title: Modulation of benzo[a]pyrene bioactivation by glucuronidation in lymphocytes and hepatic microsomes from rats with a hereditary deficiency in bilirubin UDP-glucuronosyltransferase.
abstract: UDP-glucuronosyltransferases (UGTs) play a major role in the elimination of nucleophilic metabolites of xenobiotics, such the phenols and quinols of polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BP). In this way, UGTs may prevent the further oxidation of such metabolites to toxic reactive intermediates, which may react with lipids, DNA, RNA, and protein, thereby initiating such toxicities as cellular necrosis, birth defects, and cancer. We have shown previously in vivo and in hepatic microsomes that rats with a hereditary deficiency in bilirubin UGT (Gunn and RHA strains) have decreased glucuronidation of BP metabolites and enhanced BP covalent binding to hepatic DNA and microsomal protein, and enhanced BP embryotoxicity. We further hypothesized that a similar deficiency in BP glucuronidation, with increased BP bioactivation and covalent binding, might be observed in peripheral blood lymphocytes from UGT-deficient rats, which, if true, would suggest the utility of an analogous human lymphocyte model for evaluating the toxicological relevance of human UGT deficiencies. Such an in vitro approach is essential for human studies of environmental chemicals and drugs with a high toxicologic potential. In the current study, [7,10(-14)C]BP was preincubated with NADPH and hepatic microsomes from Wistar rats induced with beta-naphthoflavone. The supernatant from this preincubation, which contained BP reactive intermediates and hydroxylated BP metabolites, was further incubated with uridine diphosphate glucuronic acid and peripheral lymphocytes from heterozygous (j/+) and homozygous (j/j) RHA rats, which are genetically deficient in bilirubin UGT, and from congenic, homozygous (+/+) UGT-normal controls. In vivo, bilirubin glucuronidation was reduced in UGT deficiency, with progressively higher plasma concentrations of unconjugated bilirubin in j/+ and j/j UGT-deficient rats compared to +/+ UGT-normal controls (p < 0.05). In in vitro studies, glucuronide conjugates of BP were measured by high-performance liquid chromatography with a radioisotope detector, and the covalent binding of BP to microsomal protein was measured by liquid scintillation spectrometry. BP glucuronidation and covalent binding, respectively, were decreased and increased to a progressively greater degree in both hepatic microsomes and lymphocytes from j/+ and j/j UGT-deficient rats compared to +/+ UGT-normal controls (p < 0.05). Reduced BP glucuronidation in lymphocytes from UGT-deficient RHA rats correlated with elevated BP covalent binding (R2 = 0.85, p < 0.005). Both BP glucuronidation and BP covalent binding in lymphocytes significantly correlated with those reactions using hepatic microsomes from the same animals. These results in UGT-deficient lymphocytes also accurately reflected the in vivo biotransformation and covalent binding of BP in UGT-deficient rats.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, University of Toronto, Canada.
authors.forename: Z
authors.initials: Z
authors.lastname: Hu
authors.forename: P G
authors.initials: PG
authors.lastname: Wells
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism / Q000493:pharmacokinetics* / Q000633:toxicity; D001711:Biotransformation; D001798:Blood Proteins / Q000378:metabolism; D003414:Crigler-Najjar Syndrome / Q000201:enzymology; D005260:Female; D005964:Glucosyltransferases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D005965:Glucuronates / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D001798:Blood Proteins; D005965:Glucuronates; D011506:Proteins; D001564:Benzo(a)pyrene; D005964:Glucosyltransferases; C035814:bilirubin UDP-glucosyltransferase