Covalent binding of phenylacetic acid to protein in incubations with freshly isolated rat hepatocytes.
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 microM) 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 microM 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-S-acyl-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 approximately 29 and approximately 33 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.
Grillo
Mark P
MP
Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, CA 94080, USA. grillo@amgen.com
Lohr
Michelle Tadano
MT
D000818:Animals; D001711:Biotransformation; D002469:Cell Separation; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D066298:In Vitro Techniques; D007850:Lauric Acids / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008862:Microsomes, Liver / Q000378:metabolism; D008970:Molecular Weight; D010648:Phenylacetates / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
D007850:Lauric Acids; D010648:Phenylacetates; C030358:lauric acid; C025136:phenylacetic acid; D005978:Glutathione