55-18-5WBNQDOYYEUMPFS-UHFFFAOYSA-NWBNQDOYYEUMPFS-UHFFFAOYSA-N
N-NitrosodiethylamineDEN
Ethanamine, N-ethyl-N-nitroso-
Diethylamine, N-nitroso-
Diethylnitrosamide
Diethylnitrosoamin
Diethylnitrosoamine
dietilnitrosoamina
N,N-Diethylnitrosoamine
N-Ethyl-N-nitrosoethanamine
Nitrosodiethylamine
N-NITROSODIAETHYLAMIN
N-Nitroso-N,N-diethylamine
NSC 132
diethylnitrosamine
DTXSID202102864-67-5DENRZWYUOJLTMF-UHFFFAOYSA-NDENRZWYUOJLTMF-UHFFFAOYSA-N
Diethyl sulfateSulfuric acid, diethyl ester
DIAETHYLSULFAT
diethyl sulphate
Diethylsulfat
NSC 56380
Sulfate de diethyle
sulfato de dietilo
Sulfuric acid diethyl ester
UN 1594
DTXSID102404562-75-9UMFJAHHVKNCGLG-UHFFFAOYSA-NUMFJAHHVKNCGLG-UHFFFAOYSA-N
N-NitrosodimethylamineDMN
Methanamine, N-methyl-N-nitroso-
DIMETHYLAMINE, N-NITROSO-
Dimethylnitrosoamin
Dimethylnitrosoamine
dimetilnitrosoamina
Nitrosodimethylamine
Nitrosodimetilamina
N-Methyl-N-nitrosomethanamine
N-NITROSODIMETHYLAMIN
N-Nitroso-N,N-dimethylamine
NSC 23226
Dimethylnitrosamine
DTXSID702102977-78-1VAYGXNSJCAHWJZ-UHFFFAOYSA-NVAYGXNSJCAHWJZ-UHFFFAOYSA-N
Dimethyl sulfateSulfuric acid, dimethyl ester
Dimethyl monosulfate
dimethyl sulphate
Dimethylsulfat
DIMETHYLSULFATE
NSC 56194
Sulfate de dimethyle
sulfato de dimetilo
Sulfuric acid dimethyl ester
SULFURIC ACID DIMETHYLESTER
UN 1595
DTXSID502405562-50-0PLUBXMRUUVWRLT-UHFFFAOYSA-NPLUBXMRUUVWRLT-UHFFFAOYSA-N
Ethyl methanesulfonateEMS
Methanesulfonic acid, ethyl ester
Ethyl mesylate
Ethyl methane sulfonate
ethyl methanesulphonate
Ethylmethansulfonat
metanosulfonato de etilo
Methanesulfonate d'ethyle
Methanesulfonic acid ethyl ester
METHANSULFONSAEURE-AETHYLESTER
METHYLSULFONATE, ETHYL
NSC 26805
O-Ethyl methylsulfonate
DTXSID6025309759-73-9FUSGACRLAFQQRL-UHFFFAOYSA-NFUSGACRLAFQQRL-UHFFFAOYSA-N
1-Ethyl-1-nitrosoureaENU
Urea, N-ethyl-N-nitroso-
N-AETHYL-N-NITROSO-HARNSTOFF
N-Ethyl-N-nitrosoharnstoff
N-ethyl-N-nitrosourea
N-Ethyl-N-nitrosouree
N-etil-N-nitrosourea
N-Nitroso-N-ethylurea
NSC 45403
Urea, 1-ethyl-1-nitroso-
DTXSID802059363885-23-4ZGONASGBWOJHDD-UHFFFAOYSA-NZGONASGBWOJHDD-UHFFFAOYSA-N
N′-Ethyl-N-nitro-N-nitrosoguanidineDTXSID3020592926-06-7SWWHCQCMVCPLEQ-UHFFFAOYSA-NSWWHCQCMVCPLEQ-UHFFFAOYSA-N
Isopropyl methanesulfonateDTXSID803149766-27-3MBABOKRGFJTBAE-UHFFFAOYSA-NMBABOKRGFJTBAE-UHFFFAOYSA-N
Methyl methanesulfonateMMS
Methanesulfonic acid, methyl ester
metanosulfonato de metilo
Methanesulfonate de methyle
methyl methanesulphonate
Methyl methylsulfonate
Methylmethansulfonat
METHYLSULFONATE, METHYL
NSC 50256
DTXSID702084570-25-7VZUNGTLZRAYYDE-UHFFFAOYSA-NVZUNGTLZRAYYDE-UHFFFAOYSA-N
Methylnitronitrosoguanidine1-Methyl-3-nitro-1-nitroso-guanidine
Guanidine, N-methyl-N'-nitro-N-nitroso-
1-Methyl-1-nitroso-2-nitroguanidine
1-Methyl-1-nitroso-3-nitroguanidine
1-Methyl-3-nitro-1-nitrosoguanidin
1-METHYL-3-NITRO-1-NITROSO-GUANIDIN
1-Methyl-3-nitro-1-nitrosoguanidine
1-metil-3-nitro-1-nitrosoguanidina
1-Nitroso-3-nitro-1-methylguanidine
Guanidine, 1-methyl-3-nitro-1-nitroso-
Methylnitronitrosoguanidine
N-Methyl-N1-nitro-N-nitrosoguanidine
N-Methyl-nitroso-N'-nitroguanidine
N-Methyl-N'-nitro-N-nitrosoguanadine
N-Methyl-N'-nitro-N-nitrosoquanidine
N-Methyl-N-nitroso-N'-nitroguanidine
N-Nitroso-N-methylnitroguanidine
N-Nitroso-N-methyl-N'-nitroguanidine
N-Nitroso-N'-nitro-N-methylguanidine
NSC 9369
DTXSID2020846CHEBI:16991deoxyribonucleic acidGO:0006305DNA alkylationGO:0006281DNA repair1increased7functional changeDiethyl nitrosamine2016-11-29T18:42:092016-11-29T18:42:09Diethyl sulfate2016-11-29T18:42:112016-11-29T18:42:11Dimethyl nitrosamine2016-11-29T18:42:112016-11-29T21:19:02Dimethyl sulfate2016-11-29T18:42:132016-11-29T18:42:13Ethyl methanesulfonate2016-11-29T18:42:142016-11-29T18:42:14Ethyl nitrosourea2016-11-29T18:42:142016-11-29T18:42:14Ethyl-N'-nitro-N-nitrosoguanidine2016-11-29T18:42:152016-11-29T18:42:15Isopropyl methanesulfonate2016-11-29T18:42:172016-11-29T18:42:17Methyl methanesulfonate2016-11-29T18:42:192016-11-29T18:42:19Methyl-l-N'-nitro-N-nitroguanidine2016-11-29T18:42:192016-11-29T18:42:19Ionizing Radiation<p>Ionizing radiation can vary in energy, dose, charge, and in the spatial distributions of energy transferred to other matter (linear energy transfer per unit length or LET) (ICRU 1970). At the same dose, low and high LET both generate energy deposition events, including many higher energy events (Goodhead and Nikjoo 1989). However, they differ in the spatial distribution and upper range of intensity of energy deposited. Lower LET such as gamma rays sparsely deposit many individual excitations or small clusters of excitations of low energy (Goodhead 1988). In contrast, high LET such as alpha particles have fewer tracks but readily transfer their energy to matter and therefore deposit their energy over a much smaller area (Goodhead 1994). Consequently, alpha and other high LET particles penetrate less deeply into tissue, interactions are densely focused on a narrow track, and individual energy depositions can be large (Goodhead 1988). These different energy deposition patterns can lead to differences in radiation effects including the pattern of DNA damage.</p>
<p>Exposure to ionizing radiation can come from natural and industrial sources. Space and terrestrial radiation includes a range of LET particles, while diagnostic radiation methods such as X-ray imaging, mammography and CT scans use low LET X-rays. Radiation therapy can use an external beam to direct radiation on a focused tissue area, or deposit solid or liquid radioactive materials in the body that release (mostly gamma) radiation internally. External radiotherapy typically uses X-rays but is moving towards higher LET charged particles such as protons and heavy ions (Durante, Orecchia et al. 2017).</p>
2019-05-03T12:36:362019-05-07T12:12:13Topoisomerase inhibitors2019-05-19T20:21:242019-05-19T20:21:24Radiomimetic compounds2019-05-19T20:21:422019-05-19T20:21:4210090mouse10036Syrian golden hamster10116rat9606Homo sapiens9913cowWikiUser_25human and other cells in cultureWCS_9606humanAlkylation, DNAAlkylation, DNAMolecular<p>The event involves DNA alkylation to form a variety of different DNA adducts (i.e., alkylated nucleotides). Alkylation occurs at various sites in DNA and can include alkylation of adenine- Nl, - N3, - N7, guanine- N3, - O6, - N7, thymine-O2, - N3, - O4, cytosine- O2, -N3, and the phosphate (diester) group (reviewed in detail in Beranek 1990). In addition, alkylation can involve modification with different sizes of alkylation groups (e.g., methyl, ethyl, propyl). It should be noted that many of these adducts are not stable or are readily repaired (discussed in more detail below). A small proportion of adducts are stable and remain bound to DNA for long periods of time.
</p><p>There is no OECD guideline for measurement of alkylated DNA, although technologies for their detection are established. Reviews of modern methods to measure DNA adducts include Himmelstein et al,. 2009 and Philips et al., 2000.
</p><p>High performance liquid chromatography (HPLC) methods can be used to measure whether an agent is capable of alkylating DNA in somatic cells. Alkyl adducts in somatic cells can be measured using immunological methods (described in Nehls et al. 1984), as well as HPLC (methods in de Groot et al. 1994) or a combination of 32P post-labeling, HPLC and immunologic detection (Kang et al. 1992). We note that mass spectrometry provides structural specificity and confirmation of the structure of DNA adducts.
</p><p>DNA alkylation can also be measured using a modified comet assay. This method involves the digestion of alkylated DNA bases with 3–methyladenine DNA glycosylase (Collins et al., 2001; Hasplova et al., 2012) followed by the standard comet assay to detect where alkyl adducts occur. The advantage of this method is that the alkaline version of the comet assay, as a core method, has an in vivo OECD guideline.
</p><p>Finally, structure-activity relationships (SARs) have been developed to predict the possibility that a chemical will alkylate DNA (e.g., Vogel and Ashby, 1994; Benigni, 2005; Dai et al., 1989; Lewis and Griffith, 1987).
</p><p><br />
Measurement of alkylation in male germ cells:
</p><p>In rodent testes, studies have detected adducts in situ by immuhistocytological staining. For example, fixed testes are incubated with O6-EtGua -specific mouse monoclonal antibody and subsequently with a labeled anti-mouse IgG F antibody. Nuclear DNA is counterstained with DAPI 4,6-diamidino- 2-phenylindole. Fluorescence signals from immunostained O6-EtGua residues in DNA are visualized by fluorescence microscopy and quantitated using an image analysis system. Methods are described in (Seiler et al. 1997). An immunoslot blot assay for detection of O6-EtGua has been described previously in (Mientjes et al. 1996).
</p><p>Alternatively, rodents have also been exposed to radio-labeled alkylating agents. Examples from the literature include [2-3H] ENU, [1-3H]di-ethyl sulfate, or [1-3H]ethyl-methane sulfonate. Following treatment with the labeled chemical, testis and other tissues of interest are removed. Germ cells are released from tubuli by pushing out the contents with forceps. Using this procedure all germ-cell stages are liberated from the tubuli, with the possible exception of part of the population of stem-cell spermatogonia that remain attached to the walls of the tubuli. DNA is then extracted from germ cells, empty testis tubuli and other tissues of interest. DNA adduct formation is determined after neutral and acid hydrolysis of DNA followed by separation of the various ethylation products using HPLC (described in van Zeeland et al. 1990).
</p><p>Alkylated DNA has been measured in somatic cells in a variety of species, from prokaryotic organisms, to rodents in vivo, to human cells in culture. Theoretically, DNA alkylation can occur in any cell type in any organism.
</p>CL:0000255eukaryotic cellHighMixedHighHighHighHigh<p><br />
Benigni, R. (2005), "Structure-activity relationship studies of chemical mutagens and carcinogens: mechanistic investigations and prediction and approaches", <i>Chem. Rev.</i>, 105: 1767-1800.
</p><p>Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", <i>Mutation Res.</i>, 231: 11-30.
</p><p>Collins, A.R., M. Dusinská and A. Horská (2001), "A Detection of alkylation damage in human lymphocyte DNA with the comet assay". <i>Acta Biochim Pol.</i>, 48: 611-4.
</p><p>Dai, Q.H. and R.G. Zhong (1989), "Quantitative pattern recognition for structure-carcinogenic activity relationship of N-nitroso compounds based upon Di-region theory", <i>Sci China B.</i>, 32:776-790.
</p><p>de Groot, A.J., J.G. Jansen, C.F. van Valkenburg and A.A. van Zeeland (1994), "Molecular dosimetry of 7-alkyl- and O6-alkylguanine in DNA by electrochemical detection", <i>Mutat Res.</i>, 307: 61-6.
</p><p>Hašplová, K., A. Hudecová, Z. Magdolénová, M. Bjøras, E. Gálová, E. Miadoková and M. Dušinská (2012), "DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD)", <i>Toxicol Lett.</i>, 208: 76-81.
</p><p>Himmelstein, M.W., P.J. Boogaard, J. Cadet, P.B. Farmer, J.J. Kim, E.A. Martin, R. Persaud and D.E. Shuker (2009), "Creating context for the use of DNA adduct data in cancer risk assessment: II. Overview of methods of identification and quantitation of DNA damage", <i>Crit. Rev. Toxicol.</i>, 39: 679-94.
</p><p>Kamino, K., F. Seiler, M. Emura, J. Thomale, M.F. Rajewsky and U. Mohr (1995), "Formation of O6-ethylguanine in spermatogonial DNA of adult Syrian golden hamster by intraperitoneal injection of diethylnitrosamine", <i>Exp. Toxicol. Pathol.</i>, 47: 443-445.
</p><p>Kang, H.I., C. Konishi, G. Eberle, M.F. Rajewsky, T. Kuroki and N.H. Huh (1992), "Highly sensitive, specific detection of O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of high-performance liquid chromatography prefractionation, 32P postlabeling, and immunoprecipitation", <i>Cancer Res.</i>, 52: 5307-5312.
