in vivo mouse model, the authors found that Fas stimulation could trigger phagocyte migration by administration of anti-Fas (Jo2) antibody into C57BL/6 mice within 10 h of anti-Fas administration. This correlated with extensive cell death in the thymus and a dramatic increase of CD11b-positive macrophages in the same tissue<a href="#cite_note-Cullen2013-1">[1]</a>. Neutrophils, on the other hand, account for about 50 70 % of all blood leukocytes in the human body <a href="#cite_note-Freitas2009-2">[2]</a><a href="#cite_note-Wessels2010-3">[3]</a>. Upon an inflammatory event, neutrophil production is upregulated, and its lifetime increases as a response to platelet activating factor (PAF), granulocyte-colony stimulating factor (G-CSF) or various pro-inflammatory cytokines, such as interleukin 1ß (IL-1ß) <a href="#cite_note-Wessels2010-3">[3]</a>. The crucial role of PMN in the human immune system is long known. In 1968, Baehner and Karnovsky described a link between a reduced PMN activity and the development of chronic granulomatous disease (CGD) <a href="#cite_note-4">[4]</a>. The important peroxidase-mediated bactericidal role of PMN and the formation of superoxide radicals as one of the main bactericial mechanisms was already described more than 30 years ago <a href="#cite_note-5">[5]</a><a href="#cite_note-6">[6]</a>. A strong negative correlation between the chemotactic ability of PMN and patients with increased bacterial sepsis was demonstrated <a href="#cite_note-7">[7]</a>, and clinical morbidity from infections is clearly increased with a reduced number of circulating PMN in the blood <a href="#cite_note-Nauseef2007-8">[8]</a>. The neutrophilic cytosol contains granules that are filled with a variety of proteins, such as defensins, bactericidal-permeability-increasing protein, proteases (e.g. elastase, cathepsins), and myeloperoxidase (MPO) that consumes hydrogen peroxide (H2O2) and generates hypochlorous acid (HOCl), the most bactericidal oxidant that is produced by PMN <a href="#cite_note-Nauseef2007-8">[8]</a><a href="#cite_note-Freitas2009-2">[2]</a>. Activated neutrophils are capable of producing a variety of pro-inflammatory cytokines, e.g. IL-1ß, IL-6, IL-12 and IL-23, and transport internalised pathogens to lymph nodes to support macrophages and dendritic cells in antigen presentation<a href="#cite_note-9">[9]</a>. Also, contact with pathogens results not only in phagocytosis, but also in the so-called oxidative burst, marked by an increased consumption of molecular oxygen and resulting production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) <a href="#cite_note-10">[10]</a>. Deregulation of this response by constant stimulation of PMNs, as could be shown for nanoparticles for example, ultimately leads to the establishment of a (chronic) inflammation. Here, also macrophages play a vital role. Resident alveolar macrophages, such as Kupffer cells in the liver, that usually phagocyte microorgansims or particles will be activated when overwhelmed by the amount of invading pathogens and in turn release inflammatory cytokines and chemokines. Consequently, neutrophils are recruited and activated as described above <a href="#cite_note-11">[11]</a><a href="#cite_note-12">[12]</a>.

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? Chemotaxis assays can be performed in vitro/ex vivo by using Chemotaxis Chambers (for example Neuro Probe Chambers). Supernatants can be added to the bottom well of the chamber and 3–8 mm nitrocellulose filters are placed on top, while the top chamber contains the inflammatory cells (for example neutrophils). After a certain time period, the number of migrated cells towards the lower chamber can be determined by staining of the cells<a href="#cite_note-Cullen2013-1">[1]</a>. Influx of inflammatory cells (mainly neutrophils) can be analysed by tissue staining by using Haematoxylin and eosin <a href="#cite_note-Huebsch2006-13">[13]</a>. In mice, neutrophil influx can be analysed using a mouse MPO ELISA kit for lysed tissue <a href="#cite_note-Kermanizadeh2012-14">[14]</a>.

<a href="#cite_note-Cullen2013-1">[1]</a>: human (cells); <a href="#cite_note-Huebsch2006-13">[13]</a>: human (tissue; representative for general application in patients, as liver inflammation is commonly found in patients with DILI) <<a href="#cite_note-Cui2011-15">[15]</a><a href="#cite_note-Kermanizadeh2012-14">[14]</a><a href="#cite_note-Ma2009-16">[16]</a>: mouse (nanomaterial-induced)

UBERON:0002107

UBERON liver

High High

<ol class="references"> <li id="cite_note-Cullen2013-1">↑ <a href="#cite_ref-Cullen2013_1-0">1.0</a> <a href="#cite_ref-Cullen2013_1-1">1.1</a> <a href="#cite_ref-Cullen2013_1-2">1.2</a> <a href="#cite_ref-Cullen2013_1-3">1.3</a> Cullen SP, Henry CM, Kearney CJ, Logue SE, Feoktistova M, Tynan GA, Lavelle EC, Leverkus M, Martin SJ. Fas/CD95-induced chemokines can serve as "find-me" signals for apoptotic cells. Mol Cell. 2013 Mar 28;49(6):1034-48 </li> <li id="cite_note-Freitas2009-2">↑ <a href="#cite_ref-Freitas2009_2-0">2.0</a> <a href="#cite_ref-Freitas2009_2-1">2.1</a> Freitas M, Lima JL, Fernandes E. Optical probes for detection and quantification of neutrophils' oxidative burst. A review. Anal Chim Acta 2009;649(1):8-23 </li> <li id="cite_note-Wessels2010-3">↑ <a href="#cite_ref-Wessels2010_3-0">3.0</a> <a href="#cite_ref-Wessels2010_3-1">3.1</a> Wessels I, Jansen J, Rink L, Uciechowski P. Immunosenescence of polymorphonuclear neutrophils. ScientificWorldJournal 2010;10:145-60 </li> <li id="cite_note-4"><a href="#cite_ref-4">↑</a> Baehner RL, Karnovsky ML. Deficiency of reduced nicotinamide-adenine dinucleotide oxidase in chronic granulomatous disease. Science 1968;162(859):1277-9 </li> <li id="cite_note-5"><a href="#cite_ref-5">↑</a> Klebanoff SJ. Iodination of bacteria: a bactericidal mechanism. J Exp Med 1967;126(6):1063-78 </li> <li id="cite_note-6"><a href="#cite_ref-6">↑</a> Klebanoff SJ, Rosen H. The role of myeloperoxidase in the microbicidal activity of polymorphonuclear leukocytes. Ciba Found Symp 1978;(65):263-84 </li> <li id="cite_note-7"><a href="#cite_ref-7">↑</a> Christou NV, Meakins JL. Neutrophil function in surgical patients: Two inhibitors of granulocyte chemotaxis associated with sepsis. J Surg Res 1979;26(4):355-364 </li> <li id="cite_note-Nauseef2007-8">↑ <a href="#cite_ref-Nauseef2007_8-0">8.0</a> <a href="#cite_ref-Nauseef2007_8-1">8.1</a> Nauseef WM. How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 2007;219:88-102 </li> <li id="cite_note-9"><a href="#cite_ref-9">↑</a> Silva MT. Neutrophils and macrophages work in concert as inducers and effectors of adaptive immunity against extracellular and intracellular microbial pathogens. J Leukoc Biol 2010;87(5):805-13 </li> <li id="cite_note-10"><a href="#cite_ref-10">↑</a> Babior BM. Phagocytes and oxidative stress. Am J Med 2000;109(1):33-44 </li> <li id="cite_note-11"><a href="#cite_ref-11">↑</a> Driscoll KE, Deyo LC, Carter JM, Howard BW, Hassenbein DG, Bertram TA. Effects of particle exposure and particle-elicited inflammatory cells on mutation in rat alveolar epithelial cells. Carcinogenesis 1997;18(2):423-30 </li> <li id="cite_note-12"><a href="#cite_ref-12">↑</a> Knaapen AM, Seiler F, Schilderman PA, Nehls P, Bruch J, Schins RP, Borm PJ. Neutrophils cause oxidative DNA damage in alveolar epithelial cells. Free Radic Biol Med 1999;27(1-2):234-40 </li> <li id="cite_note-Huebsch2006-13">↑ <a href="#cite_ref-Huebsch2006_13-0">13.0</a> <a href="#cite_ref-Huebsch2006_13-1">13.1</a> Huebscher SG. Histological assessment of non-alcoholic fatty liver disease. Histopathol. 2006;49:450–465 </li> <li id="cite_note-Kermanizadeh2012-14">↑ <a href="#cite_ref-Kermanizadeh2012_14-0">14.0</a> <a href="#cite_ref-Kermanizadeh2012_14-1">14.1</a> Kermanizadeh A, Brown DM, Hutchison GR, Stone V. Engineered Nanomaterial Impact in the Liver following Exposure via an Intravenous Route–The Role of Polymorphonuclear Leukocytes and Gene Expression in the Organ. Journal of Nanomed & Nanotechnol 2012;04(01):1–7 </li> <li id="cite_note-Cui2011-15"><a href="#cite_ref-Cui2011_15-0">↑</a> Cui Y, Liu H, Zhou M, Duan Y, Li N, Gong X, Hu R, Hong M, Hong F. Signaling pathway of inflammatory responses in the mouse liver caused by TiO2 nanoparticles. 2011; J. Biomed. Mater. Res. - Part A 96 A:221–229 </li> <li id="cite_note-Ma2009-16"><a href="#cite_ref-Ma2009_16-0">↑</a> Ma L, Zhao J, Wang J, Liu J, Duan Y, Liu H, Li N, Yan J, Ruan J, Wang H, Hong F. The Acute Liver Injury in Mice Caused by Nano-Anatase TiO2. Nanoscale Res Lett. 2009 Aug 1;4(11):1275-85 </li> </ol> AOPWiki 2016-11-29T18:41:27

