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-14C (100 μM) 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 μM 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 ∼29 and ∼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.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.026153.
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ABBREVIATIONS: PAA, phenylacetic acid; GSH, glutathione; CoA, coenzyme A; PA-CoA, phenylacetyl-S-acyl-coenzyme A thioester; PA-gly, phenylacetylglycine amide; PAGE, polyacrylamide gel electrophoresis; LC/MS/MS, liquid chromatography/tandem mass spectrometry; PA-SG, phenylacetyl-S-acyl-glutathione thioester; CBZ, carbamazepine; [1-14C]PAA, phenylacetic acid carboxy-14C; HPLC, high-performance liquid chromatography; THF, tetrahydrofuran; CID, collision-induced dissociation; MRM, multiple reaction monitoring; 2-PPA, 2-phenylproprionic acid.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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↵1 Current affiliation: School of Pharmacy, University of California at San Francisco, San Francisco, California.
- Received December 12, 2008.
- Accepted January 30, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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