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Relationship: 23


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, Heritable mutations in offspring

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 Evgeniia Kazymova (send email) Open for citation & comment WPHA/WNT Endorsed

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
Drosophila melanogaster Drosophila melanogaster 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

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.

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 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., In fact, ENU mutagenicity is a standard bench tool for genetic screens used to identify new mutations associated with a phenotype of interest.

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).

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 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.

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).

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

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.


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

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", Mutat. Res., 445(1): 73-80.

Demarini, D.M. (2012), "Declaring the existence of human germ-cell mutagens", Environ. Mol. Mutagen., 53(3): 166-172.

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", Environ. Mol. Mutagen., 49(4): 308-311.

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)", Mutat. Res., 250(1-2): 447-456.

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., 162(1): 69-80.

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., 231(1): 47-54.

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., 374(2): 193-199.

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., 249(2): 311-315.

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," Int. J. Cancer, 126(5): 1196-1205.

Marchetti, F. and A.J. Wyrobek (2005), "Mechanisms and consequences of paternally-transmitted chromosomal abnormalities", Birth Defects Res C Embryo Today, 75(2): 112-129.

Nagao, T. (1987), "Frequency of congenital defects and dominant lethals in the offspring of male mice treated with methylnitrosourea", 'Mutat. Res., 177(1): 171-178.

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", Proc. Natl. Acad. Sci. USA, 76(11): 5818-5819.

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", Proc. Natl. Acad. Sci. USA, 79(11): 3589-3591.

Russell, L.B. (2004), "Effects of male germ-cell stage on the frequency, nature, and spectrum of induced specific-locus mutations in the mouse", Genetica, 122(1): 25-36.

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., 616(1-2): 181-195.

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., 545(1-2): 81-107.

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", J. Clin. Oncol., 30(3): 239-245.

Shima, A. and A. Shimada (1994), "The Japanese medaka, Oryzias latipes, as a new model organism for studying environmental germ-cell mutagenesis", Environ. Health Perspect., 102 Suppl 12: 33-35.

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", Mutagenesis, 13(4): 375-380.

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., 231(1): 55-62.

Vilarino-Guell, C., A.G. Smith and Y.E. Dubrova (2003), "Germline mutation induction at mouse repeat DNA loci by chemical mutagens", 'Mutat. Res., 526(1-2): 63-73.

Witt, K.L. and J.B. Bishop (1996), "Mutagenicity of anticancer drugs in mammalian germ cells", 'Mutat. Res., 355(1-2): 209-234.

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", Environ. Mol. Mutagen., 36(2): 134-145.