2017-09-16T10:16:41

Activation, Macrophages

Cellular

CL:0000235

CL macrophage

AOPWiki 2016-11-29T18:41:30

2017-09-16T10:17:41

Increased, LDL uptake

Cellular

CL:0000182

CL hepatocyte

AOPWiki 2016-11-29T18:41:24

2017-09-16T10:15:14

Foam cell formation

Organ

AOPWiki 2017-06-29T02:32:56

2017-06-29T02:32:56

<upstream-id>2dbc8a02-26a1-4eec-aa1a-e4767a00d18c</upstream-id> <downstream-id>94605568-ea20-431e-beab-14b68f10d337</downstream-id> Induction of oxidative stress occurs as a result of an imbalance between the production of radical species and the antioxidant defense systems (Juan et al. 2021).  ROS can damage DNA, lipids, and proteins (Shields et al. 2021).  Superoxide dismutase is an enzyme in a common cellular defense pathway, in which superoxide dismutase converts superoxide radicals to hydrogen peroxide.  When cellular defense mechanisms are unable to mitigate ROS formation from mitochondrial respiration and stressors (biological, chemical, radiation), increased ROS levels cause oxidative stress.

This KER was identified as part of an Environmental Protection Agency effort to increase the impact of AOPs published in the peer-reviewed literature, but heretofore unrepresented in the AOP-Wiki, by facilitating their entry and update.  The originating works for this AOP were da Silva, J., Goncalves, R. V., de Melo, F. C. S. A., Sarandy, M. M., & da Matta, S. L. P. (2021). Cadmium exposure and testis susceptibility: A systematic review in murine models. Biological Trace Element Research, 199(7), 2663-2676 and Jeong and Choi (2020). This publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages. Evidence from the da Silva (2021) publication was assembled using Medline/PubMed and Scopus in September 2018.  For all databases, the search filters were based on three complementary levels: (i) animals, (ii) testis, and (iii) cadmium and studies that didn't evaluate the Cd exposure in the testicular histomorphology of murine models were excluded.

The biological plausibility linking increases in oxidative stress to reactive oxygen species (ROS) is strong.  Reactive oxygen species (ROS) are produced by many normal cellular processes (ex. cellular respiration, mitochondrial electron transport, specialized enzyme reactions) and occur in multiple chemical forms (ex. superoxide anion, hydroxyl radical, hydrogen peroxide).  Antioxidant enzymes play a major role in reducing reactive oxygen species (ROS) levels in cells (Ray et al. 2012) to prevent cellular damage to lipids, proteins, and DNA (Juan et al. 2021).  Oxidative stress occurs when antioxidant enzymes do not prevent ROS levels from increasing in cells, often induced by environmental stressors (biological, chemical, radiation).

