Neal Ingraham Callaghan

Cardiorespiratory toxicity of environmentally relevant zinc oxide nanoparticles in the freshwater fish Catostomus commersonii

Abstract The inhalation of zinc oxide engineered nanomaterials (ENMs) has been linked to cardiorespiratory dysfunction in mammalian models but the effects of aquatic ENM exposure on fish have not been fully investigated. Nano-zinc oxide (nZnO) is widely used in consumer products such as sunscreens and can make its way into aquatic ecosystems from domestic and commercial wastewater. This study examined the impact of an environmentally relevant nZnO formulation on cardiorespiratory function and energy metabolism in the white sucker (Catostomus commersonii), a freshwater teleost fish. Evidence of oxidative and cellular stress was present in gill tissue, including increases in malondialdehyde levels, heat shock protein (HSP) expression, and caspase 3/7 activity. Gill Na+/K+-ATPase activity was also higher by approximately three-fold in nZnO-treated fish, likely in response to increased epithelial permeability or structural remodeling. Despite evidence of toxicity in gill, plasma cortisol and lactate levels did not change in animals exposed to 1.0 mg L−1 nZnO. White suckers also exhibited a 35% decrease in heart rate during nZnO exposure, with no significant changes in resting oxygen consumption or tissue energy stores. Our results suggest that tissue damage or cellular stress resulting from nZnO exposure activates gill neuroepithelial cells, triggering a whole-animal hypoxic response. An increase in parasympathetic nervous signaling will decrease heart rate and may reduce energy demand, even in the face of an environmental toxicant. We have shown that acute exposure to nZnO is toxic to white suckers and that ENMs have the potential to negatively impact cardiorespiratory function in adult fish.

Spherical Gold Nanoparticles Impede the Function of Bovine Serum Albumin In vitro: A New Consideration for Studies in Nanotoxicology

Spherical Gold Nanoparticles Impede the Function of Bovine Serum Albumin In vitro: A New Suspensions of bovine serum albumin (BSA) and spherical gold nanoparticles were analyzed to determine if gold nanoparticles (nAu) affect the ligand binding properties of BSA. A range of diameters of nAu with a carboxylic acid capping agent (nAu-cap) were tested, along with nanoparticles conjugated to amine (nAu-NH3+) and carboxyl (nAu-COO-) functional groups via a covalent polymer bridge. All nAu tested were found to affect BSA conformation as determined by intrinsic tryptophan fluorescence. Smaller diameters of nAu-cap (30-50 nm), along with nAu-NH3+ and nAu-COO-, impeded the binding of 8-anilino-1-napthalenesulfonic acid (ANS) to BSA. Similarly, smaller diameters of nAu-cap tended to impede oleic acid binding to BSA, with a linear negative correlation observed between nAu-cap diameter and the dissociation constant (KD) of oleic acid over the range of 40-80 nm. 80 nm nAu-cap impeded butanoic acid binding, and necessitated a high-resolution fluorescence assay. As with oleic acid, smaller diameters of nAu-cap tended to impede ibuprofen binding, but no significance could be established.

Metabolic Adjustments to Short-Term Diurnal Temperature Fluctuation in the Rainbow Trout (Oncorhynchus mykiss)

In rainbow trout, warmer temperatures increase metabolic rate, which can be energetically stressful. Diel fluctuations in water temperatures are common in rivers, raising the question of whether fish experience metabolic preconditioning with repeated heat stress. In this study, rainbow trout (Oncorhynchus mykiss Walbaum, 1792) were subjected to three temperature treatments consisting of either a constant exposure to 16°C, a single exposure to 24°C, or three cycles between 16° and 24°C. Metabolic responses were investigated, including patterns of regulation of adenosine monophosphate–activated protein kinase and its substrates, key metabolic enzymes, and several relevant metabolites. In liver and, to a lesser extent, in heart, patterns of signal transduction suggest an increasingly anabolic phenotype with successive heat cycles. Inhibition of Raptor in the heart suggests lowered gross protein synthesis after multiple heat cycles. Fish also showed recovery of glycogen stores and lipid synthesis after multiple thermal cycles, while they maintained baseline plasma glucose levels. The animals showed no evidence of hypoxemia, and our results suggest rainbow trout exposed to repeated thermal cycles were not at risk of metabolic substrate depletion. Collectively, our data indicate that, when exposed to fluctuating but noncritical thermal cycles, rainbow trout may adopt a new metabolic phenotype to sequester readily accessible metabolic substrates in the liver in preparation for more severe or sustained thermal exposures.

