Prediction of drug-induced mitochondrial dysfunction using succinate-cytochrome c reductase activity, QSAR and molecular docking.

There is increasing evidence that links mitochondrial off-target effects with organ toxicities. For this reason, predictive strategies need to be developed to identify mitochondrial dysfunction early in the drug discovery process. In this study, as a major mechanism of mitochondrial toxicity, first, the inhibitory activity of 35 compounds against succinate-cytochrome c reductase (SCR) was investigated. This in vitro study led to the generation of consistent experimental data for a diverse range of compounds, including pharmaceutical drugs and fungicides. Next, molecular docking and protein-ligand interaction fingerprinting (PLIF) analysis were used to identify significant residues and protein-ligand interactions for the Qo site of complex III and Q site of complex II. Finally, this data was used for the development of QSAR models using a regression-based approach to highlight structural and chemical features that might be responsible for SCR inhibition. The statistically validated QSAR models from this work highlighted the importance of low aqueous solubility, low ionisation, fewer 6-membered rings and shorter hydrocarbon alkane chains in the molecular structure for increased inhibition of SCR, hence mitochondrial toxicity. PLIF analysis highlighted two key residues for inhibitory activity of the Qo site of complex III: His 161 as H-bond acceptor and Pro 270 for arene interactions. Currently, there are limited structure-activity models published in the scientific literature for the prediction of mitochondrial toxicity. We believe this study helps shed light on the chemical space for the inhibition of mitochondrial electron transport chain (ETC).

Idebenone-induced recovery of glycerol-3-phosphate and succinate oxidation inhibited by digitonin.

Digitonin solubilizes mitochondrial membrane, breaks the integrity of the respiratory chain and releases two mobile redox-active components: coenzyme Q (CoQ) and cytochrome c (cyt c). In the present study we report the inhibition of glycerol-3-phosphate- and succinate-dependent oxygen consumption rates by digitonin treatment. Our results show that the inhibition of oxygen consumption rates is recovered by the addition of exogenous synthetic analog of CoQ idebenone (hydroxydecyl-ubiquinone; IDB) and cyt c. Glycerol-3-phosphate oxidation rate is recovered to 148 % of control values, whereas succinate-dependent oxidation rate only to 68 %. We find a similar effect on the activities of glycerol-3-phosphate and succinate cytochrome c oxidoreductase. Our results also indicate that succinate-dependent oxidation is less sensitive to digitonin treatment and less activated by IDB in comparison with glycerol-3-phosphate-dependent oxidation. These findings might indicate the different mechanism of the electron transfer from two flavoprotein-dependent dehydrogenases (glycerol-3-phosphate dehydrogenase and succinate dehydrogenase) localized on the outer and inner face of the inner mitochondrial membrane, respectively.

The difference in myocardial injuries and mitochondrial damages between asphyxial and ventricular fibrillation cardiac arrests.

INTRODUCTION: Ventricular fibrillation (VF) and asphyxia account for most cardiac arrests but differ in cardiac arrest course, neurologic deficit, and myocardial damage. In VF resuscitation, cardiac mitochondria were known to be damaged via excess generation of reactive oxygen species. This study evaluated the difference of cardiac mitochondrial damages between VF and asphyxial cardiac arrests. METHODS: In the VF + electrical shock (ES) group, VF was induced and untreated for 5 minutes, followed by 1 minute of cardiopulmonary resuscitation (CPR) and 1 ES of 5 J. Animals were killed immediately after ES. In the asphyxia group, cardiac arrest was induced by airway obstruction, and then pulselessness was maintained for 5 minutes, followed by 1 minute of CPR. The animals were killed immediately after CPR. The histology and ultrastructural changes of myocardium and complex activities and respiration of mitochondria were evaluated. The mitochondrial permeability transition pore opening was measured based on mitochondrial swelling rate. RESULTS: The histopathologic examinations showed myocardial necrosis and mitochondrial damage in both cardiac arrests. Instead of regional damages of myocardium in the VF + ES group, the myocardial injury in the asphyxia group distributed diffusely. The asphyxia group demonstrated more severe mitochondrial damage than the VF + ES group, which had a faster mitochondrial swelling rate, more decreased cytochrome c oxidase activity, and more impaired respiration. CONCLUSIONS: Both VF and asphyxial cardiac arrests caused myocardial injuries and mitochondrial damages. Asphyxial cardiac arrest presented more diffuse myocardial injuries and more severe mitochondrial damages than VF cardiac arrest.