</p><p>Lewis, D.F. and V.S. Griffiths (1987), "Molecular electrostatic potential energies and methylation of DNA bases: a molecular orbital-generated quantitative structure-activity relationship", <i>Xenobiotica</i>, 17: 769-776.
</p><p>Mientjes, E.J., K. Hochleitner, A. Luiten-Schuite, J.H. van Delft, J. Thomale, F. Berends, M.F. Rajewsky, P.H. Lohman and R.A. Baan (1996), "Formation and persistence of O6-ethylguanine in genomic and transgene DNA in liver and brain of lambda(lacZ) transgenic mice treated with N-ethyl-N-nitrosourea", <i>Carcinogenesis</i>, 17: 2449-2454.
</p><p>Nehls, P., M.F. Rajewsky, E. Spiess, D. Werner (1984), "Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo", <i>EMBO J.</i>, 3:327-332.
</p><p>Phillips, D.H., P.B. Farmer, F.A. Beland, R.G. Nath, M.C. Poirier, M.V. Reddy and K.W. Turteltaub (2000), "Methods of DNA adduct determination and their application to testing compounds for genotoxicity", <i>Environ. Mol. Mutagen.</i>, 35: 222-233.
</p><p>Scherer, E., A.A. Jenner and L. den Engelse (1987), "Immunocytochemical studies on the formation and repair of O6-alkylguanine in rat tissues", <i>IARC Sci. Publ.</i>, 84: 55-58.
</p><p>Sega, G.A., C.R. Rohrer, H.R. Harvey and A.E. Jetton (1986), "Chemical dosimetry of ethyl nitrosourea in the mouse testis", <i>Mutat. Res.</i>, 159: 65-74.
</p><p>Seiler, F., K. Kamino, M. Emura, U. Mohr and J. Thomale (1997), "Formation and persistence of the miscoding DNA alkylation product O6-ethylguanine in male germ cells of the hamster", <i>Mutat. Res.</i>, 385: 205-211.
</p><p>van Zeeland, A.A., A. de Groot and A. Neuhäuser-Klaus (1990), "DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis", <i>Mutat. Res.</i> 231: 55-62.
</p><p>Vogel, E.W., Ashby, J. (1994), "Structure-activity relationships: experimental approaches." In: Methods to asses DNA Damage and repair: Interspecies comparisons. Edited by R.T. Tardiff, P.H.M. Lohman and G.N. Wogan, SCOPE, Wiley and Sons LTD.
</p>2016-11-29T18:41:222017-09-16T10:14:29Inadequate DNA repairInadequate DNA repairCellular<p>DNA lesions may result from the formation of DNA adducts (i.e., covalent modification of DNA by chemicals), or by the action of agents such as radiation that may produce strand breaks or modified nucleotides within the DNA molecule. These DNA lesions are repaired through several mechanistically distinct pathways that can be categorized as follows:</p>
<ol>
<li><strong>Damage reversal</strong> acts to reverse the damage without breaking any bonds within the sugar phosphate backbone of the DNA. The most prominent enzymes associated with damage reversal are photolyases (Sancar, 2003) that can repair UV dimers in some organisms, and O6-alkylguanine-DNA alkyltransferase (AGT) (Pegg 2011) and oxidative demethylases (Sundheim et al., 2008), which can repair some types of alkylated bases.</li>
<li><strong>Excision repair</strong> involves the removal of a damaged nucleotide(s) through cleavage of the sugar phosphate backbone followed by re-synthesis of DNA within the resultant gap. Excision repair of DNA lesions can be mechanistically divided into:
<p style="margin-left:40px"><strong>a) Base excision repair (BER)</strong><span style="font-size:1rem"> (Dianov and Hübscher, 2013), in which the damaged base is removed by a damage-specific glycosylase prior to incision of the phosphodiester backbone at the resulting abasic site. </span>This leads to an intermediate that contains a DNA strand break, whereby DNA ligase is then recruited to seal the ends of the DNA.</p>
<p style="margin-left:40px"><strong>b) Nucleotide excision repair (NER)</strong> (Schärer, 2013), in which the DNA strand containing the damaged nucleotide is incised at sites several nucleotides 5’ and 3’ to the site of damage, and a polynucleotide containing the damaged nucleotide is removed prior to DNA resynthesis within the resultant gap and sealing of the ends by DNA ligase. </p>
<p style="margin-left:40px"><strong>c) Mismatch repair (MMR)</strong> (Li et al., 2016) which does not act on DNA lesions but does recognize mispaired bases resulting from replication errors. In MMR the strand containing the misincorporated base is removed prior to DNA resynthesis.</p>
<p style="margin-left:40px">The major pathway that removes oxidative DNA damage is base excision repair (BER), which can be either monofunctional or bifunctional; in mammals, a specific DNA glycosylase (OGG1: 8-Oxoguanine glycosylase) is responsible for excision of 8-oxoguanine (8-oxoG) and other oxidative lesions (Hu et al., 2005; Scott et al., 2014; Whitaker et al., 2017). We note that long-patch BER is used for the repair of clustered oxidative lesions, which uses several enzymes from DNA replication pathways (Klungland and Lindahl, 1997). These pathways are described in detail in various reviews e.g., (Whitaker et al., 2017). </p>
</li>
<li><strong>Single strand break repair (SSBR) </strong>involves different proteins and enzymes depending on the origin of the SSB (e.g., produced as an intermediate in excision repair or due to direct chemical insult) but the same general steps of repair are taken for all SSBs: detection, DNA end processing, synthesis, and ligation (Caldecott, 2014). Poly-ADP-ribose polymerase1 (PARP1) detects and binds unscheduled SSBs (i.e., not deliberately induced during excision repair) and synthesizes PAR as a signal to the downstream factors in repair. PARP1 is not required to initiate SSBR of BER intermediates. The XRCC1 protein complex is then recruited to the site of damage where a common DNA intermediate as BER was generated, and acts as a scaffold for proteins and enzymes required for repair. Depending on the nature of the damaged termini of the DNA strand, different enzymes are required for end processing to generate the substrates that DNA polymerase β (Polβ; short patch repair) or Pol δ/ε (long patch repair) can bind to synthesize over the gap, although end processing is generally done by polynucleotide kinase. Synthesis in long-patch repair displaces a single stranded flap which is excised by flap endonuclease 1 (FEN1). In short-patch repair, the XRCC1/Lig3α complex joins the two ends after synthesis. In long-patch repair, the PCNA/Lig1 complex ligates the ends. (Caldecott, 2014). </li>
<li><strong>Double strand break repair (DSBR)</strong> is necessary to preserve genomic integrity when breaks occur in both strands of a DNA molecule. There are two major pathways for DSBR: homologous recombination (HR), which operates primarily during the S phase of dividing cell types, and nonhomologous end joining (NHEJ), which can function in both dividing and non-dividing cell types. No repair occurs in the M phase (Teruaki Iyama and David M. Wilson III, 2013). DNA repair in mitosis is controversial (Mladenov et al., 2023).</li>
</ol>
<p style="margin-left:40px">Complex lesions can be created by a single mutagen and can be more difficult to repair, as the mechanism behind how different repair pathways cooperate to address this is still unclear (Aleksandrov et al., 2018). In higher eukaryotes such as mammals, NHEJ is usually the preferred pathway for DNA DSBR. Its use, however, is dependent on the cell type, the gene locus, and the nuclease platform (Miyaoka et al., 2016). The use of NHEJ is also dependent on the cell cycle; NHEJ is generally not the pathway of choice when the cell is in the late S or G2 phase of the cell cycle, or in mitotic cells when the sister chromatid is directly adjacent to the double-strand break (DSB) (Lieber et al., 2003). In these cases, the HR pathway is commonly used for repair of DSBs. Despite this, NHEJ is still used more commonly than HR in human cells. Classical NHEJ (C-NHEJ) is the most common NHEJ repair mechanism, but alternative NHEJ (alt-NHEJ) can also occur, especially in the absence of C-NHEJ and HR.</p>
<p style="margin-left:40px">The process of C-NHEJ in humans requires at least seven core proteins: Ku70, Ku86, DNA-dependent protein kinase complex (DNA-PK<sub>cs </sub>), Artemis, X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase IV (Boboila et al., 2012). When DSBs occur, the Ku proteins, which have a high affinity for DNA ends, will bind to the break site and form a heterodimer. This protects the DNA from exonucleolytic attack and acts to recruit DNA-PK<sub>cs</sub>, the catalytic subunit, thus forming a trimeric complex on the ends of the DNA strands. Alternative NHEJ, or alt NHEJ, uses small similar sequences in two broken DNA ends to join them together. Unlike the usual repair method (cNHEJ), aNHEJ doesn't need specific proteins like LIG4 and KU. Instead, it relies on the MRN complex to process the breaks. However, alt NHEJ tends to cause mutations by adding or removing bits of DNA during the repair (Chaudhuri and Nussenzweig, 2017). The kinase activity of DNA-PK<sub>cs </sub>is then triggered, causing DNA-PK<sub>cs </sub>to auto-phosphorylate and thereby lose its kinase activity; the now phosphorylated DNA-PK<sub>cs</sub> dissociates from the DNA-bound Ku proteins. The free DNA-PK<sub>cs</sub> phosphorylates Artemis, an enzyme that possesses 5’-3’ exonuclease and endonuclease activity in the presence of DNA-PK<sub>cs</sub> and ATP. Artemis is responsible for ‘cleaning up’ the ends of the DNA. For 5’ overhangs, Artemis nicks the overhang, generally leaving a blunt duplex end. For 3’ overhangs, Artemis will often leave a four- or five-nucleotide single stranded overhang (Pardo et al., 2009; Fattah et al., 2010; Lieber et al., 2010). Next, the XLF and XRCC4 proteins form a complex which makes a channel to bind DNA and aligns the ends for efficient ligation via DNA ligase IV (Hammel et al., 2011).</p>
<p style="margin-left:40px">The process of alt-NHEJ is less well understood than C-NHEJ and is a lower fidelity mechanism. Alt-NHEJ is known to involve slightly different core proteins than C-NHEJ and required microhomology repeats, but the steps of the pathway are essentially the same between the two processes (reviewed in Chiruvella et al., 2013). It is established, however, that alt-NHEJ is more error-prone in nature than C-NHEJ, which contributes to incorrect DNA repair. Alt-NHEJ is thus considered primarily to be a backup repair mechanism (reviewed in Chiruvella et al., 2013). </p>
<p style="margin-left:40px">In contrast to NHEJ, HR takes advantage of similar or identical DNA sequences to repair DSBs and is not error-prone (Sung and Klein, 2006). The initiating step of HR is the creation of a 3’ single strand DNA (ss-DNA) overhang. Combinases such as RecA and Rad51 then bind to the ss-DNA overhang, and other accessory factors, including Rad54, help recognize and invade the homologous region on another DNA strand. From there, DNA polymerases are able to elongate the 3’ invading single strand and resynthesize the broken DNA strand using the corresponding sequence on the homologous strand.</p>
<p> </p>
<p><strong><u>Fidelity of DNA Repair</u></strong></p>
<p><br />
Most DNA repair pathways are extremely efficient. However, in principal, all DNA repair pathways can be overwhelmed when the DNA lesion burden exceeds the capacity of a given DNA repair pathway to recognize and remove the lesion. Exceeded repair capacity may lead to toxicity or mutagenesis following DNA damage. Apart from extremely high DNA lesion burden, inadequate repair may arise through several different specific mechanisms. For example, during repair of DNA containing O6-alkylguanine adducts, AGT irreversibly binds a single O6-alkylguanine lesion and as a result is inactivated (this is termed suicide inactivation, as its own action causes it to become inactivated). Thus, the capacity of AGT to carry out alkylation repair can become rapidly saturated when the DNA repair rate exceeds the de novo synthesis of AGT (Pegg, 2011).</p>
<p>A second mechanism relates to cell specific differences in the cellular levels or activity of some DNA repair proteins. For example, XPA is an essential component of the NER complex. The level of XPA that is active in NER is low in the testes, which may reduce the efficiency of NER in testes as compared to other tissues (Köberle et al., 1999). Likewise, both NER and BER have been reported to be deficient in cells lacking functional p53 (Adimoolam and Ford, 2003; Hanawalt et al., 2003; Seo and Jung, 2004). A third mechanism relates to the importance of the DNA sequence context of a lesion in its recognition by DNA repair enzymes. For example, 8-oxoguanine (8-oxoG) is repaired primarily by BER; the lesion is initially acted upon by a bifunctional glycosylase, OGG1, which carries out the initial damage recognition and excision steps of 8-oxoG repair. However, the rate of excision of 8-oxoG is modulated strongly by both chromatin components (Menoni et al., 2012) and DNA sequence context (Allgayer et al., 2013) leading to significant differences in the repair of lesions situated in different chromosomal locations.</p>
<p>DNA repair is also remarkably error-free. However, misrepair can arise during repair under some circumstances. DSBR is notably error prone, particularly when breaks are processed through NHEJ, during which partial loss of genome information is common at the site of the double strand break (Iyama and Wilson, 2013). This is because NHEJ rejoins broken DNA ends without the use of extensive homology; instead, it uses the microhomology present between the two ends of the DNA strand break to ligate the strand back into one. When the overhangs are not compatible, however, indels (insertion or deletion events), duplications, translocations, and inversions in the DNA can occur. These changes in the DNA may lead to significant issues within the cell, including alterations in the gene determinants for cellular fatality (Moore et al., 1996).</p>
<p>Activation of mutagenic DNA repair pathways to withstand cellular or replication stress either from endogenous or exogenous sources can promote cellular viability, albeit at a cost of increased genome instability and mutagenesis (Fitzgerald et al., 2017). These salvage DNA repair pathways including, Break-induced Replication (BIR) and Microhomology-mediated Break-induced Replication (MMBIR). BIR repairs one-ended DSBs and has been extensively studied in yeast as well as in mammalian systems. BIR and MMBIR are linked with heightened levels of mutagenesis, chromosomal rearrangements and ensuing genome instability (Deem et al., 2011; Sakofsky et al., 2015; Saini et al., 2017; Kramara et al., 2018). In mammalian genomes BIR-like synthesis has been proposed to be involved in late stage Mitotic DNA Synthesis (MiDAS) that predominantly occurs at so-called Common Fragile Sites (CFSs) and maintains telomere length under s conditions of replication stress that serve to promote cell viability (Minocherhomji et al., 2015; Bhowmick et al., 2016; Dilley et al., 2016). </p>
<p>Misrepair may also occur through other repair pathways. Excision repair pathways require the resynthesis of DNA and rare DNA polymerase errors during gap resynthesis will result in mutations (Brown et al., 2011). Errors may also arise during gap resynthesis when the strand that is being used as a template for DNA synthesis contains DNA lesions (Kozmin and Jinks-Robertson, 2013). In addition, it has been shown that sequences that contain tandemly repeated sequences, such as CAG triplet repeats, are subject to expansion during gap resynthesis that occurs during BER of 8-oxoG damage (Liu et al., 2009).</p>
<p>There is no test guideline for this event. The event is usually inferred from measuring the retention of DNA adducts or the creation of mutations as a measure of lack of repair or incorrect repair. These ‘indirect’ measures of its occurrence are crucial to determining the mechanisms of genotoxic chemicals and for regulatory applications (i.e., determining the best approach for deriving a point of departure). More recently, a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) has been developed to directly measure the ability of human cells to repair plasmid reporters (Nagel et al., 2014).</p>
<p><u><strong>Indirect Measurement</strong></u></p>
