HOCl causes necrotic cell death in human monocyte derived macrophages through calcium dependent calpain activation

The abundance of dead macrophages in close proximity to HOCl-modified proteins in advanced atherosclerotic plaques implicates HOCl in the killing of macrophages and the formation of the necrotic core region. The mechanism of HOCl mediated death of macrophages was unknown, so using human monocyte derived macrophages (HMDM) we here have shown that HOCl causes a rapid necrotic cell death characterized by loss of MTT reduction, cellular ATP and cell lysis without caspase-3 activation in HMDM cells. The HOCl causes a rise in cytosolic calcium level via the plasma membrane L- and T-type calcium channels and endoplasmic reticulum RyR channel. Blocking of the calcium channels or the addition of calpain inhibitors prevents the HOCl mediated loss of mitochondrial potential, lysosome failure and HMDM cell death. Blocking MPT-pore formation with cyclosporin A also prevents the loss of mitochondrial membrane potential, lysosomal destabilization and HMDM cell death. Blocking the calcium mitochondrial uniporter with ruthenium red also blocks the loss of mitochondrial potential but only at high concentrations. HOCl appears to cause HMDM cell death through destabilization of cytosolic calcium control resulting in the failure of both the mitochondria and lysosomes.     

Pharmacological intervention in oxidant-induced calcium pump dysfunction of dog heart

Micromolar concentrations of HOCl, an oxidant produced by activated neutrophils, inhibited Ca2+ uptake and Ca2+ATPase of isolated dog heart sarcoplasmic reticulum (SR). DTT antagonized completely the HOCl effect only when it was given within 5 min after the addition of HOCl. When the pharmacological intervention was delayed, the recovery with DTT was not complete, and administration of DTT 30 min after the start of HOCl's reaction with SR resulted in only a small improvement in SR Ca2+ uptake. Although H2O2 and Fe ion-chelate (a free radical-generating procedure) also inhibited Ca2+ uptake and ATPase, the concentrations required were very large. The response of cardiac sarcolemmal and skeletal muscle SR calcium pumps to oxidants was similar to that of the cardiac SR calcium pump.     

Mitochondrial metabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol

Mitochondria isolated from rat heart, liver, kidney and brain (respiratory control 4.0-6.5) release NO and H2O2 at rates that depend on the mitochondrial metabolic state: releases are higher in state 4, about 1.7-2.0 times for NO and 4-16 times for H2O2, than in state 3. NO release in rat liver mitochondria showed an exponential dependence on membrane potential in the range 55 to 180 mV, as determined by Rh-123 fluorescence. A similar behavior was reported for mitochondrial H2O2 production by [S.S. Korshunov, V.P. Skulachev, A.A. Starkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15_18.]. Transition from state 4 to state 3 of brain cortex mitochondria was associated to a decrease in NO release (50%) and in membrane potential (24-53%), this latter determined by flow cytometry and DiOC6 and JC-1 fluorescence. The fraction of cytosolic NO provided by diffusion from mitochondria was 61% in heart, 47% in liver, 30% in kidney, and 18% in brain. The data supports the speculation that NO and H2O2 report a high mitochondrial energy charge to the cytosol. Regulation of mtNOS activity by membrane potential makes mtNOS a regulable enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.     

Compartmentation of citrate in relation to the regulation of glycolysis and the mitochondrial transmembrane proton electrochemical potential gradient in isolated perfused rat heart

Subcellular fractionation of tissue in nonaqueous media was employed to study metabolite compartmentation in isolated perfused rat hearts. The mitochondrial and cytosolic concentrations of citrate and 2-oxoglutarate, total concentrations of the glycolytic intermediates and rate of glycolysis were measured in connection with changes in the rate of cellular respiration upon modulation of the ATP consumption by changes of the mechanical work load of the heart. The concentrations of citrate and 2-oxoglutarate in the mitochondria were 16- and 14-fold, respectively, greater than those in the cytosol of beating hearts. The cytosolic citrate concentration was low compared with concentrations which have been employed in demonstrations of the citrate inhibition of glycolysis. In spite of the low activities reported for the tricarboxylate carrier in heart mitochondria, the cytosolic citrate concentration reacted to perturbations of the mitochondrial citrate concentration, and inhibition of glycolysis at the phosphofructokinase step could be observed concomitantly with an increase in the cytosolic citrate concentration. The ΔpH across the inner mitochondrial membrane calculated from the 2-oxoglutarate concentration gradient and the mitochondrial membrane potential calculated from the adenylate distribution gave an electrochemical potential difference of protons compatible with chemiosmotic coupling in the intact myocardium.     

