Three models of free radical-induced cell injury

Three models of free radical-induced cell injury are presented in this review. Each model is described by the mechanism of action of few prototype toxic molecules. Carbon tetrachloride and monobromotrichloromethane were selected as model molecules for alkylating agents that do not induce GSH depletion. Bromobenzene and allyl alcohol were selected as prototypes of GSH depleting agents. Paraquat and menadione were presented as prototypes of redox cycling compounds. All these groups of toxins are converted, during their intracellular metabolism, to active species which can be radical species or electrophilic intermediates. In most cases the activation is catalyzed by the microsomal mixed function oxidase system, while in other cases (e.g. allyl alcohol) cytosolic enzymes are responsible for the activation. Radical species can bind covalently to cellular macromolecules and can promote lipid peroxidation in cellular membranes. Of course both phenomena produce cell damage as in the case of CCl4 or BrCCl3 intoxication. However, the covalent binding is likely to produce damage at the molecular site where it occurs; lipid peroxidation, on the other hand, besides causing loss of membrane structure, also gives rise to toxic products such as 4-hydroxyalkenals and other aldehydes which in principle can move from the site of origin and produce effects at distant sites. Electrophilic intermediates readily reacts with cellular nucleophiles, primarily with GSH. The result is a severe GSH depletion as in the case of bromobenzene or allyl alcohol intoxication. When the depletion reaches some threshold values lipid peroxidation develops abruptly and in an extensive way. This event is accompanied by cellular death. The reason for which lipid peroxidation develops in a cell severely depleted of GSH remains to be clarified. Probably the loss of the defense systems against a constitutive oxidative stress is not compatible with cellular life. Some free radicals generated by one-electron reduction can react with oxygen to give superoxide anions which can be converted to other more dangerous reactive oxygen species. This is the case of paraquat and menadione. Damage to cellular macromolecules is due to the direct action of these oxygen radicals and, at least in the menadione-induced cytotoxicity, lipid peroxidation is not involved. All these initial events affect the protein sulfhydryl groups in the membranes. Since some protein thiols are essential components of the molecular arrangement responsible for the Ca2+ transport across cellular membranes, loss of such thiols can affect the calcium sequestration activity of subcellular compartments, that is the capacity of mitochondria and microsomes to regulate the cytosolic calcium level.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Toxicity of 1,2-dibromoethane in isolated hepatocytes: Role of lipid peroxidation

Treatment of isolated hepatocytes with 1,2-dibromoethane (DBE) caused a concentration dependent depletion of cellular glutathione (GSH) content and a parallel increase in the covalent binding of reactive intermediates to cell proteins, as a consequence of the haloalkane activation. The reduction of the hepatocyte GSH content, induced by DBE, stimulated the onset of lipid peroxidation, as measured by malondialdehyde (MDA) accumulation. N-Acetylcysteine (1 mM) was found to partially prevent GSH loss and to inhibit MDA formation, whereas equal concentrations of cysteine and methionine were ineffective on these respects. The stimulation of the peroxidative reactions appeared to be also associated with an increase in the leakage of lactate dehydrogenase (LDH) from the cells, indicative of a severe hepatocyte injury. Antioxidants such as -tocopherol, N,N′-phenyl-phenylenediamine (DPPD) and promethazine, as well as N-acetylcysteine reduced MDA formation to various extents and also protect against LDH release, yet without interfering with the covalent binding of DBE reactive intermediates to hepatocyte proteins. These results suggest the involvement of lipid peroxidation, consequent to GSH depletion, in the pathogenesis of liver cell necrosis due to DBE.     

