Depletion of ATP but not of GSH affects viability of rat hepatocytes

The purpose of this study was to examine the role of glutathione depletion and alterations in the energy status in the induction of acute cytotoxicity to freshly isolated rat hepatocytes. Depletion of intracellular glutathione by diethyl maleate and phorone to levels below 5% of control did not induce loss of viability nor loss of intracellular ATP. Ethacrynic acid, a compound known to deplete mitochondrial GSH in addition to cytosolic GSH, induced cell killing after depletion of ATP, next to GSH depletion. The results confirmed that depletion of intracellular glutathione alone does not necessarily result in cell killing. Only when glutathione depletion is succeeded by reduction in ATP levels, loss of cell viability is observed. The relationship between alterations in the energy status and the induction of cell death was further substantiated by inhibition of glycolytic and mitochondrial ATP generation. Treatment of hepatocytes either with iodoacetic acid to inhibit glycolysis (in hepatocytes from fed rats) or with potassium cyanide to inhibit mitochondrial respiration (in hepatocytes from both fed and fasted rats) revealed that depletion of intracellular ATP could lead to lethal cell injury. The susceptibility of cells to metabolic inhibition was better reflected by the rate of reduction in the energy charge than by the reduction of ATP alone. In conclusion, our results suggest that alterations of the energy status may be a critical event in the induction of irreversible cell injury. Depletion of cellular GSH is only cytotoxic when followed by a reduction of the energy charge.     

Depletion of ATP but not of GSH affects viability of rat hepatocytes.

The purpose of this study was to examine the role of glutathione depletion and alterations in the energy status in the induction of acute cytotoxicity to freshly isolated rat hepatocytes. Depletion of intracellular glutathione by diethyl maleate and phorone to levels below 5% of control did not induce loss of viability nor loss of intracellular ATP. Ethacrynic acid, a compound known to deplete mitochondrial GSH in addition to cytosolic GSH, induced cell killing after a depletion of ATP, next to GSH depletion. The results confirmed that depletion of intracellular glutathione alone does not necessarily result in cell killing. Only when glutathione depletion is succeeded by reduction in ATP levels, loss of cell viability is observed. The relationship between alterations in the energy status and the induction of cell death was further substantiated by inhibition of glycolytic and mitochondrial ATP generation. Treatment of hepatocytes either with iodoacetic acid to inhibit glycolysis (in hepatocytes from fed rats) or with potassium cyanide to inhibit mitochondrial respiration (in hepatocytes from both fed and fasted rats) revealed that depletion of intracellular ATP could lead to lethal cell injury. The susceptibility of cells to metabolic inhibition was better reflected by the rate of reduction in the energy charge than by the reduction of ATP alone. In conclusion, our results suggest that alterations of the energy status may be a critical event in the induction of irreversible cell injury. Depletion of cellular GSH is only cytotoxic when followed by a reduction of the energy charge.     

Mitochondrial function in carbon tetrachloride-induced cirrhosis in the rat. Qualitative and quantitative defects

Mitochondrial function is impaired in patients and experimental animals with liver cirrhosis. The relationship between mitochondrial impairment and severity of cirrhosis is unknown, however. We therefore characterized the severity of cirrhosis in rats with phenobarbital/CCl4-induced cirrhosis by the aminopyrine breath test, a microsomal function test reflecting hepatocellular mass. Mitochondrial function was evaluated by measuring oxygen consumption, enzyme activities and ATP production in mitochondria isolated from cirrhotic (N = 8) and control livers (N = 4). Oxygen consumption and mitochondrial enzyme activities calculated per liver were significantly reduced in the presence of cirrhosis. This decrease corresponded to the loss of hepatocytes calculated from the reduction in aminopyrine breath test. The effect of atractylate, oligomycin and dinitrophenol on state 3 respiration was equal between the two groups. The respiratory control ratio was significantly reduced in mitochondria from cirrhotic livers with beta-hydroxybutyrate (4.01 +/- 0.94 vs 5.45 +/- 0.40), but not with succinate as substrate. The rate of ATP production was significantly decreased in mitochondria from cirrhotic rats for both substrates. In contrast, the static head (state 4) phosphate potential was fully developed after 10 min and was equal between the two groups. We conclude that cirrhosis of the liver leads to a loss of hepatocytes which is paralleled by reduced oxygen uptake and reduced mitochondrial enzyme activities.     

