Spectra of Mutations Induced by Structurally-Related Aromatic Polycyclic Carcinogens during Replication of a Shuttle Vector in Human Cells

Abstract

We are comparing the kinds and spectra of mutations induced when DNA containing covalently bound carcinogen residues (adducts) replicates in human cells. A shuttle vector, pZ189, carrying the supF gene coding for a bacterial tyrosine suppressor tRNA as the target was treated with tritiated polycyclic aromatic carcinogens and the number of adducts per plasmid was determined. The plasmids were trarsfected into human cells, and after replication had occurred, the progeny plasmids were rescued and assayed for the frequency of supF mutants. The carcinogens studied included the 7,8-diol-9,10-epoxide of benzo[a]pyrene (BPDE), 1nitrosopyrene (1-NOP), N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), and its trifluoroacetyl derivative (N-AcO-TFA-AF). BPDE binds principally to the N2 position of guanine. The other three carcinogens bind to the C8 position of guanine. Each agent caused 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 x 10^-4 to 40 x 10^-4, above a background frequency of 1.4 x 10^-4. When compared on the basis of adducts formed, BPDE was approximately four times more mutagenic than the other three carcinogens. This difference may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule, but it may also reflect differences in the rate of removal of particular adducts by nucleotide excision repair since we showed that the host cells excise BPDE induced adducts from genomic DNA at least three times slower than they excise 1-NOP induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of 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 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 70% to 80% 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 (83%) of the base substitutions were transversions, predominantly G•C→T•A.These two carcinogens produced their own spectrum of mutations. Studies to date with the N-AcO-TFA-AF have shown that the AF adduct induces predominantly base subsitutions and all of these involve guanine.