Abstract
S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a mutagenic and nephrotoxic metabolite of trichloroethylene, can be bioactivated to reactive metabolites, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS) or chlorothioketene and/or 2-chlorothionoacetyl chloride, by cysteine conjugate S-oxidase (S-oxidase) and cysteine conjugate beta-lyase (beta-lyase), respectively. Previously, we characterized the reactivity of DCVCS with Hb upon incubation of erythrocytes with DCVCS and provided evidence for the formation of distinct DCVCS-Hb monoadducts and cross-links in both isolated erythrocytes and rats given DCVCS. In the present study, we investigated DCVC bioactivation and Hb adduct formation in isolated rat erythrocytes incubated with DCVC (9 and 450 microM) at 37 degrees C and pH 7.4. The results suggested that no DCVCS monoadducts or cross-links were formed; however, LC/electrospray ionization/MS and matrix-assisted laser desorption/ionization/MS of trypsin-digested globin peptides revealed the presence of beta-lyase-derived globin monoadducts and cross-links. Adducts and cross-links in which the sulfur atom of the reactive sulfur intermediates were replaced by oxygen have also been detected. Use of SDS-PAGE provided additional evidence for globin cross-link formation in the presence of DCVC. Interestingly, the MS results suggest that the observed peptide selectivity of the beta-lyase-derived reactive sulfur/oxygen-containing species was different than that previously observed with DCVCS. While these results suggested that erythrocytes have beta-lyase but not S-oxidase activity, further support for this hypothesis was obtained using S-(2-benzothiazolyl)-L-cysteine, an alternative substrate for beta-lyases. Collectively, the results demonstrate the utility of Hb adducts and cross-links to characterize the metabolic pathway responsible for DCVC bioactivation in erythrocytes and to provide distinct biomarkers for each reactive metabolite.