N-Biotinyl-S-(1,2-dichlorovinyl)-l-cysteine Sulfoxide as a Potential Model for S-(1,2-Dichlorovinyl)-l-cysteine Sulfoxide: Characterization of Stability and Reactivity with Glutathione and Kidney Proteins in Vitro

Abstract

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.

Characterization of the chemical reactivity and nephrotoxicity of N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine sulfoxide, a potential reactive metabolite of trichloroethylene

“…Rat liver cytosol was prepared from livers of male Sprague Dawley rats (180 – 225 g) using a modified version of the method previously used to prepare rat kidney cytosol (Irving et al, 2011). GSH and other small molecules were removed by filtering cytosol (500 µL) with centrifugal filters three times, adding 500 µL phosphate buffer pH 7.4 after the first two filtration steps.…”

Toxicity mechanism-based prodrugs: glutathione-dependent bioactivation as a strategy for anticancer prodrug design

“…The conjugated system is structurally similar to α,β-unsaturated aldehyde/ketone (C=C-C=O) and thus is a Michael acceptor. Indeed, DCVCS can rapidly react with GSH under physiological conditions [43,46], and can readily modify and crosslink proteins [39,47,48].…”