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.     

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.     

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.     

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.     

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.     

Cisplatin-induced toxicity is associated with platinum deposition in mouse kidney mitochondria in vivo and with selective inactivation of the alpha-ketoglutarate dehydrogenase complex in LLC-PK1 cells

The anticancer drug cisplatin is nephrotoxic and neurotoxic. Previous data support the hypothesis that cisplatin is bioactivated to a nephrotoxicant. The final step in the proposed bioactivation is the formation of a platinum-cysteine S-conjugate followed by a pyridoxal 5'-phosphate (PLP)-dependent cysteine S-conjugate beta-lyase reaction. This reaction would generate pyruvate, ammonium, and a highly reactive platinum (Pt)-thiol compound in vivo that would bind to proteins. In this work, the cellular location and identity of the PLP-dependent cysteine S-conjugate beta-lyase were investigated. Pt was shown to bind to proteins in kidneys of cisplatin-treated mice. The concentration of Pt-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Treatment of the mice with aminooxyacetic acid (AOAA, a PLP enzyme inhibitor), which had previously been shown to block the nephrotoxicity of cisplatin, decreased the binding of Pt to mitochondrial proteins but had no effect on the amount of Pt bound to proteins in the cytosolic fraction. These data indicate that a mitochondrial enzyme catalyzes the PLP-dependent cysteine S-conjugate beta-lyase reaction. PLP-dependent mitochondrial aspartate aminotransferase (mitAspAT) is a mitochondrial enzyme that catalyzes beta-elimination reactions with cysteine S-conjugates of halogenated alkenes. We reasoned that the enzyme might also catalyze a beta-lyase reaction with the cisplatin-cysteine S-conjugate. In this study, mitAspAT was stably overexpressed in LLC-PK(1) cells. Cisplatin was significantly more toxic in confluent monolayers of LLC-PK(1) cells that overexpressed mitAspAT than in control cells containing vector alone. AOAA completely blocked the cisplatin toxicity in confluent mitAspAT-transfected cells. The Pt-thiol compound could rapidly bind proteins and inactivate enzymes in close proximity of the PLP-dependent cysteine S-conjugate beta-lyase. Treatment with 50 or 100 microM cisplatin for 3 h, followed by removal of cisplatin from the medium for 24 h, resulted in a pronounced loss of alpha-ketoglutarate dehydrogenase complex (KGDHC) activity in both mitAspAT-transfected cells and control cells. Exposure to 100 microM cisplatin resulted in a significantly greater loss of KGDHC activity in the cells overexpressing mitAspAT than in control cells. Aconitase activity was diminished in both cell types, but only at the higher level of exposure to cisplatin. AspAT activity was also significantly decreased by cisplatin treatment. By contrast, several other enzymes (both cytosolic and mitochondrial) involved in energy/amino acid metabolism were not significantly affected by cisplatin treatment in the LLC-PK(1) cells, whether or not mitAspAT was overexpressed. The susceptibility of KGDHC and aconitase to inactivation in kidney cells exposed to cisplatin metabolites may be due to the proximity of mitAspAT to KGDHC and aconitase in mitochondria. These findings support the hypothesis that a mitochondrial cysteine S-conjugate beta-lyase converts the cisplatin-cysteine S-conjugate to a toxicant, and the data are consistent with the hypothesis that mitAspAT plays a role in the bioactivation of cisplatin.     

Pentachlorobutadienyl-L-cysteine (PCBC) toxicity: the importance of mitochondrial dysfunction.

The relationship between the covalent binding, uptake, and toxicity produced by pentachlorobutadienyl-L-cysteine (PCBC) was examined in rabbit renal proximal tubules (RPT), renal basolateral membrane vesicles, and isolated renal cortical mitochondria. Renal proximal tubules rapidly metabolized PCBC to a reactive intermediate that bound to tubular protein. Approximately 70-90% of PCBC found in the cell at any given time was bound to protein. PCBC initially uncoupled oxidative phosphorylation, followed by a 45% reduction of state 3 respiration and a 90% decrease in cellular adenosine triphosphate (ATP) levels. These events preceded cell death. Isolated mitochondria also metabolized PCBC to a reactive intermediate that bound to mitochondrial protein and initiated mitochondrial toxicity. These results show that PCBC-induced mitochondrial dysfunction occurred as a result of mitochondrial bioactivation and that the mitochondrion is the critical subcellular target in PCBC toxicity. Aminooxyacetic acid (AOAA), an inhibitor of cysteine conjugate beta-lyase, reduced the covalent binding of PCBC-equivalents to tubular protein by approximately 90% and decreased but did not prevent the toxic effects produced by PCBC on RPT respiration and cellular ATP levels. AOAA delayed but had no effect on the overall extent of cell death produced by PCBC. The protective effect of AOAA was independent of any effects on PCBC uptake. These results show that AOAA decreased but did not prevent the metabolism of PCBC by cysteine conjugate beta-lyase. The partial inhibition of PCBC metabolism, and hence, PCBC-induced cell death by AOAA, may be related to limited concentrations of AOAA within the tubule cell or mitochondria.     