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.     

Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart

A method is described for the preparation of ;free' and ;synaptosomal' brain mitochondria from fractions of guinea-pig cerebral cortex respectively depleted and enriched in synaptosomes. Both preparations of mitochondria have a low membrane H(+) conductance, a high capacity to phosphorylate ADP, and a capacity to accumulate Ca(2+) at rates limited by the activity of the respiratory chain. Ca(2+) transport by ;free' brain mitochondria is compared with that of heart mitochondria. The Ca(2+) conductance of ;free' brain mitochondria was at least 20 times that for rat heart mitochondria. Ca(2+) uptake by brain mitochondria increased the pH gradient and decreased membrane potential, whereas little change occurred during the much slower uptake by heart mitochondria. In the presence of ionophore A23187, dissipative Ca(2+) cycling decreased the H(+) electrochemical potential gradient of brain mitochondria from 190 to 60mV, but caused only a slight decrease with heart mitochondria, although the ionophore lowered the pH gradient and increased membrane potential. The Ca(2+) conductance of ;free' brain mitochondria is distinctive in showing a hyperbolic dependency on free Ca(2+) concentration. In the presence of Ruthenium Red, a rapid Na(+)-dependent Ca(2+) efflux occurs. The H(+) electrochemical potential gradient is maintained during this efflux, and membrane potential increases, with both ;free' brain and heart mitochondria. The Na(+) requirement for Ca(2+) efflux appears not to be related to the high Na(+)/H(+) exchange activity but may represent a direct exchange of Na(+) for Ca(2+).     

Enterocyte mitochondrial dysfunction due to oxidative stress

The endogenous production of H2O2 in isolated rat intestinal mitochondria and oxidant induced damage to mitochondria were examined. There was an appreciable amount of H2O2 production in presence of succinate, glutamate and pyruvate, while the presence of rotenone with succinate further increased production. Superoxide generated by the X-XO system induced membrane permeability transition (MPT), calcium influx, lipid peroxidation and changes in membrane fluidity in mitochondria. A decreased mitochondrial ATPase activity and uncoupling of respiration was also observed. Spermine inhibited swelling induced by X-XO and also blocked the calcium influx and reversed the membrane fluidity changes.     

Acidification reduces mitochondrial calcium uptake in rat cardiac mitochondria

Cardiac ischemia-reperfusion (I/R) injury is accompanied by intracellular acidification that can lead to cytosolic and mitochondrial calcium overload. However, the effect of cytosolic acidification on mitochondrial pH (pHm) and mitochondrial Ca2+ (Cam2+) handling is not well understood. In the present study, we tested the hypothesis that changes in pHm during cytosolic acidification can modulate Cam2+ handling in cardiac mitochondria. pHm was measured in permeabilized rat ventricular myocytes with the use of confocal microscopy and the pH-sensitive fluorescent probe carboxyseminaphthorhodafluor-1. The contributions of the mitochondrial Na+/H+ exchanger (NHEm) and the K+/H+ exchanger (KHEm) to pHm regulation were evaluated using acidification and recovery protocols to mimic the changes in pH observed during I/R. Cam2+ transport in isolated mitochondria was measured using spectrophotometry and fluorimetry, and the mitochondrial membrane potential was measured using a tetraphenylphosphonium electrode. Cytosolic acidification (pH 6.8) resulted in acidification of mitochondria. The degree of mitochondrial acidification and recovery was found to be largely dependent on the activity of the KHEm. However, the NHEm was observed to contribute to the recovery of pHm following acidification in K+-free solutions as well as the maintenance of pHm during respiratory inhibition. Acidification resulted in mitochondrial depolarization and a decrease in the rate of net Cam2+ uptake, whereas restoration of pH following acidification increased Cam2+ uptake. These findings are consistent with an important role for cytosolic acidification in determining pHm and Cam2+ handling in cardiac mitochondria under conditions of Ca2+ overload. Consequently, interventions that alter pHm can limit Cam2+ overload and injury during I/R.     