Physiological hepatic response to zinc oxide nanoparticle exposure in the white sucker, Catostomus commersonii.

Liver toxicity of commercially relevant zinc oxide nanoparticles (nZnO) was assessed in a benthic freshwater cypriniform, the white sucker (Catostomus commersonii). Exposure to nZnO caused several changes in levels of liver enzyme activity, antioxidants, and lipid peroxidation end products consistent with an oxidative stress response. Aconitase activity decreased by ~65% but tended to be restored to original levels upon supplementation with Fe(2+), indicating oxidative inactivation of the 4Fe-4S cluster. Furthermore, glucose-6-phosphate dehydrogenase activity decreased by ~29%, and glutathione levels increased by ~56%. Taken together, these suggest that nZnO induces hepatic physiological stress. Each assay was then validated by using a single liver homogenate or plasma sample that was partitioned and treated with nZnO or Zn(2+), the breakdown product of nZnO. It was found that Zn(2+), but not nZnO, increased detected glutathione reductase activity by ~14% and decreased detected malondialdehyde by ~39%. This indicates that if appreciable nZnO dissolution occurs in liver samples during processing and assay, it may skew results, with implications not only for this study, but also for a wide range of nanotoxicology studies focusing on nZnO. Finally, in vitro incubations of cell-free rat blood plasma with nZnO failed to generate any significant increase in malondialdehyde or protein carbonyl levels, or any significant decrease in ferric reducing ability of plasma. This suggests that at the level tested, any oxidative stress caused by nZnO is the result of a coordinated physiological response by the liver.

Metabolic rate and rates of protein turnover in food-deprived cuttlefish, Sepia officinalis (Linnaeus 1758)

To determine the metabolic response to food deprivation, cuttlefish (Sepia officinalis) juveniles were either fed, fasted (3 to 5 days food deprivation), or starved (12 days food deprivation). Fasting resulted in a decrease in triglyceride levels in the digestive gland, and after 12 days, these lipid reserves were essentially depleted. Oxygen consumption was decreased to 53% and NH4 excretion to 36% of the fed group following 3-5 days of food deprivation. Oxygen consumption remained low in the starved group, but NH4 excretion returned to the level recorded for fed animals during starvation. The fractional rate of protein synthesis of fasting animals decreased to 25% in both mantle and gill compared with fed animals and remained low in the mantle with the onset of starvation. In gill, however, protein synthesis rate increased to a level that was 45% of the fed group during starvation. In mantle, starvation led to an increase in cathepsin A-, B-, H-, and L-like enzyme activity and a 2.3-fold increase in polyubiquitin mRNA that suggested an increase in ubiquitin-proteasome activity. In gill, there was a transient increase in the polyubiquitin transcript levels in the transition from fed through fasted to the starved state and cathepsin A-, B-, H-, and L-like activity was lower in starved compared with fed animals. The response in gill appears more complex, as they better maintain rates of protein synthesis and show no evidence of enhanced protein breakdown through recognized catabolic processes.

Cardioprotective mitochondrial binding by hexokinase I is induced by a hyperoxic acute thermal insult in the rainbow trout (Oncorhynchus mykiss)

Due to the substantial photosynthetic biomass in their habitat, salmonids such as the rainbow trout (Oncorhynchus mykiss) can be subject to hyperoxia in addition to high temperatures associated with climate change. Both stressful conditions increase the incidence of damaging reactive oxygen species (ROS). The mitochondrial association of hexokinase has been shown to increase in the hearts of certain fish experiencing hypoxia in a putative cardioprotective response to oxidative stress. In this study, the mitochondrial association of hexokinase I (HKI) and markers of oxidative damage and metabolic stress were probed to elucidate the cardioprotective role of hexokinase in the rainbow trout. Results showed that the co-administration of hyperoxia and hyperthermia increased the ventricular mitochondrially-bound fraction of HKI, whereas exposure to hyperthermia in normoxia had no effect; in the combined condition there was little evidence of increased stress. A second in vitro study using ventricular strips and isolated cardiomyocytes was undertaken to reconcile the cardioprotective role of HK in the rainbow trout with findings in mammalian studies, confirming that mitochondrial association of HK maintains aerobic efficiency and inhibits apoptosis. Finally, protein sequence analysis suggested that the physiological contributions of HKI and HKII in salmonids vary from those in mammals, further explaining the dynamic nature of the traditionally-inert HKI. Together, these findings help to explain the broader functions of HKI in the salmonid heart, and illustrate the role of complex environmental conditions in defining physiological responses.

