Proteomic Characterization of Metabolites, Protein Adducts, and Biliary Proteins in Rats Exposed to 1,1-Dichloroethylene or Diclofenac

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Abstract
A proteome profiling approach was used to compare effects of two toxicants, 1,1-dicloroethylene (DCE) and diclofenac, which covalently adduct hepatic proteins. Bile was examined as a potential source of protein alterations since both toxicants target the hepatic biliary canaliculus. Bile was collected before and after toxicant treatment. Biliary proteins were separated by one-dimensional SDS−PAGE and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with data-dependent scanning. Comprehensive analysis of biliary proteins was performed by using SEQUEST and BLAST database searching, in combination with de novo interpretation. Bile not subjected to tryptic digestion was analyzed for DCE metabolites. DCE treatment resulted in a marked increase in the overall number of biliary proteins, whereas few changes in the proteomic profile were apparent in bile after diclofenac treatment. This is consonant with prior observations of more profound effects of DCE on canalicular membrane integrity. LC-MS-MS analyses for DCE metabolites revealed the presence of S-carboxymethyl glutathione, S-(cysteinylacetyl)glutathione, and a product of the intramolecular rearrangement of the DCE metabolite, ClCH2COSG, not previously described in vivo. In addition, several S-carboxymethylated proteins were identified in bile from DCE-treated animals. This investigation has produced the first comprehensive baseline characterization of the content of the rat biliary proteome and the first documentation of alterations in the proteome of bile by toxicant treatment. In addition, the results provide direct in vivo evidence for DCE metabolic routes proposed in the formation of covalent adducts.