MUTAGENIC SPECIFICITY OF REDUCTIVELY ACTIVATED 1-NITROPYRENE IN ESCHERICHIA-COLI

1-Nitropyrene (1-NP), the predominant nitropolycyclic hydrocarbon foun d in diesel exhaust, is a mutagen and tumorigen. Nitroreduction is a m ajor pathway by which 1-NP is metabolized. In order to study the distr ibution of DNA adducts and the mutational specificity of reductively a ctivated 1-NP, single stranded M13mp18 DNA was treated with N-hydroxy- 1-aminopyrene generated in situ to give >95% of one major adduct, N-(d eoxyguanosin-8-yl)-1-aminopyrene. A primer was annealed to DNA contain ing different levels of adducts, and polymerase extension on these tem plates was studied. Replication inhibition, primarily at or 3' to guan ine bases, was observed. Transfection of these M13 DNA in Escherichia coli indicated a dose-dependent reduction in viability with concomitan t enhancement in mutagenesis in the lacZ gene fragment. Approximately two adducts per genome constituted one lethal hit (similar to 37% viab ility). Both survival and mutagenesis were increased when SOS function s of the host cell were induced. N-(Deoxyguanosin-8-yl)-1-aminopyrene mutagenesis appeared to be SOS-dependent. With SOS induction, one-base deletions and insertions were the major event (45%), although base su bstitutions also occurred at high frequency (44%). A major proportion of the point mutations, and particularly one-base deletions and insert ions, were detected in 5'-CG, 5'-GC, or 5'-GC sequences. Analysis of t he mutation data suggested that N-(deoxyguanosin-8-yl)-1-aminopyrene i nduced mutations occurred predominantly at the adduct site, but mutati ons at the base located next to it have been detected at a significant frequency as well. A large fraction of point mutations occurred in a hairpin loop region. If C-->T transitions are excluded, most of the po int mutations were detected at sites where polymerase arrests occurred . However, neither were the major arrest sites mutational hotspots ndr would it be possible to predict mutational sites from the polymerase arrest assay.