One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac.
Reactive metabolites have been suggested to play a role in the idiosyncratic hepatotoxicity observed with diclofenac (DF). By structural identification of the GSH conjugates formed after P450-catalyzed bioactivation of DF, it was shown that three types of reactive intermediates were formed: p-benzoquinone imines, o-imine methide and arene-oxide. Recently, detection of 2'-(glutathion-S-yl)-deschloro-diclofenac (DDF-SG), resulting from chlorine substitution, suggested the existence of a fourth type of P450-dependent reactive intermediate whose inactivation by GSH is completely dependent on presence of glutathione S-transferase. In this study, fourteen recombinant cytochrome P450s and three flavin-containing monooxygenases were tested for their ability to produce oxidative DF metabolites and their corresponding GSH conjugates. Concerning the hydroxymetabolites and their GSH conjugates, results were consistent with previous studies. Unexpectedly, all tested recombinant P450s were able to form DDF-SG to almost similar extent. DDF-SG formation was found to be partially independent of NADPH and even occurred by heat-inactivated P450. However, product formation was fully dependent on both GSH and glutathione-S-transferase P1-1. DDF-SG formation was also observed in reactions with horseradish peroxidase in absence of hydrogen peroxide. Because DDF-SG was not formed by free iron, it appears that DF can be bioactivated by iron in hemeproteins. This was confirmed by DDF-SG formation by other hemeproteins such as hemoglobin. As a mechanism, we propose that DF is subject to heme-dependent one-electron oxidation. The resulting nitrogen radical cation, which might activate the chlorines of DF, then undergoes a GST-catalyzed nucleophilic aromatic substitution reaction in which the chlorine atom of the DF moiety is replaced by GSH.
Division of Molecular Toxicology, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Jan Simon
JS
Boerma
Division of Molecular Toxicology, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Nico P E
NP
Vermeulen
Division of Molecular Toxicology, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. Electronic address: j.n.m.commandeur@vu.nl.
Jan N M
JN
Commandeur
D001205:Ascorbic Acid / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004008:Diclofenac / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D004583:Electrons; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006420:Hemeproteins / Q000494:pharmacology; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000494:pharmacology; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D010105:Oxygenases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
C586542:2'-(glutathion-S-yl)-deschlorodiclofenac; D006420:Hemeproteins; D011994:Recombinant Proteins; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D006735:Horseradish Peroxidase; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D005978:Glutathione; D001205:Ascorbic Acid
2′-(glutathion-S-yl)-deschloro-diclofenac; 4′-OH-DF; 4′-hydroxydiclofenac; 5-OH-DF; 5-hydroxydiclofenac; Bioactivation; CYP; Cytochrome P450; DDF-SG; DF; Diclofenac; FMO; GSH conjugate; Glutathione S-transferase; HRP; RM; Reactive metabolite; cytochrome P450; diclofenac; flavin-containing monooxygenase; hGST P1-1; horseradish peroxidase; human glutathione-S-transferase P1-1; reactive metabolite