Effects of clofibrate, in vitro, on mitochondrial respiration and oxidative phosphorylation

The effects of clofibrate on mitoehondrial respiration and oxidative phosphorylation were studied in vitro. Clofibrate inhibited state 3 oxidation, stimulated state 4 oxidation and lowered ADP: O ratio when 3-hydroxybutyrate, succinate and ascorbate plus tetramethylphenylenediamine (TMPD) were used as substrates. The inhibitions of state 3 oxidation were not reversed by dinitrophenol. In mitochondrial fragments, both NADH- and succinate-linked oxidations were inhibited by clofibrate but ascorbate plus TMPD oxidation was not. A higher concentration of clofibrate was required to inhibit succinate oxidation than NADH oxidation. In mitochondrial fragments, 50 per cent inhibitions of respiration, with NADH and succinate as substrates, were elicited with concentrations of 0·09 and 0·4 μmoles of clofibrate/mg of protein, respectively. It was suggested that there are at least two distinct sites at which clofibrate can inhibit respiration. One site exists between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport chain. Another, less sensitive site, exists between the interaction of succinate with suecinate dehydrogenase and cytochrome c.     

Further studies concerning the effects of clofibrate on respiration and oxidative phosphorylation of rat liver mitochondria

Clofibrate, administered in vitro, inhibited rat liver mitochondrial respiration at two sites within the respiratory chain. One site was between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport chain; another, less sensitive site, was between the interaction of succinate with succinate dehydrogenase and cytochrome c. In addition to these specific sites, clofibrate inhibited respiration by causing a depletion of pyridine nucleotides that was accompanied or followed by large-amplitude, non-energy-linked swelling. Clofibrate uncoupled oxidative phosphorylation at coupling sites II and III but not at site I. The concentrations required to cause loss of pyridine nucleotides were lower than those required to inhibit at the specific sites. p-Chlorophenoxyisobutyrate (CPIB) also inhibited succinate and β-hydroxybutyrate-linked respiration, and uncoupled oxidative phosphorylation, but at much higher concentrations (50 per cent inhibition of β-hydroxybutyrate oxidation at about 3·7 μmoles/mg of protein) than were required of clofibrate (50 per cent inhibition of β-hydroxybutyrate oxidation at about 0·17 μmole/mg of protein). Clofibrate administration to rats (100 and 300 mg/kg p.o. daily for 1 week) lowered serum lipid levels and increased the liver size, the amount of mitochondrial protein/g of liver, and the oxygen consumption of liver slices. However, mitochondria, isolated from livers of the treated rats, respired normally. A single administration of clofibrate (100 or 300 mg/kg, p.o.) did not affect liver slice respiration.     

The effects of some nutritive antibiotics on the respiration of rat liver mitochondria.

(1) Seven antibiotics used as feed additives in animal breeding were investigated for their effects on isolated rat liver mitochondria. Three were found to interfere with mitochondrial energy metabolism. (2) Zinc-bacitracin completely inhibits mitochondrial respiration in the micromolar range. as do other inhibitors known to be highly effective against electron transport system. From studies of this antibiotic on the redox state of cytochromes, as measured by split beam spectra, it is concluded that the site of inhibition is located between cytochrome b and c₁ (antimycin A site). The effect is completely reversed by chelating agents, suggesting that Zn²⁺ ions are required for full activity of the cyclic peptide antibiotic. (3) Flavomycin, a polar glycolipid, linearly stimulates oxygen consumption of mitochondria under state 4 conditions in concentrations greater than 100 μmole/gram of protein. Lower concentrations of the antibiotic inhibits respiration of coupled as well as DNP- or FCCP-uncoupled mitochondria by about 70 per cent. While the uncouplinglike effect at high concentrations of the compound can be attributed to nonspecific surface activity which might facilitate proton conductance, the inhibitory activity seen at lower concentrations is assumed to be located near the second phosphorylation site of the respiratory chain. (4) The influence of chlortetracyclin (aureomycin) on mitochondrial activity was found to be dependent on the identity of the substrate. Succinate respiration was more sensitive to chlortetracyclin (CTC) addition by comparison with NAD-linked substrate oxidation. 45 μmole of CTC/gram of protein decreased succinate respiration to half maximal values, whereas glutamate plus malate or β-hydroxybutyrate respiration were inhibited by only 25 per cent. CTC partially inhibits the dehydrogenation of succinate by the succinate dehydrogenase. Uncoupling of oxidative phosphorylation completely abolished CTC-inhibited respiration of NAD-linked substrates, while succinate respiration remained inhibited by 25 per cent. The results of these experiments are discussed in terms of two sites of action for CTC, one located close to the phosphate carrier, while the second interferes with succinate dehydrogenase.     

