Thiols, Disulfides, and Thioesters

Hua

2008

Chem. Res. Toxicol.

Ibuprofen is metabolized to chemically reactive ibuprofen-1- O-acyl-glucuronide (I-1- O-G) and ibuprofen- S-acyl-CoA (I-CoA) derivatives, which are proposed to mediate the formation of drug-protein adducts via the transacylation of protein nucleophiles. We examined the ability of ibuprofen to undergo enantioselective metabolism to ibuprofen- S-acyl-glutathione thioester (I-SG) in incubations with rat hepatocytes, where I-CoA formation is known to be highly enantioselective in favor of the (R)-(-)-ibuprofen isomer. We proposed that potential enantioselective transacylation of glutathione forming I-SG in favor of the (R)-(-)-isomer would reveal the importance of acyl-CoA formation, versus acyl glucuronidation, in the generation of reactive transacylating-type intermediates of the drug. Thus, when (R)-(-)- and (S)-(+)-ibuprofen (100 microM) were incubated with hepatocytes, the presence of I-CoA and I-SG was detected in incubation extracts by LC-MS/MS techniques. The formation of I-CoA and I-SG in hepatocyte incubations with (R)-(-)-ibuprofen was rapid and reached maximum concentrations of 2.6 microM and 1.3 nM, respectively, after 8-10 min of incubation. By contrast, incubations with (S)-(+)-ibuprofen resulted in 8% and 3.9% as much I-CoA and I-SG formation, respectively, compared to that in corresponding incubations with the (R)-(-)-isomer. Experiments with a pseudoracemic mixture of (R)-(-)-[3,3,3-(2)H3]- and (S)-(+)-ibuprofen showed that >99% of the I-SG detected in hepatocyte incubations contained deuterium and therefore was derived primarily from (R)-(-)-ibuprofen bioactivation. Inhibition of (R)-(-)-ibuprofen (10 microM) glucuronidation with (-)-borneol (100 microM) led to a 98% decrease in I-1-O-G formation; however, no decrease in I-SG production was observed. Coincubation with pivalic, valproic, or lauric acid (500 microM each) was shown to lead to a significant inhibition of I-CoA formation and a corresponding decrease in I-SG production. Results from these studies demonstrate that the reactive I-CoA derivative, and not the I-1-O-G metabolite, plays a central role in the transacylation of GSH in incubations with rat hepatocytes.

Section: Introductionmentioning

confidence: 99%

Enantioselective Formation of Ibuprofen-S-Acyl-Glutathione in Vitro in Incubations of Ibuprofen with Rat Hepatocytes

Hua

2008

Chem. Res. Toxicol.

“…In the present work, an attempt was made to determine which of these acylating derivatives may be more important in the transacylation of protein nucleophiles in vivo based on reactivity differences determined in vitro. Acyl-CoA thioesters, because of their high-energy carbon-sulfur bond (19), have been shown recently to be reactive acylating derivatives with proteins. Such studies included the covalent binding of nafenopin-CoA to proteins in vitro in incubations with rat liver microsomes plus the cofactors for acyl-CoA formation (CoA, ATP, Mg 2+ ) (8), the time-dependent acylation of proteins and peptides by palmitoyl-CoA (20), and the nonenzymatic acylation of glycine by salicyl-CoA in vitro (9).…”

Section: Introductionmentioning

confidence: 99%

Studies on the Chemical Reactivity of 2-Phenylpropionic Acid 1-O-Acyl Glucuronide andS-Acyl-CoA Thioester Metabolites

Benet

2002

Chem. Res. Toxicol.

Chemically reactive species formed from the metabolism of carboxylic acid-containing compounds have been proposed as mediators of their toxic side-effects. Two alternative metabolic pathways known to be involved in the generation of reactive acylating metabolites of carboxylic acids are acyl glucuronidation and acyl-CoA formation. Here, we present studies with 2-phenylpropionic acid focused on evaluating the relative abilities of acyl glucuronides versus acyl-CoA derivatives to transacylate the nucleophilic cysteinyl-thiol of glutathione. Thus, synthetic 2-phenylpropionyl-S-acyl-CoA (2-PPA-SCoA) and biosynthetic 2-phenylpropionyl-1-O-acyl glucuronide (2-PPA-1-O-G) were incubated separately, and at varying concentrations (15.6-500 nM as well as at 0.1 mM), with GSH (1, 5, and 10 mM) in buffer (pH 7.4, 37 degrees C), and formation of the transacylation product, 2-phenylpropionyl-S-acyl-glutathione (2-PPA-SG), was quantified by reverse-phase HPLC and LC-MS. HPLC analysis of the products from both the reaction of 2-PPA-SCoA and 2-PPA-1-O-G with GSH showed the presence of 2-PPA-SG, which was confirmed by coelution with authentic 2-PPA-SG as well as by its LC/MS mass spectrum. The formation of 2-PPA-SG was time- and concentration-dependent with a formation rate constant of (1.9 +/- 0.2) x 10(-2) M(-1) x s(-1) from reactions of GSH with 2-PPA-SCoA, and (2.7 +/- 0.4) x 10(-4) M(-1) x s(-1) from reactions of GSH with 2-PPA-1-O-G. Therefore, the reactivity of 2-PPA-SCoA with GSH was 70 times greater than the reactivity of GSH with 2-PPA-1-O-G, which was found to acyl-migrate to less reactive isomers. Analysis of the in vitro stability of 2-PPA-SCoA and 2-PPA-1-O-G in the absence of GSH showed the CoA esters to be completely stable after 24 h, whereas the acyl glucuronides decomposed by 50% in 1.3 and 2.4 h of incubation at pH 7.4 and 37 degrees C for (R)- and (S)-2-PPA-1-O-G, respectively. In addition, studies of the reactivity of 2-PPA-SCoA with bovine serum albumin showed time- and pH-dependent covalent binding to the protein in vitro. These results support the hypothesis that xenobiotic acyl-CoA thioesters are reactive acylating species that, in addition to acyl glucuronides, may contribute to xenobiotic acid-protein adduct formation in vivo.

“…In the current study, the role of the chemically reactive acyl-CoA thioester metabolite in the formation of covalent adducts to hepatocyte protein was addressed. Acyl-CoA thioester intermediary metabolites of acidic drugs are more electrophilic (Huxtable, 1986) than their corresponding acyl glucuronide derivatives and are increasingly being investigated for their contribution to the transacylation of GSH and protein (Sallustio et al, 2000;Li et al, 2002a,b;Sidenius et al, 2004;Grillo et al, 2008).…”

mentioning

confidence: 99%

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

Lohr²

2009

Drug Metab Dispos

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.