Bioassay-directed fractionation of 1-nitropyrene metabolites: generation of mutagrams by coupling reverse-phase HPLC with microsuspension mutagenicity assays.

We have performed bioassay-directed fractionation of a model complex mixture (rabbit lung S9-generated metabolites of 14C-radiolabeled 1-nitropyrene) by assaying reverse-phase HPLC fractions using two microsuspension mutagenicity assays. A forward-mutation assay measuring mutation at the gpt locus (8-azaguanine resistance) in Salmonella typhimurium TM677 was performed in a total volume of 100 microliters, and a reverse-mutation assay measuring mutation at the hisD3052 allele in S. typhimurium TA98 was performed in a total volume of 200 microliters. HPLC fractions were collected every 30 s for 45 min, resulting in 90 fractions per run. The HPLC chromatogram (absorbance at 280 nm) and the 14C profile were compared to the mutagenicity profiles (mutagrams) and to the mutagenic potencies of pure metabolites studied separately. The results indicate that a fine dissection of the mutagenic fractions can be obtained by coupling HPLC to microsuspension mutagenicity assays. Differences observed between the mutagrams generated by the two bacterial strains were most likely due to metabolic (nitroreductase) differences between the two strains. This method should be generally applicable to the bioassay-directed chemical analysis of complex mixtures.