Interaction of electron leak and proton leak in respiratory chain of mitochondria——Proton leak induced by superoxide from an electron leak pathway of univalent reduction of oxygen

By incubating the isolated rat myocardial mitochondria with xanthine-xanthine oxidase, anexogenous superoxide (O2) generating system, and by ischemia-reperfusion procedure of isolated rat heart as an endogenous O2 generating system, it was found that both sources of O2 showed the same injurious effects on mitochondrial function resulting in (i) increasing proton leak rate, lowering proton pumping activity and Ht/2e ratio of respiratory chain, and (ii) decreasing transmembrane potential of energized mitochondria] inner membrane by succinate oxidation. The injurious effects of O2 on these mitochondrial bioenergitical parameters mentioned above exhibited a dosage- or reaction time-dependent mode. (X has no effects on the electron transfer activity and transmembrane potential of nonenergized mitochondria. Being a superoxide scavenger, 3, 4-dihydroxylphenyl lactate showed obvious protection effects against damage of both exogenous superoxide sources from xanthine-xanthine oxidase system and endogenous Or sou     

Oxidant-induced mobilization of zinc from metallothionein.

Neutrophils which accumulate at sites of inflammation secrete a number of injurious oxidants which are highly reactive with protein sulfhydryls. The present study examined the possibility that this reactivity with thiols may cause protein damage by mobilizing zinc from cellular metalloproteins in which the metal is bound to cysteine. The ability of the three principal neutrophil oxidants, hypochlorous acid (HOCl), superoxide (.O2-), and hydrogen peroxide (H2O2), to cleave thiolate bonds and mobilize complexed zinc was compared using two model compounds (2,3-dimercaptopropanol and metallothionein peptide fragment 56-61), as well as metallothionein. With all compounds, 50 microM HOCl caused high rates of Zn2+ mobilization as measured spectrophotometrically with the metallochromic indicator 4-(2-pyridylazo)resorcinol. Xanthine (500 microM) plus xanthine oxidase (30 mU), which produced a similar concentration of .O2-, also effected a rapid rate of Zn2+ mobilization which was inhibited by superoxide dismutase but not catalase, indicating that .O2- is also highly reactive with thiolate bonds. In contrast, H2O2 alone was much less reactive at comparable concentrations. These data suggest that HOCl and .O2- can cause damage to cellular metalloproteins through the mobilization of complexed zinc. In view of the essential role played by zinc in numerous cellular processes, Zn2+ mobilization by neutrophil oxidants may cause significant cellular injury at sites of inflammation.     

Oxidation of proteins in rat heart and lungs by polymorphonuclear leukocyte oxidants

Summary The ability of the polymorphonuclear leukocyte (PMN) oxidants, hypochlorous acid (HOC1) and hydrogen peroxide (H₂O₂), to oxidize proteins in rat heart and lung tissues was investigated. Cardiac myocytes, heart tissue slices, isolated perfused hearts, and lung tissue slices, were treated with HOCI and H₂O₂ and the extent of methionine and cysteine oxidation was determined in the cellular proteins. Cardiac tissues were found to be highly susceptible to oxidation by physiological concentrations of HOCl. For example, in isolated hearts perfused for 60 min with 100 M HOCI, approximately 18010 of the methionine and 2801o of the cysteine residues were oxidized. Lung tissues, unlike those of the heart, were resistant to physiological concentrations of HOCI, showing no oxidation of proteins. HOCI was much more effective than H₂O₂ in oxidizing proteins, suggesting that HOCI may be the most reactive oxidant produced by activated PMN. These studies show that PMN oxidants, in particular HOC I, can cause significant oxidation of proteins in target tissues, and may therefore constitute a primary cause of tissue injury at sites of inflammation. In addition, these studies show that different tissues may have varying susceptibilities to PMN oxidants.     

Fatty acid oxidation in human and rat heart. Comparison of cell-free and cellular systems

Oxidation rates of palmitate and activities of the mitochondrial marker enzymes cytochrome c oxidase and citrate synthase have been determined in homogenates, isolated mitochondria and slices of human and rat heart and in calcium-tolerant rat cardiac myocytes. Homogenates and mitochondria from rat heart showed a 6- and 2.5-fold higher palmitate oxidation rate than the corresponding preparations from human heart. From the palmitate oxidation rates and cytochrome c oxidase and citrate synthase activities as parameters, the mitochondrial protein contents of human and rat heart were calculated to be about 18 and 45 mg/g wet weight, respectively. Based on citrate synthase activities, the fatty acid oxidation rates were about the same in homogenates and isolated mitochondria, much lower in myocytes and lowest in slices. In the cellular systems the palmitate molecule was more completely oxidized than in homogenates or isolated mitochondria. Fatty acid oxidation rates were concentration-dependent in slices, but not with myocytes. With the cellular systems, palmitate oxidation was synergistically stimulated by the addition of carnitine, coenzyme A and ATP to the incubation medium. This stimulation could be attributed only partly to an increased oxidation in damaged cells.     