The effect of n-butyl-3,5-diiodo-4-hydroxybenzoate on the function of mitochondria isolated from the rat liver and adrenal cortex

The effect of n-butyl-3,5-diiodo-4-hydroxybenzoate (DIBB), considered to be an antithyroxine drug, on the oxidative phosphorylation of mitochondria isolated from rat liver and adrenal cortex was studied.In the presence of liver mitochondria the iodinated compound inhibited state 3 oxidation, stimulated state 4 oxidation and lowered $\text{P}\text{O}$ ratio when β-hydroxybutyrate, succinate, glutamate-malate and malate-isocitrate were used as substrates. The Lineweaver-Burk plot of DIBB inhibition of the rate of O₂ consumption with variable concentrations of substrates, in the presence of ADP, produced straight lines. The inhibition was non-competitive, with succinate and competitive, with β-hydroxybutyrate. The swelling of liver mitochondria induced by DIBB was faster and more pronounced than with thyroxine at similar concentrations.Oxygen consumption by adrenal cortex mitochondria was activated by DIBB at low concentrations in respiratory states 3 and 4. When DOC replaced ADP the 11β-hydroxylation rate was decreased by the iodinated product.It is possible that there are at least two distinct sites at which DIBB can inhibit respiration; one site being probably located in the respiratory chain close to the entry of dehydrogenases and a second site in relation with the coupling of high-energy intermediates. The effects of DIBB on oxidative phosphorylation of rat mitochondria which are similar in some aspects to those observed with thyroid hormones could explain its antithyroxine activity.     

Effects of cresols (o-, m-, and p-isomers) on the bioenergetic system in isolated rat liver mitochondria

It is known that o-, m- and p-cresols exert a toxic effect on rat liver cells. However, there is little information on the mechanism for the hepatotoxicity of cresols. We, therefore, investigated the effects of o-, m-, and p-cresols on the bioenergetic system using isolated rat liver mitochondria. When o-, m- or p-cresol was added to liver mitochondria with glutamate or succinate at concentrations of 0.3 to 6.0 mumol/mg protein, each cresol isomer reduced the rate of state 3 respiration dose-dependently. Three cresol isomers at 6.0 mumol/mg protein each inhibited state 3 respiration in liver mitochondria with glutamate or succinate by about 60 or 20%, respectively. The three isomers affected NAD- and succinate-linked respirations in liver mitochondria, by which the respiratory control ratio was dose-dependently attenuated. The inhibitory effects of o-, m- and p-cresols on the NAD-linked respiration were stronger than those on the succinate-linked respiration. However, three cresol isomers had little effect on the P/O ratio in liver mitochondria with glutamate or succinate. Three cresol isomers at 15 mumol/mg protein each induced the swelling in the absence of Ca2+ in medium and accelerated the swelling of liver mitochondria in the presence of Ca2+ in medium. These results indicate that o-, m- and p-cresols inhibit liver mitochondrial respiration and induce or accelerate the swelling of liver mitochondria, and suggest that liver mitochondria may be one of the targets for the hepatotoxic actions of cresols.     

