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
N-Methyl-N'-nitro-N-nitrosoguanidine1-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 acidD009154mutationGO: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>
2019-05-03T12:36:362019-05-07T12:12:1310090Mus musculus8090medakaWCS_7227Drosophila melanogaster10090mouse10036Syrian golden hamster10116rat9606Homo sapiens9913cow8090Oryzias latipesWCS_9606humanIncrease, Heritable mutations in offspringIncrease, Heritable mutations in offspringIndividual<p>Mutations occurring in the offspring are the adverse effect. These mutations may have many eventual consequences including embryonic or fetal death, or genetic disease in the offspring. If mutations are viable, the specific sites and sequence changes of the mutations will govern the phenotypic outcome of the inherited mutation.
</p><p>DETAILS: Evolutionarily advantageous or beneficial mutations are expected to be rare. Thus, the majority of inherited mutations will be neutral, with a somewhat smaller proportion expected to be harmful. For example, Keightley (2012) used phylogenetic analysis to estimate that approximately 70 new mutations occur per generation, 2.2 of which, on average, are deleterious. These deleterious mutations affect the fitness of the organism (decreasing probability of reproducing) and thus impact the population. Alternatively, one must also consider pathogenic mutations, including those that do not affect fitness (e.g., diseases that may occur later in life and do not affect ability to reproduce). It is currently not possible to fully measure the consequences of pathogenic mutations, because we lack appropriate methods to measure their penetrance (e.g., mutations with low odds ratios, diverse phenotypes, or that contribute to multigenic disorders, etc.). Thus, we currently do not have precise mechanisms to evaluate the full impacts of de novo mutations. However, increasing use of whole genome sequencing is shedding light on the rate, spectrum, and consequences of de novo mutations. Evidence is accumulating on the major role of de novo mutations in rare Mendelian and genetically heterogeneous diseases (e.g., reviewed in Veltman and Brunner, 2012; Geschwind and Flint, 2015; Walsch et al. 2010). The rate and spectrum of human mutations is reviewed in Campbell and Eichler (2013), and potential consequences of mutations explored in Shendure and Akey (2015). Estimates indicate approximately 100 loss-of-function variants in a human genome, with as many as 20 exhibiting complete loss of gene function (McLaughlin et al. 2010). As an example, based on full genome sequencing data, paternal de novo sequence mutations are expected to account for an equal amount of the genetic burden of disease in ageing fathers as maternal aneuploidies due to ageing (Hurles, 2012). It is important to note that although mutations in coding regions are expected to have large effects on fitness, the absolute number of mutations in non-coding sequence that is under selection is actually greater than coding sequence (Green and Ewing, 2013). In general, it is widely accepted that de novo mutations contribute to the overall population genetic disease burden. The application of whole genome sequencing in the clinic is providing new knowledge on the unprecedented extent to which de novo mutations are contributing to a whole host of idiopathic human genetic disorders (e.g., Lupski et al., 2011; Ku et al., 2013; Gilissen et al., 2014).
</p><p>A heritable mutation is measured as a mutation occurring in the offspring that is not present in the parents and that is present in every cell type (the latter is not typically measured). Heritable mutations were previously measured using the Mouse Specific Locus Test (SLT) and variations on this assay (in rodents, fish and Drosophila). The Oak Ridge National Laboratory's SLT test, established by William and Lianne Russell, was the gold standard for heritable mutation screening for several decades. Transmission of mutations from exposed males to their offspring can also be measured by analysis of tandem repeat mutations, an accepted though not widely used method. No OECD guideline exists for either assay.
</p><p><br />
Mouse SLT or variations of this assay: The SLT and dominant cataract methods are no longer used today because they require too many rodents, but there is a fairly large database from the application of these methods. The SLT is based on the use of seven dominant visible trait markers in mice (Davis and Justice, 1998; Russell et al., 1979). Male mice are exposed to the mutagen and mated at varying times post-exposure to evaluate effects on different stages of spermatogenesis. Males are mated with females carrying recessive alleles at the seven loci screened in the assay. Functional mutations at the dominant (male) locus results in expression of the recessive phenotype in the offspring. These phenotypes include changes in coat colour, skeletal malformations, and other traits. Variations on this assay include looking at other visible traits including 34 common skeletal malformations and dominant cataracts. Additional variations include protein electrophoresis to explore protein changes (e.g., Lewis et al., 1991).
</p><p>Tandem repeat mutation: Tandem repeat mutations can be measured in offspring using a similar approach. Male mice are exposed to the mutagen and mated various times post-exposure to non-exposed females. DNA fingerprinting is used to measure changes in repeat length in offspring relative to their parents. This is currently the only assay that is able to measure the same mutational endpoint in sperm as in offspring, supporting that transmission of mutations from sperm to the offspring occurs. For methodologies please see Vilarino-Guell et al. (2003). A wide range of human genetic disorders are associated with de novo length change mutations in tandem repeat sequences (Mirkin, 2007). However, it should also be noted that the mutations are induced through indirect mechanisms that are likely to be associated with polymerase errors during cell cycle arrest, rather than direct lesions at the locus (Yauk et al., 2002)
</p><p>Next generation sequencing: With the advent and improvement in sequencing technologies, it is anticipated that heritable mutations will be measured by directly sequencing the offspring of males exposed to mutagenic agents. Current approaches require the exposure of parental gametes to a mutagenic agent, followed by mating and collection of offspring. Whole genome sequencing is applied to compare the genome sequences of parents and offspring to identify and haplotype (i.e., determine the parental origin) of de novo mutations (identified as mutations occurring in offspring but not their parents). Studies such as these have demonstrated that increasing paternal age causes an increase in both single nucleotide variants and tandem repeats in the offspring (Kong et al., 2012; Sun et al., 2012). Proof of principle of the ability of application of genomics tools (array comparative genome hybridization and next generation sequencing) has been published for male mice exposed to radiation (Adewoye et al., 2015). The authors show that the frequency of de novo copy number variants (CNVs) and insertion/deletion events (indels) are significantly elevated in offspring of radiation-exposed fathers. Several papers have described how research in this field should proceed (Beal and Somers, 2011; Yauk et al., 2012; Yauk et al., 2015) and propose that this will be a paradigm-changing technology.
</p><p>Note: The Dominant Lethal test (OECD TG 478) is used to measure the effects of DNA damage in sperm on dominant lethality in the offspring. The overwhelming majority of dominant lethal mutations are due to chromosomal effects rather than gene mutations (Marchetti et al., 2005). Thus, this TG is not generally suited to the measurement of inherited gene mutations.
</p><p>Heritable mutations are the basis of evolution and occur in every species.
</p>HighModerateModerate<p><br />
</p><p>Adewoye, A.B., S.J. Lindsay, Y.E. Dubrova and M.E. Hurles (2015), "The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline", <i>Nat Commun.</i>, 6: 6684.
</p><p>Beal, M.A., T.C. Glenn and C.M. Somers (2011), "Whole genome sequencing for quantifying germline mutation frequency in humans and model species: cautious optimism", <i>Mutation Research</i>, 750(2): 96-106
</p><p>Beal, M.A., A. Rowan-Carroll, C. Campbell, A. Williams, C.M. Somers, F. Marchetti and C.L. Yauk (2015), "Single-molecule PCR analysis of an unstable microsatellite for detecting mutations in sperm of mice exposed to chemical mutagens", <i>Mutat. Res.</i>, 775: 26-32.
</p><p>BEIR VII (2006), "Health Risks from Exposure to Low Levels of Ionizing Radiation", Academies NRCotN, editor. Washington, D.C.: National Academies Press.
</p><p>Campbell, C.D. and E.E. Eichler (2013), "Properties and rates of germline mutations in humans", <i>Trends Genet.</i>, 29(10): 575-584.
</p><p>Cimino, M.C. (2006), "Comparative overview of current international strategies and guidelines for genetic toxicology testing for regulatory purposes", <i>Environ. Mol. Mutagen.</i>, 47: 362–390.
</p><p>Davis, A.P. and M.J. Justice (1998), "An Oak Ridge legacy: the specific locus test and its role in mouse mutagenesis", <i>Genetics</i>, 148(1): 7-12.
</p><p>Geschwind, D.H. and J. Flint (2015), "Genetics and genomics of psychiatric disease", <i>Science</i>, 349(6255): 1489-1494
</p><p>Green, P. and B. Ewing (2013), "Comment on “Evidence of abundant purifying selection in humans for recently acquired regulatory functions”", <i>Science</i>, 340(682) discussion 682.
</p><p>Gilissen, C., J.Y. Hehir-Kwa, D.T. Thung, M. van de Vorst, B.W. van Bon, M.H. Willemsen, M. Kwint, I.M. Janssen, A. Hoischen, A. Schenck, R. Leach, R. Klein, R. Tearle, T. Bo, R. Pfundt, H.G. Yntema, B.B. de Vries, T. Kleefstra, H.G. Brunner, L.E. Vissers and J.A. Veltman (2014), "Genome sequencing identifies major causes of severe intellectual disability", <i>Nature</i>, 511(7509): 344-347.
</p><p>Eastmond, D.A., A. Hartwig, D. Anderson, W.A. Anwar, M.C. Cimino, I. Dobrev, G.R. Douglas, T. Nohmi, D.H. Phillips and C. Vickers (2009), "Mutagenicity testing for chemical risk assessment: update of the WHO/IPCS Harmonized Scheme", <i>Mutagenesis</i>, 24(4): 341-349.
</p><p>Kong, A., M.L. Frigge, G. Masson, S. Besenbacher, P. Sulem, G. Magnusson, S.A. Gudjonsson, A. Sigurdsson, A. Jonasdottir, W.S. Wong, G. Sigurdsson, G.B. Walters, S. Steinberg, H. Helgason, G. Thorleifsson, D.F. Gudbjartsson, A. Helgason, O.T. Magnusson, U. Thorsteinsdottir and K. Stefansson K. (2012), "Rate of de novo mutations and the importance of father's age to disease risk", <i>Nature</i>, 488(7412): 471-475.
</p><p>Hurles, M. (2012), Older males beget more mutations", <i>Nature Genetics</i>, 44(11): 1174-1176.
</p><p>International Conference on Harmonisation (ICH) (2011), "Guidance On Genotoxicity Testing And Data Interpretation For Pharmaceuticals Intended For Human Use S2(R1)" ICH Harmonised Tripartite Guideline, International Conference on Harmonization, Geneva, Switzerland.
</p><p>Keightley, P.D. (2012), "Rates and Fitness Consequences of New Mutations in Humans", <i>Genetics</i>, 190(2): 295–304.
</p><p>Ku, C. S., E.K. Tan and D.N. Cooper (2013), "From the periphery to centre stage: de novo single nucleotide variants play a key role in human genetic disease", <i>J. Med. Genet.</i>, 50(4): 203-211.
</p><p>Lewis, S.E., L.B. Barnett, B.M. Sadler and M.D. Shelby MD (1991), "ENU mutagenesis in the mouse electrophoretic specific-locus test, 1. Dose-response relationship of electrophoretically-detected mutations arising from mouse spermatogonia treated with ethylnitrosourea", <i>Mutat Res.</i>, 249(2): 311-5.
</p><p>Lupski, J.R., J.W. Belmont, E. Boerwinkle and R.A. Gibbs (2011), "Clan genomics and the complex architecture of human disease", <i>Cell</i>, 147(1): 32-43.
</p><p>Marchetti, F. and A.J. Wyrobek (2005), "Mechanisms and consequences of paternally-transmitted chromosomal abnormalities", <i>Birth Defects Res C Embryo Today</i>, 75(2): 112-129.
</p><p>Mirkin, S.M. (2007), "Expandable DNA repeats and human disease", <i>Nature</i>, 447(7147): 932-940.
</p><p>Russell, W.L., E.M. Kelly, P.R. Hunsicker, J.W. Bangham, S.C. Maddux and E.L. Phipps (1979), "Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse" <i>Proceedings of the National Academy of Sciences of the United States of America</i>, 76(11): 5818-5819.
</p><p>Russel, L.B. (2004), "Effects of male germ-cell stage on the frequency, nature and spectrum of induced specific-locus mutations in the mouse", <i>Genetica</i>, 122: 25-36.
</p><p>Shendure, J. and J.M. Akey (2015), "The origins, determinants, and consequences of human mutations", <i>Science</i>, 349(6255): 1478-1483.
</p><p>Sun, J.X., A. Helgason, G. Masson, S.S. Ebenesersdottir, H. Li, S. Mallick, S. Gnerre, N. Patterson, A. Kong, D. Reich and K. Stefansson (2012), "A direct characterization of human mutation based on microsatellites", <i>Nat. Genet.</i>, 44(10): 1161-1165.
</p><p>Veltman, J.A. and H.G. Brunner (2012), "De novo mutations in human genetic disease", <i>Nat. Rev. Genet.</i>, 13(8): 565-575.
</p><p>Vilarino-Guell, C., A.G. Smith and Y.E. Dubrova (2003), "Germline mutation induction at mouse repeat DNA loci by chemical mutagens" <i>Mutation Research</i>, 526(1-2): 63-73.
</p><p>Walsh, T., M.K. Lee, S. Casadei, A.M. Thornton, S.M. Stray, C. Pennil, A.S. Nord, J.B. Mandell, E.M. Swisher and M.C. King (2010) "Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing", <i>Proc Natl Acad Sci U S A.</i>, 107(28): 12629-12633.
</p><p>United Nations (UN) (2013), "Globally Harmonized System of Classification and Labelling of Chemicals (GHS)", United Nations, New York, USA.
</p><p>Yauk, C.L., Y.E. Dubrova, G.R. Grant and A.J. Jeffreys (2002), "A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus" <i>Mutation Research</i>. 500(1-2): 147-56.
</p><p>Yauk, C.L., L.J. Argueso, S.S. Auerbach, P. Awadalla, S.R. Davis, D.M. Demarini, G.R. Douglas, Y.E. Dubrova, R.K. Elespuru, T.M. Glover, B.F. Hales , M.E. Hurles, C.B. Klein, J.R. Lupski, D.K. Manchester, F. Marchetti, A. Montpetit, J.J. Mulvihill, B. Robaire, W.A. Robbins, G.A. Rouleau, D.T. Shaughnessy, C.M. Somers, J.G. Taylor 6th, J. Trasler, M.D. Waters, T.E. Wilson, K.L. Witt and J.B. Bishop (2013), "Harnessing genomics to identify environmental determinants of heritable disease" <i>Mutation Research</i>, 752(1): 6-9.
</p><p>Yauk, C.L., M.J. Aardema, J. van Benthem, J.B. Bishop, K.L. Dearfield, D.M. DeMarini, Y.E. Dubrova, M. Honma, J.R. Lupski, F. Marchetti, M.L. Meistrich, F. Pacchierotti, J. Stewart, M.D. Waters and G.R. Douglas (2015), "Approaches for Identifying Germ Cell Mutagens: Report of the 2013 IWGT Workshop on Germ Cell Assays", <i>Mutat. Res. Genet. Toxicol. Environ. Mutagen</i>. 783:36-54.
