Introduction Reactive metabolite-mediated toxicity is frequently limited to the organ where the electrophilic metabolites are generated. Some reactive metabolites however, might have the ability to translocate from their site of formation. This suggests that for these reactive metabolites, investigations into the role of organs other than the one directly affected could be relevant to understanding the mechanism of toxicity. Areas covered The authors discuss the physiological and biochemical factors that can enable reactive metabolites to cause toxicity in an organ distal from the site of generation. Furthermore, the authors present a case study which describes studies that demonstrate that S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS) and N-acetyl-S-(1,2-dichlorovinyl-L-cysteine sulfoxide (N-AcDCVCS), reactive metabolites of the known trichloroethylene metabolites S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (N-AcDCVC), are generated in the liver and translocate through the circulation to the kidney to cause nephrotoxicity. Expert Opinion The ability of reactive metabolites to translocate could be important to consider when investigating mechanisms of toxicity. A mechanistic approach, similar to the one described for DCVCS and N-AcDCVCS, could be useful in determining the role of circulating reactive metabolites in extrahepatic toxicity of drugs and other chemicals. If this is the case, intervention strategies that would not otherwise be feasible might be effective for reducing extrahepatic toxicity.

The nephrotoxicity and nephrocarcinogenicity of trichloroethylene (TCE) and tetrachloroethylene (PCE) are believed to be mediated primarily through the cysteine S-conjugate β-lyase-dependent bioactivation of the corresponding cysteine S-conjugate metabolites S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), respectively. DCVC and TCVC have previously been demonstrated to be mutagenic by the Ames Salmonella mutagenicity assay, and reduction in mutagenicity was observed upon treatment with the β-lyase inhibitor aminooxyacetic acid (AOAA). Because DCVC and TCVC can also be bioactivated through sulfoxidation to yield the potent nephrotoxicants S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS) and S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide (TCVCS), respectively, the mutagenic potential of these two sulfoxides was investigated using the Ames S. typhimuriumTA100 mutagenicity assay. The results show both DCVCS and TCVCS were mutagenic, and TCVCS exhibited 3-fold higher mutagenicity than DCVCS. However, DCVCS and TCVCS mutagenic activity was approximately 700-fold and 30-fold lower than DCVC and TCVC, respectively. DCVC and DCVCS appeared to induce toxicity in TA100, as evidenced by increased microcolony formation and decreased mutant frequency above threshold concentrations. TCVC and TCVCS were not toxic in TA100. The toxic effects of DCVC limited the sensitivity of TA100 to DCVC mutagenic effects and rendered it difficult to investigate the effects of AOAA on DCVC mutagenic activity. Collectively, these results suggest that DCVCS and TCVCS exerted a definite but weak mutagenicity in the TA100 strain. Therefore, despite their potent nephrotoxicity, DCVCS and TCVCS are not likely to play a major role in DCVC or TCVC mutagenicity in this strain.

N-Acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NA-DCVC) has been detected in the urine of humans exposed to trichloroethylene and its related sulfoxide, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (NA-DCVCS), has been detected as hemoglobin adducts in blood of rats dosed with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) or S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS). Because the in vivo nephrotoxicity of NA-DCVCS was unknown, in this study, male Sprague-Dawley rats were dosed (i.p.) with 230 µmol/kg b.w. NA-DCVCS or its potential precursors, DCVCS or NA-DCVC. At 24 h post treatment, rats given NA-DCVC or NA-DCVCS exhibited kidney lesions and effects on renal function distinct from those caused by DCVCS. NA-DCVC and NA-DCVCS primarily affected the cortico-medullary proximal tubules (S2–S3 segments) while DCVCS primarily affected the outer cortical proximal tubules (S1–S2 segments). When NA-DCVCS or DCVCS was incubated with GSH in phosphate buffer pH 7.4 at 37°C, the corresponding glutathione conjugates were detected, but NA-DCVC was not reactive with GSH. Because NA-DCVCS exhibited a longer half-life than DCVCS and addition of rat liver cytosol enhanced GSH conjugate formation, catalysis of GSH conjugate formation by the liver could explain the lower toxicity of NA-DCVCS in comparison with DCVCS. Collectively, these results provide clear evidence that NA-DCVCS formation could play a significant role in DCVC, NA-DCVC, and trichloroethylene nephrotoxicity. They also suggest a role for hepatic metabolism in the mechanism of NA-DCVC nephrotoxicity.

S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide (DCVCS) is a reactive and potent nephrotoxic metabolite of the human trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC). Because DCVCS covalent binding to kidney proteins likely plays a role in its nephrotoxicity, in this study biotin-tagged DCVCS, N-Biotinyl-DCVCS (NB-DCVCS), was synthesized and its stability in buffer alone and in the presence of rat blood or plasma was characterized in vitro. In addition, reactivity toward GSH and covalent binding to selected model enzymes and isolated kidney proteins were characterized. The half-lives of NB-DCVCS (39.6 min) and the DCVCS (diastereomer 1: 14.4 min, diastereomer 2: 6 min) in the presence of GSH were comparable. Incubating the model enzymes glutathione reductase and malate dehydrogenase with 10 µM NB-DCVCS for 3 h at 37°C followed by immunoblotting using anti-biotin antibodies demonstrated that glutathione reductase and malate dehydrogenase were extensively modified by NB-DCVCS. When rat kidney cytosol (6 µg/µL) was incubated with NB-DCVCS (312.5 nM to 5 µM) for 3 h at 37°C followed by immunoblotting, a concentration-dependent increase in signal with multiple proteins with different molecular weights was observed, suggesting NB-DCVCS binds to multiple kidney proteins with different selectivity. Incubating rat kidney cytosol with DCVCS (10 – 100 µM) prior to addition of NB-DCVCS (2.5 µM) reduced the immunoblotting signal, suggesting that NB-DCVCS and DCVCS compete for the same binding sites. A comparison of the stability of NB-DCVCS and DCVCS in rat blood and plasma was determined in vitro and NB-DCVCS exhibited higher stability than DCVCS in both media. Collectively, these results suggest NB-DCVCS shows sufficient stability, reactivity, and selectivity to warrant further investigations into its possible use as a tool for future characterization of the role of covalent modification of renal proteins by DCVCS in nephrotoxicity.