The mechanism of acute cytotoxicity of triethylphosphine gold(I) complexes. III. Chlorotriethylphosphine gold(I)-induced alterations in isolated rat liver mitochondrial function

Chlorotriethylphosphine gold(I) (TEPAu) is an organo-gold compound that has therapeutic activity in animal models of rheumatoid arthritis. Initial studies have suggested that TEPAu is a potent cytotoxic compound in vitro against a variety of cultured cell types and isolated hepatocytes. Mitochondrial dysfunction induced by this compound has been suggested as a primary biochemical alteration which may result in lethal cell injury in isolated hepatocytes. The purpose of this study was, therefore, to determine the mechanism of TEPAu-induced dysfunction of isolated rat liver mitochondria. TEPAu induced a rapid, concentration-related collapse of the mitochondrial inner membrane potential (EC50 = 24.7 +/- 2.5 microM) which was potentiated in Ca2+ loaded mitochondria (EC50 = 11.3 +/- 3.8 microM). TEPAu-induced collapse of the membrane potential was partially inhibited in the presence of ruthenium red or EGTA. TEPAu caused the rapid release of mitochondrially sequestered Ca2+ which was not inhibited by ruthenium red and, thus, was not via a reversal of the Ca2+ uniporter. TEPAu caused mitochondrial swelling, increased permeability of the inner membrane, and the oxidation/hydrolysis of endogenous mitochondrial pyridine nucleotides. Addition of exogenous ATP slightly reversed the effects of TEPAu on pyridine nucleotides. TEPAu-induced mitochondrial alterations were reversed or inhibited by exposure to the sulfhydryl reducing agent, dithiothreitol. Also, the TEPAu-induced collapse of the mitochondrial membrane potential was partially inhibited by dibucaine, a non-specific inhibitor of phospholipases. These data suggest that TEPAu-induced mitochondrial dysfunction is sulfhydryl dependent. TEPAu-induced mitochondrial dysfunction results in dissipation of the potential difference across the inner mitochondrial membrane which inhibits mitochondrial oxidative phosphorylation. The mechanism by which TEPAu induces the collapse of the membrane potential may be mediated by a sulfhydryl-dependent increase in permeability of the inner membrane to protons.     

The mechanism of acute cytotoxicity of triethylphosphine gold(I) complexes : I. Characterization of triethylphosphine gold chloride-induced biochemical and morphological changes in isolated hepatocytes

Triethylphosphine gold complexes are effective therapeutic agents used for the treatment of rheumatoid arthritis. Many of those molecules are also highly cytotoxic in vitro and can inhibit DNA and protein synthesis. Preliminary experiments have indicated that triethylphosphine gold chloride (TEPAu) may induce the peroxidative decomposition of cellular membrane lipids. The purpose of these investigations therefore was to evaluate the role of lipid peroxidation in the mechanism of acute cytotoxicity of a gold(I) coordination complex, TEPAu, and to examine the early morphological and biochemical changes induced by TEPAu in suspensions of freshly isolated rat hepatocytes. TEPAu caused a rapid loss of cell viability at concentrations above 25 microM which was significantly different from that of control by 60 min and complete by 180 min of incubation. TEPAu also depleted cells of reduced glutathione (GSH) and increased the formation of malondialdehyde (MDA) by 60 min. Incubation of cells with either of the antioxidants, N,N'-diphenyl-p-phenylenediamine (DPPD) or promethazine blocked the formation of MDA but did not alter the time course of cell death or GSH depletion induced by TEPAu. TEPAu also caused a decrease in cellular NADPH and NADH by 10 min. Electron microscopy of hepatocytes exposed to TEPAu revealed early (5 min) formation of flocculent electron-dense precipitates within condensed mitochondria. These changes characteristically preceded cell death. Energy-dispersive electron-probe microanalysis indicated that the electron-dense precipitates did not contain detectable amounts of gold. TEPAu also caused a concentration-dependent decrease in cellular ATP and oxygen consumption in isolated rat hepatocytes. These data suggest that lipid peroxidation, as indicated by the formation of MDA, is probably not a major mechanism by which triethylphosphine gold complexes lethally injure cells. These data, therefore, suggest that mitochondria may be target organelles in TEPAu-induced toxicity to isolated rat hepatocytes.     

