Subcellular interactions of dietary cadmium, copper and zinc in rainbow trout (Oncorhynchus mykiss).

Interactions of Cu, Cd and Zn were studied at the subcellular level in juvenile rainbow trout (Oncorhynchus mykiss) fed diets containing (µg/g) 500 Cu, 1000 Zn and 500 Cd singly and as a ternary mixture for 28 days. Livers were harvested and submitted to differential centrifugation to isolate components of metabolically active metal pool (MAP: heat-denaturable proteins (HDP), organelles, nuclei) and metabolically detoxified metal pool (MDP: heat stable proteins (HSP), NaOH-resistant granules). Results indicated that Cd accumulation was enhanced in all the subcellular compartments, albeit at different time points, in fish exposed to the metals mixture relative to those exposed to Cd alone, whereas Cu alone exposure increased Cd partitioning. Exposure to the metals mixture reduced (HDP) and enhanced (HSP, nuclei and granules) Cu accumulation while exposure to Zn alone enhanced Cu concentration in all the fractions analyzed without altering proportional distribution in MAP and MDP. Although subcellular Zn accumulation was less pronounced than that of either Cu or Cd, concentrations of Zn were enhanced in HDP, nuclei and granules from fish exposed to the metals mixture relative to those exposed to Zn alone. Cadmium alone exposure mobilized Zn and Cu from the nuclei and increased Zn accumulation in organelles and Cu in granules, while Cu alone exposure stimulated Zn accumulation in HSP, HDP and organelles. Interestingly, Cd alone exposure increased the partitioning of the three metals in MDP indicative of enhanced detoxification. Generally the accumulated metals were predominantly metabolically active: Cd, 67-83%; Cu, 68-79% and Zn, 60-76%. Taken together these results show both competitive and cooperative interactions dependent on the subcellular fraction, metal, exposure duration and relative metal exposure concentrations. Competitive interactions likely result from ionic mimicry with the metals displacing each other from common binding sites, whereas cooperative interactions suggest increased abundance of metal binding sites and/or existence of metal-specific non-interacting binding sites in some of the fractions. Moreover, the changes in subcellular distribution of the biometals Cu and Zn due to Cd exposure together with the shifts of the metals between MAP and MDP observed may have toxicological consequences.

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.

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.

Evaluation of antioxidant circulatory lipid-soluble vitamins and sodium as non-invasive indicators of chronic copper exposure and toxicity in rainbow trout, Oncorhynchus mykiss.

Measurement of circulatory indicators of copper (Cu) exposure and toxicity in rainbow trout revealed elevated Cu concurrent with reduced sodium (Na) concentrations in plasma of Cu-exposed fish. Using a new normal phase high performance liquid chromatography (HPLC) method developed and validated for simultaneous extraction of lipid-soluble antioxidant vitamins we found that, contrary to our original hypothesis, plasma antioxidant status was enhanced as evidenced by a linear increase in vitamin E concentration. This suggests that vitamin E was mobilized from other metabolic pools to enhance circulatory antioxidant status possibly for delivery to Cu-sensitive locales. On the other hand, plasma vitamin A was not affected by the Cu exposure although its level decreased with time concurrent with an increase in fish size suggesting increased demand for growth. Thus circulatory Cu, Na, and vitamin E, but not vitamin A, can be used as non-lethal biomarkers of chronic Cu exposure and toxicity in fish.

Bioaccumulation and hepatic speciation of copper in rainbow trout (Oncorhynchus mykiss) during chronic waterborne copper exposure.

To protect cells from toxicity, metal-sensitive cellular compartments must be insulated against essential but toxic metals [such as copper (Cu)] accumulated in excess of metabolic requirements. We measured Cu concentrations at the organ and hepatic subcellular levels in juvenile rainbow trout (Oncorhynchus mykiss) during exposure to sublethal waterborne Cu (40 microg/L) for 21 days. There was a time-dependent accumulation of Cu in the gill, liver, plasma, and carcass, with significant difference in Cu-exposed fish relative to the controls being evident by day 7. This significant accumulation of Cu was not associated with impaired growth. Copper concentrations in purity-tested liver subcellular fractions normalized to the liver protein concentration were in decreasing order: organelles > heat-stable proteins > nuclei-debris > NaOH-resistant granules > heat-labile proteins. As a proportion of the total, the majority of the hepatocellular Cu burden (60-68%) was associated with a metabolically active pool (organelles, nuclei-debris, and heat-stable proteins) and the remainder (32-40%) was associated with a metabolically detoxified pool (heat-stable proteins and NaOH-resistant granules) irrespective of the Cu-exposure regime. Because Cu concurrently accumulated in metabolically active and detoxified pools, we conclude that the spillover hypothesis of metal toxicity did not hold under the exposure conditions employed in this study. Moreover, these data suggest that rainbow trout can withstand significant above-background Cu accumulation in hepatic putative metal-sensitive compartments without chronic toxic effects at the organism level.