Impact of high glucose and transforming growth factor-ß on bioenergetic profiles in podocytes.

Diabetic nephropathy is the most common cause of chronic renal failure in industrialized countries. Depletion of podocytes plays an important role in the progression of diabetic glomerulopathy. Various factors in the diabetic milieu lead to serious podocyte stress driving the cells toward cell cycle arrest (p27(Kip1)), hypertrophy, detachment, and apoptosis. Mitochondria are responsible for oxidative phosphorylation and energy supply in podocytes. Recent studies indicated that mitochondrial dysfunction is a key factor in diabetic nephropathy. In the present study, we investigated metabolic profiles of podocytes under diabetic conditions. We examined oxygen consumption rates (OCRs) and oxidative phosphorylation complex activities in murine podocytes. Cells were exposed to high glucose for 48 hours, cultured for 10 passages under high-glucose conditions (30 mmol/L), or incubated with transforming growth factor-ß (5 ng/mL) for 24 hours. After prolonged exposure to high glucose, podocytes showed a significantly increased OCR at baseline and also a higher OCR after addition of oligomycin, indicating significant changes in mitochondrial energy metabolism. Higher OCRs after inhibition of respiration by rotenone also indicated changes in nonmitochondrial respiration. Podocytes stimulated with a proapoptotic concentration of transforming growth factor-ß displayed similar bioenergetic profiles, even with decreased citrate synthase activity. In all tested conditions, we found a higher cellular nicotinamide adenine dinucleotide content and changes in activities of respiratory chain complexes. In summary, we provide for the first time evidence that key factors of the diabetic milieu induce changes in glucose metabolism and mitochondrial function in podocytes.

Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release.

Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions. The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood. Here we addressed the underlying mechanism, explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome c (cyto.c) release. We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3). We found that cyto.c1 was cleaved at the site of D106, which is critical for binding with cyto.c, following apoptotic stresses or targeted expression of casp.3 into the mitochondrial intermembrane space. We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe. These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk. Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.

Design, syntheses, and kinetic evaluation of 3-(phenylamino)oxazolidine-2,4-diones as potent cytochrome bc1 complex inhibitors.

The cytochrome bc1 complex (EC 1.10.2.2, bc1) is one of the most promising targets for new drugs and agricultural fungicides. Among the existing bc1 complex inhibitors specifically binding to the Q(o) site, oxazolidinedione derivatives have attracted great attention. With the aim to understand the substituent effects of oxazolidinedione derivatives on the inhibition activity against the bc1 complex, a series of new oxazolidinedione derivatives were designed, synthesized, and biologically evaluated. The further inhibitory kinetics studies against porcine succinate-cytochrome c reductase (SCR) revealed that the representative compound 8d and famoxadone are both non-competitive inhibitors with respect to the substrate cytochrome c, but competitive inhibitors with respect to substrate decylubiquinol (DBH2). In addition, compound 8d and famoxadone showed, respectively, 35-fold and 15-fold greater inhibitory activity against the porcine SCR than the porcine bc1 complex, indicating that these two inhibitors not only inhibited the activity of the bc1 complex, but possibly affect the interaction between the complex II and the bc1 complex. To our knowledge, this is the first report that famoxadone and its analogs have effects on the interaction between the complex II and the bc1 complex.

Microplate-based kinetic method for assay of mitochondrial NADH-- and succinate--cytochrome c reductase activities.

This article describes a microplate-based kinetic assay for mitochondrial NADH-- and succinate--cytochrome c reductase activities in rat brain mitochondria. The assay reported here is based on the conventional spectrophotometric method and involves substrate-driven reduction of exogenous cytochrome c. Conditions regarding linearity with respect to time and protein concentration have been standardized. Furthermore, the methods were tested for inhibition of respective activities by specific inhibitors. The microplate format described here can be employed for rapid and simultaneous measurements of mitochondrial NADH-- and succinate--cytochrome c reductase activities in a large number of samples.

Late phase ischemic preconditioning preserves mitochondrial oxygen metabolism and attenuates post-ischemic myocardial tissue hyperoxygenation.

