Activation of the growth arrest and DNA damage-inducible gene gadd 153 by nephrotoxic cysteine conjugates and dithiothreitol.

The cellular and biochemical events which transduce chemical insults into signals for increased expression of the stress-responsive gene gadd 153 were investigated using nephrotoxic cysteine conjugates. In LLC-PK1 cells, cysteine conjugate toxicity is initiated by covalent binding, but depletion of cellular thiols, an increase in cytosolic free calcium, and lipid peroxidation couple the binding to cell death (Chen, Q., Jones, T. W., Brown, P. C., and Stevens, J. L. (1990) J. Biol. Chem. 265, 21603-21611; Chen, Q., Jones, T. W., and Stevens, J. L. (1991) Toxicologist 11, 101, 1991). Three different toxic cysteine conjugates induced gadd 153 mRNA. With S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the induction was both concentration and time-dependent. Preventing the metabolism of DCVC and covalent binding of DCVC-derived reactive metabolites to cellular macromolecules with the beta-lyase inhibitor (aminooxy)acetic acid blocked the induction. However, buffering free calcium with a cell permeable calcium chelator or blocking lipid peroxidation with an antioxidant did not affect the induction of gadd 153 mRNA by DCVC even though these treatments inhibit toxicity. These data suggest that covalent binding of reactive metabolites to cellular macromolecules may serve as a primary signal for the induction of gadd 153 mRNA by nephrotoxic cysteine conjugates. Interestingly, the sulfhydryl agent dithiothreitol, which was nontoxic and prevented the toxicity of DCVC, also induced an increase in gadd 153 mRNA. When both dithiothreitol and DCVC were added to cells, there were no inhibitory or additive effects on expression. Therefore, cellular thiol-disulfide status may also play a role in gadd 153 induction.     

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.     

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.     

Assessment of unscheduled DNA synthesis in a cultured line of renal epithelial cells exposed to cysteine S-conjugates of haloalkenes and haloalkanes

The ability of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFEC) and S-(2-chloroethyl)-L-cysteine (CEC) to induce DNA repair was investigated in LLC-PK1, a cultured line of porcine kidney tubular epithelial cells. DNA repair due to exposure of the cells to the S-conjugates was determined as unscheduled DNA synthesis (UDS) after inhibition of replicative DNA synthesis in confluent LLC-PK1 monolayers. DCVC, TCVC and PCBC induced dose-dependent UDS in LLC-PK1 at concentrations which did not impair the viability of the cells compared to untreated controls; higher concentrations were cytotoxic, resulting in lactate dehydrogenase leakage into the medium. Cell death was also induced by CTFEC, which failed to exert genotoxicity. CEC induced the highest response among these cysteine conjugates without impairing cell viability. Inhibition of cysteine conjugate beta-lyase with aminooxyacetic acid abolished the effects of DCVC, TCVC, PCBC and CTFEC but did not influence the genotoxicity of CEC.     

Renal cysteine conjugate beta-lyase. Bioactivation of nephrotoxic cysteine S-conjugates in mitochondrial outer membrane

Cysteine conjugate beta-lyase activity from rat kidney cortex was found in the cystosolic and mitochondrial fractions. With 2 mM S-(2-benzothiazolyl)-L-cysteine as the substrate, approximately two-thirds of the total beta-lyase activity was present in the cytosolic fraction. The kinetics of beta-lyase activity with three cysteine S-conjugates were different in the cytosolic and mitochondrial fractions, and the mitochondrial beta-lyase was much more sensitive to inhibition by aminooxyacetic acid than was the cytosolic activity. These results indicate that the beta-lyase activities in the two subcellular fractions are catalyzed by distinct enzymes. Nephrotoxic cysteine S-conjugates of halogenated hydrocarbons that require bioactivation by cysteine conjugate beta-lyase (S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, CTFC) were potent inhibitors of state 3 respiration in rat kidney mitochondria. Fractionation of mitochondria by digitonin treatment and comparison with marker enzyme distributions showed that the mitochondrial beta-lyase activity is localized in the outer mitochondrial membrane. Inhibition of the beta-lyase prevented the mitochondrial toxicity of DCVC and CTFC, and nonmetabolizable, alpha-methyl analogues of DCVC and CTFC were not toxic. Neither DCVC nor CTFC was toxic to mitoplasts, indicating that activation by the beta-lyase occurs on the outer membrane and may be essential for the expression of toxicity; in contrast, the direct acting nephrotoxin S-(2-chloroethyl)-DL-cysteine was toxic to both mitochondria and mitoplasts. Thus, the suborganelle localization of DCVC and CTFC bioactivation correlates with the observed pattern of toxicity.     