Oligomycin-induced proton uncoupling

Oligomycin is a classical mitochondrial reagent that binds to the proton channel on the F_o component of ATP synthase. As a result, oligomycin blocks mitochondrial ATP synthesis, proton translocation, and O₂ uptake. Here we show that oligomycin induces proton uncoupling subsequent to inhibition of ATP synthesis, as evidenced by recovery of O₂ uptake to near baseline levels. Uncoupling is uniquely rapid and readily observed in HepG2 cells but is also observed at longer times in the unrelated H1299 cell line. Proton fluxes plateau at oligomycin concentrations in the region 0.25–5 μM. At the plateau, fluxes are lower than expected for the classical mitochondrial permeability transition pore, although in H1229 cells, fluxes increase to levels consistent with pore opening at higher oligomycin concentrations. Uncoupling is observed in cells metabolizing either pyruvate or lactate and reversed by addition of glucose to restore ATP synthesis. Uncoupling is not sensitive to cyclosporin A and is not reversed by the ANT inhibitor bongkrekic acid. However, bongkrekic acid inhibits uncoupling if added before oligomycin, which we interpret in terms of maintenance of mitochondrial ATP levels.     

The effects of fructose on adenosine triphosphate depletion following mitochondrial dysfunction and lethal cell injury in isolated rat hepatocytes

Mitochondrial injury in aerobic mammalian cells is associated with a rapid depletion of adenosine triphosphate (ATP) which occurs prior to the onset of lethal cell injury. In this report, the relationships between ATP depletion and lethal cell injury were examined in rat hepatocytes using oligomycin as a model mitochondrial toxicant and fructose as an alternative carbohydrate source for glycolysis. Oligomycin was more potent in causing lethal cell injury in hepatocytes isolated from fasted animals than cells from fed animals. The onset of cell injury (leakage of lactate dehydrogenase) in cells from fed animals correlated with the depletion of stored glycogen and ATP. The degree and time course profile of oligomycin-induced ATP depletion could be duplicated with 50 mM fructose alone in hepatocytes from fasted animals; however, fructose did not cause lethal cell injury. Oligomycin caused marked accumulation of adenosine monophosphate (AMP) and inorganic phosphate (Pi) and a conservation of adenine nucleotides. In contrast, fructose (50 mM) caused a decrease in Pi, no persistent change in AMP, and a depletion of the adenine nucleotide pool. Fructose, at concentrations greater than 1.0 mM, protected hepatocytes from oligomycin-induced toxicity. Blockade of mitochondrial ATP synthesis with oligomycin resulted in massive ATP depletion. In the presence of oligomycin, 5.0 mM fructose maintained cellular ATP content similar to that of control cells, whereas 50 mM fructose did not, demonstrating the biphasic effect of increasing fructose concentrations on cellular ATP content. Fructose-induced protection of hepatocytes from oligomycin toxicity was due to glycolytic fructose metabolism as hepatocytes incubated with iodoacetate (30 microM), fructose, and oligomycin had reduced viability and ATP content. In conclusion, interruption of mitochondrial ATP synthesis leads to marked ATP depletion and lethal cell injury. Cell injury is clearly not due to ATP depletion alone since increased glycolytic ATP production from either glycogen or fructose can maintain cell integrity in the absence of mitochondrial ATP synthesis and at low cellular ATP levels.     

