Mitochondrial Respiratory Parameters Research Articles

Nuclear depletion of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) is an indicator of energy disruption in neurons

Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) is a multifunctional protein critical for cellular survival. Its involvement in adaptive survival responses includes key roles in redox sensing, transcriptional regulation, and repair of DNA damage via the base excision repair (BER) pathway. Ape1 is abundant in most cell types and central in integrating the first BER step catalyzed by different DNA glycosylases. BER is the main process for removal of oxidative DNA lesions in postmitotic brain cells, and after ischemic brain injury preservation of Ape1 coincides with neuronal survival, while its loss has been associated with neuronal death. Here, we report that in cultured primary neurons, diminution of cellular ATP by either oligomycin or H2O2 is accompanied by depletion of nuclear Ape1, while other BER proteins are unaffected and retain their nuclear localization under these conditions. Importantly, while H2O2 induces γH2AX phosphorylation, indicative of chromatin rearrangements in response to DNA damage, oligomycin does not. Furthermore, despite comparable diminution of ATP content, H2O2 and oligomycin differentially affect critical parameters of mitochondrial respiration that ultimately determine cellular ATP content. Taken together, our findings demonstrate that in neurons, nuclear compartmentalization of Ape1 depends on ATP and loss of nuclear Ape1 reflects disruption of neuronal energy homeostasis. Energy crisis is a hallmark of stroke and other ischemic/hypoxic brain injuries. In vivo studies have shown that Ape1 deficit precedes neuronal loss in injured brain regions. Thus, our findings bring to light the possibility that energy failure-induced Ape1 depletion triggers neuronal death in ischemic brain injuries.

Rat liver mitochondrial damage under acute or chronic carbon tetrachloride-induced intoxication: Protection by melatonin and cranberry flavonoids

In current societies, the risk of toxic liver damage has markedly increased. The aim of the present work was to carry out further research into the mechanism(s) of liver mitochondrial damage induced by acute (0.8g/kg body weight, single injection) or chronic (1.6g/ kg body weight, 30days, biweekly injections) carbon tetrachloride – induced intoxication and to evaluate the hepatoprotective potential of the antioxidant, melatonin, as well as succinate and cranberry flavonoids in rats.Acute intoxication resulted in considerable impairment of mitochondrial respiratory parameters in the liver. The activity of mitochondrial succinate dehydrogenase (complex II) decreased (by 25%, p<0.05). Short-term melatonin treatment (10mg/kg, three times) of rats did not reduce the degree of toxic mitochondrial dysfunction but decreased the enhanced NO production.After 30-day chronic intoxication, no significant change in the respiratory activity of liver mitochondria was observed, despite marked changes in the redox-balance of mitochondria. The activities of the mitochondrial enzymes, succinate dehydrogenase and glutathione peroxidase, as well as that of cytoplasmic catalase in liver cells were inhibited significantly. Mitochondria isolated from the livers of the rats chronically treated with CCl4 displayed obvious irreversible impairments. Long-term melatonin administration (10mg/kg, 30days, daily) to chronically intoxicated rats diminished the toxic effects of CCl4, reducing elevated plasma activities of alanine aminotransferase and aspartate aminotransferase and bilirubin concentration, prevented accumulation of membrane lipid peroxidation products in rat liver and resulted in apparent preservation of the mitochondrial ultrastructure. The treatment of the animals by the complex of melatonin (10mg/kg) plus succinate (50mg/kg) plus cranberry flavonoids (7mg/kg) was even more effective in prevention of toxic liver injury and liver mitochondria damage.

"MitoTea": Geranium robertianum L. decoctions decrease blood glucose levels and improve liver mitochondrial oxidative phosphorylation in diabetic Goto-Kakizaki rats.

Several chemical compounds found in plant products have proven to possess beneficial properties, being currently pointed out due to their pharmacological potential in type 2 diabetes mellitus complications. In this context, we studied the effect of Geranium robertianum L. (herb Robert) leaf decoctions in Goto-Kakizaki (GK) rats, a model of type 2 diabetes. Our results showed that oral administration of G. robertianum leaf decoctions over a period of four weeks lowered the plasma glucose levels in diabetic rats. Furthermore, the treatment with G. robertianum extracts improved liver mitochondrial respiratory parameters (state 3, state 4 and FCCP-stimulated respiration) and increased oxidative phosphorylation efficiency.