<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="background-color:#d0cece; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:138px"> Taxa </td> <td style="background-color:#d0cece; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:486px"> Support </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:138px"> Mammals </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:486px"> Deng et al. 2017; Schrinzi et al. 2017 </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:138px"> Fish </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:486px"> Lu et al. 2016; Alomar et al. 2017; Chen et al. 2017; Veneman et al. 2017; Barboza et al. 2018; Choi et al. 2018; Espinosa et al. 2018 </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:138px"> Invertebrates </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:486px"> Browne et al. 2013; Jeong et al. 2016, 2017; Paul-Pont et al. 2016; Lei et al. 2018; Yu et al. 2018 </td> </tr> </tbody> </table> The accumulation of reactive oxygen species (ROS), and resulting oxidative stress, is well-established (see Shields 2021 for overview).   In the studies listed in the above table, changes in enzyme activity and changes in gene expression are the most common oxidative stress effects detected due to increases in reactive oxygen species (see additional study details in table below).  Increases in gene expression or enzyme activity of superoxide dismutase, catalase, glutathione peroxidase, and other antioxidants are frequently used as indicators of oxidative stress. <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:114px"> Species </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:72px"> Duration </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:120px"> Dose </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:48px"> Increased ROS? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:66px"> Increased Oxidative Stress? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:116px"> Summary </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:87px"> Citation </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:114px"> Lab mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 28 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Diet exposure of 0.01, 0.1, 0.5 mg/day of 5 and 20 um polystyrene microplastic particles. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Assumed1 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Five-week old male mice showed changes in enzyme levels responsible for eliminating ROS.  Decreased catalase at 0.1/0.5 mg/day, increased glutathione peroxidase at all doses, increased superoxide dismutase at all doses. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Deng et al. (2017) </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:114px"> Human (Homo sapiens) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 48 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> In vitro exposure of 0.5, 1, 5, 10 mg/L fullerene soot, fullerol, graphene, cerium oxide, zirconium oxide, titanium oxide, aluminum oxide, silver nanoparticles, gold particles; in vitro exposure of 0.05, 0.1, 1, 10 mg/L polyethylene microspheres, polystyrene microspheres. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Cerebral and epithelial human cell lines showed measured increased percent effect of ROS (as superoxide generated) with corresponding decreases in cell viability. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Schirinzi et al. (2017) </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:114px"> Zebrafish (Danio rerio) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 7 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Aquatic exposure of 20, 200, 2000 ug/L of 5 and 20 um polystyrene microplastics. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Assumed1 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Adult five-month old fish showed changes in enzyme levels responsible for eliminating ROS.  Increased catalase at 200/2000 ug/L, increased superoxide dismutase at all doses. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Lu et al. (2016) </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:114px"> Striped red mullet (Mullus surmuletus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> NA </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Survey of wild fish with microplastic ingestion versus no microplastic ingestion. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Assumed1 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Fish showed changes in enzyme levels responsible for eliminating ROS associated with microplastic ingestion, and associated proteins.  Increased glutathione S-transferase, superoxide dismutase, catalase, malondialdehyde, only glutathione S-transferase was statistically significant </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Alomar et al. (2017) </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:114px"> Zebrafish (Danio rerio) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 72 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Aquatic exposure of 1 mg/L polystyrene microplastics (45 um) and nanoplastics (50 nm), aquatic exposure of 2, 20 ug/L positive control 17alpha-Ethinylestradiol, and mixture. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Assumed1 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Larval fish showed changes in enzyme levels responsible for eliminating ROS.  Increased catalase, increased glutathione peroxidase, increased glutathione S-transferase. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Chen et al. (2017) </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:114px"> Zebrafish (Danio rerio) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 3 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Injection exposure of 5 mg/mL of 700 nm polystyrene particles </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Assumed1 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Larva fish showed increased oxidative stress from gene ontology analysis. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Veneman et al. (2017) </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:114px"> European Seabass (Dicentrarchus labrax) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 96 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Aquatic exposure of 0.010, 0.016 mg/L of Mercury chloride, 0.26, 0.69 mg/L of 1-5 um polymer microspheres, and mixture. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Juvenile fish showed increased ROS (Brain and muscle lipid peroxidation levels) and corresponding changes in enzyme levels (increases in muscle lactate dehydrogenase, decreases in isocitrate dehydrogenase). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Barboza et al. (2018) </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:114px"> Sheepshead minnow (Cyprinodon variegatus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 4 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Aquatic exposure of 50, 250 mg/L of 150-180 um, 300-355 um polyethylene microspheres </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Adult fish showed increased ROS generation and corresponding changes in gene expression (increased catalase, increased superoxide dismutase). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Choi et al. (2018) </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:114px"> European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 24 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> In vitro exposure of 100 mg/L of polyvinylchloride and polyethylene microplastics </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Assumed1 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Fish head-kidney leucocytes showed increased gene expression of nuclear factor (nrf2), associated with oxidative stress, only statistically significant in S. aurata. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Espinosa et al. (2018) </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:114px"> Lugworms (Arenicola marina) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 10 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Aquatic exposure of nonylphenol (0.69-692.00 ug/g), phenanthrene (0.11-115.32 ug/g), PBDE (9.49-158.11 ug/g), triclosan (57.30-1097.87 ug/g) sorbed onto polyvinyl chloride, sand, or both. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Lugworms showed decreased ability to respond to ROS by ferric reducing antioxidant power (FRAP) assay, statistically significant only with phenanthrene. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Browne et al. (2013) </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:114px"> Rotifer (Brachionus koreanus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 24 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Aquatic exposure of 10 ug/mL of 0.05, 0.5, 6 um diameter polystyrene microbeads. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Rotifers showed increased ROS levels, changes in phosphorylation of MAPK signaling proteins, and  corresponding changes in enzyme and protein levels (decreased glutathione, increased superoxide dismutase, increased glutathione reductase, increased glutathione reductase, glutathione S-transferase). Enzyme statistical significance was seen most frequently with 0.05 diameter size class). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Jeong et al. (2016) </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:114px"> Copepod (Paracyclopina nana) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 24 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Aquatic exposure of 20 ug/mL of 0.05, 0.5, 6 um diameter polystyrene microbeads. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Copepods showed increased ROS for 0.05 um diameter size class only.  Corresponding increases in enzymes were also seen only in 0.05 um diameter size class (glutathione reductase, glutathione peroxidase, glutathione S-transferase, superoxide disumutase). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Jeong et al. (2017) </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:114px"> Mussel (Mytilus sp.) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 7 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Aquatic exposure of 30 ug/L fluoranthene, 32 ug/L of 2 and 6 um polystyrene microbeads, and mixture for 7 days and depuration for 7 days. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Mussels showed increased ROS production in all treatments for 7 days, changes in enzyme and gene levels were observed for catalase, superoxide dismutase, glutathione S-transferase, glutathione reductase, and lipid peroxidation, statistical significance was not always observed. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Paul-Pont et al. (2016) </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:114px"> Nematode (Caenorhabditis elegans) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 2 day </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Environmental exposure of 5.0 mg/mL of microplastic particles (polyamides (PA), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), and 0.1, 1.0, 5.0 um size polystyrene (PS)). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Assumed1 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Larval (L2) nematodes showed increased glutathione S-transferase gene expression for all but polyamide (PA) exposure. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Lei et al. (2018) </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:114px"> Crab (Eriocheir sinensis) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 21 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:120px"> Aquatic exposure of 40, 400, 4000, 40000 ug/L </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:48px"> Assumed1 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:66px"> Yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Juvenile fish showed dose-dependent changes in hepatopancreas enzyme levels (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase), protein levels (glutathione, malondialdehyde) and gene expression (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase), as well as changes in MAPK signaling gene expression.   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:87px"> Yu et al. (2018) </td> </tr> </tbody> </table> 1 Assumed: study selected stressor(s) known to elevate reactive oxygen species (ROS) levels, endpoints verified increased oxidative stress and disrupted pathway.

The reactive oxygen species (ROS) increase needed to elicit oxidative stress is highly dependent on many other variables including age, tissue, sex, nutritional status, and co-exposures to other stressors.  It is consistently characterised as an 'excess' of ROS in order to create a state of oxidative stress.  Consequently, the quantitative relationship is not easily generalized.   Some examples of normal levels have been reported at 1-8 μM (H2O2) in normal human plasma, while only 1 μM ROS present in healthy cells (Lacy et al. 2000).  Inflammatory lung diseases can cause H2O2 excesses to the level of a 20-fold increment.  It can also cause the level of H2O2 in ischemia and reperfusion to reach 160 μM (Burgoyne et al. 2013).

AP-1 and NF-κB are ROS sensing transcription factors and act as redox sensors due to the presence of a single Cys in their DNA-binding domains (Abate et al. 2006). Oxidation of these Cys residues blocks their binding to the respective consensus DNA sequences. Apurinic/apyrimidinic (AP) endonuclease 1 (APE1), functions as a reducing agent for various transcription factors (Evans et al. 2000). This ubiquitous multifunctional protein is induced by ROS (Ramana et al. 1998) and is involved in base excision repair (Demple and Sung 2005). Although reducing condition is favorable for DNA binding, both AP-1 and NF-κB can be activated by oxidative stress via induction of APE1. A Zn-finger DNA-binding protein, early growth response gene-1 (Egr-1), is activated by ROS, and a positive feedback loop between APE1 and Egr-1 regulates their early transcriptional activation after oxidative stress (Pines et al. 2005). Egr-1 also induces SOD1 and thus reduces free radical-induced damage (Minc et al. 1999).

High Unspecific

High

All life stages

High High High High

Life Stage: The life stage applicable to this key event relationship is all life stages.  Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles > embryos) due to accumulation of reactive oxygen species. Sex: This key event relationship applies to both males and females. Taxonomic: This key event relationship appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats), teleost fish, and invertebrates (cladocerans, mussels).

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I., Ale-Agha, N., & Eaton, P. (2013). Hydrogen peroxide sensing and signaling by protein kinases in the cardiovascular system. Antioxidants & redox signaling, 18(9), 1042-1052. Chen, Q., Gundlach, M., Yang, S., Jiang, J., Velki, M., Yin, D., and Hollert, H.  2017 Quantitative investigation of the mechanisms of microplastics and nanoplastics toward larvae locomotor activity.  Science of the Total Environment 584-585: 1022-1031. Choi, J.S., Jung, Y.J., Hong, N.H., Hong, S.H., and Park, J.W. 2018.  Toxicological effects of irregularly shaped and spherical microplastics in a marine teleost, the sheepshead minnow (Cyprinodon variegatus).  Marine Pollution Bulletin 129: 231-240. Demple, B., & Sung, J. S. (2005). Molecular and biological roles of Ape1 protein in mammalian base excision repair. DNA repair, 4(12), 1442-1449. 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2024-08-02T15:40:13

<upstream-id>94605568-ea20-431e-beab-14b68f10d337</upstream-id> <downstream-id>e1af03d8-91e1-4c06-b00c-11951912018b</downstream-id> 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. 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; Bonetti 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). 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-) (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).