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.     

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.     

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.     

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.     

Cytotoxicity of cysteine S-conjugates: structure-activity relationships

The cytotoxicity of cysteine S-conjugates was investigated in freshly isolated rat renal proximal tubule cells. The study was designed to determine the contribution of the thiols and of the acylating intermediates formed by cysteine conjugate beta-lyase to the initiation of cytotoxicity. Cell viability was determined by trypan blue exclusion and by lactate dehydrogenase leakage. The S-conjugates S-(1,2,2-trichlorovinyl)-L-cysteine, S-(1,2,3,3,3-pentachloro-prop-1-enyl)-L-cysteine and S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine, at a concentration of 0.2 mM, reduced cell viability compared to controls from 85% to less than 50% after 3 h. The alpha-chlorinated enethiols formed from these S-conjugates are transformed to acylating intermediates. The S-conjugate S-(2-chlorovinyl)-L-cysteine forms an enethiol, which cannot transform to an acylating intermediate and did not reduce cell viability at 0.2 mM; at 1 mM, it resulted in a very slight reduction of cell viability after 3 h. S-(pentachlorophenyl)-L-cysteine and S-benzyl-L-cysteine, which form stable thiols after metabolism by beta-lyase, were not cytotoxic at a concentration of 1 mM. The direct acting S-(2-chloroethyl)-L-cysteine (0.2 mM) reduced cell viability after 3 h from 85% to 90% (control) to 40%. The results obtained suggest that reactions of the initial thiol-metabolites with biological macromolecules do not contribute to the induction of cytotoxicity by cysteine S-conjugates and indicate that acylating intermediates formed by cysteine conjugate R-lyase induce cytotoxic effects by non-selective acylation of cellular macromolecules.     

Bacterial cysteine conjugate beta-lyase and the metabolism of cysteine S-conjugates: structural requirements for the cleavage of S-conjugates and the formation of reactive intermediates

The cysteine conjugate beta-lyase mediated metabolism and the mutagenicity of the synthetic cysteine conjugates S-(2-chloroethyl)-L-cysteine (CEC), S-(2-chlorovinyl)-L-cysteine (CVC), S-(1,2,3,3,3-pentachloroprop-1-enyl)-L-cysteine (PCPC), S-(pentachlorophenyl)-L-cysteine (PCPhC), S-(chloro-1,2,2-trifluoroethyl)-L-cysteine (CTFEC), S-benzyl-L-cysteine (SBC) and S-methyl-L-cysteine (SMC) were investigated in Salmonella typhimurium strains TA100, TA2638, TA102 and TA98 to establish structure/activity relationships. Bacterial 100,000 X g supernatants cleaved CTFEC, PCPC, CVC, PCPhC and SBC to pyruvate; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA) in all cases. Of the compounds tested, CEC, PCPC and CVC were mutagenic in the Ames-test. CTFEC, PCPhC and SBC failed to increase the number of revertants above control levels. The mutagenicity of PCPC and CVC could be inhibited by AOAA. CEC exerted a potent mutagenic effect in the Ames-test which was not affected by AOAA; CEC was not transformed to pyruvate by bacterial beta-lyase. Neither pyruvate formation nor mutagenicity were observed with SMC. These results indicate that the structure of the substituent on the sulfur atom is an important determinant for the biological activity of cysteine S-conjugates. Electronegative and/or unsaturated substituents are required for beta-lyase catalysed beta-elimination reactions. The formation of chemically unstable thiols, which may be converted to thioacylating intermediates, seems to be a prerequisite for beta-lyase dependent mutagenicity of S-conjugates.     

