Chris N. Glover

Nutritive metal uptake in teleost fish

SUMMARY Transition metals are essential for health, forming integral components of proteins involved in all aspects of biological function. However, in excess these metals are potentially toxic, and to maintain metal homeostasis organisms must tightly coordinate metal acquisition and excretion. The diet is the main source for essential metals, but in aquatic organisms an alternative uptake route is available from the water. This review will assess physiological, pharmacological and recent molecular evidence to outline possible uptake pathways in the gills and intestine of teleost fish involved in the acquisition of three of the most abundant transition metals necessary for life; iron, copper, and zinc.

Application of genomics and proteomics for study of the integrated response to zinc exposure in a non-model fish species, the rainbow trout.

The advent of DNA array technology and proteomics has revolutionised biology by allowing global analysis of cellular events. So far, the benefits from these new techniques have primarily been realised for well-characterised species. These organisms are rarely the most relevant for environmental biology and ecotoxicology. Thus, there is a need to explore new ways to exploit transcriptomics and proteomics for non-model species. In the present study, rainbow trout (Oncorhynchus mykiss) were exposed to a sublethal concentration of waterborne zinc for up to 6 days. The response in gill tissue was investigated by differential screening of a heterologous cDNA array and by protein profiling using Surface Enhanced Laser Desorption/Ionisation (SELDI). The cDNA array, which was a high-density spotted library of cDNA from Fugu rubripes gill, revealed differentially expressed genes related to energy production, protein synthesis, paracellular integrity, and inflammatory response. SELDI analysis yielded seven proteins that were consistently present only in zinc-exposed gills, and four proteins unique to gills from control fish. A further 11 proteins were differentially regulated. Identification of these proteins by bioinformatics proved difficult in spite of detailed information on molecular mass, charge and zinc-binding affinity. It is concluded that these approaches are viable to non-model species although both have clear limitations.

Calcium/cadmium interactions at uptake surfaces in rainbow trout: Waterborne versus dietary routes of exposure

Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to control, 3 microg/L waterborne Cd, or 500 mg/kg dietary Cd in combination with either a control (20 mg/g Ca2+ as CaCO3) or elevated (60 mg/g Ca2+) Ca2+ diet for 28 d. No mortality or growth effects were observed in response to either route of Cd exposure, although fish fed Ca2+-supplemented diets exhibited minor reductions in growth within the first few days of feeding. Waterborne and dietary Cd resulted in significant Cd accumulation in most tissues, with dietary uptake being far in excess of waterborne under the exposure conditions used. The order of Cd accumulation strongly reflected the exposure pathway, being gill and kidney > liver > gut > carcass (waterborne Cd); gut > kidney > liver > gill > carcass > bone (dietary Cd). On a whole-body basis, the net retention of Cd from the diet was < 1%, indicating that the gut wall forms an important protective barrier reducing Cd accumulation into internal tissues. Dietary Ca2+ supplementation reduced short-term whole-body uptake rates of waterborne Ca2+ and Cd by >50% and resulted in much lower chronic accumulation of Cd (via the water and diet) in target tissues. Results suggest that Ca2+ and Cd share common pathway(s)/transport mechanism(s) in the gill and gut and that increased gastrointestinal Ca2+ uptake likely caused downregulation of branchial and gastrointestinal Ca2+ and therefore Cd uptake pathways. Because nutrient metals other than Ca2+ may also influence Cd (and other metal) uptake, new regulatory approaches to metal toxicity (e.g., biotic ligand model) require understanding of the influence of dietary status on metal accumulation.

