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One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac.
Chem Biol Interact. 2014 Jan 25; 207:32-40.CB

Abstract

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.

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Publisher Full Text (DOI)

Authors+Show Affiliations

Boerma JS
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.
Vermeulen NP
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.
Commandeur JN
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.

MeSH

Ascorbic AcidChromatography, High Pressure LiquidCytochrome P-450 Enzyme SystemDiclofenacElectronsGlutathioneHemeproteinsHorseradish PeroxidaseHumansIronMetabolic Networks and PathwaysMicrosomes, LiverOxidation-ReductionOxygenasesRecombinant ProteinsTime Factors

Pub Type(s)

Journal Article

Language

eng

PubMed ID

24246759

Citation

Boerma, Jan Simon, et al. "One-electron Oxidation of Diclofenac By Human Cytochrome P450s as a Potential Bioactivation Mechanism for Formation of 2'-(glutathion-S-yl)-deschloro-diclofenac." Chemico-biological Interactions, vol. 207, 2014, pp. 32-40.
Boerma JS, Vermeulen NP, Commandeur JN. One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac. Chem Biol Interact. 2014;207:32-40.
Boerma, J. S., Vermeulen, N. P., & Commandeur, J. N. (2014). One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac. Chemico-biological Interactions, 207, 32-40. https://doi.org/10.1016/j.cbi.2013.11.001
Boerma JS, Vermeulen NP, Commandeur JN. One-electron Oxidation of Diclofenac By Human Cytochrome P450s as a Potential Bioactivation Mechanism for Formation of 2'-(glutathion-S-yl)-deschloro-diclofenac. Chem Biol Interact. 2014 Jan 25;207:32-40. PubMed PMID: 24246759.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac. AU - Boerma,Jan Simon, AU - Vermeulen,Nico P E, AU - Commandeur,Jan N M, Y1 - 2013/11/15/ PY - 2013/06/05/received PY - 2013/10/09/revised PY - 2013/11/05/accepted PY - 2013/11/20/entrez PY - 2013/11/20/pubmed PY - 2014/2/26/medline KW - 2′-(glutathion-S-yl)-deschloro-diclofenac KW - 4′-OH-DF KW - 4′-hydroxydiclofenac KW - 5-OH-DF KW - 5-hydroxydiclofenac KW - Bioactivation KW - CYP KW - Cytochrome P450 KW - DDF-SG KW - DF KW - Diclofenac KW - FMO KW - GSH conjugate KW - Glutathione S-transferase KW - HRP KW - RM KW - Reactive metabolite KW - cytochrome P450 KW - diclofenac KW - flavin-containing monooxygenase KW - hGST P1-1 KW - horseradish peroxidase KW - human glutathione-S-transferase P1-1 KW - reactive metabolite SP - 32 EP - 40 JF - Chemico-biological interactions JO - Chem Biol Interact VL - 207 N2 - 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. SN - 1872-7786 UR - https://www.unboundmedicine.com/medline/citation/24246759/One_electron_oxidation_of_diclofenac_by_human_cytochrome_P450s_as_a_potential_bioactivation_mechanism_for_formation_of_2'__glutathion_S_yl__deschloro_diclofenac_ DB - PRIME DP - Unbound Medicine ER -