Formation of glucuronide, sulphate and glutathione conjugates of benzo[a]pyrene metabolites in hepatocytes isolated from inbred strains of mice

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Abstract
Metabolism of benzo[a]pyrene (BP) was studied in mouse hepatocytes isolated from uninduced animals of C57BL/6 Jacobs (B6) and C3Hf/HeHa (C3) inbred strains. Conjugates with sulphate, glucuronate and glutathione were the major products of BP biotransformation in the intact cells. Their formation was measured by determining the radioactivity incorporated from [3H]BP into the appropriate metabolite, after separation on silica gel t.l.c. plates. The conjugates were identified by their susceptibility to the action of specific degrading enzymes, arylsulphatase, β-glucuronidase and γ-glutamyltransferase. Effects of inhibitors of conjugation were also examined. D-Galactosamine and diethyl maleate caused ˜50% inhibition of the formation of glucuronide and glutathione derivatives of BP, respectively. The effect of salicylamide was less specific, besides an 88% decrease in sulphation of BP metabolites, a 40% decrease in the formation of glutathione conjugates was observed in the presence of this inhibitor. In hepatocytes of B6 mouse, all the above three types of BP conjugates were formed in almost equi-molar quantities. The total formation of BP conjugates was 42% higher in B6 hepatocytes than in those of C3 strain. The most significant difference (1.7-fold) was in the production of BP glucuronides, despite an absence of observable differences between these mouse strains in the activity of microsomal UDP-glucuronosyltransferase and in the rate of 1-naphthol conjugation in isolated hepatocytes. Simultaneously, 2.5-fold higher accumulation of unconjugated BP metabolites was observed in the hepatocyte suspension of B6 than C3 strain and a 1.4-fold higher activity of aryl hydrocarbon hydroxyl-ase in hepatic microsomes of this strain. The unconjugated metabolites of BP were separated into four major fractions by h.p.l.c. The retention times of the metabolites corresponded to trans 9,10-diol; trans 7,8-diol; 9-hydroxy- and 3-hydroxy-BP. Despite quantitative differences between B6 and C3 strains of mice in BP metabolism, the same degree of covalent binding of BP metabolites to cellular DNA, was observed. The results indicate a relatively high capacity of hepatocytes from uninduced mice for conjugation of BP metabolites. Hepatocytes isolated from various strains of mice, should be useful in elucidating the role of numerous factors in metabolism and biologic activity of BP and related carcinogens.