Inhibition of lipid peroxidation by disulfiram and diethyldithiocarbamate does not prevent hepatotoxin-induced cell death in isolated rat hepatocytes. A study with allyl alcohol, tert-butyl hydroperoxide, diethyl maleate, bromoisovalerylurea and carbon tetrachloride

The relationship between lipid peroxidation and cell death, induced by a number of hepatotoxins, was studied in isolated rat hepatocytes. Disulfiram (DSF) and diethyldithiocarbamate (DDC) completely prevented lipid peroxidation, induced by allyl alcohol, tert-butyl hydroperoxide (t-BHP), diethyl maleate (DEM), bromoisovalerylurea (BIU) and carbon tetrachloride (CCl4). Lipid peroxidation was measured by the formation of both thiobarbituric acid positive material and conjugated dienes. However, DSF and DDC did not protect against cell death, induced by these hepatotoxins. In the presence of DSF or DDC, cell death occurred even earlier in time. We conclude that cell death can occur in the absence of lipid peroxidation. Therefore, lipid peroxidation is not a requisite for the induction of cell death.     

tert-Butylhydroperoxide-induced toxicity in isolated hepatocytes: contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide-induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

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.     

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.     

Lipid peroxidation, protein thiol oxidation and DNA damage in hydrogen peroxide-induced injury to endothelial cells: role of activation of poly(ADP-ribose)polymerase

These experiments are a continuation of work investigating the mechanism of oxidant-induced damage to cultured bovine pulmonary artery endothelial cells (BPEC). Earlier experiments implicated DNA strand breakage and activation of poly(ADP-ribose)polymerase as critical steps in cell injury. In the current report, a better defined model of oxidant stress was used to investigate DNA damage, lipid peroxidation and protein thiol oxidation in BPEC following oxidant stress. The dose and time response of LDH release following exposure to H2O2 were established. H2O2 was metabolized rapidly by BPEC (t1/2 = 20 min). Hydrogen peroxide-induced increases in thiobarbituric acid (TBA) reactive material were prevented by pretreatment with the lipophilic antioxidant diphenylphenylinediamine (DPPD). However, DPPD did not decrease LDH release. Conversely, pretreatment with 5 mM 3-aminobenzamide (3AB), a competitive inhibitor of poly(ADP-ribose)polymerase, prevented LDH release from BPEC following H2O2 treatment. Dithiothreitol (DTT), a sulfhydryl reducing agent, also prevented LDH release. The effects of 3AB and DTT on H2O2-induced changes in DNA strand breaks and NAD+ and ATP levels were investigated as well as the effect of H2O2 on soluble and protein-bound thiols. As DPPD inhibited peroxidation without preventing LDH release, lipid peroxidation does not appear to play a role in the loss of BPEC viability in response to oxidant stress. As protein thiol oxidation was not caused by H2O2, it does not appear to play a causative role in cytotoxicity, although DTT may protect via maintenance of soluble thiols. H2O2 induces DNA strand breaks, which activate poly(ADP-ribose)polymerase, leading to depletion of cellular NAD+ and ATP and loss in cell viability. This supports earlier studies implicating the activation of poly(ADP-ribose)polymerase in oxidant injury to cultured endothelial cells.     

tert–butylhydroperoxide—Induced toxicity in isolated hepatocytes: Contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also, occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide–induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

Glutathione depletion and the production of reactive oxygen species in isolated hepatocyte suspensions

Diethyl maleate (DEM) (5 mM) and ethyl methanesulfonate (EMS) (35 mM) treatments rapidly depleted cellular reduced glutathione (GSH) below detectable levels (1 nmol/10(6) cells), and induced lipid peroxidation and necrotic cell death in freshly isolated rat hepatocytes. In hepatocytes incubated with 2.5 mM DEM and 10 mM EMS, however, the complete depletion of cellular GSH observed was not sufficient to induce lipid peroxidation or cell death. Instead, DEM- and EMS-induced lipid peroxidation and cell death were dependent on increased reactive oxygen species (ROS) production as measured by increases in dichlorofluorescein fluorescence. The addition of antioxidants (vitamin E succinate and deferoxamine) prevented lipid peroxidation and cell death, suggesting that lipid peroxidation is involved in the sequence of events leading to necrotic cell death induced by DEM and EMS. To investigate the subcellular site of ROS generation, the cytochrome P450 inhibitor, SKF525A, was found to reduce EMS-induced lipid peroxidation but did not protect against the loss of cell viability, suggesting a mitochondrial origin for the toxic lipid peroxidation event. In agreement with this conclusion, mitochondrial electron transport inhibitors (rotenone, thenoyltrifluoroacetone and antimycin A) increased EMS-induced lipid peroxidation and cell death, while the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, blocked EMS- and DEM-mediated ROS production and lipid peroxidation. Furthermore, EMS treatment resulted in the significant loss of mitochondrial alpha-tocopherol shortly after its addition, and this loss preceded losses in cellular alpha-tocopherol levels. Treatment of hepatocytes with cyclosporin A, a mitochondrial permeability transition inhibitor, oxypurinol, a xanthine oxidase inhibitor, or BAPTA-AM, a calcium chelator, provided no protection against EMS-induced cell death or lipid peroxidation. Our results indicate that DEM and EMS induce cell death by a similar mechanism, which is dependent on the induction of ROS production and lipid peroxidation, and mitochondria are the major source for this toxic ROS generation. Cellular GSH depletion in itself does not appear to be responsible for the large increases in ROS production and lipid peroxidation observed.     

