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At excess levels, zinc (Zn) disrupts mitochondrial functional integrity and induces oxidative stress in aquatic organisms. Although much is known about the modulation of Zn toxicity by calcium (Ca) in fish, their interactions at the mitochondrial level have scarcely been investigated. Here we assessed the individual and combined effects of Zn and Ca on the relationship between mitochondrial respiration, ROS and membrane potential (ΔΨ mt ) in rainbow trout liver mitochondria. We tested if cation uptake through the mitochondrial calcium uniporter (MCU) is a prerequisite for Zn- and/or Ca-induced alteration of mitochondrial function. Furthermore, using our recently developed real-time multi-parametric method, we investigated the changes in respiration, ΔΨ mt , and reactive oxygen species (ROS, as hydrogen peroxide (H 2 O 2 )) release associated with Ca-induced mitochondrial depolarization imposed by transient and permanent openings of the mitochondrial permeability transition pore (mPTP). We found that independent of the MCU, Zn precipitated an immediate depolarization of the ΔΨ mt that was associated with relatively slow enhancement of H 2 O 2 release, inhibition of respiration and reversal of the positive correlation between ROS and ΔΨ mt . In contrast, an equitoxic dose of Ca caused transient depolarization, and stimulation of both respiration and H 2 O 2 release, effects that were completely abolished when the MCU was blocked. Contrary to our expectation that mitochondrial transition ROS Spike (mTRS) would be sensitive to both Zn and Ca, only Ca suppressed it. Moreover, Zn and Ca in combination immediately depolarized the ΔΨ mt , and caused transient and sustained stimulation of respiration and H 2 O 2 release, respectively. Lastly, we uncovered and characterized an mPTP-independent Ca-induced depolarization spike that was associated with exposure to moderately elevated levels of Ca. Importantly, we showed the stimulation of ROS release associated with highly elevated but not unrealistic Ca loads was not the cause but a result of mPTP opening in the high conductance mode. [ABSTRACT FROM AUTHOR]

Although aquatic organisms face multiple environmental stressors that may interact to alter adverse outcomes, our knowledge of stressor–stressor interaction on cellular function is limited. We investigated the combined effects of cadmium (Cd), hypoxia-reoxygenation (H-R) and temperature on mitochondrial function. Liver mitochondria from juvenile rainbow trout were exposed to Cd (0–20 μM) and H-R (0 and 5 min) at 5, 13 and 25 °C followed by measurements of mitochondrial Cd load, volume, complex І active (A) ↔ deactive (D) transition, membrane potential, ROS release and ultrastructural changes. At high temperature Cd exacerbated H-R-imposed reduction of maximal complex I (CI) respiration whereas at low temperature 5 and 10 μM stimulated maximal CI respiration post H-R. The basal respiration showed a biphasic response at high temperatures with low Cd concentrations reducing the stimulatory effect of H-R and high concentrations enhancing this effect. At low temperature Cd monotonically enhanced H-R-induced stimulation of basal respiration. Cd and H-R reduced both the P/O ratio and the RCR at all 3 temperatures. Temperature rise alone increased mitochondrial Cd load and toxicity, but combined H-R and temperature exposure reduced mitochondrial Cd load but surprisingly exacerbated the mitochondrial dysfunction. Mitochondrial dysfunction induced by H-R was associated with swelling of the organelle and blocking of conversion of CІ D to A form. However, low amounts of Cd protected against H-R induced swelling and prevented the inhibition of H-R-induced CI D to A transition. Both H-R and Cd dissipated mitochondrial membrane potential Δψ m and damaged mitochondrial structure. We observed increased reactive oxygen species (H 2 O 2 ) release that together with the protection afforded by EGTA, vitamin E and N -acetylcysteine against the Δψ m dissipation suggested direct involvement of Cd and oxidative stress. Overall, our findings indicate that mitochondrial sensitivity to Cd toxicity was enhanced by the effects of H-R and temperature, and changes in mitochondrial Cd load did not always explain this effect. [ABSTRACT FROM AUTHOR]

