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.