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Title: N-biotinyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide as a potential model for S-(1,2-dichlorovinyl)-L-cysteine sulfoxide: characterization of stability and reactivity with glutathione and kidney proteins in vitro.

Authors: Irving, Roy M; Brownfield, Mark S; Elfarra, Adnan A

Published In Chem Res Toxicol, (2011 Nov 21)

Abstract: S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide (DCVCS) is a reactive and potent nephrotoxic metabolite of the human trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC). Because DCVCS covalent binding to kidney proteins likely plays a role in its nephrotoxicity, in this study biotin-tagged DCVCS, N-biotinyl-DCVCS (NB-DCVCS), was synthesized, and its stability in buffer alone and in the presence of rat blood or plasma was characterized in vitro. In addition, reactivity toward GSH and covalent binding to selected model enzymes and isolated kidney proteins were characterized. The half-lives of NB-DCVCS (39.6 min) and the DCVCS (diastereomer 1, 14.4 min; diastereomer 2, 6 min) in the presence of GSH were comparable. Incubating the model enzymes glutathione reductase and malate dehydrogenase with 10 ýýM NB-DCVCS for 3 h at 37 ýýC followed by immunoblotting using antibiotin antibodies demonstrated that glutathione reductase and malate dehydrogenase were extensively modified by NB-DCVCS. When rat kidney cytosol (6 ýýg/ýýL) was incubated with NB-DCVCS (312.5 nM to 5 ýýM) for 3 h at 37 ýýC followed by immunoblotting, a concentration-dependent increase in signal with multiple proteins with different molecular weights was observed, suggesting that NB-DCVCS binds to multiple kidney proteins with different selectivity. Incubating rat kidney cytosol with DCVCS (10-100 ýýM) prior to the addition of NB-DCVCS (2.5 ýýM) reduced the immunoblotting signal, suggesting that NB-DCVCS and DCVCS compete for the same binding sites. A comparison of the stability of NB-DCVCS and DCVCS in rat blood and plasma was determined in vitro, and NB-DCVCS exhibited higher stability than DCVCS in both media. Collectively, these results suggest that NB-DCVCS shows sufficient stability, reactivity, and selectivity to warrant further investigations into its possible use as a tool for future characterization of the role of covalent modification of renal proteins by DCVCS in nephrotoxicity.

PubMed ID: 21988407 Exiting the NIEHS site

MeSH Terms: Animals; Binding Sites; Binding, Competitive; Biotin/chemistry; Biotin/metabolism*; Biotinylation; Blotting, Western; Cysteine/adverse effects; Cysteine/analogs & derivatives*; Cysteine/chemistry; Cysteine/metabolism; Cysteine/toxicity; Dose-Response Relationship, Drug; Drug Stability; Electrophoresis, Polyacrylamide Gel; Glutathione Reductase/blood*; Glutathione Reductase/chemistry; Half-Life; Humans; Indicators and Reagents/analysis*; Indicators and Reagents/chemistry; Kidney Neoplasms/blood*; Kidney Neoplasms/etiology; Kidney Neoplasms/pathology; Kidney/drug effects; Kidney/metabolism*; Kidney/pathology; Malate Dehydrogenase/blood*; Malate Dehydrogenase/chemistry; Protein Binding; Rats, Sprague-Dawley

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