<p>In somatic and spermatogenic cells, measurement of DNA repair is usually inferred by measuring DNA adduct formation/removal. Insufficient repair is inferred from the retention of adducts and from increasing adduct formation with dose. Insufficient DNA repair is also measured by the formation of increased numbers of mutations and alterations in mutation spectrum. The methods will be specific to the type of DNA adduct that is under study.</p>
<p>Some EXAMPLES are given below for alkylated DNA.</p>
<p>DOSE-RESPONSE CURVE FOR ALKYL ADDUCTS/MUTATIONS: It is important to consider that some adducts are not mutagenic at all because they are very effectively repaired. Others are effectively repaired, but if these repair processes become overwhelmed mutations begin to occur. The relationship (shape of dose-response curve) between exposure to mutagenic agents and mutations provide an indication of whether the removal of adducts occurs, and whether it is more efficient at low doses. Sub-linear dose-response curves (hockey stick or j-shape curves) for mutation induction indicates that adducts are not converted to mutations at low doses. This suggests the effective repair of adducts at low doses, followed by saturation of repair at higher doses (Clewell et al., 2019). Thus, measurement of a clear point of inflection in the dose-response curve for mutations suggests that repair does occur, at least to some extent, at low dosees but that reduced repair efficiency arises above the inflection point. A lack of increase in mutation frequencies (i.e., flat line for dose-response) for a compound showing a dose-dependent increase in adducts would imply that the adducts formed are either not mutagenic or are effectively repaired.</p>
<p>RETENTION OF ALKYL ADDUCTS: Alkylated DNA can be found in cells long after exposure has occurred. This indicates that repair has not effectively removed the adducts. For example, DNA adducts have been measured in hamster and rat spermatogonia several days following exposure to alkylating agents, indicating lack of repair (Seiler et al., 1997; Scherer et al., 1987).</p>
<p>MUTATION SPECTRUM: Shifts in mutation spectrum (i.e., the specific changes in the DNA sequence) following a chemical exposure (relative to non-exposed mutation spectrum) indicates that repair was not operating effectively to remove specific types of lesions. The shift in mutation spectrum is indicative of the types of DNA lesions (target nucleotides and DNA sequence context) that were not repaired. For example, if a greater proportion of mutations occur at guanine nucleotides in exposed cells, it can be assumed that the chemical causes DNA adducts on guanine that are not effectively repaired.</p>
<p><br />
<u><strong>Direct Measurement</strong></u></p>
<p>Nagel et al. (2014) we developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) to measures the ability of human cells to repair plasmid reporters. These reporters contain different types and amounts of DNA damage and can be used to measure repair through by NER, MMR, BER, NHEJ, HR and MGMT.</p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Please refer to the table below for additional details and methodologies for detecting DNA damage and repair.</span></span></p>
<table border="1" cellpadding="1" cellspacing="1" style="height:2082px; width:629px">
<tbody>
<tr>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>Assay Name</strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>References</strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>Description</strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>DNA Damage/Repair Being Measured</strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>OECD Approved Assay</strong></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Dose-Response Curve for Alkyl Adducts/ Mutations</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Lutz 1991</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif"> </span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Clewell 2016</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Creation of a curve plotting the stressor dose and the abundance of adducts/mutations; Characteristics of the resulting curve can provide information on the efficiency of DNA repair</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Alkylation,</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">oxidative damage, or DSBs</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Retention of Alkyl Adducts</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Seiler 1997</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif"> </span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Scherer 1987</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Examination of DNA for alkylation after exposure to an alkylating agent; Presence of alkylation suggests a lack of repair</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Alkylation</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Mutation Spectrum</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Wyrick 2015</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Shifts in the mutation spectrum after exposure to a chemical/mutagen relative to an unexposed subject can provide an indication of DNA repair efficiency, and can inform as to the type of DNA lesions present</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Alkylation,</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">oxidative damage, or DSBs</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSB Repair Assay (Reporter constructs)</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:times new roman,serif"><span style="font-family:arial,sans-serif">Mao</span></span><span style="font-family:arial,sans-serif"> et al., 2011</span></span></td>
<td style="text-align:center"><span style="font-size:14px">Transfection of a GFP reporter construct (and DsRed control) where the GFP signal is only detected if the DSB is repaired; GFP signal is quantified using fluorescence microscopy or flow cytometry</span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSBs</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Primary Rat Hepatocyte DNA Repair Assay</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Jeffrey and Williams, 2000</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><u><span style="font-family:arial,sans-serif"> </span></u></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Butterworth et al., 1987</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Rat primary hepatocytes are cultured with a <sup>3</sup>H-thymidine solution in order to measure DNA synthesis in response to a stressor in non-replicating cells; Autoradiography is used to measure the amount of <sup>3</sup>H incorporated in the DNA post-repair</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Unscheduled DNA synthesis in response to DNA damage</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Repair synthesis measurement by </span><sup><span style="font-family:arial,sans-serif">3</span></sup><span style="font-family:arial,sans-serif">H-thymine incorporation</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Iyama and Wilson, 2013</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Measure DNA synthesis in non-dividing cells as indication of gap filling during excision repair</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Excision repair</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Comet Assay with Time-Course</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Olive et al., 1990</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><u><span style="font-family:arial,sans-serif"> </span></u></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Trucco et al., 1998</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">-</span></span></p>
<p style="text-align:center">Dunkenberger et al., 2022 </p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Comet assay is performed with a time-course </span></span>under alkaline conditions to detect SSBs and DSBs.<span style="font-size:14px"><span style="font-family:arial,sans-serif"> Quantity of DNA in the tail should decrease as DNA repair progresses</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSBs</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif"> </span><span style="font-family:times new roman,serif"><a href="https://read.oecd-ilibrary.org/environment/test-no-489-in-vivo-mammalian-alkaline-comet-assay_9789264264885-en"><span style="font-family:arial,sans-serif">Yes</span></a></span><u><span style="font-family:arial,sans-serif"> (No. 489)</span></u></span></td>
</tr>
<tr>
<td style="text-align:center">Flow Cytometry </td>
<td>Corneo et al., 2007 </td>
<td style="text-align:center">The alt-NHEJ flow cytometer method involves utilizing an extrachromosomal substrate. Green fluorescent protein (GFP) expression is indicative of successful alt-NHEJ activity, contingent on the removal of 10 nucleotides from each end of the DNA and subsequent rejoining within a 9-nucleotide microhomology region. This approach provides a quantitative and visual means to measure the efficiency of alternative non-homologous end joining in cellular processes. </td>
<td style="text-align:center">Alt NHEJ</td>
<td style="text-align:center">No</td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Pulsed Field Gel Electro-phoresis (PFGE) with Time-Course</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:times new roman,serif"><span style="font-family:arial,sans-serif">Biedermann</span></span><u><span style="font-family:arial,sans-serif"> </span></u><span style="font-family:arial,sans-serif">et al., 1991</span></span></td>
<td style="text-align:center"><span style="font-size:14px">PFGE assay with a time-course; Quantity of small DNA fragments should decrease as DNA repair progresses</span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSBs</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Fluorescence -Based Multiplex Flow-Cytometric Host Reactivation Assay </span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">(FM-HCR)</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Nagel et al., 2014</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Measures the ability of human cells to repair plasma reporters, which contain different types and amounts of DNA damage; Used to measure repair processes including HR, NHEJ, BER, NER, MMR, and MGMT</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">HR, NHEJ, BER, NER, MMR, or MGMT</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td><span style="font-size:14px">Alkaline Unwinding Assay with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Nacci et al. 1991 </span></td>
<td style="text-align:center"><span style="font-size:14px">DNA is stored in alkaline solutions with DNA-specific dye and allowed to unwind following removal from tissue, increased strand damage associated with increased unwinding. Samples analyzed at different time points to compare remaining damage following repair opportunities </span></td>
<td style="text-align:center"><span style="font-size:14px">DSBs </span></td>
<td style="text-align:center"><span style="font-size:14px">Yes (<u><span style="font-family:arial,sans-serif">No. 489)</span></u> </span></td>
</tr>
<tr>
<td><span style="font-size:14px">Sucrose Density Gradient Centrifugation with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Larsen et al. 1982 </span></td>
<td style="text-align:center"><span style="font-size:14px">Strand breaks alter the molecular weight of the DNA piece. DNA in alkaline solution centrifuged into sugar density gradient, repeated set time apart. The less DNA breaks identified in the assay repeats, the more repair occurred </span></td>
<td style="text-align:center"><span style="font-size:14px">SSBs </span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td><span style="font-size:14px">y-H2AX Foci Staining with Time Course </span></td>
<td style="text-align:center">
<p><span style="font-size:14px">Mariotti et al. 2013 </span></p>
<p><span style="font-size:14px">Penninckx et al. 2021 </span></p>
</td>
<td style="text-align:center"><span style="font-size:14px">Histone H2AX is phosphorylated in the presence of DNA strand breaks, the rate of its disappearance over time is used as a measure of DNA repair </span></td>
<td style="text-align:center"><span style="font-size:14px">DSBs </span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td><span style="font-size:14px">Alkaline Elution Assay with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Larsen et al. 1982 </span></td>
<td style="text-align:center"><span style="font-size:14px">DNA with strand breaks elute faster than DNA without, plotted against time intervals to determine the rate at which strand breaks repair </span></td>
<td style="text-align:center"><span style="font-size:14px">SSBs </span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td><span style="font-size:14px">53BP1 foci Detection with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Penninckx et al. 2021 </span></td>
<td style="text-align:center"><span style="font-size:14px">53BP1 is recruited to the site of DNA damage, the rate at which its level decreases over time is used to measure DNA repair </span></td>
<td style="text-align:center"><span style="font-size:14px">DSBs</span></td>
<td style="text-align:center"><span style="font-size:14px">N/A </span></td>
</tr>
</tbody>
</table>
<p> </p>
<p>The retention of adducts has been directly measured in many different types of eukaryotic somatic cells (in vitro and in vivo). In male germ cells, work has been done on hamsters, rats and mice. The accumulation of mutation and changes in mutation spectrum has been measured in mice and human cells in culture. Theoretically, saturation of DNA repair occurs in every species (prokaryotic and eukaryotic). The principles of this work were established in prokaryotic models. Nagel et al. (2014) have produced an assay that directly measures DNA repair in human cells in culture.</p>
<p>NHEJ is primarily used by vertebrate multicellular eukaryotes, but it also been observed in plants. Furthermore, it has recently been discovered that some bacteria (Matthews et al., 2014) and yeast (Emerson et al., 2016) also use NHEJ. In terms of invertebrates, most lack the core DNA-PK<sub>cs</sub> and Artemis proteins; they accomplish end joining by using the RA50:MRE11:NBS1 complex (Chen et al., 2001). HR occurs naturally in eukaryotes, bacteria, and some viruses (Bhatti et al., 2016).</p>
<p><strong>Taxonomic applicability:</strong> Inadequate DNA repair is applicable to all species, as they all contain DNA (White & Vijg, 2016). </p>
<p><strong>Life stage applicability:</strong> This key event is not life stage specific as any life stage can have poor repair, though as individuals age their repair process become less effective (Gorbunova & Seluanov, 2016). </p>
<p><strong>Sex applicability: </strong>There is no evidence of sex-specificity for this key event, with initial rate of DNA repair not significantly different between sexes (Trzeciak et al., 2008). </p>
<p><strong>Evidence for perturbation by a stressor: </strong>Multiple studies demonstrate that inadequate DNA repair can occur as a result of stressors such as ionizing and non-ionizing radiation, as well as chemical agents (Kuhne et al., 2005; Rydberg et al., 2005; Dahle et al., 2008; Seager et al., 2012; Wilhelm, 2014; O’Brien et al., 2015). </p>
HighUnspecificHighAll life stagesHighModerateModerateHighLow<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Adimoolam, S. & J.M. Ford (2003), "p53 and regulation of DNA damage recognition during nucleotide excision repair" <em>DNA Repair</em> (Amst), 2(9): 947-54.</span></span></p>