The strategy for collating the evidence on radiation stressors to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Overall weight of evidence: Moderate

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).    It is broadly accepted that elevated ROS can indirectly lead to endothelial dysfunction by causing an imbalance of NO, specifically the decrease in NO bioavailability, with biologically plausible mechanisms described. 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 (O2•–) 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, Montagnani & Potenza, 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).    Oxidative stress is also established to affect 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 prostaglandin F2α (PGF2α) inhibition and therefore, vasoconstriction (Li et al., 2002).     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 also stimulate TGF-β, 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; 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).     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 that 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).     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).

Empirical evidence provides a moderate level of support to this KER. Examples of this evidence are summarized here and further in attached tables. The evidence to support the relationship between oxidative stress leading to endothelial dysfunction was gathered from studies using in vitro and in vivo 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), in vivo pig models (Li et al., 2002) and human in vitro cells (Ramadan et al., 2020). Various stressors were applied, including X-rays, hindlimb unloading (HU), heavy ions (56Fe ions), beta-rays (32P), 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 endpoints were measured including: senescence-associated β-galactosidase (SA β-gal), insulin-like growth factor-binding protein-7 (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, 4-hydroxynonenal (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.     Dose Concordance  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 at the dose when superoxide and hydrogen peroxide generation increased, vasodilation in response to acetylcholine (ACh) decreased (Hatoum et al., 2006). Another study using various doses on cerebral microvascular endothelial cells found a significant change in cellular peroxide and mitochondrial oxidative stress following a 4 Gy X-ray dose, concordant with SA β-gal showing the first large increase at 4 Gy (Ungvari et al., 2013).     Ramadan et al. (2020) used X-ray irradiation in multiple types of human endothelial cells to show 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; SA β-gal activity and endothelial apoptosis showed a response of greater magnitude following the 5 Gy dose compared to 0.1 Gy (Ramadan et al., 2020).   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 in aorta 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) showed 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).    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 56Fe 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 56Fe 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 32P radiation in pig coronary arteries.     Time Concordance  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 6 MV 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).    Incidence Concordance   There is moderate support in current literature for an incidence concordance relationship between oxidative stress and endothelial dysfunction. Three out of the 9 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).    Essentiality    Essentiality in the relationship was demonstrated in multiple 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 results 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).     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 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).    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).

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, while the 5 Gy group demonstrated an unexpected decrease before day 14

<div> <table border="1"> <tbody> <tr> <td> Modulating factor  </td> <td> Details  </td> <td> Effects on the KER  </td> <td> References  </td> </tr> <tr> <td> Drug  </td> <td> MnTBAP (a superoxide dismutase mimetic)  </td> <td> Treatment with MnTBAP after irradiation was able to reduce superoxide and peroxide levels and restore vasodilation ability  </td> <td> (Hatoum et al., 2006)  </td> </tr> <tr> <td> Drug  </td> <td> Tempol (a superoxide dismutase mimetic)  </td> <td> Treatment with tempol after irradiation was able to restore vasodilation ability  </td> <td> (Hatoum et al., 2006)  </td> </tr> <tr> <td> Drug  </td> <td> TAT-Gap19 (inhibitor of connexin 43 which is associated with atherogenesis and endothelial stiffness)  </td> <td> Treatment with TAT-Gap19 led to a decrease in ROS and SA β-gal levels after irradiation  </td> <td> (Ramadan et al., 2020)  </td> </tr> <tr> <td> Drug  </td> <td> hBMSCs (protect against vascular damage through antioxidant properties)  </td> <td> Treatment with hBMSCs after irradiation caused increased catalase and HO-1, as well as decreased oxidative damage and apoptosis  </td> <td> (Shen et al., 2018)  </td> </tr> <tr> <td> Drug  </td> <td> Oxp (can inhibit XO, a source of ROS)  </td> <td> Treatment with Oxp showed decreased XO activity and ROS production along with increased vasodilation after irradiation  </td> <td> (Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011)  </td> </tr> </tbody> </table> </div>

The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.

