Mutation spectra in salmonella of complex mixtures: Comparison of urban air to benzo[a]pyrene

Abstract

We used an ion-exchange procedure coupled to the Salmonella assay to fractionate the dichloromethane-extractable particulate organics from an urban air sample collected in Boise, Idaho. A resulting base/neutral fraction contained 81% of the mutagenic activity but only 36% of the mass of the unfractionated sample. Chemical analysis showed that polycyclic aromatic hydrocarbons (PAHs) accounted for much of the mutagenic activity of the air sample. Colony probe hybridization, PCR, and DNA sequence analysis were then used to determine the mutations induced by the complex mixtures and a model PAH, benzo[a]pyrene (BAP) in approximately 900 revertants of the frameshift hisD3052 allele and approximately 400 revertants of the base-substitution hisG46 allele. The majority (93-94%) of the mutations induced at the frameshift allele in strain TA98 by the whole or base/neutral fraction of the urban air sample was a hotspot 2-base deletion of a CG or GC within the sequence CGCGCGCG. The remaining mutations were complex frameshifts that consisted of -2 or +1 frameshifts associated with a flanking base substitution. BAP induced a somewhat similar pattern of mutations, with 70% being the hotspot mutation, 23% being complex frameshifts, and the remaining being deletions. The inferred base-substitution specificity associated with the complex frameshifts at the hisD3052 allele (primarily G.C-->T.A transversions) was consistent with the observation that this same transversion was the primary mutation induced by the whole urban air sample and BAP at the base-substitution allele in strain TA100. At the frameshift allele, adducts that promote correct incorporation/slippage could account for hotspot mutations, whereas those that promote misincorporation/slippage could account for complex frameshifts. At the base-substitution allele, a mixture of adducts or of adducts with multiple conformations could account for the observed proportion of transitions and transversions. Combined with the bioassay-directed chemical analysis, these results from the first mutation spectra of a complex mixture suggest that such spectra reflect the dominance of particular classes of chemical mutagens within the mixture.