title: The mechanism of cysteine conjugate cytotoxicity in renal epithelial cells. Covalent binding leads to thiol depletion and lipid peroxidation.
abstract: Nephrotoxic cysteine conjugates kill cells after they are metabolized by the enzyme cysteine conjugate beta-lyase to reactive fragments which bind to cellular macromolecules. We have investigated the cellular events which occur after the binding and lead ultimately to cell death in renal epithelial cells. Using S-(1,2-dichlorovinyl)-L-cysteine (DCVC) as a model conjugate, we found that the phenolic antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD), butylated hydroxyanisole, butylated hydroxytoluene, propyl galate, and butylated hydroxyquinone, and the iron chelator deferoxamine inhibited the cytotoxicity significantly. Among the five antioxidants, DPPD was most potent. DPPD blocked DCVC toxicity over an extended time period, and the rescued cells remained functional as measured by protein synthetic activity. DPPD was able to block the toxicity of two other toxic cysteine conjugates S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. In addition to LLC-PK1 cells, DPPD also protected freshly isolated rat kidney epithelial cells in suspension and in primary culture. In suspension cells, DPPD was effective at low doses of DCVC (25-50 microM) but not at high concentrations (250-500 microM). DPPD inhibition was not due to an inactivation of beta-lyase or a decrease in the binding of [35S]DCVC metabolites to cellular macromolecules and occurred at a step after the activation of the toxins. During DCVC treatment, lipid peroxidation products were detectable prior to cell death. DPPD blocked lipid peroxidation over the whole time course. Depletion of nonprotein thiols also occurred prior to cell death. DPPD did not prevent the loss of nonprotein thiols. However, the sulfhydryl-reducing agent DTT blocked lipid peroxidation and toxicity at a step after the activation of DCVC. Therefore, it appears that cysteine conjugates kill renal epithelial cells by a combination of covalent binding, depletion of nonprotein thiols, and lipid peroxidation.
authors: W. Alton Jones Cell Science Center, Lake Placid, New York 12946.
authors: Q
authors: Q
authors: Chen
authors: T W
authors: TW
authors: Jones
authors: P C
authors: PC
authors: Brown
authors: J L
authors: JL
authors: Stevens
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D013437:Carbon-Sulfur Lyases; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D003676:Deferoxamine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008054:Lipid Peroxides / Q000378:metabolism*; D008190:Lyases / Q000037:antagonists & inhibitors; D010655:Phenylenediamines / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D008054:Lipid Peroxides; D010655:Phenylenediamines; D013438:Sulfhydryl Compounds; C039961:S-(1,2-dichlorovinyl)cysteine; C004845:N,N'-diphenyl-4-phenylenediamine; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C019969:S-alkylcysteine lyase; D003676:Deferoxamine; D003545:Cysteine; D004229:Dithiothreitol