title: Characterization of cytochrome P450 isoforms involved in sequential two-step bioactivation of diclofenac to reactive p-benzoquinone imines.
abstract: Idiosyncratic drug-induced lever injury (IDILI) is a rare but severe side effect of diclofenac (DF). Several mechanisms have been proposed as cause of DF-induced toxicity including the formation of protein-reactive diclofenac-1',4'-quinone imine (DF-1',4'-QI) and diclofenac-2,5-quinone imine (DF-2,5-QI). Formation of these p-benzoquinone imines result from two-step oxidative metabolism involving aromatic hydroxylation to 4'-hydroxydiclofenac and 5-hydroxydiclofenac followed by dehydrogenation to DF-1',4'-QI and DF-2,5-QI, respectively. Although the contribution of individual cytochrome P450s (CYPs) in aromatic hydroxylation of DF is well studied, the enzymes involved in the dehydrogenation reactions have been poorly characterized. The results of the present study show that both formation of 4'-hydroxydiclofenac and it subsequent bioactivation to DF-1',4'-QI is selectively catalyzed by CYP2C9. However, the two-step bioactivation to DF-2,5-QI appears to be catalyzed with highest activity by two different CYPs: 5-hydroxylation of DF is predominantly catalyzed by CYP3A4, whereas its subsequent bioactivation to DF-2,5-QI is catalyzed with 14-fold higher intrinsic clearance by CYP2C9. The fact that both CYPs involved in two-step bioactivation of DF show large interindividual variability may play a role in different susceptibility of patients to DF-induced IDILI. Furthermore, expression levels of these enzymes and protective enzymes might be important factors determining sensitivity of in vitro models for hepatotoxicity.
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, De Boelelaan 1108, 1081HZ Amsterdam, The Netherlands.
authors.forename: Michiel W
authors.initials: MW
authors.lastname: den Braver
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, De Boelelaan 1108, 1081HZ Amsterdam, The Netherlands.
authors.forename: Shalenie P
authors.initials: SP
authors.lastname: den Braver-Sewradj
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, De Boelelaan 1108, 1081HZ Amsterdam, The Netherlands.
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, De Boelelaan 1108, 1081HZ Amsterdam, The Netherlands. Electronic address: j.n.m.commandeur@vu.nl.
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
mesh_terms: D065767:Activation, Metabolic; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism* / Q000633:toxicity; D016227:Benzoquinones / Q000378:metabolism* / Q000633:toxicity; D065729:Cytochrome P-450 CYP2C9 / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D004008:Diclofenac / Q000378:metabolism* / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D007097:Imines / Q000378:metabolism* / Q000633:toxicity; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D018570:Risk Assessment
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D016227:Benzoquinones; D007097:Imines; D011994:Recombinant Proteins; D004008:Diclofenac; C004532:quinone; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D005978:Glutathione
keywords: Bioactivation; Cytochrome P450; Diclofenac; Glutathione conjugates; Idiosyncratic liver injury; Reactive intermediates