To survive in changing environments fish utilize a wide range of biological responses that require energy. We examined the effect of warm acclimation on the electron transport system (ETS) enzymes and transcriptional responses to hypoxia and copper (Cu) exposure in fish. Rainbow trout ( Oncorhynchus mykiss ) were acclimated to cold (11 °C; control) and warm (20 °C) temperatures for 3 weeks followed by exposure to Cu, hypoxia or both for 24 h. Activities of ETS enzyme complexes I-IV (CI–CIV) were measured in liver and gill mitochondria. Analyses of transcripts encoding for proteins involved in mitochondrial respiration (cytochrome c oxidase subunits 4-1 and 2: COX4-1 and COX4-2), metal detoxification/stress response (metallothioneins A and B: MT-A and MT-B) and energy sensing (AMP-activated protein kinase α1: AMPKα1) were done in liver mitochondria, and in whole liver and gill tissues by RT-qPCR. Warm acclimation inhibited activities of ETS enzymes while effects of Cu and hypoxia depended on the enzyme and thermal acclimation status. The genes encoding for COX4-1, COX4-2, MT-A, MT-B and AMPKα1 were strongly and tissue-dependently altered by warm acclimation. While Cu and hypoxia clearly increased MT-A and MT-B transcript levels in all tissues, their effects on COX4-1, COX4-2 and AMPKα1 mRNA levels were less pronounced. Importantly, warm acclimation differentially altered COX4-2/COX4-1 ratio in liver mitochondria and gill tissue. The three stressors showed both independent and joint actions on activities of ETS enzymes and transcription of genes involved in energy metabolism, stress response and metals homeostasis. Overall, we unveiled novel interactive effects that should not be overlooked in real world situations wherein fish normally encounter multiple stress factors. [ABSTRACT FROM AUTHOR]

We investigated the interaction of temperature and copper (Cu) on mitochondrial bioenergetics to gain insight into how temperature fluctuations imposed by natural phenomena or anthropogenic activities would modulate the effects of Cu on cellular energy homeostasis. Mitochondria were isolated from rainbow trout livers and, in the first set of experiments, exposed to Cu (0-2.5 mM) at 5, 11, and 25 °C with measurement of mitochondrial complex II (mtCII)-driven respiration. In the second set of experiments, unenergized mitochondria were incubated for 30 or 60 min with lower concentrations (0-160 μM) of Cu to measure the effects on mtCII enzyme activity. Whereas maximal (state 3) respiration was inhibited by high Cu exposure, low Cu doses stimulated and high Cu doses inhibited resting (state 4) and 4 (proton leak) respirations. High temperature alone increased mitochondrial respiration in all states. The Q values for state 3, state 4, and proton leak respirations suggested active processes with state 4 respiration and proton leak exhibiting greater thermal sensitivity than state 3 respiration. The differential thermal sensitivity of resting relative to phosphorylating mitochondrial state led to uncoupling and limitation of mitochondrial oxidative capacity at both high temperature and doses of Cu. Moreover, exposure to high Cu caused loss of thermal dependence of the mitochondrial bioenergetics culminating in Q values well below unity and decreased activation energies ( E) for both maximal and resting respiration rates. In addition, mtCII activity was increased by low and decreased by high doses of Cu indicating that direct effects on this enzyme contribute to Cu-induced mitochondrial dysfunction. Taken together, it appears that the substrate oxidation (electron transport chain and tricarboxylic acid cycle) and proton leak subsystems are targets of the deleterious effects of Cu and increased temperature on mitochondrial bioenergetics. However, mitochondrial effects of Cu and temperature may not be easily distinguished because they are generally qualitatively similar. [ABSTRACT FROM AUTHOR]