Daphnetoxin interacts with mitochondrial oxidative phosphorylation and induces membrane permeability transition in rat liver

The effects of daphnetoxin on isolated rat liver mitochondria and freshly isolated rat hepatocytes were investigated. Daphnetoxin (in the microM range) increased mitochondrial state 4 respiration and decreased both state 3 and FCCP-uncoupled respiration. The transmembrane potential was strongly depressed by daphnetoxin in a concentration-dependent manner. The protonophoric activity of daphnetoxin was evidenced by the induction of mitochondrial swelling in hyposmotic K+ acetate medium in the presence of valinomycin. In isolated hepatocytes, daphnetoxin decreases intracellular ATP and simultaneously increases ADP and AMP concentrations. The addition of uncoupling concentrations of daphnetoxin to Ca2+-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Mitochondrial swelling in the presence of Ca2+ was prevented by cyclosporine A and was drastically inhibited by catalase and dithiothreitol, indicating the participation of mitochondrial generated reactive oxygen species in this process. From this study we can conclude that the bioenergetic lesion promoted by daphnetoxin seems to be sufficient to explain the lethal hapatocyte injury.     

Mitochondrial respiration and the thrombin-induced release reaction of platelets

Respiration has been monitored polarographically in suspensions of washed platelets isolated from pig blood. Induction of the release reaction by addition of thrombin to such suspensions resulted in a stimulation of respiration. A respiratory stimulation of similar magnitude was achieved by adding 2 deoxy-D-glucose or uncouplers of oxidative phosphorylation. Oligomycin caused a pronounced inhibition of respiration which could be reversed by adding uncouplers but not thrombin or deoxyglucose. Antimycin A caused a total inhibition of oxygen uptake and prevented the respiratory stimulation otherwise observed on addition of thrombin. It is concluded that the stimulation of respiration caused by thrombin reflects a response of platelet mitochondria to a change in the intracellular ratio of ATP/ADP, consequential upon ATP breakdown during the release reaction. The nature of the energy-dissipating reactions stimulated by thrombin is discussed.     

2-Ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria

2-Ethylhexanol (70 microM), a non-genotoxic carcinogen and peroxisome proliferator, stimulated oxygen uptake in the perfused rat liver by about 10% during the first 10 min of infusion. Perfusion with a higher, hepatotoxic dose of ethylhexanol (3 mM) led to a transient increase in oxygen uptake followed by a rapid inhibition of respiration of over 50% in 10 min. Lactate dehydrogenase (LDH) release, indicative of irreversible cell death, was detected in the effluent perfusate after 20 min. After 10 min of perfusion with ethylhexanol, livers were freeze-clamped, acid extracts were prepared and adenine nucleotides were measured by high-pressure liquid chromatography. Ethylhexanol decreased the ATP/ADP ratio from 2.5 to 0.9. Thus, marked decreases in hepatic energy state due to inhibition of respiration preceded cell death. To attempt to understand this phenomenon, the effect of ethylhexanol on isolated mitochondria was studied. Similar to classical uncoupling agents, ethylhexanol stimulated state-4 rates of respiration, diminished coupled rates of respiration, and decreased the P/O ratio in a dose-dependent manner in isolated mitochondria. Ethylhexanol also decreased uptake of radiolabeled 45CaCl2 by isolated mitochondria 4- to 5-fold. Therefore, we hypothesize that ethylhexanol initially uncouples oxidative phosphorylation leading to diminished ATP synthesis and collapse of ion gradients across the mitochondrial membrane.     

Cytotoxicity of butylated hydroxyanisole and butylated hydroxytoluene in isolated rat hepatocytes

The effects of the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on isolated rat hepatocytes were investigated. Both antioxidants were observed to be cytotoxic in a concentration-dependent manner at concentrations ranging from 100 to 750 microM. At equimolar concentrations BHT was more cytotoxic than BHA. Their toxicity appeared to be independent of their metabolism to reactive intermediates since inhibitors of cytochrome P-450 (metyrapone, SKF 525-A and piperonyl butoxide) had no effect on the cytotoxicity and N-acetylcysteine was also without protective effect. In addition, deuterated BHT was equitoxic with BHT. Only low temperature incubation (4 degrees), which has previously been shown to inhibit the insertion of these compounds into biomembranes, was effective in inhibiting the cytotoxic effects. Using isolated rat liver mitochondria we observed that both BHA and BHT inhibited respiratory control primarily by stimulating state 4 respiration and thus acting as membrane uncouplers. BHA and BHT also effectively dissipated membrane potential across the mitochondrial membrane and caused the release of calcium and mitochondrial swelling. These mitochondrial effects were reflected by a rapid decrease in ATP levels in intact hepatocytes which preceded cell death. These results suggest that the observed cytotoxicity of BHA and BHT to hepatocytes is related to their effects on biomembranes and mitochondrial bioenergetics.     

