Effects of phenoperidine on rat liver mitochondrial respiration.

Phenoperidine decreases rat liver mitochondrial oxidation of the following substrates in state 3: glutamate, pyruvate/malate mixture, and succinate. The intensity of inhibition is dose-dependent and substrate-dependent. With glutamate which acts at the first phosphorylation site on the respiratory chain, phenoperidine seems to have two effects on mitochondrial oxidative processes: without preincubation, phenoperidine antagonizes mitochondrial oxidation of glutamate presumably by inhibiting enzymes involved in glutamate metabolism such as glutamate dehydrogenase and glutamate oxaloacetate transaminase. Upon a three min preincubation, an additional inhibitory mechanism develops: phenoperidine prevents glutamate from penetrating into the mitochondria. Inhibition without preincubation appears to be non competitive (K_i = 7.5 × 10^?5 M) and it is assumed that it takes place at a site before the 2,4-dinitrophenol (DNP)-sensitive site on the energy transfer process. In state 4, phenoperidine increases the oxidation of these substrates. It decreases the P/O ratio: thus it acts as an uncoupler, although not as potent as DNP. When compared with phenoperidine, levorphanol and pentazocine act as uncouplers but not as energy-transfer inhibitors. DALA-enkephalinamide increases state 4 respiration on glutamate. As for morphine, etorphine, meperidine and fentanyl, they do not act on mitochondrial processes. The effects of opiates on mitochondrial respiration are irrelevant to their morphinomimetic actions because they are not antagonized by naloxone.     

Effect of styrene and other alkyl benzene derivatives on oxidation of FAD- and NAD-linked substrates in rat liver mitochondria

The effect on energetic metabolism of rat liver mitochondria (RLM) of styrene and other aliphatic benzene derivatives, i.e. toluene, ethylbenzene, alpha-methylstyrene and butylbenzene, is studied. It is shown that these compounds uncouple oxidative phosphorylation and this effect is connected with the stimulation of passive entry of protons into mitochondria. The relationship between hydrophobicity of these compounds and their biological activity and mechanism of uncoupling effect are discussed.     

Inhibition of mouse liver respiration by Chelidonium majus isoquinoline alkaloids

The alkaloids from Chelidonium majus L. which had a significant inhibitory effect in mitochondrial respiration were those which contain a positive charge due to a quaternary nitrogen atom, i.e., chelerythrine, sanguinarine, berberine and coptisine, both with malate+glutamate or with succinate as substrates. When malate+glutamate was used as substrate, chelerythrine and berberine, which contain methoxy groups, were particularly more active, since they had a strong effect even at low concentrations. In submitochondrial particles, berberine and coptisine had a marked inhibitory effect on NADH dehydrogenase activity but practically no effect on succinate dehydrogenase activity, whereas chelerythrine and sanguinarine inhibited more strongly succinate dehydrogenase than NADH dehydrogenase, which is in agreement with the results found for mitochondrial respiration. Protopine and allocryptopine, which did not inhibit mitochondrial respiration, strongly inhibited NADH dehydrogenase in submitochondrial particles, but had no effect on succinate dehydrogenase activity.     

Effect of ochratoxin A on rat liver mitochondrial respiration and oxidative phosphorylation

The in vitro effects of ochratoxin A on the membrane structure and bioenergetic functions of rat liver mitochondria were studied. It was found that when the toxin was added to the assay medium the respiratory control of the isolated mitochondria was decreased as the concentration of the toxin increased. The mitochondrial respiration was gradually uncoupled by the toxin when its concentration was raised above 1.2 X 10(-6) M, and became fully uncoupled at 6.2 X 10(-4) M. The oxidative phosphorylation was not damaged until the toxin concentration was higher than 9.3 X 10(-5) M. On the other hand, ochratoxin A inhibited the electron transfer functions of the mitochondria. At the concentration above 1.0 X 10(-4) M, ochratoxin A inhibited the succinate dehydrogenase, succinate-cytochrome c reductase, and succinate oxidase activities of the respiratory chain. Fifty percent of succinate-cytochrome c reductase and succinate oxidase activity was lost in the presence of 8.0 X 10(-4) and 6.2 X 10(-4) M ochratoxin A, respectively. The inhibition kinetic studies revealed that ochratoxin A is an uncompetitive inhibitor of both succinate-cytochrome c reductase and succinate dehydrogenase, and the inhibition constants for the 2 enzyme activities were estimated to be 4.4 X 10(-4) and 2.2 X 10(-4) M, respectively. However, the toxin did not inhibit either cytochrome oxidase or NADH dehydrogenase activity of the mitochondrial respiratory chain. It is thus concluded that ochratoxin A exerts its effect on the mitochondrial respiration and oxidative phosphorylation through the impairment of the mitochondrial membrane and inhibition of the succinate-supported electron transfer activities of the respiratory chain.     

