Ability of adducts formed in a shuttle vector by reactive metabolites of 1-nitropyrene and benzo(a)pyrene to induce mutations when the plasmid replicates in human cells.
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 human cell line 293, allowed to replicate, and the progeny plasmids rescued and assayed for the frequency of supF mutants. The agents tested were the 7,8-diol-9,10-epoxide of benzo(a)pyrene (BPDE) and 1-nitrosopyrene (1-NOP). 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 15 to 25 times higher than the background frequency of 1.4 x 10(-4). When compared on the basis of adducts formed per plasmid BPDE, which forms its principal DNA adduct at the N2 position of guanine, was approximately four times more mutagenic than 1-NOP, which binds principally at 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, but 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 three times slower than 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 derived from carcinogen-treated plasmids were base substitutions. DNA sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmid and 60 from 1-NOP-treated plasmid 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 basepairs). The majority (83%) of the base substitutions in mutants from BPDE- or 1-NOP-treated plasmid were transversions, mainly G.C----T.A. Each carcinogen produced its own spectrum of mutations.
Maher
V M
VM
Department of Microbiology, Michigan State University, East Lansing 48824-1316.
Yang
J L
JL
McCormick
J J
JJ
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D002273:Carcinogens / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000235:genetics; D004252:DNA Mutational Analysis; D004261:DNA Replication; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011721:Pyrenes / Q000378:metabolism / Q000494:pharmacology*
D002273:Carcinogens; D011721:Pyrenes; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C044707:1-nitrosopyrene; D004247:DNA