Tandem repeat mutation, global DNA methylation, and regulation of DNA methyltransferases in cultured mouse embryonic fibroblast cells chronically exposed to chemicals with different modes of action

Abstract

Mutations at expanded simple tandem repeat (ESTR) DNA sequences provide a useful tool for screening germline mutation. However, the mechanisms resulting in induced mutations are unknown and provide an impediment to the utility of the method. Induced ESTR mutations arise through a nontargeted mechanism resulting in destabilization of the repeat locus. We hypothesized that alterations in DNA methylation, or in DNA methyltransferase expression, may be associated with this indirect mechanism of mutation. DNA mutation frequency was measured in C3H/10T1/2 mouse embryonic fibroblast cells following chronic exposure to six chemicals exhibiting different modes of genotoxic action: N‐nitroso‐N‐ethylurea (ENU); benzo(a)pyrene (BaP); etoposide (ETOP); okadaic acid (OA); cisplatin (CisPt); and 5‐azacytidine (5azadC). Induced mutation ranged from 2‐fold (ENU, BaP, ETOP), to 1.3–1.4 fold (OA, 5azadC), to nonresponsive (CisPt). Global DNA methylation, measured using the cytosine extension assay, revealed hypomethylation following exposure to ENU and 5azadC, hypermethylation following BaP and OA exposure, and no change following treatment with ETOP or CisPt. DNA methyltransferase transcription (Dnmt1, Dnmt3a, Dnmt3b) was significantly affected by all treatments except ETOP, with the vast majority of changes being downregulation. There was no direct correlation between ESTR mutation, global methylation, or DNA methyltransferase transcription. However, 4/5 ESTR mutagens caused changes in global methylation, while the noninducer (CisPt) did not cause changes in methylation. We hypothesize that chemicals that modify chromatin conformation through changes in methylation may compromise the ability of mismatch repair enzymes (or other enzymes) to access and repair secondary structures that may form across ESTR loci resulting in mutation. Environ. Mol. Mutagen., 2008. Published 2008 Wiley‐Liss, Inc.

Global and gene‐specific promoter methylation changes are related to anti‐B[a]PDE‐DNA adduct levels and influence micronuclei levels in polycyclic aromatic hydrocarbon‐exposed individuals

“…13 Global DNA hypermethylation following in vitro chronic exposure to B[a]P was recently revealed in mouse embryonic fibroblasts and was associated to mutation as a consequence of genetic instability. 14,15 PBLs, although not considered target cells for PAH(B[a]P)-induced lung tumorigenesis, can be easily obtained from human subjects and their genetic (PAH(B[a]P)-DNA adducts) and epigenetic alterations (methylation) are correlated with levels in target tissues (e.g., lung). [16][17][18] Moreover, high levels of adduct and micronuclei (as markers of chromosome instability 19 ) and p53 hypomethylation in PBLs are predictive of lung cancer risk.…”

The effects of methyl-donor deficiency on mutation induction and transgenerational instability in mice

“…Germ cell mutation frequencies can be measured at the mouse expanded simple tandem repeat (ESTR) loci, which are short (4-6bp) repeat units, tandemly repeated to form long homogeneous arrays up to 20kb in length (Yauk, 2004;Yauk et al, 2008). ESTRs have a mutation rate a thousand times higher than normal protein coding genes, both in somatic and germ cells, providing a highly sensitive tool for the investigation of heritable mutation induction in laboratory mice.…”

“…It was thus hypothesised that chemicals that have the ability to change the conformation of chromatin through changes in the methylation pattern may compromise the ability of mismatch repair enzymes to mend secondary structures that may form across ESTR loci resulting in mutation (Yauk et al, 2008).…”

“…Given that ESTR mutation is a replication-based process involving polymerase slippage, there should be a relationship between the accumulation of mutation and mitotic index (Yauk, 2004;Yauk et al, 2008). If methyl-deficient diet resulted in long-term genome destabilisation, then ongoing genomic instability would lead to the accumulation of mutations in tissues with a high mitotic index such as spermatogonia.…”

“…Since ESTR mutations occur via replication slippage during DNA repair or replication processes at the pre-meiotic stages of spermatogenesis (Yauk, 2004;Yauk et al, 2008) it was anticipated that the final 4 weeks prior to sample collection for ESTR analysis would not affect the mutation outcome (Swayne et al, 2012b). The experimental design of this project is believed to effectively capture sperm derived from spermatogonial stem cells exposed to 8 weeks of methyl-donor deficiency (Test F 0 A), and a subsequent of 2 (Test F 0 B) or 6 (Test F 0 C) weeks of supplementation for germ cells committed to spermatogenesis, including 4 weeks as pre-meiotic spermatocytes (Voutounou et al, 2012).…”