The mechanism of cysteine conjugate cytotoxicity in renal epithelial cells. Covalent binding leads to thiol depletion and lipid peroxidation

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.     

Inhibition of iodoacetamide and t-butylhydroperoxide toxicity in LLC-PK1 cells by antioxidants: a role for lipid peroxidation in alkylation induced cytotoxicity

Previously we reported that thiol depletion and lipid peroxidation were associated with the cytotoxicity of nephrotoxic cysteine S-conjugates, a group of toxins which kill LLC-PK1 cells after metabolic activation and covalent binding. To determine if this is a general mechanism of cytotoxicity in these cells, we compared the effect of antioxidants, an iron chelator, and a thiol reducing agent on the toxicity of an alkylating agent, iodoacetamide (IDAM), and an organic peroxidant, t-butylhydroperoxide (TBHP). IDAM or TBHP toxicity was concentration (0.01 to 1.0 mM) and time (1 to 6 h) dependent. Both toxins caused lipid peroxidation which occurred prior to cell death as determined by leakage of lactate dehydrogenase (LDH). The alkylating agent IDAM bound to cellular macromolecules and depleted cellular non-protein thiols almost completely by 1 h, while LDH release occurred first at 2 to 3 h. The toxicity of IDAM and TBHP was inhibited by the antioxidants DPPD, BHA, BHQ, PGA, and BHT and the iron chelator deferoxamine. However, DPPD blocked TBHP- and IDAM-induced lipid peroxidation and toxicity without affecting binding and depletion of cellular nonprotein thiols. Furthermore, the thiol reducing agent dithiothreitol was able to block lipid peroxidation and toxicity. Therefore it is possible that with an alkylating agent, depletion of cellular nonprotein thiols cooperates with covalent binding and contributes to lipid peroxidation and cell death. There appear to be common elements in the toxicity of alkylating agents and organic peroxidants in LLC-PK1 cells.     

Cytotoxicity of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine in isolated rat kidney cells

S-(1,2-Dichlorovinyl)glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) produced time- and concentration-dependent cell death in isolated rat kidney proximal tubular cells. AT-125 blocked and glycylglycine potentiated DCVG toxicity, indicating that metabolism by gamma-glutamyltransferase is required. S-(1,2-Dichlorovinyl)-L-cysteinylglycine, a putative metabolite of DCVG, also produced cell death, which was prevented by 1,10-phenanthroline, phenylalanylglycine, and aminooxyacetic acid, inhibitors of aminopeptidase M, cysteinylglycine dipeptidase, and cysteine conjugate beta-lyase, respectively. Aminooxyacetic acid and probenecid protected against DCVC toxicity, indicating a role for metabolism by cysteine conjugate beta-lyase and organic anion transport, respectively. DCVC produced a small decrease in cellular glutathione concentrations and did not change cellular glutathione disulfide concentrations or initiate lipid peroxidation. DCVC caused a large decrease in cellular glutamate and ATP concentrations with a parallel decrease in the total adenine nucleotide pool; these changes were partially prevented by aminooxyacetic acid. Both DCVG and DCVC inhibited succinate-dependent oxygen consumption, but DCVC had no effect when glutamate + malate or ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine were the electron donors. DCVC inhibited mitochondrial, but not microsomal, Ca2+ sequestration. These alterations in mitochondrial function were partially prevented by inhibition of DCVG and DCVC metabolism and were strongly correlated with decreases in cell viability, indicating that mitochondria may be the primary targets of nephrotoxic cysteine S-conjugates.     

Differential cellular effects in the toxicity of haloalkene and haloalkane cysteine conjugates to rabbit renal proximal tubules

