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

Abstract

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.

Metabolic Activation of Mefenamic Acid Leading to Mefenamyl-S-Acyl-Glutathione Adduct Formation In Vitro and In Vivo in Rat

“…6A). This concentration was much lower than the C max observed for S-acyl-CoA metabolites of (R)-ibuprofen (2600 nM; Grillo and Hua, 2008) and phenylacetic acid (1300 nM; Grillo and Lohr, 2009) measured under similar incubation conditions with rat hepatocytes; however, it was similar to the C max of flunoxaprofen-S-acyl-CoA (42 nM) detected in recent studies with rat hepatocytes incubated with 100 M (R)-flunoxaprofen . In addition, the length of incubation time leading to C max (T max ) of MFA-SCoA was 30 min in the present studies, whereas the reported T max of formation of ISCoA, phenylacetyl-S-acyl-CoA, and flunoxaprofen-S-acyl-CoA was 3-to 7.5-fold shorter at 10, 4, and 6 min, respectively (Grillo, 2011).…”

“…In time-course rat hepatocyte studies, we determined the C max of MFA-SCoA detected in incubations treated with 100 M MFA to be 15 nM after 30 min of incubation, which was 170-and 90-fold lower than I-SCoA and phenylacetyl-S-acyl-CoA detected in similar studies with (R)-ibuprofen (Grillo and Hua, 2008) and phenylacetic acid (Grillo and Lohr, 2009), respectively. Differences in drug-S-acyl-CoA formation may be due to differences in the acyl-CoA synthetase(s) catalyzing their formation; however, the identity of the acyl-CoA synthetase(s) that mediates the formation of MFA-SCoA is not known.…”

Characterization of the Acyl-Adenylate Linked Metabolite of Mefenamic Acid

“…33 Stability assessment of MFA-CoA revealed it to be highly stable, with no observable hydrolysis, for at least 24 h, which is consistent with previous S -acyl-CoA stability data, in which clofibric acid- S -acyl-CoA has been shown to be highly stable in vitro in physiological buffer with a degradation half life of 21 days (~2% hydrolysis/day). 17 Similarly, 2-phenylpropionic- S -acyl-CoA 34 and phenylacetyl- S -acyl-CoA 36 have also been shown to be completely stable after one day of incubation. MFA-GSH was stable during the 24 h incubation in buffer under physiological conditions, which is expected since S -acyl-GSH thioesters are mostly chemically stable products.…”

Drug-S-Acyl-Glutathione Thioesters: Synthesis, Bioanalytical Properties, Chemical Reactivity, Biological Formation and Degradation

“…In studies with S-acyl-GSH derivatives of diclofenac (100 M; [53]), zomepirac (1 M; [40]), phenylacetic acid (1 M; [56]), and both (R)-and (S)-ibuprofen (1 M; [7]), they were shown to degrade with half-lives of 1, 0.8, 1.7, and 4-min, respectively. For each of these derivatives, no evidence for -GTmediated degradation was observed in rat hepatocyte incubations, which is consistent with studies showing insufficient activity of this enzyme in rat liver tissue [79].…”

“…One example of a carboxylic acid xenobiotic carboxylic acid that upon incubation with hepatocytes did not lead to the detection of an S-acyl-GSH adduct, even though the corresponding S-acylCoA was detected in the same incubation, was for the model arylacetic acid-containing compound, phenylacetic acid (PAA, [56]). In those studies, up to ~1.3 M PAA-S-acyl-CoA was formed in incubations of PAA (100 M) with rat hepatocytes (2 million cells/mL).…”