Mechanisms of PVP-functionalized silver nanoparticle toxicity in fish: Intravascular exposure disrupts cardiac pacemaker function and inhibits Na+/K+-ATPase activity in heart, but not gill.

Polyvinylpyrrolidone-functionalized silver nanoparticles (nAgPVP) are popular in consumer products for their colloidal stability and antimicrobial activity. Whole lake additions of nAgPVP cause long term, ecosystem-scale changes in fish populations but the mechanisms underlying this effect are unclear. We have previously shown that in fish, nAgPVP impairs cardiac contractility and Na+/K+-ATPase (NKA) activity in vitro, raising the possibility that heart dysfunction could underlie population-level exposure effects. The goal of this study was to determine if nAgPVP influences the control of heart rate (fh), blood pressure, or cardiac NKA activity in vivo. First, a dose-response curve for the effects of 5 nm nAgPVP on contractility was completed on isometrically contracting ventricular muscle preparations from Arctic char (Salvelinus alpinus) and showed that force production was lowest at 500 µg L-1 and maximum pacing frequency increased with nAgPVP concentration. Stroke volume, cardiac output, and power output were maintained in isolated working heart preparations from brook char (Salvelinus fontinalis) exposed to 700 µg L-1 nAgPVP. Both fh and blood pressure were elevated after 24 h in brook char injected with 700 µg kg body mass-1 nAgPVP and fh was insensitive to modulation with blockers of ß-adrenergic and muscarinic cholinergic receptors. Na+/K+-ATPase activity was significantly lower in heart, but not gill of nAgPVP injected fish. The results indicate that nAgPVP influences cardiac function in vivo by disrupting regulation of the pacemaker and cardiomyocyte ionoregulation. Impaired fh regulation may prevent fish from appropriately responding to environmental or social stressors and affect their ability to survive.

Intracellular taurine deficiency impairs cardiac contractility in rainbow trout (Oncorhynchus mykiss) without affecting aerobic performance.

Taurine is a non-proteinogenic sulfonic acid found in high concentrations inside vertebrate cardiomyocytes and its movement across the sarcolemmal membrane is critical for cell volume regulation. Taurine deficiency is rare in mammals, where it impairs cardiac contractility and leads to congestive heart failure. In fish, cardiac taurine levels vary substantially between species and can decrease by up to 60% in response to environmental change but its contribution to cardiac function is understudied. We addressed this gap in knowledge by generating a taurine-deficient rainbow trout (Oncorhynchus mykiss) model using a feed enriched with 3% ß-alanine to inhibit cellular taurine uptake. Cardiac taurine was reduced by 17% after 4 weeks with no effect on growth or condition factor. Taurine deficiency did not affect routine or maximum rates of O2 consumption, aerobic scope, or critical swimming speed in whole animals but cardiac contractility was significantly impaired. In isometrically contracting ventricular strip preparations, the force-frequency and extracellular Ca2+-sensitivity relationships were both shifted downward and maximum pacing frequency was significantly lower in ß-alanine fed trout. Cardiac taurine deficiency reduces sarcoplasmic reticular Ca2+-ATPase activity in mammals and our results are consistent with such an effect in rainbow trout. Our data indicate that intracellular taurine contributes to the regulation of cardiac contractility in rainbow trout. Aerobic performance was unaffected in ß-alanine-fed animals, but further study is needed to determine if more significant natural reductions in taurine may constrain performance under certain environmental conditions.

Commentary: Revisiting nanoparticle-assay interference: There's plenty of room at the bottom for misinterpretation.

Engineered nanomaterials (ENMs) are a diverse class of materials whose distinct properties make them desirable in a multitude of applications. The proliferation of nanotoxicology research has improved our understanding of ENM toxicity, but an under appreciation for their potential to interfere with biochemical assays has hampered progress in the field. The physicochemical properties of ENMs can promote their interaction with membranes or biomacromolecules (e.g. proteins, genomic material). This can influence the activity of enzymes used as biomarkers or as reagents in biochemical assay protocols, bind indicator dyes in cytotoxicity tests, and/or interfere with the cellular mechanisms controlling the uptake of such dyes. The spectral characteristics of some ENMs can cause interference with common assay chromophores, fluorophores, and radioisotope scintillation cocktails. Finally, the inherent chemical reactivity of some ENMs can short circuit assay mechanisms by directly oxidizing or reducing indicator dyes. These processes affect data quality and may lead to significant misinterpretations regarding ENM safety. We provide an overview of some ENM properties that facilitate assay interference, examples of interference and the erroneous conclusions that may result from it, and a number of general and specific recommendations for validating cellular and biochemical assay protocols in nanotoxicology studies.