Dose/incidence concordance  <table border="1"> <tbody> <tr> <td> Reference  </td> <td> Experiment Description  </td> <td> Result  </td> </tr> <tr> <td> Soucy et al. 2007   </td> <td> In vivo. Sprague-Dawley rats were whole-body irradiated with 137Cs gamma radiation at 50, 160 and 500 cGy. XO is a primary source of cardiac ROS and was used as a measure of oxidative stress. Vasodilation response to ACh was used to evaluate endothelial function.  </td> <td> At 500 cGy, XO activity was 2-fold elevated compared to control, and there was also an increase in XO quantity. Simultaneously, endothelial dysfunction was observed as a ~30 percentage point decrease in vasodilation response to ACh.  </td> </tr> <tr> <td> Soucy et al. 2010   </td> <td> In vivo. In Sprague-Dawley rats were whole-body irradiated with 137Cs gamma radiation at 5 Gy. ROS were measured using dihydroethidium fluorescence. Aortic relaxation response to ACh was also measured.  </td> <td> After 5 Gy, ROS increased 1.7-fold and relaxation decreased 0.7-fold.  </td> </tr> <tr> <td> Soucy et al. 2011   </td> <td> In vivo. Wistar rats were exposed to 0.5 and 1 Gy doses of 56Fe-ion radiation. ROS production rates were evaluated using dihydroethidium fluorescence. ACh-induced vasodilation responses were measured.  </td> <td> 56Fe-ion irradiation at 1 Gy produced a 1.8-fold increase in ROS levels. At 10-5 M ACh, aorta without irradiation relaxed by 87%, while aorta with 1 Gy irradiation had significantly lower relaxation of 76%.  </td> </tr> <tr> <td> Li et al. 2002  </td> <td> In vivo. Surgically exposed coronary arteries of yucatan pigs were irradiated with 20 Gy 32P β-irradiation. Oxidative stress was evaluated through superoxide production. Endothelial function was evaluated through endothelial-dependent vasomotor response and morphological changes.  </td> <td> Superoxide production increased 3.5-fold between the control and 20 Gy irradiated groups. Vasodilation response to KCl dropped over 50% in the irradiated group. Morphological changes were also observed, with irradiated arteries seeing enlarged endothelial cells, formation of fibrin networks, activated platelets, leukocytes exhibiting membrane protrusions and pseudopodial extensions all indicative of an inflammatory and pro-thrombotic state of endothelial dysfunction.   </td> </tr> <tr> <td> Shen et al. 2018   </td> <td> In vivo. Male mice were irradiated with 18 Gy X-rays, oxidative stress was measured with 4-HNE and 3-NE oxidative damage markers, and endothelial dysfunction was determined through apoptosis.  </td> <td> 4-HNE showed a maximum increase of ~1.8-fold and 3-NT showed a maximum increase of ~2.3-fold. Apoptosis levels peaked at a ~5-fold increase above control levels.  </td> </tr> <tr> <td> Ramadan et al. 2020   </td> <td> In vitro. Telomerase-immortalized human Coronary Artery and Microvascular Endothelial cells (TICAE) and Telomerase Immortalized human Microvascular Endothelial cells (TIME) were exposed to X-rays (0.1 and 5 Gy). ROS production was measured using CM-H2DCFDA combined with Incucyte live cell imaging.     Endothelial dysfunction was evaluated through:    Endothelial apoptosis   <ul> <li> Annexin V and Caspase 3/7 marker levels  </li> </ul> <ul> <li> Dextran fluorescein dye uptake level by necrotic cells    </li> </ul> Cell senescence   <ul> <li> SA β-gal activity  </li> </ul> <ul> <li> IGFBP-7 and GDF-15 senescence marker levels  </li> </ul> Endothelin-1 levels  </td> <td> ROS production was increased in TIME cells after 0.1 and 5 Gy dose.     In both coronary and endothelial cells, apoptosis mainly occurred after 5 Gy radiation. With coronary cells demonstrating an increase in Annexin V and Caspase 3/7 markers and endothelial cells showing elevated Annexin V and membrane leakage.      SA β-gal activity significantly increased for both 0.1 and 5 Gy doses. IGFBP-7 and GDF-15 levels were also elevated in both cell types; GDF-15 increasing at both 0.1 and 5 Gy doses, while IGFBP-7 only showed significant elevation at the 5 Gy dose. With the 0.1 Gy dose, there was a significant increase in SA β-gal activity of ~3-fold, while at 5 Gy the activity increased ~5-fold.     Endothelin-1 was significantly elevated following 5 Gy irradiation in both cell types.   </td> </tr> <tr> <td> Ungvari et al. 2013   </td> <td> In vitro. Cerebral microvascular endothelial cells (CMVECs) from F344×BN rats were harvested and cultured. Following culture, cells were irradiated with 137Cs gamma radiation at doses between 2-8 Gy.    Oxidative stress was evaluated through cellular peroxide and superoxide production.    Endothelial dysfunction was evaluated through cell senescence via SA-β-gal presence, and apoptosis via caspase 3/7 maker and ratio of apoptotic:viable cells.   </td> <td> Oxidative stress increased in a dose-dependent manner following irradiation. Change of ROS became significant after 4 Gy at a ~1.5-fold increase and reached ~3-fold increase at the highest studied dose of 8 Gy. Mitochondrial oxidative stress also became significant after 4 Gy and increased linearly for a peak of a ~1.5-fold increase at 8 Gy.    Endothelial cell senescence and apoptosis were similarly found to increase in a dose dependent manner. With ~30% of cells being SA-β-gal positive after 8 Gy irradiation, signalling premature senescence. Ratio of dead cells peaked at 10% and Caspase 3/7 peaking at a ~5.5-fold change following 18h post irradiation.  </td> </tr> <tr> <td> Hatoum et al. 2006  </td> <td> In vivo. Effect of cumulative radiation doses on rat gut microvessels was studied. Rats were exposed to 1 to 9 fractions of 250 cGy for a total dose of up to 2250 cGy. Following exposure, the animals were euthanized, and submucosal vessels isolated.    Oxidative stress was measured through superoxide and peroxide levels.    Endothelial function was assessed through ACh vasodilation response and NO bioavailability via DAF-FM fluorescence.  </td> <td> After the final cumulative dose of 2250 cGy, superoxide was ~1.6-fold elevated and peroxides were ~1.7-fold elevated compared to non-irradiated controls. ROS levels increased sharply after the second dose, immediately preceding drop in ACh vasodilation response.    Max dilation dropped from 87% to 3% between pre-irradiation and post-final radiation dose. ACh response remained within control levels following fractions 1 and 2, however following fraction 3, response dropped below 30% for all remaining doses. Following all radiation doses, NO bioavailability dropped ~0.8-fold.  </td> </tr> <tr> <td> Delp et al. 2016   </td> <td> In vivo. The effects of HU and 1 Gy dose of 56Fe radiation of the gastrocnemius muscle feed arteries and coronary arteries of C57BL/6 mice was studied.    Xanthine oxidase (XO) levels were used as a measure of ROS production and therefore oxidative stress.     Endothelial function was evaluated through vasodilation response to ACh.   </td> <td> Following 2-week HU, there were no significant changes to XO levels or vasomotor response.    Following total body irradiation with 1 Gy, there was a ~2-fold increase in XO activity in both gastrocnemius muscle feed and coronary arteries. Vasodilation response subsequently decreased ~10 percentage points.     Combined HU and total body irradiation led to a ~2.3-fold increase in XO activity in both artery types and vasodilation response decrease of ~10 percentage points.  </td> </tr> </tbody> </table>

Time concordance  <table border="1"> <tbody> <tr> <td> Reference  </td> <td> Experiment Description  </td> <td> Result  </td> </tr> <tr> <td> Soucy et al. 2007  </td> <td> In vivo. Sprague-Dawley rats were whole-body irradiated with 137Cs gamma radiation at various doses. 2 weeks after irradiation, the animals were euthanized, and aortas were harvested. XO was used as a measure of oxidative stress. Dose-dependent vasodilation response to ACh was used to evaluate endothelial function.  </td> <td> XO activity was elevated 2-fold compared to control, and there was also an increase in XO quantity. Simultaneously, endothelial dysfunction was seen with a ~30% decrease in vasodilation response to ACh.     </td> </tr> <tr> <td> Soucy et al. 2010   </td> <td> In vitro. Sprague-Dawley rats were whole-body irradiated with 137Cs gamma radiation. 2 weeks after receiving radiation dose, the animals were euthanized and aortas were harvested. ROS were measured using dihydroethidium fluorescence. Aortic relaxation response to ACh was also measured.  </td> <td> ROS increased 1.7-fold and relaxation simultaneously decreased 0.7-fold.  </td> </tr> <tr> <td> Soucy et al. 2011   </td> <td> In vivo. Wistar rats were exposed to 56Fe-ion radiation. Rats were euthanized at 4 months post-irradiation and aorta was harvested. ROS production rates evaluated using dihydroethidium along the ACh-induced vasodilation response were measured.  </td> <td> ROS levels increased by 75% 4 months post-irradiation.   ACh vasodilation response decreased by 13%.  </td> </tr> <tr> <td> Shen et al. 2018   </td> <td> In vivo. Male mice were irradiated with 18 Gy X-rays. Immunohistochemical staining assessed oxidative stress using 4-HNE and 3-NE as markers for oxidative damage. Endothelial dysfunction was determined through apoptosis.  </td> <td> Exposure to 18 Gy caused increased 4-HNE and 3-NT levels. 4-HNE showed a maximum ~1.8-fold increase at 14-days post radiation, and 3-NT showed a maximum ~2.3-fold increase 7 days post radiation.   Apoptosis levels peaked at 7 days post-irradiation with a ~5-fold increase above control levels.  </td> </tr> <tr> <td> Ramadan et al. 2020   </td> <td> In vitro. TICAE and TIME cells were exposed to X-rays (0.1 and 5 Gy).     Oxidative stress was evaluated through intracellular ROS production.     Endothelial dysfunction was evaluated through:   Endothelial apoptosis   <ul> <li> Annexin V and Caspase 3/7 marker levels  </li> </ul> <ul> <li> Dextran fluorescein dye uptake level by necrotic cells    </li> </ul> Cell senescence   <ul> <li> SA β-gal activity  </li> </ul> <ul> <li> IGFBP-7 and GDF-15 senescence marker levels  </li> </ul> Endothelin-1 levels  </td> <td> Highest response was observed for both doses at 45 minutes after irradiation followed by a decline at the 2- and 3-hour time points but remaining elevated above non-irradiated control levels. Endothelial cells studied produced more ROS than the coronary cells.     Caspase 3/7 and annexin V increased linearly until 100h.    SA β-gal activity significantly increased at 7 and 9 days. GDF-15 and IGFBP-7 were increased after 7 days.    Endothelin-1 was found to be significantly elevated after 7 days.  </td> </tr> <tr> <td> Ungvari et al. 2013   </td> <td> In vitro. Cerebral microvascular endothelial cells (CMVECs) from F344×BN rats were harvested and cultured. Following culture, cells were irradiated with 137Cs gamma radiation.    Oxidative stress was evaluated through cellular peroxide and superoxide production.    Endothelial dysfunction was evaluated through cell senescence via SA-β-gal presence, and apoptosis via caspase 3/7 maker and ratio of apoptotic:viable cells.   </td> <td> Superoxide and peroxide increased 1 day but not 14 days post-irradiation    ~30% of cells were SA-β-gal positive after 8 Gy irradiation, measured 7 days post-irradiation. 24 h after irradiation, 10% of cells were dead. Caspase 3/7 increased from 2 to 18 h, peaking at a ~5.5-fold change following 18 h post-irradiation but decreased at 24 h.  </td> </tr> <tr> <td> Hatoum et al. 2006  </td> <td> In vivo. Effect of cumulative radiation doses on rat gut microvessels was studied. Rats were exposed to 1 to 9 cGy in 3 fractions per week on alternate days for 3 successive weeks for a total dose of up to 2250 cGy over a total time of 19 days.    Oxidative stress was measured through superoxide and peroxide levels from various fluorescent markers.    Endothelial function was assessed through ACh vasodilation response.  </td> <td> After 19 days, superoxide was ~1.6-fold elevated and peroxides were ~1.7-fold elevated compared to non-irradiated controls. ROS levels increased at day 5, at the same time as a drop in ACh vasodilation response.     Max dilation dropped from 87% to 3% between day 1 and day 19.  </td> </tr> </tbody> </table>