Inhibition of respiration and gluconeogenesis by paracetamol in rat kidney preparations

Paracetamol, at concentrations up to 10 mM, caused a reversible, concentration-dependent inhibition of respiration in isolated rat-kidney tubules metabolizing glucose, glutamine, lactate or glutamate. It also strongly inhibited the synthesis of glucose from glutamine or lactate and brought about a significant fall in the cell ATP level. Paracetamol lowered both coupled and uncoupled respiration in isolated kidney mitochondria oxidizing glutamate, but had no effect on respiration supported by succinate. Experiments with submitochondrial particles revealed that the drug did not influence the activity of NADH dehydrogenase but slowed the rate at which electrons were transferred from reduced NADH dehydrogenase to cytochrome b. The implications of these findings for paracetamol cytoxicity are discussed.     

Influence of propolis water solution on heart mitochondrial function

The effect of propolis water solution (PWS) on the respiration of rat heart mitochondria with NAD-linked (pyruvate + malate), FAD-linked (succinate) substrates and fatty acids (palmitoyl-L-carnitine) was investigated in this study. PWS at the lowest concentration of 4 microg mL(-1) of phenolic compounds (PC) had no effect on mitochondrial respiration with all investigated substrates. PWS at concentrations of 63 and 125 microg mL(-1) of PC caused a significant decrease of basal (24 and 54%) and maximal (58 and 70%) respiration rates with succinate as substrate. At these PWS concentrations the oxidation of pyruvate + malate and palmitoyl-L-carnitine was diminished to a lower degree: the basal respiration rate decreased by 13-18% and the maximal respiration rate by 15-28%. Succinate oxidation was affected, probably because of the inhibition of succinate dehydrogenase by the 1,2-benzenedicarboxylic acid esters found in PWS. The PWS-caused decrease in the mitochondrial respiration rate with pyruvate + malate and fatty acids could be due to diminished activities of respiratory chain complexes and/or ADP/ATP translocator.     

The effects of cadmium on succinate and NADH-linked substrate oxidations in rat hepatic mitochondria

Low concentrations of cadmium (3.3-40 microM) inhibited State 3 NADH-linked respiration in rat hepatic mitochondria, but failed to release oligomycin (1 microgram) inhibited State 3 respiration, or to significantly change the State 4 rate. In the presence of succinate, 40 microM cadmium inhibited State 3 respiration by 89%, while concentrations between 3.3 and 13.3 microM stimulated State 4 respiration. Higher concentrations caused marked inhibition. In the presence of succinate, cadmium released oligomycin inhibited State 3 respiration. Cadmium (0.001-1.0 mM) did not stimulate mitochondrial ATPase activity or inhibit ferricyanide reduction, but stimulated NAD+ linked mitochondrial dehydrogenase activities and NADH oxidation. These results indicate that cadmium interacts with either the NADH dehydrogenase complex or other NADH-dependent enzymes and not solely by an uncoupling action.     

Inhibition of respiration by phenacetin in isolated tubules and mitochondria of rat kidney

Phenacetin. an analgesic drug thought to exert nephrotoxic effects in vivo, was found to inhibit respiration in isolated rat kidney tubules metabolizing endogenous substrate or exogenous glutamine, glucose or lactate. With isolated rat kidney mitochondria the oxidation of glutamate or succinate was strongly inhibited by phenacetin; in each case State 3 respiration and State 3u (uncoupled) respiration were affected to the same extent, indicating that phenacetin exerted its influence directly on the respiratory chain. The effects of phenacetin on the oxidation of NADH and succinate by submitochondrial particles in the presence of various electron acceptors suggested that at least two oxidoreduction reactions of the respiratory chain were susceptible to inhibition by phenacetin. One of these reactions was that catalysed by succinate dehydrogenase, while the other probably lay between reduced NADH dehydrogenase and coenzyme Q. The possibility that impairment to the oxygen-metabolising capacity of the kidney cell might contribute to the perceived cytotoxicity of phenacetin is discussed.     