Inhibitory effect of Arctium minus on mitochondrial bioenergetics in diabetic Goto-Kakizaki rats

Despite an increasing use of medicinal plants in diabetes mellitus therapy, their use is usually based on empirical knowledge. This work aims to evaluate the effects of decoctions prepared from Arctium minus Bernh. on glycaemic control of goto-kakizaki (GK) rats, a type 2 diabetes mellitus animal model, and access the potential toxicity of A. minus decoctions, using liver mitochondrial preparations. GK rats were drinking ad libitum A. minus root decoction for an interval of 4 weeks. During this time, food and beverage ingested and weight increase were controlled. Occasional glycaemias were also evaluated twice a week. Intraperitoneal glucose tolerance was determined at the beginning and at the end of the experiment. Four weeks later, GK rats liver mitochondria were isolated and respiratory parameters evaluated. A. minus root decoctions did not significantly affect the glycaemic control and long-lasting treatments induced noxious effects, considering that the liver mitochondria isolated from A. minus treated GK rats presented a significant decrease in mitochondrial respiratory parameters (RCR and FCCP stimulated respiration). This study showed that long lasting treatments with A. minus tea induced deleterious effects on cellular metabolism and, thus, should be avoided in type 2 diabetes long-term therapy.

Cardiac MuRF1 expression alters mitochondrial oxidative phosphorylation function in vivo

Muscle ring finger 1 (MuRF1) is a cardiac‐specific ubiquitin ligase regulating cardiac atrophy and fatty acid metabolism. Here, we examined the effects of cardiac‐specific over expression (MuRF1 Tg+) and ablation (MuRF1 −/−) of MuRF1 on mitochondrial bioenergetics in the heart. Cardiac mitochondrial respiration parameters were measured using a Clark‐type oxygen electrode using a combination of malate (M), palmitoyl‐L‐carnitine (CP), pyruvate (P) and succinate (S) as oxidative substrates. The oligomycin‐insensitive basal proton leak was augmented in the MuRF1 Tg+ cardiac mitochondria by 40 ± 2.4% (n=5; p&lt;0.05 vs. wild‐type control) when M+P were used as oxidative substrates. The respiratory control index (RCI), a marker of mitochondrial viability, was increased in the MuRF1 −/− mice by 48 ± 4.5% (n=5; p&lt;0.05 vs. wild type control) when succinate was employed as an oxidative substrate. No significant changes were observed in the other mitochondrial parameters measured. Our data demonstrate that increased cardiac MuRF1 expression results in impaired mitochondrial oxidative phosphorylation as a result of increased basal proton leak, which may explain the increased susceptibility to heart failure we have identified in MuRF1 Tg+ hearts.

MITOCHONDRIAL ISCHEMIA-REPERFUSION INJURY OF THE TRANSPLANTED RAT HEART

Cold ischemia time and preservation of organs are limited by I/R injury leading to primary nonfunction of the graft. In a rat heart transplant model, we compared cardioplegic St Thomas (ST) to histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin preservation solutions in terms of contractile function, and mitochondrial respiratory and enzymatic defects after prolonged cold ischemia and reperfusion. Contractile function was scored after transplantation and 24 h of reperfusion. Mitochondrial function was investigated by high-resolution respirometry of permeabilized myocardial fibers. Graft performance in terms of contractile function declined with the duration of cold storage. Recovery was significantly improved after 10 h of cold storage in HTK compared with ST (cardiac scores, 3.3+/-0.5 and 1.8+/-0.8, respectively). Tissue lactate dehydrogenase was better preserved in HTK than ST. Increase of tissue water content (edema) was less pronounced in HTK than ST (3.33+/-0.14 and 3.73+/-0.21 mg/mg dry weight, respectively). Similar cardiac scores (2.6+/-0.9 and 2.9+/-1.2, respectively) and mitochondrial respiratory parameters were obtained after preservation in HTK and University of Wisconsin. Decline in contractile function of individual grafts correlated well with loss of mitochondrial respiratory capacity, whereas citrate synthase activity remained largely preserved, indicating specific damage of respiratory complexes. Our data provide evidence for the superiority of preservation solutions versus a cardioplegic solution for prolonged cold storage of the heart. The correlation of graft performance and mitochondrial function indicates the potential of high-resolution respirometry for quantitative assessment of myocardial injury upon cold I/R, providing a basis for diagnostic approaches and evaluation of improved preservation solutions for heart transplantation.