Toxic injury from mercuric chloride in rat hepatocytes

The relationship between cytosolic free Ca2+, mitochondrial membrane potential, ATP depletion, pyridine nucleotide fluorescence, cell surface blebbing, and cell death was evaluated in rat hepatocytes exposed to HgCl2. In cell suspensions, 50 microM HgCl2 oxidized pyridine nucleotides between 1/2 and 2 min, caused ATP depletion between 2 and 5 min, and produced an 89% loss of cell viability after 20 min. Rates of cell killing were identical in high (1.2 mM) and low (2.6 microM) Ca2+ buffers. Cytosolic free Ca2+ was determined in 1-day cultured hepatocytes by ratio imaging of Fura-2 employing multiparameter digitized video microscopy. In high Ca2+ medium, HgCl2 caused a 3-4-fold increase of free Ca2+ beginning after 6-7 min, but free Ca2+ did not change in low Ca2+ medium. Bleb formation occurred after about 4-5 min in both buffers prior to any increase of free Ca2+. Subsequently, in high Ca2+ medium, blebs became hot spots of free Ca2+ (greater than 600 nM). After about 2 min of exposure to HgCl2, rhodamine 123 fluorescence redistributed from mitochondrial to cytosolic compartments signifying collapse of the mitochondrial membrane potential. The results taken together demonstrate that bleb formation, ATP depletion, and the onset of cell death are not dependent on an increase of cytosolic free Ca2+. HgCl2 toxicity appears to be a consequence of inhibition of oxidative phosphorylation leading to ATP depletion and cell death.     

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.     

The role of mitochondrial glutathione and cellular protein sulfhydryls in formaldehyde toxicity in glutathione-depleted rat hepatocytes

Depletion of cellular GSH by diethyl maleate (DEM) potentiates CH2O toxicity in isolated rat hepatocytes and it was postulated that this increase in toxicity is due to the further decrease in GSH caused by CH2O in DEM-pretreated hepatocytes (1). The present investigation was conducted to investigate further the effects of CH2O, DEM, and acrolein (a compound which is structurally related to CH2O and DEM) on subcellular GSH pools and on protein sulfhydryl groups (PSH). CH2O caused a decrease in cytosolic GSH but had no effect on mitochondrial GSH either in previously untreated hepatocytes or in DEM-pretreated hepatocytes in which GSH was approximately 25% of control. DEM decreased both cytosolic and mitochondrial GSH but it did not produce toxicity. Neither CH2O (up to 7.5 mM) nor DEM (20 mM) decreased PSH. However, in cells pretreated with 1 mM DEM, CH2O (7.5 mM) decreased PSH and this effect preceded cell death. Acrolein decreased both cytosolic and mitochondrial GSH and it also decreased PSH significantly prior to causing cell death. CH2O and acrolein stimulated phosphorylase alpha activity, indicative of an increase in cytosolic free Ca2+, by a PSH-independent and PSH-dependent mechanism, respectively. These results suggest that the further depletion of cellular GSH by CH2O in DEM-pretreated cells is not due to the depletion of mitochondrial GSH. CH2O toxicity in DEM-pretreated cells is, however, correlated with depletion of PSH. The critical sulfhydryl protein(s) responsible for cell death remain to be more clearly defined.     

Toxicity of S-pentachlorobutadienyl-L-cysteine studied with isolated rat renal cortical mitochondria

