Cysteine Conjugate β-Lyase Activity of Rat Erythrocytes and Formation of β-Lyase-Derived Globin Monoadducts and Cross-Links after in Vitro Exposure of Erythrocytes to S-(1,2-Dichlorovinyl)-l-cysteine

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 β-lyase (β-lyase), respectively. Previously, we characterized reactivity of DCVCS with Hb upon incubation of erythrocytes with DCVCS and provided evidence for formation of distinct DCVCS-Hb monoadducts and cross-links both in isolated erythrocytes and in 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 μM) at 37 °C, pH 7.4. The results suggested no DCVCS monoadducts or cross-links were formed; however, LC/ESI/MS and MALDI/MS of trypsin digested globin peptides revealed presence of β-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 the observed peptide selectivity of the β-lyase–derived reactive sulfur/oxygen-containing species was different than that previously observed with DCVCS. While these results suggested erythrocytes have β-lyase but not S-oxidase activity, further support for this hypothesis was obtained using S-(2-benzothiazolyl)-L-cysteine, an alternative substrate for β-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.

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

“…Although we were unable to obtain MS/MS fragmentation of peptides modified by the S-oxidase-derived intermediates, we obtained conclusive evidence for the presence of the β-lyase-derived monoadduct type 3 (oxygen-containing intermediate) on Cys93 and Cys125 of β chains in one of the rats treated subacutely with 30 μmol/kg DCVC. Similar to DCVCS, cross-linking between globin chains was more prevalent than formation of monoadducts at all dosing regimens consistent with our in vitro results when RBCs were incubated with 9 μM DCVC (33). Involvement of nucleophilic residues other than cysteines, (i. e. lysines, histidines, methionines, or arginines) is evident because of the presence of multiple monoadducts on the same peptides suggesting less selectivity than DCVCS which is only reactive with cysteines (19, 40).…”

“…Previously, we established the presence of β-lyase activity in RBCs (33). Because RBCs represent an additional compartment for DCVC metabolism via the β-lyase pathway, the detected adducts with chlorothioketene/chloroketene and 2-chlorothionoacetyl chloride/chloroacetyl chloride could be due to bioactivation of DCVC within RBCs or outside of the circulation.…”

“…Mass Spectral Analyses of Tryptic Peptides. The analyses of the digest for the specific monoadducts and cross-links shown in Figure 1 were performed as described previously (32,33). The search for modified cysteine-containing peptides (including those with one and two missed cleavages) included the following DCVCS-derived monoadducts: DCVCS (+194.9757 Da, addition of DCVCS moiety and loss of HCl), DCVCS-GSH (+466.0828 Da, addition of DCVCS-GSH conjugate and loss of two HCl), N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (NA-DCVCS) (+236.9862 Da, addition of NA-DCVCS and loss of HCl), and NA-DCVCS-GSH (+508.0933 Da, addition of NA-DCVCS-GSH conjugate and loss of two HCl).…”

“…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. In the present study, we used mass spectrometry techniques to analyze trypsin digested globin peptides for monoadducts and cross-links due to S-oxidase and β-lyase-derived reactive metabolites after single or multiple dosing of rats with DCVC.…”

Role of reactive metabolites in the circulation in extrahepatic toxicity

“…Given that no S-oxidase activity was detectable in red blood cells [85], the results from these studies provide in vivo evidence for bioactivation of DCVC to DCVCS and formation of N-AcDCVCS outside of the circulation. The high reactivity of DCVCS and N-AcDCVCS toward sulfhydryl groups suggests that when excreted from the liver into the circulation, a fraction is likely to be present in the form of a GSH conjugate, either due to spontaneous GSH conjugation or GSH S-transferase-mediated conjugation.…”