Lipid peroxidation-dependent and -independent protein thiol modifications in isolated rat hepatocytes: differential effects of vitamin E and disulfiram

Exposure of isolated rat hepatocytes to allyl alcohol (AA), diethyl maleate (DEM) and bromoisovalerylurea (BIU) induced lipid peroxidation, depletion of free protein thiols to about 50% of the control value and cell death. Vitamin E completely prevented lipid peroxidation, protein thiol depletion and cell death. A low concentration (0.1 mM) of the lipophylic disulfide, disulfiram (DSF), also prevented the induction of lipid peroxidation by the hepatotoxins; however, in the presence of DSF, protein thiol depletion and cell death occurred more rapidly. Incubation of cells with a high concentration (10 mM) of DSF alone led to 100% depletion of protein thiols and cell death, which could not be prevented by vitamin E. The level of free protein thiols in cells, decreased to 50% by exposure to AA, DEM and BIU, could be reversed to 75% of the initial level by dithiothreitol (DTT) treatment, indicating that the protein thiols were partially modified into disulfides and partially into other, stable thiol adducts. The 100% depletion of protein thiols by DSF was completely reversed by DTT treatment. The involvement of lipid peroxidation in protein thiol depletion was studied by measuring the effect of a lipid peroxidation product, 4-hydroxynonenal (4-HNE), on protein thiols in a cell free liver fraction. 4-HNE did not induce lipid peroxidation in this system, but protein thiols were depleted to 30% of the initial value, irrespective of the presence of vitamin E. DTT treatment could reverse this for only 25%. Similar, DSF-induced protein thiol depletion could be reversed completely by DTT. We conclude that (at least) two types of protein thiol modifications can occur after exposure of hepatocytes to toxic compounds: one due to interaction of endogeneously generated lipid peroxidation products with protein thiols, which is not reversible by the action of DTT, and one due to a disulfide interchange between disulfides like DSF and protein thiols, which can be reversed by the action of DTT.     

Oxidative cell injury in the killing of cultured hepatocytes by allyl alcohol

The killing of cultured hepatocytes by allyl alcohol depended on the metabolism of this hepatotoxin by alcohol dehydrogenase to the reactive electrophile, acrolein. An inhibitor of alcohol dehydrogenase, pyrazole, prevented both the toxicity of allyl alcohol and the rapid depletion of GSH. Treatment of the hepatocytes with a ferric iron chelator, deferoxamine, or an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), prevented the cell killing but not the metabolism of allyl alcohol and the resulting depletion of GSH. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sensitized the hepatocytes to allyl alcohol, an effect that was not attributable to the reduction in GSH with BCNU. The cell killing with allyl alcohol was preceded by the peroxidation of cellular lipids as evidence by an accumulation of malondialdehyde in the cultures. Deferoxamine and DPPD prevented the lipid peroxidation in parallel with their protection from the cell killing. These data indicate that acrolein produces an abrupt depletion of GSH that is followed by lipid peroxidation and cell death. Such oxidative cell injury is suggested to result from the inability to detoxify endogenous hydrogen peroxide and the ensuing iron-dependent formation of a potent oxidizing species. Oxidative cell injury more consistently accounts for the hepatotoxicity of allyl alcohol than does the covalent binding of acrolein to cellular macromolecules.     

Reduced glutathione protection against rat liver microsomal injury by carbon tetrachloride. Dependence on O2.

Rat liver microsomal membranes contain a reduced-glutathione-dependent protein(s) that inhibits lipid peroxidation in the ascorbate/iron microsomal lipid peroxidation system. It appears to exert its protective effect by scavenging free radicals. The present work was carried out to assess the effect of this reduced-glutathione-dependent mechanism on carbon tetrachloride-induced microsomal injury and on carbon tetrachloride metabolism because they are known to involve free radicals. Rat liver microsomes were incubated at 37 degrees C with NADPH, EDTA and carbon tetrachloride. The addition of 1 mM-reduced glutathione (GSH) markedly inhibited lipid peroxidation and glucose 6-phosphatase inactivation and, to a lesser extent, inhibited cytochrome P-450 destruction. GSH also inhibited covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. These results indicate that a GSH-dependent mechanism functions to protect the microsomal membrane against free-radical injury in the carbon tetrachloride system as well as in the iron-based systems. Under anaerobic conditions, GSH had no effect on chloroform formation, carbon tetrachloride-induced destruction of cytochrome P-450 or covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. Thus, the GSH protective mechanism appears to be O2-dependent. This suggests that it may be specific for O2-based free radicals. This O2-dependent GSH protective mechanism may partly underlie the observed protection of hyperbaric O2 against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity.     

