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Zinc and calcium have highly interwoven functions that are essential for cellular homeostasis. Here we first present a novel real-time flow cytometric technique to measure mitochondrial redox state and show it is modulated by zinc and calcium, individually and combined. We then assess the interactions of zinc and calcium on mitochondrial H2O2 production, membrane potential (ΔΨm), morphological status, oxidative phosphorylation (OXPHOS), complex I activity, and structural integrity. Whereas zinc at low doses and both cations at high doses individually and combined promoted H2O2 production, the two cations individually did not alter mitochondrial redox state. However, when combined at low and high doses the two cations synergistically suppressed and promoted, respectively, mitochondrial shift to a more oxidized state. Surprisingly, the antioxidants vitamin E and N-acetylcysteine showed pro-oxidant activity at low doses, whereas at high antioxidant doses NAC inhibited OXPHOS and dyscoupled mitochondria. Individually, zinc was more potent than calcium in inhibiting OXPHOS, whereas calcium more potently dissipated the ΔΨm and altered mitochondrial volume and ultrastructure. The two cations synergistically inhibited OXPHOS but antagonistically dissipated ΔΨm and altered mitochondrial volume and morphology. Overall, our study highlights the importance of zinc and calcium in mitochondrial redox regulation and functional integrity. Importantly, we uncovered previously unrecognized bidirectional interactions of zinc and calcium that reveal distinctive foci for modulating mitochondrial function in normal and disease states because they are potentially protective or damaging depending on conditions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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 Q10 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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 Q10 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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 4ol (proton leak) respirations. High temperature alone increased mitochondrial respiration in all states. The Q10 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 Q10 values well below unity and decreased activation energies (E a) 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.

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.

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 (IC(50)) 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 IC(50) 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.

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 © 2011 Elsevier Ltd. All rights reserved.)

Rainbow trout were exposed to sublethal waterborne Cd (5 and 10 μg L(-1)) and dietary Ca (60 mg g(-1)), individually and in combination, for 30 d 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 © 2011 Elsevier Ltd. All rights reserved.)

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 (5-100 microM) of Cd and Ca singly and in combination at 15 degrees 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 (1 microM) 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 5 min, 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, 100 microM) 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., (2009 Elsevier B.V. All rights reserved.)

Sampling of a population is frequently required to understand trends and patterns in natural resource management because financial and time constraints preclude a complete census. A rigorous probability-based survey design specifies where to sample so that inferences from the sample apply to the entire population. Probability survey designs should be used in natural resource and environmental management situations because they provide the mathematical foundation for statistical inference. Development of long-term monitoring designs demand survey designs that achieve statistical rigor and are efficient but remain flexible to inevitable logistical or practical constraints during field data collection. Here we describe an approach to probability-based survey design, called the Reversed Randomized Quadrant-Recursive Raster, based on the concept of spatially balanced sampling and implemented in a geographic information system. This provides environmental managers a practical tool to generate flexible and efficient survey designs for natural resource applications. Factors commonly used to modify sampling intensity, such as categories, gradients, or accessibility, can be readily incorporated into the spatially balanced sample design.

Molecular diagnostics may be a more efficient method to manage resources; but most Microbiology laboratories have not introduced them into routine use due to the specialized training required. Using vancomycin resistant enterococci (VRE) screening during a comparison of a multiplex PCR (MPCR) and conventional biochemical testing (CBT) we studied 3 objectives: 1) to develop a molecular diagnostics in-house training program, 2) to assess the training program outcomes for competency and confidence, and 3) to determine laboratory payback. A training program for 14 technologists using multiple adult learning methods was implemented. Methods to minimize technical errors were introduced and included: use of a calibrated loop to deliver sample; prealiquotting reagents; increasing volume of specimen; addition of gel loading dye directly into reaction tubes; and establishment of an equivocal zone. In our laboratory MPCR costs $7.06 less than CBT, therefore the payback period for training and implementation would be approximately 3 years.