</p>2016-11-29T18:41:242016-11-29T19:06:24Alkylation, 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>2016-11-29T18:41:222017-09-16T10:14:29Increase, MutationsIncrease, MutationsMolecular<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">A mutation is a change in DNA sequence. Mutations can thus alter the coding sequence of genes, potentially leading to malformed or truncated proteins. Mutations can also occur in promoter regions, splice junctions, non-coding RNA, DNA segments, and other functional locations in the genome. These mutations can lead to various downstream consequences, including alterations in gene expression. There are several different types of mutations including missense, nonsense, insertion, deletion, duplication, and frameshift mutations, all of which can impact the genome and its expression in unique ways. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Missense mutations are the substitution of one base in the codon with another. This change is significant because the three bases in a codon code for a specific amino acid and the new combination may signal for a different amino acid to be formed. Nonsense mutations also result from changes to the codon bases, but in this case, they cause the generation of a stop codon in the DNA strand where there previously was not one. This stop codon takes the place of a normal coding triplet, preventing its translation into an amino acid. This will cause the translation of the strand to prematurely stop. Both missense and nonsense mutations can result from substitutions, insertions, or deletions of bases (Chakarov et al. 2014). </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Insertion and deletion mutations are the addition and removal of bases from the strand, respectively. These often accompany a frameshift mutation, as the alteration in the number of bases in the strand causes the frame of the base reader to shift by the added or reduced number, altering the amino acids that are produced if that number is not devisable by three. Codons come in specific orders, sectioned into groups of three. When the boundaries of which three bases are included in one group are changed, this can change the whole transcriptional output of the strand (Chakaroy et al. 2014). </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Mutations can be propagated to daughter cells upon cellular replication. Mutations in stem cells (versus terminally differentiated non-replicating cells) are the most concerning, as these will persist in the organism. The consequence of the mutation, and thus the fate of the cell, depends on the location (e.g., coding versus non-coding) and the type (e.g., nonsense versus silent) of mutation.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Mutations can occur in somatic cells or germ cells (sperm or egg).</span></span></p>
<p> <span style="color:#0000ff">M</span>utations can be measured using a variety of both OECD and non-OECD mutagenicity tests. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</p>
<p><strong>Somatic cells:</strong> The Salmonella mutagenicity test (Ames Test) is generally used as part of a first tier screen to determine if a chemical can cause gene mutations. This well-established test has an OECD test guideline (OECD TG 471, 2020). A variety of bacterial strains are used, in the presence and absence of a metabolic activation system (e.g., rat liver microsomal S9 fraction), to determine the mutagenic potency of chemicals by dose-response analysis. A full description is found in Test No. 471: Bacterial Reverse Mutation Test (OECD, 2016).</p>
<p>A variety of in vitro mammalian cell gene mutation tests are described in OECD’s Test Guidelines 476 (2016) and 490 (2015). TG 476 (2016) is used to identify substances that induce gene mutations at the hprt (hypoxanthine-guanine phosphoribosyl transferase) gene, or the transgenic xprt (xanthine-guanine phosphoribosyl transferase) reporter locus. The most commonly used cells for the HPRT test include the CHO, CHL and V79 lines of Chinese hamster cells, L5178Y mouse lymphoma cells, and TK6 human lymphoblastoid cells. The only cells suitable for the XPRT test are AS52 cells containing the bacterial xprt (or gpt) transgene (from which the hprt gene was deleted).</p>
<p>The new OECD TG 490 (2015) describes two distinct in vitro mammalian gene mutation assays using the thymidine kinase (tk) locus and requiring two specific tk heterozygous cells lines: L5178Y tk+/-3.7.2C cells for the mouse lymphoma assay (MLA) and TK6 tk+/- cells for the TK6 assay. The autosomal and heterozygous nature of the thymidine kinase gene in the two cell lines enables the detection of cells deficient in the enzyme thymidine kinase following mutation from tk+/- to tk-/-.</p>
<p>It is important to consider that different mutation spectra are detected by the different mutation endpoints assessed. The non-autosomal location of the hprt gene (X-chromosome) means that the types of mutations detected in this assay are point mutations, including base pair substitutions and frameshift mutations resulting from small insertions and deletions. Whereas, the autosomal location of the transgenic xprt, tk, or gpt locus allows the detection of large deletions not readily detected at the hemizygous hprt locus on X-chromosomes. Genetic events detected using the tk locus include both gene mutations (point mutations, frameshift mutations, small deletions) and large deletions.</p>
<p>The transgenic rodent mutation assay (OECD TG 488, 2020) is the only assay capable of measuring gene mutation in virtually all tissues in vivo. Specific details on the rodent transgenic mutation reporter assays are reviewed in Lambert et al. (2005, 2009). The transgenic reporter genes are used for detection of gene mutations and/or chromosomal deletions and rearrangements resulting in DNA size changes (the latter specifically in the lacZ plasmid and Spi- test models) induced in vivo by test substances (OECD, 2009, OECD, 2011; Lambert et al., 2005). Briefly, transgenic rodents (mouse or rat) are exposed to the chemical agent sub-chronically. Following a manifestation period, genomic DNA is extracted from tissues, transgenes are rescued from genomic DNA, and transfected into bacteria where the mutant frequency is measured using specific selection systems.</p>
<p>The Pig-a (phosphatidylinositol glycan, Class A) gene on the X chromosome codes for a catalytic subunit of the N-acetylglucosamine transferase complex that is involved in glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Cells lacking GPI anchors, or GPI-anchored cell surface proteins are predominantly due to mutations in the Pig-a gene. Thus, flow cytometry of red blood cells expressing or not expressing the Pig-a gene has been developed for mutation analysis in blood cells from humans, rats, mice, and monkeys. The assay is described in detail in Dobrovolsky et al. (2010). Development of an OECD guideline for the Pig-a assay is underway. In addition, experiments determining precisely what proportion of cells expressing the Pig-a mutant phenotype have mutations in the Pig-a gene are in progress (e.g., Nicklas et al., 2015, Drobovolsky et al., 2015). A recent paper indicates that the majority of CD48 deficient cells from 7,12-dimethylbenz[a]anthracene-treated rats (78%) are indeed due to mutation in Pig-a (Drobovolsky et al., 2015).</p>
<p><br />
<strong>Germ cells:</strong> Tandem repeat mutations can be measured in bone marrow, sperm, and other tissues using single-molecule PCR. This approach has been applied most frequently to measure repeat mutations occurring in sperm DNA. Isolation of sperm DNA is as described above for the transgenic rodent mutation assay, and analysis of tandem repeats is done using electrophoresis for size analysis of allele length using single-molecule PCR. For expanded simple tandem repeat this involved agarose gel electrophoresis and Southern blotting, whereas for microsatellites sizing is done by capillary electrophoresis. Detailed methodologies for this approach are found in Yauk et al. (2002) and Beal et al. (2015).</p>
<p>Mutations in rodent sperm can also be measured using the transgenic reporter model (OECD TG 488, 2020). A description of the approach is found within this published TG. Further modifications to this protocol have been made as of 2022 for the analysis of germ cells. Detailed methodology for detecting mutant frequency arising in spermatogonia is described in Douglas et al. (1995), O'Brien et al. (2013); and O'Brien et al. (2014). Briefly, male mice are exposed to the mutagen and killed at varying times post-exposure to evaluate effects on different phases of spermatogenesis. Sperm are collected from the vas deferens or caudal epididymis (the latter preferred). Modified protocols have been developed for extraction of DNA from sperm.</p>
<p>A similar transgenic assay can be used in transgenic medaka (Norris and Winn, 2010).</p>
<p><br />
Please note, gene mutations that occur in somatic cells in vivo (OECD Test. No. 488, 2020) or in vitro (OECD Test No. 476: In vitro Mammalian Cell Gene Mutation Test, 2016), or in bacterial cells (i.e., OECD Test No. 471, 2020) can be used as an indicator that mutations in male pre-meiotic germ cells may occur for a particular agent (sensitivity and specificity of other assays for male germ cell effects is given in Waters et al., 1994). However, given the very unique biological features of spermatogenesis relative to other cell types, known exceptions to this rule, and the small database on which this is based, inferring results from somatic cell or bacterial tests to male pre-meiotic germ cells must be done with caution. That mutational assays in somatic cells may predict mutations in germ cells has not been rigorously tested empirically (Singer and Yauk, 2010). The IWGT working group on germ cells specifically addressed this gap in knowledge in their report (Yauk et al., 2015) and recommended that additional research address this issue. Mutations can be directly measured in humans (and other species) through the application of next-generation sequencing. Although single-molecule approaches are growing in prevalence, the most robust approach to measure mutation using next-generation sequencing today requires clonal expansion of the mutation to a sizable proportion (e.g., sequencing tumours; Shen et al., 2015), or analysis of families to identify germline derived mutations (reviewed in Campbell and Eichler, 2013; Adewoye et al., 2015).</p>
<p><span style="font-size:14px"><span style="font-family:arial,sans-serif">Please refer to the table below for additional details and methodologies for measuring mutations. </span></span></p>
<table border="1" cellpadding="1" cellspacing="1" style="height:2351px; width:633px">
<tbody>
<tr>
<td style="background-color:#eeeeee; text-align:center">A<strong>ssay Name</strong></td>
<td style="background-color:#eeeeee; text-align:center"><strong>References </strong></td>
<td style="background-color:#eeeeee; text-align:center"><strong>Description </strong></td>
<td style="background-color:#eeeeee; text-align:center"><strong>OECD Approved Assay</strong></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Assorted Gene Loci Mutation Assays</span></td>
<td>
<p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Tindall et al., 1989; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:times new roman,serif; font-size:12pt"><a href="file://ncr-a_hecsbc6s/hecsbc6/share/CCRPB/Radbiology/Vinita/AOP/assay%20summary%20table%20papers/MANY%20OTHER%20gene%20loci%20example%202.pdf"><span style="font-family:arial,sans-serif; font-size:11pt">Kruger</span></a></span><span style="font-family:arial,sans-serif; font-size:11pt"> et al., 2015</span></span></span></p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">After exposure to a chemical/mutagen, mutations can be measured by the ability of exposed cells to form colonies in the presence of specific compounds that would normally inhibit colony growth; Usually only cells -/- for the gene of interest are able to form colonies</span></td>
<td>N/A</td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">TK Mutation Assay</span></td>
<td>
<p style="text-align:center"><a href="file://ncr-a_hecsbc6s/hecsbc6/share/CCRPB/Radbiology/Vinita/AOP/assay%20summary%20table%20papers/TK%20mutation%20assay%20use.pdf"><span style="font-family:arial,sans-serif; font-size:11pt">Yamamoto</span></a><span style="font-family:arial,sans-serif; font-size:11pt"> et al., 2017; <span style="font-family:arial,sans-serif; font-size:11pt"><a href="file://ncr-a_hecsbc6s/hecsbc6/share/CCRPB/Radbiology/Vinita/AOP/assay%20summary%20table%20papers/TK%20mutation%20assay%20protocol.pdf"><span style="font-family:arial,sans-serif; font-size:11pt">Liber</span></a><span style="font-family:arial,sans-serif; font-size:11pt"> et al., 1982; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Lloyd and Kidd, 2012</span></span></span></span></span></p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">After exposure to a chemical/mutagen, mutations are detected at the thymidine kinase (TK) loci of L5178Y wild-type mouse lymphoma TK (+/-) cells by measuring resistance to lethaltriflurothymidine (TFT); Only TK-/- cells are able to form colonies</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 490)</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">HPRT Mutation Assay</span></td>
<td>
<p style="text-align:center"><a href="file://ncr-a_hecsbc6s/hecsbc6/share/CCRPB/Radbiology/Vinita/AOP/assay%20summary%20table%20papers/HPRT%20mutation%20assay%20use.pdf"><span style="font-family:arial,sans-serif; font-size:11pt">Ayres</span></a><span style="font-family:arial,sans-serif; font-size:11pt"> et al., 2006; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Parry and Parry, 2012</span></span></span></p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Similar to TK Mutation Assay above, X-linked HPRT mutations produced in response to chemical/mutagen exposure can be measured through colony formation in the presence of 6-TG or 8-azoguanine; Only HPRT-/- cells are able to form colonies</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 476)</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Salmonella Mutagenicity Test (Ames Test)</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">OECD, 1997</span></td>
<td>After exposure to a chemical/mutagen, point mutations are detected by analyzing the growth capacity of different bacterial strains in the presence and absence of various metabolic activation systems </td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 471)</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">PIG-A / PIG-O Assay</span></td>
<td>
<p style="text-align:center"><a href="file://ncr-a_hecsbc6s/hecsbc6/share/CCRPB/Radbiology/Vinita/AOP/assay%20summary%20table%20papers/MANY%20OTHER%20gene%20loci%20example%202.pdf"><span style="font-family:arial,sans-serif; font-size:11pt">Kruger</span></a><span style="font-family:arial,sans-serif; font-size:11pt"> et al., 2015; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Nakamura, 2012; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Chikura, 2019</span></span></span></span></span></p>
</td>
<td>After exposure to a chemical/mutagen, mutations in PIG-A or PIG-O (which decrease the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor protein) are assessed by the colony-forming capabilities of cells after <em>in vitro</em> exposure, or by flow cytometry of blood samples after <em>in vivo </em>exposure</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">N/A</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Single Molecule PCR</span></td>
<td>
<p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Kraytsberg & Khrapko, 2005; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Yauk, 2002</span></span></span></p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">This PCR technique uses a single DNA template, and is often employed for detection of mutations in microsatellites, recombination studies, and generation of polonies</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">N/A</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">ACB-PCR</span></td>
<td>
<p>Myers et al., 2014 (Textbook, pg 345-363); Banda et al., 2013; Banda et al., 2015; Parsons et al., 2017</p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Using this PCR technique, single base pair substitution mutations within oncogenes or tumour suppressor genes can be detected by selectively amplifying specific point mutations within an allele and selectively blocking amplification of the wild-type allele </span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">N/A</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Transgenic Rodent Mutation Assay </span></td>
<td>
<p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">OECD 2013; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Lambert 2005; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Lambert 2009</span></span></span></span></span></p>
</td>
<td>This <em>in vivo</em> test detects gene mutations using transgenic rodents that possess transgenes and reporter genes; After<em> in vivo</em> exposure to a chemical/mutagen, the transgenes are analyzed by transfecting bacteria with the reporter gene and examining the resulting phenotype</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 488)</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Conditionally inducible transgenic mouse models</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Parsons 2018 (Review)</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Inducible mutations linked to fluorescent tags are introduced into transgenic mice; Upon exposure of the transgenic mice to an inducing agent, the presence and functional assessment of the mutations can be easily ascertained due to expression of the linked fluorescent tags </span></td>
<td>N/A</td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Error</span><span style="font-family:arial,sans-serif; font-size:12pt">-</span><span style="font-family:arial,sans-serif; font-size:11pt">Corrected Next Generation Sequencing (NGS)</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Salk 2018 (Review)</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">This technique detects rare subclonal mutations within a pool of heterogeneous DNA samples through the application of new error-correction strategies to NGS; At present, few laboratories in the world are capable of doing this, but commercial services are becoming available (e.g., Duplex sequencing at TwinStrand BioSciences) </span></td>
<td>N/A </td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>Taxonomic applicability:</strong> Mutations can occur in any organism and in any cell type, and are the fundamental material of evolution. The test guidelines described above range from analysis from prokaryotes, to rodents, to human cells in vitro. Mutations have been measured in virtually every human tissue sampled in vivo.</p>
<p><strong>Life stage applicability:</strong> This key event is not life stage specific as all stages of life have DNA that can be mutated; however, baseline levels of mutations are seen to increase with age (Slebos et al., 2004; Kirkwood, 1989). </p>
<p><strong>Sex applicability:</strong> This key event is not sex specific as both sexes undergo mutations. Males have a higher mutation rate than females (Hedrick, 2007). </p>
<p><strong>Evidence for perturbation by a stressor:</strong> Many studies demonstrate that increased mutations can occur as a result of ionizing radiation (Sankaranarayanan & Nikjoo, 2015; Russell et al., 1957; Winegar et al., 1994; Gossen et al., 1995). </p>