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Treatment of isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to promote lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the cytochrome P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production.Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack.In isolated mouse liver cells paracetamol does not require preliminary cytochrome P-450 induction to stimulate MDA formation, even at concentrations ineffective in rat cells.However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not promote MDA formation either in the rat or in the mouse hepatocytes. This effect may be due to the ability of paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems.Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA formation is inhibited by high doses of the drug or by addition of antioxidants such as α-tocopherol and diphenylphenylenediamine (DPPD).Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.     

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.     

Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation

GSH is an important cellular defense against oxidant injury. Its effect in the rat liver microsomal lipid peroxidation system has been examined. Incubation of fresh rat liver microsomes with ascorbic acid and ADP-chelated iron leads to the peroxidation of microsomal lipids (production of thiobarbituric acid-reactive substances and destruction of polyunsaturated fatty acids) following a 2 to 5 min lag. Addition of 0.1 mM GSH to the system lengthened the lag period by 5 to 15 min without affecting the rate or the extent of lipid peroxidation. GSH could not be replaced in prolonging the lag by cysteine, mercaptoethanol, dithiothreitol, propylthiouracil, or GSSG. The GSH effect on the lag was abolished by heating or trypsin digestion of the microsomes, indicating that microsomal protein is required for its expression. Progressively longer lags were observed as the GSH concentration was increased from 0.1 to 5 mM, but there was no evidence of GSH oxidation as a consequence of the protection against lipid peroxidation. GSH protected against heat inactivation of the microsomal protein responsible for the GSH effect. Experiments with an oxygen electrode revealed that the GSH protection did not alter the ratio of O₂ consumed to thiobarbituric acid-reactive substances produced. This implicated free radical scavenging as the mechanism of protection. These results indicate the existence of a GSH-dependent rat liver microsomal protein which scavenges free radical. This protein may be an important defense against free radical injury to the microsomal membrane.     

Modification of hepatic vitamin E stores in vivo. I. Alterations in plasma and liver vitamin E content by methyl ethyl ketone peroxide

Since experiments with freshly isolated rat hepatocytes have shown that cellular vitamin E is consumed in response to insult by compounds that induce an oxidative stress only after cellular glutathione (GSH) concentrations have been substantially depleted, experiments were performed to determine whether this sequence of events occurred in response to oxidative insult in vivo. The role that plasma vitamin E plays in the response to chemically induced oxidative injury in vivo was also assessed. Treatments with 40 mg/kg of methyl ethyl ketone peroxide (MEKP) quickly induced lipid peroxidation in vivo and from one to 4 h after treatment caused a depression in the plasma content of vitamin E and the liver content of GSH, as well as signs of toxicity (elevations in serum activities of alanine and aspartate aminotransferases). At these time points however, the liver content of vitamin E was either indistinguishable from or slightly elevated from controls. By 12 to 24 h after treatment the liver content of vitamin E was reduced by 20-25% whereas values for all other indicators had returned toward control levels. Pretreatment of rats with L-buthionine-S,R-sulfoximine, an inhibitor of GSH by 4 or 24 h after treatment, did not alter the time course or extent of hepatic vitamin E depletion that was observed after treatment with MEKP. Other compounds that induce oxidative stress and lipid peroxidation to the liver, carbon tetrachloride and menadione, did not provoke an alteration in hepatic vitamin E levels as compared to controls 1 day after treatment. These findings indicate that depletion of hepatic vitamin E may not occur as an immediate consequence of oxidative insult to the liver and that the depletion of hepatic vitamin E levels may not be related to the extent of prior GSH depletion. Moreover, these findings suggest that alterations in the plasma concentration of vitamin E may not reflect concurrent alterations in hepatic vitamin E levels. A mechanism whereby liver vitamin E stores are mobilized for the maintenance of plasma vitamin E levels is proposed.     

