Renal cysteine S-conjugate beta-lyase-mediated cytotoxicity and genotoxicity of haloalkenes

Abstract

Haloalkenes, such as hexachlorobutadiene, are selectively nephrocytotoxic through a metabolic bioactivation pathway involving glutathione conjugation, metabolism to the cysteine conjugate or mercapturic acid analogue, translocation to the kidney, and metabolism by the enzyme renal cysteine conjugate {dollar}\beta{dollar}-lyase, to a reactive intermediate. Chlorinated alkenes are also mutagenic in the Ames assay, and weakly renal carcinogenic in mammalian in vitro and in vivo test systems. This dissertation demonstrates the relevance of haloalkene cytotoxicity to human health, an issue which had not been previously documented, and investigates the poorly understood mechanism through which haloalkenes induce genotoxicity. To document the relevance to human health, primary human renal proximal tubular cell cultures were exposed to cysteine and glutathione conjugates over a wide range of doses. Time- and dose-dependent {dollar}\beta{dollar}-lyase-mediated conjugate toxicity was observed in human renal cell cultures in association with macromolecular covalent binding. Two approaches were used to evaluate the genotoxic potential of haloalkenyl cysteine conjugates in primary cultures of rat proximal tubule cells. When primary rat proximal tubule cell cultures were dosed with cysteine conjugates over a wide range of times and concentrations, no significant unscheduled DNA synthesis was observed. However, DNA single (SSB) and double strand breaks, as measured by alkaline and neutral elution, in rat renal cell cultures were induced through {dollar}\beta{dollar}-lyase bioactivation of cysteine conjugates in a time- and dose-dependent manner. The degree of SSB's closely correlated with the degree of cytotoxicity, as measured by lactate dehydrogenase release. This was true for both chlorinated and fluorinated conjugates (previously not shown to be mutagenic in prokaryotic cell systems). Calcium chelation or the presence of the antioxidant, N,N{dollar}\sp\prime{dollar}-diphenyl-p-phenylenediamine, inhibited cytotoxicity and SSB's without affecting macromolecular binding. These data suggest that secondary processes such as cytotoxicity-induced lipid peroxidation leads to SSB's in injured and/or neighboring cells. We conclude that haloalkenyl conjugates are cytotoxic to human renal cells and that conjugates induce carcinogenesis through a largely nongenotoxic mechanism closely associated with cytotoxicity, involving Ca2+ deregulation, oxidative stress and intracellular activation and/or release of secondary factors that mediate DNA damage.