10.1007/s11827-008-1007-6

Respiration parameters of liver mitochondria (MCh) in rats fed with amaranth seed oil for 3 weeks have been evaluated. Thirty minutes before decapitation, adrenaline was injected intraperitoneally at a low dose (350 μg/kg body weight) to both control and experimental animals. It was shown that in animals that were injected with adrenaline and did not receive oil, the rate of phosphorylating respiration increased by 32% and phosphorylation time decreased by 22% upon oxidation of succinate; upon oxidation of α-ketoglutarate in the presence of the succinate dehydrogenase inhibitor malonate, phosphorylating respiration was activated by 23%. The respiration of MCh upon oxidation of succinate + glutamate and α-ketoglutarate in the absence of malonate was not affected by adrenaline. The intake of oil markedly activated almost all parameters of mitochondrial respiration in experimental rats upon oxidation of all above-listed substrates in both coupled and uncoupled MCh. However, phosphorylation time was close to the control value (upon oxidation of succinate) or increased (upon oxidation of α-ketoglutarate in the presence and absence of malonate). The injection of adrenaline to animals receiving oil did not affect the oil-activated respiration of MCh oxidizing the substrates used; however, phosphorylation time in all groups of animals decreased. Ca2+ capacity of MCh in rats receiving amaranth oil did not change. Thus, our data show that feeding of rats with amaranth oil activates mitochondrial respiration and prevents MCh hyperactivation induced by adrenaline.

EFFECT OF PREPARATION PROCEDURE ON THE FUNCTION AND MORPHOLOGY OF LIVER MITOCHONDRIA FROM CONTROL AND ENDOTOXIC RATS

Background: Endotoxic shock is a condition in which cells fail to extract oxygen adequately from blood. The mitochondria are responsible for oxygen extraction from the blood. It is important to determine adequately mitochondrial function during pathological states. The isolation of mitochondria can result in a loss of a mitochondrial population (e.g. pathologically changed mitochondria). Aim: We determined whether the preparation procedure can influence mitochondrial functions and morphology or not. Methods: Rats received LPS (8 mg/kg i.v.). Untreated rats were used as a control. After 16 hours the livers were extracted, homogenized, mitochondria were prepared and used in three experiments. A) Comparison of mitochondrial morphology in liver tissue and isolated mitochondria by means of electron microscopy. B) Comparison of mitochondrial respiratory parameters using the high-resolution respirometry. C), comparison of the quality of mitochondrial preparations by the analysis of respiratory chain markers (cyt b, cyt c1, cyt c and cyt a). Results: We found A) no difference in swelling of mitochondria, observed in liver slices during endotoxic shock and in isolated mitochondria (no swelling in isolated control mitochondria), B) no difference between mitochondrial inrespiratory parameters in homogenate and isolated mitochondria, C) no significant difference in the quality of mitochondria in homogenates or isolated. Conclusions: All these data together suggest that the used isolation procedure of rat liver mitochondria from control and LPS treated rats does not significantly influence mitochondrial morphology and function and represent the full mitochondrial pool in liver tissue.

