Kinds of mutations found when a shuttle vector containing adducts of 1,6-dinitropyrene replicates in human cells

Abstract

To gain insight into the mechanisms by which carcinogens induce mutations in human cells, we have been comparing the frequency and spectrum of mutations induced when a shuttle vector, pS189, carrying covalently bound residues of structurally related carcinogens, replicates in human 293 cells. In the present study, we investigated the mutagenic effects of N-hydroxy-1-amino-6-nitropyrene, a partially reduced derivative of 1,6-dinitropyrene (1,6-DNP). The results were compared with what was found previously in the same assay with N-hydroxy-1-aminopyrene, the partially reduced derivative of 1-nitropyrene. The shuttle vector plasmids were exposed to tritiated 1-nitro-6-nitrosopyrene for 1 h in the presence of ascorbic acid, which served as a reducing agent to generate N-hydroxy-1-amino-6-nitropyrene. 32P-Postlabeling showed that only a single adduct was formed, i.e. N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene. There was a linear increase in the number of adducts per plasmid as a function of applied concentration and also in the frequency of supF mutants as a function of adducts per plasmid, reaching 58.8 x 10(-4) above a background of 0.8 x 10(-4). When the frequency of mutants induced when plasmids carrying residues of 1,6-DNP replicated in the human cells was compared with that induced by 1-NP residues, the former was 1.8 times more mutagenic than the latter. Both carcinogens induced mainly base substitutions, primarily G.C----T.A transversions; but 1,6-DNP adducts produced a significant fraction of -1 frameshifts, with most of these located in a unique run of five Gs in the gene. Polymerase termination reactions indicated that 1,6-DNP adducts were formed at every guanine, but not elsewhere in the gene. The 'hot spots' for adduct formation were not perfectly correlated with 'hot spots' for mutation induction. This indicates that the ultimate biological effect of the chemical depends not only on the number of adducts originally formed, but also on such processes as cellular DNA repair, which may remove such adducts from the plasmids before DNA replication occurs, as well as on the structure of the neighboring bases at the site of the adduct.