The goal of the present study was to elucidate the modulatory effects of cadmium (Cd) on hypoxia/reoxygenation-induced mitochondrial dysfunction in light of the limited understanding of the mechanisms of multiple stressor interactions in aquatic organisms. Rainbow trout (Oncorhynchus mykiss) liver mitochondria were isolated and energized with complex I substrates (malate–glutamate), and exposed to hypoxia (0>PO2<2 Torr) for 0–60 min followed by reoxygenation and measurement of coupled and uncoupled respiration and complex I enzyme activity. Thereafter, 5 min hypoxia was used to probe interactions with Cd (0–20 μmol l−1) and to test the hypothesis that deleterious effects of hypoxia/reoxygenation on mitochondria were mediated by reactive oxygen species (ROS). Hypoxia/reoxygenation inhibited state 3 and uncoupler-stimulated (state 3u) respiration while concomitantly stimulating states 4 and 4ol (proton leak) respiration, thus reducing phosphorylation and coupling efficiencies. Low doses of Cd (≤5 μmol l−1) reduced, while higher doses enhanced, hypoxia-stimulated proton leak. This was in contrast to the monotonic enhancement by Cd of hypoxia/reoxygenation-induced reductions of state 3 respiration, phosphorylation efficiency and coupling. Mitochondrial complex I activity was inhibited by hypoxia/reoxygenation, hence confirming the impairment of at least one component of the electron transport chain (ETC) in rainbow trout mitochondria. Similar to the effect on state 4 and proton leak, low doses of Cd partially reversed the hypoxia/reoxygenation-induced complex I activity inhibition. The ROS scavenger and sulfhydryl group donor N-acetylcysteine, administrated immediately prior to hypoxia exposure, reduced hypoxia/reoxygenation-stimulated proton leak without rescuing the inhibited state 3 respiration, suggesting that hypoxia/reoxygenation influences distinct aspects of mitochondria via different mechanisms. Our results indicate that hypoxia/reoxygenation impairs the ETC and sensitizes mitochondria to Cd via mechanisms that involve, at least in part, ROS. Moreover, we provide, for the first time in fish, evidence for a hormetic effect of Cd on mitochondrial bioenergetics – the attenuation of hypoxia/reoxygenation-stimulated proton leak and partial rescue of complex I inhibition by low Cd doses. [ABSTRACT FROM AUTHOR]

Impairment of the electron transport chain (ETC) is implicated in cadmium (Cd)- and calcium (Ca)-induced mitochondrial dysfunction. To localize the sites of the impairment, effects of 0–50μM Cd and Ca, singly and in combination, on complex I- to IV-driven respirations were investigated using isolated rainbow trout liver mitochondria. Mitochondrial Cd/Ca accumulation and respiration rates were measured following sequential inhibition and activation of complexes I, II, III, and IV. Mitochondrial adenosine triphosphate (ATP) synthesis was measured on exposure to (micromolar) 20 Cd and 50 Ca, singly and combined, whereas malondialdehyde (MDA) was measured on incubation with 0–1μM Cd and/or Ca. We show that mitochondrial accumulation of Cd and Ca and the states 3 and 4 rates of respiration depended on the active ETC complex. Although complex IV was highly recalcitrant to Cd and/or Ca, dose-dependent inhibitions of complex I-, II-, and III-driven state 3 respiration rates were observed with half maximal inhibitory concentrations (IC50) of (micromolar) 12.4, 12, and 13.7 (Cd); 57.1, 46.1, and 26.2 (Ca); and 8.3, 13.5, and 5.1 (Cd + Ca), respectively. The lower IC50 values for complex I- and III-mediated respirations in the Cd + Ca treatment suggests that these complexes are the sites of cooperative actions of Cd and Ca. State 4 respiration rates were unaffected by Cd and/or Ca exposure but reduced mitochondrial coupling was apparent from the lower respiratory control and adenosine diphosphate/O ratios except in mitochondria oxidizing complex IV substrate. Additionally, there was reduced ATP synthesis in complex I substrates–energized mitochondria and increased MDA concentrations symptomatic of membrane lipid peroxidation. [ABSTRACT FROM PUBLISHER]

Abstract: Molecular features of cadmium (Cd) and calcium (Ca) uptake and toxicity in rainbow trout liver mitochondria were studied using modulators of mitochondrial permeability transition pore (MPTP), mitochondrial calcium uniporter (MCU) and rapid uptake mode (RaM). Malate-glutamate energized mitochondria were exposed to 20μM Cd and 50μM Ca, singly and in combination, with and without addition of ruthenium red (RR), cyclosporin A (CsA), bongkrekic acid (BKA) or dithiothreitol (DTT). State 3 mitochondrial respiration was inhibited by 50% by either Cd or Ca, and by 70% when the two cations were added simultaneously. All the modulators tested reduced the inhibition of state 3 respiration with DTT completely reversing the Cd effect. While state 4 respiration was unaffected by Ca and/or Cd, 1.5–3 fold stimulation was observed on addition of the modulators. Uncoupler-stimulated respiration was inhibited by Cd, Ca and Cd+Ca with complete (DTT) and partial (RR, CsA, BKA) protection of the Cd and Cd+Ca effects. All the modulators completely reversed the Ca-induced inhibition. Swelling, the hallmark of MPTP, measured following incubation of mitochondria with 0–100μM of the two cations, singly and in combination, was abolished by all the modulators. Overall these data show the existence of membrane channels in rainbow trout liver mitochondria with some characteristics similar to mammalian MPTP, MCU and RaM. Moreover, entry of Ca and Cd into mitochondria is important in the toxicity of these cations. [Copyright &y& Elsevier]

