Oxygen free radicals are highly reactive species that damage DNA and cause mutations. We determined the mutagenic spectrum of oxygen free radicals produced by the aerobic incubation of single-stranded M13mp2 DNA with Fe2+. The Fe2(+)-treated DNA was transfected into component Escherichia coli, and mutants within the nonessential lac Z alpha gene for beta-galactosidase were identified by decreased alpha-complementation. The frequency of mutants obtained with 10 microM Fe2+ was 20- to 80-fold greater than that obtained with untreated DNA. Mutagenesis was greater after the host cells were exposed to UV irradiation to induce the SOS "error-prone" response. The ability of catalase, mannitol, and superoxide dismutase to diminish mutagenesis indicates the involvement of oxygen free radicals. The sequence data on 94 of the mutants establish that mutagenesis results primarily from an increase in single-base substitutions. Ninety-four percent of the mutants with detectable changes in nucleotide sequence were single-base substitutions, the most frequent being G----C transversions, followed by C----T transitions and G----T transversions. The clustering of mutations at distinct gene positions suggests that Fe2+/oxygen damage to DNA is nonrandom. This mutational spectrum provides evidence that a multiplicity of DNA lesions produced by oxygen free radicals in vitro are promutagenic and could be a source of spontaneous mutations.

Reactive oxygen species are generated by a variety of cellular processes. These endogenously generated, reactive intermediates produce a multiplicity of DNA alterations and mutations and have been implicated in the pathogenesis of several human diseases. We report here that treatment of single-stranded M13mp2 bacteriophage DNA with methylene blue and white light generates increased levels of 8-hydroxydeoxyguanosine and that mutagenesis is both highly specific and dependent on the SOS response. Lesions produced block the progression of DNA synthesis one base preceding template guanines. In SOS-induced Escherichia coli, 97% of all methylene blue-induced mutations in the lacZa gene of M13mp2 DNA are single-base substitutions opposite template guanines. The most frequent mutations are G -+ C transversions. The G -+ T transversions expected from the presence of 8-hydroxydeoxyguanosine in the template strand occur, but at a lower frequency. Sequence data together with SOS dependency and the presence of replication blockage demonstrate that while 8-hydroxydeoxyguanosine may serve as an important marker to monitor oxygen-induced DNA damage in humans, it does not account for either the observed blockage to replication or the mutagenesis by methylene blue plus light in SOS-induced E. coli. Instead, an as yet unidenti lesion generated by active oxygen species is a more potent mutagenic event.In cells, oxygen is metabolized by a series of one-electron reductions. A result ofoxygen metabolism is the formation of free radicals and related species including hydroxyl radicals, hydrogen peroxide, superoxide ions, and singlet oxygen. These highly reactive molecules can damage many cellular macromolecules, including proteins, lipids, RNA, and DNA (1, 2), and the resultant deleterious effects have been hypothesized to contribute to the pathogenesis of many agerelated human diseases, including cancer, atherosclerosis, and joint diseases (3, 4). To elucidate the relationship of DNA damage by reactive oxygen species to the pathogenesis of diseases, methods are required to quantitate the different types of DNA alterations produced and to determine the mutagenic potential of each alteration. However, the multiplicity and complexity of reactions that generate reactive oxygen species in cells have complicated these analyses. First, there are multiple processes in cells that generate oxygen free radicals (2, 5), and these radicals undergo further reactions to form additional reactive species. Second, based on the multiplicity of nucleotide alterations produced by oxygen free radicals in vitro, it can be estimated that in DNA more than 100 different nucleotide adducts can be formed (6-9). Third, certain molecules that generate oxygen free radicals preferentially bind to specific nucleotide sequences in DNA (10, 11). Fourth, long-lived DNA damage and mutagenesis result from inadequate DNA repair and altered incorporation by DNAreplicating enzymes, respectively; these mechanisms, in eukaryotic cells, have yet to be characterized (12)....

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