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Relationship: 24


A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Alkylation, DNA leads to Inadequate DNA repair

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Downstream event
The responding Key Event (KE) in a Key Event Relationship (KER). More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name Adjacency Weight of Evidence Quantitative Understanding Point of Contact Author Status OECD Status
Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations adjacent High Moderate Evgeniia Kazymova (send email) Open for citation & comment WPHA/WNT Endorsed
Alkylation of DNA leading to cancer 1 adjacent High Moderate Arthur Author (send email) Open for adoption
Alkylation of DNA leading to reduced sperm count adjacent Brendan Ferreri-Hanberry (send email) Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER.  More help
Term Scientific Term Evidence Link
Syrian golden hamster Mesocricetus auratus Moderate NCBI
mouse Mus musculus Moderate NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER.  More help

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

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.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings.  Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help
Biological Plausibility
Addresses the biological rationale for a connection between KEupstream and KEdownstream.  This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured.   More help

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).

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.

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).

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).

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).

Uncertainties and Inconsistencies
Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

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.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references.  More help
Response-response Relationship
Provides sources of data that define the response-response relationships between the KEs.  More help
Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help
Known Feedforward/Feedback loops influencing this KER
Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

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.


List of the literature that was cited for this KER description. More help

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.

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", Cancer Research, 51(19): 5188-5197.

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", Cancer Research, 52(7): 2008-2011.

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", DNA Repair, 3(11): 1389-1407.

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", Proc. Natl. Acad. Sci. USA, 99(26): 16660-16665.

Fang, Q., S. Kanugula, J.L. Tubbs, T.A. Tainer and A.E. Pegg (2010), "Repair of O4-alkylthymine by O6-alkylguanine-DNA alkyltransferases", J. Biol. Chem. 12(285): 885-895.

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", Biochem. J., 174(3): 1031-1044.

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.

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", Mol. Cell, 47(1): 50-60.

Muller, L., E. Gocke, T. Lave and T. Pfister (2009), "Ethyl methanesulfonate toxicity in Viracept – a comprehensive assessment based on threshold data for genotoxicity", Toxicology Letters, 190: 317-329.

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.

Scherer, E., A.A. Jenner and L. den Engelse (1987), "Immunocytochemical studies on the formation and repair of O6-alkylguanine in rat tissues", IARC Sci. Publ., 84: 55-8.

Sega, G.A., C.R. Rohrer, H.R. Harvey and A.E. Jetton (1986), "Chemical dosimetry of ethyl nitrosourea in the mouse testis", Mutat. Res., 159(1-2): 65-74.

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", Mutat. Res., 385(3): 205-211.

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.

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", Mutat. Res., 231(1):55-62.

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", Bioessays, 21(8): 668-676.

York S.J. and P. Modrich (2006), "Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts", J. Biol. Chem., 281(32): 22674-22683.