PHARMACOLOGICAL PROPERTIES OF THE NATURAL MARINE PRODUCT FUROSPONGIN-1

1. The natural marine product, furospongin-1 (6, 12 and 24.5 μmol/L) significantly inhibited contractions of segments of guinea-pig ileum induced by submaximal concentrations (0.1 μmol/L) of acetylcholine (ACh) and histamine. Furospongin-1 (24.5 and 36.7 μmol/L) reduced both the phasic and tonic components of a contraction induced by 30 mmol/L K⁺ solution in the absence and presence of atropine (1 μmol/L), mepyramine (1 μmol/L) and phentolamine (1 μmol/L). Furospongin-1 also decreased basal tension and the amplitude of spontaneous phasic contractions of guinea-pig ileum. 2. The mitochondrial ATP synthase inhibitor oligomycin (0.3, 1 and 3 μmol/ L) had a similar concentration-dependent action, reducing basal activity and contractions evoked by histamine and ACh. Oligomycin also reduced both the phasic and tonic components of a contraction induced by 30 mmol/L K⁺ solution in the absence and presence of atropine (1 μmol/L), mepyramine (1 μmol/L) and phentolamine (1 μmol/L). 3. Furospongin-l (6 and 37.6 μmol/L) and oligomycin (3 μmol/L) had no effect on contractions of chemically skinned guinea-pig ileum longitudinal muscle segments. In this same tissue, furospongin-1 (6, 12 and 24.5 μmol/L) and oligomycin (0.3, 1 and 3 μmol/L) concentration-dependently reduced tissue levels of ATP. 4. In lyzed bovine mitochondria, oligomycin (0.1, 0.3, 1 and 3 μmol/L) inhibited conversion of ATP to ADP whilst furospongin-1 (6, 12 and 24.5 μmol/L) and carbonyl cyanide m-chlorophenylhydrazone (0.5 mmol/L) had no significant effect on ATP breakdown. 5. Furospongin-1 (12 and 24.5 μmol/L) and oligomycin (3 μmol/L) had no effect on State 4 mitochondrial respiration of whole bovine mitochondria, but converted State 3 back to State 4 respiration. Carbonyl cyanide m-chlorophenyl-hydrazone (0.5 mmol/L) increased oxygen consumption in both situations. 6. These results suggest that furospongin-1 may inhibit the activity of inner mitochondrial membrane ADP-ATP translocases or other membrane transporters to inhibit ATP synthesis.     

Levosimendan is a mitochondrial K(ATP) channel opener.

Levosimendan, a new inodilator developed for the treatment of heart failure has been shown to have a vasodilatory effect via opening of K(ATP) channels in the plasma membrane of vascular smooth muscle cells. In this study, we investigated the effects of levosimendan on the mitochondrial K(ATP) channel. This compound did not influence mitochondrial transmembrane potential (DeltaPsi), and at up to 2.2 microM had no effect on the respiration rate of rat liver mitochondria, respiring on 5 mM succinate (+5 microM rotenone). A sensitive method was developed for assessing K(ATP) channel opening activity employing rat liver mitochondria, respiring only on endogenous substrates in the presence of 400 microM ATP and 1 microg oligomycin/mg mitochondrial protein. In this model, levosimendan (0.7-2.6 microM) decreased DeltaPsi by 6.5-40.4% (n=3, incubation time 15 min). This effect was dependent on the K+ concentration in the incubation medium and was abolished by the selective blocker of the mitochondrial K(ATP) channel-5-hydroxydecanoate (200 microM). Our results indicate that levosimendan opens mitochondrial K(ATP) channels.     

Effects of bepridil on heart mitochondrial membrane and the isolated rat heart preparation

Bepridil (Cordium) was found to activate rat heart mitochondrial membrane-bound ATPase at concentrations of 10 nmol/l-10 mumol/l. By contrast, oligomycin-sensitive ATPase from beef heart mitochondria was inhibited at concentrations of 1-10 mumol/l. In both systems sensitivity toward the inhibitor oligomycin was reduced. Under the influence of the drug, RCR (coupling degree of electron transport to ATP synthesis), ST3 (oxygen uptake in presence of substrate and ADP) and OPR (oxidative phosphorylation rate, amount of ATP synthesized in mitochondrial metabolic state ST3) values are reduced, indicating partial inhibition of oxidative phosphorylation. At 0.25 mumol/l concentration of bepridil, in the isolated normoxic working rat heart preparation aortic flow was reduced to zero. No changes in oxidative phosphorylation parameters were found in mitochondria isolated from these preparations. In the isolated, working rat heart preparation bepridil at a concentration of 0.05 mumol/l reduced aortic flow to about 75% of its original value. In this preparation, no cardioprotective effects (neither on aortic flow nor on mitochondrial function) could be demonstrated during postischemic reperfusion. It is suggested, that in vitro mitochondrial activities of bepridil are not related to in vivo action of the drug.     

