CHEMICALLY-INDUCED MUTATIONS IN MITOCHONDRIAL-DNA OF HUMAN-CELLS - MUTATIONAL SPECTRUM OF N-METHYL-N'-NITRO-N-NITROSOGUANIDINE

We have observed a reproducible mitochondrial mutational spectrum in t he MT1 human lymphoblastoid line treated with N-methyl-N'-nitro-N-nitr osoguanidine (MNNG), The MNNG spectrum was distinct from the spontaneo us mutational spectrum. However, our ability to observe MNNG-induced m itochondrial mutations above the high level of accumulated spontaneous mutations was dependent on the MT1 phenotype. MT1 cells are markedly resistant to the cytotoxicity but not the mutagenicity of MNNG, presum ably as a result of inactivation of both copies of the hMSH6 (GTBP) mi smatch repair gene, Thus, we were able to use conditions of treatment that yielded induced mitochondrial mutant fractions beyond the practic al limits for human cell experiments in mismatch-proficient human cell lines. In contradistinction, when MT1 cells were treated repeatedly w ith maximum tolerated concentrations of (+/-) anti-benzo(a)pyrene diol -epoxide, no induced mitochondrial mutations above the spontaneous bac kground were observed. A single dose of 4 mu M MNNG (survival, 0.85) i nduced a mutant fraction of 8 x 10(-3) in the nuclear hypoxanthine-gua nine phosphoribosyltransferase gene, and a clear and reproducible patt ern of seven MNNG-induced hotspot mutations was observed within the mi tochondrial DNA target sequence studied (mitochondrial bp 10,030-10,13 0). All of the MNNG-induced hotspot mutations were G:C to A:T transiti ons present at frequencies between 6 x 10(-5) and 30 x 10(-5) Addition al experiments supported the conclusion that MNNG-induced hotspot muta tions observed were generated in living cells as a result of MNNG trea tment and not from mismatch intermediates or DNA adducts converted int o mutations during the PCR process.