High Male Low Female Low Unspecific

Moderate

Adult

Low

Not Otherwise Specified

Low Moderate High Low

The evidence is derived from rat in vivo and in vitro models. Mice cell-derived studies were also available but less in-vivo 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.

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(2006), “Radiation Induces Endothelial Dysfunction in Murine Intestinal Arterioles via Enhanced Production of Reactive Oxygen Species”, Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 26/2, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/01.ATV.0000198399.40584.8c" rel="noreferrer noopener" target="_blank">https://doi.org/10.1161/01.ATV.0000198399.40584.8c</a>.   Hughson, R.L., A. Helm and M. Durante (2018), “Heart in space: Effect of the extraterrestrial environment on the cardiovascular system”, Nature Reviews Cardiology, Vol. 15/3, Nature Portfolio, London, <a href="https://doi.org/10.1038/nrcardio.2017.157" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/nrcardio.2017.157</a>.   Krüger-Genge, A. et al. (2019), “Vascular Endothelial Cell Biology: An Update”, International Journal of Molecular Sciences, Vol. 20/18, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/IJMS20184411" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/IJMS20184411</a>.   Li, J. et al. (2002), “Endovascular irradiation impairs vascular functional responses in noninjured pig coronary arteries”, Cardiovascular Radiation Medicine, Vol. 3/3–4, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/S1522-1865(03)00096-9" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/S1522-1865(03)00096-9</a>.   Marasciulo, F., M. Montagnani and M. Potenza (2006), “Endothelin-1: The Yin and Yang on Vascular Function”, Current Medicinal Chemistry, Vol. 13/14, Bentham Science Publishers, Sharjah, <a href="https://doi.org/10.2174/092986706777441968" rel="noreferrer noopener" target="_blank">https://doi.org/10.2174/092986706777441968</a>.   Nagane, M. et al. 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(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>.   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>.  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>.   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" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RR2598.1</a>.  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>.  Soucy, K. G. et al. (2007), “Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta”, Radiation and Environmental Biophysics, Vol. 46/2, Springer, New York, <a href="https://doi.org/10.1007/s00411-006-0090-z" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/s00411-006-0090-z</a>.   Sylvester, C. B. et al. (2018), “Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer”, Frontiers in Cardiovascular Medicine, 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>.   Tapio, S. (2016), “Pathology and biology of radiation-induced cardiac disease”, Journal of Radiation Research, Vol. 57/5, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jrr/rrw064" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jrr/rrw064</a>.   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>.   Venkatesulu, B. P. et al. (2018), “Radiation-Induced Endothelial Vascular Injury”, 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>.   Wang, Y., M. Boerma and D. Zhou (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, Radiation research, 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>.  AOPWiki 2022-09-28T12:43:32

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AOPWiki 2024-11-25T19:21:53

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Activation of reactive oxygen species leading the atherosclerosis

Activation of ROS leading the atherosclerosis

Hiromi Ohara

Hiromi Ohara 1, Shigeaki Ito 1 1 Japan Tobacco Inc. 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa, 227-8512, Japan

BY-SA

2.5

The pathogenesis of atherosclerosis is initiated by the production of reactive oxygen species (ROS: MIE), which elicit oxidative stress in the vasculature (KE1). Oxidative stress responses elicit further endothelial dysfunction (KE2). This impairs the endothelium readily causing the penetration of low-density lipoprotein (LDL) into the intima, which is directly oxidized by ROS, forming oxidized LDL (KE5). The impaired endothelium has an increased expression of adhesion molecules, which recruit blood monocytes, which adhere and then infiltrate into the intima region (KE3). The microenvironment of the impaired endothelium induces the differentiation of monocytes into macrophages (KE4). Macrophages in the intima uptake oxidized LDL intracellularly, forming foam cells (KE6) and the accumulation of lipid-rich foam cells and their debris after necrosis form plaques (i.e., lipid core, KE7)). These conditions promote the migration of fibroblasts and trans-differentiation of smooth muscle cells into myofibroblasts, which form a fibrous cap on the apical side of the plaque (KE7). Then, the increased expression of collagenase causes thinning of the fibrous cap by the degradation of collagen in the fibrous cap (KE8). These plaques are unstable and this eventually leads to rapture. The aggregation of platelets occurs around the raptured endothelium leading to the formation of three-dimensional clots (i.e. thrombosis, AO).

adjacent

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High

Adults

Life Stage Applicability Age is a significant independent risk factor for CVD because it is associated with an increased likelihood of developing any number of other additional cardiac risk factors, including obesity and diabetes [1]. The prevalence of most types of CVDs is considerably higher among older adults compared with the general population [2]. An increase in the production of ROS occurs with advancing age [3, 4], and is linked to persistent inflammation and progression to chronic disease status. Therefore, conditions that induce ROS activation and result in thrombosis may be more applicable to adults. Sex Applicability Estrogen was shown to have a cardioprotective role and to be directly associated with a lower overall incidence of CVD in premenopausal women compared with age-matched men [5-7]. The decline of sex hormones has an important role in the development of CVD with advanced age, in men and women [6]. Therefore, ROS activation resulting in thrombosis may be moderate in premenopausal women.