Abstract: Rainbow trout were exposed to sublethal waterborne Cd (5 and 10μgL−1) and dietary Ca (60mgg−1), individually and in combination, for 30d to elucidate the interactive effects and evaluate the toxicological significance of mitochondrial responses to these cations in vivo. Indices of fish condition and mortality were measured and livers, centers of metabolic homeostasis, were harvested to assess mitochondrial function and cation accumulation. All indices of condition assessed (body weight, hepatosomatic index and condition factor) were reduced in all the treatment groups. Mortality occurred in the Cd-exposed groups with dietary Ca partly protecting against and enhancing it in the lower and higher Cd exposure, respectively. State 3 mitochondrial respiration was inhibited by 30%, 35% and 40% in livers of fish exposed to Ca, Cd and Cd+Ca, respectively, suggesting reduced ATP turnover and/or impaired substrate oxidation. While the phosphorylation efficiency was unaffected, state 4 and state 4+ (+ oligomycin) respirations were inhibited by all the exposures. Mitochondrial coupling was reduced and transiently restored denoting partially effective compensatory mechanisms to counteract Cd/Ca toxicity. The respiratory dysfunction was associated with accumulation of both Cd and Ca in the mitochondria. Although fish that survived acute effects of Cd and Ca exposure apparently made adjustments to energy generation such that liver mitochondria functioned more efficiently albeit at reduced capacity, reduced fitness was persistent possibly due to increased demands for maintenance and defense against toxicity. Overall, interactions between Cd and Ca on condition indices and mitochondrial responses were competitive or cooperative depending on exposure concentrations and duration. [Copyright &y& Elsevier]

Abstract: The interactive effects of cadmium (Cd) and calcium (Ca) on energy metabolism in rainbow trout liver mitochondria were studied to test the prediction that Ca would protect against Cd-induced mitochondrial liability. Isolated rainbow trout liver mitochondria were energized with malate and glutamate and exposed to increasing concentrations (5–100μM) of Cd and Ca singly and in combination at 15°C. Accumulation of Cd and Ca in the mitochondria and mitochondrial respiration (oxygen consumption) rates were measured. Additionally, un-energized mitochondria were incubated with low doses (1μM) of Cd and Ca singly and in combination, with time-course measurements of cation accumulation/binding and oxygen consumption rates. In energized actively phosphorylating mitochondria, the uptake rates of both Cd and Ca were dose-dependent and enhanced when administered concurrently. Upon low-dose incubation, Cd accumulation was rapid and peaked in 5min, while no appreciable uptake of Ca occurred. Functionally, the resting (state 4, ADP-limited) respiration rate was not affected in the dose–response exposure, but it decreased remarkably on low-dose incubation. Adenosine diphosphate (ADP)-stimulated respiration (state 3) rate was impaired dose-dependently with maximal inhibitions (at the highest dose, 100μM) of 32, 64 and 73% for Ca, Cd, and combined exposures, respectively. The combined effects of Ca and Cd suggested synergistic (more than additive) action and partial additivity of effects at low and higher doses of the two cations, respectively. Moreover, on a molar basis, Cd was twice as toxic as Ca to rainbow trout liver mitochondria and when combined, approximately 90% of the effects were attributable to Cd. The coupling efficiency, as measured by respiratory control ratio (RCR) and phosphorylation efficiency, measured as ADP/O ratio, both decreased as the exposure dosage and duration increased. In addition, Cd and Ca exposure decreased mitochondrial proton leak (state 4+ respiration) rates on prolonged exposure possibly by inhibiting processes that generate mitochondrial membrane potential, the force that drives proton leak. Overall these data suggest that the widely accepted theme that Ca and Cd are competitive antagonists does not hold for mitochondrial effects and that Cd and Ca cooperate to impair oxidative phosphorylation in rainbow trout liver mitochondria. [Copyright &y& Elsevier]