HighUnspecificHighAll life stagesHighModerateHighModerate<p>Adewoye, A.B. et al. (2015), "The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline", <em>Nat. Commu.</em>, 6:6684. Doi: 10.1038/ncomms7684.</p>
<p>Ayres, M. F. et al. (2006), “Low doses of gamma ionizing radiation increase hprt mutant frequencies of TK6 cells without triggering the mutator phenotype pathway”, <em>Genetics and Molecular Biology</em>. 2(3): 558-561. Doi:10.1590/S1415-4757200600030002.</p>
<p>Banda M, Recio L, and Parsons BL. (2013), “ACB-PCR measurement of spontaneous and furan-induced H-ras codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver”, <em>Environ Mol Mutagen</em>. 54(8):659-67. Doi:10.1002/em.21808.</p>
<p>Banda, M. et al. (2015), “Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation”, <em>Mutat Res Genet Toxicol Environ Mutagen</em>. 789-790:53-60. Doi: 10.1016/j.mrgentox.2015.07.003</p>
<p>Campbell, C.D. & E.E. Eichler (2013), "Properties and rates of germline mutations in humans", <em>Trends Genet</em>., 29(10): 575-84. Doi: 10.1016/j.tig.2013.04.005</p>
<p>Chakarov, S. et al. (2014), “DNA damage and mutation. Types of DNA damage”, BioDiscovery, Vol.11, Pensoft Publishers, Sofia, https://doi.org/10.7750/BIODISCOVERY.2014.11.1.</p>
<p>Chikura, S. et al. (2019), “Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society”, <em>Genes Environ</em>. 27:41-5. Doi: 10.1186/s41021-019-0121-z.</p>
<p>Dobrovolsky, V.N. et al. (2015), "CD48-deficient T-lymphocytes from DMBA-treated rats have de novo mutations in the endogenous Pig-a gene. CD48-Deficient T-Lymphocytes from DMBA-Treated Rats Have De Novo Mutations in the Endogenous Pig-a Gene", Environ. Mol. Mutagen., (6): 674-683. Doi: 10.1002/em.21959.</p>
<p>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.</p>
<p>Gossen, J.A. et al. (1995), "Spontaneous and X-ray-induced deletion mutations in a LacZ plasmid-based transgenic mouse model", Mutation Research, 331/1, Elsevier, Amsterdam, https://doi.org/10.1016/0027-5107(95)00055-N. </p>
<p>Hedrick, P.W. (2007), “Sex: Differences In Mutation, Recombination, Selection, Gene Flow, And Genetic Drift”, Evolution, Vol.61/12, Wiley, Hoboken, https://doi.org/10.1111/j.1558-5646.2007.00250.x. </p>
<p>Kirkwood, T.B.L. (1989), “DNA, mutations and aging”, Mutation Research, Vol.219/1, Elsevier B.V., Amsterdam, https://doi.org/10.1016/0921-8734(89)90035-0</p>
<p>Kraytsberg,Y. & Khrapko, K. (2005), “Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations”, <em>Expert Rev Mol Diagn</em>. 5(5):809-15. Doi: 10.1586/14737159.5.5.809.</p>
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2016-11-29T18:41:232023-01-10T19:00:51Inadequate 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></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. </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 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. 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 S phase in dividing cells, and nonhomologous end joining (NHEJ), which can function in both dividing and non-dividing cells (Teruaki Iyama and David M. Wilson III, 2013). </li>
</ol>
<p style="margin-left:40px">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>, thus forming a trimeric complex on the ends of the DNA strands. 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. Alt-NHEJ is known to involve slightly different core proteins than C-NHEJ, 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 (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 between exposure to mutagenic agents and the presence of adducts (determined as adducts per nucleotide) provide an indication of whether the removal of adducts occurs, and whether it is more efficient at low doses. A sub-linear DNA adduct curve suggests that less effective repair occurs at higher doses (i.e., repair processes are becoming saturated). A sub-linear shape for the dose-response curves for mutation induction is also suggestive of repair of adducts at low doses, followed by saturation of repair at higher doses. Measurement of a clear point of inflection in the dose-response curve for mutations suggests that repair does occur, at least to some extent, but reduced repair efficiency arises above the breakpoint. 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><span style="color:#0000cd">Assay Name</span></strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong><span style="color:#0000cd">References</span></strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong><span style="color:#0000cd">Description</span></strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong><span style="color:#0000cd">DNA Damage/Repair Being Measured</span></strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><span style="color:#0000cd"><strong>OECD Approved Assay</strong></span></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>
</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; 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"><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>
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<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>
2016-11-29T18:41:232023-04-24T11:01:39d60ba6ec-566a-4f74-8d93-2ea82d846bba1e13b1ea-e7ca-491e-ac53-6b15bd9871a2<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>
2016-11-29T18:41:332019-12-10T10:43:251e13b1ea-e7ca-491e-ac53-6b15bd9871a23deb5d18-e5b1-4f35-befc-e8ee8d950e0b<p><span style="font-size:12px">Insufficient repair results in the retention of damaged DNA that is then used as a template during DNA replication. During replication of damaged DNA, incorrect nucleotides may be inserted, and upon replication these become ‘fixed’ in the cell. Further replication propagates the mutation to additional cells.</span></p>
<p><span style="font-size:12px">For example, it is well established that replication of alkylated DNA can cause insertion of an incorrect base in the DNA duplex (i.e., mutation). Replication of non-repaired O4 thymine alkylation leads primarily to A:T→G:C transitions. Retained O6 guanine alkylation causes primarily G:C→A:T transitions.</span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">For repairing DNA double strand breaks (DSBs), non-homologous end joining (NHEJ) is one of the repair mechanisms used in human somatic cells (Petrini et al., 1997; Mao et al., 2008). However, this mechanism is error-prone and may create mutations during the process of DNA repair (Little, 2000). NHEJ is considered error-prone because it does not use a homologous template to repair the DSB. The NHEJ mechanism involves many proteins that work together to bridge the DSB gap by overlapping single-strand termini that are usually less than 10 nucleotides long (Anderson, 1993; Getts & Stamato, 1994; Rathmell & Chu, 1994). Inherent in this process is the introduction of errors that may result in mutations such as insertions, deletions, inversions, or translocations.</span></span></p>
<p><span style="font-size:12px">Overall Weight of Evidence: High </span></p>
<p><span style="font-size:12px">If DNA repair is able to correctly and efficiently repair DNA lesions introduced by a genotoxic stressor, then no increase in mutation frequency will occur.</span></p>
<p><span style="font-size:12px">For example, for alkylated DNA, efficient removal by O<sup>6</sup>-alkylguanine DNA alkyltransferase will result in no increases in mutation frequency. However, above a certain dose AGT becomes saturated and is no longer able to efficiently remove the alkyl adducts. Replication of O-alkyl adducts leads to mutation. The evidence demonstrating that replication of unrepaired O-alkylated DNA causes mutations is extensive in somatic cells and has been reviewed (Basu and Essigmann 1990; Shrivastav et al. 2010); specific examples are given below.</span></p>
<p><span style="font-size:12px">It is important to note that not all DNA lesions will cause mutations. It is well documented that many are bypassed error-free. For example, N-alkyl adducts can quite readily be bypassed error-free with no increase in mutations (Philippin et al., 2014).</span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><strong>Inadequate repair of DSB</strong></span></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Collective data from tumors and tumor cell lines has emerged that suggests that DNA repair mechanisms may be error-prone (reviewed in Sishc et al., 2017) (Sishc & Davis, 2017). NHEJ, the most common pathway used to repair DSBs, has been described as error-prone. The error-prone nature of NHEJ, however, is thought to be dependent on the structure of the DSB ends being repaired, and not necessarily dependent on the NHEJ mechanism itself (Bétermier et al., 2014). Usually when perfectly cohesive ends are formed as a result of a DSB event, ligase 4 (LIG4) will have limited end processing to perform, thereby keeping ligation errors to a minimum (Waters et al., 2014). When the ends are difficult to ligate, however, the resulting repair may not be completed properly; this often leads to point mutations and other chromosomal rearrangements. It has been shown that approximately 25 - 50% of DSBs are misrejoined after exposure to ionizing radiation (Löbrich et al., 1998; Kuhne et al., 2000; Lobrich et al., 2000). Defective repair mechanisms can increase sensitivity to agents that induce DSBs and lead eventually to genomic instability (reviewed in Sishc et al., (2017)).</span></span></p>
<p><span style="font-size:12px">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). </span> </p>
<p><strong>I<span style="font-size:12px">NSUFFICIENT REPAIR OF ALKYLATED DNA</span></strong></p>
<p><span style="font-size:12px">Evidence in somatic cells</span></p>
<p><span style="font-size:12px">Empirical evidence to support this KER is primarily from studies in which synthetic oligonucleotides containing well-characterized DNA lesions were genetically engineered in viral or plasmid genomes and subsequently introduced into bacterial or mammalian cells. Mutagenicity of each lesion is ascertained by sequencing, confirming that replication of alkylated DNA (i.e., unrepaired DNA) causes mutations in addition to revealing the important DNA repair pathways and polymerases involved in the process. For example, plasmids containing O6-methyl or O6-ethylguanine were introduced into AGT deficient or normal Chinese hamster ovary cells (Ellison et al. 1989). Following replication, an increase in mutant fraction to 19% for O6-methylguanine and 11% for O6-ethylguanine adducts was observed in AGT deficient cells versus undetectable levels for control plasmids. The relationship between input of alkylated DNA versus recovered mutant fractions revealed that a large proportion of alkyl adducts were converted to mutations in the AGT deficient cells (relationship slightly sublinear, with more adducts than mutations). The primary mutation occurring was G:C-A:T transitions. The results indicate that replication of the adducted DNA caused mutations and that this was more prevalent with reduced repair capacity. The number of mutations measured is less than the unrepaired alkyl adducts transfected into cells, supporting that insufficient repair occurs prior to mutation. Moreover, the alkyl adducts occur prior to mutation formation, demonstrating temporal concordance.</span></p>
<p><span style="font-size:12px">Various studies in cultured cells and microorganisms have shown that the expression of <span style="color:#27ae60">O<sup>6</sup>-methylguanine DNA methyltransferase (</span>AGT/MGMT) (repair machinery – i.e., decrease in <span style="color:#27ae60">DNA strand breaks</span>) greatly reduces the incidence of mutations caused by exposure to methylating agents such as MNU and MNNG (reviewed in Kaina et al. 2007; Pegg 2011). Thomas et al. (2013) used O6-benzylguanine to specifically inhibit MGMT activity in AHH-1 cells. Inhibition was carried out for one hour prior to exposure to MNU, a potent alkylating agent. Inactivation of MGMT resulted in increased MNU-induced HPRT (hypoxanthine-guanine phosphoribosyltransferase) mutagenesis and shifted the concentrations at which induced mutations occurred to the left on the dose axis (10 fold reduction of the lowest observed genotoxic effect level from 0.01 to 0.001 µg/ml). The ratio of mutants recovered in DNA repair deficient cells was 3-5 fold higher than repair competent cells at concentrations below 0.01 µg/ml, but was approximately equal at higher concentrations, indicating that repair operated effectively to a certain concentration. Only at this concentration (above 0.01 µg/ml when repair machinery is overwhelmed and repair becomes deficient) do the induced mutations in the repair competent cells approach those of repair deficient. Thus, induced mutation frequencies in wild type cells are suppressed until repair is overwhelmed for this alkylating agent. The mutations prevented by MGMT are predominantly G:C-A:T transitions caused by O6-methylguanine.</span></p>
<p><br />
<span style="font-size:12px">Evidence in germ cells</span></p>
<p><span style="font-size:12px">That saturation of repair leads to mutation in spermatogonial cells is supported by work using the OECD TG488 rodent mutation reporter assay in sperm. A sub-linear dose-response was found using the lacZ MutaMouse assay in sperm exposed as spermatogonial stem cells, though the number of doses was limited (van Delft and Baan 1995). This is indirect evidence that repair occurs efficiently at low doses and that saturation of repair causes mutations at high doses. Lack of additional data motivated a dose-response study using the MutaMouse model following both acute and sub-chronic ENU exposure by oral gavage (O’Brien et al. 2015). The results indicate a linear dose-response for single acute exposures, but a sub-linear dose-response occurs for lower dose sub-chronic (28 day) exposures, during which mutation was only observed to occur at the highest dose. This is consistent with the expected pattern for dose-response based on the <span style="color:#27ae60">hypothetical</span> AOP. Thus, this sub-linear curve for mutation at low doses following sub-chronic ENU exposure suggests that DNA repair in spermatogonia is effective in preventing mutations until the process becomes overwhelmed at higher doses.</span></p>
<p><span style="font-size:12px">Mutation spectrum: Following exposure to alkylating agents, the most mutagenic adducts to DNA in pre-meiotic male germ cells include O6-ethylguanine, O4-ethylthymine and O2-ethylthymine (Beranek 1990; Shelby and Tindall 1997). Studies on sperm samples collected post-ENU exposure in transgenic rodents have shown that 70% of the observed mutations are at A:T sites (Douglas et al. 1995). The mutations observed at G:C base pairs are almost exclusively G:C-A:T transitions, presumably resulting from O6-ethylguanine. It is proposed that the prevalence of mutations at A:T basepairs is the result of efficient removal of O6-alkylguanine by AGT in spermatogonia, which is consistent with observation in human somatic cells (Bronstein et al. 1991; Bronstein et al. 1992). This results in the majority of O6-ethylguanine adducts being removed, leaving O4- and O2-ethylthymine lesions to mispair during replication. Thus, lack of repair predominantly at thymines and guanines at increasing doses leads to mutations in these nucleotides, consistent with the concordance expected between diminished repair capabilities at these adducts and mutation induction (i.e., concordance relates to seeing these patterns across multiple studies, species and across the data in germ cells and offspring).</span></p>
<p> </p>
<p><span style="font-size:12px"><u>Inadequate repair of oxidative DNA lesions: In vitro studies</u></span></p>
<ul>
<li><span style="font-size:12px">AS52 Chinese hamster ovary cells (wild type and OGG1-overexpressing) were exposed to kJ/m<sup>2 </sup>UVA radiation (Dahle et al., 2008).</span>
<ul style="list-style-type:circle">
<li><span style="font-size:12px">Mutations in the gpt gene were quantified in both wild type and OGG1+ cells by sequencing after 13-15 days following 400 kJ/m<sup>2 </sup>UVA irradiation</span>
<ul>
<li><span style="font-size:12px">G:C-A:T mutations in UVA-irradiated OGG1+ cells were completely eliminated</span></li>
<li><span style="font-size:12px">G:C-A:T mutation frequency in wild type cells increased from 1.8 mutants/million cells to 3.8 mutants/million cells following irradiation – indicating incorrect repair or lack of repair of accumulated 8-oxo-dG</span></li>
<li><span style="font-size:12px">Elevated levels of OGG1 was able to prevent G:C-A:T mutations, while the OGG1 levels in wild type cells was insufficient, leading to an increase in mutants (demonstrates inadequate repair leading to mutations)</span></li>
</ul>
</li>
</ul>
</li>
<li><span style="font-size:12px">Xeroderma pigmentosum complementation group A (XPA) knockout (KO) and wild type TSCER122 human lymphoblastoid cells were transfected with TK gene-containing vectors with no adduct, a single 8-oxo-dG, or two 8-oxo-dG adducts in tandem (Sassa et al., 2015).</span>
<ul style="list-style-type:circle">
<li><span style="font-size:12px">XPA is a key protein in nucleotide excision repair (NER) that acts as a scaffold in the assembly the repair complex.</span></li>
<li><span style="font-size:12px">Mutation frequency was determined by the number of TK-revertant colonies</span></li>
<li><span style="font-size:12px">Control vector induced a mutation frequency of 1.3% in both WT and XPA KO</span></li>
<li><span style="font-size:12px">Two 8-oxo-dG in tandem on the transcribed strand were most mutagenic in XPA KO, inducing 12% mutant frequency compared to 7% in WT</span></li>
<li><span style="font-size:12px">For both XPA KO and WT, G:C-A:T transversion due to 8-oxo-dG was the most predominant point mutation in the mutants </span></li>
<li><span style="font-size:12px">The lack of a key factor in NER leading to increased 8-oxo-dG-induced transversions demonstrates insufficient repair leading to increase in mutations </span></li>
</ul>
</li>
</ul>
<p> </p>
<p><span style="font-size:12px"><u>Inadequate repair of oxidative DNA lesions: In vivo studies in mice</u></span></p>
<ul>
<li><span style="font-size:12px">Spontaneous mutation frequencies in the liver of Ogg1-deficient (-/-) Big Blue mice was measured at 10 weeks of age (Klungland et al., 1999).</span>
<ul style="list-style-type:circle">
<li><span style="font-size:12px">Mutation frequencies were 2- to 3-fold higher in the <em>Ogg1</em>-/- mice than in wild type</span></li>
<li><span style="font-size:12px">Of the 16 base substitutions detected in <em>Ogg1</em> -/- mutant plaques analyzed by sequencing, 10 indicated G:C-A:T transversions consistent with the known spectrum of mutation</span></li>
<li><span style="font-size:12px">The results support that insufficient repair of oxidized bases leads to mutation.</span></li>
</ul>
</li>
<li><span style="font-size:12px"><em>Ogg1 </em>knockout (<em>Ogg1</em>-/-) in C57BL/6J mice resulted in 4.2-fold and 12-fold increases in the amount of 8-oxo-dG in the liver compared to wild type at 9 and 14 weeks of age, respectively (Minowa et al., 2000).</span>
<ul style="list-style-type:circle">
<li><span style="font-size:12px">In these mice, there was an average of 2.3-fold increase in mutation frequencies in the liver (measured between 16-20 weeks)</span>
<ul>
