Renal cysteine conjugate beta-lyase-mediated toxicity studied with primary cultures of human proximal tubular cells

Abstract

The beta-lyase pathway has been shown to mediate the nephrotoxicity of S-cysteine conjugates of a variety of haloalkenes in a number of animal models in vitro and in vivo. However, there is no information available concerning this mechanism of bioactivation in human tissues. In this investigation a well-characterized model of human proximal tubule epithelial cells, the presumed target cell, was used to investigate the toxicity of a series of glutathione and cysteine conjugates of nephrotoxic haloalkenes. Both S-(1,2-dichlorovinyl)-glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) caused dose-dependent toxicity over a range of 25 to 500 microM. DCVC was consistently found to be more toxic than DCVG, but the inclusion of gamma-glutamyltransferase (0.5 U/ml) increased the toxicity of DCVG to that observed with an equimolar concentration of DCVC, indicating that metabolism to the cysteine conjugate is an important rate-limiting step in this in vitro model. S-(1,2,3,4,4-Pentachlorobutadienyl)-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine were also found to be toxic to human proximal tubular cells. Incubation with [35S]DCVC resulted in covalent binding of 35S-label, which increased linearly to a final level of 1.05 nmol/mg protein at 6 hr. Aminooxyacetic acid (250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as beta-lyase, protected the cells from the toxicity of all of the cysteine conjugates and inhibited the covalent binding of 35S-label from [35S]DCVC to cellular macromolecules. The results of the present study provide the first evidence that human proximal tubular cells are sensitive to the toxicity of glutathione and/or cysteine conjugates of a variety of chloro- and fluoroalkenes which are activated via the beta-lyase pathway. The implications for human health are discussed.