<p>Aleksandrov, Radoslav et al. (2018), “Protein Dynamics in Complex DNA Lesions.” Molecular cell,69(6): 1046-1061.e5. doi:10.1016/j.molcel.2018.02.016 </p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Allgayer, J. et al. (2013), "Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence", <em>Nucleic Acids Res</em>, 41(18): 8559-8571. Doi: <a href="https://doi.org/10.1093/nar/gkt620" target="_blank">10.1093/nar/gkt620</a>.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", <em>Mutation Research</em>, 231(1): 11-30. Doi: 10.1016/0027-5107(90)90173-2.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Bhatti, A. et al., (2016), “Homologous Recombination Biology.”, <em>Encyclopedia Britannica</em>.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Bhowmick, R., S. et al. (2016), "RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress", <em>Mol Cell</em>, 64:1117-1126. Doi: 10.1016/j.molcel.2016.10.037.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Biedermann, A. K. et al. (1991), “SCID mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair”, <em>Cell Biology</em>, 88(4): 1394-7. Doi: 10.1073/pnas.88.4.1394.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Boboila, C., F. W. Alt & B. Schwer. (2012), “Classical and alternative end-joining pathways for repair of lymphocyte-specific and general DNA double-strand breaks.” <em>Adv Immunol</em>, 116, 1-49. doi:10.1016/B978-0-12-394300-2.00001-6</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Bronstein, S.M. et al. (1991), "Toxicity, mutagenicity, and mutational spectra of N-ethyl-N-nitrosourea in human cell lines with different DNA repair phenotypes", <em>Cancer Research</em>, 51(19): 5188-5197.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Bronstein, S.M. et al. (1992), "Efficient repair of O6-ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells", <em>Cancer Research</em>, 52(7): 2008-2011. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Brown, J.A. et al. (2011), "Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis", <em>DNA Repair (Amst).</em>, 10(1):24-33.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Butterworth, E. B. et al., (1987), A protocol and guide for the in vitro rat hepatocyte DNA-repair assay. <em>Mutation Research</em>. 189, 113-21. Doi: 10.1016/0165-1218(87)90017-6.</span></span></p>
<p>Caldecott, K. W. (2014), "DNA single-strand break repair", Exp Cell Res, 329(1): 2-8.</p>
<p>Chaudhuri, R.A. and Nussenzweig, A. (2017), “The multifaceted roles of PARP1 in DNA repair and chromatin remodelling”. Nat Rev Mol Cell Biol 18, 610–621. https://doi.org/10.1038/nrm.2017.53 </p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Chen, L. et al., (2001), Promotion of DNA ligase IV-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. <em>Mol Cell</em>. 8(5), 1105-15.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Chiruvella, K. K., Z. Liang & T. E. Wilson, (2013), Repair of Double-Strand Breaks by End Joining. <em>Cold Spring Harbor Perspectives in Biology</em>, 5(5):127-57. Doi: 10.1101/cshperspect.a012757.</span></span></p>
<p>Clewell, R. A. et al. (2019). “Dose-dependence of chemical carcinogenicity: Biological mechanisms for thresholds and implications for risk assessment”. Chem Biol Interact. 2019 Mar 1;301:112-127. doi: 10.1016/j.cbi.2019.01.025. </p>
<p>Corneo, B. et al., 2007, "Rag mutations reveal robust alternative end joining”. Nature 449, 483–486 (2007). https://doi.org/10.1038/nature06168 </p>
<p><span style="font-size:14px">Dahle, J., et al. (2008), “Overexpression of human OGG1 in mammalian cells decreases ultraviolet A induced mutagenesis”, Cancer Letters, Vol.267, Elsevier, Amsterdam, https://doi.org/10.1016/j.canlet.2008.03.002. </span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Deem, A. et al. (2011), "Break-Induced Replication Is Highly Inaccurate.", <em>PLoS Biol</em>. 9:e1000594. Doi: 10.1371/journal.pbio.1000594.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Dianov, G.L. & U. Hübscher (2013), "Mammalian base excision repair: the forgotten archangel", <em>Nucleic Acids Res.</em>, 41(6):3483-90. Doi: 10.1093/nar/gkt076.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Dilley, R.L. et al. Greenberg (2016), "Break-induced telomere synthesis underlies alternative telomere maintenance", <em>Nature</em>, 539:54-58. Doi: 10.1038/nature20099.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Douglas, G.R. et al. (1995), "Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice", <em>Proceedings of the National Academy of Sciences of the United States of America</em>, 92(16):7485-7489. Doi: 10.1073/pnas.92.16.7485.</span></span></p>
<p>Dunkenberger, Logan et al. (2022), “Comet Assay for the Detection of Single and Double-Strand DNA Breaks.” Methods in molecular biology (Clifton, N.J.), 2422: 263-269. doi:10.1007/978-1-0716-1948-3_18 </p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Fattah, F. et al., (2010), Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells. PLoS Genet, 6(2), doi:10.1371/journal.pgen.1000855</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Fitzgerald, D.M., P.J. Hastings, and S.M. Rosenberg (2017), "Stress-Induced Mutagenesis: Implications in Cancer and Drug Resistance", Ann Rev Cancer Biol, 1:119-140. Doi: 10.1146/annurev-cancerbio-050216-121919.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Gorbunova, V. and A. Seluanov. (2016), “DNA double strand break repair, aging and the chromatin connection”, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol.788/1-2, Elsevier, Amsterdam, http://dx.doi.org/10.1016/j.mrfmmm.2016.02.004. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Hammel, M. et al., (2011), XRCC4 protein interactions with XRCC4-like factor (XLF) create an extended grooved scaffold for DNA ligation and double strand break repair. J Biol Chem, 286(37), 32638-32650. doi:10.1074/jbc.M111.272641.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Hanawalt, P.C., J.M. Ford and D.R. Lloyd (2003), "Functional characterization of global genomic DNA repair and its implications for cancer", <em>Mutation Research</em>, 544(2-3): 107–114.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Harbach, P. R. et al., (1989), “The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes”, <em>Mutation Research</em>, 216(2):101-10. Doi:10.1016/0165-1161(89)90010-1.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Iyama, T. and D.M. Wilson III (2013), "DNA repair mechanisms in dividing and non-dividing cells", <em>DNA Repair</em>, 12(8): 620– 636.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Jeffrey, M. A.& M. G. Williams, (2000), “Lack of DNA-damaging Activity of Five Non-nutritive Sweeteners in the Rat Hepatocyte/DNA Repair Assay”, <em>Food and Chemical Toxicology</em>, 38: 335-338. Doi: 10.1016/S0278-6915(99)00163-5.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Köberle, B. et al. (1999), "Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours", <em>Curr. Biol.</em>, 9(5):273-6. Doi: <a href="https://doi.org/10.1016/s0960-9822(99)80118-3" target="_blank">10.1016/s0960-9822(99)80118-3</a>.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Kozmin, S.G. & S. Jinks-Robertson S. (2013), “The mechanism of nucleotide excision repair-mediated UV-induced mutagenesis in nonproliferating cells”, <em>Genetics</em>, 193(3): 803-17. Doi: 10.1534/genetics.112.147421.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Kramara, J., B. Osia, and A. Malkova (2018), "Break-Induced Replication: The Where, The Why, and The How", <em>Trends Genet</em>, 34:518-531. Doi: 10.1016/j.tig.2018.04.002.</span></span></p>
<p><span style="font-size:14px">Kuhne, M., G. Urban and M. Lo (2005), "DNA Double-Strand Break Misrejoining after Exposure of Primary Human Fibroblasts to CK Characteristic X Rays, 29 kVp X Rays and 60Co γ Rays", Radiation. Research, Vol.164/5, Radiation Research Society, Indianapolis, https://doi.org/10.1667/RR3461.1. </span></p>
<p><span style="font-size:14px">Larsen, K.H. et al. (1982), “DNA repair assays as tests for environmental mutagens: A report of the U.S. EPA gene-tox program”, Mutation Research, Vol.98/3, Elsevier, Amsterdam, https://doi.org/10.1016/0165-1110(82)90037-9. </span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Li Z, A. H. Pearlman, and P. Hsieh (2016), "DNA mismatch repair and the DNA damage response", <em>DNA Repair (Amst)</em>, 38:94-101.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Lieber, M. R., (2010), “The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway.” <em>Annu Rev Biochem</em>. 79:181-211. doi:10.1146/annurev.biochem.052308.093131.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Lieber, M. R. et al., (2003), “Mechanism and regulation of human non-homologous DNA end-joining”, <em>Nat Rev Mol Cell Biol</em>. 4(9):712-720. doi:10.1038/nrm1202.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Liu, Y. et al. (2009), "Coordination between polymerase beta and FEN1 can modulate CAG repeat expansion", J. Biol. Chem., 284(41): 28352-28366. Doi: 10.1074/jbc.M109.050286.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Mao, Z. et al., (2011), “SIRT6 promotes DNA repair under stress by activating PARP1”, <em>Science</em>. 332(6036): 1443-1446. doi:10.1126/science.1202723.</span></span></p>
<p><span style="font-size:14px">Mariotti, L.G. et al. (2013), “Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures”, PLoS ONE, Vol.8/11, PLoS, San Francisco, https://doi.org/10.1371/journal.pone.0079541. </span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Matthews, L. A., & L. A. Simmons, (2014), “Bacterial nonhomologous end joining requires teamwork”, <em>J Bacteriol</em>. 196(19): 3363-3365. doi:10.1128/JB.02042-14.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Menoni, H. et al. (2012), "Base excision repair of 8-oxoG in dinucleosomes", <em>Nucleic Acids Res.</em> ,40(2): 692-700. Doi: <a href="https://doi.org/10.1093/nar/gkr761" target="_blank">10.1093/nar/gkr761</a>.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Minocherhomji, S. et al. (2015), "Replication stress activates DNA repair synthesis in mitosis", <em>Nature</em>, 528:286-290. Doi: 10.1038/nature16139.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Miyaoka, Y. et al., (2016), “Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing”, <em>Sci Rep</em>, 6, 23549. doi:10.1038/srep23549/.</span></span></p>
<p>Mladenov. et al. (2023), . “New Facets of DNA Double Strand Break Repair: Radiation Dose as Key Determinant of HR versus c-NHEJ Engagement”. International journal of molecular sciences, 24(19), 14956. https://doi.org/10.3390/ijms241914956 </p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Moore, J. K., & J. E. Haber, (1996), “Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae”, <em>Molecular and Cellular Biology</em>, 16(5), 2164–73. Doi: 10.1128/MCB.16.5.2164.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Nacci, D. et al. (1992), “Application of the DNA alkaline unwinding assay to detect DNA strand breaks in marine bivalves”, Marine Environmental Research, Vol.33/2, Elsevier BV, Amsterdam, https://doi.org/10.1016/0141-1136(92)90134-8. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Nagel, Z.D. et al. (2014), "Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis", <em>Proc. Natl. Acad. Sci. USA</em>, 111(18):E1823-32. Doi: 10.1073/pnas.1401182111.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">O’Brien, J.M. et al. (2015), "Sublinear response in lacZ mutant frequency of Muta™ Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea", <em>Environ. Mol. Mutagen.</em>, 56(4): 347-55. Doi: 10.1002/em.21932.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Olive, L. P., J. P. Bnath & E. R. Durand, (1990), “Heterogeneity in Radiation-Induced DNA Damage and Repairing Tumor and Normal Cells Measured Using the "Comet" Assay”, <em>Radiation Research</em>. 122: 86-94. Doi: 10.1667/rrav04.1.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Pardo, B., B. Gomez-Gonzalez & A. Aguilera, (2009), “DNA repair in mammalian cells: DNA double-strand break repair: how to fix a broken relationship<em>”, Cell Mol Life Sci</em>, 66(6), 1039-1056. doi:10.1007/s00018-009-8740-3.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Pegg, A.E. (2011), "Multifaceted roles of alkyltransferase and related proteins in DNA repair, DNA damage, resistance to chemotherapy, and research tools", <em>Chem. Res. Toxicol.</em>, 4(5): 618-39. Doi: 10.1021/tx200031q.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Penninckx, S. et al. (2021), “Quantification of radiation-induced DNA double strand break repair foci to evaluate and predict biological responses to ionizing radiation”, NAR Cancer, Vol.3/4, Oxford University Press, Oxford, https://doi.org/10.1093/narcan/zcab046. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Rydberg, B. et al. (2005), "Dose-Dependent Misrejoining of Radiation-Induced DNA Double-Strand Breaks in Human Fibroblasts: Experimental and Theoretical Study for High- and Low-LET Radiation", Radiation Research, Vol.163/5, Radiation Research Society, Indianapolis, https://doi.org/10.1667/RR3346. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Sancar, A. (2003), "Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors", <em>Chem Rev.</em>, 103(6): 2203-37. Doi: 10.1021/cr0204348.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Saini, N. et al. (2017), "Migrating bubble during break-induced replication drives conservative DNA synthesis", <em>Nature</em>, 502:389-392. Doi: 10.1038/nature12584.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Sakofsky, C.J. et al. (2015), "Translesion Polymerases Drive Microhomology-Mediated Break-Induced Replication Leading to Complex Chromosomal Rearrangements", <em>Mol Cell</em>, 60:860-872. Doi: 10.1016/j.molcel.2015.10.041.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Schärer, O.D. (2013), "Nucleotide excision repair in eukaryotes", <em>Cold Spring Harb. Perspect. Biol.</em>, 5(10): a012609. Doi: 10.1101/cshperspect.a012609.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Scherer, E., A.A. Jenner and L. den Engelse (1987), "Immunocytochemical studies on the formation and repair of O6-alkylguanine in rat tissues", <em>IARC Sci Publ.</em>, 84: 55-8.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Seiler, F., K. Kamino, M. Emura, U. Mohr and J. Thomale (1997), "Formation and persistence of the miscoding DNA alkylation product O6-ethylguanine in male germ cells of the hamster", <em>Mutat Res.</em>, 385(3): 205-211. Doi: 10.1016/s0921-8777(97)00043-8.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Shelby, M.D. and K.R. Tindall (1997), "Mammalian germ cell mutagenicity of ENU, IPMS and MMS, chemicals selected for a transgenic mouse collaborative study",<em> Mutation Research</em>, 388(2-3): 99-109. Doi: 10.1016/s1383-5718(96)00106-4.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Seo, Y.R. and H.J. Jung (2004), "The potential roles of p53 tumor suppressor in nucleotide excision repair (NER) and base excision repair (BER)", <em>Exp. Mol. Med.</em>, 36(6): 505-509. Doi: 10.1038/emm.2004.64.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Sundheim, O. et al. (2008), "AlkB demethylases flip out in different ways",<em> DNA Repair (Amst)</em>., 7(11): 1916-1923. Doi: <a href="https://doi.org/10.1016/j.dnarep.2008.07.015" target="_blank">10.1016/j.dnarep.2008.07.015</a>.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Sung, P., & H. Klein, (2006), “Mechanism of homologous recombination: mediators and helicases take on regulatory functions”, <em>Nat Rev Mol Cell Biol</em>, 7(10), 739-750. Doi:10. 1038/nrm2008.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Trucco, C., et al., (1998), “DNA repair defect i poly(ADP-ribose) polymerase-deficient cell lines”, Nucleic Acids Research. 26(11): 2644–2649. Doi: 10.1093/nar/26.11.2644.</span></span></p>
<p><span style="font-size:14px">Trzeciak, A.R. et al. (2008), “Age, sex, and race influence single-strand break repair capacity in a human population”, Free Radical Biology & Medicine, Vol. 45, Elsevier, Amsterdam, https://doi.org/10.1016/j.freeradbiomed.2008.08.031. </span></p>
