Relationships among benzo(a)pyrene metabolism, benzo(a)pyrene-diol-epoxide:DNA adduct formation, and sister chromatid exchanges in human lymphocytes from smokers and nonsmokers.

In the present study, benzo(a)pyrene (BP) metabolism, DNA adduct formation, ethoxyresorufin-O-deethylase activity, and sister chromatid exchange induction by BP were compared in human lymphocytes prepared from whole blood of smokers and nonsmokers following an in vitro incubation with BP. There was an approximate 7- to 10-fold variation in all parameters measured. To determine the source of this variation, participants were resampled, the assays were repeated, and all the data were analyzed to assess (a) smoking-related effects, (b) differences in multiple samples from the same individual, and (c) intraindividual, experimental, and interindividual variation. No smoking-related effects were observed except for baseline sister chromatid exchange frequency. The variation observed for BP-related DNA adducts and ethoxyresorufin-O-deethylase activity was primarily due to interindividual variation. For example, in vitro formation of DNA adducts did not change when samples were obtained at different times from the same individual and were not influenced significantly by culture conditions. No significant correlation existed between DNA adduct formation and BP metabolism [correlation coefficient (r) = 0.27] for either the total population or when segregated based on smoking status. Furthermore, no correlation was seen between DNA adducts and sister chromatid exchange induction by BP. Our studies have compared a number of commonly used lymphocyte markers and conclude that it is difficult to predict changes in one marker based on changes in another. However, in vitro formation, of PB-derived DNA adducts is consistent over time for individuals.

Involvement of cytochrome P-450c in alpha-naphthoflavone metabolism by rat liver microsomes.

Metabolism of alpha-naphthoflavone (ANF) is increased markedly in rat liver microsomes by 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two inducers of cytochromes P-450c and P-450d (P-450c and P-450d). Although several indirect lines of evidence in the literature suggest that ANF is metabolized by P-450c, Vyas et al. [J. Biol. Chem. 258:5649-5659 (1983)] reported that ANF metabolism by 3-MC-induced rat liver microsomes was only partially inhibited by antibodies against P-450c. Our laboratory has previously reported clastogenic effects of metabolites of ANF, and in the present study we reexamined the role of P-450c in ANF metabolism by both uninduced and TCDD-induced rat liver microsomes, using monospecific polyclonal antibodies to P-450c and P-450d. ANF metabolism was inhibited to different extents in TCDD-induced microsomes by different preparations of anti-P-450c. One lot of anti-P-450c produced only 50% inhibition of ANF metabolism in TCDD-induced microsomes, whereas another lot of anti-P-450c inhibited ANF metabolism by 80%. Anti-P-450d had no effect on ANF metabolism. Neither anti-P-450c nor anti-P-450d inhibited ANF metabolism in uninduced rat liver microsomes. In a reconstituted enzyme system, purified P-450c metabolized ANF 47 and 510 times more rapidly than P-450d and P-450b, respectively. Metabolites resulting from oxidation at 7,8- or 5,6-positions (7,8-dihydro-7,8-dihydroxy-ANF, 5,6-dihydro-5,6-dihydroxy-ANF, 5,6-oxide-ANF, and 6-hydroxy-ANF) were formed by all preparations of microsomes. An unknown toxic ANF metabolite was formed only with a reconstituted P-450c system and with 3-MC- or TCDD-induced microsomes. Our results indicate that P-450c is responsible for the majority of the metabolism of ANF in TCDD-induced microsomes, whereas other constitutive isozymes are responsible for the metabolism seen in uninduced liver microsomes. The variable inhibition of ANF metabolism with different lots of anti-P-450c probably reflects the differences in the proportion of antibodies to different epitopes important in the binding or metabolism of this substrate.