Cerium oxide nanoparticles exhibit minimal cardiac and cytotoxicity in the freshwater fish Catostomus commersonii.

Metal oxide nanomaterials can cause oxidative, cardiorespiratory, and osmoregulatory stress in freshwater fish. In contrast, cerium oxide nanoparticles (nCeO2) can have antioxidant effects but their aquatic toxicity has not been fully characterized. Heart rate and heart rate variability were followed in white sucker (Catostomus commersonii) acutely exposed to 1.0 mg L(-1) nCeO2 for 25 h. Malondialdehyde (MDA) was measured to assess oxidative tissue damage, and plasma cortisol, glucose, lactate, and osmolality were assessed as indicators of physiological and osmoregulatory stress. There was no MDA accumulation in gill or heart of fish exposed to nCeO2 and heart function was unchanged over the 25 h treatment. Plasma cortisol increased 6-fold but there was no change in plasma glucose or lactate. Cellular osmoregulatory toxicity was studied using an isolated red blood cell (RBC) model. In vitro exposure to 1.0 mg L(-1) nCeO2 for 1h had no effect on cell morphological parameters and did not sensitize RBCs to hemolysis under hypotonic stress. Overall, there were no indications of oxidative, cardiorespiratory, or osmoregulatory stress following acute exposure to nCeO2. Elevated plasma cortisol levels suggest that nCeO2 may exert mild toxicity to tissues outside of the cardiorespiratory system.

β‐Adrenergic augmentation of cardiac contractility is dependent on PKA‐mediated phosphorylation of myosin‐binding protein C and troponin I