<p><span style="font-size:14px">White, R.R. and J. Vijg. (2016), “Do DNA Double-Strand Breaks Drive Aging?”, Molecular Cell, Vol.63, Elsevier, Amsterdam, http://doi.org/10.1016/j.molcel.2016.08.004. </span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Wyrick, J.J. & S. A. Roberts, (2015), “Genomic approaches to DNA repair and mutagenesis”, DNA Repair (Amst). 36:146-155. doi: 10.1016/j.dnarep.2015.09.018.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">van Zeeland, A.A., A. de Groot and A. Neuhäuser-Klaus (1990), "DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis", <em>Mutat. Res.</em>, 231(1): 55-62.</span></span></p>
2016-11-29T18:41:232024-03-08T12:15:51Increase, DNA strand breaksIncrease, DNA strand breaksMolecular<p>DNA strand breaks are a type of damage resulting from the hydrolysis of phosphodiester groups in the backbone of DNA molecules (Gates, 2009) and can occur on a single strand (single strand breaks; SSBs) or both strands (double strand breaks; DSBs) (Cannan and Pederson, 2016; Tamanoi and Yoshikawa, 2022; Tripathy et al., 2021). SSBs arise when the phosphate backbone connecting adjacent nucleotides in DNA is broken on one strand. DSBs are generated when both strands are simultaneously broken at sites that are sufficiently close to one another that base-pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. As a consequence, the two DNA ends generated by a DSB can physically dissociate from one another, becoming difficult to repair and increasing the chance of inappropriate recombination with other sites in the genome (Jackson, 2002). SSBs can also turn into DSBs if the replication fork stalls at the lesion leading to fork collapse. It is also worth noting that there are error-prone and error-free forms of DSB repair (Jackson, 2002), and that the SSB repair pathway are distinct form the DSB repair pathways.</p>
<p>Strand breaks are intermediates in various biological events, including DNA repair (e.g., base excision repair), V(D)J recombination in developing lymphoid cells and chromatin remodeling in both somatic cells and germ cells. The spectrum of damage can be intricate, resulting in complex lesions, leading to mutations, and clustered damage defined as two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns. These lesions are more difficult to repair and have been studied in many types of models (Barbieri et al., 2019 and Asaithamby et al., 2011)<span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">. </span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">DSBs and complex lesions are of particular concern, as they are considered the most lethal and deleterious type of DNA lesion. If misrepaired or left unrepaired, DSBs may drive the cell towards genomic instability, apoptosis or tumorigenesis (Beir, 1999).</span></span></p>
<p> </p>
<p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Please refer to the table below for details regarding these and other methodologies for detecting DNA DSBs.</span></span></p>
<p style="text-align:center"> </p>
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<p style="text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Assay Name</span></span></strong></p>
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<p style="text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">References</span></span></strong></p>
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<p style="text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Description</span></span></strong></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">OECD </span></span></strong><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Approved Assay</span></span></strong></p>
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<p style="margin-left:10px; margin-right:9px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Comet Assay (Single Cell Gel Eletrophoresis - Alkaline)</span></span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Collins, 2004; Olive </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">and Banath, 2006; Platel et al., 2011; Nikolova et al., 2017</span></span></p>
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<p style="margin-left:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect SSBs or DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at an alkaline pH (pH >13); DNA fragments are forced to move, forming a "comet"-like </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">appearance</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">Yes (No. 489)</span></p>
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<p style="margin-left:11px; margin-right:10px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Comet Assay (Single Cell Gel Eltrophoresis - Neutral)</span></span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Collins, 2014; Olive </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">and Banath, 2006; Anderson and Laubenthal, 2013; Nikolova et al., 2017</span></span></p>
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<p style="margin-left:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at a neutral pH; DNA fragments, which are not denatured at the neutral pH, are forced to move, forming a "comet"-</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">like appearance</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:1px; margin-right:-1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX Foci Quantification - Flow </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Cytometry</span></span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Rothkamm and Horn, 2009; Bryce et al., </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">2016</span></span></p>
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<p style="margin-left:26px; margin-right:-1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Measurement of <span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX immunostaining in cells by flow cytometry, normalized to total levels of H2AX</span></span></p>
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<p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></p>
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<p style="margin-left:12px; margin-right:11px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX Foci Quantification - </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Western Blot</span></span></p>
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<p style="margin-left:9px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Burma et al., 2001; Revet et al., 2011</span></span></p>
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<p style="margin-left:14px; margin-right:9px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Measurement of <span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX immunostaining in cells by Western blotting, normalized to total levels of H2AX</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:12px; margin-right:11px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX Foci Quantification - Microscopy</span></span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Redon et al., 2010; Mah et al., 2010; Garcia-Canton et al., </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">2013</span></span></p>
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<p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Quantification of <span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX immunostaining by counting <span style="font-family:"MS UI Gothic",sans-serif">γ</span>- H2AX foci visualized with a microscope</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:12px; margin-right:11px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX Foci Detection -</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif"> </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">ELISA and flow cytometry</span></span></p>
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<p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Ji et al., 2017; </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Bryce et al., 2016</span></span></p>
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<p style="margin-right:-1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Detection of <span style="font-family:"MS UI Gothic",sans-serif">γ</span>-H2AX in cells by ELISA, normalized to total levels of H2AX; γH2AX foci detection can be high-throughput and automated using flow cytometry-based immunodetection.</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">Pulsed Field Gel Electrophoresis (PFGE)</span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Ager et al., 1990; Gardiner et al., 1985; Herschleb et al., 2007; Kawashima et </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">al., 2017</span></span></p>
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<p style="margin-left:9px; margin-right:8px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect DSBs, cells are embedded and lysed in agarose, and the released DNA undergoes gel electrophoresis in which the direction of the voltage is periodically alternated; Large DNA fragments are thus </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">able to be separated by size</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">The TUNEL </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">(Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) Assay</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">Loo, 2011</span></p>
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<p style="margin-left:5px; margin-right:4px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect strand breaks, dUTPs added to the 3’OH end of a strand break by the DNA polymerase terminal deoxynucleotidyl transferase (TdT) are tagged with a fluorescent dye or a reporter enzyme to allow visualization (We note that this method is typically used to measure apoptosis)</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<p style="margin-left:7px; margin-right:6px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><em>In Vitro </em>DNA Cleavage Assays using </span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Topoisomerase</span></span></p>
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<p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Nitiss, 2012</span></span></p>
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<p style="margin-left:15px; margin-right:15px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Cleavage of DNA can be achieved using purified topoisomerase; DNA strand breaks can then be separated and quantified using gel electrophoresis</span></span></p>
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<p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
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<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Figueroa‑González & Pérez‑Plasencia, 2017 </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">Assay of strand breaks through the observation of DNA amplification prevention. Breaks block Taq polymerase, reducing the number of DNA templates, preventing amplification </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
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<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">Sucrose density gradient centrifuge </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Raschke et al. 2009 </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">Division of DNA pieces by density, increased fractionation leads to lower density pieces, with the use of a sucrose cushion </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
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<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">Alkaline Elution Assay </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Kohn, 1991 </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">Cells lysed with detergent-solution, filtered through membrane to remove all but intact DNA </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
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<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">Unwinding Assay </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Nacci et al. 1992 </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">DNA is stored in alkaline solutions with DNA-specific dye and allowed to unwind following removal from tissue, increased strand damage associated with increased unwinding </span></strong></td>
<td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
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<p><span style="font-size:11px"><strong>Taxonomic applicability: </strong>DNA strand breaks are relevant to all species, including vertebrates such as humans, that contain DNA (Cannan & Pederson, 2016). </span></p>
<p><span style="font-size:11px"><strong>Life stage applicability: </strong>This key event is not life stage specific as all life stages display strand breaks. However, there is an increase in baseline levels of DNA strand breaks seen in older individuals though it is unknown whether this change due to increased break induction or a greater retention of breaks due to poor repair (White & Vijg, 2016). </span></p>
<p><span style="font-size:11px"><strong>Sex applicability:</strong> This key event is not sex specific as both sexes display evidence of strand breaks. In some cell types, such as peripheral blood mononuclear cells, males show higher levels of single strand breaks than females (Garm et al., 2012). </span></p>
<p><span style="font-size:11px"><strong>Evidence for perturbation by a stressor: </strong>There are studies demonstrating that increased DNA strand breaks can result from exposure to multiple stressor types including ionizing & non-ionizing radiation, chemical agents, and oxidizing agents (EPRI, 2014; Hamada, 2014; Cencer et al., 2018; Cannan & Pederson, 2016; Yang et al., 1998). </span></p>
HighUnspecificHighAll life stagesNot Specified<p>Ager, D. D. et al. (1990). “Measurement of Radiation- Induced DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis.” Radiat Res. 122(2), 181-7.</p>
<p>Anderson, D. & Laubenthal J. (2013), “Analysis of DNA Damage via Single-Cell Electrophoresis. In: Makovets S, editor. DNA Electrophoresis. Totowa.”, NJ: Humana Press. p 209-218.</p>
<p>Asaithamby, A., B. Hu and D.J. Chen. (2011) Unrepaired clustered DNA lesions induce chromosome breakage in human cells. Proc Natl Acad Sci U S A 108(20): 8293-8298 .</p>
<p>Barbieri, S., G. Babini, J. Morini et a l (2019). . Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures. Scientific Reports 9(1): 14019</p>
<p>Beir, V. et al. (1999), “The Mechanistic Basis of Radon-Induced Lung Cancer”, in Health Risks of Exposure to Radon: BEIR VI, National Academy Press, Washington, D.C., https://doi.org/10.17226/5499 </p>
<p>Bryce, S. et al. (2016), “Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.”, Environ Mol Mutagen. 57:171-189. Doi: 10.1002/em.21996.</p>
<p>Burma, S. et al. (2001), “ATM phosphorylates histone H2AX in response to DNA double-strand breaks.”, J Biol Chem, 276(45): 42462-42467. doi:10.1074/jbc.C100466200</p>
<p>Cannan, W.J. and D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", Journal of Cellular Physiology, Vol.231/1, Wiley, New York, https://doi.org/10.1002/jcp.25048. </p>
<p>Cannan, W.J. and D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", Journal of Cellular Physiology, Vol.231/1, Wiley, New York, https://doi.org/10.1002/jcp.25048. </p>
<p>Cencer, C. et al. (2018), “PARP-1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light”, Photochemistry and Photobiology, Vol.94/1, Wiley-Blackwell, Hoboken, https://doi.org/10.1111/php.12814. </p>
<p>Charlton, E. D. et al. (1989), “Calculation of Initial Yields of Single and Double Stranded Breaks in Cell Nuclei from Electrons, Protons, and Alpha Particles.”, Int. J. Radiat. Biol. 56(1): 1-19. doi: 10.1080/09553008914551141.</p>
<p>Collins, R. A. (2004), “The Comet Assay for DNA Damage and Repair. Molecular Biotechnology.”, Mol Biotechnol. 26(3): 249-61. doi:10.1385/MB:26:3:249</p>
<p>Durdik, M et al. (2015), “Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes.” Cytometry. Part A. 87(12): 1070-8. Doi:10.1002/cyto.a.22731 </p>
<p>EPRI (2014), Epidemiology and mechanistic effects of radiation on the lens of the eye: Review and scientific appraisal of the literature, EPRI, California. </p>
