Collins Kamunde

Early subcellular partitioning of cadmium in gill and liver of rainbow trout (Oncorhynchus mykiss) following low-to-near-lethal waterborne cadmium exposure.

Non-essential metals such as cadmium (Cd) accumulated in animal cells are envisaged to partition into potentially metal-sensitive compartments when detoxification capacity is exceeded. An understanding of intracellular metal partitioning is therefore important in delineation of the toxicologically relevant metal fraction for accurate tissue residue-based assessment of toxicity. In the present study, the early intracellular Cd accumulation was studied to test the prediction that it conforms to the spillover model of metal toxicity. Juvenile rainbow trout (10-15g) were exposed for 96h to three doses of cadmium (5, 25 and 50microg/l) and a control (nominal 0microg/l Cd) in hard water followed by measurement of the changes in intracellular Cd concentrations in the gill and liver, and carcass calcium (Ca) levels. There were dose-dependent increases in Cd concentration in both organs but the accumulation pattern over time was linear in the liver and biphasic in the gill. The Cd accumulation was associated with carcass Ca loss after 48h. Comparatively, the gill accumulated 2-4x more Cd than the liver and generally the subcellular compartments reflected the organ-level patterns of accumulation. For the gill the rank of Cd accumulation in subcellular fractions was: heat-stable proteins (HSP)>heat-labile proteins (HLP)>nuclei>microsomes-lysosomes (ML)>/=mitochondria>resistant fraction while for the liver it was HSP>HLP>ML>mitochondria>nuclei>resistant fraction. Contrary to the spillover hypothesis there was no exposure concentration or internal accumulation at which Cd was not found in potentially metal-sensitive compartments. The proportion of Cd bound to the metabolically active pool (MAP) increased while that bound to the metabolically detoxified pool (MDP) decreased in gills of Cd-exposed fish but remained unchanged in the liver. Because the Cd concentration increased in all subcellular compartments while their contribution to the total increased, decreased or remained unchanged following Cd exposure, use of percentage data to infer spillover requires caution.

The influence of ration size on copper homeostasis during sublethal dietary copper exposure in juvenile rainbow trout, Oncorhynchus mykiss.

The influence of ration size on homeostasis and sublethal toxicity of copper (Cu) was assessed in rainbow trout (Oncorhynchus mykiss) during dietary Cu exposure in synthetic soft water. A constant dietary dose of 0.24 micromol Cu per g fish per day as CuSO(4).5H(2)O was delivered via diets containing 15.75, 7.87, and 5.24 micromol Cu g(-1) fed at 1.5, 3.0, and 4.5% wet body weight daily ration, respectively. Juvenile rainbow trout showed clear effects of ration but not Cu on growth suggesting that growth is hardly a sensitive endpoint for detection of sublethal dietary Cu exposure. All Cu-exposed fish accumulated the same total metal load when expressed on a per fish basis. This suggests that differences in tissue and whole-body Cu concentrations among the treatments reflected the differences in the fish size rather than total Cu accumulation, and demonstrate that absorption and accumulation of Cu from the gut during dietary exposure are independent of the food quantity in which the Cu is delivered. Fish fed a high ration exhibited greater mass-specific unidirectional uptake of waterborne Cu than fish fed a low ration indicating an increased need for Cu for growth processes in rapidly growing fish. Stimulated excretion of Cu was indicated by greater Cu accumulation in the bile of the Cu-exposed fish. Branchial Na(+), K(+)-ATPase was not affected by dietary Cu exposure or ration but gut Na(+), K(+)-ATPase activities showed stimulatory effects of increasing ration but not of Cu exposure. The 96-h LC50 for waterborne Cu (range 0.17-0.21 micromol l(-1) (10.52-13.20 microg l(-1)) was the same in all treatment groups indicating that ration size was unimportant and that dietary Cu did not induce an increase in tolerance to waterborne Cu. Taken together these results suggest that the nutritional status, fish size, and growth rates should be considered when comparing whole-body and tissue Cu concentration data for biomonitoring and risk assessment. Moreover, expressing the exposure as total metal dose rather than metal concentration in the diet is more appropriate.

Bioaccumulation and subcellular partitioning of zinc in rainbow trout (Oncorhynchus mykiss): cross-talk between waterborne and dietary uptake.

Zinc homeostasis was studied at the tissue and gill subcellular levels in rainbow trout (Oncorhynchus mykiss) following waterborne and dietary exposures, singly and in combination. Juvenile rainbow trout were exposed to 150 or 600microgl(-1) waterborne Zn, 1500 or 4500microgg(-1) dietary Zn, and a combination of 150microgl(-1) waterborne and 1500microgg(-1) dietary Zn for 40 days. Accumulation of Zn in tissues and gill subcellular fractions was measured. At the tissue level, the carcass acted as the main Zn depot containing 84-90% of whole body Zn burden whereas the gill held 4-6%. At the subcellular level, the majority of gill Zn was bioavailable with the estimated metabolically active pool being 81-90%. Interestingly, the nuclei-cellular debris fraction bound the highest amount (40%) of the gill Zn burden. There was low partitioning of Zn into the detoxified pool (10-19%) suggesting that sequestration and chelation are not major mechanisms of cellular Zn homeostasis in rainbow trout. Further, the subcellular partitioning of Zn did not conform to the spill-over model of metal toxicity because Zn binding was indiscriminate irrespective of exposure concentration and duration. The contribution of the branchial and gastrointestinal uptake pathways to Zn accumulation depended on the tissue. Specifically, in plasma, blood cells, and gill, uptake from water was dominant whereas both pathways appeared to contribute equally to Zn accumulation in the carcass. Subcellularly, additive uptake from the two pathways was observed in the heat-stable proteins (HSP) fraction. Toxicologically, Zn exposure caused minimal adverse effects manifested by a transitory inhibition of protein synthesis in gills in the waterborne exposure. Overall, subcellular fractionation appears to have value in the quest for a better understanding of Zn homeostasis and interactions between branchial and gastrointestinal uptake pathways.