Polyvinylpyrolidone-functionalized silver nanoparticles do not affect aerobic performance or fractional rates of protein synthesis in rainbow trout (Oncorhynchus mykiss).

Aerobic performance in fish is linked to individual and population fitness and can be impacted by anthropogenic contaminants. Exposure to some engineered nanomaterials, including silver nanoparticles (nAg), reduces rates of oxygen consumption in some fish species, but the underlying mechanisms remain unclear. In addition, their effects on swim performance have not been studied. Our aim was to quantify the impact of exposure to functionalized nAg on aerobic scope and swim performance in rainbow trout (Oncorhychus mykiss) and to characterize the contribution of changing rates of protein synthesis to these physiological endpoints. Fish were exposed for 48 h to 5 nm polyvinylpyrolidone-functionalized nAg (nAgPVP; 100 µg L-1) or 0.22 µg L-1 Ag+ (as AgNO3), which was the measured quantity of Ag released from the nAgPVP over that time period. Aerobic scope, critical swimming speed (Ucrit), and fractional rates of protein synthesis (Ks), were then assessed, along with indicators of osmoregulation and cardiotoxicity. Neither nAgPVP, nor Ag+ exposure significantly altered aerobic scope, its component parts, or swim performance. Ks was similarly unaffected in 8 tissue types, though it tended to be lower in liver of nAgPVP treated fish. The treatments tended to decrease gill Na+/K+-ATPase activity, but effects were not significant. The latter results suggest that a longer or more concentrated nAgPVP exposure may induce significant effects. Although this same formulation of nAgPVP is bioactive in other fish, it had no effects on rainbow trout under the conditions tested. Such findings on common model animals like trout may thus misrepresent the safety of nAg to more sensitive species.

Functionalized silver nanoparticles depress aerobic metabolism in the absence of overt toxicity in brackish water killifish, Fundulus heteroclitus.

Engineered nanomaterials (ENMs) tend to precipitate in saline waters so the majority of aquatic toxicity studies have focused on freshwaters, where bioavailability is presumed to be higher. Recent studies have illustrated that some ENM formulations are bioavailable and bioactive in salt water and that their effects are more pronounced at the physiological than biochemical level. These findings raise concerns regarding the effects of ENMs on marine organisms. Therefore, our goal was to characterize the effects of polyvinylpyrolidone-functionalized silver ENMs (nAg) on aerobic performance in the killifish (Fundulus heteroclitus), a common euryhaline teleost. Fish were exposed to 80 µg L-1 of 5 nm nAg for 48 h in brackish water (12 ppt) and routine (MO2min) and maximum (MO2max) rates of oxygen consumption were quantified. Silver dissolution was minimal and nAg remained well dispersed in brackish water, with a hydrodynamic diameter of 21.0 nm, compared to 19.3 in freshwater. Both MO2min and MO2max were significantly lower (by 53 and 30%, respectively) in killifish exposed to nAg and a reduction in MO2 variability suggested spontaneous activity was suppressed. Neither gill Na+/K+-ATPase activity, nor various other biochemical markers were affected by nAg exposure. The results illustrate that a common ENM formulation is bioactive in salt water and, as in previous studies on functionalized copper ENMs, that effects are more pronounced at the whole animal than the biochemical level.

Cardiorespiratory and tissue adenosine responses to hypoxia and reoxygenation in the short-horned sculpin Myoxocephalus scorpius.

Adenosine is a product of adenylate phosphate breakdown that can exert protective effects on tissues during energy limitation. Accumulation of cardiac adenosine under hypoxia is well documented in mammals but has not been shown in fish. Adenosine content was measured in heart and brain tissue from short-horned sculpin Myoxocephalus scorpius L. exposed to acute hypoxia and to graded hypoxia and reoxygenation at 8 degrees C. Cardiorespiratory parameters were recorded along with plasma lactate, K(+), Ca(2+) and Na(+) levels and their relationship to adenosine levels investigated. Sculpin exhibited a large bradycardia during hypoxia, with a concomitant drop in cardiac output that recovers fully with reoxygenation. Ventilation rate also declined with hypoxia, suggesting a depression of activity. Plasma lactate concentration was significantly elevated after 4 h at 2.0 mg l(-1) dissolved oxygen while K(+) levels increased during acute hypoxia. Adenosine levels were maintained in heart under acute and graded hypoxia. Brain levels fluctuated under hypoxia and showed no change with reoxygenation. It is concluded that a depression of cardiac activity in conjunction with an adequate anaerobic metabolism allow sculpin to avoid excessive adenosine accumulation under conditions of moderate hypoxia. Cardiac adenosine levels decreased and plasma K(+) levels and heart rate increased significantly at reoxygenation.