Sensitivity of oligomycin-inhibited respiration of isolated rat liver mitochondria to perfluidone, a fluorinated arylalkylsulfonamide

Oxygen electrode polarographic measurements of the rate of oxygen consumption by isolated rat liver mitochondria revealed that oligomycin inhibition of respiration was offset to different degrees by varying concentrations of perfluidone (1,1,1-trifluoro-N-(2 methyl-4-(phenylsulfonyl) methanesulfonamide). Using any of pyruvate-malate, succinate or ascorbate-TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) as substrate, this herbicidal and anti-inflammatory agent at 100 microM concentration caused a 5-fold stimulation of oligomycin-inhibited respiration. Higher concentrations of the herbicide (greater than or equal to 120 microM) gave lower stimulatory effects. Similar stimulatory effects were obtained with 1 microM FCCP (carbonylcyanide p-trifluoromethyoxyphenyl-hydrazone), a classical protonophore. Our results also show an enhanced oligomycin-sensitive ATPase action in intact mitochondria incubated with ATP and varying concentrations of perfluidone. Maximum enhancement effect (111.3%) was obtained at 120 microM perfluidone. FCCP (1 microM) stimulated this ATPase action by 130%. An initial inhibition of respiration by oligomycin is due to an interaction with the proton well of FOF1-ATP synthetase (Lardy, H.A. et al., Arch. Biochem. Biophys., 78 (1953) 587). Perfluidone probably increases the proton conductance of mitochondrial inner membrane in the same manner as FCCP and thus causes an increase in mitochondrial respiratory rate. As protons move into the matrix, delta mu H+, the proton electrochemical potential gradient becomes very small and the F0F1-ATP synthetase functions in the direction of hydrolysis of ATP rather than its shnthesis (Mitchell, P., Eur. J. Biochem., 95 (1979) 1). These findings therefore indicate that perfluidone acts in a way similar to FCCP, a classical uncoupler and protonophore.     

KATP channel openers have opposite effects on mitochondrial respiration under different energetic conditions

Mitochondrial (m) KATP channel opening has been implicated in triggering cardiac preconditioning. Its consequence on mitochondrial respiration, however, remains unclear. We investigated the effects of two different KATP channel openers and antagonists on mitochondrial respiration under two different energetic conditions. Oxygen consumption was measured for complex I (pyruvate/malate) or complex II (succinate with rotenone) substrates in mitochondria from fresh guinea pig hearts. One of two mKATP channel openers, pinacidil or diazoxide, was given before adenosine diphosphate in the absence or presence of an mKATP channel antagonist, glibenclamide or 5-hydroxydecanoate. Without ATP synthase inhibition, both mKATP channel openers differentially attenuated mitochondrial respiration. Neither mKATP channel antagonist abolished these effects. When ATP synthase was inhibited by oligomycin to decrease [ATP], both mKATP channel openers accelerated respiration for both substrate groups. This was abolished by mKATP channel blockade. Thus, under energetically more physiological conditions, the main effect of mKATP channel openers on mitochondrial respiration is differential inhibition independent of mKATP channel opening. In contrast, under energetically less physiological conditions, mKATP channel opening can be evidenced by accelerated respiration and blockade by antagonists. Therefore, the effects of mKATP channel openers on mitochondrial function likely depend on the experimental conditions and the cell's underlying energetic state.     

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: ensuing energetic and oxidative stress implications

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 μM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca²⁺ efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP⁺ transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds.     

Glutathione-dependent reduction of arsenate by glycogen phosphorylase a reaction coupled to glycogenolysis.

Arsenate (As(V)) is reduced in the body to the more toxic arsenite (As(III)). We have shown that two enzymes catalyzing phosphorolytic cleavage of their substrates, namely purine nucleoside phosphorylase and glyceraldehyde-3-phosphate dehydrogenase, can reduce As(V) in presence of an appropriate thiol and their substrates. Another phosphorolytic enzyme that may also reduce As(V) is glycogen phosphorylase (GP). With inorganic phosphate (P(i)), GP catalyzes the breakdown of glycogen to glucose-1-phosphate; however, it also accepts As(V). Testing the hypothesis that GP can reduce As(V), we incubated As(V) with the phosphorylated GPa or the dephosphorylated GPb purified from rabbit muscle and quantified the As(III) formed from As(V) by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. In the presence of adenosine monophosphate (AMP), glycogen, and glutathione (GSH), both GP forms reduced As(V) at rates increasing with enzyme and As(V) concentrations. The As(V) reductase activity of GPa was 10-fold higher than that of GPb. However, incubating GPb with GP kinase and ATP (that converts GPb to GPa) increased As(V) reduction by phosphorylase up to the rate produced by GPa incubated under the same conditions. High concentration of inorganic sulfate, which activates GPb like phosphorylation, also promoted reduction of As(V) by GPb. As(V) reduction by GPa (like As(V) reduction in rats) required GSH. It also required glycogen (substrate for GP) and was stimulated by AMP (allosteric activator of GP) even at low micromolar concentrations. P(i), substrate for GP competing with As(V), inhibited As(III) formation moderately at physiological concentrations. Glucose-1-phosphate, the product of GP-catalyzed glycogenolysis, also decreased As(V) reduction. Summarizing, GP is the third phosphorolytic enzyme identified capable of reducing As(V) in vitro. For reducing As(V) by GP, GSH and glycogen are indispensable, suggesting that the reduction is linked to glycogenolysis. While its in vivo significance remains to be tested, further characterization of the GP-catalyzed As(V) reduction is presented in the adjoining paper.     