Minocycline exerts uncoupling and inhibiting effects on mitochondrial respiration through adenine nucleotide translocase inhibition

The present study was aimed to provide a better understanding of the mitochondria-targeted actions of minocycline (MC), a second-generation tetracycline which has cytoprotective effects. Although the specific mechanisms underlying its activity remained elusive, considerable amounts of data indicated mitochondria as the primary pharmacological target of MC. Previous reports have shown that MC affects the oxygen-uptake rate by isolated mitochondria in different respiratory states. Here, we report on the effect of MC, in the range 50-200μM, on mitochondrial respiration. State 3 respiration titration with carboxyatractyloside revealed that MC inhibits the adenine nucleotide translocase. Furthermore, we analyze MC channel-forming capacity in the lipid membrane bilayer. Our results confirmed the crucial role of Δψ and showed a dependence on Ca(2+) for MC to have an effect on mitochondria. Our data also indicated that outer and inner mitochondrial membranes contribute differently to this effect, involving the presence of Δψ (the inner membrane) and VDAC (the outer membrane). Data from three isosmotic media indicate that MC does not increase the permeability of the inner membrane to protons or potassium. In addition, by using mitoplasts and ruthenium red, we showed that Ca(2+) uptake is not involved in the MC effect, suggesting involvement of VDAC in the MC interaction with the outer membrane. Our data contribute to unravel the mechanisms behind the mitochondria-targeted activity of the cytoprotective drug MC.     

Glucocorticoid inhibition of inflammation-induced metallothionein synthesis in mouse liver.

The effects of glucocorticoid treatment on the induction of hepatic metallothionein (MT) during inflammation initiated by turpentine oil (TUR) or endotoxin (LPS) were studied in mice. The administration of TUR increased concentrations of hepatic MT as well as that of plasma fibrinogen. Although hepatic MT was modestly induced by dexamethasone (DEX) alone, pretreatment with DEX (12.5 to 100 mg/kg, sc) inhibited the increases both in hepatic MT and in plasma fibrinogen from a subsequent dose of TUR 6 hr after DEX administration. The concentration of hepatic MT in the DEX-pretreated (25 mg/kg) group was higher than that in the nonpretreated group 4 hr after administration of TUR, but after 24 hr, the MT concentration in the DEX-pretreated group was inhibited to 20% of that in the nonpretreated group. These inhibitory effects were also observed by prednisolone (PRE) but not by salicylic acid. The inhibitory effect of DEX on the induction of MT synthesis during inflammation was observed after administration of the exudate obtained from inflamed tissue. When inflammation was initiated by an injection of LPS as well as TUR, pretreatment of either DEX or PRE inhibited the increase of hepatic MT. Pretreatment of DEX did not affect the induction of hepatic MT synthesis by cadmium. In contrast to inflammation initiated by TUR or LPS, pretreatment of DEX caused an additive increase of hepatic MT concentration after administration of zinc. These results suggest that glucocorticoids, despite being direct inducers of MT, inhibit the induction of MT synthesis during inflammation by the suppression of cytokine production.     

Acetaminophen toxicity induces mitochondrial complex I inhibition in human liver tissue

Acetaminophen (APAP) is used worldwide and is regarded as safe in therapeutic concentrations but can cause acute liver failure in higher doses. High doses of APAP have been shown to inhibit complex I and II mitochondrial respiratory capacity in mouse hepatocytes, but human studies are lacking. Here, we studied mitochondrial respiratory capacity in human hepatic tissue ex vivo with increasing doses of APAP. Hepatic biopsies were obtained from 12 obese patients who underwent a Roux‐en‐Y gastric bypass (RYGB) or a sleeve gastrectomy surgery. Mitochondrial respiration was measured by high‐resolution respirometry. Therapeutic concentrations (≤0.13 mmol/L) of APAP did not inhibit state 3 complex I‐linked respiration. APAP concentrations of ≥2.0 mmol/L in the medium significantly reduced hepatic mitochondrial respiration in a dose‐dependent manner. Complex II‐linked mitochondrial respiration was not inhibited by APAP. We conclude that the mitochondrial respiratory capacity is affected by a hepato‐toxic effect of APAP, which involved complex I, but not complex II.     