Effect of triclosan (TRN) on energy-linked functions of rat liver mitochondria

Bisphenols are a class of compounds that exhibit a broad spectrum of antimicrobial activity. One of the most widely used member of this group is triclosan (TRN). TRN is a synthetic, non-ionic, broad-spectrum antimicrobial agent, which is incorporated into several products, including hand soaps and detergents and those of skin care and oral hygiene. The effects of TRN on mitochondrial respiratory parameters and the inner mitochondrial membrane potential (Δ Ψ) are described. That of TRN (up to 60 nmol mg −1 protein) on isolated liver mitochondria decreased oxygen consumption of state 3 respiration, as well as Δ Ψ, but increased oxygen consumption of state 4 respiration, characteristic of an uncoupler effect. Analysis of segments of the respiratory chain suggested that the TRN inhibition site is located between complexes II and III. Mitochondrial swelling, energized or driven by the K + diffusion potential using valinomycin, was also inhibited by TRN, the former being completely inhibited at concentrations greater than 10 nmol TRN mg −1 protein, suggesting that it is also able to interfere with fluidity of the inner mitochondrial membrane. These results suggest that, besides its antibacterial effect, TRN can also impair the mitochondrial function of animal cells.

Functional coupling of adenine nucleotide translocase and mitochondrial creatine kinase is enhanced after exercise training in lung transplant skeletal muscle

Mechanisms responsible for limitation of exercise capacity in lung transplant recipients (LR) and benefits gained by exercise training were studied. Mitochondrial respiration parameters, energy transfer, and cell structure were assessed in vastus lateralis biopsies using the permeabilized fiber technique with histochemical and morphometric measurements. Twelve male controls (C) and 12 LR performed exercise training over 12 wk. Before exercise training, there were strong correlations between exercise capacity (maximal O(2) consumption and endurance time at 70% maximal power output) and cellular events, as assessed by percentage of type I fibers and apparent K(m) for exogenous ADP. Anticalcineurins were not involved in LR exercise limitation, since there were no differences in maximal mitochondrial rate of respiration before exercise training and no abnormalities in respiratory chain complexes compared with C. Training resulted in a significant increase in physiological parameters both at the cellular (apparent K(m) for exogenous ADP and stimulating effect of creatine) and integrated (maximal O(2) consumption, power output at ventilatory threshold, maximal power output, and endurance time at 70% maximal power output) levels in LR and C. After the training period, improvements in maximal O(2) consumption and in maximal mitochondrial rate of respiration were noted, as well as changes in endurance time and percentage of type I fibers. Because there were no changes in diameters and fiber types, baseline alteration of apparent K(m) for exogenous ADP and its improvement after training might be related to changes within the intracellular energetic units. After the training period, intracellular energetic units exhibited a higher control of mitochondrial respiration by creatine linked to a more efficient functional coupling adenine nucleotide translocase-mitochondrial creatine kinase, resulting in better exercise performances in C and LR.

Mitochondrial energy metabolism is markedly impaired by d-2-hydroxyglutaric acid in rat tissues

Tissue accumulation of high amounts of d-2-hydroxyglutaric acid (DGA) and l-2-hydroxyglutaric acid (LGA) is the biochemical hallmark of the inherited neurometabolic disorders d-2-hydroxyglutaric aciduria (DHGA) and l-2-hydroxyglutaric aciduria (LHGA), respectively. Patients affected by DHGA predominantly present neurological and cardiomuscular symptoms, while those with LHGA have mainly severe neurological symptoms. Lactic aciduria and/or lactic acidemia may also occur in both disorders, suggesting mitochondrial dysfunction. We have previously reported that cytochrome c oxidase (COX) activity is severely inhibited by DGA in rat cerebral cortex and human skeletal muscle. In the present study, we initially evaluated the role of DGA and LGA on the mitochondrial respiratory chain complex activities, as well as CO 2 on production in cardiac and skeletal muscle from 30-day-old Wistar rats. DGA significantly inhibited COX and ATP synthase ( F 0 F 1-ATP synthase) activities, in contrast to the other activities of the respiratory chain enzymes which were not affected by DGA in both muscular tissues. In addition, CO 2 production was also markedly reduced by DGA in rat skeletal and cardiac muscles. On the other hand, LGA did not interfere with any of the respiratory chain complex activities studied, neither with CO 2 generation. We also measured mitochondrial respiratory parameters in rat brain mitochondrial preparations in the presence of DGA and LGA. Both metabolites significantly lowered the respiratory control ratio in the presence of glutamate/malate and succinate. Since the metabolites stimulated oxygen consumption in state IV and compromised ATP formation, it can be presumed that these organic acids might act as endogenous uncouplers of mitochondria respiration. Moreover, COX activity linked to TMPD-ascorbate was significantly reduced by DGA in the brain mitochondrial enriched fractions. Finally, DGA and LGA reduced cell viability of rat cerebral cortex slices, as determined by the MTT assay. In case our in vitro data also occur in vivo, it may be presumed that impairment of energy metabolism may contribute to the understanding of the clinical features mainly of patients affected by DHGA.