Inhibition of mitochondrial respiration by flavoskyrin, a toxic metabolite of Penicillium islandicum Sopp

The effects of flavoskyrin, a toxic bianthraquinoid compound from Penicillium islandicum Sopp., on the DNA repair system in rat and mouse hepatocytes and on the ATP biosynthesis system in rat liver mitochondria were studied to gain insight into the mechanism for its cytotoxicity. Flavoskyrin did not elicit unscheduled DNA synthesis (UDS) in hepatocytes at all, implying non-genotoxicity of this compound. Flavoskyrin was found to uncouple oxidative phosphorylation in mitochondria, significantly decreasing both respiratory control (RC) index and P/O ratio. In addition to its uncoupling effect, flavoskyrin produced a marked depression of state-3 respiration, showing 50% inhibition at about 20 microM. These reactions lead to the inhibition of ATP biosynthesis in mitochondria, which may reflect one of the mechanisms of flavoskyrin cytotoxicity.     

The regulation of mitochondrial respiration by opening of mKCa channels is age-dependent

The protective potency of ischemic preconditioning decreases with increasing age. A key step in ischemic preconditioning is the opening of mitochondrial Ca(2+) sensitive K(+) (mK(Ca)) channels, which causes mild uncoupling of mitochondrial respiration. We hypothesized that aging reduces the effects of mK(Ca) channel opening on mitochondrial respiration. We measured the effects of mK(Ca) channel opener NS1619 (30 microM) on mitochondrial respiration in isolated heart mitochondria from young (2-3 months) and old (22-26 months) Wistar rats. Oxygen consumption was monitored online after addition of 250 microM ADP (state 3 respiration), and after complete phosphorylation of ADP to ATP (state 4 respiration) in the presence or absence of the mK(Ca) channel blocker paxilline (5 microM). The respiratory control index (RCI) was calculated as state 3/state 4. In mitochondria from young rats, NS1619 increased state 4 respiration by 11.9+/-4.1% (mean+/-S.E.M.), decreased state 3 respiration by 7.6+/-2.5%, and reduced the RCI from 2.6+/-0.03 (control) to 2.1+/-0.06 (all P<0.05, n=12 for all groups). Paxilline blocked the effect of NS1619 on state 4 respiration (0.7+/-2.8%), but did not affect the decrease in state 3 respiration; paxilline blunted the decrease of RCI. In mitochondria from old rats, NS1619 had neither effect on state 4 (0.4+/-1.6%), and state 3 respiration (-7.4+/-1.5%), nor on RCI (3.0+/-0.13 vs. 3.2+/-0.11, n=12). Increasing age reduced the effects of mK(Ca) opening on mitochondrial respiration. This might be one underlying reason of the decreased protective potency of ischemic preconditioning in the aged myocardium.     

Mercuric chloride toxicity in rat liver mitochondria and isolated hepatocytes

The effects of mercuric chloride on isolated rat liver mitochondria and freshly isolated rat hepatocytes were investigated. Mercuric chloride (in the μM range) depresses state 3 respiration, suggesting a strong effect at the level of the phosphorylation system. It also stimulates state 4 respiration and decreases mitochondrial membrane potential, suggesting an uncoupling action. In isolated hepatocytes, mercuric chloride causes a dose- and time-dependent cell death, accompanied by depletion of intracellular glutathione. Furthermore, mercuric chloride decreases intracellular ATP and ADP levels simultaneously with increasing AMP concentration, indicating fast hydrolysis of ATP without adequate rephosphorylation. From this study we can conclude that the bioenergetic lesion promoted by mercuric chloride seems to be sufficient to explain lethal hepatocyte injury.     