In vitro and in vivo effect of methyl isocyanate on rat liver mitochondrial respiration.

Previous work has shown that irrespective of the route of exposure methyl isocyanate (MIC) caused acute lactic acidosis in rats (Jeevaratnam et al., Arch. Environ. Contam. Toxicol. 19, 314-319, 1990) and the hypoxia was of stagnant type due to tissue hypoperfusion resulting from hypovolemic hypotension in rabbits administered MIC subcutaneously (Jeevarathinam et al., Toxicology 51, 223-240, 1988). The present study was designed to investigate whether MIC could induce histotoxic hyperoxia through its effects on mitochondrial respiration. Male Wistar rats were used for liver mitochondrial and submitochondrial particle (SMP) preparation. Addition of MIC to tightly coupled mitochondria in vitro resulted in stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/O ratio, and inhibition of state 3 oxidation. The oxidation of NAD(+)-linked substrates (glutamate + malate) was more sensitive (five- to sixfold) to the inhibitory action of MIC than succinate while cytochrome oxidase remained unaffected. MIC induced twofold delay in the onset of anerobiosis, and cytochrome b reduction in SMP with NADH in vitro confirms inhibition of electron transport at complex I region. MIC also stimulated the ATPase activity in tightly coupled mitochondria while lipid peroxidation remained unaffected. As its hydrolysis products, methylamine and N,N'-dimethylurea failed to elicit any change in vitro; these effects reveal that MIC per se acts as an inhibitor of electron transport and a weak uncoupler. Administration of MIC sc at lethal dose caused a similar change only with NAD(+)-linked substrates, reflecting impairment of mitochondrial respiration at complex I region and thereby induction of histotoxic hypoxia in vivo.     

Relationship between inhibition of mitochondrial respiration by naphthoquinones, their antitumor activity, and their redox potential

The physicochemical properties of a series of 1,4-naphthoquinones were correlated with their activities against Sarcoma-180 by Hodnett et al. [J. med. Chem. 26, 570 (1983)]. Redox potential was the most important molecular parameter determining antitumor activity in this series of compounds, suggesting that interference with electron transport contributes to their cytotoxicity. We evaluated this same series of quinones for their abilities to inhibit the beef heart mitochondrial succinoxidase and NADH-oxidase enzyme systems. They exhibited a broad range of inhibitory potencies. There was a strong relationship between succinoxidase inhibition, antitumor activity (T/C ratio), and redox potential. The redox potentials of the quinones which inhibited succinoxidase lay within the narrow range of endogenous components of the respiratory chain. In contrast, inhibition of NADH-oxidase was related to redox potential but did not significantly predict antitumor activity. These results suggest that inhibition of mitochondrial succinoxidase may be a useful preliminary screen for antitumor activity.     

Enhancement of NAD-linked isocitrate dehydrogenase activity in rat liver by clofibrate feeding

After feeding rats for 3 weeks with clofibrate specific activities of the liver mitochondrial enzymes glycerol-3-phosphate dehydrogenase (G3PDH) and NAD-linked isocitrate dehydrogenase (NAD-ICDH) were found to be increased in a large particle fraction 6-fold and 1.6-fold, respectively, whereas the activity of NADP-linked isocitrate dehydrogenase (NADP-ICDH) remained unchanged. Possibly these effects contribute to the hypolipidemic action of clofibrate.     

Effect of ryanodine on mitochondrial respiration.

Ryanodine is a pharmacological agent that stimulates calcium leakage into the cytoplasm resulting in an increase in tension. In skeletal muscle, ryanodine acts primarily on the sarcoplasmic reticulum whereas in smooth muscle, the sites of action are less clear. Visually, the increase in tension is slow and the time course can be mimicked by mitochondrial poisoning and the resultant leak of mitochondrial calcium into the cytoplasm. Although it has been reported that ryanodine has no effect on calcium flow into or from mitochondria, the effect of ryanodine on mitochondrial oxidative function was not studied. In the current investigation direct measurements of the effect of ryanodine on mitochondrial oxygen utilization were made. The results demonstrate that ryanodine, even at high concentrations, has no effect (stimulatory or inhibitory) on mitochondrial oxidation.     

