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.