<li><span style="font-size:12px">57% of the observed base substitutions were G:C-A:T transversions, while 35% in wild type mice corresponded to this transversion.</span></li>
<li><span style="font-size:12px">Approximately 70% of the increase in mutation frequency was due to G to T transversions.</span></li>
</ul>
</li>
<li><span style="font-size:12px">Concordantly, KBrO3 treatment resulted in a 2.9-fold increase in mutation frequency in the kidney of <em>Ogg1 </em>-/- mice compared to KBrO3-treated wild type (Arai et al., 2002).</span>
<ul>
<li><span style="font-size:12px">G:C-A:T transversions made up 50% of the base substitutions in the <em>Ogg1-/- </em>mice.</span></li>
</ul>
</li>
<li><span style="font-size:12px">Heterozygous <em>Ogg1 </em>mutants (<em>Ogg1</em>+/-) retained the original repair capacity, where no increase in 8-oxo-dG lesions was observed in the liver at 9 and 14 weeks (Minowa et al., 2000).</span>
<ul>
<li><span style="font-size:12px">This observation was consistent even after KBrO3 treatment of the mice (Arai et al., 2002).</span></li>
</ul>
</li>
<li><span style="font-size:12px">From these results, we can infer that OGG1 proteins are present in excess and that one functional copy of the gene is sufficient in addressing endogenous and, to a certain degree, chemical-induced oxidative DNA lesions.</span></li>
</ul>
</li>
</ul>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><span style="color:#0000cd"><strong><u>Inadequate Repair of </u><u>DSB</u></strong></span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><span style="color:#0000cd">Empirical data obtained for this KER moderately supports the idea that inadequate DNA repair increases the frequency of mutations. The evidence presented below related to the inadequate repair of DSBs is summarized in table 5, <a href="https://docs.google.com/spreadsheets/d/1iehBBqhFFSOhgis-0U3tasQwJ50bZJPVmenWUiR4vmA/edit?usp=sharing" target="_blank">here (click link)</a>. The review article by Sishc & Davis (2017) provides an overview of NHEJ mechanisms with a focus on the inherently error-prone nature of DSB repair mechanisms, particularly when core proteins of NHEJ are knocked-out. Another review also provides an overview of DSB induction, the repair process and how mutations may result, as well as the biological relevance of misrepaired or non-repaired DNA damage (Sage & Shikazono, 2017).</span></span></span></p>
<p><br />
<span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><u><span style="color:#0000cd"><span style="color:#0000cd"><strong>Dose and Incidence Concordance</strong></span></span></u></span></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><span style="color:#0000cd">There is evidence in the literature suggesting a dose/incidence concordance between inadequate DNA repair and increases in mutation frequencies. Evidence presented below related to the dose-response of mutation frequencies is summarized in table 2, <a href="https://docs.google.com/spreadsheets/d/1iehBBqhFFSOhgis-0U3tasQwJ50bZJPVmenWUiR4vmA/edit?usp=sharing" target="_blank">here (click link)</a>. In response to increasing doses from a radiation stressor, dose-dependent increases in both measures of inadequate DNA repair and mutation frequency have been found. In an analysis that amalgamated results from several different studies conducted using in vitro cell-lines, the rate of DSB misrepair was revealed to increase in a dose-dependent fashion from 0 - 80 Gy, with the mutation rate also similarly increasing from 0 - 6 Gy (Mcmahon et al., 2016). Additionally, using a plant model, it was shown that increasing radiation dose from 0 - 10 Gy resulted in increased DNA damage as a consequence of inadequate repair. Mutations were observed 2 - 3 weeks post-irradiation (Ptácek et al., 2001). Moreover, increases in mutation densities were found in specific genomic regions of cancer samples (namely promoter DNAse I-hypersensitive sites (DHS) and 100 bp upstream of transcription start sites (TSS)) that were also found to have decreased DNA repair rates attributable to inadequate nucleotide excision repair (NER) (Perera et al., 2016).</span></span></span><br />
</p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><span style="color:#0000cd">Interestingly, mutation rates have been shown to increase as the required DNA repair becomes more complex. Upon completion of DSB repair in response to radiation and treatment with restriction enzymes, more mutations were found in cases where the ends were non-complementary and thus required more complex DNA repair (1 - 4% error-free) relative to cases where ends were complementary (34 - 38% error-free) (Smith et al., 2001).</span></span></span></p>
<p><span style="font-size:12px"><u><span style="font-family:arial,helvetica,sans-serif"><span style="color:#0000cd"><strong>Temporal Concordance</strong></span></span></u></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><span style="color:#0000cd">There is evidence in the literature suggesting a time concordance between the initiation of DNA repair and the occurrence of mutations. For simple ligation events, mutations were not evident until 12 - 24 hours, whereas DSB repair was evident at 6 -12 hours. For complex ligation events, however, mutations and DSB repair were both evident at 12 - 24 hours. As the relative percent of DNA repair increased over time, the corresponding percent of error-free rejoining decreased over time in both ligation cases, suggesting that overall DNA repair fidelity decreases with time ((Smith et al., 2001).</span></span></span></p>
<p><span style="font-size:12px"><u><span style="font-family:arial,helvetica,sans-serif"><span style="color:#0000cd"><strong>Essentiality</strong></span></span></u></span></p>
<p><span style="font-size:12px"><span style="color:#27ae60"><span style="font-family:arial,helvetica,sans-serif">Inadequate DNA repair has been found to increase mutations above background levels. There is evidence from knock-out/knock-down studies suggesting that there is a strong relationship between the adequacy of DNA repair and mutation frequency. In all examined cases, deficiencies in proteins involved in DNA repair resulted in altered mutation frequencies relative to wild-type cases. There were significant decreases in the frequency and accuracy of DNA repair in cell lines deficient in LIG4 (DNA ligase 4, a DNA repair protein) (Smith et al., 2003) and Ku80 (Feldmann et al., 2000). Rescue experiments performed with these two cell lines further confirmed that inadequate DNA repair was the cause of the observed decreases in repair frequency and accuracy (Feldmann et al., 2000; Smith et al., 2003). In primary Nibrin-deficient mouse fibroblasts, there was increased spontaneous DNA damage relative to wild-type controls, suggestive of inadequate DNA repair. Using the corresponding Nibrin-deficient and wild-type mice, in vivo mutation frequencies were also found to be elevated in the Nibrin-deficient animals (Wessendorf et al., 2014). Furthermore, mutation densities were differentially affected in specific genomic regions in cancer patients depending on their Xeroderma pigmentosum group C (XPC) gene status. Specifically, mutation frequencies were increased in XPC-wild-type patients at DNase I-hypersensitive site (DHS) promoters and 100 bp upstream of TSS relative to cancer patients lacking functional XPC (Perera et al., 2016). Lastly, in a study using WKT1 cells with less repair capacity, radiation exposure induced four times more mutations in these cells than in TK6 cell, which had a normal repair capacity (Amundson and Chen, 1996). </span></span></span></p>
<p><span style="font-size:12px">Repair of alkylated DNA</span></p>
<p><span style="font-size:12px">There were no inconsistencies in the empirical data reviewed or in the literature relating to biological plausibility. Much of the support for this KER comes predominantly from data in somatic cells and in prokaryotic organisms. We note that all of the data in germ cells used in this KER are produced exclusively from ENU exposure. Data on other chemicals are required. We consider the overall weight of evidence of this KER to be strong because of the obvious biological plausibility of the KER, and documented temporal association and incidence concordance based on studies over-expressing and repressing DNA repair in somatic cells.</span></p>
<p><span style="font-size:12px">Repair of oxidative lesions</span></p>
<ul>
<li><span style="font-size:12px">Thresholded concentration-response curve of mutation frequency was observed in AHH-1 human lymphoblastoid cells after treatment with pro-oxidants (H<sub>2</sub>O<sub>2 </sub>and KBrO<sub>2</sub>) known to cause oxidative DNA damage (Seager et al., 2012), suggesting that cells are able to tolerate low levels of DNA damage using basal repair. However, increase in 8-oxo-dG lesions and up-regulation of DNA repair proteins were not observed under the same experimental condition.</span></li>
<li><span style="font-size:12px">Mutagenicity of oxidative DNA lesions other than 8-oxo-dG, such as FaPydG and thymidine glycol, has not been as extensively studied and there are mixed results regarding the mutagenic outcome of these lesions.</span></li>
</ul>
<p><span style="font-size:12px">Repair of double strand breaks </span></p>
<ul>
<li><span style="font-size:12px">One review paper found that DNA DSBs are repaired more efficiently at low dose (≤0.1 Gy) compared to high dose (>1 Gy) X-rays, but delayed mutation induction and genomic instability have also been demonstrated to occur at low doses (<1 cGy) of ionizing radiation (Preston et al., 2013). </span></li>
</ul>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Overall</span></span></p>
<ul>
<li><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Mutation induction is stochastic, spontaneous, and dependent on the cell type as well as the individual’s capability to repair efficiently (NRC, 1990; Pouget & Mather, 2001).</span></span></li>
</ul>
<p><span style="font-size:12px">Thresholds for mutagenicity indicate that the response at low doses is modulated by the DNA repair machinery, which is effectively able to remove alkylated DNA at low doses [Gocke and Muller 2009; Lutz and Lutz 2009; Pozniak et al. 2009]. Kinetics of DNA repair saturation in somatic cells is described in Muller et al. [Muller et al. 2009].</span></p>
<p><span style="font-size:12px">For O-methyl adducts, once the primary repair process is saturated, in vitro data suggest that misreplication occurs almost every time a polymerase encounters a methylated guanine [Ellison et al. 1989; Singer et al. 1989]; however, it should be noted that this process can be modulated by flanking sequence. This conversion of adducts to mutations also appears to be reduced substantially in vivo [Ellison et al. 1989]. The probability of mutation will also depend on the type of adduct (e.g., O-alkyl adducts are more mutagenic than N-alkyl adducts; larger alkyl groups are generally more mutagenic, etc.). Overall, a substantive number of factors must be considered in developing a quantitative model.</span></p>
<p><span style="font-size:12px"><u>Inadequate repair of oxidative </u><u>lesions</u></span></p>
<p><span style="font-size:12px">The relationship between the quantity/activity of repair enzymes such as OGG1 in the cell and the quantity of oxidative lesions need to be better understood to define a threshold on the quantity of oxidative lesions exceeding basal repair capacity. Moreover, the proportion of oxidative lesions formed that lead to mutation versus strand breaks is not clearly understood.</span></p>
<p><span style="font-size:12px">Mutations resulting from oxidative DNA damage can occur via replicative polymerases and translesion synthesis (TLS) polymerases during replication, and during attempted repair. However, an in vitro study on TLS in yeast has shown that bypass of 8-oxo-dG by TLS polymerases during replication is approximately 94-95% accurate. Therefore, the mutagenicity of 8-oxo-dG and other oxidative lesions may depend on their abundance, not on a single lesion (Rodriguez et al., 2013). Applicability of this observation in mammalian cells needs further investigation. Information on the accuracy of 8-oxo-dG bypass in mammalian cells is limited. </span></p>
<p><span style="font-size:12px">The most notable example of mutation arising from inadequate repair of DNA oxidation is G to T transversion due to 8-oxo-dG lesions. Previous studies have demonstrated higher mutation frequency of this lesion compared to other oxidative lesions; for example, Tan et al. (1999) compared the mutation rate of 8-oxo-dG and 8-oxo-dA in COS-7 monkey kidney cells and reported that under similar conditions, 8-oxo-dG was observed to be four times more likely to cause base substitution (Tan et al., 1999). </span></p>
<p><span style="font-size:12px"><strong><span style="color:#0000cd"><u>Inadequate Repair of DSB</u></span></strong></span></p>
<p><span style="font-size:12px"><span style="color:#0000cd">Quantitative understanding of this linkage is derived from the studies that examined DSB misrepair rates or mutation rates in response to a radiation stressor. In general, combining results from these studies suggests that increased mutations can be predicted when DNA repair is inadequate. At a radiation dose of 10 Gy, the rate of DSB misrepair was found to be approximately 10 - 15% (Lobrich et al., 2000); this rate increased to 50 - 60% at a radiation exposure of 80 Gy (Kuhne et al., 2000; Lobrich et al., 2000; McMahon et al., 2016). For mutation rates in response to radiation across a variety of models and radiation doses, please refer to the example table below.</span></span></p>
<table border="1" cellpadding="1" cellspacing="1" style="height:158px; width:645px">
<tbody>
<tr>
<td style="text-align:center; width:150px"><span style="font-size:12px"><strong>Reference</strong></span></td>
<td style="text-align:center"><span style="font-size:12px"><strong>Summary</strong></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="color:#0000cd">Matuo et al., 2018</span></span></td>
<td><span style="font-size:12px"><span style="color:#0000cd">Yeast cells (saccharomyces cerevisiae) exposed to high LET cardbon ions (25 keV/um) and low LET carbon ions (13 keV/um) between 0-200 Gy induces a 24-fold increase overbaseline of mutations (high LET) and 11-fold increase over baseline mutations (low LET).</span></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="color:#0000cd">Nagashima et al., 2018</span></span></td>
<td><span style="font-size:12px"><span style="color:#0000cd">Hamster cells (GM06318-10) exposed to x-rays in the 0-1 Gy. Response of 19.0 ± 6.1 mutants per 10<sup>9</sup> survivors.</span></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="color:#0000cd">Albertini et al., 1997</span></span></td>
<td><span style="font-size:12px"><span style="color:#0000cd">T-lymphcytes isolated from human peripheral blood exposed to low LET gamma-rays (0.5-5 Gy) and high LET radon gas (0-1 Gy). Response of 7.0x10<sup>-6</sup> mutants/Gy (Gamma-rays 0-2 Gy), 54x10<sup>-6</sup> mutants/Gy (Gamma-rays 2-4 Gy) and 63x10<sup>-6</sup> mutants/Gy (0-1 Gy).</span></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="color:#0000cd">Dubrova et al., 2002</span></span></td>
<td><span style="font-size:12px"><span style="color:#0000cd">Observation of paternal ESTR mutation rates in CBAH mice following exposure to acute low LET X-rays (0-1 Gy), chronic low LET gamma-rays (0-1 Gy) and chronic high LET neutrons (0-0.5 Gy). Modelled response of y = mx + C, values of (m,C): X-rays: (0.338, 0.111), Gamma-rays: (0.373±0.082, 0.110), Neutrons: (1.135±0.202, 0.136).</span></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px"><span style="color:#0000cd">McMahon et al., 2016</span></span></td>
<td><span style="font-size:12px"><span style="color:#0000cd">Study of HPRT gene in Chinese hamster cells following exposure to radiation of 1-6 Gy. Observation of 0.2 mutations in HPRT gene per 10<sup>4</sup> cells and 0.1 point mutations per 10<sup>4</sup> cells (1 Gy). At 6 Gy, observation of 1.5 mutations in the HPRT gene per 10<sup>4</sup> cells and 0.4 point mutations per 10<sup>4</sup> cells.</span></span></td>
</tr>
</tbody>
</table>
<p> </p>
HighUnspecificHighAll life stagesHighHighHigh<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">This KER is plausible in all life stages, sexes, and organisms with DNA. The majority of the evidence is from in vivo adult mice and male human, and mice in vitro models. </span></span></p>
<p><span style="font-size:12px">All organisms, from prokaryotes to eukaryotes, have DNA repair systems. Indeed, much of the empirical evidence on the fundamental principles described in this KER are derived from prokaryotic models. DNA adducts can occur in any cell type with DNA, and may or may not be repaired, leading to mutation. While there are differences among DNA repair systems across eukaryotic taxa, all species develop mutations following excessive burdens of DNA lesions like DNA adducts. Theoretically, any sexually reproducing organism (i.e., producing gametes) can also acquire DNA lesions that may or may not be repaired, leading to mutations in gametes.</span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Albertini, R.J. et al. (1997), "Radiation Quality Affects the Efficiency of Induction and the Molecular Spectrum of HPRT Mutations in Human T Cells", 148(5 Suppl):S76-86.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Amundson, S.A. & D.J. Chen (1996), "Ionizing Radiation-Induced Mutation of Human Cells With Different DNA Repair Capacities.", Adv. Space Res. 18(1-2):119-126.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Anderson, C.W. 1993, "DNA damage and the DNA-activated protein kinase.", Trends Biochem. Sci. 18(11):433–437. doi:10.1016/0968-0004(93)90144-C.</span></span></p>
<p><span style="font-size:12px">Arai, T., Kelly, V.P., Minowa, O., Noda, T., Nishimura, S. (2002), High accumulation of oxidative DNA damage, 8-hydroxyguanine, in Mmh/Ogg1 deficient mice by chronic oxidative stress, Carcinogenesis, 23:2005-2010.</span></p>
<p><span style="font-size:12px">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.</span></p>
<p><span style="font-size:12px">Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", <em>Mutation Research</em>, 231(1): 11-30.</span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Bétermier, M., P. Bertrand & B.S. Lopez (2014), "Is Non-Homologous End-Joining Really an Inherently Error-Prone Process?", PLoS Genet. 10(1). doi:10.1371/journal.pgen.1004086.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Bhowmick, R., S. Minocherhomji & I.D. Hickson (2016), "RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress", Mol. Cell., 64(6):1117-1126.</span></span></p>
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<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Wilson, T.E. & M.R. Lieber (1999), "Efficient Processing of DNA Ends during Yeast Nonhomologous End Joining.", J. Biol. Chem. 274(33):23599–23609. doi:10.1074/jbc.274.33.23599.</span></span></p>
2016-11-29T18:41:332023-01-10T19:12:17d60ba6ec-566a-4f74-8d93-2ea82d846bba3deb5d18-e5b1-4f35-befc-e8ee8d950e0b<p>Alkylated DNA may be ‘misread’ during DNA replication, leading to insertion of incorrect nucleotides. Upon replication, these changes become fixed as mutations. Subsequent replication propagates these mutations to daughter cells. Mutations in stem cells are of the greatest concern, as these will persist throughout the organism’s lifetime. Thus, increased mutations will be found in the cells of organisms that possess alkylated DNA.