Dual actions of the metabolic inhibitor, sodium azide on K(ATP) channel currents in the rat CRI-G1 insulinoma cell line

1. The effects of various inhibitors of the mitochondrial electron transport chain on the activity of ATP-sensitive K+ channels were examined in the Cambridge rat insulinoma G1 (CRI-G1) cell line using a combination of whole cell and single channel recording techniques. 2. Whole cell current clamp recordings, with 5 mM ATP in the pipette, demonstrate that the mitochondrial uncoupler sodium azide (3 mM) rapidly hyperpolarizes CRI-G1 cells with a concomitant increase in K+ conductance. This is due to activation of K(ATP) channels as the sulphonylurea tolbutamide (100 microM) completely reversed the actions of azide. Other inhibitors of the mitochondrial electron transport chain, rotenone (10 microM) or oligomycin (2 microM) did not hyperpolarize CRI-G1 cells or increase K+ conductance. 3. In cell-attached recordings, bath application of 3 mM sodium azide (in the absence of glucose) resulted in a rapid increase in K(ATP) channel activity, an action readily reversible by tolbutamide (100 microM). Application of sodium azide (3 mM), in the presence of Mg-ATP, to the intracellular surface of excised inside-out patches also increased K(ATP) channel activity, in a reversible manner. 4. In contrast, rotenone (10 microM) or oligomycin (2 microM) did not increase K(ATP) channel activity in either cell-attached, in the absence of glucose, or inside-out membrane patch recordings. 5. Addition of sodium azide (3 mM) to the intracellular surface of inside-out membrane patches in the presence of Mg-free ATP or the non-hydrolysable analogue 5'-adenylylimidodiphosphate (AMP-PNP) inhibited, rather than increased, K(ATP) channel activity. 6. In conclusion, sodium azide, but not rotenone or oligomycin, directly activates K(ATP) channels in CRI-G1 insulin secreting cells. This action of azide is similar to that reported previously for diazoxide.     

Pulmonary mixed-function oxidation: stimulation by glucose and the effects of metabolic inhibitors

Substrate requirements for pulmonary mixed-function oxidation of p-nitroanisole to p-nitrophenol were evaluated using the isolated perfused rabbit lung and a lung microsomal fraction. Addition of glucose (5 mM) to the lung perfusate (Krebs bicarbonate buffer) increased the mean rate of p-nitroanisole oxidation by 25–55 per cent; addition of pyruvate (5 mM) or palmitate (0.5 mM) gave similar results. Sucrose (5 mM) had no effect. Antimycin A, KCN, oligomycin and bis-hexafluoroacetonyl acetone (an uncoupling agent) markedly depressed p-nitroanisole metabolism by the isolated lung. KCN also inhibited p-nitroanisole metabolism by lung microsomes, but antimycin A was without effect. These results indicate that pulmonary mixed-function oxidation requires substrate for intermediary metabolism as well as ATP, for maintenance of maximal rates. Glucose and mitochondrial substrates are equally effective in providing the energy requirements and the reducing potential for this reaction.     