The relationship between the covalent binding, uptake, and toxicity produced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) was investigated in suspensions of rabbit renal proximal tubules (RPT). The DCVC and TFEC at concentrations of 25 μM produced a time-dependent (1–6 hours) loss of RPT viability. The TFEC was bio-transformed rapidly by β-lyase to a reactive metabolite which bound covalently to tubular protein. Approximately 63% of the TFEC-equivalents inside the cell were bound to protein. Covalent binding of TFEC-equivalents was associated with a 30% decrease in tubular basal and state 3 respiration, a sevenfold increase in lipid peroxidation, and, ultimately, cell death. The DCVC was biotransformed rapidly to a reactive metabolite which bound covalently to tubular protein. Approximately 90% of the DCVC-equivalents inside the cell were bound covalently to tubular protein. Following exposure to 25 μM DCVC, the binding of DCVC-equivalents was associated with a 17-fold increase in lipid peroxidation but, in contrast to TFEC, had no effect on tubular respiration. However, exposure of RPT to 100 μM DCVC resulted in a ninefold increase in the binding of DCVC- equivalents and a 30% decrease in tubular state 3 respiration. The β-lyase inhibitor aminooxyacetic acid (AOAA) blocked the covalent binding, mitochondrial dysfunction, lipid peroxidation, and cell death produced by TFEC. The AOAA decreased the covalent binding and the lipid peroxidation produced by DCVC by approximately 60–70% but had no effect on cell death. These results suggest that mitochondria! bioactivation of TFEC by β-lyase is critical for TFEC-induced mitochondrial dysfunction and the resulting cell death. These results also suggest that cytosolic bioactivation and binding, but not mitochondrial bioactivation and dysfunction, are important in the toxicity produced by DCVC to rabbit RPT. The lack of protection against DCVC toxicity by AOAA may be related to incomplete inhibition of DCVC metabolism or bioactivation of DCVC by pathways other than β-lyase.     

Evidence for trichloroethylene bioactivation and adduct formation in the rat epididymis and efferent ducts

Recent studies indicate that trichloroethylene (TCE) may be a male reproductive toxicant. It is metabolized by conjugation with glutathione and cytochrome p450-dependent oxidation. Reactive metabolites produced along both pathways are capable of forming protein adducts and are thought to be involved in TCE-induced liver and kidney damage. Similarly, in situ bioactivation of TCE and subsequent binding of metabolites may be one mechanism by which TCE acts as a reproductive toxicant. Cysteine-conjugate beta-lyase (beta-lyase) bioactivates the TCE metabolite dichlorovinyl cysteine (DCVC) to a reactive intermediate that is capable of binding cellular macromolecules. In the present study, Western blot analysis indicated that the soluble form of beta-lyase, but not the mitochondrial form, was present in the epididymis and efferent ducts. Both forms of beta-lyase were detected in the kidney. When rats were dosed with DCVC, no protein adducts were detected in the epididymis or efferent ducts, although adducts were present in the proximal tubule of the kidney. Trichloroethylene can also be metabolized and form protein adducts through a cytochrome p450-mediated pathway. Western blot analysis detected the presence of cytochrome p450 2E1 (CYP2E1) in the efferent ducts. Immunoreactive proteins were localized to efferent duct and corpus epididymis epithelia. Metabolism of TCE was demonstrated in vitro using microsomes prepared from untreated rats. Metabolism was inhibited 77% when efferent duct microsomes were preincubated with an antibody to CYP2E1. Dichloroacetyl adducts were detected in epididymal and efferent duct microsomes exposed in vitro to TCE. Results from the present study indicate that the cytochrome p450-dependent formation of reactive intermediates and the subsequent covalent binding of cellular proteins may be involved in the male reproductive toxicity of TCE.     

Inhibition of S-(1,2-dichlorovinyl)-L-cysteine-induced lipid peroxidation by antioxidants in rabbit renal cortical slices: Dissociation of lipid peroxidation and toxicity

Precision-cut, rabbit renal slices were used to examine the effects of three novel antioxidants (U-74006, U-74500, and U-78517) on S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced lipid peroxidation and toxicity. Slices exposed to DCVC showed a dose- and time-dependent increase in lipid peroxidation (TBARS) and a decrease in cellular viability, as evidenced by the loss of intracellular potassium, during the course of a 3 hour incubation. Subsequent studies employed DCVC concentrations of 100 μM. Microemulsion formulations of U-78517, U-74500, and U-74006 (100 μM) inhibited DCVC-induced lipid peroxidation by 100±, 50±, and <5% (not significant), respectively. However, none of these antioxidants had a significant effect on DCVC-dependent cytotoxicity, as indicated by intracellular potassium release. The effects of U-78517, the most potent of the three antioxidants, were similar to those observed with two model antioxidants, diphenyl-p-phenylenedi-amine (DPPD) and the iron chelator, deferoxamine. Aminooxyacetic (AOAA), an inhibitor of renal cysteine conjugate β-lyase, had only a minimal effect on DCVC-induced lipid peroxidation, and no effect on toxicity. These data represent the first report of DCVC-induced lipid peroxidation in rabbit renal cortical slices, a system which has been widely used to investigate mechanisms of nephrotoxicity, including that induced by DCVC. Our results demonstrate that DCVC-induced lipid peroxidation in renal slices can be inhibited by a variety of antioxidant compounds operating by different mechanisms. Because inhibition of lipid peroxidation had minimal effect on DCVC-dependent cytotoxicity, the data suggest that DCVC-induced lipid peroxidation is not a major mechanism in the cytotoxicity induced by this compound.     

