Metabolic Activation and Immunochemical Localization of Liver Protein Adducts of the Nonsteroidal Anti-inflammatory Drug Diclofenac

Hargus, Sally J.; Amouzedeh, Hamid R.; Pumford, Neil R.; Myers, Timothy G.; McCoy, S; Pohl, Lance R.

doi:10.1021/tx00040a014

Cited by 108 publications

(89 citation statements)

References 35 publications

(52 reference statements)

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“…In previously reported studies with diclofenac (Hargus et al, 1994), the importance of acyl-CoA formation has been overlooked because in such studies the covalent binding of the acidic drug to protein was not detected. We propose that the presence of millimolar amounts of DTT, although often necessary for the formation of acyl-CoA thioesters, would compete with protein nucleophiles for covalent binding.…”

Section: Fig 9 Time-dependent Reaction Of S-acyl-glutathione Derivamentioning

confidence: 99%

Studies on the Reactivity of Clofibryl-S-Acyl-CoA Thioester with Glutathione in Vitro

Grillo¹,

Benet²

2002

Drug Metab Dispos

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This paper is available online at http://dmd.aspetjournals.org ABSTRACT:Clofibric acid (p-chlorophenoxyisobutyric acid) is metabolized in vivo to a thioester-linked glutathione conjugate, S-(p-chlorophenoxyisobutyryl)glutathione (CA-SG). The formation of this metabolite is presumed to occur via transacylation reactions between glutathione (GSH) and reactive acyl-linked metabolite(s) of the drug. The present study examines the chemical reactivity of clofibryl-S-acyl-CoA (CA-SCoA), an acyl-CoA thioester intermediary metabolite of clofibric acid, with GSH to form the CA-SG in vitro. Incubations of CA-SCoA (1 mM) with GSH (5 mM) were carried out at pH 7.5 and 37°C, with analysis of the formed reaction products by isocratic reverse-phase high-performance liquid chromatography (HPLC). Results showed a time-dependent and linear formation of CA-SG up to 4 h (50 M CA-SG formed/h), and after a 1-day incubation, the reaction mixture contained 0.7 mM CA-SG. The identity of CA-SG was confirmed by analysis of HPLC-purified material by tandem mass spectrometry. The rate of CA-SG formation was found to be increased 3-fold in incubations containing rat liver glutathione S-transferases (4 mg/ml). Analysis of the chemical stability of CA-SCoA in buffer at 37°C and varying pH showed the derivative to be stable under mildly acidic and basic aqueous conditions but to hydrolyze at pH values greater than 10 after a 1-day incubation (t 1/2 ‫؍‬ ϳ1 day at pH 10.5). Results from these studies show that CA-SCoA is a reactive thioester derivative of clofibric acid and is able to acylate GSH and other thiol-containing nucleophiles in vitro and, therefore, may be able to acylate protein thiols in vivo, which could contribute to the toxic side effects of the drug.

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Section: Fig 9 Time-dependent Reaction Of S-acyl-glutathione Derivamentioning

confidence: 99%

Studies on the Reactivity of Clofibryl-S-Acyl-CoA Thioester with Glutathione in Vitro

Grillo¹,

Benet²

2002

Drug Metab Dispos

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“…6 -9 Adducts appear to be localized on the bile canalicular plasma membrane of hepatocytes. 9,10 Diclofenactreated hepatocytes carry antigenic determinants that are recognized by T cell and non-T cell-enriched splenocytes derivedfromdiclofenac/keyholelimpethemocyanin(KLH)-immunized mice, resulting in immunomediated destruction of target hepatocytes. 11 Antibodies directed against 4Ј-hydroxydiclofenac glucuronide have been detected in the serum of a patient with diclofenac-induced immune hemolytic anemia, 12 raising the possibility that humoral immune mechanisms might also be involved in diclofenac hepatotoxicity.…”