AIMS: Late phase ischemic preconditioning (LPC) protects the heart against ischemia-reperfusion (I/R) injury. However, its effect on myocardial tissue oxygenation and related mechanism(s) is unknown. The aim of the current study is to determine whether LPC attenuates post-ischemic myocardial tissue hyperoxygenation through preserving mitochondrial oxygen metabolism. MAIN METHODS: C57BL/6 mice were subjected to 30 min coronary ligation followed by 60 min or 24 h reperfusion with or without LPC (3 cycles of 5 min I/5 min R): Sham, LPC, I/R, and LPC+I/R group. Myocardial tissue Po(2) and redox status were measured with electron paramagnetic resonance (EPR) spectroscopy. KEY FINDINGS: Upon reperfusion, tissue Po(2) rose significantly above the pre-ischemic level in the I/R mice (23.1 ± 2.2 vs. 12.6 ± 1.3 mmHg, p<0.01). This hyperoxygenation was attenuated by LPC in the LPC+I/R mice (11.9 ± 2.0 mmHg, p<0.01). Activities of NADH dehydrogenase (NADH-DH), succinate-cytochrome c reductase (SCR) and cytochrome c oxidase (CcO) were preserved or increased in the LPC group, significantly reduced in the I/R group, and conserved in the LPC+I/R group. Manganese superoxide dismutase (Mn-SOD) protein expression was increased by LPC in the LPC and LPC+I/R mice compared to that in the Sham control (1.24 ± 0.01 and 1.23 ± 0.01, p<0.05). Tissue redox status was shifted to the oxidizing state with I/R (0.0268 ± 0.0016/min) and was corrected by LPC in the LPC+I/R mice (0.0379 ± 0.0023/min). Finally, LPC reduced the infarct size in the LPC+I/R mice (10.5 ± 0.4% vs. 33.3 ± 0.6%, p<0.05). SIGNIFICANCE: Thus, LPC preserved mitochondrial oxygen metabolism, attenuated post-ischemic myocardial tissue hyperoxygenation, and reduced I/R injury.

The effect of aging and increasing ascorbate concentrations on respiratory chain activity in cultured human fibroblasts.

The specific activities of Complexes I-III, II-III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 micromol 1(-1)) were added. Results indicated that ascorbate at 100 micromol 1(-1) was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 micromol 1(-1) however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I-III and II-III activities which required 300 micromol 1(-1) ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations.

Effect of cadmium on resumption of respiration in cotyledons of germinating pea seeds.

Pea seeds (Pisum sativum L.) were germinated by soaking in H2O or 5 mM CdCl2 during a 5-day period. Enzyme activities involved in respiratory metabolism were studied in cotyledons. Mitochondrial cytochrome c oxidase and NADH- and succinate-cytochrome c reductase activities were inhibited by cadmium treatment. The effects of Cd were performed in vivo and in vitro allowing to distinguish between the direct inhibition of the enzyme activities and the influence on the same enzymes into the cell environment. However, Cd exposure stimulated an enzyme activity of fermentation and inhibited the capacity of the enzyme inactivator (alcohol dehydrogenase inactivator). Moreover, the enzyme activities of NAD(P)H-recycling dehydrogenases via secondary pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases, were enhanced in Cd-stressed seeds. These disturbances suggest that cadmium may inflict a serious injury on renewal of respiration. The findings will help clarify the overall mechanisms that underlie cadmium-mediated toxicity in germinating seeds.

Changes in mitochondrial respiratory enzyme activity after ischemia-reperfusion injury in living-donor liver transplantation.

BACKGROUND: Ischemia-reperfusion (I-R) injury plays an important role in the immediate graft function in living-donor liver transplantation (LDLT). There is growing evidence that mitochondria play a pivotal role in I-R injury. Our aim was to evaluate changes in mitochondrial respiratory enzyme activities after I-R injury in LDLT. METHODS: Specimens from 8 donor recipient pairs enrolled in this study were obtained from the donor livers before harvest (before I-R injury) and after vascular anastomosis in the recipient (after I-R injury). Histidine-tryptophan-ketoglutarate solution was used to perfuse the organ during the cold ischemic period between harvesting and transplantation. We correlated changes in mitochondrial respiratory enzyme complex activity (succinate cytochrome c reductase [SCCR]; NADH cytochrome c reductase [NCCR]) after I-R injury with clinical data and graft status. RESULTS: NCCR and SCCR activities did not uniformly decrease after I-R injury. Two of 8 recipients experienced graft dysfunction after transplantation. The decrease in neither NCCR nor SCCR activity correlated with graft dysfunction in these 2 patients. Among the clinical factors, grafts from older donors tended to show decreased NCCR activity after I-R injury. CONCLUSIONS: In this study, changes in mitochondrial respiratory enzyme activity failed to predict the severity of I-R injury in LDLT. The organ preservation solution may play a protective role on mitochondrial respiratory enzymes during I-R injury.

