Glucuronidation of 3-hydroxybenzo(a)pyrene in liver microsomes.
A simple and sensitive fluorimetric method is described for evaluation of 3-hydroxybenzo(a)pyrene conjugation with UDPglucuronic acid. It is less expensive than a radiochemical method and suitable for routine use. 3-Hydroxybenzo(a)pyrene is readily conjugated in isolated liver microsomes in the presence of UDPglucuronic acid. Activity in rat liver microsomes was 0.90--1.20 nmol.min-1.mg-1 microsomal protein. The activity in homozygous and heteroxygous Gunn rats was considerably lower than in Wistar rats. Activity in guinea pigs was 2.5--3 nmol.min-1.mg-1 protein. 3-Methylcholanthrene pretreatment (20 mg/kg of body weight for 4 consecutive days) of rats enhanced the hepatic UDPglucuronosyltransferase activity 2--5 fold. In untreated microsomal membranes of rat liver the apparent Km for 3-hydroxybenzo(a)pyrene was 0.09 mM and for Udpglucuronic acid 4.6 mM. Conjugation with UDPglucose did not occur. 4-Nitrophenol and 4-nitrophenyl-beta-D-glucuronide behaved like non competitive inhibitors. In contrast to 4-nitrophenol conjugation, both ionic (cholic acid) and non-ionic (Triton X-100, digitonin) surfactants had no effect or inhibited the glucuronic acid conjugation of 3-hydroxybenzo(a)pyrene in rat liver microsomes as also did the treatment of microsomal membranes with phospholipases A and C. Trypsin was almost without an effect on UDPglucuronosyltransferase activity when 3-hydroxybenzo(a)pyrene was used as substrate.
D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D009596:Nitrophenols / Q000494:pharmacology; D010741:Phospholipases A / Q000494:pharmacology; D051381:Rats; D014357:Trypsin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D001580:Benzopyrenes; D009596:Nitrophenols; D014539:Uridine Diphosphate Sugars; D014535:Uridine Diphosphate Glucuronic Acid; D008748:Methylcholanthrene; D014453:Glucuronosyltransferase; D010741:Phospholipases A; D010738:Type C Phospholipases; D014357:Trypsin; D014532:Uridine Diphosphate Glucose