During times of increased circulatory demand, cardiac β-adrenergic stimulation serves to maximize cardiac output through the activation of protein kinase A (PKA) (Layland et al. 2005; Gresham et al. 2014; Gresham & Stelzer, 2016). PKA phosphorylates troponin I (TnI) and myosin-binding protein C (MyBP-C), which has been associated with increased cardiac contractility and output. In heart failure patients, changes in PKA expression and activation patterns resulted in lowered TnI and MyBP-C phosphorylation, as well as in ventricular wall remodelling and contractile dysfunction (Han et al. 2013; Gresham & Stelzer, 2016). Previous examination of one of the PKA-mediated phosphorylations of MyBP-C (Ser) suggested that the event was critical in achieving the contractile kinetics associated with the β-adrenergic response (Gresham et al. 2014). The authors attributed the role of MyBP-C phosphorylation in the observed changes in force development kinetics and the resulting haemodynamics to a relieving of sterical constraint on myosin heads, allowing them to crosslink more efficiently with actin. Before a recent study in The Journal of Physiology (Gresham & Stelzer, 2016), the relative contributions of PKA-specific TnI and MyBP-C phosphorylation in contractility and morphology were unknown. This study appears to be the first to examine the role of both TnI and MyBP-C phosphorylation together. The authors of this study provide an extensive and convincing argument for the role of PKA-phosphorylated MyBP-C but not TnI in the regulation of ventricular morphology and in β-adrenergic cardiac tone. The effects of MyBP-C phosphorylation are well interpreted and supported by previous studies, but there is also an indication of subtle TnI effects in contractile dynamics, as found in other work, that merits further investigation. In this study, mouse strains that were unphosphorylatable at either TnI Ser and Ser (TnIPKA−) or MyBP-C Ser, Ser, and Ser (MyBPPKA−) by PKA due to serine-to-alanine substitutions were crossed to produce a TnI and MyBP-C phosphorylation-ablated strain (DBLPKA−). The success of each ablation was confirmed using total protein content and phospho-specific Western blot analysis for TnI, MyBP-C and troponin T in myofibrillar extracts exposed to active PKA. Echocardiography was employed to assess the effect of these respective phosphorylation ablations on cardiac function by comparing each of the three strains to a wild-type (WT) control. Both MyBPPKA− and DBLPKA− but not TnIPKA− hearts were increased from the WT in mass relative to body weight, left-ventricular posterior wall thickness, and end-systolic and diastolic volumes. The authors observed lowered ejection fractions in MyBPPKA− and DBLPKA− lines, which did not significantly increase under β-adrenergic-stimulated (induced with dobutamine (DOB)) conditions as they did in WT and TnIPKA− strains. MyBPPKA− and DBLPKA− lines also decreased in fractional shortening, under dobutamine induction of the β-adrenergic response (DOB+) and control (DOB−) conditions. Haemodynamic parameters were further assessed using pressure–volume (P–V) loop analysis using DOB− and DOB+ cannulated left ventricles from each strain. Peak ejection rates were impaired in MyBPPKA− and DBLPKA− lines when exposed to DOB compared to the WT strain, as were the maximum rate of pressure development and the maximum ventricular power. Diastolic parameters were also impaired in MyBPPKA− and DBLPKA− strains; the difference in end-diastolic pressure was significantly larger in TnIPKA−, MyBPPKA−, and DBLPKA− lines than the wild type. The difference between sham and DOB responses from WT and TnlPKA− lines was significant in the peak filling rate, the rate of diastolic pressure decrease, and the relaxation time constant tau. Furthermore, the MyBPPKA− and DBLPKA− but not the TnIPKA− lines exhibited impaired early relaxation kinetics and time to minimum rate of pressure development when compared to the WT strain. Maximum systolic pressure and heart rate in either DOB− or DOB+ treatments did not vary between treatments. After DOB activation of the β-adrenergic response, MyBPPKA− and DBLPKA− strains increased in systolic duration while decreasing in diastolic duration. Specifically, the largest differences in phase duration in these lines were observed in ejection and filling. These effects translated to similar in vivo changes. Predictably, blood pressure monitoring in MyBPPKA− and DBLPKA− animals exposed to DOB showed decreased systolic blood pressure, and an increased time to peak pressure during systole without changing the heart rate. These results clearly show that MyBP-C phosphorylation during the β-adrenergic response is responsible for accelerated pressure generation and diastolic filling and relaxation. Given its subtle effects, the authors understandably focus the discussion away from the contributions of TnI phosphorylation to β-adrenergic tone and kinetics. TnI phosphorylation contributes to a decreased affinity for calcium, and thus a positive lusitropic effect (Layland et al. 2005). The provided diastolic relaxation curves suggest a certain baseline level of TnI phosphorylation, as the TnIPKA− and DBLPKA− but not so much MyBPPKA− lines show a decreased rate of ventricular pressure dissipation in DOB− conditions when compared to the WT strain. Equally importantly, the slope of the curve remains constant even at low levels of ventricular pressure. In all other strains, the slope increases as pressure decreases, suggesting a key role of TnI in the systolic–diastolic transition. Why a similar trend is not present in the DBLPKA− strain is not immediately evident, but these results suggest that MyBP-C and TnI function are interrelated in diastolic relaxation. A supporting role of TnI is evident in the phase duration data, as the ablation of TnI phosphorylation tends to extend ejection (systole) and shorten filling (diastole). As

Nanoparticulate-specific effects of silver on teleost cardiac contractility

Silver nanoparticles (nAg), due to their biocidal properties, are common in medical applications and are used in more consumer products than any other engineered nanomaterial. This growing abundance, combined with their ability to translocate across the epithelium and bioaccumulate, suggests that internalized nAg may present a risk of toxicity to many organisms in the future. However, little experimentation has been devoted to cardiac responses to acute nAg exposure, even though nAg is known to disrupt ion channels even when ionic Ag+ does not. In this study, we examined the cardiac response to nAg exposure relative to a sham and an ionic AgNO3 control across cardiomyocyte survival and homeostasis, ventricular contractility, and intrinsic pacing rates of whole hearts. Our results suggest that nAg, but not Ag+ alone, inhibits force production by the myocardium, that Ag in any form disrupts normal pacing of cardiac contractions, and that these responses are likely not due to cytotoxicity. This evidence of nanoparticle-specific effects on physiology should encourage further research into nAg cardiotoxicity and other potential sublethal effects.