The subcellular mechanism of alkenyl halide S-conjugate-induced nephrotoxicity was studied in mitochondria isolated from rat kidney cortex in vitro using the cysteine conjugate of hexachloro-1,3-butadiene, i.e., S-pentachlorobutadienyl-L-cysteine (PCBC) as a model substrate. Respiring mitochondria exposed to various concentrations of PCBC exhibited a dose-dependent loss of ability to retain calcium. This phenomenon was associated with a sudden collapse of the mitochondrial membrane potential. PCBC caused a slow nonenzymatic depletion of mitochondrial glutathione. This was not due to oxidation or formation of mixed disulfides, and was efficiently counteracted by preincubation with aminooxyacetic acid, an inhibitor of cysteine-conjugate beta-lyase activity. PCBC inhibited state 3 respiration in the presence of succinate as substrate, which indicates that the activity of succinate dehydrogenase was affected. Thus, the present data confirm that impairment of mitochondrial function is a feature of nephrotoxicity mediated by alkenyl halide S-conjugates. We suggest a pathway involving interaction of beta-lyase-dependent reactive metabolite with the mitochondrial inner membrane, loss of membrane potential, disturbance of Ca2+ homeostasis, and subsequent respiratory insufficiency as a mechanism for renal tubular cytotoxicity.     

Primary alveolar epithelial cell surface membrane microdomain function is required for Pneumocystis β-glucan-induced inflammatory responses

Intense lung inflammation characterizes respiratory failure associated with Pneumocystis pneumonia. Our laboratory has previously demonstrated that alveolar epithelial cells (AECs) elaborate inflammatory cytokines and chemokines in response to the Pneumocystis carinii cell wall constituent β-(1→3)-glucan (PCBG), and that these responses require lactosylceramide, a prominent glycosphingolipid constituent of certain cell membrane microdomains. The relevance of membrane microdomains, also termed plasma membrane lipid rafts, in cell signaling and macromolecule handling has been increasingly recognized in many biologic systems, but their role in P. carinii-induced inflammation is unknown. To investigate the mechanisms of microdomain-dependent P. carinii-induced inflammation, we challenged primary rat AECs with PCBG with or without pre-incubation with inhibitors of microdomain function. Glycosphingolipid and cholesterol rich microdomain inhibition resulted in significant attenuation of P. carinii-induced expression of TNF-α and the rodent C-X-C chemokine MIP-2, as well as their known inflammatory secondary signaling pathways. We have previously shown that protein kinase C (PKC) is activated by PCBG challenge and herein show that PKC localizes to AEC microdomains. We also demonstrate by conventional microscopy, fluorescence microscopy, confocal microscopy and spectrophotofluorimetry that AECs internalize fluorescently-labeled PCBG by microdomain-mediated mechanisms, and that anti-microdomain pretreatments prevent internalization. Taken together, these data suggest an important role for AEC microdomain function in PCBG-induced inflammatory responses. This offers a potential novel target for therapeutics for a condition that continues to exert unacceptable morbidity and mortality among immunocompromised populations.     

Isolation and characterization of a rat liver enzyme with both cysteine conjugate beta-lyase and kynureninase activity

Cysteine conjugate beta-lyase is a name applied to enzymes which cleave the S-cysteine conjugates of some xenobiotics to pyruvate, ammonia, and a thiol. Recently, several laboratories have characterized these enzymes from kidney, liver, and bacterial sources in an effort to understand their role in the genesis of novel sulfur-containing metabolites of xenobiotics and in the toxicity of some S-cysteine conjugates. Kynureninase is an enzyme which plays a key role in the biosynthesis of nicotinamide ribonucleotides. This investigation demonstrates that rat hepatic cysteine conjugate beta-lyase is the same enzyme as kynureninase. Both activities copurify on ion exchange, hydroxylapatite, and molecular exclusion chromatography. The subunit composition of enzyme prepared by two different methods is identical, Mr = 55,000. The Km values for 3-OH-kynurenine and kynurenine are 13 and 400 microM, respectively. Kynurenine and 3-hydroxykynurenine inhibit cysteine conjugate beta-lyase activity. Inactivation of the enzyme by substrates which undergo beta-elimination results in loss of kynureninase activity, but kynurenine does not inactivate the enzyme. Both enzyme activities react with anti-cysteine conjugate beta-lyase antibody. Product inhibitors of kynureninase, anthranilate, and 3-hydroxyanthranilate are also inhibitors of cysteine conjugate beta-lyase. Heat inactivation at 70 degrees C produced coincident loss of both activities. The enzyme has an absorption maximum at 432 nm suggesting a bound pyridoxal phosphate. These data show that at least one cysteine conjugate beta-lyase is a pyridoxal phosphate enzyme with a biological function other than xenobiotic metabolism. The enzyme can catalyze two distinct types of reactions, i.e. beta-elimination and the kynureninase reaction.     

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.     

Mitochondrial damage and its role in causing hepatocyte injury during stimulation of lipid peroxidation by iron nitriloacetate.