Thiol depletion induces lethal cell injury in cultured cardiomyocytes.

Treatment of cultured neonatal cardiomyocytes with ethacrynic acid (EA) induced a rapid depletion of glutathione (GSH) that preceded a gradual elevation of cytosolic Ca2+ (monitored by phosphorylase a activation), a loss of protein thiols, and a marked inactivation of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD). A subsequent decline of mitochondrial transmembrane potential (delta psi) and ATP occurred prior to the onset of lipid peroxidation which closely paralleled a loss of cardiomyocyte viability. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented lipid peroxidation and cell death but had no effect on elevated cytosolic Ca2+, delta psi loss, GSH depletion, or G3PD inactivation. Pretreatment with the iron chelator, deferoxamine, decreased both lipid peroxidation and cell death. EA-induced lipid peroxidation and cell damage were also diminished by preincubation with acetoxymethyl esters of the Ca2+ chelators Quin-2 and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, even though cytosolic Ca2+ remained elevated. The extent of GSH depletion was unaltered by either chelator; however, Quin-2 did protect G3PD from inactivation by EA. An inhibitor of the mitochondrial respiratory chain, antimycin A, decreased EA-induced lipid peroxidation and cell death but had no effect on thiol depletion or elevated cytosolic Ca2+. These data suggest that cardiomyocyte thiol status may be linked to intracellular Ca2+ homeostasis and that peroxidative damage originating in the mitochondria is a major event in the onset of cell death in this cardiomyocyte model of thiol depletion.     

A role of vitamin E in protection against cell injury. Maintenance of intracellular glutathione precursors and biosynthesis

The depletion of cell calcium from isolated rat hepatocytes results in stimulated lipid peroxidation, loss of intracellular and mitochondrial GSH (reduced glutathione), and enhancement of both efflux and oxidation of GSH. These events are followed by cell injury and enhance the susceptibility of the cells to toxic chemicals. It is shown herein that an initial event in the generation of such injury is the depletion of cellular alpha-tocopherol. alpha-Tocopheryl succinate addition (25 microM) to the calcium-depleted cells markedly elevated the alpha-tocopherol content of the cells, inhibited the associated lipid peroxidation, and maintained intracellular GSH levels without affecting its efflux or redox status. This resulted in an enhanced formation of total glutathione after a 5-h incubation, which correlated with the alpha-tocopherol content of the cells, and was greater than that expected by a direct sparing action of vitamin E. Inhibition of hepatocyte glutathione biosynthesis by buthionine sulfoximine (0.5 mM) eliminated the enhancement of GSH formation by vitamin E. Analysis of endogenous and 35S-labelled precursors of glutathione biosynthesis by high-performance liquid chromatography demonstrated that the depletion of cellular alpha-tocopherol resulted in the efflux of glutathione precursors. It is concluded that cell injury associated with alpha-tocopherol depletion is partly the result of the efflux of glutathione precursors, and hence diminished biosynthesis and intracellular levels of GSH. These losses and resultant cell injury are preventable by maintenance of cellular alpha-tocopherol levels.     

