Mutation spectrum of 4-nitroquinoline 1-oxide-damaged single-stranded shuttle vector DNA transfected into monkey cells

Abstract

4-Nitroquinoline 1-oxide (4NQO) is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. We recently determined the mutation spectrum induced by the ultimate metabolite of 4NQO, acetoxy-4-aminoquinoline 1-oxide in the M13 lacZ′/E. coli lacZΔM15 α-complementation assay. Our data suggested that dGuo-C8-AQO induces (per se or via AP sites) G to Pyr transversions. Here we report our study on 4NQO mutagenesis in monkey cells. 4NQO lesions were induced in vitro on a single-stranded (ss) DNA shuttle vector carrying the supF tRNA gene. This vector was able to replicate both in mammalian cells and in bacteria. The mutations induced in monkey cells were screened by the white/blue β-galactosidase activity assay in E. coli. We took advantage of the peculiar feature of ss supF DNA in which the extent of secondary structure may be a function of the temperature, with the dependence of the 4NQO-specific adduct spectrum on DNA secondary structure. We reasoned that mutational spectra derived from damage induced in the presence (20°C) or absence (70°C) of DNA secondary structure should be different. The result of sequencing a total of 89 induced and spontaneous mutants confirmed that the spectra are statistically different. These data suggest that the two 4NQO guanine adducts may induce different mutations.