Metabolism and cytotoxicity of bisphenol A and other bisphenols in isolated rat hepatocytes

The relation between the metabolism and the cytotoxic effects of bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane) has been studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria. The incubation of hepatocytes with BPA (0.25–1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by losses of intracellular ATP and total adenine nucleotide pools. BPA at a low-toxic level (0.25 mM) in the hepatocyte suspensions was rapidly converted to its major conjugate, BPA-glucuronide, and other minor products without marked loss of cell viability, although at a toxic level (0.5 mM), more than 65% of the compound presented in an unaltered form 2 h after the incubation. Addition of salicylamide (2 mM), non-toxic to hepatocytes during the incubation period, enhanced BPA-induced cytotoxicity and reduced the loss of BPA and the formation of BPA-glucuronide. The addition of BPA to isolated hepatic mitochondria caused a concentration (0–0.5 mM)-dependent increase in the rate of state 4 oxygen consumption in the presence of an FAD-linked substrate (succinate), indicating an uncoupling effect, whereas the rate of state 3 oxygen consumption was inhibited by BPA. Further, the addition of BPA (0.25 mM) reduced state 3 respiration with NAD⁺-linked substrates (pyruvate plus malate) and/or with the FAD-linked substrate, whereas state 3 respiration with ascorbate plus tetramethyl-p-phenylenediamine (cytochrome oxidase-linked respiration) was not significantly affected by BPA. A comparative study of the toxic effects of BPA and some bisphenols on cell viability (at 1.0 mM) and mitochondrial respiration (at 0.25 mM) revealed that 4,4′-(1,2-diethyl-1,2-ethenediyl)bisphenol (diethylstilbestrol) was more toxic than BPA, followed by 4,4′-methylenediphenol and 4,4′-biphenol. These results indicate that the onset of cytotoxicity caused by BPA may depend on the intracellular energy status and that mitochondria are important targets of the compound. The toxicity caused by the inhibition of ATP synthesis may be related to the concentration of unmetabolised free BPA remaining in the cell suspensions. In addition, the toxic potency of bisphenols to hepatocytes and mitochondria depends on the relative elongation and/or molecular size of the hydrocarbon bridge between the phenolic groups. Received: 25 October 1999 / Accepted: 31 January 2000     

Oxidant-induced changes in the cellular energy homeostasis. A study with 3,5-dimethyl N-acetyl-p-benzoquinone imine and isolated hepatocytes

Exposure of isolated hepatocytes to 400 microM 3,5-dimethyl N-acetyl-p-benzoquinone imine (3,5-diMe NAPQI), rapidly induced the formation of plasma membrane blebs. More than 50% of the viable cells were affected after 1 min incubation with 3,5-diMe NAPQI. Rapid loss of mitochondrial ATP, and sequential increases in ADP and AMP accompanied hepatocyte blebbing. 3,5-diMe NAPQI also induced a pronounced elevation of mitochondrial NADP level, whereas the NAD concentration was unaffected. Similar alterations in the adenine and pyridine nucleotide pools were found to occur in the cytosol, although at slower rates. During the initial phase of ATP loss and NADP production, there was also a concomitant decrease in the oxygen uptake of the hepatocytes. The decreases in energy substrates occurred in parallel to an increased uptake of trypan blue into the cells. Treatment of the hepatocytes with dithiothreitol, following 4 min exposure of the cells to 3,5-diMe NAPQI, reversed the quinone imine-induced changes in nucleotide levels and reduced the cytotoxicity. It is concluded that alteration of mitochondrial function, which results in changes in the cellular energy homeostasis, is an important event in the development of cytotoxicity caused by 3,5-diMe NAPQI.     

Effects of in vivo treatment of rats with trimethyltin chloride on respiratory properties of rat liver mitochondria