Mitochondria play an important role in adenosine-induced ATP release from Madin-Darby canine kidney cells

We previously found that adenosine stimulates ATP release from Madin-Darby canine kidney (MDCK) cells, by activating an Ins(1,4,5)P(3) sensitive-calcium (Ca(2+)) pathway through the stimulation of A(1) receptors. Thus, we investigated the intracellular pathway of ATP efflux after the rise in intracellular Ca(2+) in MDCK cells. Adenosine evoked an increase in mitochondrial Ca(2+) using Rhod-2/AM, a mitochondrial Ca(2+) indicator. Adenosine-induced ATP release was inhibited by mitochondrial modulators, such as oxidative phosphorylation modulators (carbonyl cyanide 3-chlorophenylhydrazone and oligomycin), mitochondrial ADP/ATP carrier inhibitors (N-ethylmaleimide, carboxyatractyloside and bongkrekic acid), a mitochondrial Na(+)-Ca(2+) exchange inhibitor (CGP-37157). In addition, mitochondrial modulators significantly reduced intracellular ATP content. On the other hand, 2-deoxy-glucose (2-DG) induced a greater decrease in intracellular ATP content than mitochondrial modulators. ATP release was still induced by adenosine in the presence of 5mM 2-DG. These results suggest that mitochondria play an important role in the signaling pathway of adenosine-triggered ATP release in MDCK cells.     

Protection by apple peel polyphenols against indometacin‐induced oxidative stress,mitochondrial damage and cytotoxicity in Caco‐2 cells

Objectives Exposure of Caco‐2 cells to indometacin can be a useful model to assess some of the cytotoxic events that appear to underlie the gastrointestinal lesions associated with the use of this anti‐inflammatory agent. Using such a cellular model, we addressed here the cytoprotective potential of a recently standardized apple peel polyphenol extract, APPE. Methods We firstly characterized APPE in terms of its free radical scavenging and antioxidant properties, and subsequently investigated its potential to protect Caco‐2 cells against the deleterious effects of indometacin on cellular oxidative status (redox state, malondialdehyde, glutathione (GSH) and oxidized glutathione (GSSG) levels), mitochondrial function (ATP and mitochondrial membrane potential) and cell viability (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) leakage). For comparative purposes, the free radical scavenging properties and reducing capacity of quercetin, epicatechin and rutin were also estimated. Key findings In the absence of APPE, indometacin induced mitochondrial perturbations (reducing ATP and the mitochondrial membrane potential), enhanced the oxidative status (decreasing the GSH/GSSG ratio and increasing dichlorofluorescein oxidation and malondialdehyde) and lowered the cell viability (decreasing MTT reduction and increasing LDH leakage). APPE, whether pre‐added or co‐incubated with indometacin, concentration‐dependently prevented these mitochondrial, oxidative and cell viability alterations. Prompted by the recently recognized ability of indometacin to enhance the mitochondrial formation of reactive oxygen species, APPE was also characterized in terms of its free radical‐scavenging capacity. APPE was found to actively scavenge O₂·^‐, HO· and peroxyl radicals. Such free radical‐scavenging activity of APPE suggests that its ability to protect mitochondria and prevent the oxidative and lytic damage induced by indometacin arises from its potent antioxidant capacity. Conclusions In Caco‐2 cells APPE prevented mitochondrial oxidative and cell viability alterations induced by indometacin possibly through its ability to scavenge reactive oxygen species. These findings are of interest in view of the high prevalence of gastrointestinal side‐effects associated with the use of conventional anti‐inflammatory agents.     

Zinc toxicity alters mitochondrial metabolism and leads to decreased ATP production in hepatocytes

Although zinc (Zn) is a known environmental toxicant, its impact on the cellular energy-producing machinery is not well established. This study investigated the influence of this divalent metal on the oxidative ATP producing network in human hepatocellular carcinoma (HepG2) cells. Zn-challenged cells contained more oxidized proteins and lipids compared with control cells. Zn severely impeded mitochondrial functions by inhibiting aconitase, alpha-ketoglutarate dehydrogenase, isocitrate dehydrogenase-NAD+ dependent, succinate dehydrogenase and cytochrome C oxidase Zn-exposed cells had a disparate mitochondrial metabolism compared with the control cells and produced significantly less ATP. However, the expression of isocitrate dehydrogenase-NADP+ dependent was more prominent in cells treated with Zn. Hence, Zn-induced pathologies may be due to the inability of the mitochondria to generate energy effectively.     