</p><p>Alkylating agents can cause a variety of adducts and DNA damage (e.g., alkali labile sites, DNA strand breaks, etc.) that are potentially mutagenic and clastogenic. This KER focuses on the probability that an alkyl DNA adduct will lead to a mutation.
</p><p>Not all adducts are equally mutagenic. Very generally, chemicals that preferentially cause O-alkylation in DNA induce DNA sequence changes, whereas chemicals that cause N-alkylation of DNA are more efficient inducers of structural chromosomal aberrations (reviewed in Beranek 1990). Indeed, a review of the biological significance of N7 alkyl-guanine adducts concluded that these adducts simply be used to confirm exposure to target tissue (Boysen et al., 2009), because the vast majority of studies shows that these adducts do not cause mispairing. A variety of work has demonstrated that N7-alkylguanine adducts can be bypassed essentially error free (e.g., Philippin et al., 2014; Shrivastav et al., 2010). Moreover, alkylation can involve modification with different sizes of alkylation groups (e.g., methyl, ethyl, propyl). Although response to these is qualitatively similar with respect to the key events, in general, larger alkylating groups tend to be more mutagenic (Beranek, 1990). It is widely known that chemicals that preferentially cause O-alkylation in DNA induce mutations. ENU (N-ethyl-N-nitrosourea) is a prototypical O-alkylating agent and the most studied male germ cell mutagen.
</p><p>Alkylating agents are prototypical somatic and male germ cell mutagens.
</p><p>Evidence in somatic cells
It is well established that transfection of cells with alkylated DNA leads to mutation at the sites of alkyl damage. The design of these experiments requires waiting for cellular replication in order to produce the mutation, confirming the temporal concordance of DNA alkylation and subsequent mutation. A summary of the empirical data to support this is reviewed in Shrivastav et al. (2010).
</p><p>Various studies have examined the dose-response of DNA adduct levels and mutations. These studies demonstrate that alkyl adducts can be seen at lower doses in the absence of increased mutations both in vitro and in vivo, or demonstrate equal or increased incidence of adducts relative to mutations at the same doses. For example, following exposure of AHH-1 cells to increasing concentrations of MMS, a linear increase in alkylated DNA is measured with significant increases occurring in adduct levels at 0.25 µg/ml (Swenberg et al. 2008). Significant increases in mutations in the HPRT gene in the same cells are not measurable until 1.25 µg MMS/ml (Swenberg et al. 2008) (Figure 1). In vivo, time-series analysis of λlacZ transgenic mice exposed to a single dose of either ENU or DEN, demonstrate that global and lacZ-specific O6-EtGua adducts occur within hours of exposure in the liver, with the bulk of adducts removed by three days post-exposure (Mientjes et al. 1996 and 1998). In contrast, mutant frequency does not begin to significantly increase until three days post-exposure, demonstrating temporal concordance of adduct and mutation formation (see Figure 1 in Mientjes et al. 1998). Levels of O6-EtGua adducts are also consistently higher than the induced mutation frequency per nucleotide. In the bone marrow, DEN is not metabolized and thus is unable to create O6-EtGua adducts. The finding of lack of O6-alkyl adducts in bone marrow is consistent with the lack of an increase in mutations observed in this tissue (Mientjes et al. 1998). This is in contrast to ENU exposure (a direct acting mutagen that does not require metabolic activation), where both adducts and mutations increase in a concordant fashion in the bone marrow.
</p><p>This pattern of adduct incidence versus mutation incidence is consistent for somatic tissues in rodents in vivo for other types of adducts (we were not able to find suitable dose-response studies to compare oxygen-alkyl adducts to mutation frequencies in vivo). For example, MutaMouse males were exposed to increasing doses of the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (forms mutagenic bulky DNA adducts) for 28 days (followed by a 3 day break for mutation fixation) following OECD protocol TG488 (Malik et al. 2013). Significant increases in hepatic DNA adducts were found at 25 mg/kg, but increases in lacZ mutant frequency in liver did not occur until 50 mkg/kg. Bulky DNA adducts in both the livers and lungs of MutaMouse males exhibit an order of magnitude greater incidence per nucleotide than mutations in the lacZ gene using a similar experimental design following exposure to benzo[a]pyrene (Labib et al. 2012; Malik et al. 2012). Lower tissue adduct burden is correlated with lower tissue-specific gene mutation frequencies in Big Blue mice exposed to benzo[a]pyrene (Skopek et al. 1996). Thus, adducts in DNA occurs at lower doses than mutations and are correlated with mutation burden in somatic tissues for different types of DNA adducts.
</p><p>Evidence in germ cells:
No study has compared dose-response for adduct formation and mutation in a single experiment on germ cells. However, it is possible to look across experiments. It is important to note that adducts are measured immediately following exposure because they are relatively quickly repaired. However, analysis of lacZ mutation requires collection of mature sperm from the caudal epididymis. Thus, sperm is collected 42 or 49 days post-exposure (OECD TG488). This is because spermatogonia can not be sampled directly for these purposes. Therefore, comparison of adducts to mutations in pre-meiotic male germ cells requires sampling at different time points for these endpoints (early for adducts, much later for mutations), which is consistent with the expected temporal order of events, with adducts occurring before mutations.
</p><p>Dose-response for alkyl adduct levels has been very well characterized in mouse testicular DNA for ENU, EMS and DES (van Zeeland et al. 1990). A summary of dose-response data for mouse exposure to ENU and mutation analysis using the transgenic rodent mutation assay in sperm is given in the attached Table I and Figure 2. These studies involve acute injections or oral gavage studies only. Alkyl adducts are evident in gonadal tissues within 2 hours of exposure (van Zeeland et al. 1990) and are fairly efficiently removed within days of exposure in the absence of continued exposure. For this analysis, transgene mutant frequencies were converted to per nucleotide mutation frequency by dividing mutant frequency by the length of the lacZ gene (3096 bp). The data demonstrate that alkyl adduct incidence at low doses is much greater per nucleotide than transgene mutations in the lacZ locus. Adducts are observed to increase substantially at the lowest exposure dose (10 mg/kg), whereas mutation increases in lacZ are marginal at 25 mg/kg. Alkyl adducts in mouse testes following ENU exposure were in the range of approximately 40 in 10E7 nucleotides for 80 mg/kg ENU exposure (van Zeeland et al. 1990). Conversion of the data in O’Brien et al. (2015) to mutations per nucleotide (by dividing mutant frequency by the length of the lacZ locus, which is 3096 bp) produces an estimated induced mutation frequency in spermatogonia of approximately 4 mutations per 10E7 nucleotides for the highest dose (100 mg/kg ENU), an increase of approximately 2 in 10E7 above controls. This suggests that the incidence of adducts is an order of magnitude greater than incidence of mutations. Similarly, exposure to a single dose of 250 mg/kg EMS leads to an over 10-fold increase in the number of alkyl adducts (although the majority are on nitrogen atoms, with only a small proportion on oxygen) (van Zeeland et al. 1990), but only a marginal 2-fold increase in lacZ mutation frequency [van Delft et al. 1997]. Indeed, Van Zeeland et al. (1990) estimate that approximately 10 O6-ethylguanine adducts are required in the gene-coding region to generate a mutation.
</p><p>Analysis of germ cell mutation during a sub-chronic exposure was carried out by O’Brien et al. (2015). The lower doses used in that study revealed that significant increases in mutations occurred only after 28 days of exposure to the highest dose of 5 mg/kg ENU (cumulative dose of 140 mg/kg), again supporting that higher adduct burdens are required to lead to mutations.
</p><p>In general, many studies in different mouse strains have used similar experimental designs to conclusively demonstrate that exposure to a variety of alkylating agents causes mutations in spermatogonia (Brooks and Dean 1997; Douglas et al. 1995; Katoh et al. 1997; Katoh et al. 1994; Liegibel and Schmezer 1997; Mattison et al. 1997; O'Brien et al. 2014; O’Brien et al. 2015; Renault et al. 1997; Suzuki et al. 1997; Swayne et al. 2012; Tinwell et al. 1997; van Delft et al. 1997). These studies have been done using a single dose and thus do not enable further comparison of the concordance of dose-response. We also note that ENU exposure of pre-meiotic male germ cells in fish (transgenic medaka) also causes significant increases in mutations observed in spermatozoa (Norris and Winn 2010), supporting the effects of alkylating agents on mutations in male pre-meiotic germs across taxa using similar experimental designs.
</p><p>As described above, not all alkyl adducts are mutagenic. The proportion of oxygen-alkylation and the type of mutation (with ethylation > methylation) will govern mutagenicity, but there are few empirical data to support this. There are no inconsistencies or uncertainties for ENU or iPMS; other alkylating agents (EMS, MMS) have yielded some discrepancies in the transgenic rodent mutation assay. However, the experimental protocols applied were sub-standard (the OECD TG for this analysis was revised and published in 2013). Overall, more work is needed on alkylating agents other than ENU to fill important data gaps.
</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>The shape of the dose-response curve for alkyl adduct formation versus mutation demonstrates that a threshold exists whereby alkyl adducts can be seen at low doses in the absence of increased mutations occurring. For example, following exposure of AHH-1 cells to increasing concentrations of MMS, a linear increase in alkylated DNA is measured. However, a hockey-stick shaped curve was found for mutations at HPRT in the same cells (Thomas et al. 2013). Thus, alkylation of DNA occurs at lower doses than mutation, and above a certain dose (where repair is saturated), mutation frequencies increase.
</p><p>That DNA alkylation leads to mutation in spermatogonia in a similar hockey stick-shaped response (implying that a minimal dose must be exceeded) is supported by work using the LacZ Muta™Mouse assay. Exposure of male mice to the prototypical agent ENU was used to examine effects on spermatogonial stem cells, though the number of doses was limited (van Delft and Baan 1995). This analysis revealed that mutations did not occur at the lowest dose, where adducts are known to be measurable in other studies (van Zeeland et al., 1990). This data gap motivated a dose-response study using the Muta™Mouse model following both acute and sub-chronic ENU exposure by oral gavage at Health Canada (O’Brien et al., 2015). These data indicate a clear dose-response for single acute exposures, whereas a hockey stick-shaped dose-response occurs for lower dose sub-chronic (28 day) exposures. At the single acute high doses where the DNA repair machinery is expected to be overwhelmed (and thus higher levels of alkylation occur), significantly more mutations occur relative to the same dose spread out over 28 daily oral gavage exposures (O’Brien et al., 2015).
</p><p>Additional contributors to the probability that an adduct will cause mutation include the site of alkylation, with agents that cause O-alkyl lesions being the primary mutagens, and the size of the alkyl group, with larger alkyl groups generally being more mutagenic.
</p><p>A computational model to describe the mutational efficiency of different alkyl adducts has not yet been developed to our knowledge.
</p>HighModerate<p>Alkylating agents are well-established to cause mutation in virtually any cell type in any organism.
</p><p><br />
Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", <i>Mutation Research</i>, 231(1): 11-30.
</p><p>Boysena, G., B.F. Pachkowski, J. Nakamura and J.A. Swenberg (2009), "The formation and biological significance of N7-guanine adducts", <i>Mutation Research</i>, 678: 76–94.
</p><p>Brooks, T.M. and S.W. Dean (1997), "The detection of gene mutation in the tubular sperm of Muta Mice following a single intraperitoneal treatment with methyl methanesulphonate or ethylnitrosourea", <i>Mutat. Res.</i>, 388(2-3): 219-222.
</p><p>Douglas, G.R., J. Jiao, J.D. Gingerich, J.A. Gossen and L.M. Soper (1995), "Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice", <i>Proc. Natl. Acad. Sci. USA</i>, 92(16): 7485-7489.
</p><p>Katoh, M., N. Horiya and R.P. Valdivia (1997), "Mutations induced in male germ cells after treatment of transgenic mice with ethylnitrosourea", Mutat Res. 1997 Feb 14;388(2-3):229-37.
</p><p>Katoh, M., T. Inomata, N. Horiya, F. Suzuki, T. Shida, K. Ishioka and T. Shibuya (1997), "Studies on mutations in male germ cells of transgenic mice following exposure to isopropyl methanesulfonate, ethylnitrosourea or X-ray", <i>Mutat. Res.</i>, 388(2-3):213-8.
</p><p>Liegibel, U.M. and P. Schmezer (1994), "Detection of the two germ cell mutagens ENU and iPMS using the LacZ/transgenic mouse mutation assay" <i>Mutat. Res.</i>, 341(1):17-28.
</p><p>Mattison, J.D., L.B. Penrose and B. Burlinson (1997), "Preliminary results of ethylnitrosourea, isopropyl methanesulphonate and methyl methanesulphonate activity in the testis and epididymal spermatozoa of Muta Mice", <i>Mutat. Res.</i> 388(2-3): 123-7.
</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(11): 2449-2454.
</p><p>Mientjes, E.J., A. Luiten-Schuite, E. van der Wolf, Y. Borsboom, A. Bergmans, F. Berends, P.H. Lohman, R.A. Baan RA, J.H. van Delft (1998), "DNA adducts, mutant frequencies, and mutation spectra in various organs of lambda lacZ mice exposed to ethylating agents", <i>Environ. Mol. Mutagen.</i>, 31(1): 18-31
</p><p>Norris, M.B. and R.N. Winn (2010), "Isolated spermatozoa as indicators of mutations transmitted to progeny", <i>Mutat. Res.</i>, 688(1-2): 36–40.
</p><p>Labib, S., C. Yauk, A. Williams, V.M. Arlt, D.H. Phillips, P.A. White and S. Halappanavar (2012)," Subchronic oral exposure to benzo(a)pyrene leads to distinct transcriptomic changes in the lungs that are related to carcinogenesis. Toxicol Sci 129(1):213-224.
</p><p>Malik, A.I., A. Williams, C.L. Lemieux, P.A. White and C.L. Yauk (2012), "Hepatic mRNA, microRNA, and miR-34a-target responses in mice after 28 days exposure to doses of benzo(a)pyrene that elicit DNA damage and mutation", <i>Environ. Mol. Mutagen.</i>, 53(1): 10-21.
</p><p>Malik, A.I., A. Rowan-Carroll, A. Williams, C.L. Lemieux, A.S. Long, V.M. Arlt, D.H. Phillips, P.A. White and C.L. Yauk (2013), "Hepatic genotoxicity and toxicogenomic responses in MutaMouse males treated with dibenz[a,h]anthracene", <i>Mutagenesis</i>, 28(5): 543-554.
</p><p>O'Brien, J.M., M.A. Beal, J.D. Gingerich, L. Soper L, G.R. Douglas, C.L. Yauk and F. Marchetti (2014), "Transgenic rodent assay for quantifying male germ cell mutant frequency", <i>J. Vis. Exp.</i>, (90): e51576.
</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", <i>Environ. Mol. Mutagen.</i>, 56(4): 347-55.
</p><p>Renault, D., D. Brault and V. Thybaud (1997), "Effect of ethylnitrosourea and methyl methanesulfonate on mutation frequency in MutaMouse germ cells (seminiferous tubule cells and epididymis spermatozoa)", <i>Mutat. Res.</i>, 388(2-3): 145-153.