Glutathione depleting agents and lipid peroxidation

The mechanisms by which glutathione (GSH) depleting agents produce cellular injury, particularly liver cell injury have been reviewed. Among the model molecules most thoroughly investigated are bromobenzene and acetaminophen. The metabolism of these compounds leads to the formation of electrophilic reactants that easily conjugate with GSH. After substantial depletion of GSH, covalent binding of reactive metabolites to cellular macromolecules occurs. When the hepatic GSH depletion reaches a threshold level, lipid peroxidation develops and severe cellular damage is produced. According to experimental evidence, the cell death seems to be more strictly related to lipid peroxidation rather than to covalent binding. Loss of protein sulfhydryl groups may be an important factor in the disturbance of calcium homeostasis which, according to several authors, leads to irreversible cell injury. In the bromobenzene-induced liver injury loss of protein thiols as well as impairment of mitochondrial and microsomal Ca2+ sequestration activities are related to lipid peroxidation. However, some redox active compounds such as menadione and t-butylhydroperoxide produce direct oxidation of protein thiols.     

S-(1,2-dichlorovinyl)-L-homocysteine-induced cytotoxicity in isolated rat kidney cells

Incubation of isolated, rat kidney cells with S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) caused time-dependent cell death. Cytotoxicity of DCVHC was potentiated by addition of alpha-ketobutyrate, indicating the involvement of pyridoxal phosphate-dependent enzymes. A second addition of DCVHC to cells produced increased cytotoxicity, indicating that the bioactivating ability is not lost after exposure to the conjugate. DCVHC decreased cellular glutathione concentrations by 52% and substantially inhibited glutathione biosynthesis from precursors. In contrast, the cysteine analog S-(1,2-dichlorovinyl)-L-cysteine (DCVC) failed to decrease cellular glutathione concentrations and only partially inhibited glutathione biosynthesis. As with DCVC, DCVHC did not increase cellular glutathione disulfide concentrations and did not initiate lipid peroxidation, indicating that it does not produce an oxidative stress. DCVHC and DCVC produced similar alterations in mitochondrial function: Cellular ATP concentrations were decreased by 57% and cellular ADP and AMP concentrations were increased twofold, thereby decreasing the ATP/ADP ratio from 2.8 to 0.6 and the cellular energy charge from 0.80 to 0.56; DCVHC was a potent inhibitor of succinate-dependent oxygen consumption, but had little effect on respiration linked to oxidation of glutamate + malate or ascorbate + N,N,N'N'-tetramethyl-p-phenylenediamine. DCVHC was a potent inhibitor of mitochondrial Ca2+ sequestration and, in contrast to DCVC, also inhibited microsomal Ca2+ sequestration. These DCVHC-induced alterations in cellular metabolism were prevented by addition of propargylglycine or aminooxyacetic acid, and the alpha-methyl analog S-(1,2-dichlorovinyl)-DL-alpha-methylhomocysteine was not toxic. These results support a role for pyridoxal phosphate-dependent bioactivation of DCVHC and indicate that the greater nephrotoxic potency of DCVHC as compared to DCVC is partially due to the presence of both mitochondrial and extramitochondrial targets for DCVHC.     

Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia.     

