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

Abstract

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 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 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 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 both in vitro and in vivo. Thus, hemoglobin adducts and cross-links may be useful biomarkers to investigate possible presence of DCVCS in the circulation after DCVC or trichloroethylene exposure.

Globin Monoadducts and Cross-Links Provide Evidence for the Presence of S-(1,2-Dichlorovinyl)-l-cysteine Sulfoxide, Chlorothioketene, and 2-Chlorothionoacetyl Chloride in the Circulation in Rats Administered S-(1,2-Dichlorovinyl)-l-cysteine

“…These results are consistent with our previous findings when we detected globin cross-links due to N-acetylation of DCVCS after rats were dosed with DCVCS (32). Although the presence of DCVCS monoadducts and cross-links provided evidence for bioactivation of DCVC by FMO3 in vivo, formation of NA-DCVCS may also be dependent on another S-oxidase (CYP450 3A1/2) (38), i.e.…”

“…Although our previous DCVCS study led to detecting globin adducts with 4 out of 5 cysteine residues per αβ-heterodimer (32), all 5 cysteine sites appeared to be involved in DCVCS-derived adduct formation after DCVC administration. Overall, two and three out of four rats exhibited S-oxidase-derived globin modifications at both high acute and subacute DCVC doses (460 and 30 μmol/kg) and at low subacute DCVC dose (3 μmol/kg), respectively (Table 8).…”

“…Because such cross-links were also detected after treatment of rats with DCVCS (23 and 230 μmol/kg) (32), these peptide cross-links could serve as reliable biomarkers of TCE exposure. Interestingly, both α(Cys104) and β(Cys93) residues of rat Hb occupy the same positions in human Hb with β(Cys93) considered as the most reactive sulfhydryl site (39).…”

“…Separation of peptides was then accomplished using Vydac C 18 protein and peptide HPLC column (5 μ, 4.6 mm × 25 cm) at a flow rate of 1 mL/min and monitored at A 220 as previously described (32). Briefly, mobile phases were: pump A, 0.1% TFA in doubly deionized H 2 O and pump B, 0.1% TFA in ACN.…”

“…Previously, evidence for formation of DCVCS-derived monoadducts and cross-links with cysteine residues of globin was provided both in vitro when incubating red blood cells (RBCs) with DCVCS and in vivo upon dosing Sprague-Dawley rats with DCVCS (32). Because incubation of RBCs with DCVC had also resulted in detection of β-lyase-derived monoadducts and cross-links (33), globin adducts could be used to investigate the presence of DCVCS and chlorothioketene and/or 2-chlorothioacetyl chloride in the circulation after DCVC exposure.…”