Selective inhibition of glucuronidation by 2,2,2-triphenylethyl-UDP in isolated rat hepatocytes: conjugation of harmol, 3,3',5-triiodothyronine, and N-hydroxy-2-acetylaminofluorene.

2,2,2-Triphenylethyl-UDP (TPEU) was synthesized as an analogue of the transition state of the glucuronidation reaction catalyzed by UDP-glucuronosyltransferase; it contains both a uridine and an acceptor substrate moiety. It inhibits rat liver microsomal UDP-glucuronosyltransferase [Eur. J. Biochem. 188:309-312 (1990)]. In the present work, TPEU was tested as an inhibitor of glucuronidation in intact rat hepatocytes. Two phenols (harmol and 3,3',5-triiodothyronine) and a hydroxamic acid (N-hydroxy-2-acetylaminofluorene) were used as substrates for glucuronidation. The glucuronidation of these substrates was strongly decreased by TPEU at 0.3-5 mM. Up to 5 mM TPEU did not kill the cells, as shown by unimpaired trypan blue exclusion at the end of the incubation. When glucuronidation was inhibited, the sulfation of harmol increased, as did the production of reactive species generated from N-hydroxy-2-acetylaminofluorene that bind to cellular macromolecules. This indicates that a decreased substrate consumption by loss of glucuronidation leads to increased conversion by competing pathways. The results show, therefore, that TPEU is an effective inhibitor of glucuronidation in this cellular system in vitro.