Intracellular calcium and the relationship to contractility in an avian model of heart failure

Global contractile heart failure was induced in turkey poults by furazolidone feeding (700 ppm). Abnormal calcium regulation appears to be a key factor in the pathophysiology of heart failure, but the cellular mechanisms contributing to changes in calcium fluxes have not been clearly defined. Isolated ventricular myocytes from non-failing and failing hearts were therefore used to determine whether the whole heart and ventricular muscle contractile dysfunctions were realized at the single cell level. Whole cell current- and voltage-clamp techniques were used to evaluate action potential configurations and L-type calcium currents, respectively. Intracellular calcium transients were evaluated in isolated myocytes with fura-2 and in isolated left ventricular muscles using aequorin. Action potential durations were prolonged in failing myocytes, which correspond to slowed cytosolic calcium clearing. Calcium current-voltage relationships were normal in failing myocytes; preliminary evidence suggests that depressed transient outward potassium currents contribute to prolonged action potential durations. The number of calcium channels (as measured by radioligand binding) were also similar in non-failing and failing hearts. Isolated ventricular muscles from failing hearts had enhanced inotropic responses, in a dose-dependent fashion, to a calcium channel agonist (Bay K 8644). These data suggest that changes in intracellular calcium mobilization kinetics and longer calcium-myofilament interaction may be able to compensate for contractile failure. We conclude that the relationship between calcium current density and sarcoplasmic reticulum calcium release is a dynamic process that may be altered in the setting of heart failure at higher contraction rates. Accepted: 1 March 2000     

Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes.

There is evidence that myocardial injury, as would occur on post-ischemic reperfusion, may be caused by the generation of oxygen radicals, as well as by the induction of intracellular calcium overload; however, the relationship between these two mechanisms of injury is not known. To test the hypothesis that oxidants and oxygen radicals can cause cardiac myocyte injury and intracellular calcium overload, isolated adult rat ventricular myocytes were exposed to H2O2 (1-10 mM) and Fe3(+)-nitrilotriacetate. EPR measurements confirmed the production of the highly reactive .OH radical by this system. The oxygen radical generating system initially caused a transient augmentation of twitch amplitude in single field stimulated myocytes. This was followed by contractile oscillations occurring during the twitch prior to full cell relaxation, and spontaneous mechanical oscillations occurring between electrically stimulated contractions. Eventually, cells became inexcitable and abruptly underwent contracture. In the presence of lower bathing calcium concentrations, these oxidant-induced alterations were prevented or delayed. However, cells exposed to the radical generating system in the absence of extracellular calcium still eventually underwent contracture but stimulated contractions or mechanical oscillations were not seen. Measurements in single myocytes loaded with the fluorescent probe of intracellular calcium, Indo-1, demonstrated a rise in both systolic and diastolic fluorescence ratio, as well as oscillations and widening of the fluorescence transient, suggestive of cellular calcium loading, following exposure to the radical generating system. Injured myocytes did not take up trypan blue dye. Contractile dysfunction and calcium channel blocker, nitrendipine. NMR measurements of cellular [ATP] demonstrated that these alterations in cellular calcium preceded the depletion of ATP. Subsequent depletion of ATP was accompanied by the appearance of increased concentrations of sugar phosphates indicative of a block in glycolysis and ATP depletion correlated with cellular rigor. Thus, oxygen free radicals can cause cardiac myocyte injury with contractile abnormalities which occur due to myocyte calcium loading. The mechanism of oxidant-induced calcium loading is not due to nonspecific membrane damage, or energy depletion, but rather due to increased calcium influx through voltage gated calcium channels. This early calcium overload state as well as oxidant induced block of glycolysis result in cellular energy depletion and cell death with the induction of contracture.     

Co-oxidation of luminol by hypochlorite and hydrogen peroxide implications for neutrophil chemiluminescence

Stimulated neutrophils produce several potent oxidants including H2O2, O2- and HOCl. Previous studies have revealed all of these compounds to be capable of oxidizing luminol, a reagent often used to indicate, by its chemiluminescence, the oxidative burst of neutrophils. Data presented in this paper indicate that H2O2 and HOCl spontaneously react at physiologic pH to produce luminol-dependent chemiluminescence 100 times the sum of the chemiluminescence of either reagent alone. This enhancement is due to a co-oxidation by HOCl and H2O2, or to a novel oxidant generated by the interaction of HOCl and H2O2. The HOCl scavenger, taurine, inhibits the chemiluminescence. Evidence is presented against the participation of hydroxyl radical, O2- or singlet oxygen in the oxidation of luminol by HOCl and H2O2. These findings have implications for potential anti-inflammatory compounds.     

