Indirect cytotoxicity of flucloxacillin toward human biliary epithelium via metabolite formation in hepatocytes.
Flucloxacillin, an isoxazolyl-penicillin, causes cholestasis and biliary epithelium injury. The aim of the study was to determine whether flucloxacillin, either directly or through metabolite formation, may induce cytotoxicity in hepatic or biliary cells. Cytotoxicity was assessed by lactate dehydrogenase 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 resonance spectroscopy, and mass spectrometry. While flucloxacillin induced no direct cytotoxicity in any of the hepatocyte (n = 12) and BEC (n = 19) preparations, the conditioned media from cultured hepatocytes preincubated with flucloxacillin (50-500 mg/L) triggered a significant increase in lactate dehydrogenase release over controls in approximately 50% of BEC preparations (7/12), and this effect depended upon flucloxacillin concentration. Remaining BEC preparations exhibited no toxic response. Cytotoxicity in BEC preparations (9/13) was also induced by the supernatants of human liver microsomes and of recombinant human cytochrome P450 (CYP)3A4 preincubated with flucloxacillin (500 mg/L). Supernatants from both liver microsome and CYP3A4 preparations contained one major metabolite which was identified as 5'-hydroxymethylflucloxacillin. The production of this metabolite was inhibited following CYP3A4 inhibition by troleandomycin in human liver microsomes, and markedly enhanced following CYP3A induction by dexamethasone in rat liver microsomes. As opposed to BEC, cultured hepatocytes displayed significant CYP3A activity 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 conclusion, hepatocytes mainly via CYP3A4 activity, generate flucloxacillin metabolite(s) including 5'-hydroxymethylflucloxacillin that may induce cytotoxicity in susceptible BEC. These metabolic events may contribute to the pathogenesis of drug-induced cholangiopathies.
Lakehal
F
F
Unité INSERM U402, Faculté de Médecine Saint-Antoine, Hôpital Saint-Antoine, Paris, France.
Dansette
P M
PM
Becquemont
L
L
Lasnier
E
E
Delelo
R
R
Balladur
P
P
Poupon
R
R
Beaune
P H
PH
Housset
C
C
D001659:Biliary Tract / Q000166:cytology / Q000201:enzymology / Q000473:pathology*; D018929:Cell Culture Techniques; D002779:Cholestasis / Q000139:chemically induced; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004848:Epithelium / Q000187:drug effects / Q000473:pathology; D005436:Floxacillin / Q000009:adverse effects* / Q000378:metabolism; D005704:Gallbladder / Q000166:cytology / Q000473:pathology; D022781:Hepatocytes / Q000201:enzymology; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000473:pathology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010406:Penicillins / Q000009:adverse effects* / Q000378:metabolism
D010406:Penicillins; D005436:Floxacillin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human