Cysteine conjugate beta-lyase activities in rat kidney cortex: subcellular localization and relationship to the hepatic enzyme

Kidney cortex cysteine conjugate beta-lyase enzymes were characterized using S-(2-benzothiazolyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-cysteine as substrates. The contribution of the hepatic form of cysteine conjugate beta-lyase to renal metabolism of these S-cysteine conjugates is not substantial. No cysteine conjugate beta-lyase activity was found in kidney cortex brush border membrane vesicles. Two cysteine conjugate beta-lyase activities with densities corresponding to the mitochondrial and soluble fractions were separated on Percoll gradients.     

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.     

Bacterial beta-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene

The metabolism of beta-lyase and the mutagenicity of the synthetic cysteine conjugates S-1,2-dichlorovinylcysteine (DCVC), S-1,2,2-trichlorovinylcysteine (TCVC), S-1,2,3,4,4-pentachlorobuta-1,3-dienylcysteine (PCBC) and S-3-chloropropenylcysteine (CPC) were investigated in Salmonella typhimurium strains TA100, TA2638 and TA98. The bacteria contained significantly higher concentrations of beta-lyase than mammalian subcellular fractions. Bacterial 100,000 X g supernatants cleaved benzthiazolylcysteine to equimolar amounts of mercaptobenzthiazole and pyruvate. DCVC, TCVC and PCBC produced a linear time-dependent increase in pyruvate formation when incubated with bacterial 100,000 X g supernatants; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA). CPC was not cleaved by bacterial enzymes to pyruvate. DCVC, TCVC and PCBC were mutagenic in three strains of S. typhimurium (TA100, TA2638 and TA98) in the Ames-test without addition of mammalian subcellular fractions; their mutagenicity was decreased by the addition of AOAA to the preincubation mixture. CPC was not mutagenic in any of the strains of bacteria tested. These results indicate that beta-lyase plays a key role in the metabolism and mutagenicity of haloalkenylcysteines when tested in S. typhimurium systems. The demonstrated formation in mammals of the mutagens DCVC, TCVC and PCBC during biotransformation of trichloroethylene (Tri), tetrachloroethylene (Tetra) and hexachlorobutadiene (HCBD) may provide a molecular explanation for the nephrocarcinogenicity of these compounds.     

Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains.

An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma-methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS)     

A purified cysteine conjugate beta-lyase from rat kidney cytosol. Requirement for an alpha-keto acid or an amino acid oxidase for activity and identity with soluble glutamine transaminase K

Cysteine conjugate beta-lyase has been purified from rat kidney cytosol. The enzyme is a 100,000-dalton dimer of two 55,000-dalton subunits and has an absorption maximum at 432 nm. The enzyme has phenylalanine alpha-keto-gamma-methiolbutyrate transaminase activity and appears to be identical to rat kidney cytosolic glutamine transaminase K. Metabolism of S-1,2-dichlorovinyl-L-cysteine (DCVC) by the purified enzyme was dependent on the presence of either alpha-keto-gamma-methiolbutyrate or a protein factor which is present in the cytosolic fraction of rat kidney cortex. The protein factor was identified as a flavin containing L-amino acid oxidase which oxidized DCVC to S-(1,2-dichlorovinyl)-3-mercapto-2-oxopropionic acid. S-(1,2-Dichlorovinyl)-3-mercapto-2-oxopropionic acid has not been previously reported as a metabolite of DCVC. The data also show that rat kidney cytosolic glutamine transaminase K catalyzes both a beta-elimination and a transamination reaction with DCVC when alpha-keto-gamma-methiolbutyrate is present and that amino acid oxidase and alpha-keto-gamma-methiolbutyrate stimulate the enzyme activity by providing amino acceptors. When incubations were done with DCVC as substrate in the presence of excess alpha-keto-gamma-methiolbutyrate, the beta-lyase catalyzed beta-elimination and transamination in a ratio of 1:1.3, respectively. Under conditions where most of the alpha-keto-gamma-methiolbutyrate was consumed, the beta-elimination predominated indicating that the S-1,2-dichlorovinyl-3-mercapto-2-oxopropionic acid pool was consumed by transamination after the alpha-keto-gamma-methiolbutyrate had been depleted. The data are discussed with regard to the importance of these pathways as regulators or participants in the toxicity of S-cysteine conjugates.     