• Cu reduces mitochondrial H 2 O 2 emission induced by Cd or Zn. • Binary mixtures of Cu, Cd, and Zn reduce mitochondrial H 2 O 2 consumption. • Cu, Cd, and Zn singly and in binary mixtures inhibit thioredoxin reductase. • Cu singly and as a mixture with Cd or Zn stimulates glutathione peroxidase. • Binary mixtures of Cu, Cd, and Zn act additively or antagonistically. For continuous pumping of blood, the heart needs a constant supply of energy (ATP) that is primarily met via oxidative phosphorylation in the mitochondria of cardiomyocytes. However, sustained high rates of electron transport for energy conversion redox reactions predisposes the heart to the production of reactive oxygen species (ROS) and oxidative stress. Mitochondrial ROS are fundamental drivers of responses to environmental stressors including metals but knowledge of how combinations of metals alter mitochondrial ROS homeodynamics remains sparse. We explored the effects and interactions of binary mixtures of copper (Cu), cadmium (Cd), and zinc (Zn), metals that are common contaminants of aquatic systems, on ROS (hydrogen peroxide, H 2 O 2) homeodynamics in rainbow trout (Oncorhynchus mykiss) heart mitochondria. Isolated mitochondria were energized with glutamate-malate or succinate and exposed to a range of concentrations of the metals singly and in equimolar binary concentrations. Speciation analysis revealed that Cu was highly complexed by glutamate or Tris resulting in Cu2+ concentrations in the picomolar to nanomolar range. The concentration of Cd2+ was 7.2–7.5 % of the total while Zn2+ was 15 % and 21 % of the total during glutamate-malate and succinate oxidation, respectively. The concentration-effect relationships for Cu and Cd on mitochondrial H 2 O 2 emission depended on the substrate while those for Zn were similar during glutamate-malate and succinate oxidation. Cu + Zn and Cu + Cd mixtures exhibited antagonistic interactions wherein Cu reduced the effects of both Cd and Zn, suggesting that Cu can mitigate oxidative distress caused by Cd or Zn. Binary combinations of the metals acted additively to reduce the rate constant and increase the half-life of H 2 O 2 consumption while concomitantly suppressing thioredoxin reductase and stimulating glutathione peroxidase activities. Collectively, our study indicates that binary mixtures of Cu, Zn, and Cd act additively or antagonistically to modulate H 2 O 2 homeodynamics in heart mitochondria. [ABSTRACT FROM AUTHOR]

The naphthenic acid fraction compound (NAFC), 3,5-dimethyladamantane-1-acetic acid, was tested for its ability to uncouple mitochondrial oxidative phosphorylation. Mitochondria isolated from rainbow trout (Oncorhynchus mykiss) liver were exposed to 3,5-dimethyladamantane-1-acetic acid in state 3 and 4 respiration, and mitochondrial membrane potential were quantified. Electron transport chain (ETC) protein complexes were isolated using pharmacological agents and inhibitors, and their activities measured. The NAFC compound completely inhibited states 3 and 4 respiration with an IC50 of 0.77 and 1.01 mM, respectively. The NAFC compound partially uncoupled mitochondrial membrane potential in state 3 and 4 respiration with an IC50 of 2.19 and 1.73 mM, respectively. The NAFC impaired the activities of ETC protein complexes with a 9.5-fold range in sensitivity. The relative inhibitory effect of the ETC protein complexes to NAFC was CIV≥CI>CIII>CII. The impairment of mitochondrial oxidative phosphorylation by adamantane 3,5-dimethyladamantane-1-acetic acid is mediated via its inhibition of ETC protein complexes. • Potency of a naphthenic acid as a respiratory toxicant was examined in mitochondria. • The adamantane was capable of completely inhibiting respiration. • The adamantane compound could only partially reduce membrane potential. • Complexes I and IV were more sensitive as compared to II and III by 10-fold. [ABSTRACT FROM AUTHOR]