Liver mitochondria isolated from rats treated in vivo with trimethyltin chloride show stimulation of respiration using glutamate/malate as substrate, and a transient inhibition on rates of respiration using palmitoyl-L-carnitine as substrate. This phenomenon was observed with both ADP- and FCCP-stimulated respiration. In contrast, rates of respiration by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride, following prior treatment with clofibrate, were inhibited when glutamate/malate was respiratory substrates. With palmitoyl-L-carnitine no effect of trimethyltin chloride was observed. In vitro treatment of rat liver mitochondria, or of rat liver homogenates, led to the expected, powerful inhibition of respiration. The synthesis of ATP by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride was not inhibited compared to mitochondria isolated from control rats. Similarly, ATP synthesis by mitochondria isolated from rats treated with clofibrate, before treatment with trimethyltin chloride, was not inhibited. We, therefore, conclude that the powerful inhibitory effects of trimethyltin found in vitro, is not expressed in vivo during the first 36 hr following administration. In vivo treatment of rats with trimethyltin chloride caused a marked increase in hepatic levels of taurine and glycine, while levels of glutathione and glutamine were diminished. This is consistent with an enhanced oxidative stress in the liver. Our findings lead to the conclusion that increased oxidative stress, rather than inhibition of the mitochondrial ATPase, is a likely major cause of the in vivo toxic effects due to trimethyltin chloride.     

Influence of the anesthetic 2,6-diisopropylphenol on the oxidative phosphorylation of isolated rat liver mitochondria

Isolated rat liver mitochondria have been incubated in the presence of the general anesthetic 2,6-diisopropylphenol (0-100 microM) and the efficiency of oxidative phosphorylation has been evaluated by measuring the respiratory rates, the rates of ATP synthesis or hydrolysis and the magnitude of the transmembrane electrical potential. The results obtained indicate that: (a) in mitochondria energized either by succinate or by ATP, 2,6-diisopropylphenol decreased the transmembrane electrical potential and increased the rates of either electron transfer or ATP hydrolysis; (b) in succinate-energized mitochondria 2,6-diisopropylphenol, at concentrations causing substantial depression of the transmembrane electrical potential, did not modify either the rate of phosphorylation of added ADP or the rate of ADP-stimulated respiration: (c) in succinate-energized mitochondria 2,6-diisopropylphenol caused a concentration-dependent inhibition of the uncoupler-stimulated rate of succinate oxidation. These findings suggest that under the experimental conditions reported 2,6-diisopropylphenol affected the generation and/or maintenance of the transmembrane electrical potential while leaving unchanged the coupling between the electron flow in the respiratory chain and the synthesis of ATP.     

Effects of SC-26096 [2-methyl-3-oxo-2-azabicyclo (2.2.2.) octan-6-exo-yl 5-(4-biphenylyl)-3-methyl valerate], a new hypolipidemic agent,on mitochondrial respiration and oxidative phosphorylation

The effects of SC-26096 [2-methyl-3-oxo-2-azabicyclo (2.2.2.) octan-6-exo-yl 5-(4-biphenylyl)-3-methyl valerate] on respiration and oxidative phosphorylation of rat liver mitochondria were studied in vitro. It was found that SC-26096 is an inhibitor of electron transport. In preparations of intact and sonicated mitochondria, the drug effectively inhibited NAD-linked respiration at low concentration and inhibited succinate-linked respiration at much higher concentration. In intact mitochondria, dinitrophenol partially reversed the inhibition of NAD-linked state 3 repiration but did not reverse the inhibition of succinate-linked respiration. At the SC-26096 concentrations which were required to inhibit succinate oxidation. ADP:O ratio of phosphorylation site III was lowered and state 4 oxidation rate and ATPase activities were raised. At the drug concentrations which were tested. SC-26096 did not alter the ADP:O ratio of phosphorylation site II. 3-Hydroxybutyrate oxidation by mitochondria which were pretreated with SC-26096 remained inhibited after washing with 0.25 M sucrose, but this inhibition was relieved by adding albumin. The effects of SC-26096 were primarily dependent upon the ratio of drug:mitochondrial protein and not upon the initial molarity of the drug in the incubation medium. The results suggest that SC-26096 is bound to mitochondria in vitro and that the bound drug produces the mitochondrial effects. At least two sites of inhibition, within the respiratory chain, are indicated. One site lies between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport system. A second, less sensitive, site lies between the interaction of succinate with succinate dehydrogenase and cytochrome c. A third inhibitory site appears to be present in the site I phosphorylation system at or distal to the dinitrophenol-sensitive site.     

