MUTAGENICITY OF NITRIC-OXIDE IS NOT CAUSED BY DEAMINATION OF CYTOSINEOR 5-METHYLCYTOSINE IN DOUBLE-STRANDED DNA

Several human tumors of diverse histological origin have a high incide nce of C:G to T:A transition mutations at methylated CpG sites in tumo r suppressor genes. We used a sensitive genetic assay to examine the a bility of nitric oxide (NO), a physiological intra- and intercellular messenger molecule, to promote these transitions by deaminating cytosi ne (C) or methylcytosine (5mC) in double-stranded DNA. Exposure of a t est 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 resu lt in a significant increase in reversion frequency. In addition, expo sure of E.coli transformed with the target plasmid to an NO-releasing spermine-NO complex during log-phase growth did not result in larger n umbers of revertants, whereas Salmonella typhimurium strain TA1535 sho wed a dose-responsive increase in reversion frequency when treated in the same way. We conclude that genotoxicity of NO is not caused by dea mination of C or 5mC to U or T, respectively, in double-stranded DNA. This is supported by the finding that extracts of TA1535 contained hig h uracil-DNA glycosylase activity, suggesting that the difference in m utagenesis between the strains is not due to a lack of uracil repair. Therefore, mutational hot-spots seen in human tumor tissues at CpG sit es are probably not due to the action of NO at 5mC.