Sites of inhibition of mitochondrial electron transport by rhein

The effect of rhein on the oxygen consumption, oxidative phosphorylation, ATPase activity and redox state of electron carriers of rat liver mitochondria has been studied. Rhein inhibits ADP- and uncoupler-stimulated respiration on various NAD-linked substrates and succinate, but stimulates state 4 respiration of mitochondria respiring on succinate. Experiments on specific segments of the respiratory chain showed that rhein does not inhibit electron flow through cytochrome oxidase. Electron flow through site 2, the ubiquinone-cytochrome b-cytochrome c1 complex, was also unaffected by rhein, which failed to inhibit the oxidation of duroquinol. Rhein affects oxidative phosphorylation by inhibiting both electron transfer and ADP-driven H+ uptake. The inhibition of succinate oxidation by rhein was found to take place at a point between succinate and ubiquinone, perhaps at the level of succinic dehydrogenase. Spectroscopic evidence demonstrated that rhein induces a NAD(P)H oxidation in mitochondria respiring either on endogenous substrates or on glutamate + malate, and an inhibition of the cytochrome b reduction by succinate. These observations, together with other evidence, suggest that rhein inhibits electron transport in rat liver mitochondria at the dehydrogenase-coenzyme level, particularly when the electron carriers are in a relatively oxidized state and/or when the inner membrane-matrix compartment is in the condensed state.     

Effects of pyrazole, 4-bromopyrazole and 4-methylpyrazole on mitochondrial function

Pyrazole, an inhibitor of alcohol dehydrogenase, has been widely used in studies of ethanol metabolism. Since its specificity has recently been questioned, we studied the effects of pyrazole, methylpyrazole and bromopyrazole on mitochondrial function. These compounds inhibited oxidative phosphorylation, the ATP^?32P exchange reaction, and energy dependent and independent calcium uptake. With α-ketoglutarate as substrate, state 3 (coupled) respiration was inhibited, whereas state 4 (resting) respiration was not affected. By contrast, state 4 respiration was stimulated when succinate or ascorbate served as the substrate, while state 3 respiration was slightly inhibited. Regardless of the substrate, the respiratory control ratio was depressed. The activities of succinic dehydrogenase and cytochrome oxidase were stimulated by pyrazole and its derivatives, which may explain the stimulation of succinate and ascorbate oxidation. The inhibitory effects of these compounds were reversed by washing the mitochondria, indicating that no permanent damage to mitochondria had occurred. This is supported by the lack of stimulation of latent ATPase activity and the unchanged barrier to the penetration of NADH. Pyrazole and its derivatives decreased the uptake of citrate and glutamate, but stimulated that of phosphate and malate. Methylpyrazole and bromopyrazole inhibited the transport of reducing equivalents into the mitochondria, as catalyzed by the malate-aspartate, fatty acid and α-glycerophosphate shuttles. The data mandate caution in advocating the therapeutic use of pyrazole or its derivatives in man, and suggest that the use of pyrazole to assess ethanol metabolism and its sequelae in vivo may have limitations.     

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.     

The effect of p,p'-DDT (dicophane) and its main metabolites on the respiration of rat liver mitochondria

The effects of DDT, DDE, DDOH and DDA on the oxidation of NADH, glutamate, -hydroxybutyrate and sucoinate by rat liver mitochondria were investigated. The influence of these compounds on the activity of purified liver glutamate dehydrogenase was also checked. It was found that DDT and all those of its metabolites investigated inhibited oxidation of NAD-linked substrates by both intact and sonicated mitochondria. The water-soluble metabolites of DDT (DDOH and DDA) stimulated succinate oxidation by intact but not by sonicated mitochondria, and inhibited the activity of glutamate dehydrogenase. It is concluded that DDT and its metabolites may affect mitochondrial respiratory chain between NADH and CoQ, inhibit glutamate dehydrogenase, and uncouple oxidative phosphorylation.     