Mutagenicity of amino acid and glutathione S-conjugates in the Ames test

The mutagenicity of the glutathione S-conjugate S-(1,2-dichlorovinyl)glutathione (DCVG), the cysteine conjugates S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2-dichlorovinyl)-DL-alpha-methylcysteine (DCVMC), and the homocysteine conjugates S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) and S-(1,2-dichlorovinyl)-DL-alpha-methylhomocysteine (DCVMHC) was investigated in Salmonella typhimurium strain TA2638 with the preincubation assay. DCVC was a strong, direct-acting mutagen; the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid decreased significantly the number of revertants induced by DCVC; rat renal mitochondria (11,000 X g pellet) and cytosol (105,000 X g supernatant) with high beta-lyase activity increased DCVC mutagenicity at high DCVC concentrations. DCVG was also mutagenic without the addition of mammalian activating enzymes; the presence of low gamma-glutamyltransferase activity in bacteria, the reduction of DCVG mutagenicity by aminooxyacetic acid, and the potentiation of DCVG mutagenicity by rat kidney mitochondria and microsomes (105,000 X g pellet) with high gamma-glutamyltransferase activity indicate that gamma-glutamyltransferase and beta-lyase participate in the metabolism of DCVG to mutagenic intermediates. The homocysteine conjugate DCVHC was only weakly mutagenic in the presence of rat renal cytosol, which exhibits considerable gamma-lyase activity, this mutagenic effect was also inhibited by aminooxyacetic acid. The conjugates DCVMC and DCVMHC, which are not metabolized to reactive intermediates, were not mutagenic at concentrations up to 1 mumole/plate. The results demonstrate that gamma-glutamyltransferase and beta-lyase are the key enzymes in the biotransformation of cysteine and glutathione conjugates to reactive intermediates that interact with DNA and thereby cause mutagenicity.     

Expression of the pro-apoptotic gene gadd153/chop is elevated in liver with aging and sensitizes cells to oxidant injury

Aging is generally accompanied by reduced tolerance to oxidative stress and altered responsiveness to proliferative signals. We have shown that hepatocytes derived from aged rats (24-26 months) exhibit greater sensitivity to H(2)O(2) treatment and reduced proliferation following epidermal growth factor (EGF) treatment than cells of young adult rats (5-6 months). Here we examined the effects of aging and calorie restriction (CR) on expression of the oxidative stress-inducible and pro-apoptotic gene gadd153 (chop) in these hepatocytes, and we investigated its influence on sensitivity to oxidants. We show that aging was associated with elevated expression of gadd153, both basally and in response to H(2)O(2) treatment. CR, which attenuates age-associated declines in stress tolerance, prevented the age-related increase in gadd153 expression. EGF treatment also resulted in gadd153 induction in old cells. This effect was absent in young cells and in old cells of CR rats. gadd153 induction by EGF was reactive oxygen species-dependent and correlated with heightened sensitivity to subsequent H(2)O(2) treatment, suggesting that elevated Gadd153 contributes to the greater sensitivity of EGF-pretreated old cells to oxidative stress. Additional support for this hypothesis was provided by experiments with Rat1 fibroblasts in which conditional expression of Gadd153 conferred increased sensitivity to H(2)O(2). We propose a model whereby the diminished ability of old hepatocytes to overcome an EGF-triggered reactive oxygen species load leads to induction of the proapoptotic gene gadd153, which, in turn, sensitizes the cells to oxidant injury. Our findings point to gadd153 expression levels as an important factor in liver aging.     

The role of glutathione conjugate metabolism and cysteine conjugate beta-lyase in the mechanism of S-cysteine conjugate toxicity in LLC-PK1 cells

A cell line derived from pig kidney, LLC-PK1, was grown in a culture system in which the cells express morphological and biochemical characteristics of the proximal tubule. This model was used to investigate the mechanism of S-cysteine conjugate toxicity and the role of glutathione conjugate metabolism. LLC-PK1 cells have the degradative enzymes of the mercapturate pathway, and S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-glutathione are toxic. S-(1,2-Dichlorovinyl)-L-glutathione is not toxic when the cells are pretreated with AT-125, an inhibitor of gamma-glutamyl transpeptidase. The cells respond to a variety of toxic cysteine conjugates. Cysteine conjugate beta-lyase activity is not detectable by standard assays, but can be measured using radiolabeled S-(1,2-dichlorovinyl)-L-cysteine. Pyruvate stimulates the beta-elimination reaction with S-(1,2-dichlorovinyl)-L-cysteine as substrate 2-3-fold. The data suggest that a side transamination reaction regulates the flux of substrate through the beta-elimination pathway; therefore, cysteine conjugate beta-lyase in LLC-PK1 cells may be regulated by transamination, and measurement of lyase activity in some systems may require the presence of alpha-ketoacids. Aminoxyacetic acid blocks both the metabolism of S-(1,2-dichlorovinyl)-L-cysteine to a reactive species which covalently binds to cellular macromolecules and toxicity. Glutathione inhibits the binding of the sulfur containing cleavage fragment to acid insoluble material in vitro. The data provide direct evidence that S-(1,2-dichlorovinyl)-L-cysteine is metabolized to a reactive species which covalently binds to cellular macromolecules, and the binding is proportional to toxicity.