Identification of a ryanodine receptor in rat heart mitochondria

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.     

Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants

An important aspect of myocardial injury is the role of neutrophils in post-ischemic damage to the heart. Stimulated neutrophils initiate a series of reactions that produce toxic oxidizing agents. Superoxide rapidly dismutases to H2O2 and neutrophils contain myeloperoxidase which catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HOCl). The highly reactive HOCl combines non-enzymatically with nitrogenous compounds to generate long-lived, non-radical oxidants, monochloramine and taurine N-monochloramine. We investigated the role of oxygen radicals and long-lived oxidants on cardiac sarcoplasmic reticulum function, which plays a major role in the regulation of intracellular Ca2+ and thereby in the generation of force. Incubation of sarcoplasmic reticulum with phorbol myristate acetate (PMA)-stimulated neutrophils (4 x 10(6) cells/ml) significantly decreased calcium uptake rate (0.85 +/- 0.11 to 0.11 +/- 0.06 mumol/min per mg) and Ca2+-ATPase activity (1.67 +/- 0.08 to 0.46 +/- 0.10 mumol/min per mg). Inclusion of myeloperoxidase inhibitors (cyanide, sodium azide and 3-amino-1,2,4-triazole), catalase, superoxide dismutase plus catalase, and alpha-tocopherol significantly protected (P less than 0.01) calcium uptake rates and Ca2+-ATPase activity of sarcoplasmic reticulum. Superoxide dismutase (10 microgram/ml) alone or deferoxamine (1 mM) had no protective effect in this system. The maximum inhibition of sarcoplasmic reticulum function was observed with (3-4) x 10(6) cells/ml in 4-6 min. HOCl and NH2Cl inhibited calcium uptake rate and Ca2+-ATPase activity of sarcoplasmic reticulum in a dose-dependent manner (2-20 microM), whereas H2O2 damaged sarcoplasmic reticulum at concentrations ranging from 5 to 25 mM. HOCl (20 microM) inhibited 80-90% of Ca2+-uptake rate and Ca2+-ATPase activity and L-methionine (0.1-1 mM) provided complete protection. We conclude that stimulated neutrophils damage cardiac sarcoplasmic function by generation of myeloperoxidase-catalyzed oxidants.     

Inactivation of poly (ADP-ribose) polymerase by hypochlorous acid.

The effects of the phagocyte-derived reactive oxidants hydrogen peroxide (H2O2) and hypochlorous acid (HOC1) on the activity of poly(ADP-ribose) polymerase (pADP RP), an enzyme involved in DNA repair, and on the induction and repair of DNA strand breaks in human mononuclear leukocytes (MNL) have been investigated in vitro. Exposure of MNL to reagent H2O2 was accompanied by DNA damage and activation of pADP RP. Addition of reagent HOCl (25 microM) was not associated with DNA strand breaks. However, when combined with 150 microM H2O2, HOCl potentiated H2O2-mediated DNA damage, and compromised the repair process. Furthermore, HOCl caused a dose-related decrease in the activity of pADP RP in both control and H2O2-exposed MNL. Interactions between the phagocyte-derived reactive oxidants H2O2 and HOCl are probably involved in the etiology of inflammation-related cancer.     

Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury.

HepG2 cells were transfected with vectors containing human catalase cDNA and catalase cDNA with a mitochondrial leader sequence to allow comparison of the effectiveness of catalase overexpressed in the cytosolic or mitochondrial compartments to protect against oxidant-induced injury. Overexpression of catalase in cytosol and in mitochondria was confirmed by Western blot, and activity measurement and stable cell lines were established. The intracellular level of H(2)O(2) induced by exogenously added H(2)O(2) or antimycin A was lower in C33 cell lines overexpressing catalase in the cytosol and mC5 cell lines overexpressing catalase in the mitochondria as compared with Hp cell lines transfected with empty vector. Cell death caused by H(2)O(2), antimycin A, and menadione was considerably suppressed in both the mC5 and C33 cell lines. C33 and mC5 cells were also more resistant to apoptosis induced by H(2)O(2) and to the loss of mitochondrial membrane potential induced by H(2)O(2) and antimycin A. In view of the comparable protection by catalase overexpressed in the cytosol versus the mitochondria, catalase produced in both cellular compartments might act as a sink to decompose H(2)O(2) and move diffusable H(2)O(2) down its concentration gradient. The present study suggests that catalase in cytosol and catalase in mitochondria are capable of protecting HepG2 cells against cytotoxicity or apoptosis induced by oxidative stress.