Role of vicinal protein thiols in radiation and cytotoxic responses

Glutathione (GSH) and more recently protein thiols (P-SH) have been found to play a major role in cellular radiation response. However, the effects of protein vicinal thiols, which are important for the functions of several major enzymes, on cellular responses to radiation have not been clearly delineated. Here we investigated the effects of depleting GSH and protein vicinal thiols (HS-P-SH) and P-SH on cell toxicity and radiation response. We used hydroxyethyldisulfide (HEDS, beta-mercaptoethanol-disulfide) alone and in combination with phenylarsine oxide (PAO) to alter P-SH, HS-P-SH and GSH. HEDS, a direct substrate for thioredoxin reductase and an indirect substrate for glutaredoxin (thioltransferase), did not alter protein vicinal thiols in cells. However, PAO, which specifically forms a covalent adduct with vicinal thiols, blocked bioreduction of HEDS; there was a concomitant and yet unexplained decrease in K1 cell GSH in the presence of HEDS and PAO. G6PD+ (K1) and G6PD- (E89) cells treated with L-buthionine sulfoximine (L-BSO) for 72 h to deplete GSH followed by PAO showed an increased cytotoxic response. However, the surviving E89 cells showed a 10,000-fold greater radiation lethality than the K1 cells. The effects of rapid depletion of GSH by a combination of L-BSO and dimethyfumarate (DMF), a glutathione-S-transferase substrate, were also investigated. Under these conditions, PAO radiosensitized the E89 cells more than 1000-fold over the K1 cells. The potential mechanisms for the altered response may be related to the inhibition of thioredoxin reductase and glutaredoxin. Both are key enzymes involved in DNA synthesis, protein homeostasis and cell survival. With GSH removed, vicinal thiols appear to play a critical role in determining cell survival and radiosensitivity. Decreasing P-SH and removing GSH and vicinal thiols is extremely toxic to K1 and E89 cells. We conclude that radiation sensitivity and cell survival are dependent on vicinal thiol and GSH. In the former and latter cases, the protein thiols are also important.     

4-Hydroxy-2,3-trans-nonenal stimulates microsomal lipid peroxidation by reducing the glutathione-dependent protection

Glutathione (GSH) protects liver microsomes against lipid peroxidation. This is probably due to the reduction of vitamin E radicals by GSH, a reaction catalyzed by a membrane-bound protein. Pretreatment of liver microsomes with 0.1 or 1mM 4-hydroxy-2,3-trans-nonenal (HNE), a major product of lipid peroxidation, reduces the GSH-dependent protection. GSH and vitamin E concentrations are not affected by this pretreatment. Pretreatment with 0.1 mM N-ethyl maleimide (NEM), a synthetic sulfhydryl reagent, resulted in a reduction similar to that with HNE of the GSH-dependent protection against lipid peroxidation. The reduction of the GSH-dependent protection by HNE and NEM is probably the result of inactivation of the membrane-bound protein by covalent binding to an essential SH group on the protein. If the GSH-dependent protection would proceed via the microsomal GSH transferase, pretreatment with NEM, which activates the microsomal GSH transferase, should enhance the GSH-dependent protection. Actually a decrease in the GSH-dependent protection is found. Apparently the GSH-dependent protection does not proceed via the microsomal GSH transferase. Also the microsomal phospholipase A2 is not involved, since addition of 0.1 mM mepacrine, an inhibitor of phospholipase A2, did not preclude the GSH-dependent protection. Once the process of lipid peroxidation, either in vivo or in vitro, has started, the protection of liver microsomes by GSH is less effective. This might be the result of formed HNE. In this way an endproduct of lipid peroxidation stimulates the process that generates this product.     

The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membrane-lipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between alpha-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with alpha-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, alpha-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.     

Protective effects of N-acetyl-L-cysteine against acute carbon tetrachloride hepatotoxicity in rats

