Tyson J. MacCormack

Inhibition of enzyme activity by nanomaterials: Potential mechanisms and implications for nanotoxicity testing

Abstract The objective of this study was to investigate whether nanoparticle-exposure affects enzyme function and to determine the mechanisms responsible. Silicon, Au, and CdSe nanoparticles were synthesized in house and their physicochemical properties were characterized. The activity of purified lactate dehydrogenase (LDH) was inhibited or abolished by all nanoparticles tested. Inhibition was dependent upon particle core and surface-functional group composition. Inhibition of LDH was absent in crude tissue homogenates, in the presence of albumin, and at the isoelectric point of the protein, indicating that nanoparticles bind non-specifically to abundant proteins via a charge interaction. Circular dichroism spectroscopy suggests that the structure of LDH may be altered by nanoparticles in a manner different from that of bulk controls. We present new data on the specific physicochemical properties of nanoparticles that may lead to bioactivity and highlight a number of potentially serious problems with common nanotoxicity testing methods.

Silver nanoparticles inhibit sodium uptake in juvenile rainbow trout (Oncorhynchus mykiss).

The silver ion (Ag(+)) is well documented to be a potent inhibitor of sodium (Na(+)) transport in fish. However, it has not been determined whether silver nanoparticles (Ag NPs) elicit this same effect and, if so, if the NP itself and/or the dissociation of ionic Ag(+) causes this effect. Citrate-capped Ag NPs were dialyzed in water to determine the dissolution rate of ionic Ag(+) from the NPs and the maximum concentration of free Ag(+) released from the NPs was used as a paired Ag(+) control to distinguish NP effects from ionic metal effects. The maximum concentration of ionic Ag(+) released from these NPs over 48 h was 0.02 μg l(-1). Juvenile rainbow trout were exposed to 1.0 mg l(-1) citrate-capped Ag NPs and dialyzed citrate-capped Ag NPs or 10 μg l(-1) and 0.02 μg l(-1) ionic Ag(+) (as AgNO(3)) as controls. Both nondialyzed and dialyzed Ag NPs and 10 μg l(-1) ionic Ag(+) significantly inhibited unidirectional Na(+) influx by over 50% but had no effect on unidirectional Na(+) efflux. Na(+),K(+)-ATPase was significantly inhibited by the Ag NPs with no discernible effect on carbonic anhydrase activity. This study is the first to show that sodium regulation is disrupted by the presence of citrate-capped Ag NPs, and the results suggest that there are nanospecific effects.

Mechanistic insights into the effect of nanoparticles on zebrafish hatch.

Abstract Aquatic organisms are susceptible to waterborne nanoparticles (NP) and there is only limited understanding of the mechanisms by which these emerging contaminants may affect biological processes. This study used silicon (nSi), cadmium selenide (nCdSe), silver (nAg) and zinc NPs (nZnO) as well as single-walled carbon nanotubes (SWCNT) to assess NP effects on zebrafish (Danio rerio) hatch. Exposure of 10 mg/L nAg and nCdSe delayed zebrafish hatch and 100 mg/L of nCdSe as well as 10 and 100 mg/L of uncoated nZnO completely inhibited hatch and the embryos died within the chorion. Both the morphology and the movement of the embryos were not affected, and it was determined that the main mechanism of hatch inhibition by NPs is likely through the interaction of NPs with the zebrafish hatching enzyme. Furthermore, it was concluded that the observed effects arose from the NPs themselves and not their dissolved metal components.

Large-Scale Proteome Profile of the Zebrafish (Danio rerio) Gill for Physiological and Biomarker Discovery Studies

Zebrafish are an important model in vertebrate genetics, developmental biology, physiology, and toxicology. In this study, we established the first large-scale proteome profile of a teleost fish tissue using a shotgun method based on two-dimensional liquid chromatography-electrospray ionization tandem mass spectrometry. Proteome coverage was significantly improved with the application of a sequential protein solubilization method for protein fractionation and a precursor ion exclusion method for improving peptide and protein identification efficiency. Five thousand seven hundred sixteen proteins were identified with an estimated false-positive matching rate of 1.34%, and the proteome exhibited excellent coverage of important biochemical pathways relevant to the function of the gill in respiration, ion and acid-base homeostasis, and energy metabolism. Numerous established and potential biomarkers of stress, disease, and environmental contamination were also expressed in the gill. Annotation information was completely lacking for >30% of the detected proteins, highlighting the need for advancements in bioinformatics analysis techniques to complement this research. Nevertheless, the results provide important insights into the physiological function of the gill as well as its role as an environmental interface. We discuss the significance of these findings in the context of exploratory physiological and toxicological studies.

The regulation and importance of glucose uptake in the isolated Atlantic cod heart: rate-limiting steps and effects of hypoxia

