In Vitro Covalent Binding of Nafenopin–CoA to Human Liver Proteins

ABSTRACT:Phenylacetic acid (PAA) represents a substructure of a class of nonsteroidal anti-inflammatory carboxylic acid-containing drugs capable of undergoing metabolic activation in the liver to acylcoenzyme A (CoA)-and/or acyl glucuronide-linked metabolites that are proposed to be associated with the formation of immunogenic, and hence potentially hepatotoxic, drug-protein adducts. Herein, we investigated the ability of PAA to undergo phenylacetyl-S-acyl-CoA thioester (PA-CoA)-mediated covalent binding to protein in incubations with freshly isolated rat hepatocytes in suspension. Thus, when hepatocytes were incubated with phenylacetic acid carboxy-14 C (100 M) and analyzed for PA-CoA formation and covalent binding of PAA to protein and over a 3-h time period, both PA-CoA formation and covalent binding to protein increased rapidly, reaching 1.3 M and 291 pmol equivalents/mg protein after 4 and 6 min of incubation, respectively. However, the covalent binding of PAA to protein was reversible and decreased by 72% at the 3-h time point. After 3 h of incubation, PAA was shown to be metabolized primarily to phenylacetyl-glycine amide (84%). No PAA-acyl glucuronide was detected in the incubation extracts. PA-CoA reacted readily with glutathione in buffer, forming PA-Sacyl-glutathione; however, this glutathione conjugate was not detected in hepatocyte incubation extracts. Coincubation of hepatocytes with lauric acid led to a marked inhibition of PA-CoA formation and a corresponding inhibition of covalent binding to protein. SDS-polyacrylamide gel electrophoresis analysis showed the formation of two protein adducts having molecular masses of ϳ29 and ϳ33 kDa. In summary, PA-CoA formation in rat hepatocytes leads to the highly selective, but reversible, covalent binding to hepatocyte proteins, but not to the transacylation of glutathione.

Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Covalent Binding of Phenylacetic Acid to Protein in Incubations with Freshly Isolated Rat Hepatocytes

Grillo

Lohr²

2009

“…In a recent study, it has been shown that the pyrrole moiety of Tol may undergo bioactivation to a reactive arene oxide (Chen et al, 2006). Other reports have shown that acyl-coenzyme A thioesters (acyl-CoAs) are also reactive electrophilic metabolites (Sallustio et al, 2000;Grillo and Benet, 2002;Sidenius et al, 2004). Acyl-CoAs are intermediates in amino acid conjugation, carnitine conjugation, fatty acid synthesis, and ␤-oxidation.…”

Section: Introductionmentioning

confidence: 99%

Studies on the Metabolism of Tolmetin to the Chemically Reactive Acyl-Coenzyme A Thioester Intermediate in Rats

Olsen

Li²,

Skonberg³

et al. 2007

ABSTRACT:Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of tolmetin (Tol) to reactive metabolites and the subsequent formation of Tol-protein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg i.p.), tolmetin acyl-CoA (Tol-CoA) was identified by liquid chromatography-tandem mass spectrometry in liver homogenates. Similarly, the acyl-CoA-dependent metabolites tolmetin-taurine conjugate (Tol-Tau) and tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day i.p. for 7 days) before dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acidtreated rats. Similarly, levels of Tol-CoA increased from 0.6 nmol/g to 4.4 nmol/g liver after pretreatment with clofibric acid, whereas the formation of Tol-O-G and Tol-Tau was unaffected by clofibric acid treatment. However, Tol-Car levels increased from 0.08 to 0.64 nmol/g after clofibric acid treatment. Collectively, these results confirm that Tol-CoA is formed in vivo in the rat and that this metabolite can have important consequences in terms of covalent binding to liver proteins.Drugs with a carboxylate functional group can be metabolized to chemically reactive metabolites that react with nucleophilic amino acids of proteins to form drug-protein adducts (Boelsterli, 2002;Bailey and Dickinson, 2003). Tolmetin (Tol; 5-[4Ј-methylbenzoyl]-1-methylpyrrole-2-acetic acid) is an example of a carboxylic acidcontaining drug which, in humans, is partly metabolized to the acyl glucuronide (Hyneck et al., 1987). In vitro studies have shown that the tolmetin-acyl glucuronide (Tol-O-G) is a reactive metabolite that reacts with the amino acid side chains of lysine, arginine, and serine present in human serum albumin (Ding et al., 1993(Ding et al., , 1995.Covalent binding of Tol has also been studied in humans, where Tol-protein adducts have been detected in plasma (Hyneck et al., 1988;Zia-Amirhosseini et al., 1994). Tol-protein adducts in plasma were believed to result from reaction of the acyl glucuronide with plasma proteins, since a good correlation between covalent plasmaprotein adducts and exposure to Tol-O-G was observed (Hyneck et al., 1988). In the liver, other metabolic pathways may contribute to Tol-protein adduct formation. In a recent study, it has been shown that the pyrrole moiety of Tol may undergo bioactivation to a reactive arene oxide (Chen et al., 2006). Other reports have shown that acyl-coenzyme A thioesters (acyl-CoAs) a...