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confidence: 99%

Hepatic adducts, circulating antibodies, and cytokine polymorphisms in patients with diclofenac hepatotoxicity

et al. 2004

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Diclofenac is a nonsteroidal anti-inflammatory drug that causes rare but serious hepatotoxicity, the mechanism of which is unclear. The purpose of the present study was to explore the potential role played by the immune processes. Antibodies to diclofenac metabolite-modified liver protein adducts were detected in the sera of seven out of seven patients with diclofenac-induced hepatotoxicity, 12 of 20 subjects on diclofenac without hepatotoxicity, and none of four healthy controls. The antibodies recognized adducts expressed in livers from rats treated with multiple doses of diclofenac, but not in those given single doses. In addition, several potential diclofenac adducts were identified in the liver of a patient with diclofenac-induced hepatic failure, but not from a normal human donor liver, by immunoblotting with an adduct-selective rabbit antiserum. To determine whether or not polymorphisms in genes encoding cytokine-related proteins influence susceptibility to hepatotoxicity, genotyping for the polymorphisms -627 in the interleukin (

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“…The formation of protein adducts, which was suggested to be one of the causal factors for DCF-induced ulcer formation in the intestine, may either directly impair cellular signal transduction cascades or indirectly cause tissue damage by eliciting an immune response (Atchison et al, 2000;Boelsterli, 2003). Several DCF protein adducts were detected previously in mice and rats (Pumford et al, 1993;Hargus et al, 1994;Ware et al, 1998), including adducts with liver plasma membrane proteins (110, 140, and 200 kDa in size), formed presumably through UGT-mediated formation of DCF-G as the proximate toxicant, and an adduct with a liver microsomal protein (50 kDa in size), formed apparently via P450-catalyzed formation of reactive DCF intermediates. In the current study, a 50-kDa protein adduct band was also detected in vitro and in vivo in SI microsomes from WT mice; this adduct was not detected in SI microsomes from the IE-Cpr-null mice, thus supporting the notion that intestinal P450-catalyzed metabolic activation of DCF is responsible for the formation of this protein adduct.…”

Section: Discussionmentioning

confidence: 99%

Role of Intestinal Cytochrome P450 Enzymes in Diclofenac-Induced Toxicity in the Small Intestine

Zhu¹

2012

J Pharmacol Exp Ther

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The aim of this study was to determine the role of small intestinal (SI) cytochrome P450 (P450) enzymes in the metabolic activation of diclofenac (DCF), a widely used nonsteroidal antiinflammatory drug, and DCF-induced intestinal toxicity. DCF induces intestinal ulcers in humans and mice, but the underlying mechanisms, including the necessity for drug bioactivation in the target tissues and the sources and identities of reactive intermediates, are not fully understood. We found that the number of DCF-induced (at 50 mg/kg p.o.) intestinal ulcers was significantly smaller in an intestinal epithelium (IE)-specific P450 reductase (CPR) knockout (IE-Cpr-null) mouse model, which has little P450 activity in the IE, than in wild-type (WT) mice, determined at 14 h after DCF administration. The involvement of intestinal P450 enzymes was confirmed by large reductions (Ͼ80 -90%) in the rates of in vitro formation, in SI microsomal reactions, of hydroxylated DCF metabolites and reactive intermediates, trapped as DCFglutathione (GSH) conjugates, in the IE-Cpr-null, compared with WT mice. The SI levels of DCF-GSH conjugates (at 4 h after dosing) and DCF-protein adducts (at 14 h after dosing) were significantly lower in IE-Cpr-null than in WT mice. In additional experiments, we found that pretreatment of mice with grapefruit juice, which is known to inhibit SI P450 activity, ameliorated DCF-induced intestinal toxicity in WT mice. Our results not only strongly support the notion that SI P450 enzymes play an important role in DCF-induced intestinal toxicity, but also illustrate the possibility of preventing DCF-induced intestinal toxicity through dietary intervention.

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Metabolic Activation and Immunochemical Localization of Liver Protein Adducts of the Nonsteroidal Anti-inflammatory Drug Diclofenac

Cited by 108 publications

References 35 publications

Studies on the Reactivity of Clofibryl-S-Acyl-CoA Thioester with Glutathione in Vitro

Studies on the Reactivity of Clofibryl-S-Acyl-CoA Thioester with Glutathione in Vitro

Hepatic adducts, circulating antibodies, and cytokine polymorphisms in patients with diclofenac hepatotoxicity

Role of Intestinal Cytochrome P450 Enzymes in Diclofenac-Induced Toxicity in the Small Intestine

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