</p><p>Shrivastav, N., D. Li and J.M. Essigmann (2010), "Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation", <i>Carcinogenesis</i>, 31(1): 59-70.
</p><p>Skopek, T.R., K.L. Kort, D.R. Marino, L.V. Mittal, D.R. Umbenhauer, G.M. Laws and S.P. Adams (1996), "Mutagenic response of the endogenous hprt gene and lacI transgene in benzo[a]pyrene-treated Big Blue B6C3F1 mice", <i>Environ. Mol. Mutagen.</i>, 28(4): 376-384.
</p><p>Suzuki, T., S. Itoh, N. Takemoto, N. Yajima, M. Miura, M. Hayashi, H. Shimada and T. Sofuni (1997), "Ethyl nitrosourea and methyl methanesulfonate mutagenicity in sperm and testicular germ cells of lacZ transgenic mice (Muta Mouse)", <i>Mutat. Res.</i>, 388(2-3): 155-163.
</p><p>Swenberg, J.A., E. Fryar-Tita, Y. Jeong, G. Boysen, T. Starr, V.E. Walker and R.J. Albertini (2008), "Biomarkers in toxicology and risk assessment: informing critical dose-response relationships", <i>Chem. Res. Toxicol.</i>, 21(1): 253-265.
</p><p>Swayne, B.G., A. Kawata, N.A. Behan, A. Williams, M.G. Wade, A.J. Macfarlane and C.L. Yauk (2012), "Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice", <i>Mutat. Res.</i>, 737(1-2): 1-7.
</p><p>Tinwell, H., P. Lefevre, C.V. Williams and J. Ashby (1997), "The activity of ENU, iPMS and MMS in male mouse germ cells using the Muta Mouse positive selection transgenic mutation assay", <i>Mutat. Res.</i>, 388(2-3): 179-185.
</p><p>van Delft J.H., A. Bergmans and R.A. Baan RA (1997), "Germ-cell mutagenesis in lambda lacZ transgenic mice treated with ethylating and methylating agents: comparison with specific-locus test", <i>Mutat. Res.</i>, 388(2-3): 165-173.
</p><p>van Delft, J.H. and R.A. Baan (1995), "Germ cell mutagenesis in lambda lacZ transgenic mice treated with ethylnitrosourea; comparison with specific-locus test", <i>Mutagenesis</i>, 10(3): 209-214.
</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(1):55-62.
</p>2016-11-29T18:41:332016-11-29T19:53:41d60ba6ec-566a-4f74-8d93-2ea82d846bba35950b57-6313-41be-982b-011ff85be96b<p>Pre-meiotic male germ cells are outside of the blood-testis barrier and thus are exposed if there is systemic distribution. Exposure of pre-meiotic male germ cells to DNA alkylating agents results in DNA alkyl adducts. Replication of DNA with alkyl adducts thus can cause mutations in these cells. Fertilization of an egg by sperm containing mutations causes an increase in the number of mutations that are transmitted to their offspring.
</p><p>Alkylating agents are prototypical mutagens in laboratory animals. It is established that these agents, especially those chemicals that preferentially cause O-alkylation in DNA, induce heritable mutations. ENU (N-ethyl-N-nitrosourea) is a prototypical agent used to derive offspring with de novo mutations inherited from exposed males (e.g., <a rel="nofollow" target="_blank" class="external free" href="http://ja.brc.riken.jp/lab/mutants/genedriven.htm">http://ja.brc.riken.jp/lab/mutants/genedriven.htm</a>). In fact, ENU mutagenicity is a standard bench tool for genetic screens used to identify new mutations associated with a phenotype of interest.
</p><p>A variety of alkylating agents are positive in the mouse specific locus test demonstrating that they cause heritable mutations in offspring as a result of exposure of pre-meiotic male germ cells. These agents include ENU, methyl nitrosourea (MNU), procarbazine and melphalan. This has been thoroughly reviewed by Marchetti and Wyrobek (2005) and Witt and Bishop (1996). It should be noted that procarbazine and melphalan predominantly cause N-alkyl adducts and yield a weaker response in the specific locus test assay in male pre-meiotic germs (these agents yield higher responses in post-meiotic stages of spermatogenesis).
</p><p>Dose-response: No study has directly compared alkyl adducts in sperm and mutations in offspring within a single experiment. However, comparisons can be made across experiments. The shape of the dose-response curve for adducts in testes (van Zeeland et al. 1990) and offspring with mutations (using the SLT – described in Favor et al. 1990) are similarly sub-linear, and incidence of adducts exceeds mutations at similar doses. For example, alkyl adducts in spermatogonia of mice exposed to 80 mg/kg bw ENU (van Zeeland et al. 1990) were in the range of approximately 1 in 10E6 nucleotides (range 0.23 to 1.92 x 10E-6). Conversion of the data in the specific locus test to per bp mutation (using exon sizes published in (Russell 2004) reveals a control mutation rate of 8.23 per 10E11 nucleotides, and 1.47 in 10E9 nucleotides for mice treated with 80 mg/kg ENU (from Favor et al. 1990) (see Table I and Figure 2 for summary of data). These are conservative estimates of mutation rate because they only account for functional mutations. However, it is clear that the incidence of adducts is much greater than the increased incidence of offspring with mutations. Moreover, alkyl adducts occur within hours of exposure in spermatogonia (van Zeeland et al. 1990), whereas studies in mice that focus on spermatogonial stem cells wait a minimum of 49 days prior to mating to confirm that this is the phase of spermatogenesis that was affected. Thus, alkylation of DNA occurs prior to the mutations in the offspring supporting temporal concordance of these events. Mutations in the offspring of males exposed to alkylating agents occur in tandem repeat DNA sequences (Dubrova et al. 2008; Vilarino-Guell et al. 2003) and genes associated with visible phenotypic traits in mice (Ehling and Neuhäuser-Klaus 1991; Favor 1986; Russell et al. 1979; Selby et al. 2004). Specific locus mutations in the offspring of males exposed to alkylating agents has also been demonstrated in Drosophila (Tosal et al. 1998) and medaka (fish) (Shima and Shimada 1994). A substantive number of studies have demonstrated inherited mutations caused by exposure to ENU, but data also exist showing increases incidence of mutations in offspring with increasing doses of the two alkylating agents MNU and iPMS (Ehling and Neuhäuser-Klaus 1991; Nagao 1987; Russell et al. 2007; Vilarino-Guell et al. 2003).
</p><p>As described above, not all alkylating agents cause heritable mutations as a result of mutations arising in spermatogonia. O-alkylation is critical, and the size of the alkyl group is important, with ENU exhibiting an order of magnitude greater response than MNU. Although there are no inconsistencies based on knowledge of the spectrum of adducts expected for specific alkylating agents, the database on which this KER is assessed is nearly exclusively centered on ENU. Moreover, a key data gap includes evidence of the effect of alkylating agents in the offspring of exposed humans.
</p><p>Very little data are available on exposed humans despite the fact that humans may be exposed to high doses of alkylating agents during chemotherapy. Thus far the evidence has not supported that the cancer treatments pose heritable mutagenic hazards based on assessment of cancer (Madanat-Harjuoja et al., 2011), minisatellite mutations (Tawn et al., 2013) and congenital anomalies (Signorello et al., 2012) in offspring, or minisatellite mutation analysis in sperm ( Zheng et al., 2000; Armour et al., 199). However, cancer therapies are complex combinations of drugs that include agents that generally induce N-alkylation rather than O-alkylation. It has been suggested that the search for human germ cell mutagens has been flawed by lack of appropriate power, focus on the wrong agents, and using the wrong tools (DeMarini, 2012).
</p><pre>As with mutations arising in sperm, it is established that the levels of O-alkylation must exceed a specific threshold before mutations begin to measurably increase in frequency above controls in the descendants of exposed males [Favor et al. 1990; Russell et al. 1982]. In addition, fractionation of the dose reduces the recovery of mutations, indicating that more of the DNA damage is repaired [Favor et al. 1997]. A quantitative model has not been developed because of insufficient data.
</pre>HighModerate<p>That alkylation of DNA causes heritable mutations has been demonstrated specifically in flies, fish, and rodents. However, it is assumed that alkylating agents would act broadly on virtually any DNA sequence, in any organism, in any cell type. Thus, as long as the species has male germ cells, this KER would be relevant to that species.
</p><p><br />
Armour, J.A., M.H. Brinkworth and A. Kamischke (1999), "Direct analysis by small-pool PCR of MS205 minisatellite mutation rates in sperm after mutagenic therapies", <i>Mutat. Res.</i>, 445(1): 73-80.
</p><p>Demarini, D.M. (2012), "Declaring the existence of human germ-cell mutagens", <i>Environ. Mol. Mutagen.</i>, 53(3): 166-172.
</p><p>Dubrova, Y.E., P. Hickenbotham, C.D. Glen, K. Monger, H.P. Wong and R.C. Barber (2008), "Paternal exposure to ethylnitrosourea results in transgenerational genomic instability in mice", <i>Environ. Mol. Mutagen.</i>, 49(4): 308-311.
</p><p>Ehling, U.H. and A. Neuhäuser-Klaus (1991), "Induction of specific-locus and dominant lethal mutations in male mice by 1-methyl-1-nitrosourea (MNU)", <i>Mutat. Res.</i>, 250(1-2): 447-456.
</p><p>Favor, J. (1986), "The frequency of dominant cataract and recessive specific-locus mutations in mice derived from 80 or 160 mg ethylnitrosourea per kg body weight treated spermatogonia." 'Mutat. Res.<i>, 162(1): 69-80.</i>
</p><p>Favor, J., M. Sund, A. Neuhauser-Klaus and U.H. Ehling (1990), "A dose-response analysis of ethylnitrosourea-induced recessive specific-locus mutations in treated spermatogonia of the mouse", 'Mutat. Res.<i>, 231(1): 47-54.</i>
</p><p>Favor, J., A. Neuhäuser-Klaus, U.H. Ehling, A. Wulff and A.A. van Zeeland (1997), "The effect of the interval between dose applications on the observed specific-locus mutation rate in the mouse following fractionated treatments of spermatogonia with ethylnitrosourea", 'Mutat. Res.<i>, 374(2): 193-199. </i>
</p><p>Lewis, S.E., L.B. Barnett, B.M. Sadler and M.D. Shelby (1991), "ENU mutagenesis in the mouse electrophoretic specific-locus test, 1. Dose-response relationship of electrophoretically-detected mutations arising from mouse spermatogonia treated with ethylnitrosourea", 'Mutat. Res.<i>, 249(2): 311-315.</i>
</p><p>Madanat-Harjuoja, L.M., N. Malila, P. Lähteenmäki, E. Pukkala, J.J. Mulvihill, J.D. Boice Jr and R. Sankila (2010), "Risk of cancer among children of cancer patients - a nationwide study in Finland," <i>Int. J. Cancer</i>, 126(5): 1196-1205.
</p><p>Marchetti, F. and A.J. Wyrobek (2005), "Mechanisms and consequences of paternally-transmitted chromosomal abnormalities", <i>Birth Defects Res C Embryo Today</i>, 75(2): 112-129.
</p><p>Nagao, T. (1987), "Frequency of congenital defects and dominant lethals in the offspring of male mice treated with methylnitrosourea", 'Mutat. Res.<i>, 177(1): 171-178.</i>
</p><p>Russell, W.L., E.M. Kelly, P.R. Hunsicker, J.W. Bangham, S.C. Maddux and E.L. Phipps (1979), "Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse", <i>Proc. Natl. Acad. Sci. USA</i>, 76(11): 5818-5819.
</p><p>Russell, W.L., P.R. Hunsicker, G.D. Raymer, M.H. Steele, K.F. Stelzner and H.M. Thompson HM (1982), "Dose-response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia", <i>Proc. Natl. Acad. Sci. USA</i>, 79(11): 3589-3591.
</p><p>Russell, L.B. (2004), "Effects of male germ-cell stage on the frequency, nature, and spectrum of induced specific-locus mutations in the mouse", <i>Genetica</i>, 122(1): 25-36.
</p><p>Russell, L.B., P.R. Hunsicker and W.L. Russell (2007), "Comparison of the genetic effects of equimolar doses of ENU and MNU: while the chemicals differ dramatically in their mutagenicity in stem-cell spermatogonia, both elicit very high mutation rates in differentiating spermatogonia", 'Mutat. Res.<i>, 616(1-2): 181-195.</i>
</p><p>Selby, P.B., V.S. Earhart, E.M. Garrison and G. Douglas Raymer (2004), "Tests of induction in mice by acute and chronic ionizing radiation and ethylnitrosourea of dominant mutations that cause the more common skeletal anomalies", 'Mutat. Res.<i>, 545(1-2): 81-107.</i>
</p><p>Signorello, L.B., J.J. Mulvihill, D.M. Green, H.M. Munro, M. Stovall, R.E. Weathers, A.C. Mertens, J.A. Whitton, L.L. Robison and J.D. Boice Jr. (2012), "Congenital anomalies in the children of cancer survivors: a report from the childhood cancer survivor study", <i>J. Clin. Oncol.</i>, 30(3): 239-245.
</p><p>Shima, A. and A. Shimada (1994), "The Japanese medaka, Oryzias latipes, as a new model organism for studying environmental germ-cell mutagenesis", <i>Environ. Health Perspect.</i>, 102 Suppl 12: 33-35.
</p><p>Tosal, L., M.A. Comendador and L.M. Sierra (1998), "N-ethyl-N-nitrosourea predominantly induces mutations at AT base pairs in pre-meiotic germ cells of Drosophila males", <i>Mutagenesis</i>, 13(4): 375-380.
</p><p>Van Zeeland, A.A., A. de Groot and A. Neuhauser-Klaus (1990), "DNA adduct formation in mouse testis by ethylating agents: a comparison with germ cell mutagenesis", 'Mutat. Res.<i>, 231(1): 55-62.</i>
</p><p>Vilarino-Guell, C., A.G. Smith and Y.E. Dubrova (2003), "Germline mutation induction at mouse repeat DNA loci by chemical mutagens", 'Mutat. Res.<i>, 526(1-2): 63-73.</i>
</p><p>Witt, K.L. and J.B. Bishop (1996), "Mutagenicity of anticancer drugs in mammalian germ cells", 'Mutat. Res.<i>, 355(1-2): 209-234.</i>
</p><p>Zheng, N., D.G. Monckton, G. Wilson, F. Hagemeister, R. Chakraborty, T.H. Connor, M.J. Siciliano, M.L. Meistrich (2000), "Frequency of minisatellite repeat number changes at the MS205 locus in human sperm before and after cancer chemotherapy", <i>Environ. Mol. Mutagen.</i>, 36(2): 134-145.
</p>2016-11-29T18:41:332016-11-29T19:53:313deb5d18-e5b1-4f35-befc-e8ee8d950e0b35950b57-6313-41be-982b-011ff85be96b<p>If a mutation arises in spermatogonial stem cells and does not influence cellular function, the mutation can become fixed and/or propagated within the stem cell population. Mutations that do not affect sperm maturation will persist through the succeeding stages of spermatogenesis and will be found in the mature sperm of the organism throughout its reproductive lifespan. Mutations can also occur in differentiating spermatogonia; however, once the sperm generated by these dividing spermatogonia are ejaculated there will be no ‘record’ of the induced mutation. Mutations that impair spermatogenesis or the viability of the cell will be lost via apoptosis and cell death, potentially contributing to decreased fertility. Mutations that do not impact sperm motility, morphology or ability to penetrate the zona pellucida (or other important sperm parameters), and that are present in mature sperm, may be transmitted to the egg resulting in the development of an offspring with a mutation. Thus, increased incidence of mutations in germ cells leads to increased incidence of mutations in the offspring. There is a great deal of evidence demonstrating that exposure to a variety of DNA alkylating agents induces mutations in male spermatogenic cells.
</p><p>Evolution requires heritable mutations that are transmitted to offspring via parental gametes. This is the primary mechanism by which an offspring would have a sequence variant in every single one of its cells that is not found in its parents. Indeed, as stated in a recent review in Science by Shendura and Aikey "Germline mutations are the principal cause of heritable disease and the ultimate source of evolutionary change." Thus, this KER is supported by substantive understanding of genetics and evolution, with heritable germ cell mutations forming the basis for the selective evolution of species.