Effects of extensively oxidized low-density lipoprotein on mitochondrial function and reactive oxygen species in porcine aortic endothelial cells.

Atherosclerotic cardiovascular disease is the leading cause of mortality in the Western world. Dysfunction of the mitochondrial respiratory chain and overproduction of reactive oxygen species (ROS) are associated with atherosclerosis and cardiovascular disease. Oxidation increases the atherogenecity of LDL. Oxidized LDL may be apoptotic or nonapoptotic for vascular endothelial cells (EC), depending on the intensity of oxidation. A previous study demonstrated that nonapoptotic oxidized LDL increased activity of mitochondrial complex I in human umbilical vein EC. The present study examined the impact of extensively oxidized LDL (eoLDL) on oxygen consumption and the activities of key enzymes in the mitochondrial respiratory chain of cultured porcine aortic EC. Oxygraphy detected that eoLDL significantly reduced oxygen consumption in various mitochondrial complexes. Treatment with eoLDL significantly decreased NADH-ubiquinone dehydrogenase (complex I), succinate cytochrome c reductase (complex II/III), ubiquinone cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV) activities and the NAD+-to-NADH ratio in EC compared with mildly oxidized LDL, LDL, or vehicle. Butylated hydroxytoluene, a potent antioxidant, normalized eoLDL-induced reductions in complex I and III enzyme activity in EC. Mitochondria-associated intracellular ROS and release of ROS from EC were significantly increased after eoLDL treatment. These findings suggest that eoLDL impairs enzyme activity in mitochondrial respiratory chain complexes and increases ROS generation from mitochondria of arterial EC. Collectively, these effects could contribute to vascular injury and atherogenesis under conditions of hypercholesterolemia and oxidative stress.

Effect of treatment with cadmium on structure-function relationships in rat liver mitochondria: studies on oxidative energy metabolism and lipid/phospholipids profiles.

Effects of treatment with a single intraperitoneal injection of cadmium (Cd) on oxidative energy metabolism and lipid/phospholipid profiles of rat liver mitochondria were examined at the end of 1 week and 1 month. Following Cd treatment the body weight increased only in the 1 month group, whereas the liver weight increased in both groups. State 3 and 4 respiration rates in general decreased significantly, with the maximum effect being seen with succinate. The 1 week Cd group showed decreased respiratory activity with glutamate, pyruvate + malate, and succinate as the substrates. In the 1 month Cd-treated group respiration rates recovered with glutamate and pyruvate + malate but not with succinate. All cytochrome contents decreased in the 1 week Cd-treated group but recovered in the 1 month group. ATPase activity registered an increase in both Cd-treated groups. Dehydrogenase activities increased in the 1 week group but decreased in the 1 month Cd-treated group. The mitochondrial cholesterol content increased in the 1 week Cd-treated group. In the 1 week Cd-treated group the lysophospholipid (Lyso), sphingomyelin (SPM), and diphosphatidylglycerol (DPG) components increased. By contrast, the phosphatidylethanolamine (PE) component decreased. In the 1 month Cd-treated group the phosphatidylinositol, phosphatidylserine, and DPG components increased, whereas the Lyso, SPM, and phosphatidylcholine components decreased. The results demonstrate that single-dose Cd treatment can have adverse effects on liver mitochondrial oxidative energy metabolism and lipid/phosphopholipid profiles, which in turn can affect membrane structure-function relationships.

Abnormal mitochondrial function in locomotor and respiratory muscles of COPD patients.

Several cellular and molecular alterations have been described in skeletal and respiratory muscles of patients with chronic obstructive pulmonary disease (COPD), but information on potential abnormalities of mitochondrial function is scarce. The aim of the present study was to investigate mitochondrial function in the vastus lateralis (VL) and external intercostalis (EI) of COPD patients. Biopsies from VL and EI were obtained during surgery for lung cancer in 13 patients with mild to moderate COPD (age 68+/-6 yrs, forced expiratory volume in one second (FEV(1)) 66+/-15% predicted) and 19 control subjects (age 67+/-9 yrs, FEV(1) 95+/-18% pred). State 3 and 4 mitochondrial oxygen consumption (V'(O(2),m)), ATP synthesis, citrate synthase, cytochrome oxidase (COX) and complex I-III activities, as well as reactive oxygen species (ROS) production, were determined. In COPD patients, in both muscles, COX activity (VL: COPD 3.0+/-0.8 versus control 2.0+/-0.8; EI: 3.7+/-1.6 versus 2.4+/-0.9 micromol min(-1) mg(-1)) and ROS production (VL: 1,643+/-290 versus 1,285+/-468; EI: 1,033+/-210 versus 848+/-288 arbitrary units) were increased, whereas state 3 V'(O(2),m) was reduced (VL: 2.9+/-0.3 versus 3.6+/-0.4; EI: 3.6+/-0.3 versus 4.1+/-0.4 mmol min(-1) kg(-1)). Skeletal muscle mitochondria of patients with chronic obstructive pulmonary disease show electron transport chain blockade and excessive production of reactive oxygen species. The concurrent involvement of both vastus lateralis and external intercostalis suggests a systemic (rather than a local) mechanism(s) already occurring in relatively early stages (Global Initiative for Chronic Obstructive Lung Disease stage II) of the disease.