Role for an episulfonium ion in S-(2-chloroethyl)-DL-cysteine-induced cytotoxicity and its reaction with glutathione

The cysteine S conjugate of 1,2-dichloroethane, S-(2-chloroethyl)-DL-cysteine (CEC), is hepatotoxic, nephrotoxic, and mutagenic. To determine the cellular and chemical mechanisms involved in CEC-induced toxicity and to assess the role of an episulfonium ion, the effect of CEC on the viability of isolated rat hepatocytes was studied. CEC addition resulted in both a time- and concentration-dependent loss of cell viability. Depletion of intracellular glutathione concentrations (greater than 70%) and inhibition of microsomal Ca2+ transport and Ca2+-ATPase activity preceded the loss of cell viability, and initiation of lipid peroxidation paralleled the loss of viability. The depletion of glutathione concentrations was partially attributable to a reaction between glutathione and CEC to form S-[2-(DL-cysteinyl)ethyl]glutathione, which was identified by NMR and mass spectrometry. N-Acetyl-L-cysteine, vitamin E, and N,N'-diphenyl-p-phenylenediamine protected against the loss of cell viability. N,N'-Diphenyl-p-phenylenediamine inhibited CEC-initiated lipid peroxidation but did not protect against cell death at 4 h, indicating that lipid peroxidation was not the cause of cell death. The analogues S-ethyl-L-cysteine, S-(3-chloropropyl)-DL-cysteine, and S-(2-hydroxyethyl)-L-cysteine, which cannot form an episulfonium ion, were not cytotoxic, thus demonstrating a role for an episulfonium ion in the cytotoxicity associated with exposure to CEC and, possibly, 1,2-dichloroethane. These results show that an alteration in Ca2+ homeostasis and the generation of an electrophilic intermediate may be involved in the mechanism of cell death.     

The effects of haloalkene cysteine conjugates on cytosolic free calcium levels in suspensions of rat renal proximal tubules

Disturbances in intracellular calcium homeostasis may play a role in the injury induced by various haloalkene cysteine conjugates. The effects of S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L-cysteine (PCBC), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) on cytosolic free calcium levels were examined in suspensions of rat renal proximal tubules. Cytosolic free calcium levels, measured with fura 2, in control tubules, were 112 +/- 3 nM and increased more than 200% within 1 minute after exposure to the calcium ionophore ionomycin (0.005 mM). PCBC (0.1 mM) increased cytosolic free calcium levels 18% after 5 minutes, while tubular oxygen consumption was unaffected. DCVC (1 mM) did not alter tubular cytosolic free calcium levels or oxygen consumption under similar conditions. TFEC (1 mM) increased cytosolic free calcium levels 36%, had no effect on basal oxygen consumption, and decreased nystatin-stimulated oxygen consumption 30% after 5 minutes. TFEC increased cytosolic free calcium levels in tubules incubated in a nominally calcium-free buffer but not in a calcium containing buffer in the presence of EGTA. The data suggest that the TFEC-induced increase in cytosolic free calcium levels may result from an influx of extracellular calcium or from inhibition of calcium efflux. The increase in cytosolic free calcium levels preceded changes in basal oxygen consumption in tubules exposed to PCBC and TFEC. This study shows that an increase in cytosolic free calcium levels is an early event following PCBC and TFEC but not DCVC exposure.