Metal-metal interactions of dietary cadmium, copper and zinc in rainbow trout, Oncorhynchus mykiss

The influence of metal-metal interactions on uptake, accumulation, plasma transport and chronic toxicity of dietary Cu, Cd and Zn in rainbow trout (Oncorhynchus mykiss) was explored. Juvenile rainbow trout were fed diets supplemented with (μg/g) 500 Cu, 1000 Zn and 500 Cd singly and as a ternary mixture at 2.5% body weight daily ration for 28 days. Complex interactions among the metals dependent on the tissue/organ, metals ratios and duration of exposure were observed. While Zn did not accumulate, whole-body Cd and Cu concentrations increased following linear and saturation patterns, respectively. Early enhanced whole-body Cu accumulation in fish exposed to the metals mixture was correlated with reduced Cd concentration whereas late enhancement of Cd accumulation corresponded with elevated Cd concentration. This suggests early mutual antagonism and late cooperation between Cd and Cu probably due to interactions at temporally variable metal accumulation sites. At the level of uptake, Cd and Cu were either antagonistic or mutually increased the concentrations of each other depending on the duration of exposure and section of the gut. At the level of transport, enhanced Cd accumulation in plasma was closely correlated with reduced concentrations of both Zn and Cu indicating competitive binding to plasma proteins and/or antagonism at uptake sites. Compared to the Cu alone exposure, Cu concentrations were either lower (gills and carcasses) or higher (liver and kidney) in fish exposed to the metals mixture. On the other hand, Cd accumulation was enhanced in livers and carcasses of fish exposed to the mixture compared to those exposed to Cd alone, while Zn stimulated Cu accumulation in gills. Chronic toxicity was demonstrated by elevated malondialdehyde levels in livers and reduced concentrations of Zn and Cu in plasma. Overall, interactions of Cd, Cu and Zn are not always consistent with the isomorphous competitive binding theory.

Differential Inhibition of Electron Transport Chain Enzyme Complexes by Cadmium and Calcium in Isolated Rainbow Trout (Oncorhynchus mykiss) Hepatic Mitochondria

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.

Reciprocal enhancement of uptake and toxicity of cadmium and calcium in rainbow trout (Oncorhynchus mykiss) liver mitochondria.

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.

Effects of hypoxia–cadmium interactions on rainbow trout (Oncorhynchus mykiss) mitochondrial bioenergetics: attenuation of hypoxia-induced proton leak by low doses of cadmium

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.

Interactions of copper and thermal stress on mitochondrial bioenergetics in rainbow trout, Oncorhynchus mykiss.

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.

Effect of humic acid during concurrent chronic waterborne exposure of rainbow trout (Oncorhynchus mykiss) to copper, cadmium and zinc

The effects of commercial dissolved organic carbon (DOC) in moderating accumulation, biochemical responses and toxicity of a waterborne mixture of copper (Cu), cadmium (Cd) and zinc (Zn) were investigated during a chronic exposure. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to a ternary metals mixture containing (nominal concentrations in μg/l): Cu 30, Cd 15, and Zn 150 in hard water (260 mg/l as CaCO(3)) with and without addition of 5 mg/l DOC as Aldrich humic acid (HA) for 28 days. Mortality, growth, metals accumulation, ionoregulatory impairment, and oxidative stress response were measured. While growth was unaffected, 19% mortality occurred during the first week of the exposure in fish exposed to the metals mixture without added HA. The early mortality was associated with transitory whole-body sodium (Na) loss and inhibition of branchial Na(+), K(+)-ATPase activity. Although these ionoregulatory responses mechanistically suggested that Cu was the more potent toxicant than either Cd or Zn, they were not correlated uniquely with elevated tissue Cu concentrations. The effects of HA on accumulation were metal-specific and depended on the organ examined and exposure duration. Specifically, Zn accumulation occurred only in the gill early in the exposure and HA reversed it, while protection against accumulation was absent or complete for Cu and absent or partial for Cd, dependent on tissue and exposure duration. The computed ambient free metal ion activities could explain the Cd but not the Cu and Zn accumulation indicating the involvement of physiological regulatory mechanisms in defining accumulation of essential metals. Surprisingly, the metals mixture (with and without added HA) reduced the concentrations of malondialdehyde (MDA) in gill suggesting induction of reductive rather than oxidative stress. Overall these data indicate that the free metal ion activity alone is not universally a good predictor of metals mixture accumulation and chronic effects nor does consideration of the mechanisms of toxicity unambiguously identify the more potently toxic metal in a mixture.

Modulation of cadmium-induced mitochondrial dysfunction and volume changes by temperature in rainbow trout (Oncorhynchus mykiss).

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.