The toxicity of disulphides to isolated hepatocytes and mitochondria

The disulfide metabolites of thiono-sulfur drugs were found to be about 50 to 100 times more toxic to isolated rat hepatocytes than the corresponding parent drugs. The order of decreasing cytotoxicity for the disulfide metabolites was disulfiram greater than propylthiouracil disulfide greater than formamidine disulfide greater than phenylthiourea disulfide greater than thiobenzamide disulfide greater than cystamine. Depletion of intracellular GSH levels preceded cytotoxicity. GSH could be restored and cytotoxicity averted by adding the thiol reducing dithiothreitol. Depletion of GSH with diethylmaleate potentiated the toxicity of disulfides 3 to 4-fold confirming the protective role of GSH in disulfide toxicity. The toxicity of disulfiram was increased 4-fold in cells pretreated with ATP (0.8 mM) to effect a transient increase in cytosolic Ca2+ suggesting an impairment of Ca2+ homeostasis by the toxicant. Disulfiram (200 microM) rapidly depleted hepatocyte ATP levels within 15 minutes which suggests that ATP production is inhibited. The disulfide effectiveness at causing mitochondrial Ca2+ release was similar to their effectiveness at inducing hepatocyte cytotoxicity. These results suggest that hepatocyte toxicity is the result of oxidative inactivation of membrane protein thiols that regulate intracellular Ca2+ homeostasis.     

Effects of mitochondrial inhibitors on cell viability in U937 monocytes under glucose deprivation

We studied cytotoxic mechanism of mitochondrial inhibitors in U937 cells. U937 cells were sensitive to cytotoxicity of mitochondrial inhibitors under glucose deprivation condition, whereas PC12 neuronal cells were not. In glucose deprivation condition, intracellular ATP content is decreased and thereby AMP-activated protein kinase (AMPK) is activated. And also activation of JNK, inactivation of ERK, and enhanced expression of Bcl-2 were observed. Mitochondrial inhibitors such as rotenone, TTFA, antimycin A, sodium azide, oligomycin, and valinomycin were used in this study. Inhibitors did not much influence intracellular ATP contents and activity of AMPK under glucose deprivation condition. Activities of Akt and p38 MAPK, however, were decreased by the inhibitors under glucose deprivation condition except TTFA. Furthermore, intracellular Ca2+ concentration was also greatly increased by the inhibitors. Finally, mitochondrial membrane potential was decreased by the inhibitors but TTFA increase the potential and oligomycin maintains it. In the present study, results suggest that under glucose deprivation condition mitochondrial inhibitors may induce severe cytotoxicity of U937 cells through inhibition of Akt and p38 MAPK, increase of [Ca2+]i, and decrease of MMP, but not through inhibition of ATP production and activation of AMPK.     

Protection of rats against 3-butene-1,2-diol-induced hepatotoxicity and hypoglycemia by N-acetyl-l-cysteine

3-Butene-1,2-diol (BDD), an allylic alcohol and major metabolite of 1,3-butadiene, has previously been shown to cause hepatotoxicity and hypoglycemia in male Sprague-Dawley rats, but the mechanisms of toxicity were unclear. In this study, rats were administered BDD (250 mg/kg) or saline, ip, and serum insulin levels, hepatic lactate levels, and hepatic cellular and mitochondrial GSH, GSSG, ATP, and ADP levels were measured 1 or 4 h after treatment. The results show that serum insulin levels were not causing the hypoglycemia and that the hypoglycemia was not caused by an enhancement of the metabolism of pyruvate to lactate because hepatic lactate levels were either similar (1 h) or lower (4 h) than controls. However, both hepatic cellular and mitochondrial GSH and GSSG levels were severely depleted 1 and 4 h after treatment and the mitochondrial ATP/ADP ratio was also lowered 4 h after treatment relative to controls. Because these results suggested a role for hepatic cellular and mitochondrial GSH in BDD toxicity, additional rats were administered N-acetyl-l-cysteine (NAC; 200 mg/kg) 15 min after BDD administration. NAC treatment partially prevented depletion of hepatic cellular and mitochondrial GSH and preserved the mitochondrial ATP/ADP ratio. NAC also prevented the severe depletion of serum glucose concentration and the elevation of serum alanine aminotransferase activity after BDD treatment without affecting the plasma concentration of BDD. Thus, depletion of hepatic cellular and mitochondrial GSH followed by the decrease in the mitochondrial ATP/ADP ratio was likely contributing to the mechanisms of hepatotoxicity and hypoglycemia in the rat.