Alkylation-Induced Oxidative Cell Injury of Renal Proximal Tubular Cells: Involvement of Glutathione Redox-Cycle Inhibition

Abstract

The nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is an alkylating agent that causes oxidative stress and subsequently death of renal proximal tubular cells (PTC). In this paper the role of inhibition of the glutathione redox cycle (GSH-reductase (GRd) and -peroxidase (GPx) in the development of DCVC-induced oxidative cell injury is described. DCVC inhibited both GRd and GPx activity in PTC. Inhibition occurred already after 10 min incubation while at that time point lipid peroxidation and cell death had not started yet; the antioxidant N,N-diphenyl-p-phenylenediamine did not prevent inhibition of GRd and Gpx- inhibition of L-cysteine S-conjugate beta-lyase-mediated formation of reactive metabolites using aminooxyacetic acid, which prevented covalent binding to cellular macromolecules, was associated with prevention of the DCVC-induced inhibition of both enzymes. Covalent binding of reactive metabolites of [35S]DCVC to several cellular proteins was found, including proteins which had molecular weights identical to subunits of GPx and GRd. An inhibitor of GRd, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), potentiated the oxidative cell injury caused by DCVC, whereas BCNU itself did not use oxidative stress and cell death. The thiol-reducing compound dithiothreitol prevented the oxidative cell injury whereas oxidation of cellular thiols with diamide potentiated the DCVC-induced oxidative stress and cell death. Moreover, incubation with (R,S)-3-hydroxy-4-pentenoic acid (HPA), which depletes mitochondrial GSH, potentiated the DCVC-induced oxidative cell injury. Neither diamide nor HPA affected the covalent binding and inhibition of the GSH-redox cycle. Together, the data suggest that the inhibition of GRd and GPx, presumably caused by binding of reactive metabolites of DCVC, impairs the cellular antioxidant system, which seems causally related to DCVC-induced oxidative cell injury.