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Detection of Multiple Globin Monoadducts and Cross-Links after in Vitro Exposure of Rat Erythrocytes to S-(1,2-Dichlorovinyl)-l-cysteine Sulfoxide and after in Vivo Treatment of Rats with S-(1,2-Dichlorovinyl)-l-cysteine Sulfoxide

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journal contribution
posted on 15.09.2008 by Nella Barshteyn, Adnan A. Elfarra
S-(1,2-Dichlorovinyl)-l-cysteine sulfoxide (DCVCS), a Michael acceptor produced by an FMO3-mediated oxidation of the trichloroethylene metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. Because DCVCS incubations with N-acetyl-l-cysteine at pH 7.4, 37 °C resulted in the formation of three diastereomeric monoadducts and one diadduct, globin monoadducts and cross-links formed after in vitro incubations of rat erythrocytes with DCVCS (0.9−450 μM) for 2 h and those present at 30 min after in vivo treatment of rats with DCVCS (23 and 230 μmol/kg) were characterized. ESI/MS of intact globin chains revealed adduction of 1 DCVCS moiety on the β2 chain at the three lowest DCVCS concentrations and on the β1 chain after the in vivo treatment with 230 μmol/kg DCVCS. Interestingly, intact globin dimers and trimers were detectable by ESI/MS with all DCVCS concentrations in vitro (also by SDS−PAGE) and in vivo. LC/MS and MALDI/FTICR of trypsin digested peptides from globin samples obtained after in vitro (450 μM DCVCS) or in vivo exposure to DCVCS (230 μmol/kg) suggested the formation of DCVCS monoadducts not only with Cys93 and Cys125 of the β chains but also with Cys13 of the α chains, whereas no monoadducted peptides were detected at lower DCVCS concentrations in vitro or in vivo. However, LC/MS and MALDI-TOF/TOF suggested the presence of several DCVCS-derived peptide cross-links both in vivo and in vitro at all DCVCS exposure levels. Collectively, the results indicate at least 4 out of the 5 cysteine moieties of the rat hemoglobin heterodimer may be alkylated by DCVCS, in reactions that could also lead to the formation of multiple cross-links. DCVCS- and N-acetyl-DCVCS (NA-DCVCS)-derived globin cross-links containing GSH and Cys were also detected by mass spectrometry, providing strong evidence for the reactivity and/or cross-linking ability of DCVCS, NA-DCVCS, and their GSH or Cys conjugates in both the in vitro and the in vivo. Thus, hemoglobin adducts and cross-links may be useful biomarkers to investigate the possible presence of DCVCS in circulation after DCVC or trichloroethylene exposure.