Induction of mitochondrial mutations in human cells by methotrexate

Inhibition of dihydrofolate reductase by the folate analog, methotrexate (MTX) results in a depletion of tetrahydrofolate dependent one carbon transfer reactions in amino acid and nucleic acid biosynthesis. When human cells (either HeLa or normal skin fibroblasts) are exposed to MTX in a defined medium containing dialyzed fetal calf serum, essential and non-essential amino acids, and purine source, the thymidylate pools alone are depleted. Under these conditions exposure to 10(-6) M MTX induces mitochondrial mutagenesis, measured as an increase in the frequency of chloramphenicol resistant (CAPR) colonies, without altering the rate of nuclear mutation monitored by determining the frequency of 6-thioguanine resistance (TGr). The occurrence of CAPR mutations is time, and MTX concentration dependent and the frequency of CAPR can be decreased quantitatively by adding thymidine to the culture medium. This mitochondrial specific mutagenesis can also be achieved using the thymidylate synthetase inhibitor, 5-fluorodeoxyuridine further implicating thymidylate pools as the mediator of this effect. During the course of exposure to 10(-6) M MTX the thymidine kinase deficient HeLa BU25 cell line exhibits a progressive depletion and degradation of mitochondrial DNA suggesting that the mutagenesis and DNA degradation represent portions of a progressive process. The basis for the selective sensitivity of the mitochondrial genome to thymidylate depletion mutagenesis may be the consequence of its differences from the nuclear genome in mechanisms of DNA replication or repair.