Mitochondrial thioredoxin reductase inhibition by gold(I) compounds and concurrent stimulation of permeability transition and release of cytochrome c

The effects of auranofin, chloro(triethylphosphine)gold(I) (TEPAu), and aurothiomalate on mitochondrial respiration, pyridine nucleotide redox state, membrane permeability properties, and redox enzymes activities were compared. The three gold(I) derivatives, in the submicromolar range, were extremely potent inhibitors of thioredoxin reductase and stimulators of the mitochondrial membrane permeability transition (MPT). Auranofin appeared as the most effective one. In the micromolar range, it inhibited respiratory chain and glutathione peroxidase activity only slightly if not at all. TEPAu and aurothiomalate exhibited effects similar to auranofin, although TEPAu showed a moderate inhibition on respiration. Aurothiomalate inhibited glutathione peroxidase at concentrations where auranofin and TEPAu were without effect. Under nonswelling conditions, the presence of auranofin and aurothiomalate did not alter the redox properties of the mitochondrial pyridine nucleotides indicating that membrane permeability transition occurred independently of the preliminary oxidation of pyridine nucleotides. Under the same experimental conditions, TEPAu showed a moderate stimulation of pyridine nucleotides oxidation. Mitochondrial total thiol groups, in the presence of the gold(I) derivatives, slightly decreased, indicating the occurrence of an oxidative trend. Concomitantly with MPT, gold(I) compounds determined the release of cytochrome c that, however, occurred also in the presence of cyclosporin A and, partially, of EGTA, indicating its independence of MPT. It is concluded that the specific inhibition of thioredoxin reductase by gold(I) compounds may be the determinant of MPT and the release of cytochrome c.     

Protective effects of verapamil and diltiazem against inorganic phosphate induced impairment of oxidative phosphorylation of isolated heart mitochondria

The effects of verapamil and diltiazem on oxidative phosphorylation of isolated rabbit heart mitochondria were related to the experimental conditions employed. In an assay medium containing 250 mM sucrose, 1 mM pyruvate and 5 mM potassium phosphate buffer (pH 7) at 37° (sucrose medium), only a high concentration of verapamil (200–800 μM) or diltiazem (400–600 μM) affected mitochondria. State 4 respiration was stimulated, state 3 respiration was inhibited, and the ADP: O ratio was decreased by these drugs in sucrose medium. These effects resulted in a depression of the respiratory control index (RCI) and oxidative phosphorylation rate (OPR). On the other hand, in an assay medium containing 150 mM KCl, 1 mM pyruvate and 2 mM potassium phosphate buffer (pH 7) at 37° (KCl medium), the high rate of state 3 respiration and the normal value of the ADP: O ratio were not influenced significantly by diltiazem (400–800 μM) or verapamil (200–400 μM). These data indicate that neither verapamil nor diltiazem has an effect on the normal, functioning, isolated mitochondria in KCl medium. Elevation of inorganic phosphate (P₁) from 2 to 5 mM in the KCl medium induced a swelling of the mitochondria, inhibition of state 3 respiration, and a decrease in the ADP: O ratio, RCI and OPR. Under these conditions, a low concentration of verapamil (25–200 μM) or diltiazem (50–800 μM) inhibited the swelling effect of P_i and at the same time prevented the P_i-induced decrease in state 3 respiration, and the ADP: O ratio, RCI and OPR. In a medium containing 150 mM KCl, 1 mM pyruvate, 2 mM ADP and 10 μM palmitoyl-CoA, the addition of 5 mM P_i induced swelling of mitochondria and a decreased rate of state 3 respiration. Under these conditions, even a low concentration of verapamil (6–200 μM) or diltiazem (25–400 μM) inhibited swelling and prevented the inhibition of state 3 respiration. It is concluded that low concentrations of verapamil and diltiazem had no effect on unswollen heart mitochondria. An increase in the free P_i concentration induced swelling of mitochondria and resulted in an inhibition of oxidative phosphorylation, provided that the extramitochondrial potassium concentration was as high as that normally found in the cytosol. Under these conditions, a low concentration of verapamil and diltiazem was able to affect the mitochondrial membranes so as to prevent P_i-induced swelling and the related inhibition of oxidative phosphorylation.     