</p><p>In addition, in toxicology, a large body of literature demonstrates that exposure to specific genotoxic chemicals during pre-meiotic stages of spermatogenesis leads to mutations in both the sperm and the offspring, supporting that mutations occurring in sperm fertilize the egg and result in offspring with mutations (reviewed in Demarini 2012; Marchetti and Wyrobek 2005; Yauk et al. 2012). Indeed, ENU is used as a tool in genetics to create offspring carrying mutations for the purposes of understanding gene function ( e.g., <a rel="nofollow" target="_blank" class="external free" href="http://www.riken.jp/en/research/labs/brc/mutagen_genom">http://www.riken.jp/en/research/labs/brc/mutagen_genom</a>). In these studies, male mice are mutagenized by exposure to ENU and mated to females. The offspring of these males have a much higher incidence of mutation; the function of new mutations occurring in genes in these offspring is used to study gene function.
</p><p>Thus, overall this KER is biologically plausible and widely understood.
</p><p>Identification of mutations in sperm requires the destruction of the sperm. Thus, tracking a mutated spermatogonial stem cell through to fertilization and characterization of the mutation in the offspring is not possible, and the empirical evidence to support this KER is weak. No single study has looked at the dose-response relationship of the same mutation endpoint in germ cells and offspring because technologies are not currently available to do this. We caution that comparing mutation rates across different genes or genetic loci is imprecise, because factors intrinsic to specific loci govern mutation rates (e.g., length, GC content, transcribed versus non-transcribed, coding versus non-coding, chromatin structure, DNA methylation, sequence, etc.).
</p><p>Increased numbers of germ cell mutations occur in mature sperm 42+ days post-exposure in mice (indicating that mutations arose in pre-meiotic male germ cells). Mating in this time interval to produce offspring also results in increased incidence of mutation in the descendants of exposed males, indicating temporal concordance. By virtue of the required experimental designs, mutations measured in the offspring occur after the mutations in germ cells. For example, mutations identified in proteins via electrophoresis (a variation of the SLT test) are found in the offspring of male mice mated 10+ weeks post-exposure to ENU (Lewis et al. 1991). These inherited changes are the result of mutations in stem cells that persist through spermatogenesis and are transmitted to offspring.
</p><p>The only assay that presently can measure mutations in both sperm and offspring is the tandem repeat mutation assay. A single study on one dose of radiation (1 Gy X-ray) against matched controls has shown that increases in mutation frequencies in exposed sperm are similar to the increases observed in the offspring of exposed males for tandem repeats (Yauk et al. 2002), suggesting that tandem repeat mutations in sperm are transmitted to offspring. Alkylating agents cause similar increases in tandem repeat mutations in both sperm and in the offspring through comparison across studies (Dubrova et al. 2008; Swayne et al. 2012; Vilarino-Guell et al. 2003), but dose-response studies have not been conducted. It is advisable that dose-response experiments in sperm for tandem repeats be conducted in the future to address this gap.
</p><p>Many studies have shown the induction of transgene mutations recovered in mature sperm derived from toxicant-exposed pre-meiotic male germ cells in transgenic mutation reporter mice (e.g., Brooks and Dean 1997; Liegibel and Schmezer 1997; Mattison et al. 1997). One study measured transgene mutations in the offspring of mice exposed to three single i.p. doses of 100 mg/kg ENU (in 7 day intervals) (Barnett et al. 2002). Four inherited transgene mutations were found from 280 mice (confirmed in multiple somatic tissues), for a mutant frequency of 35.7 x 10E-5 per locus. There is no comparable study on the sperm of lacI transgenic mice, or a similar exposure in another transgenic strain for comparison. However, conversion of the per locus lacI offspring mutant frequency to per nucleotide reveals a mutant frequency of 3.31 x 10-7 per nucleotide. LacZ mutant frequencies for 150 mg/kg (half the exposure level of the lacI transgenic mice) exposures of MutaMouse males to ENU results in a per nucleotide mutation frequency ranging from 0.37 to 2.21 x 10E-7 (Liegibel and Schmezer 1997; van Delft and Baan 1995). Thus, the induced mutation frequency in sperm and offspring are within the same range, despite higher doses in the offspring study, supporting incidence concordance for these events.
</p><p>Finally, it has been documented that DNA damage and mutation accumulates as human males age (reviewed in Paul and Robaire 2013), which is concordant with increased incidence of mutation in the offspring of ageing fathers (Kong et al. 2012; Sun et al. 2012). Comparison of the dose-response characteristics of this relationship is not possible because of differences in the mutagenic endpoints measured in sperm versus offspring.
</p><p>There are no inconsistencies in the data for this KER, although the data are limited. There is a possibility that mutations can arise in the early embryo instead of in the spermatogenic cells. However, given the study designs for this type of work (where > 42 days pass prior to sperm collection or mating – see OECD TG488 for the time-series required for transgene mutation analysis in sperm), it is unlikely that this contributes significantly. Limitations in technology currently prevent the analyses required to describe the incidence of mutations in sperm versus offspring, but this is a future research direction. It should be noted that the locations and types of mutations would influence the probably of transmission; this relationship has not been confirmed empirically and limits extrapolation across studies applying different endpoints.
</p><p>Mutations conferring a selective disadvantage to sperm or to the embryo will not be measured in live born offspring and will be eliminated. Thus, mutation frequency in sperm should be equal to mutation rate derived by measuring mutations in the offspring for non-selective loci (as is seen in the rodent tandem repeat and transgene mutation examples described above); or, sperm mutation frequency should be greater than mutation rate measured by identifying mutations in the offspring. However, quantitative data to demonstrate this are lacking because of current technical limitations to study this. It is anticipated that improved models will be developed to predict the likely outcome of increased rates of heritable mutation from sperm mutation frequency data when more data are available from studies applying next generation sequencing technologies in sperm and pedigrees.
</p>HighHigh<p>Mutation is the underlying source of evolution and occurs in every species. Theoretically, any sexually reproducing organism (i.e., producing gametes) can acquire mutations in their gametes and transmit these to descendants. Thus, the present KER is relevant to any species producing sperm.
</p><p><br />
Barnett, L.B., R.W. Tyl, B.S. Shane, M.D. Shelby and S.E. Lewis (2002), "Transmission of mutations in the lacI transgene to the offspring of ENU-treated Big Blue male mice", <i>Environ. Mol. Mutagen.</i>, 40(4): 251-257.
</p><p>Brooks, T.M. and S.W. Dean (1997), "The detection of gene mutation in the tubular sperm of Muta Mice following a single intraperitoneal treatment with methyl methanesulphonate or ethylnitrosourea", <i>Mutat. Res.</i>, 388(2-3): 219-222.
</p><p>Demarini, D.M. (2012), "Declaring the existence of human germ-cell mutagens", <i>Environ. Mol. Mutagen.</i>, 53(3): 166-172.
</p><p>Dubrova, Y.E., P. Hickenbotham, C.D. Glen, K. Monger, H.P. Wong and R.C. Barber (2008), "Paternal exposure to ethylnitrosourea results in transgenerational genomic instability in mice", <i>Environ. Mol. Mutagen.</i>, 49(4): 308-311.
</p><p>Kong, A., M.L. Frigge, G. Masson, S. Besenbacher, P. Sulem, G. Magnusson, S.A. Gudjonsson, A. Sigurdsson, A. Jonasdottir, W.S. Wong, G. Sigurdsson, G.B. Walters, S. Steinberg, H. Helgason, G. Thorleifsson, D.F. Gudbjartsson, A. Helgason, O.T. Magnusson, U. Thorsteinsdottir and K. Stefansson K. (2012), "Rate of de novo mutations and the importance of father's age to disease risk", <i>Nature</i>, 488(7412): 471-475.
</p><p>Lewis, S.E., L.B. Barnett, B.M. Sadler and M.D. Shelby (1991), "ENU mutagenesis in the mouse electrophoretic specific-locus test, 1. Dose-response relationship of electrophoretically-detected mutations arising from mouse spermatogonia treated with ethylnitrosourea", 'Mutat. Res.<i>, 249(2): 311-315.</i>
</p><p>Liegibel, U.M. and P. Schmezer (1994), "Detection of the two germ cell mutagens ENU and iPMS using the LacZ/transgenic mouse mutation assay" <i>Mutat. Res.</i>, 341(1):17-28.
</p><p>Marchetti, F. and A.J. Wyrobek (2005), "Mechanisms and consequences of paternally-transmitted chromosomal abnormalities", <i>Birth Defects Res C Embryo Today</i>, 75(2): 112-129.
</p><p>Mattison, J.D., L.B. Penrose and B. Burlinson (1997), "Preliminary results of ethylnitrosourea, isopropyl methanesulphonate and methyl methanesulphonate activity in the testis and epididymal spermatozoa of Muta Mice", <i>Mutat. Res.</i> 388(2-3): 123-7.
</p><p>O'Brien, J.M., A. Williams, J. Gingerich, G.R. Douglas, F. Marchetti and C.L. Yauk (2013), "No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea", <i>Mutat Res.</i>, 741-742:11-7
</p><p>Paul,C. and B. Robaire (2013), "Ageing of the male germ line", <i>Nat. Rev. Urol.</i>, 10(4): 227-234.
</p><p>Shendura, J. and M. Akey (2015), "The origins, determinants, and consequences of human mutations", <i>Science</i>, 349(6255): 1478-1483.
</p><p>Sun, J.X., A. Helgason, G. Masson, S.S. Ebenesersdottir, H. Li, S. Mallick, S. Gnerre, N. Patterson, A. Kong, D. Reich and K. Stefansson (2012), "A direct characterization of human mutation based on microsatellites", <i>Nat. Genet.</i>, 44(10): 1161-1165.
</p><p>Swayne, B.G., A. Kawata, N.A. Behan, A. Williams, M.G. Wade, A.J. Macfarlane and C.L. Yauk (2012), "Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice", <i>Mutat. Res.</i>, 737(1-2): 1-7.
</p><p>Vilarino-Guell, C., A.G. Smith and Y.E. Dubrova (2003), "Germline mutation induction at mouse repeat DNA loci by chemical mutagens", 'Mutat. Res.<i>, 526(1-2): 63-73.</i>
</p><p>Yauk, C.L., Y.E. Dubrova, G.R. Grant and A.J. Jeffreys (2002), "A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus", <i>Mutat Res.</i>, 500(1-2): 147-156.
</p><p>Yauk, C.L., L.J. Argueso, S.S. Auerbach, P. Awadalla, S.R. Davis, D.M. Demarini, G.R. Douglas, Y.E. Dubrova, R.K. Elespuru, T.M. Glover, B.F. Hales , M.E. Hurles, C.B. Klein, J.R. Lupski, D.K. Manchester, F. Marchetti, A. Montpetit, J.J. Mulvihill, B. Robaire, W.A. Robbins, G.A. Rouleau, D.T. Shaughnessy, C.M. Somers, J.G. Taylor 6th, J. Trasler, M.D. Waters, T.E. Wilson, K.L. Witt and J.B. Bishop (2013), "Harnessing genomics to identify environmental determinants of heritable disease" <i>Mutation Research</i>, 752(1): 6-9.
</p>2016-11-29T18:41:332016-11-29T19:59:38Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutationsAlkylation of DNA leading to heritable mutations<p>Carole Yauk (1)*</p>
<p>Iain Lambert (2)</p>
<p>Francesco Marchetti (1)</p>
<p>George Douglas (1)</p>
<p><br />
(1) Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON, Canada</p>
<p>(2) Dept. of Biology, Carleton University, Ottawa, ON, Canada</p>
<ul>
<li>Communicating author: carole.yauk@canada.ca</li>
</ul>
Open for citation & commentWPHA/WNT EndorsedIncluded in OECD Work Plan1.11<p>Germ cell/heritable mutations are important regulatory endpoints for international agencies interested in protecting the health of future generations. However, germ cell mutation analysis has been hampered by a lack of efficient tools. With the publication of the OECD test guideline TG488 (rodent transgene mutation assay) and new technologies (including next generation sequencing) this field is experiencing renewed focus. Indeed, regulatory approaches to assess germ cell mutagenicity were the focus of an IWGT workshop (Yauk et al., 2013). Of particular concern is the inability to address this endpoint through high-throughput screening assays (because spermatogenesis cannot be carried out in culture), and mutagenesis is an important gap in existing high-throughput tests. The motivation for developing this AOP was to provide context for new assays in this field, identify research gaps and facilitate the development of new methods.</p>
<p>In this AOP, a compound capable of alkylating DNA is delivered to the testes causing germ cell mutations and subsequent mutations in the offspring of the exposed parents. The AOP requires uptake of the parent compound or metabolite in spermatogonia and interaction with DNA in those cells. DNA alkylation in male pre-meiotic germ cells is the molecular initiating event. A variety of different DNA adducts are formed that are subject to DNA repair; however, at high doses the repair machinery becomes saturated or overwhelmed. The fate of remaining adducts includes: (1) attempted DNA repair by alternative DNA repair machinery, or (2) no repair. Key event (KE) 1 is insufficient or incorrect DNA repair. Lack of repair can lead to replication of adducted DNA and ensuing mutations in male pre-meiotic germ cells (KE2). Mutations that do not impair spermatogenic processes will persist in these cells and eventually be present in the mature sperm. Thus, the mutations can be transmitted to the offspring (adverse outcome – inherited mutations). It is well documented that mice and other animals exposed to alkylating agents develop mutations in male pre-meiotic germ cells that are then found in sperm, resulting in the transmission of mutations to their offspring. There is a significant amount of empirical evidence supporting the AOP and the overall weight of evidence is strong. Although there are some gaps surrounding some mechanistic aspects of this AOP, the overarching AOP is widely accepted and applies broadly to any species that produces sperm.</p>
<p>De novo germ cell mutations are changes in the DNA sequence of sperm or egg that can be inherited by offspring. De novo mutations contribute to a wide range of human disorders including cancer, infertility, autism, schizophrenia, intellectual disability, and epilepsy (Girirajan et al. 2010; Hoischen et al. 2010; Ku et al. 2012; Lupski 2010; Morrow 2010; Vissers et al. 2010). Each child inherits, on average, approximately one de novo mutation per 100 million nucleotides delivered via the parental egg and sperm (Conrad et al. 2011; Kong et al. 2012; O'Roak et al. 2012; Roach et al. 2010). The precise locations and types of mutations in the genomic DNA sequence govern the outcome of these mutations (e.g., protein coding versus intergenic sequences, conserved versus non-conserved mutations, etc.). Although a large portion of human DNA is of unknown function, recent literature suggests that at least 80% of the genome is transcribed, and most DNA is expected to have a biological function (Bernstein et al. 2012). It has been estimated that the proportion of coding and splice-site base substitutions that result in truncating mutations is ~5% (Kryukov et al. 2007), and that as many as 30% of missense mutations are also likely to be highly deleterious due to loss of function (Boyko et al. 2008). When they occur in functional sites, de novo mutations can cause embryonic or fetal lethality, or if viable, can produce a broad spectrum of inherited genetic disorders. Recent estimates suggest that a human genome contains approximately 100 loss-of-function variants, with as many as 20 exhibiting complete loss of gene function (McLaughlin et al. 2010). Therefore, de novo mutations contribute to the overall population genetic disease burden. The present AOP focuses on <a href="/wiki/index.php/DNA_alkylation_in_spermatogonia" title="DNA alkylation in spermatogonia">DNA alkylation in spermatogonia</a> that causes inherited mutation transmitted via sperm, arguably one of the most well characterized modes of action in genetic toxicology. Humans are exposed to alkylating agents from external (e.g., abiotic plant materials, tobacco smoke, combustion products, chemotherapeutic agents) and internal (e.g., byproducts of oxidative damage and cellular methyl donors) sources.</p>
<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><p>Heritable mutations are an important regulatory endpoint for most agencies around the world (reviewed in Yauk et al. 2015). Strategies and guidelines for regulatory toxicology testing in various national regulatory jurisdictions, including requirements for germ cell mutation assays, have been described extensively by Cimino (2006), and have not changed significantly. While no jurisdiction requires germ cell testing per se in an initial test battery, many regulatory authorities can request germ cell tests for follow-up studies, e.g. in the U.S.A (U.S. EPA), Canada (Health Canada), the United Kingdom (Committee on Mutagenicity: COM), and Europe (Registration, Evaluation, Authorization and Restriction of Chemicals, i.e. REACH). For example, within the REACH strategy a substance that is genotoxic in somatic cells is evaluated from the literature to see if it is a potential germ cell mutagen based on bioavailability to the germ cells and appropriate in vivo data. If such an evaluation shows that the literature is insufficient to determine whether the agent is or is not a potential germ cell mutagen, then that agent can be tested in a suitable germ cell genotoxicity assay. Although germ cell testing is not specifically required under the Canadian Environmental Protection Act (CEPA) New Substances Notification Regulations, germ cell mutation tests are requested and evaluated when necessary. For new chemical assessments under CEPA from 1994 to 2012, a total of 19 chemicals have been evaluated for germ cell mutagenicity (12 for which the test was submitted, plus 7 for which the test was referenced on the MSDS); importantly, this is comparable to the number for which testing in rodent cancer assays was evaluated (i.e. total of 20; 17 for which the test was submitted, plus 3 for which test was referenced on the MSDS) (Personal Communication, New Substances Assessment and Control Bureau, Health Canada). These examples illustrates the regulatory importance of heritable mutations as an adverse outcome.
