Instability of expanded simple tandem repeats is induced in cell culture by a variety of agents: N-Nitroso-N-ethylurea, benzo(a)pyrene, etoposide and okadaic acid.
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AbstractExpanded simple tandem repeat (ESTR) sequences have proven useful biomarkers to detect genotoxicity in vivo. Their high sensitivity has been used to assess environmentally relevant doses of mutagens such as ionizing radiation, DNA alkylating agents and airborne particulate pollution, for germline mutations in mouse assays. The mutagenic response involves size alteration of these ESTR loci induced by agents causing a variety of cellular damage. The mechanistic aspects of this induced instability remain unclear and have not been studied in detail. Mechanistic knowledge is important to help understand the relevance of increased ESTR mutation frequencies. In this study, we applied a murine cell culture system to examine induced response to four agents exhibiting different modes of toxic action including: N-nitroso-N-ethylurea (ENU), benzo(a)pyrene (BaP), okadaic acid and etoposide at slightly sub-toxic levels. We used single-molecule-polymerase chain reaction (SM-PCR) to assess the relative mutant frequency after 4-week chemical treatments at the Ms6-hm ESTR sequence of cultured C3H/10T1/2 cells (a mouse embryonic cell line). Increased mutation was observed with both 0.64 mM ENU (1.95-fold increase, P<0.0001), 1 microM benzo(a)pyrene (1.87-fold increase, P=0.0006) and 3 nM etoposide (1.89-fold increase, P=0.0003). The putative ESTR mutagen okadaic acid (1.27-fold increase, P=0.2289), administered at 0.5 nM, did not affect the C3H/10T1/2 Ms6-hm locus. Therefore, agents inducing small and bulky adducts, and indirectly causing strand breaks through inhibition of topoisomerase, caused similar induction of instability at an ESTR locus at matched toxicities. As size spectra for induced mutations were identical, the data indicate that although these chemicals exhibit distinct modes of action, a similar indirect process is influencing ESTR instability. In contrast, a potent tumour promoter that is a kinase inhibitor does not contribute to induced ESTR instability in cell culture.
CitationMutat. Res. 2006, 598 (1-2):73-84
DescriptionBiomarkers of exposure & effect:: validationBiomarker: Expanded simple tandem repeat (ESTR) sequencesExposure/effect represented: ENU, BaP, okadaic acid and etoposide/genotoxicityStudy type (in vitro, animals, humans): in vitroMethod of analysis: SM-PCR (single molecule -PCR)Sensitivity (LOD): allows 40-60% of amplifications to be visibleAccuracy: it needs 10-30pg DNAIntralaboratory repeatability: 4 biological replicatesDose-response: okadaic acid 1.3-fold increase, P=0.2289, Etoposide 1.9-fold, P=0.0003, ENU 2-fold, P<0.0001, BaP 1.9-fold, P=0.0006. KEYWORDS CLASSIFICATION: Animals;biomarkers of exposure & effect: validation;Benzo(a)pyrene;Canada;Cell Division;Cell Line;drug effects;Environmental Health;Ethylnitrosourea;Etoposide;Genomic Instability;mechanisms of carcinogenesis;Mice;Mice,Inbred C3H;Mutagenesis;Mutagens;Okadaic Acid;Polymerase Chain Reaction;Research;toxicity;Tandem Repeat Sequences;Toxicology.