Effects of organic solvent vehicles on benzo[a]pyrene metabolism in rabbit lung microsomes

In order to study the metabolism of benzo[a]pyrene (BP), it must be dissolved in an organic solvent vehicle for delivery to the tissue. We studied the effects of five organic solvent vehicles, i.e. dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, and ethyl acetate, on benzo[a]pyrene hydroxylase activity and the BP metabolite profile in rabbit lung microsomes. Fluorescence detection of 3- and 9-OH-BP was used to evaluate benzo[a]pyrene hydroxylase activity, and the BP metabolite profile was obtained by HPLC analysis. All solvent vehicles inhibited benzo[a]pyrene hydroxylase in a dose-dependent manner. When the smallest volume of each solvent (10 microliter/ml reaction mixture) was employed, the resulting enzyme activities as related to solvent type, from highest to lowest, were DMSO greater than or equal to methanol greater than ethanol greater than or equal to acetone greater than ethyl acetate. HPLC analysis of BP metabolites formed in the presence of the five solvent vehicles showed that production of all metabolites was greatest when DMSO was used and that linearity of product formation was retained longer with DMSO. The metabolites produced when DMSO was used as the solvent were BP-9,10-diol, BP-4,5-diol, BP-7,8-diol, BP-1,6-quinone, BP-3,6-quinone and 3-OH-BP. A similar metabolite profile was obtained when reactions were carried out with methanol as the solvent vehicle, although the magnitude of production was less than with DMSO. When acetone was used, there were greater amounts of BP-4,5-diol and BP quinone formation and lesser amounts of 3-OH-BP formed than with DMSO or methanol. When ethanol or ethyl acetate was used as a solvent, BP-9,10-diol and 3-OH-BP were the only metabolites produced. These results indicate that all solvent vehicles studied inhibit benzo[a]pyrene hydroxylase from rabbit lung microsomes in a dose-dependent manner and that the magnitudes and types of metabolites formed are highly dependent upon the specific solvent used as the vehicle. The study also indicates that DMSO is probably the solvent vehicle of choice for study of BP metabolism in rabbit lung microsomes.