Effect of Stryphnodendron adstringens (barbatimão) on energy metabolism in the rat liver

The action of a barbatim?o extract on hepatic energy metabolism was investigated using isolated mitochondria and the perfused rat liver. In mitochondria the barbatim?o extract inhibited respiration in the presence of ADP and succinate. Stimulation occurred, however, after ADP phosphorylation (state IV respiration). The ADP/O and respiratory control ratios were reduced. The activities of succinate-oxidase, NADH-oxidase and the oxidation of ascorbate were inhibited. The ATPase of intact mitochondria was stimulated, but the ATPases of uncoupled and disrupted mitochondria were inhibited. In perfused livers the extract caused stimulation of oxygen consumption, inhibition of gluconeogenesis and stimulation of glycolysis. Glucose release due to glycogenolysis was stimulated shortly after the introduction of the extract, but inhibition gradually developed as the infusion was continued. Apparently the barbatim?o extract impairs the hepatic energy metabolism by three mechanisms: (1) uncoupling of oxidative phosphorylation, (2) inhibition of mitochondrial electron transport, and (3) inhibition of ATP-synthase.     

Studies on the effect of capsaicin on metabolic reactions of isolated rat liver mitochondria.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) was found to inhibit the respiratory response of rat liver mitochondria, respiring in the presence of glutamate as substrate, to the additions of ADP(+P_i), 2,4-dinitrophenol(DNP), and CaCl₂(+P_i). The inhibitory effect of capsaicin on mitochondrial oxidative phosphorylation can also be observed with other NAD-linked substrates such as β-hydroxybutyrate and malate plus pyruvate and was partially reversed by adding bovine serum albumin to the reaction mixtures. When the mitochondria were respiring with succinate as substrate, capsaicin was less effective in inhibiting state 3 and DNP-stimulated respiration but exerted an uncoupling action on mitochondrial respiration. This uncoupling effect was evident at the doses of capsaicin much higher than that required to depress oxidative phosphorylation when NAD-linked substrates were used. Capsaicin was also found to depress the DNP-activated adenosine triphosphatase (ATPase) activity of rat liver mitochondria. These results indicate that capsaicin has profound effect on the energy-linked functions of isolated mitochondria.     

Effects of indomethacin on respiration and the alpha-glycerolphosphate shuttle in rat kidney mitochondria.

The anti-inflammatory drug indomethacin was found to stimulate State 4 respiration in rat kidney mitochondria, indicating an uncoupler activity which was maximal at a concentration of 0.1–0.2mM. Indomethacin also inhibited State 3 respiration in mitochondria oxidizing glutamate or succinate, but not in mitochondria oxidizing ascorbate together with tetramethylphenylene diamine. This inhibition was not relieved by 2,4-dinitrophenol and suggested that indomethacin directly inhibited electron transport along the respiratory chain at a point prior to cytochrome c.At concentrations one order of magnitude lower than that required for substantial uncoupling or respiratory inhibition, indomethacin severely restricted the transfer of reducing equivalents from extramitochondrial NADH to the respiratory chain via a reconstructed α-glycerolphosphate shuttle. It was found that the drug exerted a strong inhibitory effect on mitochondrial α-glycerolphosphate dehydrogenase activity, and that this inhibition was relatively specific since indomethacin had little effect on the activity of succinate dehydrogenase, another FAD-linked enzyme.The inhibition of the α-glycerolphosphate shuttle is discussed in relation to the previously observed effects of indomethacin on glucose metabolism in isolated rat kidney tubules.     

Mechanism of S-(1,2-dichlorovinyl)-L-cysteine- and S-(1,2-dichlorovinyl)-L-homocysteine-induced renal mitochondrial toxicity

The mechanism by which the nephrotoxic S-conjugates S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) produce toxicity in rat kidney mitochondria was studied by examining their effects on mitochondrial function, structural integrity, and metabolism. Both S-conjugates inhibited succinate-linked state 3 respiration and impaired the ability of mitochondria to retain Ca2+ and to generate a membrane potential; 30-60 min were required for maximal expression of these functional changes. Mitochondrial structure was damaged, as indicated by enhanced polyethylene glycol-induced shrinkage of matrix volume and by leakage of protein and malic dehydrogenase from the matrix; 60-120 min were required for maximal expression of these structural changes. Much shorter incubation times (15-30 min) were required for DCVC and DCVHC to decrease ATP concentrations, to alter the concentrations of several citric acid cycle intermediates, and to inhibit succinate:cytochrome c oxidoreductase and isocitrate dehydrogenase activities. Lipid peroxidation and oxidation of glutathione to glutathione disulfide also occurred. The relative time courses of these pathological changes indicate that the initial effects of DCVC and DCVHC in renal mitochondria are the inhibition of energy metabolism and the oxidation of glutathione. These changes then lead to alterations in mitochondrial function and ultimately to irreversible damage to mitochondrial structure.     

