Mutagenicity of nitric oxide is not caused by deamination of cytosine or 5-methylcytosine in double-stranded DNA.

Several human tumors of diverse histological origin have a high incidence of C:G to T:A transition mutations at methylated CpG sites in tumor suppressor genes. We used a sensitive genetic assay to examine the ability of nitric oxide (NO), a physiological intra- and intercellular messenger molecule, to promote these transitions by deaminating cytosine (C) or methylcytosine (5mC) in double-stranded DNA. Exposure of a test double-stranded plasmid containing C or 5mC at the target site to NO in phosphate-buffered solution at pH 7.4 followed by transformation into Escherichia coli ung- strain to avoid repair of U did not result in a significant increase in reversion frequency. In addition, exposure of E. coli transformed with the target plasmid to an NO-releasing spermine-NO complex during log-phase growth did not result in larger numbers of revertants, whereas Salmonella typhimurium strain TA1535 showed a dose-responsive increase in reversion frequency when treated in the same way. We conclude that genotoxicity of NO is not caused by deamination of C or 5mC to U or T, respectively, in double-stranded DNA. This is supported by the finding that extracts of TA1535 contained high uracil-DNA glycosylase activity, suggesting that the difference in mutagenesis between the strains is not due to a lack of uracil repair. Therefore, mutational hot-spots seen in human tumor tissues at CpG sites are probably not due to the action of NO at 5mC.