Upstream eventN/A, Insufficient or incorrect DNA repair
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations||adjacent||High||Moderate|
|Alkylation of DNA leading to cancer 2||adjacent||High||Moderate|
|Alkylation of DNA leading to cancer 1||non-adjacent||High||Moderate|
Life Stage Applicability
Key Event Relationship Description
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.
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.
Evidence Supporting this KER
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.
For example, for alkylated DNA, efficient removal by AGT 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.
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).
INSUFFICIENT REPAIR OF ALKYLATED DNA
Evidence in somatic cells
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.
Various studies in cultured cells and microorganisms have shown that the expression of AGT/MGMT (repair machinery – i.e., decrease in KE1) 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.
Evidence in germ cells
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 hypothesized 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.
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).
Uncertainties and Inconsistencies
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.
Quantitative Understanding of the Linkage
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].
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.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
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, 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.
Basu, A.K. and J.M. Essigmann (1990), "Site-specific alkylated oligodeoxynucleotides: Probes for mutagenesis, DNA repair and the structure effects of DNA damage", Mutation Research, 233: 189-201.
Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", Mutation Research, 231(1): 11-30.
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", Proc. Natl. Acad. Sci. USA, 92(16): 7485-7489.
Ellison, K.S., E. Dogliotti, T.D. Connors, A.K. Basu and J.M. Essigmann (1989), "Site-specific mutagenesis by O6-alkyguanines located in the chromosomes of mammalian cells: Influence of the mammalian O6-alkylguanine-DNA alkyltransferase", Proc. Natl. Acad. Sci. USA, 86: 8620-8624.
Gocke, E. and L. Muller (2009), "In vivo studies in the mouse to define a threhold for the genotoxicity of EMS and ENU", Mutat. Res., 678, 101-107.
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", DNA Repair, 6: 1079–1099.
Muller, L., E. Gocke, T. Lave and T. Pfister (2009), "Ethyl methanesulfonate toxicity in Viracept – A comprehensive human risk assessment based on threshold data for genotoxicity", Toxicology Letters, 190: 317-329.
O'Brien, J.M., A. Williams, J. Gingerich, G.R. Douglas, F. Marchetti and C.L. Yauk CL. (2013), "No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea", Mutat. Res., 741-742:11-7
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", Environ. Mol. Mutagen., 56(4): 347-55.
Pegg, A.E., (2011), "Multifaceted roles of alkyltransferase and related proteins in DNA repair, DNA damage, resistance to chemotherapy, and research tools", Chem. Res. Toxicol., 24(5): 618-639.
Philippin, G., J. Cadet, D. Gasparutto, G. Mazon, R.P. Fuchs (2014), "Ethylene oxide and propylene oxide derived N7-alkylguanine adducts are bypassed accurately in vivo", DNA Repair (Amst), 22:133-6.
Pzoniak, A., L. Muller, M. Salgo, J.K. Jone, P. Larson and D. Tweats (2009), "Elevated ethyl methansulfonate in nelfinavir mesylate (Viracept, Roche): overview", Aids Research and Therapy, 6: 18.
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. Mutation Research 388(2-3):99-109.
Shrivastav, N., D. Li and J.M. Essignmann (2010), "Chemical biology of mutagenesis and DNA repair: cellular response to DNA alkylation", Carcinogenesis, 31(1): 59-70.
Singer, B., F. Chavez, M.F. Goodman, J.M. Essigman and M.K. Dosanjh (1989), "Effect of 3' flanking neighbors on kinetics of pairing of dCTP or dTTP opposite O6-methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used", Proc. Natl. Acad. Sci. USA, 86(21): 8271-8274.
Thomas, A.D., G.J. Jenkins, B. Kaina, O.G. Bodger, K.H. Tomaszowski, P.D. Lewis, S.H. Doak and G.E. Johnson (2013), "Influence of DNA repair on nonlinear dose-responses for mutation", Toxicol. Sci., 132(1): 87-95.
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", Mutagenesis, 10(3): 209-214.