Cellular effects of reactive intermediates: Nephrotoxicity of S-conjugates of amino acids

Several cysteine S-conjugates are potent nephrotoxins and require enzymatic activation to produce cytotoxicity. Strategies based on the knowledge that renal cysteine conjugate -lyase is apparently a pyridoxal phosphate (PLP)-dependent enzyme have been exploited to test the hypothesis that a -lyase-dependent activation is required for the expression of cysteine S-conjugate-induced toxicity. First, the toxicity of the model conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is blocked both in vivo and in isolated, renal proximal tubular cells by aminooxyacetic acid, an inhibitor of PLP-dependent enzymes. Second, the nonmetabolizable -methyl analogue S-(1,2-dichlorovinyl)-DL--methylcysteine is not toxic. Third, to test the hypothesis that the toxicity of DCVC is associated with the metabolic formation of a reactive thiol, S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC), which may undergo a PLP-dependent -elimination reaction to produce an identical thiol, was studied. DCVHC is a potent nephrotoxin, and, similar to DCVC, its toxicity was blocked by aminooxyacetic acid and the -methyl analogue S-(1,2-dichlorovinyl)-DL--methylhomocysteine was not toxic. Moreover, exposure of renal proximal tubular cells to propargylglycine, a suicide substrate for PLP-dependent enzymes that catalyze -elimination reactions, blocked the toxicity of DCVHC. Fourth, the renal mitochondrial -lyase is localized in the outer membrane; therefore, although DCVC was toxic to mitochondria, no toxicity was produced in mitoplasts, which shows that a suborganelle site of activation is involved in the mitochondrial toxicity of DCVC. Finally, the toxicity of both DCVC and DCVHC was blocked by probenecid, indicating a role for the anion transport system. DCVC and DCVHC inhibit cellular and mitochondrial respiration, indicating that mitochondria are primary intracellular targets for nephrotoxic S-conjugates. Thus, the nephrotoxicity of cysteine and homocysteine S-conjugates is dependent on enzymatic activation to produce a reactive thiol, which is involved in the production of cytotoxicity.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthdayThis research was supported by National Institute of Environmental Health Sciences grant ES03127 to M. W. A.L. H. L. was supported by N. I. E. H. S. Institutional Research Service Award ES07026