Comparing the frequency and spectra of mutations induced when an SV-40 based shuttle vector containing covalently bound residues of structurally-related carcinogens replicates in human cells

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An SV40-based shuttle vector, pZ189, carrying a bacterial suppressor tRNA target gene ( supF) was treated with radiolabeled polycyclic aromatic carcinogens and the number of covalently bound residues (adducts) per plasmid was determined. The plasmids were transfected into the human embryonic kidney cell lien 293 and allowed to replicate. The progeny plasmids were rescued and assayed for the frequency of supF mutants by being used to transform indicator bacteria carrying an amber mutation in the β-galactosidase gene. The agents tested were the 7,8-diol-9,10-epoxide of benzo[ a]pyrene (BPDE); 1-nitrosopyrene (1-NOP); N-acetoxy-2-acetylaminofluorene (N-AcO-AAF); and its trifluoro-derivative (N-AcO-F 3-AAF) which yields deacetylated adducts. With each agent there was a linear increase in the frequency of supF mutants as a function of the number of DNA adducts formed, reaching frequencies as high as 20 × 10 −4 to 40 × 10 −4, with a background frequency of 1.4 × 10 −4. When compared on the basis of adducts formed per plasmid, BPDE, which forms its principal DNA adduct at the N 2 position of guanine, was approximately 4 times more mutagenic than 1-NOP, N-AcO-AAF and N-AcO-F 3-AAF, which bind principally or exclusively to the C8 position of guanine. This difference in mutagenic effectiveness may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule. It could also reflect a difference in the rate of removal of particular adducts by nucleotide excision repair since the 293 host cell line excised BPDE-induced adducts from genomic DNA at least 3 times slower than 1-NOP-induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of 35 mutants derived from untreated plasmids showed that the majority (70%) involved deletions, insertions, or altered gel mobility (gross rearrangements). In contrast, the majority of those derived from carcinogen-treated plasmids were base-substitutions. DNA-sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmids and 60 from 1-NOP-treated plasmids indicated that 60% and 80%, respectively, contained a single base-substitution, 5–10% had two base-substitutions, and 4–10% had small insertions or deletions (one or two base pairs). The majority (84–86%) of the base-substitutions in mutants from BPDE- or 1-NOP-treated plasmids were transversions, with 73% of these being G· C → T · A. These two carcinogens produced their own spectrum of mutations, i.e., of the 7 hot spots for base-substitution methods produced with BPDE, two were the same as seen with 1-NOP-treated plasmid. However, 4 of the other hot spots were cold spots for 1-NOP-treated plasmids. Conversely, the 3 other hot spots seen with 1-NOP-treated plasmids were cold spots for BPDE-treated plasmids. The spectra produced by AF and AAF adducts are still being determined.