<p>Figueroa‑González, G. and C. Pérez‑Plasencia. (2017), “Strategies for the evaluation of DNA damage and repair mechanisms in cancer”, Oncology Letters, Vol.13/6, Spandidos Publications, Athens, https://doi.org/10.3892/ol.2017.6002. </p>
<p>Garcia-Canton, C. et al. (2013), “Assessment of the in vitro p-H2AX assay by High Content Screening asa novel genotoxicity test.”, Mutat Res. 757:158-166. Doi: 10.1016/j.mrgentox.2013.08.002</p>
<p>Gardiner, K. et al. (1986), “Fractionation of Large Mammalian DNA Restriction Fragments Using Vertical Pulsed-Field Gradient Gel Electrophoresis.”, Somatic Cell and Molecular Genetics. 12(2): 185-95.Doi: 10.1007/bf01560665.</p>
<p>Garm, C. et al. (2012), “Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells”, Aging Cell, Vol.12/1, Blackwell Publishing Ltd, Oxford, https://doi.org/10.1111/acel.12019. </p>
<p>Guo, X. et al. (2018), “Acetylation of 53BP1 dictates the DNA double strand break repair pathway.” Nucleic acids research. 46(2): 689-703. doi:10.1093/nar/gkx1208 </p>
<p>Hamada, N. (2014), “What are the intracellular targets and intratissue target cells for radiation effects?”, Radiation research, Vol. 181/1, The Radiation Research Society, Indianapolis, https://doi.org/10.1667/RR13505.1. </p>
<p>Herschleb, J. et al. (2007), “Pulsed-field gel electrophoresis.”, Nat Protoc. 2(3): 677-684. doi:10.1038/nprot.2007.94</p>
<p>Jackson, S. (2002). “Sensing and repairing DNA double-strand breaks.”, Carcinogenesis. 23:687-696. Doi:10.1093/carcin/23.5.687.</p>
<p>Ji, J. et al. (2017), “Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay.”, PLoS One. 12(2): e0171582. doi:10.1371/journal.pone.0171582</p>
<p>Kawashima, Y.(2017), “Detection of DNA double-strand breaks by pulsed-field gel electrophoresis.”, Genes Cells 22:84-93. Doi: 10.1111/gtc.12457.</p>
<p>Khoury, L. et al. (2013), “Validation of high-throughput genotoxicity assay screening using cH2AX in-cell Western assay on HepG2 cells.”, Environ Mol Mutagen, 54:737-746. Doi: 10.1002/em.21817.</p>
<p>Khoury, L. et al. (2016), “Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening.”, Mutagenesis, 31:83-96. Doi: <a href="https://doi.org/10.1093/mutage/gev058" target="_blank">10.1093/mutage/gev058</a>.</p>
<p>Kohn, K.W. (1991), “Principles and practice of DNA filter elution”, Pharmacology & Therapeutics, Vol.49/1, Elsevier, Amsterdam, https://doi.org/10.1016/0163-7258(91)90022-E. </p>
<p>Loo, DT. (2011), “In Situ Detection of Apoptosis by the TUNEL Assay: An Overview of Techniques. In: Didenko V, editor. DNA Damage Detection In Situ, Ex Vivo, and In Vivo. Totowa.”, NJ: Humana Press. p 3-13.doi: <a href="https://doi.org/10.1007/978-1-60327-409-8_1" target="_blank">10.1007/978-1-60327-409-8_1</a>.</p>
<p>Mah, L. J. et al. (2010), “Quantification of gammaH2AX foci in response to ionising radiation.”, J Vis Exp(38). doi:10.3791/1957.</p>
<p>Nacci, D. et al. (1992), “Application of the DNA alkaline unwinding assay to detect DNA strand breaks in marine bivalves”, Marine Environmental Research, Vol.33/2, Elsevier BV, Amsterdam, https://doi.org/10.1016/0141-1136(92)90134-8. </p>
<p>Nikolova, T., F. et al. (2017), “Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.”, Mutat Res 822:10-18. Doi: <a href="https://doi.org/10.1016/j.mrgentox.2017.07.004" target="_blank">10.1016/j.mrgentox.2017.07.004</a>.</p>
<p>Nitiss, J. L. et al. (2012), “Topoisomerase assays. ”, Curr Protoc Pharmacol. Chapter 3: Unit 3 3.</p>
<p>OECD. (2014). Test No. 489: “In vivo mammalian alkaline comet assay.” OECD Guideline for the Testing of Chemicals, Section 4 .</p>
<p>Olive, P. L., & Banáth, J. P. (2006), “The comet assay: a method to measure DNA damage in individual cells.”, Nature Protocols. 1(1): 23-29. doi:10.1038/nprot.2006.5.</p>
<p>Platel A. et al. (2011), “Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the <em>in vitro </em>modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels.”, Mutat Res 726:151-159. Doi: 10.1016/j.mrgentox.2011.09.003.</p>
<p>Popp, H. D. et al. (2017), “Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks”, Journal of visualized experiments, 129: 56617, doi:10.3791/56617 </p>
<p>Raschke, S., J. Guan and G. Iliakis. (2009), “Application of alkaline sucrose gradient centrifugation in the analysis of DNA replication after DNA damage”, Methods in Molecular Biology, Vol.521, Humana Press, Totowa, https://doi.org/10.1007/978-1-60327-815-7_18. </p>
<p>Redon, C. et al. (2010), “The use of gamma-H2AX as a biodosimeter for total-body radiation exposure in non-human primates.”, PLoS One. 5(11): e15544. doi:10.1371/journal.pone.0015544</p>
<p>Revet, I. et al. (2011), “Functional relevance of the histone γH2Ax in the response to DNA damaging agents.” Proc Natl Acad Sci USA.108:8663-8667. Doi: 10.1073/pnas.1105866108</p>
<p>Rogakou, E.P. et al. (1998), “DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139.” , J Biol Chem, 273:5858-5868. Doi: 10.1074/jbc.273.10.5858</p>
<p>Rothkamm, K. & Horn, S. (2009), “γ-H2AX as protein biomarker for radiation exposure.”, Ann Ist Super Sanità, 45(3): 265-71.</p>
<p>Tamanoi, F., & Yoshikawa, K. (2022), “Overview of DNA damage and double-strand breaks”, The Enzymes, Vol.51, 1–5. https://doi.org/10.1016/bs.enz.2022.08.001 </p>
<p>Tripathy, B. K., Pal, K., Shabrish, S., & Mittra, I. (2021), “A New Perspective on the Origin of DNA Double-Strand Breaks and Its Implications for Ageing” Genes, Vol.12/2, 163. <a href="https://doi.org/10.3390/genes12020163" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/genes12020163</a> </p>
<p>White, R.R. and J. Vijg. (2016), “Do DNA Double-Strand Breaks Drive Aging?”, Molecular Cell, Vol.63, Elsevier, Amsterdam, http://doi.org/10.1016/j.molcel.2016.08.004. </p>
<p>Yang, Y. et al. (1998), “The effect of catalase amplification on immortal lens epithelial cell lines”, Experimental Eye Research, Vol.67/6, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1998.0560. </p>
2019-05-19T16:33:202024-03-08T12:05:51Increase, ApoptosisIncrease, ApoptosisCellular2017-04-15T16:17:342017-04-15T16:17:34Reduce, Sperm countReduce, Sperm countIndividual2020-04-06T09:49:032020-04-06T11:40:28529654da-b29b-4747-bb32-136aa8bc262d6ea1aee1-fc63-4430-8b5c-188a3045182b<p>Alkylated DNA may be tolerated and/or repaired error-free by a variety of DNA repair pathways. However, at high doses, it is established that the primary DNA repair pathway (O6-Alkylguanine-DNA alkyltransferase: AGT) responsible for removing alkylated DNA becomes saturated. This may lead to several potential adduct fates: (i) error-free repair of the DNA adduct using alternative DNA repair mechanisms; (ii) no repair (DNA damage is retained); or (iii) instability in the DNA duplex leading to DNA strand breaks and possibly activation of DNA damage signaling. For repair of alkyl adducts it is well established that the O6-alkylguanine-DNA alkyltransferase pathway becomes saturated at high doses leading to insufficient repair at high doses.</p>
<p>General details: The weight of evidence for this KER is strong. It is widely accepted that damaged DNA is subject to repair, and that in the absence of DNA repair, mutations will ensue. Specifically, AGT (Damage Reversal DNA repair: pathway #1 in KE155), also known as O6-methylguanine-DNA methyltransferase (MGMT), reverses alkylation damage by directly transferring alkyl groups from the O6 position of guanine to a cysteine residue on the AGT (or MGMT) molecule, restoring the DNA in a single step. However, transfer of the alky group to AGT results in concomitant inactivation of AGT (Pegg 2011). The mammalian protein is also active on O6-ethylguanine and can remove only one ethyl group from DNA, following which the protein is degraded. Thus, high levels of alkylation damage overwhelm the cellular AGT capacity to remove lesions. In mammalian cells, O4-ethylthymine and O2-ethylthymine are poor substrates for AGT (Fang et al. 2010) and no other DNA repair pathway has been identified that is able to efficiently repair these lesions; consequently, these lesions are extremely persistent in cells. Reviews on this topic have been published (Kaina et al. 2007; Pegg 2011). In the absence of the AGT/MGMT pathway, other DNA repair pathways may be invoked, but the relative efficiency of these pathways is not well understood (further details described below).</p>
<p><br />
The role of nucleotide excision repair (NER; excision repair pathways: #2 in KE155) in alkylation damage repair in mammalian cells remains unclear. Earlier studies using human cell lines suggested that both AGT and NER may be involved in the repair of O6-ethylguanine (Bronstein et al. 1991; Bronstein et al. 1992). Very recently, an alkyltransferase like protein (ATL1) that has homology to AGT has been identified in a range of prokaryotes and lower eukaryotes. This protein has no alkyltransferase activity but can couple O6-alkylguanine damage to NER (Latypov et al. 2012). ATL1 proteins have not yet been identified in mammals.</p>
<p><br />
Some alkyl adducts, such as N7-ethylguanine and N3-ethyladenine, are inherently unstable and may depurinate (i.e., hydrolytic cleavage of the glycosidic bond, which releases adenine or guanine). The resultant abasic sites are normally repaired through error-free pathways although they may occasionally be transformed to DNA strand breaks. In mammals, N-methylpurine DNA glycosylases, such as alkyladenine DNA glycosylase (AAG), have a wide range of substrates including N7-alkylguanine and N3-alkyladenine derivatives (Wyatt et al. 1999). However, there are no specific reports in the literature that the ethylated derivatives are AAG substrates. Glycosylases such as AAG yield abasic sites that are processed as described above. An alternative repair mechanism for repairing minor lesions such as N3-ethylcytosine and N1-ethyladenine is through oxidative dealkylation catalyzed by AlkB and mammalian homologs (Drabløs et al. 2004). This pathway is an error-free damage reversal pathway that releases the oxidized ethyl group as acetaldehyde (Duncan et al. 2002).</p>
<p><br />
A final mechanism through which DNA repair pathways may influence the fate of alkylation damage is through futile cycling of the mismatch repair (MMR; excision repair pathways: #2 in KE155) system at an O6-alkyl G:T mispair. In this scenario, unrepaired O6-alkylguanine is able to mispair with T, and the mispair is recognized by MMR enzymes resulting in the removal of the newly incorporated thymine from the nascent strand opposite the O6-alkyguanine adduct. During DNA repair synthesis, O6-alkylguanine preferentially pairs once again with thymine, reinitiating the repair/synthesis cycle. This iteration of excision and synthesis may produce strand breaks and activate damage signaling pathways (York and Modrich 2006).</p>
<p> </p>
<p>If the pathways described above become saturated or do not operate properly, the alkylated DNA will not be repaired and will provide a template for replication of this damaged DNA. This is widely understood and accepted. Many studies have demonstrated that the introduction of plasmids or vectors with alkylated DNA (i.e., unrepaired lesions) into prokaryotic and eukaryotic cells, followed by replication, results in the formation of mutations at the alkylated sites, and that the probability of a mutation occurring at the alkylated site is modified by specific DNA repair genes/pathways (reviewed in Basu and Essigmann 1990; Shrivastav et al. 2010).</p>
<p>Insufficient repair is inferred from the formation and retention of adducts, and the formation of increased numbers of mutations above background (i.e., KE185 - methodologies described therein).</p>
<p>A variety of studies show that alkylated DNA persists for prolonged periods of time post-exposure. For example, persistence of different alkylated nucleotides was shown in livers and brains of C57BL mice exposed to N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulfonate using high-performance liquid chromatography several days post-exposure (Frei et al., 1978). The stability of methyl and ethyl adducts in somatic tissues for various adduct types is summarized in Beranuk, 1990. The in vivo liver half life of methyl adducts ranges from 0-3 days, and liver ethyl adduct half lives can be up to 17 days, indicating poorer repair of oxygen-bound ethyl adducts. This prolonged retention of adducts indicates that there is insufficient repair by AGT or other DNA repair pathways of these adducts.</p>
<p>Studies in both hamsters and rats show persistence of alkylated nucleotides several days post-exposure, indicating lack of DNA repair of some adducts (Scherer et al. 1987; Seiler et al. 1997). For example, 101xC3H mouse hybrid testes exhibited DNA adducts within 1 hour of exposure to ENU (10 or 100 mg/kg by i.p.), but some adducts remained unrepaired six days post-exposure (Sega et al. 1986). O6-ethylguanine adducts were also found in hamster spermatogonia DNA up to four days after exposure to DEN (100 µg/g body weight) (Seiler et al. 1997). O6-ethylguanine adducts were found in spermatogonia 1.5 hours post-exposure to ENU in Syrian Golden hamsters (Seiler et al. 1997). Approximately 30% persisted in spermatogonia four days post-exposure. Moreover, the amount of O6-ethylguanine recovered after a 100 mg ENU/kg exposure was 40% greater than predicted from a linear extrapolation of the amount of O6-ethylguanine recovered after exposure to 10 mg/kg. The data suggest that the high dose exposure to ENU results in depletion of AGT within the testis and permits O6-ethylguanine to persist at higher levels than would be predicted from lower exposure. The relationship between dose and formation of DNA adducts in tubular germ cells is non-linear, indicating relatively rapid repair at low doses that becomes saturated at higher doses (van Zeeland et al. 1990). Thus, with increasing dose, increasing incidence of KE1 (insufficient repair) occurs. This implies that mouse spermatogonia are capable of repairing a major part of the DNA damage at low doses. However, at higher doses the repair process is saturated and mutations begin to occur. Indeed, the dose-response curve for mutations in spermatogonia measured in sperm of exposed males is sub-linear with a clear point of inflection at low sub-chronic doses of ENU (O’Brien et al. 2015).</p>
<p>Finally, both alkyl adducts and mutations increase with increasing doses of alkylating agents in somatic cells and in male germ cells, indicating that DNA repair processes are not operating to remove all of the damage (ability to remove adducts and prevent mutations).</p>
<p>DNA repair is not generally measured directly; thus, insufficient repair is inferred from the retention of adducts or the induction of increases in mutation frequencies post-exposure. In addition, various sizes of alkylation groups (e.g., methyl, ethyl, propyl) can be involved. Although it appears that the larger alkyl adducts tend to be more mutagenic (Beranek, 1990), this is not completely established and there are insufficient data to establish that this is true for germ cells. However, in general, this KER is biologically plausible, broadly accepted for alkyl adducts and has few uncertainties. The direct measurement of insufficient repair can be considered a data gap.</p>
<p><em>Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships? </em></p>
<p>There is a clear need to exceed a specific dose to overwhelm the DNA repair process. Kinetics of DNA repair saturation in somatic cells is described in Muller et al. (2009). The shapes of the dose-response curve for mutation induction in male germ cells is sub-linear, supporting that this effect occurs in both somatic cells and spermatogonia. There is a general understanding that methyl adducts are more readily repaired that ethyl adducts, which contributes to quantitative differences between chemicals in their genotoxic potency. There are no models that exist for this to our knowledge.</p>
ModerateModerate<p>DNA adducts can occur in any cell type. While there are differences across taxa, all species have some DNA repair systems in place and it is common to extrapolate conclusions across eukaryotic species.</p>
