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Relationship: 25


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 Increase, Mutations

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 non-adjacent High Moderate Carole Yauk (send email) Open for citation & comment WPHA/WNT Endorsed
Alkylation of DNA leading to cancer 1 non-adjacent High Moderate Carole Yauk (send email) Open for adoption

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
mouse Mus musculus High NCBI
medaka Oryzias latipes 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 ‘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.

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

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.

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.

Alkylating agents are prototypical somatic and male germ cell mutagens.

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

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.

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

Alkylating agents are well-established to cause mutation in virtually any cell type in any organism.


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

Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", Mutation Research, 231(1): 11-30.

Boysena, G., B.F. Pachkowski, J. Nakamura and J.A. Swenberg (2009), "The formation and biological significance of N7-guanine adducts", Mutation Research, 678: 76–94.

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", Mutat. Res., 388(2-3): 219-222.

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.

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.

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", Mutat. Res., 388(2-3):213-8.

Liegibel, U.M. and P. Schmezer (1994), "Detection of the two germ cell mutagens ENU and iPMS using the LacZ/transgenic mouse mutation assay" Mutat. Res., 341(1):17-28.

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", Mutat. Res. 388(2-3): 123-7.

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", Carcinogenesis, 17(11): 2449-2454.

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", Environ. Mol. Mutagen., 31(1): 18-31

Norris, M.B. and R.N. Winn (2010), "Isolated spermatozoa as indicators of mutations transmitted to progeny", Mutat. Res., 688(1-2): 36–40.

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.

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", Environ. Mol. Mutagen., 53(1): 10-21.

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", Mutagenesis, 28(5): 543-554.

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", J. Vis. Exp., (90): e51576.

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.

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)", Mutat. Res., 388(2-3): 145-153.

Shrivastav, N., D. Li and J.M. Essigmann (2010), "Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation", Carcinogenesis, 31(1): 59-70.

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", Environ. Mol. Mutagen., 28(4): 376-384.

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)", Mutat. Res., 388(2-3): 155-163.

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", Chem. Res. Toxicol., 21(1): 253-265.

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", Mutat. Res., 737(1-2): 1-7.

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", Mutat. Res., 388(2-3): 179-185.

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", Mutat. Res., 388(2-3): 165-173.

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.

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.