Toxic consequence of the abrupt depletion of glutathione in cultured rat hepatocytes

Cultured hepatocytes were exposed to two chemicals, dinitrofluorobenzene (DNFB) and diethyl maleate (DEM), that abruptly deplete cellular stores of glutathione. Upon the loss of GSH, lipid peroxidation was evidenced by an accumulation of malondialdehyde in the cultures followed by the death of the hepatocytes. Pretreatment of the hepatocytes with a ferric iron chelator, deferoxamine, or the addition of an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), to the culture medium prevented both the lipid peroxidation and the cell death produced by either DNFB or DEM. However, neither deferoxamine nor DPPD prevented the depletion of GSH caused by either agent. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or inhibition of catalase by aminotriazole sensitized the hepatocytes to the cytotoxicity of DNFB. In a similar manner, pretreatment with BCNU potentiated the cell killing by DEM. DPPD and deferoxamine protected hepatocytes pretreated with BCNU and then exposed to DNFB or DEM. These data indicate that an abrupt depletion of GSH leads to lipid peroxidation and cell death in cultured hepatocytes. It is proposed that GSH depletion sensitizes the hepatocyte to its constitutive flux of partially reduced oxygen species. Such an oxidative stress is normally detoxified by GSH-dependent mechanisms. However, with GSH depletion these activated oxygen species are toxic as a result of the iron-dependent formation of a potent oxidizing species.     

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.     

Biochemical mechanism of irreversible cell injury caused by free radical-initiated reactions

Effects of oxidative stress on isolated rat ventricular myocytes were studied. Myocyte viability was determined by the ability of these cells to retain rod-shaped morphology and to exclude trypan blue. The mean life time of myocytes was quantitated using the Weibull distribution function. Superfusion with 200 M tert-butyl hydroperoxide (t-BHP) led to a time-dependent loss of cell viability, generation of the products of lipid peroxidation, oxidation of protein and non-protein thiols, a decrease in [ATP]_i and in the cellular energy charge. Dithiothreitol (DTT, 5 mM) prolonged survival of myocytes exposed to t-BHP, attenuated oxidation of protein and non-protein thiols, and preserved the energy charge. Exposure to DTT did not affect the concentration of t-BHP-generated lipid peroxidation products. Promethazine (1 M) prevented t-BHP-induced increase in the concentration of lipid peroxidation products, but did not prevent either loss of thiols or loss of cell viability. Superfusion with N-ethylmaleimide (NEM, 5 M) also led to loss of cell viability, with accompanying decreases in protein and non-protein thiols, ATP and energy charge without the accumulation of the products of lipid peroxidation. Superfusion with FeSO₄ (400 M) and ascorbate (1 mM), (Fe-Asc) did not result in loss of cell viability or a decrease protein thiols or the energy charge. Superfusion with Fe-Asc, did, however, lead to a slight decrease in the concentration of non-protein thiols and ATP and a large increase in the concentration of lipid peroxidation products. Accumulation of lipid peroxidation products induced by Fe-Asc was prevented by promethazine. These results indicate that free radical-induced irreversible cell injury results from a loss of protein thiols. Changes in the cellular energy charge and lipid peroxidation do not bear a simple relationship to the survival of cardiac myocytes under oxidative stress.     

Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.     

Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts

Here we report on the marked protective effect of resveratrol on 4-hydroxynonenal (4-HNE) induced oxidative stress and apoptotic death in Swiss 3T3 fibroblasts. 4-HNE, one of the major aldehydic products of the peroxidation of membrane w-6 polyunsaturated fatty acids, has been suggested to contribute to oxidant stress mediated cell injury. Indeed, in vitro treatment of 3T3 fibroblasts with 4-HNE induced a condition of oxidative stress as monitored by the oxidation of dichlorofluorescein diacetate; this reaction was prevented when cells were pretreated with resveratrol. Further, 4-HNE-treated fibroblasts eventually underwent apoptotic death as determined by differential staining and internucleosomal DNA fragmentation. Resveratrol pretreatment also prevented 4-HNE induced DNA fragmentation and apoptosis. These observations are consistent with a potential role of lipid peroxidation-derived products in programmed cell death and demonstrate that resveratrol can counteract this effect by quenching cell oxidative stress.     

Glutathione depleting agents and lipid peroxidation

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

Redox regulation of the signaling pathways leading to eNOS phosphorylation

Oxidative stress mediates positive and negative effects on physiological processes. Recent reports show that H(2)O(2) induces phosphorylation and activation of endothelial nitric oxide synthase (eNOS) through an Akt-phosphorylation-dependent pathway. In this study, we assessed activation of eNOS and Akt by determining their phosphorylation status. Whereas moderate levels of H(2)O(2) (100 microM) activated the Akt/eNOS pathway, higher levels (500 microM) did not, suggesting differential effects by differing levels of oxidative stress. We then found that two pro-oxidants with activity on sulfhydryl groups, 1-chloro-2,4-dinitrobenzene (CDNB) and diethyl maleate (DEM), blocked the phosphorylation events induced by 100 microM H(2)O(2). GSH was not a target thiol in this system because buthionine sulfoximine did not inhibit this phosphorylation. However, down-regulation of cell membrane surface and intracellular free thiols was associated with the inhibition of phosphorylation, suggesting that oxidation of non-GSH thiols inhibits the H(2)O(2)-induced phosphorylation of eNOS and Akt. DTT reversed the inhibitory effects of CDNB and DEM on Akt phosphorylation and concomitantly restored cell surface thiol levels more efficiently than it restored intracellular thiols, suggesting a more prominent role for the former. Similarly, DEM and CDNB inhibited TNF-alpha-induced Akt and eNOS phosphorylation, suggesting that thiol modification is involved in eNOS inductive pathways. Our findings suggest that eNOS activation is exquisitely sensitive to regulation by redox and that cell surface thiols, other than glutathione, regulate signal transduction leading to phosphorylation of Akt and eNOS.     

Changes in protein and nonprotein thiol contents in bladder, kidney and liver of mice by the pesticide sodium-o-phenylphenol and their possible role in cellular toxicity.

Acute treatment of mice with Na-o-phenylphenol or phenylbenzoquinone, an electrophilic metabolite of o-phenylphenol, resulted in differential depletion of contents of protein and nonprotein thiols in bladder, kidney and liver. Maximum decrease in the levels of protein and nonprotein reduced thiols was observed in bladder (by both agents) and was followed by kidney (by both agents) and liver (phenylbenzoquinone only). The reason for this differential changes in reduced thiol contents remains to be understood. The content of protein and nonprotein disulfides was higher in bladder of mice treated with Na-o-phenylphenol compared to that observed in untreated mice bladder. Phenyl 2,5'-p-benzoquinone mediated in vivo depletion of nonprotein and protein thiols suggests that Na-o-phenylphenol treatment may decrease in vivo thiols via the formation of phenylbenzoquinone. Increased disulfide formation is considered to represent an index of oxidative stress produced by chemical. Increases in the level of protein and nonprotein disulfides in bladder suggest as observed in this study that administration of Na-o-phenylphenol to mice produced oxidative stress in bladder. Products of redox cycling of xenobiotics are known to cause cellular toxicity via altering the homeostasis of thiol status. Therefore, it is concluded that decreases in protein thiol contents either via alkylation and/or oxidation of sulfhydryl groups of proteins and increases in disulfide contents presumably by products of redox cycling of Na-o-phenylphenol may play a role in Na-o-phenylphenol-induced cellular toxicity.     

