Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.

Chlorophyllin (CHL), a mixture of water soluble derivatives of chlorophyll, has been shown to be an effective inhibitor of aflatoxin B(1) (AFB(1)) carcinogenesis and AFB(1)-DNA adduct formation in rainbow trout and rats [Breinholt, V., Hendricks, J., Pereira, C., Arbogast, D., and Bailey, G. (1995) Cancer Res. 55, 57-62; Kensler, T. W., Groopman, J. D., and Roebuck, B. D. (1998) Mutat. Res. 402, 165-172]. The chemopreventive action of CHL has been previously attributed to molecular complexing. In 1997, a randomized, double-blind clinical trial of CHL was conducted in Qidong, Jiangsu Province, People's Republic of China. At the completion of the study, when serum samples were regrouped by subject identification number, it was noted that many of the participant samples were green in color. Using HPLC, ESI/MS, and MS/MS techniques, serum samples from individuals receiving CHL were found to contain previously unreported copper chlorin e(4) ethyl ester (CuCle(4) ethyl ester) as well as copper chlorin e(4) (CuCle(4)). Both chlorins originated in the study tablet, were absorbed into the bloodstream, and conferred a green color to the sera. This initial finding of in vivo absorption and bioavailability of two chlorin derivatives suggests that the mechanism of CHL chemoprevention may lie in the actions of these two components in vivo in addition to preventing carcinogen absorption from the gut.

Three different pathways have been proposed for the metabolic activation of the ubiquitous polycyclic aromatic hydrocarbon, benzo[a]pyrene (BP). The most widely accepted activation mechanism is based on ring oxidation to diol epoxides; the other two relatively less studied pathways involve radical cation formation and benzylic electrophilic ester formation arising from a chain of substitution reactions. The present study was undertaken to test for the existence of the latter mechanism in vivo. Female Sprague-Dawley weanling rats were injected subcutaneously with 320 mumol of BP/kg body weight, and the formation of DNA adducts was examined. 32P-Postlabeling analysis of the subcutaneous tissue DNA under newly developed chromatography conditions exhibited two sets of adduct profiles: one resulting from alkyl substitution and the other from ring oxidation. One major and two minor aralkyl-DNA adducts were detected. The relative adduct labeling (adducts/10(10) nucleotides) remained constant at around 2 during the first 5 days of treatment and then increased to 6.4 +/- 2.6 at day 7. The corresponding total values of the known ring oxidation (e.g., diol epoxide) adducts were 15-50 times higher. When animals were injected with 6-methyl-BP, 6-(hydroxymethyl)-BP, and 6-(acetoxymethyl)-BP, the known or proposed intermediates in the benzylic ester pathway, each of these and the parent compound showed chromatographically identical profiles of aralkyl-DNA adducts. Cochromatography in multiple solvents of these in vivo adducts with standards prepared by reaction of 6-(bromomethyl)-BP with individual nucleotides showed that the predominant in vivo aralkyl-DNA adduct was derived from guanine while the second major adduct was from adenine.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

The major product of the S-adenosyl-L-methionine methyltransferase dependent substitution reaction (bioalkylation) of benzo[a]pyrene in preparations of rat liver cytosol was isolated by solvent extraction and high performance liquid chromatography and its structure determined by GC/MS and NMR. The results indicate that the methyl group is introduced at the center of highest chemical reactivity (or carbon atom at which it is easiest to localize pi electrons) in the anthracene nucleus, giving rise to 6-methylbenzo[a]pyrene. These data, unequivocally demonstrate the site of substitution of the methyl group in the bioalkylation of benzo[a]pyrene.