Effect of chlordane on hepatic mitochondrial respiration

In order to clarify the cytotoxicity of chlordane, an industrial product used as an insecticide, its effect on oxidative phosphorylation in rat hepatic mitochondria was studied. The respiration rate, RCI and ADP/O ratios were inhibited by chlordane-related compounds; the degree of inhibition was in the descending order of trans-chlordane, cis-chlordane, heptachlor and heptachlorepoxide. Of the indices indicating various respiratory activities, state 3 respiration was the most sensitively inhibited by these compounds, suggesting that they inhibit energy transfer. However, electron transport was inhibited also by high concentrations of chlordane constituents. The inhibitory effect of the chlordane constituents on respiratory activity varied depending on the species of respiratory substrate, suggesting site-specificity of these compounds. The release of K+ ions paralleled the results of the respiratory activity study. Heptachlorepoxide, a metabolic product of heptachlor, had less effect on mitochondria than heptachlor.     

Epoxide hydrolase activity in the mitochondrial and submitochondrial fractions of mouse liver

Distribution of epoxide hydrolase activity in subcellular fractions of livers from male Swiss-Webster mice and Sprague-Dawley rats was monitored with trans-β-ethylstyrene oxide, trans-stilbene oxide and benzo[a]pyrene 4,5-oxide following differential centrifugation. With the former two substrates the highest activity was encountered in the cytosolic fraction; however, significant activity was found in the mitochondrial fraction. These fractions hydrated benzo[a]pyrene 4,5-oxide very slowly, and the major benzo[a]pyrene 4,5-oxide hydrolyzing activity was recovered in the microsomal fraction. Using Triton WR-1339-treated mice, it was shown that trans-β-ethylstyrene oxide hydrolyzing activity was predominantly localized in the mitochondria rather than in lysosomes and peroxisomes. Subsequent separation of the mitochondrial fraction into submitochondrial components by swelling, shrinking, and sonication, followed by sucrose density gradient centrifugation, showed that most of the epoxide hydrolyzing activity was present in the matrix and intermembrane space fraction. Significant activity was also present in the outer and inner membrane fractions. However, epoxide hydrolyzing activity in these fractions was reduced if either increased sonication times were used or the fractions were washed, indicating possible contamination of these fractions by the matrix and intermembrane space enzyme(s). The epoxide hydrolase activity in the mitochondrial and cytosolic fractions in mice appeared similar with regard to inhibition, molecular weight, and substrate selectivity.     

Effect of epinephrine and alprenolol on ethanol metabolism, liver cell respiration and mitochondrial function.

Chronic administration of epinephrine to adult male rats resulted in a significant increase in the rate of ethanol elimination, when given alone or together with the beta-adrenergic blocker alprenolol. This effect was observed concomitantly with an increased hepatic oxygen utilization and no changes in mitochondrial respiratory functions. Epinephrine given acutely did not modify the rate of ethanol metabolism. Blood glucose levels were enhanced in these conditions, but were unaffected in rats treated with epinephrine plus alprenolol. These results suggest that chronic epinephrine treatment induces an increased oxidative capacity in the liver characterized by enhanced rates of oxygen uptake and ethanol metabolism, which is not related to its beta-adrenergic actions.     

Effects of endosulfan and its metabolites on rat liver mitochondrial respiration and enzyme activities in vitro

Endosulfan (E) is an organochloric insecticide, which is quickly metabolized and eliminated from the body system. Toxic effects of E and its metabolites have been reported. The influence of E and its metabolites, viz. endosulfan sulfate (ES), endosulfan diol (ED) and endosulfan lactone (EL), has been examined on rat liver mitochondria in vitro. Endosulfan stimulated state-4 respiration at lower concentrations and inhibited it at higher ones, whereas state-3 respiration was inhibited at all the concentrations used, i.e. 5-100 micrograms/ml. A maximal 25-fold activation of latent Mg2+-ATPase was achieved at a concentration that caused maximal stimulation of state-4 respiration. Activities of the respiratory chain-linked enzymes were inhibited at levels which corresponded to the concentrations of endosulfan used in vitro. Both the respiratory control ratio (RCR) and the ADP:O ratio fell sharply at endosulfan concentrations above 10 micrograms/ml. ES and ED exerted similar effects on mitochondrial oxidation of beta-hydroxybutyrate, but at more than double the concentration of the parent compound, while EL proved least effective. The effects of the latter compound on mitochondrial enzyme activities were negligible. Our results suggest that endosulfan possesses dual properties, that of an uncoupler of oxidative phosphorylation and of an inhibitor of the electron transport chain, and that the in vivo cytotoxic/insecticidal effects of endosulfan and its metabolites might, therefore, be the consequence of impaired mitochondrial bioenergetics.