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.