Disodium tetrachloropalladate (Na2PdCl4), an inhibitor of rat liver mitochondrial electron transport

The effects of Na2PdCl4 were studied on isolated rat liver mitochondrial electron transport and oxidative phosphorylation in vitro. Significant reductions in ADP-stimulated respiration were observed with increasing Na2PdCl4 concentrations with both succinate and NADH-linked substrate oxidations. Concentration necessary for half-maximal inhibition of oxygen uptake (EC50) for an NADH-linked substrate system was 18 muM while with succinate as substrate the EC50 was 15 muM. At 64 muM both systems were inhibited maximally at 60 and 80%, respectively. At concentrations of Na2PdCl4 sufficient to inhibit acceptor-stimulated oxygen uptake, there was a concomitant decrease in the rate of ADP phosphorylation as measured by proton absorption. Uncoupling agents had no effect on Na2PdCl4 inhibited mitochondria. Mg-ATPase activity and phosphate acceptor limited (State 4) respiratory activity were not stimulated by any Na2PdCl4 concentration used in these investigations. Data from these experiments indicate that Na2PdCl4 inhibits the mitochondrial respiratory chain in vitro.     

In vitro effects of 10,10′-oxybisphenoxarsine on isolated rat liver mitochondria

10,10′-Oxybisphenoxarsine (OBPA), a trivalent, heterocyclic organoarsenical, is a potent, broad spectrum antimicrobial agent often incorporated into plasticized polyvinyl chlorides. Although highly toxic, the mechanism of OBPA toxicity is unknown. Since it is hepatotoxic, we investigated the in vitro effects of OBPA on rat liver mitochondria. Isolated mitochondria were incubated with 1 to 8 μm OBPA and oxygen consumption was monitored polarographically. State 3 respiration of α-ketoglutarate, pyruvate, isocitrate, and succinate was rapidly inhibited. OBPA also uncoupled oxidative phosphorylation, although this was masked, at higher concentrations, by progressive respiratory inhibition. The electron transport chain appeared unaffected by OBPA, as measured by NADH oxidation, but several tricarboxcylic acid cycle dehydrogenases were inhibited. This inhibition was prevented and, in some cases, reversed by glutathione. It is likely that OBPA, like other trivalent organoarsenicals, exerts its toxic effects by reacting with vital mitochondrial sulfhydryl groups.     

Oxidative phosphorylation and respiration by liver mitochondria from polychlorinated biphenyl-intoxicated rats

Respiration and oxidative phosphorylation by intact hepatic mitochondria isolated from rats fed polychlorinated biphenyls (PCBs) were studied polarographically using glutamate and succinate as substrates. High levels of PCBs fed for 3 weeks resulted in an increase in respiration and in ADP/O ratios. However, when PCBs were fed to rats for 20 weeks at 100 ppm, there was no significant difference from control values. Gas Chromatographic determination of PCB levels in mitochondrial fractions revealed 2–3 μg/mg of protein for treated rats in the acute study and about 0·3 μg/mg in the chronic study. Addition of PCBs in vitro to control rat liver mitochondria caused an inhibition of oxidative phosphorylation and respiration at or above a concentration of 50 μg/mg of mitochondrial protein. Since 2,4-dinitrophenol did not initiate uncontrolled respiration in PCB-inhibited rat liver mitochondria, and since inhibition occurred with both substrates, it appears that PCB inhibition in vitro occurs at a site along the respiratory chain.