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

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 degrees 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 mu M) for 2 h and those present at 30 min after in vivo treatment of rats with DCVCS (23 and 230 mu mol/kg) were characterized. ESI/MS of intact globin chains revealed adduction of I DCVCS moiety on the 02 chain at the three lowest DCVCS concentrations and on the beta 1 chain after the in vivo treatment with 230 mu mol/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,M DCVCS) or in vivo exposure to DCVCS (230 mu mol/kg) suggested the formation of DCVCS monoadducts not only with Cys93 and Cys125 of the beta chains but also with Cys13 of the alpha 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.