“…Their studies showed the chemical reactivity of xenobiotic acyl-CoAs and their potential to undergo nonenzymatic protein acylation. Sallustio et al (2000) were the first to show a direct relationship between xenobiotic-CoA formation (in this case, nafenopin-CoA) and acylation of human liver proteins. No acyl-CoA synthase-dependent BNX, FLX, or IBP adduct formation was characterized in the present study because of a lack of specific and potent inducers or inhibitors of this metabolic pathway.…”

Section: Discussionmentioning

confidence: 99%

Glucuronidation and Covalent Protein Binding of Benoxaprofen and Flunoxaprofen in Sandwich-Cultured Rat and Human Hepatocytes

Dong

Smith²

2009

ABSTRACT:Benoxaprofen (BNX), a nonsteroidal anti-inflammatory drug (NSAID) that was withdrawn because of hepatotoxicity, is more toxic than its structural analog flunoxaprofen (FLX) in humans and rats. Acyl glucuronides have been hypothesized to be reactive metabolites and may be associated with toxicity. Both time-and concentration-dependent glucuronidation and covalent binding of BNX, FLX, and ibuprofen (IBP) were determined by exposing sandwich-cultured rat hepatocytes to each NSAID. The levels of glucuronide and covalent protein adduct measured in cells followed the order BNX > FLX > IBP. These results indicate that 1) BNXglucuronide (G) is more reactive than FLX-G, and 2) IBP-G is the least reactive metabolite, which support previous in vivo studies in rats. The proportional increases of protein adduct formation for BNX, FLX, and IBP as acyl glucuronidation increased also support the hypothesis that part of the covalent binding of all three NSAIDs to hepatic proteins is acyl glucuronide-dependent. Moreover, theses studies confirmed the feasibility of using sandwich-cultured rat hepatocytes for studying glucuronidation and covalent binding to hepatocellular proteins. These studies also showed that these in vitro methods can be applied using human tissues for the study of acyl glucuronide reactivity. More BNX-protein adduct was formed in sandwich-cultured human hepatocytes than FLX-protein adduct, which not only agreed with its relative toxicity in humans but also was consistent with the in vitro findings using rat hepatocyte cultures. These data support the use of sandwich-cultured human hepatocytes as an in vitro screening model of acyl glucuronide exposure and reactivity.Many types of acidic drugs form acyl glucuronides, and other xenobiotics are metabolized to carboxylic acids (Phase I metabolites), which subsequently undergo Phase II conjugation to form acyl glucuronides. Often such a glucuronide conjugate constitutes the major metabolite. The major site of conjugation for most compounds in humans is believed to be the liver. Modification of critical hepatic proteins by covalent binding of acidic drugs through reactive acyl glucuronides may provide a basis for direct hepatocyte toxicity or immune-mediated adverse reactions (Gillette, 1974;Faed, 1984;Boelsterli, 2002;Bailey and Dickinson, 2003).Some pharmaceutical companies have been conducting in vitro experiments by incubating acyl glucuronides with model proteins or peptides (Wang et al., 2004) to determine their reactivity, and hence possibly predict the relative extent of covalent protein binding in vivo in humans. This method requires chemical synthesis or biosynthesis of individual glucuronides for each drug candidate, a process that can be tedious and costly. In addition, hepatic tissue proteins are not present in the incubation, which makes it less likely to correlate covalent protein binding with hepatotoxicity. In vitro models involving hepatic materials, including liver homogenates, microsomal subcellular preparation, and isolated hepatocyte s...