Incubation of isolated rat hepatocytes with 0.1 mM iron nitrilotriacetic acid (FeNTA) caused a rapid rise in lipid peroxidation followed by a substantial increase in trypan blue staining and lactate dehydrogenase release, but did not affect the protein and non-protein thiol content of the cells. Hepatocyte death was preceded by the decline of mitochondrial membrane potential, as assayed by rhodamine 123 uptake, and by the depletion of cellular ATP. Chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid or inhibition of Ca2+ cycling within the mitochondria by LaCl3 or cyclosporin A did not prevent the decline of rhodamine 123 uptake. On the other hand, a dramatic increase in the conjugated diene content was observed in mitochondria isolated from FeNTA-treated hepatocytes. Oxidative damage of mitochondria was accompanied by the leakage of matrix enzymes glutamic oxalacetic aminotransferase (GOT) and glutamate dehydrogenase (GLDH). The addition of the antioxidant N,N'-diphenylphenylene diamine (DPPD) completely prevented GOT and GLDH leakage, inhibition of rhodamine 123 uptake, and ATP depletion induced by FeNTA, indicating that Ca(2+)-independent alterations of mitochondrial membrane permeability consequent to lipid peroxidation were responsible for the loss of mitochondrial membrane potential. DPPD addition also protected against hepatocyte death. Similarly hepatocytes prepared from fed rats were found to be more resistant than those obtained from starved rats toward ATP depletion and cell death caused by FeNTA, in spite of undergoing a comparable mitochondrial injury. A similar protection was also observed following fructose supplementation of hepatocytes isolated from starved rats, indicating that the decline of ATP was critical for the development of FeNTA toxicity. From these results it was concluded that FeNTA-induced peroxidation of mitochondrial membranes impaired the electrochemical potential of these organelles and led to ATP depletion which was critical for the development of irreversible cell injury.     

Pentachlorobutadienyl-l-cysteine (PCBC) toxicity: The importance of mitochondrial dysfunction

The relationship between the covalent binding, uptake, and toxicity produced by pentachlorobutadienyl-L-cysteine (PCBC) was examined in rabbit renal proximal tubules (RPT), renal basolateral membrane vesicles, and isolated renal cortical mitochondria. Renal proximal tubules rapidly metabolized PCBC to a reactive intermediate that bound to tubular protein. Approximately 70–90% of PCBC found in the cell at any given time was bound to protein. PCBC initially uncoupled oxidative phosphorylation, followed by a 45% reduction of state 3 respiration and a 90% decrease in cellular adenosine triphosphate (ATP) levels. These events preceded cell death. Isolated mitochondria also metabolized PCBC to a reactive intermediate that bound to mitochondrial protein and initiated mitochondrial toxicity. These results show that. PCBC-induced mitochondrial dysfunction occurred as a result of mitochondrial bioactivation and that the mitochondrion is the critical subcellular target in PCBC toxicity. Aminooxyacetic acid (AOAA), an inhibitor of cysteine conjugate β-lyase, reduced the covalent binding of PCBC-equivalents to tubular protein by approximately 90% and decreased but did not prevent the toxic effects produced by PCBC on RPT respiration and cellular ATP levels. AOAA delayed but had no effect on the overall extent of cell death produced by PCBC. The protective effect of AOAA was independent of any effects on PCBC uptake. These results show that AOAA decreased but did not prevent the metabolism of PCBC by cysteine conjugate β-lyase. The partial inhibition of PCBC metabolism, and hence, PCBC-induced cell death by AOAA, may be related to limited concentrations of AOAA within the tubule cell or mitochondria.     

Hydrogen peroxide removal and glutathione mixed disulfide formation during metabolic inhibition in mesencephalic cultures