Assessing the sensitivity of Atlantic salmon (Salmo salar) to dietary endosulfan exposure using tissue biochemistry and histology

The incorporation of plant-based ingredients, and the possible carry-over of pesticides such as endosulfan, in fish feeds may present new toxicological challenges to aquacultural species. Biological responses of Atlantic salmon (Salmo salar) to a 35-day dietary endosulfan exposure at levels ranging from 4 to 710 microgkg(-1) were assessed using tissue histology and biochemistry. Liver 7-ethoxyresorufin-O-deacetylase (EROD) activity was significantly elevated in the highest exposure group (710 microgkg(-1)) by day 35. Other hepatic indicators of stress impacts and responses (glutathione-S-transferase and glutathione peroxidase activities and hepatic alpha-tocopherol content) remained unchanged. Branchial Na(+), K(+)-ATPase activity was significantly reduced at day 14 in the highest exposure group, but returned to control levels by day 35. Conversely, intestinal Na(+), K(+)-ATPase activity was significantly inhibited at day 35, but again only at the highest exposure level. In contrast to the biochemical results, hepatic and intestinal histology revealed effects of exposure even at the lowest dose tested (4 microgkg(-1)). In the posterior intestine, pathology was characterised by vacuolation and fusion of villi, and in the most severe cases, loss of epithelial integrity in villi tips. In the liver the primary effects were glycogen depletion and lipidosis. These changes were typical of a generalised stress response. While histology endpoints may prove to be the most sensitive indicators of dietary endosulfan exposure, the organismal relevance of these structural changes must be considered in the absence of effects in other biomarkers at dietary levels less than 710 microgkg(-1).

Zinc uptake across the apical membrane of freshwater rainbow trout intestine is mediated by high affinity, low affinity, and histidine-facilitated pathways.

Zinc is both a vital nutrient and an important toxicant to aquatic biota. In order to understand the interplay between nutrition and toxicity, it will be important to determine the mechanisms and the factors that regulate zinc uptake. The mechanism of apical intestinal Zn(II) uptake in freshwater rainbow trout and its potential modification by the complexing amino acid histidine was investigated using brush-border membrane vesicles (BBMVs). Following characterisation of the BBMV preparation, zinc uptake in the absence of histidine was both time- and concentration-dependent and consisted of two components. A saturable phase of uptake was described by an affinity constant of 57+/-17 microM and a transport capacity of 1867+/-296 nmol mg membrane protein(-1) min(-1). At higher zinc levels (>500 microM) a linear, diffusive component of uptake was evident. Zinc transport was also temperature-dependent, with Q10 values suggesting zinc uptake was a carrier-mediated process. Zinc uptake by vesicles in the presence of histidine was correlated to a mono-histidine species (Zn(His)+) at all Zn(II) concentrations examined.

Humic Substances Influence Sodium Metabolism in the Freshwater Crustacean Daphnia magna

Humic substances are ubiquitous components of natural waters with important roles in alleviating metal toxicity to aquatic organisms. Recent literature reports suggest that humic substances may also exert direct influences on biota. This study investigated the influence of two commercially available humic substances on sodium metabolism in Daphnia magna, a hyperregulating freshwater crustacean. Environmentally realistic levels of Suwannee River natural organic matter (SRN) and Aldrich humic acid (AHA) significantly enhanced sodium transport. This effect was described as an uncompetitive stimulation of sodium influx, as characterised by an increased maximal sodium transport rate (Jmax), accompanied by a decreased uptake affinity (increased Km). SRN exposure also significantly promoted the unidirectional loss of sodium from the daphnids to the water, an effect not observed in the presence of AHA. A 24‐h preexposure to AHA before influx measurement had no effect on AHA‐induced stimulation of sodium influx. Conversely, 24‐h preexposure to SRN resulted in influx values that returned to control (humic‐free) levels. Whole‐body sodium levels reduced by SRN exposure were also restored to control levels following 24‐h SRN preexposure. The significance and potential mechanisms of these actions are discussed, and the toxicological implications of these findings are assessed.