Disruption of hepatic mitochondrial bioenergetics is not a primary mechanism for the toxicity of methoprene - relevance for toxicological assessment.

Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) is an insect growth regulator generally used to control insect populations by preventing insect maturation. So far, the effects of the insecticide on mitochondrial bioenergetics were not investigated. In the present work, liver mitochondria from Wistar rats were isolated and features of mitochondrial physiology were characterized in the presence of methoprene. High concentrations of methoprene, in the range of 40-100 nmol/mg of protein could decrease the transmembrane electric potential (Delta Psi) developed by mitochondria and, at the highest concentration, methoprene prevented complete Delta Psi repolarization after ADP addition. The effect was more evident using succinate than with ascorbate+TMPD as substrate. State 3 respiration was approximately 60% inhibited by 80 nmol of methoprene/mg of protein, while state 4 respiration, within the same range of methoprene concentrations, showed a slight increase, when both glutamate-malate and succinate were used as substrates. Additionally, FCCP-stimulated respiration was inhibited to an extent comparable to the effect on state 3, which suggests an interaction of methoprene with the respiratory chain, more evident with glutamate/malate as substrate. The activity of complex I (NADH-ubiquinone oxidorreductase) and that of the segment comprehending complexes II and III (succinate-cytochrome c reductase) were decreased in the presence of methoprene (approximately 60% and 85% of inhibition, respectively, with 300 nmol of methoprene/mg of protein), while the activities of cytochrome c oxidase and ATPase do not seem to be affected. Furthermore, the action of methoprene on the mitochondrial permeability transition was also studied, showing that the insecticide (in the range of 30-80 nmol mg(-1) of protein) decreases the susceptibility of liver mitochondria to the opening of the transition pore, even in non-energized mitochondria. These results lead to the conclusion that methoprene interference with hepatic mitochondrial function occurs only for high concentrations, which implies that the noxious effects of the insecticide reported for a number of non-target organisms are not fully attributable to mitochondrial effects. Therefore, it seems that mitochondrial activity does not represent the primary target for methoprene toxic action.

Evaluation of olive oil mill wastewater toxicity on the mitochondrial bioenergetics after treatment with Candida oleophila.

In a previous work the ability of Candida oleophila to use phenolic compounds as sole carbon and energy source at high concentrations without an additional carbon source was reported. C. oleophila grown in bioreactor batch cultures in a diluted and sterilized olive oil mill wastewater (OMW) caused a significant decrease in the total tannins content but no significant alteration was observed in phenolic acid and fatty acid content. Both treated and untreated OMWs were tested to evaluate the capacity in interfering with mitochondrial bioenergetics. Mitochondrial respiration was not affected by treated OMW on the range of used concentrations, contrary to the untreated OMW. Furthermore, mitochondrial membrane potential and respiratory complexes were always significantly less affected by treated OMW in comparison with untreated OMW. However, supplementary treatment should be applied before OMW could be considered non-toxic.

Early mitochondrial dysfunction in electron transfer activity and reactive oxygen species generation after cardiac arrest.