Evidence for the essentiality of Key Events (KEs) has been confirmed mostly by the attenuation of KEs by inhibitory substances, targeted gene silencing. Attenuation of KEs by inhibitory substances and modification of target gene expression (i.e., knockdown, knockout, and overexpression). Rationale for essentiality includes: Reactive oxygen species (ROS) lead to oxidative stress [High] Reactive oxygen species (ROS) are a group of highly reactive molecules derived from O2 metabolism [8]. Members of the ROS family include superoxide (O2-), alkoxyl radical (RO-), peroxyl radical (ROO-), hydroxyl radical (OH-), peroxynitrate (ONOO-), hydrogen peroxide (H2O2), ozone (O3), and hypochlorous acid (HOCl). Although physiological concentrations of ROS are important signaling molecules that maintain vascular homeostasis, excessive ROS production can lead to oxidative stress and the progression of vascular disease. ROS maintain vascular cell homeostasis by regulating the phenotype and fate of multiple cell types, including endothelial cells (ECs), vascular smooth muscle cells (SMCs), outer membrane cells, bone marrow cells, and resident stem/progenitor cells [9, 10]. The administration of the antioxidant N-acetylcysteine, a known inhibitor of oxidative stress, reduced the severity of atherosclerosis [11]. Oxidative stress leads to endothelial dysfunction [High] Endothelial dysfunction is considered an early indicator of atherosclerosis characterized by the overexpression of adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) [12]. Extracellular as well as intracellular ROS functions as signaling molecules that, produced either intracellularly, extracellularly or through ligand-receptor interactions, function as signaling molecules that activate ICAM-1 and regulate immune cells migration through the vascular endothelium to sites of inflammation and injury [13]. Endothelial dysfunction leads to monocyte infiltration [Moderate] Vascular endothelial functions are critical; thus, genetic modification to disrupt their function is lethal. Maintaining nitric oxide (NO) bioavailability is one of the key functions of the vascular endothelium and is achieved by the expression of endothelial NO synthase (eNOS). The overexpression of eNOS in a diet-induced atherosclerosis model resulted in a significant reduction in atherosclerotic lesions [14]. Monocyte infiltration is associated with the induction of ICAM-1 on the endothelial cell surface, which captures circulating blood monocytes. A deficiency of ICAM-1 in apolipoprotein E deficient mice significantly reduced atherosclerotic lesions [15]. Monocyte infiltration leads to macrophage differentiation [Moderate] The monocyte subpopulations, CC chemokine receptor 2 (CCR2)highLy6C+ inflammatory monocytes and CCRlowLy6C− resident monocytes, are generally thought to preferentially differentiate into M1 inflammatory macrophages and M2 anti-inflammatory macrophages, respectively, in early inflammation [16]. Ly6C− monocytes dominate the early phase of myocardial infarction and exhibit phagocytic, proteolytic, and inflammatory functions, as well as digesting damaged tissues. However, Ly6C− monocytes, recruited at a later phase of inflammation, have attenuated inflammatory properties and differentiate toward M2 macrophages and contribute to angiogenesis, genesis of myofibroblasts, and collagen deposition [17]. Oxidative stress leads to LDL oxidation [Moderate] Under oxidative stress, the oxidation of LDL occurs by lipid peroxidation, primarily involving phospholipid molecules. Under pathological conditions, apolipoprotein B-containing lipoproteins in the plasma penetrate through the damaged endothelium into the vascular subendothelial intima where they are oxidized by ROS [18, 19]. Macrophage differentiation leads to foam cell formation [Moderate] Monocytes migrate into the intima guided by chemokines [20] and differentiate into macrophages. These macrophages then take up modified lipoproteins and form foam cells as they accumulate excess lipids [21]. LDL oxidation leads to foam cell formation [Moderate] Oxidized LDL (ox-LDL) is the archetypal source of cholesterol and inducer of foam cell formation [22]. The formation of fatty streaks is a major characteristic of atherosclerosis caused by the conversion of macrophages into foam cells. Foam cell formation is characterized by an accumulation of lipids, predominantly cholesterol esters [22, 23]. Foam cell formation leads to plaque formation [Moderate] Subsequent cell apoptosis and necroptosis, complicated by failed efferocytosis (dead cell removal by phagocytes), lead to the formation of a lipid-rich necrotic core (NC) and production of thrombogenic tissue factors [24]. NC components and inflammatory cells promote the degradation of plaque-stabilizing extracellular fibrous matrix-like collagen and proteoglycans and thinning of the fibrous cap [25]. Hypoxia-inducible factors produced by cells contained in the NC promote pathologic neoangiogenesis, which favors intraplaque hemorrhage and further expansion of the NC. Unresolved inflammation triggers plaque calcification, which reduces further the mechanical stability of the plaque [25]. Plaque formation leads to plaque instability [Moderate] After foam cell formation, the release of substances including matrix metalloproteinases (MMPs) increases monocyte mobilization and promotes the degradation of extracellular matrix proteins including collagen and fibronectin [26]. This process leads to plaque instability and eventually plaque rupture [27, 28]. Plaque instability leads to thrombosis [Moderate] Mature atherosclerotic plaques are composed of a lipid core that is separated from the vessel lumen by a cap composed of fibrillar collagen [29]. Disruption of this cap exposes the plaque’s underlying thrombogenic core to the bloodstream, resulting in thromboembolism. This process of ‘plaque rupture’ is the main cause of acute coronary syndromes [30-33] and ischemic cerebral events [34-36].

