We have observed a reproducible mitochondrial mutational spectrum in the MT1 human lymphoblastoid line treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The MNNG spectrum was distinct from the spontaneous mutational spectrum. However, our ability to observe MNNG-induced mitochondrial 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, presumably as a result of inactivation of both copies of the hMSH6 (GTBP) mismatch repair gene. Thus, we were able to use conditions of treatment that yielded induced mitochondrial mutant fractions beyond the practical limits for human cell experiments in mismatch-proficient human cell lines. In contradistinction, when MT1 cells were treated repeatedly with maximum tolerated concentrations of (±) anti-benzo(a)pyrene diol-epoxide, no induced mitochondrial mutations above the spontaneous background were observed.

A single dose of 4 µm MNNG (survival, 0.85) induced a mutant fraction of 8 × 10-3 in the nuclear hypoxanthine-guanine phosphoribosyltransferase gene, and a clear and reproducible pattern of seven MNNG-induced hotspot mutations was observed within the mitochondrial DNA target sequence studied (mitochondrial bp 10,030–10,130). All of the MNNG-induced hotspot mutations were G:C to A:T transitions present at frequencies between 6 × 10-5 and 30 × 10-5.

Additional experiments supported the conclusion that MNNG-induced hotspot mutations observed were generated in living cells as a result of MNNG treatment and not from mismatch intermediates or DNA adducts converted into mutations during the PCR process.


This work was supported by Grant PO1-E503926 from the National Institute for Environmental Health Sciences, Genetics and Toxicology Program, Grant P42-ES04675 from the Superfund Hazardous Substances Basic Research Program, and Grants NIH R01-CA 77044-01 and P30 AG 13314.

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