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.

Previously, our laboratory has shown that S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS), a Michael acceptor produced by a flavin-containing monooxygenase 3 (FMO3)-mediated oxidation of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. In the present study, we characterized reactions of DCVCS with nucleophilic amino acids. DCVCS incubations with N-acetyl-L-cysteine (NAC) at pH 7.4 and 37 degrees C for 1 h resulted in the formation of three monoadducts and one diadduct characterized by LC/MS, 1H NMR, and 1H-detected heteronuclear single quantum correlation. The formation of all adducts (with relative ratios of 29, 31, 24, and 12%, respectively) was rapid and time-dependent; the half-lives of the two DCVCS diastereomers in the presence of NAC were 13.8 (diastereomer I) and 9.4 min (diastereomer II). Adducts 1 and 2 were determined to be diastereomers of S-[1-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed by Michael addition of NAC to the terminal vinylic carbon of DCVCS followed by loss of HCl. Adduct 4 was determined to be S-[2-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed from the initial Michael addition product followed by a less favorable loss of HCl and/or by a rearrangement of adduct 2 through the formation of a cyclic chloronium ion. The addition of another molecule of NAC to monoadducts 1, 2, or 4 resulted in the formation of the novel diadduct, S-[2,2-( N-acetyl-L-cystein-S-yl)vinyl]-L-cysteine sulfoxide (adduct 3), whose detection in relatively large amount suggests that DCVCS could act as a cross-linking agent. DCVCS was not reactive with N-acetyl-L-lysine or L-valinamide at similar incubation conditions. Collectively, the results suggest selective reactivity of DCVCS toward protein sulfhydryl groups. Furthermore, the cross-linking properties of DCVCS may in part explain its high nephrotoxic potency.

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S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a mutagenic and nephrotoxic metabolite of trichloroethylene is bioactivated to S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS) and chlorothioketene and/or 2-chlorothionoacetyl chloride by cysteine conjugate S-oxidase (S-oxidase) and cysteine conjugate β-lyase (β-lyase), respectively. Previously, we identified DCVCS-globin monoadducts and cross-links upon treating rats with DCVCS or incubating erythrocytes with DCVCS. In this study, formation of DCVC-derived reactive intermediates was investigated after rats were given a single (230 or 460 μmol/kg, i.p.) or multiple (3 or 30 μmol/kg daily for 5 days) DCVC doses. LC/ESI/MS of trypsin digested globin peptides revealed both S-oxidase and β-lyase-derived globin monoadducts and cross-links consistent with in vivo DCVC bioactivation by both pathways. MS/MS analyses of trypsin-digested fractions of globin from one of the rats treated with multiple 30 μmol/kg DCVC doses led to identification of β-lyase-derived monoadducts on both Cys93 and Cys125 of the β chains. While rats dosed with the 230 μmol/kg DCVC dose exhibited β-lyasedependent monoadducts and cross-links only (4 out of 4 rats), rats given the 460 μmol/kg DCVC dose (2 out of 4), and rats administered the multiple DCVC doses (2 out of 4) exhibited both β-lyase and S-oxidase-derived monoadducts and cross-links. Since previous incubations of erythrocytes with DCVC did not result in detection of DCVCS-derived monoadducts or cross-links and had only resulted in detection of β-lyase-derived monoadducts and cross-links, the DCVCS-globin monoadducts and cross-links detected in this study are likely the result of DCVC bioactivation outside the circulation and subsequent translocation of DCVCS and N-acetylated DCVCS into the erythrocytes.

Previously, our laboratory has shown that hydroxymethylvinyl ketone (HMVK), a Michael acceptor oxidation product of the 1,3-butadiene metabolite, 3-butene-1,2-diol, readily reacts with hemoglobin at physiological conditions and that mass spectrometry of trypsin digested peptides suggested adduct formation with various nucleophilic amino acids. In the present study, we characterized reactions of HMVK (3 mM) with three model nucleophilic amino acids (6 and/or 15 mM): N-acetyl-L-cysteine (NAC), L-valinamide, and N-acetyl-L-lysine (NAL). NAC was the most reactive toward HMVK followed by L-valinamide and NAL. HMVK incubations with each amino acid at pH 7.4, 37°C resulted in formation of a mono-Michael adduct. In addition, HMVK incubated with NAL gave rise to two additional bis-Michael adducts characterized by LC/MS, LC/MS/MS, 1H NMR, and 1H-detected heteronuclear single quantum correlation (HSQC). The relative ratios of areas of NAL monoadduct (adduct 1) and diadducts (adducts 2 and 3) at 6 h were 49, 21, and 30% of total product area, respectively. Formation of adduct 2 was dependent upon the presence of both adduct 1 and HMVK whereas formation of adduct 3 was dependent upon presence of adduct 2 only. Monoadducts were formed by a Michael addition reaction of one HMVK moiety with nucleophilic amino acid whereas NAL diadducts were products of two Michael addition reactions of 2 HMVK moieties followed by enolization and formation of an octameric cyclic product. NAL diadduct (adduct 3) was formed by loss of a water molecule from adduct 2 followed by autooxidation of one of the hydroxy groups yielding a diketone conjugated system. Collectively, our results provide strong evidence that HMVK can react with various nucleophilic residues and form different types of adducts suggesting that a variety of proteins may be subjected to these modifications which could result in loss of protein function.