Interactive effects of temperature, cadmium, and hypoxia on rainbow trout (Oncorhynchus mykiss) liver mitochondrial bioenergetics.

Fish in their natural environments possess elaborate mechanisms that regulate physiological function to mitigate the adverse effects of multiple environmental stressors such as temperature, metals, and hypoxia. We investigated how warm acclimation affects mitochondrial responses to Cd, hypoxia, and acute temperature shifts (heat shock and cold snap) in rainbow trout. We observed that state 3 respiration driven by complex I (CI) was resistant to the stressors while warm acclimation and Cd reduced complex I +II (CI + II) driven state 3 respiration. In contrast, state 4 (leak) respirations for both CI and CI + II were consistently stimulated by warm acclimation resulting in reduced mitochondrial coupling efficiency (respiratory control ratio, RCR). Warm acclimation and Cd exacerbated their individual effect on leak respiration to further reduce the RCR. Moreover, the effect of warm acclimation on mitochondrial bioenergetics aligned with its inhibitory effect on activities of citrate synthase and both CI and CII. Unlike the Cd and warm acclimation combined exposure, hypoxia alone and in combination with warm acclimation and/or Cd abolished the stimulation of CI and CI + II powered leak respirations resulting in partial recovery of RCR. The response to acute temperature shifts indicated that while state 3 respiration returned to pre-acclimation level, the leak respiration did not. Overall, our findings suggest a complex in vivo interaction of multiple stressors on mitochondrial function that are not adequately predicted by their individual effects.

Interactions of binary mixtures of metals on rainbow trout (Oncorhynchus mykiss) heart mitochondrial H2O2 homeodynamics.

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, H2O2) 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 H2O2 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 H2O2 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 H2O2 homeodynamics in heart mitochondria.

A model naphthenic acid decouples oxidative phosphorylation through selective inhibition of mitochondrial complex activity.

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.

Effect of skeletal muscle mitochondrial phenotype on H2O2 emission.

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 H2O2) 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 H2O2 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 H2O2 emission rates were highest at the lowest protein concentration tested and decreased exponentially thereafter. However, the absolute values of H2O2 emission rates depended on the calibration curves used to convert fluorescent signals to H2O2 while the trends depended on the normalization strategy. We found substantial qualitative and quantitative differences between RMM and WMM in the H2O2 emission rates depending on the substrates being oxidized and their concentrations. Similarly, pharmacological modulators of the ETS altered the magnitudes and trends of the H2O2 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 H2O2 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 H2O2 emission rates.