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Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites.
Drug Metab Dispos. 1999 Mar; 27(3):365-72.DM

Abstract

The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.

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Authors+Show Affiliations

Tang W
Department of Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey 07065, USA. wei_tang@merck.com
Stearns RA
No affiliation info available
Bandiera SM
No affiliation info available
Zhang Y
No affiliation info available
Raab C
No affiliation info available
Braun MP
No affiliation info available
Dean DC
No affiliation info available
Pang J
No affiliation info available
Leung KH
No affiliation info available
Doss GA
No affiliation info available
Strauss JR
No affiliation info available
Kwei GY
No affiliation info available
Rushmore TH
No affiliation info available
Chiu SH
No affiliation info available
Baillie TA
No affiliation info available

MeSH

AnimalsAnti-Inflammatory Agents, Non-SteroidalBileBiotransformationCells, CulturedChromatography, LiquidCytochrome P-450 Enzyme SystemDiclofenacFemaleGlutathioneHumansMaleMass SpectrometryMicrosomes, LiverRatsRats, Sprague-Dawley

Pub Type(s)

Journal Article

Language

eng

PubMed ID

10064567

Citation

Tang, W, et al. "Studies On Cytochrome P-450-mediated Bioactivation of Diclofenac in Rats and in Human Hepatocytes: Identification of Glutathione Conjugated Metabolites." Drug Metabolism and Disposition: the Biological Fate of Chemicals, vol. 27, no. 3, 1999, pp. 365-72.
Tang W, Stearns RA, Bandiera SM, et al. Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites. Drug Metab Dispos. 1999;27(3):365-72.
Tang, W., Stearns, R. A., Bandiera, S. M., Zhang, Y., Raab, C., Braun, M. P., Dean, D. C., Pang, J., Leung, K. H., Doss, G. A., Strauss, J. R., Kwei, G. Y., Rushmore, T. H., Chiu, S. H., & Baillie, T. A. (1999). Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 27(3), 365-72.
Tang W, et al. Studies On Cytochrome P-450-mediated Bioactivation of Diclofenac in Rats and in Human Hepatocytes: Identification of Glutathione Conjugated Metabolites. Drug Metab Dispos. 1999;27(3):365-72. PubMed PMID: 10064567.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites. AU - Tang,W, AU - Stearns,R A, AU - Bandiera,S M, AU - Zhang,Y, AU - Raab,C, AU - Braun,M P, AU - Dean,D C, AU - Pang,J, AU - Leung,K H, AU - Doss,G A, AU - Strauss,J R, AU - Kwei,G Y, AU - Rushmore,T H, AU - Chiu,S H, AU - Baillie,T A, PY - 1999/3/4/pubmed PY - 1999/3/4/medline PY - 1999/3/4/entrez SP - 365 EP - 72 JF - Drug metabolism and disposition: the biological fate of chemicals JO - Drug Metab Dispos VL - 27 IS - 3 N2 - The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury. SN - 0090-9556 UR - https://www.unboundmedicine.com/medline/citation/10064567/Studies_on_cytochrome_P_450_mediated_bioactivation_of_diclofenac_in_rats_and_in_human_hepatocytes:_identification_of_glutathione_conjugated_metabolites_ L2 - http://dmd.aspetjournals.org/cgi/pmidlookup?view=long&pmid=10064567 DB - PRIME DP - Unbound Medicine ER -