Biological plausibility and empirical support for KERs Although ROS are generated under normal daily activity, they can be eliminated by homeostasis. Dysfunctional homeostasis and unhealthy daily habits can promote the persistent generation of ROS, resulting in chronic and high-level oxidative stress, which eventually leads to thrombosis. In general, the biological plausibility of causal linkage from reactive oxygen species as well as oxidative stress through various diseases including thrombosis is well established. Support for the biological plausibility of KERs is summarized in the table below.   <table cellspacing="0" class="Table" style="border-collapse:collapse; border:none; width:100%"> <tbody> <tr> <td colspan="3" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:40px; width:100%"> Support for biological plausibility of KERs </td> </tr> <tr> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> MIE => KE 1 </td> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> ROS generally activates the anti-oxidant system to maintain homeostasis of cellular functions. An imbalance between ROS and the anti-oxidant system leads to cellular oxidative stress, whereby cellular components are oxidized to be malfunctional. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of the MIE => KE1 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:46px; width:28%"> There is a well-established mechanistic understanding between MIE-->KE1. </td> </tr> <tr> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:203px; width:15%"> KE 1 => KE 2 </td> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:203px; width:55%"> Oxidative stress promotes an intracellular oxidative environment, which causes an uncoupling of endothelial nitric oxide synthase (eNOS). NO bioavailability is crucial to maintain vascular tone. Oxidative stress also alters the barrier integrity of endothelial cells and irregular expression of adhesion molecules such as ICAM-1. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:203px; width:28%"> Biological plausibility of KE1 => KE2 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:57px; width:28%"> Oxidative stress is one cause of endothelial dysfunction. Inflammatory responses are also involved in endothelial dysfunction. However, a direct relationship between KE1 and KE2 is consistent with current biological knowledge. </td> </tr> <tr> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:43px; width:15%"> KE 1 => KE 5 </td> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:43px; width:55%"> Oxidative stress leads to the generation of excess ROS, which causes the lipid peroxidation of LDL as well as apoB modification. The final product is oxidized LDL. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:43px; width:28%"> Biological plausibility of KE1 => KE5 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Strong relationship between KE1 => KE5 is well-established and consistent with current biological knowledge. </td> </tr> <tr> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 2 => KE 3 </td> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Endothelial dysfunction enables the easy access of circulating immune cells to the vascular intima, accompanied by the upregulation of adhesion molecules and disruption of the barrier integrity. Circulating monocytes attached to dysfunctional endothelial cells via adhesion molecules then penetrate into the intima. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE2 => KE3 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> The functional relationship between KE 2 and KE 3 is consistent with current biological knowledge. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 3 => KE 4 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Infiltrated monocytes are differentiated into macrophages by autocrine and paracrine mechanisms. Dysfunctional endothelial cells allow various immune cells to penetrate into the intima region. Oxidative stress also contributes to the activation of these immune cells, leading to the secretion of inflammatory cytokines, which promote the differentiation of monocytes into macrophages </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE3 => KE4 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 4 and KE 5=> KE 6 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Activated macrophages express receptors for LDL uptake. Representative receptors are the scavenger receptor and LOX-1. Macrophages uptake lipoproteins, especially oxidized-LDL, which accumulates inside the cells, termed foam cells. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE4 => KE5 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE6 =>KE7 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Accumulated foam cells then die via necrosis. Cell debris and lipids released from the necrotic foam cells form plaques. During plaque formation, smooth muscle cells migrate underneath endothelial cells and express extracellular matrix to form a fibrous cap. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE5 => AO is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE7 =>KE8 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Cell death of the migrated smooth muscle cells causes a thinning of the fibrous cap. Extracellular matrix in the fibrous cap is also degraded by proteinases including matrix metalloproteinases. An unstable fibrous cap causes vulnerable plaques. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE6 => AO is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE8 =>KE9 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Vulnerable plaques finally rupture and components in the plaques are eroded. Platelets accumulate and form a thrombus. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE7 => AO is high. </td> </tr> <tr> <td colspan="3" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:46px; width:100%"> Empirical Support for KERs </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> MIE => KE 1 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Oxidative stress is a condition whereby excess intracellular ROS is not scavenged. The source of ROS varies, for example, chemical substances induce ROS via biological processes including metabolism and mitochondrial activity. ROS inhibitors such as N-acetylcysteine attenuate oxidative stress. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between MIE and KE1 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 1 => KE 2 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Treatment with the causative substances of ROS induction as well as oxidative stress, including hydrogen peroxide, causes endothelial dysfunction. For example, the increased expression of adhesion molecules and monocyte-endothelial adhesion are observed. These phenomena are caused by oxidative stress-induced inflammatory responses, and the scavenging of exogenous ROS with inhibitors such as N-acetylcysteine ameliorates oxidative stress-inducible endothelial dysfunction. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE1 and KE2 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 1 => KE 5 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Circulating native LDL infiltrates into the intima region of the vasculature, where ROS oxidizes LDL to form oxidized-LDL (Ox-LDL). ROS inhibitors (e.g., NAC, resveratrol, ascorbate) attenuate the oxidative modification of LDL. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE1 and KE5 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 2 => KE 3 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Monocyte infiltration is initiated by monocyte-endothelial adhesion via adhesion molecules expressed on the apical surface of endothelial cells. Adherent monocytes then infiltrate into the intima region of the vasculature. Increased expression of adhesion molecules and a leaky endothelial barrier are representative features of endothelial dysfunction. ICAM-1 deficiency in ApoE KO mice resulted in fewer atherosclerotic lesions, possibly because of the reduced recruitment of monocytes into the intima. However, few studies have provided direct evidence for a relationship between endothelial dysfunction and monocyte infiltration. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE2 and KE3 is low. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 3 => KE 4 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Monocytes differentiate into macrophages. Many studies reported that various stimuli such as proinflammatory cytokines promote this differentiation. Atherogenic vasculature produces proinflammatory cytokines; thus, theoretically, the infiltrated monocytes should be differentiated into macrophages in the intima. An increase in macrophage-like cells is observed in atherosclerotic lesions; however, direct evidence demonstrating infiltrated monocyte differentiation is limited. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE3 and KE4 is moderate. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 4 and KE 5=> KE 6 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Cumulative evidence suggests macrophages change their appearance related to the intracellular accumulation of lipids, leading to the formation of foam cells. The uptake of lipids, especially ox-LDL, is crucial for atherosclerotic lesions and is promoted by its receptors LOX-1 and scavenger receptor (CD36). A deficiency in these receptors results in reduced areas of lipid staining in arteries. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE4-KE5 and KE6 is moderate. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE6 =>KE7 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Inhibition of foam cell formation via the attenuation of scavenger receptors resulted in atherosclerotic plaque formation in ApoE KO mice. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE6 and KE7 is moderate. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE7 =>KE8 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Plaque instability is caused by several metalloproteinases, and their inhibition was reported to prevent vulnerable atherosclerotic plaques [1]. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE7 and KE8 is moderate. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE8 =>AO </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Various blood components aggregate at the site of endothelial rupture. Tissue factors induced by inflammation and eroded by endothelial rupture have a critical role in this region, and their inhibition effectively reduced thrombosis [37]. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE8 and AO is moderate. </td> </tr> </tbody> </table> Concordance of dose-response relationships Studies presenting a clear dose-response relationship in the late stages of this AOP are limited because the later KEs are caused by the cumulative or constitutive effects of the earlier stages. Much evidence of dose-response relationships in the earlier KEs was reported; however, data of the later stages are sparse. In brief, oxidative stress (MIE) is caused by an imbalance between oxidants and their scavenging by anti-oxidant systems such as glutathione. The disruption of an anti-oxidant system was reported to be dose-dependent. Similarly, endothelial dysfunction was also dose-dependent; oxidative and proinflammatory chemical substances lead to eNOS instability, impaired barrier integrity, and increased adhesion molecule expression. However, the acute incidence of the early phase KEs including endothelial dysfunction is normally cleared by the homeostatic capacity of the vasculature; therefore, elicitation of the later KEs needs the consecutive or frequent occurrence of the earlier KEs rather than the strength of stimuli. Monocyte infiltration (KE3) requires the expression of adhesion molecules on the apical surface of endothelial cells, but at the appropriate time. Although macrophage differentiation (KE4) is promoted by proinflammatory cytokines, this occurs in a consecutive manner, not by a single stimulus. In this sense, consecutive inflammation in the vasculature is necessary for the differentiation of infiltrated monocytes into macrophages. LDL oxidation appears to be dose-dependent because the level of oxidants determines the fate of LDL. However, LDL is generally oxidized in the intima region of the vasculature; therefore, the causative oxidants are probably generated in the intima under oxidative conditions. KE7 through AO is a biological event that is cumulative of the earlier Kes; therefore, a dose-response relationship between apical exposure to chemicals and biological events is not obvious. Nevertheless, strong stimulation of the vascular system requires a long time to clear the early key events; thus, consecutive unhealthy conditions in the vasculature are likely to occur in a dose-dependent manner. Temporal concordance among the key events and adverse effects Few studies have evaluated the temporal concordance throughout this AOP. However, key event relationships between the KEs acutely elicited by stressors are well studied and established. Endothelial dysfunction (KE1) is strongly associated with oxidative stress, because excess ROS is generated in cells under oxidative stress conditions, and ROS decreased nitric oxide, inflammation, and apoptosis in the vasculature, which eventually impair endothelial functions. Temporal concordance of the relationship between MIE and KE1 has been elucidated by time-course analyses and ROS scavenging. Temporal concordance among other key events is empirically or theoretically understood; however, robust evidence for most KERs is lacking from the viewpoint of time-course analyses or inhibition of specific KEs. Uncertainties, inconsistencies, and data gaps Uncertainties underlying this AOP include individual variability in the anti-oxidant capacity. As mentioned previously, oxidative stress is a condition whereby excess ROS is generated intracellularly, leading to the oxidation of intracellular components. Intracellular ROS levels are generally determined by the balance between ROS and the anti-oxidant capacity of cells. However, the anti-oxidant capacity varies between individuals related to their age, dietary behavior, smoking habits, and exercise.

<div> <table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td> </td> <td> </td> <td> </td> </tr> </tbody> </table> </div>

ROS induction is well-understood in terms of the dose-response relationship and various studies elucidated the quantitative relationship between intracellular ROS levels, endothelial dysfunction, and stressors [38]. However, this AOP is based upon chronic exposure to stressors, and not a single exposure. In addition, the level of stressors should be varied over time. Therefore, the progress of the AOP is influenced by environmental conditions and individual homeostatic capacity. Because there are many confounding factors for this AOP, a quantitative understanding of KERs is needed to determine how chronic elevation of intracellular ROS levels induced by a stressor could influence the downstream KEs and AO. Currently, there is a good quantitative understanding of how ROS activation influences oxidative stress, endothelial dysfunction, monocyte infiltration, macrophage differentiation, LDL oxidation, and foam cell formation. In addition, in most of the previous studies, the summary evidence indicates dose-response relationships, time-response relationships, and causality for ROS activation leading to increased oxidative stress, lending strong support for these KERs. However, quantitative knowledge is lacking with respect to the identity of thrombosis undergoing plaque formation and plaque instability, which makes empirical support for these KERs weak. Furthermore, data on plaque formation and plaque instability at the biological level were mainly obtained from surrogate measures, which are accepted in clinical practice as indicators of thrombosis, although they may not adequately reflect quantitative values. Taken together, quantitative evidence for the KERs at the tissue and organism levels is moderate at best.

Not Specified

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