Temperature fluctuations, hypoxia and metals pollution frequently occur simultaneously or sequentially in aquatic systems and their interactions may confound interpretation of their biological impacts. With a focus on energy homeostasis, the present study examined how warm acclimation influences the responses and interactions of acute temperature shift, hypoxia and copper (Cu) exposure in fish. Rainbow trout ( Oncorhynchus mykiss ) were acclimated to cold (11 °C; control) and warm (20 °C) temperature for 3 weeks followed by exposure to environmentally realistic levels of Cu and hypoxia for 24 h. Subsequently, mitochondrial electron transport system (ETS) respiratory activity supported by complexes I–IV (CI–IV), plasma metabolites and condition indices were measured. Warm acclimation reduced fish condition, induced aerobic metabolism and altered the responses of fish to acute temperature shift, hypoxia and Cu. Whereas warm acclimation decelerated the ETS and increased the sensitivity of maximal oxidation rates of the proximal (CI and II) complexes to acute temperature shift, it reduced the thermal sensitivity of state 4 (proton leak). Effects of Cu with and without hypoxia were variable depending on the acclimation status and functional index. Notably, Cu stimulated respiratory activity in the proximal ETS segments, while hypoxia was mostly inhibitory and minimized the stimulatory effect of Cu. The effects of Cu and hypoxia were modified by temperature and showed reciprocal antagonistic interaction on the ETS and plasma metabolites, with modest additive actions limited to CII and IV state 4. Overall, our results indicate that warm acclimation came at a cost of reduced ETS efficiency and increased sensitivity to added stressors. [ABSTRACT FROM AUTHOR]

We investigated how temperature modulates cadmium (Cd)-induced mitochondrial bioenergetic disturbances, metal accumulation and volume changes in rainbow trout ( Oncorhynchus mykiss ). In the first set of experiments, rainbow trout liver mitochondrial function and Cd content were measured in the presence of complex I substrates, malate and glutamate, following exposure to Cd (0–100 μM) at three (5, 13 and 25 °C) temperatures. The second set of experiments assessed the effect of temperature on Cd-induced mitochondrial volume changes, including the underlying mechanisms, at 15 and 25 °C. Although temperature stimulated both state 3 and 4 rates of respiration, the coupling efficiency was reduced at temperature extremes due to greater inhibition of state 3 at low temperature and greater stimulation of state 4 at the high temperature. Cadmium exposure reduced the stimulatory effect of temperature on state 3 respiration but increased that on state 4, consequently exacerbating mitochondrial uncoupling. The interaction of Cd and temperature yielded different responses on thermal sensitivity of state 3 and 4 respiration; the Q 10 values for state 3 respiration increased at low temperature (5–13 °C) while those for state 4 increased at high temperature (13–25 °C). Importantly, the mitochondria accumulated more Cd at high temperature suggesting that the observed greater impairment of oxidative phosphorylation with temperature was due, at least in part, to a higher metal burden. Cadmium-induced mitochondrial volume changes were characterized by an early phase of contraction followed by swelling, with temperature changing the kinetics and intensifying the effects. Lastly, using specific modulators of mitochondrial ion channels, we demonstrated that the mitochondrial volume changes were associated with Cd uptake via the mitochondrial calcium uniporter (MCU) without significant contribution of the permeability transition pore and/or potassium channels. Overall, it appears that high temperature exacerbates Cd-induced mitochondrial dysfunction and volume changes in part by increasing metal uptake through the MCU. [ABSTRACT FROM AUTHOR]