<p>Basu, A.K. and J.M. Essigmann (1990), "Site-specific alkylated oligodeoxynucleotides: Probes for mutagenesis, DNA repair and the structure effects of DNA damage", <em>Mutation Research</em>, 233: 189-201.</p>
<p>Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", <em>Mutation Research</em> , 231(1): 11-30.</p>
<p>Bronstein, S.M., J.E. Cochrane, T.R. Craft, J.A. Swenberg and T.R. Skopek (1991), "Toxicity, mutagenicity, and mutational spectra of N-ethyl-N-nitrosourea in human cell lines with different DNA repair phenotypes", <em>Cancer Research</em>, 51(19): 5188-5197.</p>
<p>Bronstein, S.M., T.R. Skopek and J.A. Swenberg (1992), "Efficient repair of O6-ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells", <em>Cancer Research</em>, 52(7): 2008-2011.</p>
<p>Drabløs, F., E. Feyzi, P.A. Aas, C.B. Vaagbø, B. Kavli, M.S. Bratlie, J. Peña-Diaz, M. Otterlei, G. Slupphaug and H.E. Krokan (2004), "Alkylation damage in DNA and RNA--repair mechanisms and medical significance", <em>DNA Repair</em>, 3(11): 1389-1407.</p>
<p>Duncan, T., S.C. Trewick, P. Koivisto, P.A. Bates, T. Lindahl and B. Sedgwick B (2002), "Reversal of DNA alkylation damage by two human dioxygenases", <em>Proc. Natl. Acad. Sci. USA</em>, 99(26): 16660-16665.</p>
<p>Fang, Q., S. Kanugula, J.L. Tubbs, T.A. Tainer and A.E. Pegg (2010), "Repair of O4-alkylthymine by O6-alkylguanine-DNA alkyltransferases", <em>J. Biol. Chem.</em> 12(285): 885-895.</p>
<p>Frei, J.V., D.H .Swenson, W. Warren, P.D. Lawley (1978), "Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography", <em>Biochem. J.</em>, 174(3): 1031-1044.</p>
<p>Kaina, B., M. Christmann, S. Naumann and W.P. Roos (2007), "MGMT: Key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents", <em>DNA Repair</em>, 6: 1079–1099.</p>
<p>Latypov, V.F., J.L. Tubbs, A.J. Watson, A.S. Marriott, G. McGown, M. Thorncroft, O.J. Wilkinson, P. Senthong, A. Butt, A.S. Arvai, C.L. Millington, A.C. Povey, D.M. Williams, M.F. Santibanez-Koref, J.A. Tainer and G.P. Margison GP (2012), "Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines", <em>Mol. Cell</em>, 47(1): 50-60.</p>
<p>Muller, L., E. Gocke, T. Lave and T. Pfister (2009), "Ethyl methanesulfonate toxicity in Viracept – a comprehensive assessment based on threshold data for genotoxicity", <em>Toxicology Letters</em>, 190: 317-329.</p>
<p>O’Brien, J.M., M. Walker, A. Sivathayalan, G.R. Douglas, C.L. Yauk and F. Marchetti (2015), "Sublinear response in lacZ mutant frequency of Muta™ Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea", <em>Environ. Mol. Mutagen.</em>, 56(4): 347-55.</p>
<p>Pegg, A.E. (2011), "Multifaceted roles of alkyltransferase and related proteins in DNA repair, DNA damage, resistance to chemotherapy, and research tools", <em>Chem. Res. Toxicol.</em>, 24(5): 618-639.</p>
<p>Scherer, E., A.A. Jenner and L. den Engelse (1987), "Immunocytochemical studies on the formation and repair of O6-alkylguanine in rat tissues", <em>IARC Sci. Publ.</em>, 84: 55-8.</p>
<p>Sega, G.A., C.R. Rohrer, H.R. Harvey and A.E. Jetton (1986), "Chemical dosimetry of ethyl nitrosourea in the mouse testis", <em>Mutat. Res.</em>, 159(1-2): 65-74.</p>
<p>Seiler, F., K. Kamino, M. Emura, U. Mohr and J. Thomale (1997), "Formation and persistence of the miscoding DNA alkylation product O6-ethylguanine in male germ cells of the hamster", <em>Mutat. Res.</em>, 385(3): 205-211.</p>
<p>Shrivastav, N., D. Li and J.M. Essignmann (2010), "Chemical biology of mutagenesis and DNA repair: cellular response to DNA alkylation", <em>Carcinogenesis</em>, 31(1): 59-70.</p>
<p>van Zeeland, A.A., A. de Groot and A. Neuhäuser-Klaus (1990), "DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis", <em>Mutat. Res.</em>, 231(1):55-62.</p>
<p>Wyatt, M.D., J.M. Allan, A.Y. Lau, T.E. Ellenberger, L.D. Samson (1999), "3-methyladenine DNA glycosylases: structure, function, and biological importance", <em>Bioessays</em>, 21(8): 668-676.</p>
<p>York S.J. and P. Modrich (2006), "Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts", <em>J. Biol. Chem.</em>, 281(32): 22674-22683.</p>
2016-11-29T18:41:332019-12-10T10:43:256ea1aee1-fc63-4430-8b5c-188a3045182bbb9246a5-873d-4837-9f40-76c82c90ed10<p>Inadequate repair of DNA damage includes incorrect repair (i.e., incorrect base insertion), incomplete repair (i.e., accumulation of repair intermediates such as strand breaks, stalled replications forks, and/or abasic sites), and absent repair resulting in the retention of DNA damage.</p>
<p>It is well-established that DNA excision repair pathways require DNA strand breakage for removing the damaged sites; for example, base excision repair (BER) of oxidative lesions involves removal of oxidized bases by glycosylases followed by cleavage of the DNA strand 5’ from the abasic site. If the repair process is disrupted at this point, repair intermediates including single strand breaks (SSB) may persist in the DNA. A SSB can turn into a double strand break (DSB) if it occurs sufficiently close to another SSB on the opposite strand. SSBs can be converted into DSBs when helicase unwinds the DNA strands during replication. Furthermore, SSBs and abasic sites can act as replication blocks causing the replication fork to stall and collapse, giving rise to DSBs <!--[if supportFields]><span
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<p>The two most common DSB repair mechanisms are non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is may favoured over HR and has also been shown to be 10<sup>4</sup> times more efficient than HR in repairing DSBs (Godwin et al., 1994; Benjamin and Little, 1992). There are two subtypes of NHEJ: canonical NHEJ (C‐NHEJ) or alternative non-homologous end joining (alt-NHEJ). During C-NHEJ, broken ends of DNA are simply ligated together. In alt‐NHEJ, one strand of the DNA on either side of the break is resected to repair the lesion (Betermeir et al., 2014). Although both repair mechanisms are error‐prone (Thurtle‐Schmidt and Lo, 2018), alt-NHEJ is considered more error-prone than C-NHEJ (Guirouil-Barbat et al., 2007; Simsek and Jasin, 2010). While NHEJ may prevent cell death due to the cytotoxicity of DSBs, it may lead to mutations and genomic instability downstream. </p>
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<p>1. DNA strand breaks generated due to faulty attempted repair</p>
<p>Excision repair pathways require the induction of SSB as part of damage processing. Increases in DNA lesions may lead to the accumulation of intermediate SSB. Attempted excision repair of lesions on opposite strands can turn into DSBs if the two are in close proximity (Eccles et al., 2010). Generation of DSBs has been observed in both nucleotide excision repair (NER) and BER (Ma et al., 2009; Wakasugi et al., 2014).</p>
<p>Previous studies have demonstrated that an imbalance in one of the multiple steps of BER can lead to an accumulation of repair intermediates and failed repair. It is highly likely that a disproportionate increase in oxidative DNA lesions compared to the level of available BER glycosylases leads to an imbalance between lesions and the initiating step of BER <!--[if supportFields]><span class=Geen><span
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Brenerman,B. 2014}}<span style='mso-element:field-separator'></span></span></span><![endif]-->(Brenerman et al., 2014)<!--[if supportFields]><span class=Geen><span
style='font-size:12.0pt;font-family:"Times New Roman","serif";mso-bidi-font-family:
Calibri'><span style='mso-element:field-end'></span></span></span><![endif]-->. Accumulation of oxidative lesions, abasic sites, and SSBs generated from OGG1, NTH1, and APE1 activities would be observed as a result. Moreover, studies have reported accumulation of SSB due to OGG1- and NHT1-overexpression <!--[if supportFields]><span
class=Geen><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
mso-bidi-font-family:Calibri'><span style='mso-element:field-begin'></span>ADDIN
RW.CITE{{260 Yang,N. 2004; 311 Yoshikawa,Y. 2015; 255 Wang,R. 2018}}<span
style='mso-element:field-separator'></span></span></span><![endif]-->(Yang et al., 2004; Yoshikawa et al., 2015; Wang et al., 2018)<!--[if supportFields]><span
class=Geen><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
mso-bidi-font-family:Calibri'><span style='mso-element:field-end'></span></span></span><![endif]-->. BER repair intermediates have been observed to interfere with transcription as well <!--[if supportFields]><span
class=Geen><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
mso-bidi-font-family:Calibri'><span style='mso-element:field-begin'></span>ADDIN
RW.CITE{{408 Kitsera,N. 2011}}<span style='mso-element:field-separator'></span></span></span><![endif]-->(Kitsera et al., 2011)<!--[if supportFields]><span class=Geen><span
style='font-size:12.0pt;font-family:"Times New Roman","serif";mso-bidi-font-family:
Calibri'><span style='mso-element:field-end'></span></span></span><![endif]-->. While overexpression may lead to imbalanced lyase activities that generate excessive SSB intermediates, deficiency of these enzymes is also known to cause an accumulation of oxidative lesions that could lead to strand breaks downstream. Hence, both the overexpression and deficiencies of repair enzymes can lead to strand breaks due to excessive activity or inadequate repair, respectively.</p>
<p>2. DNA strand breaks generated due to replication stress caused by accumulated DNA lesions</p>
<p>Retention of DNA lesions (i.e., damaged bases and SSB) can interfere with the progression of the replication fork. Thymidine glycol is an example of an oxidative DNA lesion that acts as a replication block (Dolinnaya et al., 2013). Persistent replication fork stalling and dissociation of replication machinery are known to cause the replication fork to collapse, which generates highly toxic DSBs (Zeman and Cimprich, 2014; Alexander and Orr-Weaver, 2016). Fork stalling also increases the risk of two replication forks colliding with each other, generating DSBs.</p>
<p>In addition, the replication fork can collide with SSBs generated during BER, hindering the completion of repair and giving rise to DSBs (Ensminger et al., 2014).</p>
<div>
<div>
<div>
<p> </p>
</div>
</div>
</div>
<p>In vitro studies with empirical evidence are shown below for select DNA repair pathways. These studies build in elements of essentiality (modulation of DNA repair), as well as dose and incidence concordance. The primary evidence is essentiality, where repair is genetically modulated in some way. Because multiple lines of evidence are considered within individual studies, we present the data by source of evidence (in vitro versus in vivo) rather than by type of empirical evidence (dose, incidence, or temporal concordance; essentiality) to avoid repetitive use of the same studies.</p>
<p> </p>
<p><u>Inadequate repair of oxidative lesions</u></p>
<ul>
<li>Concentration concordance of strand breaks in repair-deficient and –proficient cells (insufficient repair) (Wu et al., 2008)
<ul style="list-style-type:circle">
<li>In a study using A549 human adenocarcinoma cells, DNA strand breaks in hOGG1-proficient and hOGG1-deficient cells were compared following exposure to increasing concentrations of bleomycin.</li>
<li>Strand breaks were measured as DNA migration length in alkaline comet assay after 3 hours of exposure to six increasing concentrations (0.05, 0.25, 0.5, 1, 5, and 10 mg/L).</li>
<li>Concentration-dependent increase in strand breaks was observed in both cell types; however, at all concentrations significantly more strand breaks (p<0.05) were present in the hOGG1-deficient cells than in the proficient cells, demonstrating insufficient repair of oxidative lesions leading to DNA strand breaks.</li>
<li>Thus, this evidence supports the essentiality of inadequate DNA repair as a modulator of the downstream KE.</li>
</ul>
</li>
<li>Incomplete OGG1-initiated base excision repair (BER) leads to DNA strand breaks (Wang et al., 2018):
<ul>
<li>In a study using mouse embryonic fibroblasts (MEF), Ogg1+/+ and Ogg1-/- cells were treated with increasing concentrations of H2O2 for varying durations<br />
Higher levels of 8-oxodG were detected in Ogg1-/- cells compared to Ogg1+/+ cells after treatment with 400 µM H2O2 at all time points (5, 15, 30, 60, and 90 min)
<ul>
<li>Demonstrates insufficient removal of 8-oxo-dG in OGG1-deficient cells</li>
</ul>
</li>
<li>Significantly more strand breaks, as indicated by the higher % of TUNEL-positive cells (p<0.001), were detected in Ogg1+/+ cells compared to Ogg1-/- cells after exposure to 400 µM H2O2 for 3 hours
<ul>
<li>Both cell types showed a very similar increase in DNA strand breaks at lower concentrations (50, 100, and 200<br />
µM) and there was no significant difference between Ogg1+/+ and Ogg1-/- cells at these concentrations – this suggests that up to a certain level of oxidative damage, OGG1-initiated BER does not exacerbate strand breaks but when oxidative stress is excessive (at 400µM in this study), OGG1-initiated BER is compromised and leads to increased strand breaks (incomplete repair)</li>
</ul>
</li>
<li>Finally, DNA strand breaks in both cell types were measured using both alkaline and neutral comet assay after a 30- minute exposure to 400µM H2O2; while there was an increase in the olive tail moment (indicating DNA strand breaks) in both cell types compared to the control, the increase of strand breaks in Ogg1+/+ cells was significantly larger than in Ogg1-/- cells in both assays (p<0.001)</li>
</ul>
</li>
</ul>
<p> </p>
<p><u>Inadequate repair of alkylated DNA</u></p>
<ul>
<li>Interference of N-methylpurine DNA glycosylase (MPG)-initiated BER by replication leading to strand breaks (Ensminger et al., 2014)
<ul style="list-style-type:circle">
<li>A549 human alveolar basal epithelial cells were exposed to increasing concentrations of methylmethane sulfonate (MMS) for 1 hour and replicating cells were labeled using a thymidine analogue, 5-ethynyl-2’-desoxyuridine (EdU).</li>
<li>In S-phase cells, MMS concentration-dependent increase in γH2AX foci was detected (70 foci/cell at the highest concentration). In contrast, γH2AX foci were not detected G1- and G2-phase cells until the highest concentration (15 foci/cell).</li>
<li>MPG-depleted cells in S-phase showed no significant increase in γH2AX foci, while the control cells showed significant MMS concentration-dependent increases.</li>
<li>These results suggest interference of MPG-initiated BER by replication, leading to DSBs, and that the depletion of MPG decreases the probability of strand breaks in S-phase (evidence of essentiality of ‘inadequate repair’ to KEdown). </li>
</ul>
</li>
</ul>
<p><br />
</p>
<p><u>Inadequate mismatch repair </u></p>
<ul>
<li>Incomplete/incorrect mismatch repair (MMR) leads to DNA strand breaks (Peterson-Roth et al., 2005):
<ul style="list-style-type:circle">
<li>MLH1 (MMR protein)-deficient and -proficient HCT116 human colon cancer cells were treated with 30µM K<sub>2</sub>CrO<sub>4</sub> (DNA crosslinking, Cr adducts, protein-DNA crosslinking, DNA oxidation) for 3, 6, and 12 hours and γH2AX foci (biomarker of DNA DSB) were scored by fluorescence microscopy</li>
<li>At 6 and 12 hours, MLH1+ cells had higher percentage of γH2AX foci than MLH1- cells</li>
<li>The futile repair model of MMR suggests that strand breaks arise from MMR attempting repeatedly to repair the newly synthesized strand opposite adducts in S and G2 phases; approximately 80% of the γH2AX-positive MLH1+ cells were in G2 phase 12 hours after a 3-hour exposure to 20 µM Cr(VI), while the level was five times lower in MLH1- cells, suggesting that the MMR-induced DSB occurred following DNA synthesis; this supports the futile repair model and demonstrates inadequate repair</li>
</ul>
</li>
</ul>
<p><br />
</p>
<p><u>Inadequate Repair of DSBs </u></p>
<ul>
<li>Rydberg et al. [2005] exposed GM38 primary human dermal fibroblasts to increasing doses of linear electron transfer (LET) radiation of helium and iron ions (Rydberg et al., 2005).