Biological Evaluation of Some Selected Cyclic Imides: Mitochondrial Effects and in vitro Cytotoxicity

Cyclic imides such as succinimides, maleimides, glutarimides, phthalimides and their derivatives contain an imide ring and a general structure -CO-N(R)-CO- that confers hydrophobicity and neutral characteristic. A diversity of biological activities and pharmaceutical uses have been attributed to them, such as antibacterial, antifungal, antinociceptive, anticonvulsant, antitumor. In spite of these activities, much of their action mechanisms at molecular and cellular levels remain to be elucidated. We now show the effects of several related cyclic imides: maleimides (S2, S2.1, S2.2, S3), glutarimides (S4, S5, S6), 4-aminoantipyrine derivatives (L1, F1, AL1, F1.14, F1.2) and sulfonated succinimides (RO1, FA, FE, FD, MC, DMC) on isolated rat liver mitochondria, B16-F10 melanoma cell line, peritoneal macrophages and different bacterial streams. The effects on mitochondrial respiratory parameters, cell viability and antibacterial activity were also evaluated. The results indicated that S3, S5 and S6 caused an increased oxygen consumption in the presence of ADP (state III) or its absence (state IV), while all other compounds decreased those parameters at different degrees of inhibition. All the compounds decreased the respiratory control coefficient (RCC). Loss of cell viability of peritoneal macrophages and the B16-F10 cell line was observed, L1 and S2.1 being more effective. S1, S2, S3, L1 and F1 compounds showed antibacterial activity at experimental concentrations.

Effects of deltamethrin on functions of rat liver mitochondria and on native and synthetic model membranes

Deltamethrin (DTM) is a synthetic pyrethroid insecticide used wideworld in agriculture, home pest control, protection of foodstuff, and disease vector control. It has widespread applications in Brazilian agriculture. The effects of DTM on mitochondrial respiratory parameters and on the organization of artificial and native membranes are described. DTM (200 nmol mg −1 protein) on isolated liver mitochondria decreased oxygen consumption of both, state III and state IV, as well as the inner mitochondrial membrane potential (Δ ψ). Analysis of segments of the respiratory chain suggested that the DTM inhibition site is located between complex II and complex III. Mitochondrial swelling, energized or driven by the K + diffusion potential using valinomycin, were partially inhibited by DTM (200 nmol mg −1 protein). Fluorescence polarization of DPH and DPH-PA, probing the core and outer regions, respectively, of dimyristoylphosphatidylcholine (DMPC) and native mitochondrial membranes, indicated that DTM shifts the midpoint phase transition to lower values, besides broadening the phase transition. DTM decreased the lipid order of DMPC bilayers, at temperatures lower than the transition temperature and also caused a disordering effect on native membranes. However at temperatures above the transition temperature, the pesticide increased the rigidity of the membrane. These results suggest that DTM causes perturbations in lipid-lipid and lipid-protein interactions, interferes in transport mechanisms operating at the membrane level, and causes alterations of membrane permeability and mitochondrial enzyme activities. These effects could be associated with the toxicity of deltamethrin.

Does angiotensin-converting enzyme inhibition improve the energetic status of cardiac and skeletal muscles in heart failure induced by aortic stenosis in rats?

