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Research ArticleArticle

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

Mark P. Grillo and Michelle Tadano Lohr
Drug Metabolism and Disposition May 2009, 37 (5) 1073-1082; DOI: https://doi.org/10.1124/dmd.108.026153
Mark P. Grillo
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Michelle Tadano Lohr
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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

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.108.026153.

  • 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.

  • ↵ Embedded Image The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

  • ↵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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Drug Metabolism and Disposition: 37 (5)
Drug Metabolism and Disposition
Vol. 37, Issue 5
1 May 2009
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Research ArticleArticle

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

Mark P. Grillo and Michelle Tadano Lohr
Drug Metabolism and Disposition May 1, 2009, 37 (5) 1073-1082; DOI: https://doi.org/10.1124/dmd.108.026153

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Research ArticleArticle

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

Mark P. Grillo and Michelle Tadano Lohr
Drug Metabolism and Disposition May 1, 2009, 37 (5) 1073-1082; DOI: https://doi.org/10.1124/dmd.108.026153
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