<ul style="list-style-type:circle">
<li>The cells were allowed to recover for 16 hours following irradiation.</li>
<li>Unrepaired DSBs were measured after recovery using PFGE.</li>
<li>There was a dose-dependent increase in unrepaired DSBs due to both ion exposures.</li>
<li>Increase in persistent unrepaired DSBs with increasing dosage indicates exceeded repair capacity.</li>
</ul>
</li>
<li>DSB repair was also monitored by measuring γH2AX foci 0.05 - 24 hours after irradiation.
<ul>
<li>DSBs decreased over time and less than 1 foci per cell on average remained in MRC-5 cells 24hours after 0.02, 0.2 and 2 Gy exposures.</li>
<li>Repair was slower in 180BR cells, particularly for the 2 Gy exposure, where 20 foci per cell remained after 24 h. </li>
<li>A follow-up study by the same group, found similar results for MRC-5 and 180BR cells exposed to 0.02 and 0.2 Gy of X-rays (Kühne et al., 2004). </li>
</ul>
</li>
<li>Rothkamm and Löbrich (2003) exposed MRC-5 primary human lung fibroblasts (repair-proficient) and 180BR DNA ligase IV-deficient human fibroblasts to 10 and 80 Gy of X-rays (Rothkamm and Lobrich, 2003).
<ul style="list-style-type:circle">
<li>DNA ligase IV deficiency results in impaired NHEJ</li>
<li>DSB repair was monitored using PFGE by measuring the % of DSBs remaining after 0.25, 2, and 24 h following irradiation.</li>
<li>DSBs decreased over time and, eventually, less than 10% of the DSBs remained in MRC-5 cells after 24h following both 80 and 10 Gy exposures.</li>
<li>Repair was noticeably slower in 180BR cells, where the clearance of DSBs was hindered and approximately 40 and 20% of the DSBs remained at 24 hours following 80 and 10 Gy exposures, respectively.</li>
<li>The above demonstrates defective DNA repair leading to persistent DSBs.</li>
</ul>
</li>
</ul>
<p> </p>
<ul>
<li>A variety of confounding factors and genetic characteristics (i.e., SNPs) may modulate which repair pathways are invoked and the degree to which they are inadequate. These have yet to be fully defined.</li>
<li>Both protective and damaging effects of OGG1 against strand breaks have been described in the literature. As demonstrated in the section above, the effect of OGG1-deficiency (BER-initiating enzyme) is observed to be different in different cell types; Wang et al. (2018) demonstrated strand breaks exacerbated by excessive OGG1 activity, while Wu et al. (2008) and Shah et al. (2018) demonstrated increased strand breaks due to lack of repair in mammalian cells in culture (Shah et al., 2018; Wu et al., 2008; Wang et al., 2018). Cell cycle and replication may influence the effect of DNA repair on exacerbating strand breaks. </li>
<li>Dahle et al. (2008) exposed wild type and OGG1-overexpressing Chinese hamster ovary cells, AS52, to UVA. While OGG1-overexpression prevented the accumulation of Fpg-sensitive lesions (e.g., 8-oxo-dG and FaPyG) that were observed in wild type cells 4 hours after irradiation, there was no difference in the amount of strand breaks in the two cell types at 4h <!--[if supportFields]><span class=Geen><span
style='font-size:12.0pt;line-height:107%;font-family:"Times New Roman","serif";
mso-fareast-font-family:"Malgun Gothic";mso-fareast-theme-font:minor-fareast;
mso-bidi-theme-font:minor-bidi;mso-fareast-language:KO;mso-bidi-language:AR-SA'><span
style='mso-element:field-begin'></span>ADDIN RW.CITE{{326 Dahle,J. 2008}}<span
style='mso-element:field-separator'></span></span></span><![endif]-->(Dahle et al., 2008)<!--[if supportFields]><span
class=Geen><span style='font-size:12.0pt;line-height:107%;font-family:"Times New Roman","serif";
mso-fareast-font-family:"Malgun Gothic";mso-fareast-theme-font:minor-fareast;
mso-bidi-theme-font:minor-bidi;mso-fareast-language:KO;mso-bidi-language:AR-SA'><span
style='mso-element:field-end'></span></span></span><![endif]-->. </li>
<li>A recent study suggests that the NHEJ may be more accurate than previously thought (reviewed in Betermier et al., 2014). The accuracy of NHEJ may be dependent on the structure of the termini. The termini processing rather than the NHEJ itself is thus argued to be error-prone process (Betemier et al., 2014).</li>
</ul>
Not SpecifiedUnspecificNot SpecifiedAll life stagesNot SpecifiedNot SpecifiedNot Specified<p>This KER applies to any cell type that has DNA repair capabilities.</p>
<p style="margin-left:22.5pt">Alexander, J., Orr-Weaver, T. (2016), Replication fork instability and the consequences of fork collisions from rereplication, Genes Dev, 30:2241-2252.</p>
<p style="margin-left:22.5pt">Brenerman, B., Illuzzi, J., Wilson III, D. (2014), Base excision repair capacity in informing healthspan, Carcinogenesis, 35:2643-2652.</p>
<p style="margin-left:22.5pt">Dahle, J., Brunborg, G., Svendsrud, D., Stokke, T., Kvam, E. (2008), Overexpression of human OGG1 in mammalian cells decreases ultraviolet A induced mutagenesis, Cancer Lett, 267:18-25.</p>
<p style="margin-left:22.5pt">Dolinnaya, N., Kubareva, E., Romanova, E., Trikin, R., Oretskaya, T. (2013), Thymidine glycol: the effect on DNA molecular structure and enzymatic processing, Biochimie, 95:134-147.</p>
<p style="margin-left:22.5pt">Eccles, L., Lomax, M., O’Neill, P. (2010), Hierarchy of lesion processing governs the repair, double-strand break formation and mutability of three-lesion clustered DNA damage, Nucleic Acids Res, 38:1123-1134.</p>
<p style="margin-left:22.5pt">Ensminger, M., Iloff, L., Ebel, C., Nikolova, T., Kaina, B., Lobrich, M. (2014), DNA breaks and chromosomal aberrations arise when replication meets base excision repair, J Cell Biol, 206:29.</p>
<p style="margin-left:22.5pt">Kitsera, N., Stathis, D., Luhnsdorf, B., Muller, H., Carell, T., Epe, B., Khobta, A. (2011), 8-Oxo-7,8-dihydroguanine in DNA does not constitute a barrier to transcription, but is converted into transcription-blocking damage by OGG1, Nucleic Acids Res, 38:5926-5934.</p>
<p style="margin-left:22.5pt">Kühne, M., E. Riballo, N. Rief, K. Rothkamm, P. Jeggo, & M. Löbrich (2004), "A Double-Strand Break Repair Defect in ATM-Deficient Cells Contributes to Radiosensitivity", Cancer Res, 64(2): 500-508.</p>
<p style="margin-left:22.5pt">Ma, W., Panduri, V., Sterling, J., Van Houten, B., Gordenin, D., Resnick, M. (2009), The Transition of Closely Opposed Lesions to Double-Strand Breaks during Long-Patch Base Excision Repair Is Prevented by the Coordinated Action of DNA Polymerase and Rad27/Fen1 , Mol Cell Biol, 29:1212-1221.</p>
<p style="margin-left:22.5pt">Minko, I., Jacobs, A., de Leon, A., Gruppi, F., Donley, N., Harris, T., Rizzo, C., McCullough, A., Lloyd, R.S. (2016), Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites, Sci Rep, 6.</p>
<p style="margin-left:22.5pt">Peterson-Roth, E., Reynolds, M., Quievryn, G., Zhitkovich, A. (2005), Mismatch Repair Proteins Are Activators of Toxic Responses to Chromium-DNA Damage, Mol Cell Biol, 25:3596-3607.</p>
<p style="margin-left:22.5pt">Rothkamm, K., Lobrich, M. (2003), Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses, Proc Natl Acad Sci USA, 100:5057-5062.</p>
<p style="margin-left:22.5pt">Rydberg, B., Cooper, B., Cooper, P., Holley, W., Chatterjee, A. (2005), Dose-Dependent Misrejoining of Radiation-Induced DNA Double-Strand Breaks in Human Fibroblasts: Experimental and Theoretical Study for High- and Low-LET Radiation, Radiat Res, 163:526-534.</p>
<p style="margin-left:22.5pt">Shah, A., Gray, K., Figg, N., Finigan, A., Starks, L., Bennett, M. (2018), . Defective Base Excision Repair of Oxidative DNA Damage in Vascular Smooth Muscle Cells Promotes Atherosclerosis, Circulation, 138:1446-1462.</p>
<p style="margin-left:22.5pt">Wakasugi, M., Sasaki, T., Matsumoto, M., Nagaoka, M., Inoue, K., Inobe, M., Horibata, K., Tanaka, K., Matsunaga, T. (2014), Nucleotide Excision Repair-dependent DNA Double-strand Break Formation and ATM Signaling Activation in Mammalian Quiescent Cells, J Biol Chem, 289:28730-28737.</p>
<p style="margin-left:22.5pt">Wang, R., Li, C., Qiao, P., Xue, Y., Zheng, X., Chen, H., Zeng, X., Liu, W., Boldogh, I., Ba, X. (2018), OGG1-initiated base excision repair exacerbates oxidative stress-induced parthanatos, Cell Death and Disease, 9:628.</p>
<p style="margin-left:22.5pt">Whitaker, A., Schaich, M., Smith, M.S., Flynn, T., Freudenthal, B. (2017), Base excision repair of oxidative DNA damage: from mechanism to disease, Front Biosci, 22:1493-1522.</p>
<p style="margin-left:22.5pt">Wu, M., Zhang, Z., Che, W. (2008), Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line, Toxicol App Pharmacol, 228:395-402.</p>
<p style="margin-left:22.5pt">Yang, N., Galick, H., Wallace, S. (2004), Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks, DNA Repair, 3:1323-1334.</p>
<p style="margin-left:22.5pt">Yoshikawa, Y., Yamasaki, A., Takatori., K., Suzuki, M., Kobayashi, J., Takao, M., Zhang-Akiyama, Q. (2015), Excess processing of oxidative damaged bases causes hypersensitivity to oxidative stress and low dose rate irradiation, Free Radic Res, 49:1239-1248.</p>
<p style="margin-left:22.5pt">Zeman, M., Cimprich, K. (2014), Causes and Consequences of Replication Stress, Nat Cell Biol, 12:2-9.</p>
<p> </p>
<p> </p>
2019-05-19T16:36:022023-01-09T20:56:58bb9246a5-873d-4837-9f40-76c82c90ed10d7e6221c-be67-49ec-ab91-e3b70c7611672020-04-06T11:45:272020-04-06T11:45:27d7e6221c-be67-49ec-ab91-e3b70c76116799e550d7-7eb4-4713-aaa7-9c085b5da1152020-04-06T11:45:422020-04-06T11:45:42Alkylation of DNA leading to reduced sperm countAlkylation of DNA leading to reduced sperm countUnder development: Not open for comment. Do not cite<p>Alkylating agents are prototypical DNA-reactive compounds and have been extensively studied for decades (reviewed in Beranek 1990). The chemicals can be direct-acting electrophiles, or can be converted from non-reactive substances to reactive metabolites via metabolism. A prototypical alkylating agent is N-ethyl-N-nitrosourea (chemical formula C3H7N3O2) (ENU). ENU is rapidly absorbed following oral exposure and intraperitoneal injections and distributed widely across the tissues. ENU is unstable and readily reacts with somatic and germ cell DNA in mice, rats, flies and hamsters, to alkylate DNA. Very generally, mono-functional (referring to the transfer of a single alkyl group) alkylating agents include:
1. Alkyl sulfates: e.g., diethyl (DES) and dimethyl sulfate (DMS);
2. Alkyl alkanesulfonates: e.g., methyl (MMS) and ethyl methanesulfonate (EMS);
3. Nitrosamides: e.g., methyl (MNU) and ethyl nitrosourea (ENU), methyl- (MNNG) and ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and the indirect-acting (i.e., requiring metabolic activation) dimethyl (DMN) and diethyl nitrosamines (DEN).
</p><p><br />
ENU is the most widely studied and understood alkylating agent and as such has been instrumental in contributing to the knowledgebase in this field. Immunohistochemistry studies clearly indicate the presence of alkylated DNA following exposure to ENU in both somatic cells and spermatogonia (Kamino et al. 1995; Seiler et al. 1997; van Zeeland et al. 1990).
</p>adjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot Specified2020-03-31T15:19:282023-04-29T16:03:02