Upstream eventAlkylation, DNA
Increase, Heritable mutations in offspring
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||non-adjacent||High||Moderate|
|Drosophila melanogaster||Drosophila melanogaster||Moderate||NCBI|
Life Stage Applicability
Key Event Relationship Description
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 Supporting this KER
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., http://ja.brc.riken.jp/lab/mutants/genedriven.htm). 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).
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).
Uncertainties and Inconsistencies
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).
Quantitative Understanding of the Linkage
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.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
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.
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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.
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