Recently, we have demonstrated that heart failure in rats is associated with a myopathy altering energy metabolism in different muscles, but the origin of this myopathy is still unknown. Here, we studied the possible involvement of increased angiotensin II (Ang II) by treatment with perindopril, an inhibitor of angiotensin-converting enzyme (ACE). The beneficial effects of ACE inhibition could result either from vasodilatation-induced cardiac unloading or from inhibition of the direct angiotensin action on the muscle cells. The model of aortic banding with persisting left ventricular (LV) overload where the cardiac unloading does not occur allows to distinguish between the two effects of ACE inhibition. Four months after aortic clipping (just before the treatment), echocardiographic study showed an impairment of the systolic function (decrease of the LV shortening by 30% and ejection fraction by 21%). Ten-week treatment with perindopril dramatically decreased Ang II plasma level but did not reduce LV hypertrophy though a significant decrease in right ventricular (RV) hypertrophy occurred. Perindopril did not improve alterations in activities of energy metabolism enzymes (creatine kinase, citrate synthase, cytochrome c oxidase, lactate dehydrogenase) either in ventricular or in skeletal (gastrocnemius) muscle. Similarly, ACE inhibition did not improve the main parameters of mitochondrial respiration in permeabilized muscle fibers. These data suggest that the generalized metabolic myopathy induced by the hemodynamic abnormalities conditioned by the continuous LV overload (aorta clipping) does not result from the increase in Ang II level per se. Correction of hemodynamic parameters and LV unloading seem to be the prerequisite for the improvement of muscle energy metabolism abnormalities.

Caffeine Enhances the Calcium-Dependent Cardiac Mitochondrial Permeability Transition: Relevance for Caffeine Toxicity

Caffeine (1,3,7-trimethylxanthine), a compound present in beverages such as tea and coffee, is known to be toxic at high concentrations. Some of the observed clinical conditions include cardiovascular disease and reproductive disorders, among others. The possible toxic effects of caffeine on heart mitochondria are still poorly understood. The influence of caffeine on the mitochondrial permeability transition has not been clarified so far. The objective of this study was to investigate whether caffeine, at toxic concentrations, had any stimulating effect on the permeability transition of heart mitochondria isolated from Wistar rats, as well as whether it influenced mitochondrial respiratory parameters. Our results show that caffeine reduced mitochondrial ability to accumulate calcium by increasing the susceptibility of heart mitochondria to the opening of the transition pore. Caffeine not only hindered mitochondrial capacity to recover membrane potential after calcium addition but also increased the rate of calcium-dependent mitochondrial swelling and calcium-induced calcium release. The increased swelling was also observed in nonenergized mitochondria. Caffeine also showed a complex array of effects on heart mitochondrial bioenergetics, as evaluated by respiratory parameter measurements. We observed an increase in state 4 respiration and a depression in state 3 respiration, although no effect was observed on succinate-sustained mitochondrial membrane potential in the absence of calcium. Our work may be relevant to cardiovascular problems linked to caffeine toxicity and also to in vitro experiences involving caffeine-induced calcium release from the sarcoplasmic reticulum and uptake by mitochondria.

Resistance of isolated pulmonary mitochondria during in vitro anoxia/reoxygenation

The aim of the study was to investigate the effect of in vitro anoxia/reoxygenation on the oxidative phosphorylation of isolated lung mitochondria. Mitochondria were isolated after harvesting from fresh pig lungs flushed with Euro-Collins solution. Mitochondrial respiratory parameters were determined in isolated mitochondria before anoxia (control), after 5–45 min anoxia followed by 5 min reoxygenation, and after 25 or 40 min of in vitro incubation in order to follow the in vitro aging of mitochondria during respiratory assays. Respiratory parameters measured after anoxia/reoxygenation did not show any oxidative phosphorylation dysfunction, indicating a high resistance of pulmonary mitochondria to in vitro anoxia/reoxygenation (up to 45 min anoxia). These results indicate that mitochondria are not directly responsible of their oxidative phosphorylation damage observed after in vivo ischemia (K. Willet et al., Transplantation 69 (2000) 582) but are a target of others cellular injuries leading to mitochondrial dysfunction in vivo.

Effects of cold and warm ischemia on the mitochondrial oxidative phosphorylation of swine lung.