In recent years, N-acetyl-L-cysteine (NAC) has been widely investigated as a potentially useful protective and antioxidative agent to be applied in many pathological states. The aim of the present work was further evaluation of the mechanisms of the NAC protective effect under carbon tetrachloride-induced acute liver injuries in rats. The rat treatment with CCl4 (4 g/kg, intragastrically) caused pronounced hepatolysis observed as an increase in blood plasma bilirubin levels and hepatic enzyme activities, which agreed with numerous previous observations. The rat intoxication was accompanied by an enhancement of membrane lipid peroxidation (1.4-fold) and protein oxidative damage (protein carbonyl group and mixed protein-glutathione disulphide formations) in the rat liver. The levels of nitric oxide in blood plasma and liver tissue significantly increased (5.3- and 1.5-fold, respectively) as blood plasma triacylglycerols decreased (1.6-fold). The NAC administration to control and intoxicated animals (three times at doses of 150 mg/kg) elevated low-molecular-weight thiols in the liver. The NAC administration under CCl4-induced intoxication prevented oxidative damage of liver cells, decreased membrane lipid peroxidation, protein carbonyls and mixed protein-glutathione disulphides formation, and partially normalized plasma triacylglycerols. At the same time the NAC treatment of intoxicated animals did not produce a marked decrease of the elevated levels of blood plasma ALT and AST activities and bilirubin. The in vitro exposure of human red blood cells to NAC increased the cellular low-molecular-weight thiol levels and retarded tert-butylhydroperoxide-induced cellular thiol depletion and membrane lipid peroxidation as well as effectively inhibited hypochlorous acid-induced erythrocyte lysis. Thus, NAC can replenish non-protein cellular thiols and protect membrane lipids and proteins due to its direct radical-scavenging properties, but it did not attenuate hepatotoxicity in the acute rat CCl4-intoxication model.     

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.     

Therapeutic efficacy of green tea polyphenols on cellular thiols in 4-Nitroquinoline 1-oxide-induced oral carcinogenesis

In cancer, a high flux of oxidants not only depletes the cellular thiols, but damages the whole cell as well. Epidemiological studies suggest green tea may mitigate cancers in human and animal models for which several mechanisms have been proposed. In the present investigation, the levels of cellular thiols such as reduced glutathione (GSH), oxidised glutathione (GSSG), protein thiols (PSH), total thiols, lipid peroxidation product conjugated dienes and the activity of gamma glutamyl transferase (GGT) were assessed in tongue and oral cavity. In 4-Nitroquinoline 1-oxide- (4-NQO) induced rats, there was a decrease in the levels of GSH, PSH and total thiols and an increase in the levels of GSSG, conjugated dienes and the activity of GGT. On supplementation of green tea polyphenols (GTP) for 30 days (200 mg/kg) for the oral cancer-induced rats, there was a moderate increase in the levels of GSH, PSH and total thiols and a decrease in the levels of GSSG, conjugated dienes and the activity of GGT. Thus, GTP reduces the oxidant production thereby maintains the endogenous low molecular weight cellular thiols in oral cancer-induced rats. From the results, it can be concluded that GTP supplementation enhances the cellular thiol status thereby mitigate oral cancer.     

Vitamin E protection against chemical-induced cell injury. I. Maintenance of cellular protein thiols as a cytoprotective mechanism

Vitamin E protection against chemical-induced toxicity to isolated hepatocytes was examined during an imbalance in the thiol redox system. Intracellular reduced glutathione (GSH) was depleted by two chemicals of distinct mechanisms of action: adriamycin, a cancer chemotherapeutic agent that undergoes redox cycling, producing reactive oxygen species that consume GSH, and ethacrynic acid, a direct depleter of GSH. The experimental system used both nonstressed vitamin E-adequate isolated rat hepatocytes and compromised hepatocytes subjected to physiologically induced stress, generated by incubation in calcium-free medium. At doses whereby intracellular GSH was near total depletion, cell injury induced by either chemical was found to follow the depletion of cellular alpha-tocopherol, regardless of the status of the GSH redox system. Changes in protein thiol contents of the cells closely paralleled the changes in alpha-tocopherol contents throughout the incubation period. Supplementation of the calcium-depleted hepatocytes with alpha-tocopheryl succinate (25 microM) markedly elevated their alpha-tocopherol content and prevented the toxicities of both drugs. The prevention of cell injury and the elevation in alpha-tocopherol contents were both associated with a prevention of the loss in cellular protein thiols in the near total absence of intracellular GSH. The mechanism of protection by vitamin E against chemical-induced toxicity to hepatocytes may therefore be an alpha-tocopherol-dependent maintenance of cellular protein thiols.