Mitochondrial reactive oxygen species (ROS) have been implicated in organ damage caused by environmental stressors, prompting studies on the effect of oxygen deprivation and metal exposure on ROS metabolism. However, how anoxia and copper (Cu) jointly influence heart mitochondrial ROS metabolism is not understood. We used rainbow trout heart mitochondria to probe the effects of anoxia-reoxygenation and Cu on hydrogen peroxide (H 2 O 2) emission during oxidation of palmitoylcarnitine (PC), succinate, or glutamate-malate. In addition, we examined the influence of anoxia-reoxygenation and Cu on site-specific H 2 O 2 emission capacities and key antioxidant enzymes, glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Results showed that anoxia-reoxygenation suppressed H 2 O 2 emission regardless of substrate type or duration of anoxia. Anoxia-reoxygenation reduced mitochondrial sensitivity to Cu during oxidation of succinate or glutamate-malate whereas high Cu concentration additively stimulated H 2 O 2 emission in mitochondria oxidizing PC. Prolonged anoxia-reoxygenation stimulated H 2 O 2 emission from sites O F and I F , inhibited emission from sites I Q , II F and III Qo , and disparately altered the sensitivity of the sites to Cu. Interestingly, anoxia-reoxygenation increased GPx and TrxR activities, more prominently when reoxygenation followed a short duration of anoxia. Cu did not alter GPx but reduced TrxR activity in normoxic and anoxic-reoxygenated mitochondria. Overall, our study revealed potential mechanisms that may reduce oxidative damage associated with anoxia-reoxygenation and Cu exposure in heart mitochondria. The increased and decreased H 2 O 2 emission from NADH/NAD+ and QH 2 /Q isopotential sites, respectively, may represent a balance between H 2 O 2 required for oxygen deprivation-induced signaling and prevention of ROS burst associated with anoxia-reoxygenation. [Display omitted] • Anoxia-reoxygenation suppresses H 2 O 2 emission regardless of substrate type and duration of anoxia exposure. • Prolonged anoxia-reoxygenation stimulates H 2 O 2 emission from sites I F and O F , but inhibits emission from other sites. • Anoxia-reoxygenation attenuates or intensifies the effect of Cu depending on site and duration of anoxia exposure. • Anoxia-reoxygenation increases GPx and TrxR activities whereas Cu reduces TrxR activity but does not alter GPx. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are a key output of the skeletal muscle mitochondrial information processing system both at rest and during exercise. In skeletal muscle, mitochondrial ROS release depends on multiple factors; however, fiber-type specific differences remain ambiguous in part owing to the use of mitochondria from mammalian muscle that consist of mixed fibers. To elucidate fiber-type specific differences, we used mitochondria isolated from rainbow trout (Oncorhynchus mykiss) red and white skeletal muscles that consist of spatially distinct essentially pure red and white fibers. We first characterized the assay conditions for measuring ROS production (as H 2 O 2) in isolated fish red and white skeletal muscle mitochondria (RMM and WMM) and thereafter compared the rates of emission during oxidation of different substrates and the responses to mitochondrial electron transport system (ETS) pharmacological modulators. Our results showed that H 2 O 2 emission rates by RMM and WMM can be quantified using the same protein concentration and composition of the Amplex UltraRed-horseradish peroxidase (AUR-HRP) detection system. For both RMM and WMM, protein normalized H 2 O 2 emission rates were highest at the lowest protein concentration tested and decreased exponentially thereafter. However, the absolute values of H 2 O 2 emission rates depended on the calibration curves used to convert fluorescent signals to H 2 O 2 while the trends depended on the normalization strategy. We found substantial qualitative and quantitative differences between RMM and WMM in the H 2 O 2 emission rates depending on the substrates being oxidized and their concentrations. Similarly, pharmacological modulators of the ETS altered the magnitudes and trends of the H 2 O 2 emission differently in RMM and WMM. While comparable concentrations of substrates elicited maximal albeit quantitively different emission rates in RMM and WMM, different concentrations of pharmacological ETS modulators may be required for maximal H 2 O 2 emission rates depending on muscle fiber-type. Taken together, our study suggests that biochemical differences exist in RMM compared with WMM that alter substrate oxidation and responses to ETS modulators resulting in fiber-type specific mitochondrial H 2 O 2 emission rates. • H 2 O 2 calibration is altered by fluorescence quenching and biological activity. • Fiber-type specific H 2 O 2 emission varies with substrate type and concentration. • ETS pharmacological modulators alter fiber-type specific H 2 O 2 emission differently. • Fiber-type specific H 2 O 2 emission measurement may require different conditions. [ABSTRACT FROM AUTHOR]