Mechanism of sulfite cytotoxicity in isolated rat hepatocytes

Sulfite (SO(3)(2-)) has been widely used as preservative and antimicrobial in preventing browning of foods and beverages. SO(2), a common air pollutant, also is capable of producing sulfite and bisulfite depending on the pH of solutions. A molybdenum-dependent mitochondrial enzyme, sulfite oxidase, oxidizes sulfite to inorganic sulfate and prevents its toxic effects. In the present study, sulfite toxicity towards isolated rat hepatocytes was markedly increased by partial inhibition of cytochrome a/a(3) by cyanide or by putting rats on a high-tungsten/low-molybdenum diet, which result in inactivation of sulfite oxidase. Sulfite cytotoxicity was accompanied by a rapid disappearance of GSSG followed by a slow depletion of reduced glutathione (GSH). Depleting hepatocyte GSH beforehand increased cytotoxicity of sulfite. On the other hand, dithiothreitol (DTT), a thiol reductant, added even 1h after the addition of sulfite to hepatocytes, prevented cell death and restored hepatocyte GSH levels. Sulfite cytotoxicity was also accompanied by an increase of oxygen uptake, reactive oxygen species (ROS) formation and lipid peroxidation. Cytochrome P450 inhibitors, metyrapone and piperonyl butoxide also prevented sulfite-induced cytotoxicity and lipid peroxidation. Desferroxamine and antioxidants also protected the cells against sulfite toxicity. These findings suggest that cytotoxicity of sulfite is mediated by free radicals as ROS formation increases by sulfite and antioxidants prevent its toxicity. Reaction of sulfite or its free radical metabolite with disulfide bonds of GSSG and GSH results in the compromise of GSH/GSSG antioxidant system leaving the cell susceptible to oxidative stress. Restoring GSH content of the cell or protein-SH groups by DTT can prevent sulfite cytotoxicity.     

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.     

Fluorescent localization of thiols and disulfides in marsupial spermatozoa by bromobimane labelling

The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membranepermeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa. The nucleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley-Liss, Inc.     

Cocaine-induced biochemical changes and cytotoxicity in hepatocytes isolated from both mice and rats

The mechanism of cocaine-induced cytotoxicity was investigated in hepatocytes isolated from both male C3H mice and male Sprague-Dawley rats. Cocaine was more cytotoxic to mouse hepatocytes than rat and induced reduced glutathione (GSH) depletion prior to marked increases in cytotoxicity in both systems. In both mouse and rat cells, GSH depletion was accompanied by GSSG production, but in rat cells, quantitative measures suggested that other mechanisms contributed to GSH depletion. No cocaine-induced depletion of protein-thiol groups or generation of protein-glutathione mixed disulfides could be detected in rat cells. Cocaine induced lipid peroxidation, using malondialdehyde (MDA) production as an index of the peroxidation process, in both mouse and rat hepatocytes. Inhibition of MDA production to below control levels using the antioxidant N,N'-diphenyl-phenylene diamine (DPPD) however, had no inhibitory effect on cocaine-induced cytotoxicity in either mouse or rat cells. These data suggest that neither generalized protein thiol depletion nor lipid peroxidation are critical determinants of cocaine-induced cytotoxicity in cellular systems.     

Palladacycles catalyse the oxidation of critical thiols of the mitochondrial membrane proteins and lead to mitochondrial permeabilization and cytochrome c release associated with apoptosis

Permeabilization of the mitochondrial membrane has been extensively associated with necrotic and apoptotic cell death. Similarly to what had been previously observed for B16F10-Nex2 murine melanoma cells, PdC (palladacycle compounds) obtained from the reaction of dmpa (N,N-dimethyl-1-phenethylamine) with the dppe [1,2-ethanebis(diphenylphosphine)] were able to induce apoptosis in HTC (hepatoma, tissue culture) cells, presenting anticancer activity in vitro. To elucidate cell site-specific actions of dmpa:dppe that could respond to the induction of apoptosis in cancer cells in the present study, we investigated the effects of PdC on isolated RLM (rat liver mitochondria). Our results showed that these palladacycles are able to induce a Ca2+-independent mitochondrial swelling that was not inhibited by ADP, Mg2+ and antioxidants. However, the PdC-induced mitochondrial permeabilization was partially prevented by pre-incubation with CsA (cyclosporin A), NEM (N-ethylmaleimide) and bongkreic acid and totally prevented by DTT (dithiothreitol). A decrease in the content of reduced thiol groups of the mitochondrial membrane proteins was also observed, as well as the presence of membrane protein aggregates in SDS/PAGE without lipid and GSH oxidation. FTIR (Fourier-transform IR) analysis of PdC-treated RLM demonstrated the formation of disulfide bonds between critical thiols in mitochondrial membrane proteins. Associated with the mitochondrial permeabilization, PdC also induced the release of cytochrome c, which is sensitive to inhibition by DTT. Besides the contribution to clarify the pro-apoptotic mechanism of PdC, this study shows that the catalysis of specific protein thiol cross-linkage is enough to induce mitochondrial permeabilization and cytochrome c release.