2,5,2',5'-Tetrachlorobiphenyl impairs the bioenergetic functions of isolated rat liver mitochondria

The effects of 2,5,2',5'-tetrachlorobiphenyl (25TCB) on parameters related to the bioenergetic functions of isolated rat liver mitochondria were investigated. State 3 respiration was inhibited by 25TCB with both succinate and glutamate/malate as the respiratory substrates. The extent of inhibition with succinate was larger than that observed with glutamate/malate. The concentration of 25TCB required to cause 50% inhibition for succinate was 51 microM, but with glutamate/malate, only 53% inhibition was observed at 200 microM. 25TCB stimulated state 4 respiration after 1-2 min lag period; state 4 respiration in the presence of glutamate/malate was more intensely stimulated by 25TCB than in the presence of succinate. 25TCB dissipated the membrane potential across the mitochondrial membranes. Isolated rat liver mitochondria accumulate large amounts of Ca2+ at the expense of respiration-linked energy (substrate oxidation) or of that provided by the hydrolysis of ATP by the mitochondrial ATPase. The Ca2+ accumulation by mitochondria was severely depressed by 25TCB when the energy was supplied by respiration. Furthermore, the inhibition of Ca2+ accumulation by 25TCB with succinate was greater than that produced with glutamate/malate. On the other hand, with ATP as the source of energy, 25TCB inhibited Ca2+ accumulation at high concentrations. 25TCB also released Ca2+ from mitochondria that had already accumulated Ca2+, indicating that mitochondrial membrane integrity was damaged by the intercalation of 25TCB. These results show that 25TCB impairs mitochondrial energy production, and inhibits Ca2+ sequestration by mitochondria.     

Inhibition of rat heart mitochondrial respiration by cadmium chloride

Mitochondria were isolated from hearts obtained from adult male Sprague-Dawley rats by two-part differential centrifugation of heart homogenates. Time-dependent (0-120 sec) and concentration-dependent (0-10 microM CdCl2) effects of cadmium on pyruvate-malate-supported state 3 and state 4 respiration were measured in a constant temperature reaction chamber at 37 degrees C, according to established procedures. The ID50 for cadmium chloride on state 3 respiration was determined to be 4.2 microM. The inhibition produced by cadmium chloride in heart mitochondria was compared, using identical procedures, to the effects induced by two compounds, sodium atractyloside and potassium cyanide, which are known to alter mitochondrial respiration at specific sites. The calculated ID50 values for these agents in heart mitochondria were 1.8 and 16 microM, respectively. The concentration-dependent inhibition of mitochondrial respiration induced by either cadmium chloride or potassium cyanide was maintained in the presence of 50 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP), a known uncoupling agent. In contrast, sodium atractyloside did not block the uncoupling effect of 50 microM CCCP. In addition cadmium chloride was also shown to inhibit CCCP-uncoupled mitochondrial respiration. The cadmium-induced inhibition of mitochondrial respiration was reversed partially by cysteine and completely by 2,3-dimercaptopropanol. The results of the present study indicate that, at all concentrations, cadmium chloride acted solely as an inhibitor of rat heart pyruvate-malate-supported mitochondrial respiration. These findings suggest a possible mechanism for the reported disturbances in myocardial metabolism and function that occur in conjunction with acute and chronic cadmium exposure in humans and experimental animals.     

Effects of inhibitors of platelets aggregation on oxidative phosphorylation in rat liver mitochondria in vitro.

The effects of inhibitors of platelets aggregation used in clinical practice (nicergoline, sulphinpyrazone, dipyridamole and aspirin) on respiration and phosphorylation of rat liver mitochondria in vitro are investigated. All the drugs studied, except aspirin, act as “inhibitors-uncouplers” in a same concentration range, 50–300 nmol/mg protein: they stimulate state 4 respiration, inhibit simultaneously state 3 oxidation (inhibition not reversed by 2–4 dinitrophenol), reduce the ADP/O ratio and respiratory control index (RCI) and stimulate the latent ATPase activity. The latest stimulation is inhibited by oligomycin 6 μg/mg protein. In presence of 200–800 nmol of aspirin/mg protein, the state 3 oxidation is only inhibited with succinate as substrate; aspirin would be a DNP-like uncoupler. It is observed that Triton X 100 acts in the same qualitative way and some similarities are suggested between inhibitors of platelets aggregation and detergents. Correlation between anti-aggregating properties and drug effects on mitochondrial functions are discussed.     

Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction

Indirubin, a red colored 3,2′-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3′-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3′-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3′-oxime incubated with isolated rat liver mitochondria, at concentrations above 10μM, significantly depresses the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3′-oxime at up to 25μM stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3′-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3′-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3′-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3′-oxime effects on cell function.