OBJECTIVE: Mitochondrial biology appears central to many conditions that progress to death but remains poorly characterized after cardiac arrest. Mitochondrial dysfunction in electron transfer and reactive oxygen species leakage during ischemia may lead to downstream events including mitochondrial protein oxidation, tyrosine nitrosylation, cytochrome c loss, and eventual death. We sought to better define early fixed alterations in these mitochondrial functions after whole animal cardiac arrest. METHODS: We used a murine model of 8 mins of untreated KCl-induced cardiac arrest followed by resuscitation and return of spontaneous circulation to study mitochondrial functions in four groups of animals: 1) after 8 min cardiac arrest (CA8) but no resuscitation, 2) 30 min postreturn of spontaneous circulation (R30), 3) 60 min postreturn of spontaneous circulation (R60), and in 4) shams. Heart mitochondria were immediately harvested, isolated, and stored at -80 degrees C for later spectrophotometric measurements of electron transfer activities and reactive oxygen species leakage using appropriate substrates and inhibitors. Mitochondrial cytochrome c content and tyrosine nitration were analyzed by Western blot and densitometry. RESULTS: A significant reactive oxygen species leakage from complex I was evident after just 8 min of cardiac arrest (CA8 group, p < .05), which was followed by a progressive reduction in complex I electron transfer activity (CA8 > R30 > R60). In contrast, complex II and II-III activities appeared more resistant to ischemia at the time points evaluated. Early changes in a approximately 50 kDa and approximately 25 kDa protein were observed in tyrosine nitration along with a loss of cytochrome c. CONCLUSIONS: A relatively "orderly" process of mitochondrial dysfunction progresses during ischemia and reperfusion. Changes in mitochondrial reactive oxygen species generation and electron transfer from complex I occur along with tyrosine nitrosylation and loss of cytochrome c; these may represent important new targets for future human therapies.

Mitochondria a key role in microcystin-LR kidney intoxication.

Microcystins (MCs) are a group of closely related cyclic heptapeptides produced by a variety of common cyanobacteria. These toxins have been implicated in both human and livestock mortality. Microcystin-LR could affect renal physiology by altering vascular, glomerular and urinary parameters, indicating that MC-LR could act directly on the kidney. The aim of the current work was to examine the effect of MC-LR on mitochondrial oxidative phosphorylation of rat kidney isolated mitochondria.Furthermore, microcystin-LR decreased both state 3 and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled respiration. The transmembrane potential was strongly depressed by MC-LR in a concentration dependent manner, pointing to an uncoupling effect; however, microcystin-LR did not increase the permeability of the inner mitochondria membrane to protons. Therefore, the transmembrane decrease was a consequence of a strong inhibitory effect on redox complexes. The addition of uncoupling concentrations of MC-LR to Ca(2+)-loaded mitochondria treated with ruthenium red resulted in mitochondrial permeability transition pore (MPTP) opening, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Mitochondrial swelling in the presence of Ca(2+) was prevented by cyclosporin A and was drastically inhibited by catalase and dithiothreitol, indicating the participation of mitochondrial generated reactive oxygen species in this process. From this study it can be concluded that the bioenergetic lesion promoted by microcystin-LR seems to be sufficient to explain renal injury.

Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement.

The deleterious effects of H202 on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H2O2 resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H2O2 on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H2O2 on respiratory complexes was almost completely recovered by beta-mercaptoethanol treatment. H2O2 treatment resulted in full resistance to Qo site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Qo) of complex III is the target for H2O2. H2O2 did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H2O2 addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.

Mitochondrial metabolism during daily torpor in the dwarf Siberian hamster: role of active regulated changes and passive thermal effects.

During daily torpor in the dwarf Siberian hamster, Phodopus sungorus, metabolic rate is reduced by 65% compared with the basal rate, but the mechanisms involved are contentious. We examined liver mitochondrial respiration to determine the possible role of active regulated changes and passive thermal effects in the reduction of metabolic rate. When assayed at 37 degrees C, state 3 (phosphorylating) respiration, but not state 4 (nonphosphorylating) respiration, was significantly lower during torpor compared with normothermia, suggesting that active regulated changes occur during daily torpor. Using top-down elasticity analysis, we determined that these active changes in torpor included a reduced substrate oxidation capacity and an increased proton conductance of the inner mitochondrial membrane. At 15 degrees C, mitochondrial respiration was at least 75% lower than at 37 degrees C, but there was no difference between normothermia and torpor. This implies that the active regulated changes are likely more important for reducing respiration at high temperatures (i.e., during entrance) and/or have effects other than reducing respiration at low temperatures. The decrease in respiration from 37 degrees C to 15 degrees C resulted predominantly from a considerable reduction of substrate oxidation capacity in both torpid and normothermic animals. Temperature-dependent changes in proton leak and phosphorylation kinetics depended on metabolic state; proton leakiness increased in torpid animals but decreased in normothermic animals, whereas phosphorylation activity decreased in torpid animals but increased in normothermic animals. Overall, we have shown that both active and passive changes to oxidative phosphorylation occur during daily torpor in this species, contributing to reduced metabolic rate.