The mutagenic potentials of ethylmethane sulfonate, N-methyl-N′-nitrosoguanidine, and benzo(a)pyrene diol-epoxide in human mitochondria were determined by cloning and nucleotide sequencing of mitochondrial (mt) DNA from HeLa cells treated with these mutagens. Mutagen concentrations that reduced cell survival to approximately 0.1% of untreated cultures were used. Mitochondrial DNA was prepared 2 to 3 weeks after mutagen treatment, at which time the treated cell population had regrown to 10 times the starting cell number. In one series of experiments, a portion of the D-loop region of mtDNA from treated or control HeLa cells was cloned into the bacteriophage vector M13mp19, and the nucleotide sequences of 102 independent clones were determined. Only a single G:C base pair deletion was observed in 1 of 12 clones derived from HeLa cells treated 6 times with ethylmethane sulfonate. From benzo(a)pyrene diol-epoxide-treated HeLa cells, G:C base pair deletions were found in 14 of 63 clones. All 14 of these G:C deletion mutations occurred at the same position in independent clones, however, and thus could be the progeny of a single mutational event. In a second series of experiments, a method for the selection of mtDNA mutants was utilized. Mutations in an “uncloneable” fragment of human mtDNA render the fragment cloneable and thus provide a selection for mutations in this region of human mtDNA. No enhancement in the cloning efficiency of this region of mtDNA was observed after exposure of cells to toxic concentrations of either MNNG or benzo(a)pyrene diol-epoxide. Moreover, the site and types of nucleotide sequence alterations observed after mutagen treatment were similar to those obtained in the absence of drug treatment. The results of both type of experiments suggest that mutagenesis of human mtDNA is an infrequent event, even after extensive treatment of HeLa cells with potent mutagens that can covalently modify mtDNA.
This work was supported by the NIH (R35-CA-39903, AG-07151) and the United States Environmental Protection Agency (R809-623-010).