Indirect cytotoxicity of flucloxacillin toward human biliary epithelium via metabolite formation in hepatocytes

Flucloxacillin, an isoxazolyl-penicillin, causes cholestasis and biliary ep ithelium injury. The aim of the study was to determine whether flucloxacill in, either directly or through metabolite formation, may induce cytotoxicit y in hepatic or biliary cells. Cytotoxicity was assessed by lactate dehydro genase release in primary cultures of human hepatocytes and of gallbladder- derived biliary epithelial cells (BEC). Metabolite production in microsome and cell preparations was analyzed by chromatography, nuclear magnetic reso nance spectroscopy, and mass spectrometry. While flucloxacillin induced no direct cytotoxicity in any of the hepatocyte (n 12) and BEC (n = 19) prepar ations, the conditioned media from cultured hepatocytes preincubated with f lucloxacillin (50-500 mg/L) triggered a significant increase in lactate deh ydrogenase release over controls in similar to 50% of BEC preparations (7/1 2), and this effect depended upon flucloxacillin concentration. Remaining B EC preparations exhibited no toxic response. Cytotoxicity in BEC preparatio ns (9/13) was also induced by the supernatants of human liver microsomes an d of recombinant human cytochrome P450 (CYP)3A4 preincubated with flucloxac illin (500 mg/L). Supernatants from both liver microsome and CYP3A4 prepara tions contained one major metabolite which was identified as 5'-hydroxymeth ylflucloxacillin. The production of this metabolite was inhibited following CYP3A4 inhibition by troleandomycin in human liver microsomes, and markedl y enhanced following CYP3A induction by dexamethasone in rat liver microsom es. As opposed to BEG, cultured hepatocytes displayed significant CYP3A act ivity and produced low amounts of this metabolite. The purified metabolite (0.01-5 mg/L) exerted toxic effects in BEC but not in hepatocytes. In concl usion, hepatocytes mainly via CYP3A4 activity, generate flucloxacillin meta bolite(s) including 5'-hydroxymethylflucloxacillin that may induce cytotoxi city in susceptible BEG. These metabolic events may contribute to the patho genesis of drug-induced cholangiopathies.