Fish expend significant amounts of energy to handle the numerous potentially stressful biotic and abiotic factors that they commonly encounter in aquatic environments. This universal requirement for energy singularizes mitochondria, the primary cellular energy transformers, as fundamental drivers of responses to environmental change. Our study probed the interacting effects of thermal stress, hypoxia-reoxygenation (HRO) and copper (Cu) exposure in rainbow trout to test the prediction that they act jointly to impair mitochondrial function. Rainbow trout were acclimated to 11 (controls) or 20 °C for 2 months. Liver mitochondria were then isolated and their responses in vitro to Cu (0–20 μM) without and with HRO were assessed. Sequential inhibition and activation of mitochondrial electron transport system (ETS) enzyme complexes permitted the measurement of respiratory activities supported by complex I–IV (CI–IV) in one run. The results showed that warm acclimation reduced fish and liver weights but increased mitochondrial protein indicating impairment of energy metabolism, increased synthesis of defense proteins and/or reduced liver water content. Whereas acute rise (11 → 20 °C) in temperature increased mitochondrial oxidation rates supported by CI–IV, warm acclimation reduced the maximal (state 3) and increased the basal (state 4) respiration leading to global uncoupling of oxidative phosphorylation (OXPHOS). HRO profoundly inhibited both maximal and basal respiration rates supported by CI–IV, reduced RCR for all except CII and lowered CI:CII respiration ratio, an indication of decreased OXPHOS efficiency. The effects of Cu were less pronounced but more variable and included inhibition of CII–IV maximal respiration rates and stimulation of both CI and CIII basal respiration rates. Surprisingly, only CII and CIII indices exhibited significant 3-way interactions whereas 2-way interactions of acclimation either with Cu or HRO were portrayed mostly by CIV, and those of HRO and Cu were most common in CI and II respiratory indices. Our study suggests that warm acclimation blunts sensitivity of the ETS to temperature rise and that HRO and warm acclimation impose mitochondrial changes that sensitize the ETS to Cu. Overall, our study highlights the significance of the ETS in mitochondrial bioenergetic dysfunction caused by thermal stress, HRO and Cu exposure. [ABSTRACT FROM AUTHOR]

Thermal stress may influence how organisms respond to concurrent or subsequent chemical, physical and biotic stressors. To unveil the potential mechanisms via which thermal stress modulates metals-induced bioenergetic disturbances, the interacting effects of temperature and copper (Cu) were investigated in vitro. Mitochondria isolated from rainbow trout livers were exposed to a range of Cu concentrations at three temperatures (5, 15 and 25 °C) with measurement of mitochondrial complex I (mtCI)-driven respiratory flux indices and uncoupler-stimulated respiration. Additional studies assessed effects of temperature and Cu on mtCI enzyme activity, induction of mitochondrial permeability transition pore (MPTP), swelling kinetics and mitochondrial membrane potential (MMP). Maximal and basal respiration rates, as well as the proton leak, increased with temperature with the Q 10 effects being higher at lower temperatures. The effect of Cu depended on the mitochondrial functional state in that the maximal respiration was monotonically inhibited by Cu exposure while low and high Cu concentrations stimulated and inhibited the basal respiration/proton leak, respectively. Importantly, temperature exacerbated the effects of Cu by lowering the concentration of the metal required for toxicity and causing loss of thermal dependence of mitochondrial respiration. Mitochondrial complex I activity was inhibited by Cu but was not affected by incubation temperature. Compared with the calcium (Ca) positive control, Cu-imposed mitochondrial swelling exhibited variable kinetics depending on the inducing conditions, and was highly temperature-sensitive. A partial reversal of the Cu-induced swelling by cyclosporine A was observed suggesting that it is in part mediated by MPTP. Interestingly, the combination of high Cu and high temperature not only completely inhibited mitochondrial swelling but also greatly increased the respiratory control ratio (RCR) relative to the controls. Copper exposure also caused marked MMP dissipation which was reversed by N-acetyl cysteine and vitamin E suggesting a role of reactive oxygen species (ROS) in this response. Taken together, Cu impairs oxidative phosphorylation in part by inhibiting the electron transport chain (ETC), stimulating proton leak, inducing MPTP and dissipating MMP, with high temperature exacerbating these effects. Thus environmental temperature rise due to natural phenomenon or global climate change may sensitize fish to Cu toxicity by exacerbating mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]