Molecular analysis of mitochondrial DNA mutations from bleomycin-treated rats.
In our previous studies, we have shown the mutagenicity of bleomycin (BLM) at the nuclear hprt locus. In the present study we have analyzed mutagenic effects of BLM in mitochondrial DNA (mtDNA) using short extension-PCR (SE-PCR) method for detection of low-copy deletions. Fisher 344 rats were treated with a single dose of BLM and total DNA preparations from splenic lymphocytes were processed in SE-PCR assay. Spontaneous deletions were typically flanked by direct repeats (78.5%), while the in BLM-treated group, direct repeats were found in only 46.6% of breakpoints. The ratio between deletions based on direct repeats and random sequence deletions changed from 3.67 in control group to 0.87 in BLM-treated animals, which corresponds to an approximate 1.7-fold increase in the deletion mutation frequency. Furthermore, 62.5% of deletions not flanked by direct repeats in the treated group contained cleavage sites for BLM. The localization of breakpoints was not entirely random. We have found four clusters containing deletions from both groups indicative of deletion hot spots. The results indicate that BLM exposure may be associated with the induction of mtDNA mutations, and suggest the utility of SE-PCR method for evaluating drug-induced genotoxicity.
Khaidakov
Magomed
M
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson Laboratories of the FDA, Jefferson, AR 72079, USA. mkhaidakov@nctr.fda.gov
Manjanatha
Mugimane G
MG
Aidoo
Anane
A
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000633:toxicity*; D001483:Base Sequence; D001761:Bleomycin / Q000633:toxicity*; D003001:Cloning, Molecular; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics*; D017353:Gene Deletion; D014644:Genetic Variation; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D051381:Rats; D011916:Rats, Inbred F344; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D013601:T-Lymphocytes / Q000187:drug effects
D000964:Antimetabolites, Antineoplastic; D004272:DNA, Mitochondrial; D009153:Mutagens; D001761:Bleomycin; D007041:Hypoxanthine Phosphoribosyltransferase