Compromised mitochondrial energy metabolism and oxidative stress have been associated with the pathophysiology of Parkinson's disease. Our previous experiments exemplified the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. This study further defines the role of oxidative stress during energy inhibition and begins to unravel the mechanisms by which GSH and other antioxidants may contribute to cell survival. Treatment of mesencephalic cultures with 10 microM buthionine sulfoximine for 24 h depleted total GSH by 60%, whereas 3 h exposure to 5 mM 3-amino-1,2,4-triazole irreversibly inactivated catalase activity by 90%. Treatment of GSH-depleted cells with malonate (40 mM) for 6, 12 or 24 h both potentiated and accelerated the time course of malonate toxicity, however, inhibition of catalase had no effect. In contrast, concomitant treatment with buthionine sulfoximine plus 3-amino-1,2,4-triazole in the presence of malonate significantly potentiated toxicity over that observed with malonate plus either inhibitor alone. Consistent with these findings, GSH depletion enhanced malonate-induced reactive oxygen species generation prior to the onset of toxicity. These findings demonstrate that early generation of reactive oxygen species during mitochondrial inhibition contributes to cell damage and that GSH serves as a first line of defense in its removal. Pre-treatment of cultures with 400 microM ascorbate protected completely against malonate toxicity (50 mM, 12 h), whereas treatment with 1 mM Trolox provided partial protection. Protein-GSH mixed disulfide formation during oxidative stress has been suggested to either protect vulnerable protein thiols or conversely to contribute to toxicity. Malonate exposure (50 mM) for 12 h resulted in a modest increase in mixed disulfide formation. However, exposure to the protective combination of ascorbate plus malonate increased membrane bound protein-GSH mixed disulfides three-fold. Mixed disulfide levels returned to baseline by 72 h of recovery indicating the reversible nature of this formation. These results demonstrate an early role for oxidative events during mitochondrial impairment and stress the importance of the glutathione system for removal of reactive oxygen species. Catalase may serve as a secondary defense as the glutathione system becomes limiting. These findings also suggest that protein-GSH mixed disulfide formation under these circumstances may play a protective role.     

Mitochondrial aconitase modification,functional inhibition,and evidence for a supramolecular complex of the TCA cycle by the renal toxicant S-(1,1,2,2-tetrafluoroethyl)-L-cysteine

Metabolism of the common industrial gas tetrafluoroethylene in mammals results in the formation of S-(1,1,2,2)-tetrafluoroethyl-L-cysteine (TFEC), which can be bioactivated by a mitochondrial C-S lyase commonly referred to as beta-lyase. The resultant "reactive intermediate", difluorothioacetyl fluoride (DFTAF), is a potent thioalkylating and protein-modifying species. Previously, we have identified mitochondrial HSP70, HSP60, aspartate aminotransferase, and the E2 and E3 subunits of the alpha-ketoglutarate dehydrogenase (alphaKGDH) complex as specific proteins structurally modified during this process. Moreover, functional alterations to the alphaKGDH complex were also detected and implicated in the progression of injury. We report here the identification, by tandem mass spectrometry, and functional characterization of the final remaining major protein species modified by DFTAF, previously designated as P99(unk), as mitochondrial aconitase. Aconitase activity was maximally inhibited by 56.5% in renal homogenates after a 6 h exposure to TFEC. In comparison to alphaKGDH, aconitase inhibition (up to 79%) in a cell culture model for TFEC-mediated cytotoxicity was greater and preceded alphaKGDH inhibition, indicating that aconitase modification may constitute an early event in TFEC-mediated mitochondrial damage and cell death. These findings largely define the initial lesion of TFEC-mediated cell death and also have implications for the modeling of mitochondrial enzymatic architecture and the localization and identity of renal mitochondrial cysteine S-conjugate beta-lyase.     

Nonxenobiotic manipulation and sulfur precursor specificity of human endothelial cell glutathione

Confluent human umbilical vein endothelial (HUVE) cells were readily (within 1 h) depleted of their glutathione (GSH) by diethylmaleate (0.1-1.0 mM), but dose-dependent cell detachment was noted. Buthionine sulfoximine (BSO, 25 microM) depleted cell GSH with sigmoidal kinetics, showing an initial half-life of depletion of 4-6 h and greater than 95% depletion by 48 h without morphological changes to the cells. However, BSO-dependent depletion of cell GSH was only partially reversible by cell washing and reincubation with complete medium. Likewise, incubation of the cells in sulfur-free medium depleted cell GSH again without morphological changes to the cells. However, unlike with BSO, these cells readily resynthesized GSH when resupplied with complete medium, fresh plasma, or whole blood, with a characteristic overloading of cell GSH (up to 200%) by 12 h. By use of the sulfur-free medium, it was shown that both cystine and cysteine are effective precursors to GSH synthesis in HUVE cells in culture and that cystine is the most likely precursor in vivo. During cystine-supported resynthesis of GSH, high levels of cysteine accumulated in the cells (up to 10% of total soluble free thiol). Physiologically relevant concentrations of extracellular GSH were not as effective as cystine or cysteine in stimulating GSH biosynthesis, whereas nonphysiologically high (mM) concentrations resulted in substantial elevation of GSH levels above those of control cells in a BSO-insensitive manner. These findings provide a simple methodology for the manipulation of HUVE cell GSH in studies of endothelial-specific oxidant toxicity and the sulfur dependence of the biochemistry and turnover of GSH in these human cells.