SUMMARY This study investigated the regulation of glucose uptake in Atlantic cod (Gadus morhua) hearts. Isolated hearts were perfused with or without glucose in the medium, under either normoxic or severely hypoxic conditions. Working at basal levels, hearts did not require extracellular glucose to maintain power under aerobic conditions. However, cardiac performance was significantly reduced without exogenous glucose under oxygen-limiting conditions. The addition of the glucose transporter inhibitor cytochalasin B caused hypoxic hearts to fail early, and hearts perfused with a glucose analogue, 2-deoxyglucose (2-DG), increased glucose uptake 3-fold under hypoxia. The uptake of 2-DG was only partially inhibited when cytochalasin B was added to the medium. Isolated ventricle strips were also incubated in the presence of 2-DG and the extracellular marker mannitol. Glucose uptake (glucose transport plus intracellular phosphorylation) was assessed by measuring the initial rate of 2-deoxyglucose-6-phosphate (2-DG-6-P) accumulation. At 1 mmol l-1 2-DG, the rate of 2-DG uptake remained linear for 60 min, and 2-DG-6-P, but not free 2-DG, accumulation was increased. The fact that intracellular 2-DG did not increase indicates that glucose transport is the rate-limiting step for glucose utilization in non-stimulated cardiac tissue. Replacement of Na+ by choline in the incubation medium did not affect 2-DG uptake, providing evidence that Na+-coupled glucose transport is absent in cod cardiac tissue. Similar to cytochalasin B, glucose uptake was also inhibited by phloridzin, suggesting that facilitated, carrier-mediated glucose transport occurs in cod hearts. Under the conditions employed in these experiments, it is clear that (1) activation of glucose transport is required to support hypoxic performance, (2) the rate-limiting step for glucose utilization is glucose transport rather than glucose phosphorylation, (3) 2-DG uptake accurately reflects glucose transport activity and (4) glucose uptake in cod hearts does not involve an Na+-dependent mechanism.

Sequence and expression of a constitutive, facilitated glucose transporter (GLUT1) in Atlantic cod Gadus morhua.

SUMMARY A putative glucose transporter, GLUT1, is reported for Atlantic cod Gadus morhua. A combination of RT-PCR, RLM-RACE and genome walking were used to articulate a 4560 bp cDNA (GenBank accession number AY526497). It contains a 149 bp 5′ UTR, a 1470 bp open reading frame and a 2941 bp 3′ UTR. At the nucleotide level, the cod GLUT1 ORF shares 78.2% sequence identity to human GLUT1 and the deduced amino acid sequence clusters with GLUT1s from rainbow trout and carp. GLUT1 transcript is highly expressed in brain, gill, heart and kidney and expressed to a lower level in at least six other tissues. Expression is evident immediately upon fertilization of eggs. Six hours of hypoxia at 40% DO2 did not alter expression levels in brain, gill, heart or kidney. The level of expression is not substantially altered in heart during low temperature challenge, although there is a suggestion that colder temperature could lead to lower levels of expression, consistent with the concept that the cold-acclimated heart has a reduced dependence upon glucose as a metabolic fuel. Two months of starvation did not significantly alter the level of expression of GLUT1 in heart. This is in marked contrast to the rat heart where fasting leads to a substantial decrease in GLUT1 levels. Overall, there is a ubiquitous tissue distribution of GLUT1, consistent with other species, and the level of gene expression, especially in heart, is relatively constant over a range of physiological conditions.

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

SUMMARY 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°C. Cardiorespiratory parameters were recorded along with plasma lactate, K+, Ca2+ 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.

Intracellular Glucose and Binding of Hexokinase and Phosphofructokinase to Particulate Fractions Increase under Hypoxia in Heart of the Amazonian Armored Catfish (Liposarcus pardalis)

Armored catfish (Liposarcus pardalis), indigenous to the Amazon basin, have hearts that are extremely tolerant of oxygen limitation. Here we test the hypothesis that resistance to hypoxia is associated with increases in binding of selected glycolytic enzymes to subcellular fractions. Preparations of isolated ventricular sheets were subjected to 2 h of either oxygenated or hypoxic (via nitrogen gassing) treatment during which time the muscle was stimulated to contract. The bathing medium contained 5 mM glucose and was maintained at 25°C. Initial experiments revealed increases in anaerobic metabolism. There was no measurable decrease in glycogen level; however, hypoxic treatment led to a twofold increase in heart glucose and a 10‐fold increase in lactate content. It is suggested that the increase in heart glucose content is a result of an enhanced rate of facilitated glucose transport that exceeds the rate of phosphorylation of glucose. Further experiments assessed activities of metabolic enzymes in crude homogenates and subsequently tracked the degree of enzyme binding associated with subcellular fractions. Total maximal activities of glycolytic enzymes (hexokinase [HK], phosphofructokinase [PFK], aldolase, pyruvate kinase, lactate dehydrogenase), and a mitochondrial marker, citrate synthase, were not altered with the hypoxic treatment. A substantial portion (≥50%) of HK is permanently bound to mitochondria, and this level increases under hypoxia. The amount of HK that is bound to the mitochondrial fraction is at least fourfold higher in hearts of L. pardalis than in rat hearts. Hypoxia also resulted in increased binding of PFK to a particulate fraction, and the degree of binding is higher in hypoxia‐tolerant fish than in hypoxia‐sensitive mammalian hearts. Such binding may be associated with increased glycolytic flux rates through modulation of enzyme‐specific kinetics. The binding of HK and PFK occurs before any significant decrease in glycogen level.

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.

Mitochondrial KATP channels and sarcoplasmic reticulum influence cardiac force development under anoxia in the Amazonian armored catfish Liposarcus pardalis

The contribution of alterations in mitochondrial K(ATP) channel activity and the sarcoplasmic reticulum (SR) to anaerobic cardiac function in the anoxia tolerant armored catfish Liposarcus pardalis were assessed. K(ATP) channels contribute to hypoxic cardioprotection in mammals, but little is known of their action in more hypoxia tolerant animals. Anoxia resulted in a decrease in force in isometrically contracting ventricle strips to approximately 40% of the pre-anoxic level. This was maintained for at least 2 h. Upon reoxygenation, hearts recovered to the same level as control preparations. Treatment with 5-hydroxydecanoic acid (5HD), a specific mitochondrial K(ATP) blocker significantly increased force in preparations during anoxia and caused hypercontracture at reoxygenation. Ryanodine, a specific inhibitor of SR function, significantly increased force loss in ventricle preparations under anoxia. Results show that mitochondrial K(ATP) channel activity and SR function are important in anaerobic and post-anaerobic contractility in armored catfish heart.