</p><p>For pharmaceuticals, the ICH Technical Requirements for Registration of Pharmaceuticals for Human Use does not require germ cell tests and assumes that in vivo somatic tests and carcinogenicity data will provide sufficient predictivity/protection for germ cell effects (ICH, 2011)
</p><p>The World Health Organization (WHO)/International Programme on Chemical Safety (IPCS) has developed a harmonized scheme for mutagenicity testing. In this document the relationship between somatic cell mutagenicity and germ cell risk is summarized in the following statement. “For substances that give positive results for mutagenic effects in somatic cells in vivo, their potential to affect germ cells should be considered. If there is toxicokinetic or toxicodynamic evidence that germ cells are actually exposed to the somatic mutagen or its bioactive metabolites, it is reasonable to assume that the substance may also pose a mutagenic hazard to germ cells and thus a risk to future generations.” (Eastmond et al. 2009).
</p><p>The Global Harmonization Scheme (GHS; UN, 2013) is a germ cell mutation classification system developed by the United Nations that identifies them according to the categories noted in the Table below. To date over 60 countries have implemented this programme, and are in the process of integrating it into their relevant regulations. To date its implementation is focussed on product labelling legislation in the respective countries and regulatory jurisdictions.
</p><p>Categorization of mutagens by GHS
Category Description
1A Chemicals known to induce heritable mutations in germ cells of humans
1B Chemicals that should be regarded as if they induce heritable mutations in germ cells of humans
2 Chemicals that cause concern for induction of heritable mutations in germ cells of humans
</p><p>Finally, we note that mouse data obtained with the specific locus test and other recessive mutation analyses played an important role in estimating radiation dose risk in the human population (BEIR VII 2006).
</p>adjacentModerateHighadjacentModerateHighadjacentModerateHighnon-adjacentModerateHighnon-adjacentModerateHigh<p>Essentiality was not directly tested for all of the KEs. The MIE cannot be ‘blocked’ in any way to our knowledge (e.g., as you might block a receptor-binding MIE). However, as described in the KERs, enhanced DNA repair of alkylated DNA reduces mutation frequencies and reduction in repair increases mutation frequencies, supporting the essentiality of KE1 (i.e., moderate support). Correct repair of the alkylated DNA (i.e., a block of KE1) will not lead to mutation. For example, MGMT overexpression protects mgt1 mutant yeast against alkylation-induced mutation (Xiao and Fontanie 1995). In addition, Big Blue® mice over-expressing human AGT exhibit greatly reduced O6-methylguanine-mediated lacI and K-ras mutations in the thymus following treatment with MNU (Allay et al. 1999) relative to wild type Big Blue® mice. Insufficient DNA repair is well-established to lead to mutations. In addition, inactivation of MGMT sensitizes cells to alkylation-induced mutagenesis resulting in an increased number of mutations per adduct (Thomas et al. 2013).</p>
<p>The remainder of the AOP requires transmission of mutations in sperm to offspring. There are no means to study the essentiality of mutations in sperm. Once mutations occur in male pre-meiotic germ cells, they cannot be removed to observe whether occurrence in offspring is decreased. In addition, mutations that occur in stem cells are propagated clonally and can become fixed in the spermatogonial cell population. Thus, waiting a longer period of time, or removing the exposure, is not effective in causing a decline in the mutation frequency. Therefore, the essentiality of this KE is inferred by the biology of the pathway and cannot be addressed directly with experimental evidence.</p>
HighMaleHighAdultHighHighLowLow<p>Before developing this AOP a review of the literature was undertaken to identify studies in which male germ cells were exposed to alkylating agents and measures of DNA adducts, DNA repair and mutations, as well as mutations in offspring, were evaluated. The focus of this AOP (as described in the KERs) is on O-alkylating agents, which are signficantly more mutagenic than N-alkylation chemicals. Studies where sufficient information relating to the chemicals used, dose, tissue, time-point, animal model, experimental procedures and experimental results were available were considered to assess empirical data in germ cells for each of the KEs and KERs in the AOP. The germ cell database on which the AOP was based is found in Supplementary Table I (<a href="https://aopwiki.org/system/dragonfly/production/2017/05/19/1qoq9ky7zb_AOP15_supporting_evidence.pdf" title="File:SupplementalTablesAop-15.pdf">SupplementalTablesFigures</a>) and is comprised of 32 studies. No study measured multiple KEs within it; however, for each KE there were at least two dose-response and time-series analyses for at least one alkylating agent. We consider this overall number of high quality studies to be fairly extensive evidence of the ability of O-alkylating agents to cause adducts and mutations in germ cells, and mutations in offspring, although no studies were ideally suited to establish the empirical linkages between the KERs. We thus compared results across studies where possible to attempt to do this. All of the studies either used ENU as the primary study compound, or applied ENU as one of the positive controls to assess other alkylating agents. Strong dose-response data for mutations occurring in exposed pre-meiotic germ cells and mutations in offspring are only available for ENU. The other alkylating agents show varying degrees mutagenicity, but single doses were used in most studies. Thus, the evaluation of concordance of the dose-response could only be undertaken with ENU for in vivo germ cell and heritable effects. However, where possible we used information from research on somatic cells to provide additional support for the KERs. In particular, experiments in somatic cells were necessary to assess the involvement of DNA repair in removing adducts and preventing mutations. Overall, we note that the rationale for claiming high confidence in this AOP and its KERs is based primarily on the more influential Bradford Hill consideration of biological plausibility, with decades of research having been done in somatic and germ cells on DNA damage, repair and mutation. Much of the data, then, supporting AOP evaluation derives from historical studies from the 1990’s, with less recent evidence. As noted, a primary motivation for developing this AOP was the recent release of TG 488, and newly available whole generation sequencing methods, which we expect to be increasingly applied. Thus, additional well-designed experiments that dissect the relationships between alkyl adducts, mutations in sperm, and mutations in offspring to assess essentiality and empirical support are expected in the future through application of these improved approaches. Below we describe each KE and KER in detail, using the wiki entries as a guide to the order of presentation and the content described.</p>
<p> </p>
<p>This AOP is relevant exclusively to mature males and their pre-meiotic germ cells. Although not considered in this AOP, progenitor germ cells from earlier life stages may also be susceptible to induced mutations from alkylating agents, which could then be transmitted to offspring after sexual maturity. Relevant endpoints have been characterized across different taxa: (1) alkyl adduct levels in this AOP were from hamsters, mice and rats; (2) repair of alkylated DNA has been studied in prokaryotes to higher eukaryotes, including human cells in culture (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); (3) mutations in male germ cells were measured in mice and fish; and (4) mutations in offspring were measured in Drosophila, Japanese Medaka and mice. Quite generally, the AOP applies to any species that produces sperm. The similarity in spermatogenesis and in DNA repair of alkyl adducts is well documented across rodents and humans (Adler 1996). Heritable mutations are the basis of evolution and occur in every species. That mutations in sperm are transmitted to offspring in humans is best demonstrated by studies exploring the effects of ageing. Significant increases are observed in the amount of DNA damage and mutation as human males age (reviewed in Paul and Robaire 2013). Similarly, increased incidence of single nucleotide mutations and microsatellite mutation in the offspring of ageing fathers has recently been measured by advanced genomics technologies (Kong et al. 2012; Sun et al. 2012). Lifestyle factors including smoking and lower income brackets in human fathers in associated with increased minisatellite mutations in their offspring (LinSchooten et al., 2013).</p>
<p>Essentiality was not directly tested for all of the KEs. The MIE cannot be ‘blocked’ in any way to our knowledge (e.g., as you might block a receptor-binding MIE). However, as described in the KERs, enhanced DNA repair of alkylated DNA reduces mutation frequencies and reduction in repair increases mutation frequencies, supporting the essentiality of KE1 (i.e., moderate support). Correct repair of the alkylated DNA (i.e., a block of KE1) will not lead to mutation. For example, MGMT overexpression protects mgt1 mutant yeast against alkylation-induced mutation (Xiao and Fontanie 1995). In addition, Big Blue® mice over-expressing human AGT exhibit greatly reduced O6-methylguanine-mediated lacI and K-ras mutations in the thymus following treatment with MNU (Allay et al. 1999) relative to wild type Big Blue® mice. Insufficient DNA repair is well-established to lead to mutations. In addition, inactivation of MGMT sensitizes cells to alkylation-induced mutagenesis resulting in an increased number of mutations per adduct (Thomas et al. 2013).</p>
<p>The remainder of the AOP requires transmission of mutations in sperm to offspring. There are no means to study the essentiality of mutations in sperm. Once mutations occur in male pre-meiotic germ cells, they cannot be removed to observe whether occurrence in offspring is decreased. In addition, mutations that occur in stem cells are propagated clonally and can become fixed in the spermatogonial cell population. Thus, waiting a longer period of time, or removing the exposure, is not effective in causing a decline in the mutation frequency. Therefore, the essentiality of this KE is inferred by the biology of the pathway and cannot be addressed directly with experimental evidence.</p>
<p>Biological plausibility of the KERs: Strong. There is extensive understanding of the ability of alkylating agents to cause DNA adducts, the requirement for overcoming DNA repair, and the resulting mutations that arise in both somatic and germ cells. It is established that exposure to alkylating agents produced mutations in germ cells – ENU is used in genetic screening to produce mutations to derive new phenotypes for research.</p>
<p>Empirical support for the KERs: Across the KERs the degree of support ranges from weak to strong (<a href="/wiki/index.php/File:AssessmentSummaryAop-15.pdf" title="File:AssessmentSummaryAop-15.pdf">File:AssessmentSummaryAop-15.pdf</a> - Table II). Support from somatic cells in culture contributes to moderate calls for the relationships between adduct formation, insufficient DNA repair and mutation. The weak call is based on lack of empirical data to support that mutations in germ cells are transmitted to offspring. However, increased mutation frequencies in germ cells occur following exposure to the same types of chemicals that cause increased mutations in the offspring. It should be noted that biological plausibility for this KER is strong as it is based on understanding of molecular biology and evolution. The strongest support is associated with the indirect KER linking alkylation of DNA to mutation in germ cells (KER4). This is primarily based on extensive evidence in both somatic and germ cells demonstrating that chemicals that alkylate DNA cause mutations, that alkyl adducts occur at a greater incidence than mutations at matching doses, and that alkyl adducts precede mutations. In somatic cells, work has been done on many different chemicals, whereas the germ cell data were primarily for the chemical ENU (but data were also available for a few select other chemicals) (<a href="/wiki/index.php/File:AssessmentSummaryAop-15.pdf" title="File:AssessmentSummaryAop-15.pdf">File:AssessmentSummaryAop-15.pdf</a> - Table I, Figure 2). In addition, data are available for multiple species to support this indirect KER. There is a large degree of consistency in the germ cell literature to show that a variety of O-alkylating agents cause male germ cell mutations in many species (Drosophila, fish and rodent) and that these effects occur at many mutational loci (e.g., mutations in genes that are inherited measured with the Specific Locus Test, sperm mutations in tandem repeat DNA sequences, tandem repeat mutations in offspring, transgene mutations in sperm). Many alkylating agents have been tested to show that they create adducts in male rodent germ cells (e.g., DEN, ENU, EMS, DES), mutations in male mouse germ cells (ENU, IPMS and MNU) and mutations in the offspring of exposed male mice (ENU, MNU and IPMS). In summary, we consider the overall empirical data supporting the AOP to be MODERATE (the median call). Rank order (provided in the overall assessment Table - <a href="/wiki/index.php/File:AssessmentSummaryAop-15.pdf" title="File:AssessmentSummaryAop-15.pdf">File:AssessmentSummaryAop-15.pdf</a>):</p>
<p>Rank order of the KERs and the weight of evidence for the essentiality all point to the overall weight of evidence for this AOP as strong. Biological plausibility is strong for all KERs, with primarily moderate evidence for KER linkages and relatively few uncertainties or inconsistencies.</p>
<p>As described above, it is established that alkyl adducts, mutations in spermatogonia and mutations in offspring all increase with dose in a manner that is consistent with the AOP. Alkylation must exceed a threshold (determined by saturation of the relevant DNA repair pathways) before alkyl DNA lesions persist, and mutations subsequently begin to occur. However, the precise quantitative relationship has not been modeled. Existing data published in the literature could be mined to do this and thresholds for specific adduct types (i.e., estimates of how many adducts are needed to cause a mutation in a gene on average) have been published for certain cell types, which should theoretically correlate with germ cell mutagenicity for ENU and other alkylating agents.</p>
<p>The quantitative relationship between mutations in sperm and mutations in the offspring has not been determined and will be locus- and mutation-type specific (e.g., stronger selection against coding mutations than non-coding mutations, which will influence transmission probability); however, although many mutations will lead to embryonic loss, a large subset of mutations is expected to be heritable and viable. It is expected that quantitative understanding of this relationship will increase as advanced single cell sequencing technologies are more developed to query mutations in sperm versus offspring. For non-coding sites (e.g., transgenic reporter genes and non-coding DNA like tandem repeats), the relationship is expected to approach 1:1.</p>
<p><br />
Overall, the variables that could be used to predict whether a heritable mutation is probable following exposure to an alkylating agent are the number and types of adducts per nucleotide (and knowledge of their repair efficiency). Generally, the probability of a mutation occurring is highly dependent on the type of adduct formed (mutagenicity of the adduct is based on repair efficiency and probability of error-free replication over the lesion) and abundance of the adducts, and could be modeled using existing published data.</p>
<p>The information provided in this AOP will provide context for understanding how to interpret new data produced from the rodent transgene mutation assay applied to sperm (OECD TG 488) [OECD 2013], which is being increasingly applied, as well as data produced using tandem repeat mutation assays. In addition, it is envisioned that next generation sequencing technologies will enable the analysis of germ cell mutations in human populations and the eventual discovery of human germ cell mutagens. It is important to note that the regulation of chemicals that can induce heritable effects has, to date, been based heavily on extrapolation from somatic cell data. Although regulatory agencies around the world have policies in place for germ cell mutagens, risk management based on an agent that is classified as a germ cell mutagen has not yet occurred because of lack of solid evidence that these exist. This AOP demonstrates strong evidence to support the existence of male rodent germ cell mutagens, supported by data in other species (fish, flies, birds), and strongly implies that such mutagens will also affect human germ cells.</p>
<p>Allay, E., M. Veigl and S.L. Gerson (1999), "Mice over-expressing human O6 alkylguanine-DNA alkyltransferase selectively reduce O6 methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea", <em>Oncogene</em>, 18(25): 3783-3787.</p>
<p>Bernstein, B.E., E. Birney, I. Dunham, E.D. Green, C. Gunter and M. Snyder (2012), "An integrated encyclopedia of DNA elements in the human genome", <em>Nature</em>, 489(7414): 57-74.</p>
<p>Boyko, A.R., S.H. Williamson, A.R. Indap, J.D. Degenhardt, R.D. Hernandez, K.E. Lohmueller, M.D. Adams, S. Schmidt, J.J. Sninsky, S.R. Sunyaev, T.J. White, R. Nielsen, A.G. Clark and C.D. Bustamante (2008), "Assessing the evolutionary impact of amino acid mutations in the human genome", <em>PLoS Genetics</em>, 4: e1000083.</p>
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<p>Girirajan, S., C.D. Campbell and E.E. Eichler (2010) "Human Copy Number Variation and Complex Genetic Disease", <em>Annual Review of Genetics</em>, 45: 203-226.</p>
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