Covalent Binding of 2-Phenylpropionyl-S-Acyl-Coa Thioester to Tissue Proteins in Vitro

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:In this study, we investigated the possible involvement of acylCoA, reactive intermediary metabolites of 2-arylpropionic acids (profens), in protein adduct formation in rat liver homogenate and in human serum albumin ( 2-Arylpropionic acids (profens) are a commonly used class of nonsteroidal anti-inflammatory drugs, widely prescribed as analgesic, antipyretic, and anti-inflammatory agents. Conjugation with glucuronic acid is a major route for the biotransformation and elimination of profen drugs, such as ibuprofen, carprofen, fenoprofen, and naproxen (Spahn-Langguth et al., 1997). Studies have shown that these acyl-linked glucuronides are chemically reactive species that undergo hydrolysis to regenerate the pharmacologically active parent drug and that also undergo intramolecular acyl migration to yield ␤-glucuronidase-resistant isomers (Spahn-Langguth and Benet, 1992;Spahn-Langguth et al., 1997;. More importantly, these electrophilic metabolites have been shown to bind covalently to serum albumins in vitro and to plasma and tissue proteins in vivo (Spahn-Langguth and Benet, 1992;; and, therefore, metabolic activation of profen drugs by acyl glucuronidation has been proposed as a metabolic route to mediate the idiosyncratic liver toxicity associated with the use of profen drugs (Boelsterli et al., 1995).Another metabolic route of profen drugs is the acyl-CoA thioester pathway, recently recognized also as yielding reactive metabolites of acidic drugs. Sallustio et al. (2000) demonstrated that covalent binding of nafenopin to human liver proteins is directly associated with formation of a nafenopin acyl-CoA thioester intermediate. A number of studies on protein fatty acylation have shown that endogenous acyl-CoAs, including palmitoyl-CoA and arachidonoyl-CoA, can react nonenzymatically with sulfhydryl groups on proteins and peptides in vitro in a time-and concentration-dependent fashion (Bharadwaj and Bizzozero, 1995;Duncan and Gilman, 1996). Our recent studies with 2-phenylpropionic acid (2-PPA 3 ) demonstrated that 2-phenylpropionyl-S-acyl-CoA (2-PPA-CoA) was able to acylate glutathione sulfhydryl to form 2-PPA-S-acyl-glutathione at a rate that was approximately 70-fold more rapid than the similar reactions with 2-PPA-1-O-acyl glucuronides . The present study was designed to examine the covalent binding of 2-PPA-CoA to human serum albumin and rat liver homogenate in vitro. 2-PPA was chosen because it is the simplest congener of profen drugs and is capable of forming 2-PPA-CoA, as inferred from the unidirectional chiral inversion of 2-PPA in vivo (Fournel and Caldwell, 1986). Results from these in vitro studies strongly suggest that acyl-CoA thioesters of profen drugs are chemically reactive electrophiles and are able to bind covalently to tissue proteins in vitro, which may, therefore, contribute significantly to covalent adduct formation of profen drugs in vivo, in addition to acyl glucuronides. Materials and Methods Materials. (R)-(Ϫ)-

Differential Effects of Fibrates on the Metabolic Activation of 2-Phenylpropionic Acid in Rats

“…Other acyl-CoA synthetases that are more susceptible to fibrate induction may also play a role. This is consistent with our previous kinetic studies in rat liver homogenate, which indicated that more than one acyl-CoA synthetase is involved in 2-PPA-CoA formation (Li et al, 2003c).…”

“…Perfused livers were rapidly removed, weighed, and frozen immediately in liquid nitrogen. Rat liver homogenate was prepared as described previously (Li et al, 2003c). To determine (R)-2-PPA-CoA synthetase activity, (R)-2-PPA (1 mM) was incubated with 0.25 mg of liver homogenate protein, 0.05% Triton X-100, 1 mM DTT, 2 mM EDTA, 1.2 mM CoA, 6.2 mM MgCl 2 , and 2.5 mM ATP in a final volume of 0.5 ml (Li et al, 2003c).…”

“…Rat liver homogenate was prepared as described previously (Li et al, 2003c). To determine (R)-2-PPA-CoA synthetase activity, (R)-2-PPA (1 mM) was incubated with 0.25 mg of liver homogenate protein, 0.05% Triton X-100, 1 mM DTT, 2 mM EDTA, 1.2 mM CoA, 6.2 mM MgCl 2 , and 2.5 mM ATP in a final volume of 0.5 ml (Li et al, 2003c). After a 3-min preincubation, reactions were initiated by addition of ATP, allowed to proceed for 30 min at 37°C, and then stopped by addition of 50 l of perchloric acid (7%).…”

Metabolic activation of carboxylic acids

“…Due to their physicochemical properties, acyl-CoA derivatives are not excreted from the cell but can only be detected in (for example) hepatocytes, or in liver homogenates from in-vivo experiments. Recent examples of in-vitro and in-vivo detection and identification of xenobiotic acyl-CoAs include tolmetin [12,13] , zomepirac [14] , and 2-phenyl propionic acid [15,16] . Acyl-CoAs act as intermediates in a number of phase II conjugation reactions, which include amino acid conjugation (mainly glycine conjugation), taurine conjugation and carnitine ester formation.…”

Interaction of the Electrophilic Ketoprofenyl-Glucuronide and Ketoprofenyl-Coenzyme A Conjugates with Cytosolic Glutathione S-Transferases

“…The lower inhibitory potency observed in the presence of cytosolic extracts than with purified GST is likely to be due to the buffering effect of additional proteins interacting with the two acyl metabolites of KPF. Various proteins such as albumin (Li et al, 2003b), sulfotransferases (Tulik et al, 2002), COX (Levoin et al, 2004), hepatocyte nuclear factor-4␣ (Hertz et al, 2001), and glucose-6-phosphate dehydrogenase (Asensio et al, 2007) have indeed been reported to interact with CoA thioester derivatives. It was found previously that long-chain saturated fatty acyl-CoAs and peroxisome proliferator-CoA thioesters exert high affinity toward GST (Silva et al, 1999).…”