The aim of the study was to investigate the consequence of warm and cold ischemia on lung mitochondria in order to define bioenergetic limits within lung could be suitable for pulmonary transplantation. Twenty-two pigs underwent lung harvesting after lung flush with Euro-Collins solution. Mitochondria were isolated from fresh lungs, from lungs submitted to 24 or 48 hr of cold ischemia, to 30 or 45 min of warm ischemia, and to 30 min of warm ischemia followed by 24 or 48 hr of cold ischemia. Mitochondrial oxidative phosphorylation parameters were determined in isolated mitochondria by in vitro measurement of oxygen consumption. Relative to controls, mitochondria submitted to cold ischemia showed an alteration in the oxidoreductase activities of the respiratory chain but no membrane permeability alteration. After 48 hr of cold ischemia, there was a decrease in the yield of the oxidative phosphorylation. Thirty minutes of warm ischemia did not alter the mitochondrial respiratory parameters. However, lung submitted to 45 min of warm ischemia showed mitochondrial damage as a decrease in the oxidative phosphorylation efficiency and ADP availability but no change in the oxidoreductase activities. Relative to cold ischemia alone, 30 min of warm ischemia preceding cold ischemia promoted no significant change in the respiratory parameters. On bioenergetic basis, lung submitted to warm ischemia could be suitable for transplantation if the warm ischemia duration does not exceed 30 min. This could be a major concern in lung procurement from non-heart beating donors.

Generation of superoxide anion by mitochondria and impairment of their functions during anoxia and reoxygenation in vitro

A small portion of the oxygen consumed by aerobic cells is converted to superoxide anion at the level of the mitochondrial respiratory chain. If produced in excess, this harmful radical is considered to impair cellular structures and functions. Damage at the level of mitochondria have been reported after ischemia and reperfusion of organs. However, the complexity of the in vivo system prevents from understanding and describing precise mechanisms and locations of mitochondrial impairment. An in vitro model of isolated-mitochondria anoxia-reoxygenation is used to investigate superoxide anion generation together with specific damage at the level of mitochondrial oxidative phosphorylation. Superoxide anion is detected by electron paramagnetic resonance spin trapping with POBN-ethanol. Mitochondrial respiratory parameters are calculated from oxygen consumption traces recorded with a Clark electrode. Respiring mitochondria produce superoxide anion in unstressed conditions, however, the production is raised during postanoxic reoxygenation. Several respiratory parameters are impaired after reoxygenation, as shown by decreases of phosphorylating and uncoupled respiration rates and of ADP/O ratio and by increase of resting respiration. Partial protection of mitochondrial function by POBN suggests that functional damage is related and secondary to superoxide anion production by the mitochondria in vitro.

Submitochondrial particles asin vitro biosensors of heavy metal toxicity

The effects on mitochondrial respiratory parameters of heavy metals, such as Cu, Ni, Pb, Cd, Zn, Ag, Hg, were recorded by using thein vitro response of submitochondrial particles (SMP) from beef heart mitochondria.

Submitochondrial particles as toxicity biosensors of chlorophenols

AbstractAn in vitro bioassay procedure was used to investigate the toxic action of chlorophenols on mitochondrial respiratory parameters The toxicity of these compounds was evaluated by determining their effects on the energy coupled reverse electron transfer (RET) in submitochondrial particles (SMPs) from beef heart mitochondria The bioassay procedure is based on the spectrophotometric recording of the effects of toxicants on the rate of NAD+ reduction, induced by ATP and succinate at the first site level of the respiratory chain The toxicity end point was expressed as the toxicant concentration that causes 50% inhibition of NAD+ reduction rate (EC50). The EC50 values determined for the 14 tested chlorophenols ranged from 17 mg/L for 2 chlorophenol to 0 081 mg/L for pentachlorophenol, indicating a general trend of increasing toxicity with increasing chlorine substitution Among chlorophenol isomers, which have the same number of chlorine atoms, a lesser toxicity was associated with ortho‐substituted chlorophenols, whereas meta substituted chlorophenols were much more toxic The EC50 values were compared with the toxicity data for a variety of bioassays, by means of linear regression analysis High degrees of correlation obtained with toxicity tests involving different freshwater species demonstrate the ability of SMPs to reproduce the toxic effects of the tested compounds upon aquatic organisms This supports the assessment that the respiratory chain is the main target of this class of toxicants Results obtained with chlorophenols and, in previous studies, with other environmental contaminants confirm the suitability of the SMP bioassay as a prescreening or complementary short term test for monitoring aquatic toxicity