Derivation of a toxicity-based model to predict how water chemistry influences silver toxicity to invertebrates ☆

The effect of altering water chemistry on acute silver toxicity to three invertebrate species, two Daphnids, Daphnia magna and Daphnia pulex, as well as an amphipod Gammarus pulex was assessed. In addition, the physiological basis of Ag(I) toxicity to G. pulex was examined. Daphnia magna and D. pulex were more sensitive than G. pulex and 48 h LC(50) values in synthetic ion poor water were 0.47, 0.65 and 2.1 microg Ag(I) l(-1), respectively. Increasing water [Cl(-)] reduced Ag(I) toxicity in all species, and increasing water [Ca(2+)] from 50 to 1,500 microM reduced Ag(I) toxicity in G. pulex. Whole body Na(+) content, but not K(+) or Ca(2+) was significantly reduced in G. pulex exposed to 6 microg Ag(I) l(-1) for 24 h, but there was no inhibition of whole body Na(+)/K(+)-ATPase activity. Both increasing water [Cl(-)] and [Ca(2+)] reduced this Ag(I)-induced Na(+) loss. For D. magna, the presence of 10 mg l(-1) humic acid or 0.5 microM 3-mercaptoproprionic acid (3-MPA) increased the 48 h LC(50) values by 5.9 and 58.5-fold, respectively, and for D. pulex the presence of 1 microM thiosulfate increased the 48 h LC(50) value by four-fold. The D. magna toxicity data generated from this study were used to derive a Daphnia biotic ligand model (BLM). Analysis of the measured LC(50) values vs. the predicted LC(50) values for toxicity data from the present and published results where water Cl(-), Ca(2+), Na(+) or humic acid were varied showed that 91% of the measured toxicity data fell within a factor of two of the predicted LC(50) values. However, the daphnid BLM could not accurately predict G. pulex toxicity. Additionally, the Daphnia BLM was under-protective in the presence of the organic thiols 3-MPA or thiosulphate and predicted an increase in the LC(50) value of 114- and 74-fold, respectively. The Daphnia toxicity based BLM derived from the present data set is successful in predicting Daphnia toxicity in laboratory data sets in the absence of sulfur containing compounds, but shows its limitations when applied to waters containing organic thiols or thiosulphate.

The skin of fish as a transport epithelium: a review.

The primary function of fish skin is to act as a barrier. It provides protection against physical damage and assists with the maintenance of homoeostasis by minimising exchange between the animal and the environment. However in some fish, the skin may play a more active physiological role. This is particularly true in species that inhabit specialised environmental niches (e.g. amphibious and air-breathing fish such as the lungfish), those with physiological characteristics that may subvert the need for the integument as a barrier (e.g. the osmoconforming hagfish), and/or fish with anatomical modifications of the epidermis (e.g. reduced epithelial thickness). Using examples from different fish groups (e.g. hagfishes, elasmobranchs and teleosts), the importance of fish skin as a transport epithelium for gases, ions, nitrogenous waste products, and nutrients was reviewed. The role of the skin in larval fish was also examined, with early life stages often utilising the skin as a surrogate gill, prior to the development of a functional branchial epithelium.

The Disruption of Daphnia magna Sodium Metabolism by Humic Substances: Mechanism of Action and Effect of Humic Substance Source

Humic substances have important functions in aquatic systems. While these roles are primarily indirect, influencing the physicochemical environment, recent evidence suggests these materials may also have direct biological actions. This study investigated the mechanism by which humic substances perturb sodium metabolism in a freshwater invertebrate, the water flea Daphnia magna. Aldrich humic acid (AHA) stimulated the maximal rate of whole‐body sodium influx (Jmax) when experimental pH was 6 and water calcium content was 0.5 mM. This effect persisted at pH 8 and 1 mM calcium but not at pH 8 in the absence of calcium. An indirect action of AHA on apical transporter activity was proposed to explain this effect. At pH 4 AHA promoted a linear sodium uptake kinetic relationship, attributed to altered membrane permeability due to enhanced membrane binding of humic substances at low pH. In contrast, a real‐world natural organic matter sample had no consistent action on sodium influx, suggesting that impacts on sodium metabolism may be limited to commercially available humic materials. These findings question the applicability of commercially available humic substances for laboratory investigations and have significant implications for the study of environmental metal toxicity.

Adaptations to in situ feeding: novel nutrient acquisition pathways in an ancient vertebrate

During feeding, hagfish may immerse themselves in the body cavities of decaying carcasses, encountering high levels of dissolved organic nutrients. We hypothesized that this feeding environment might promote nutrient acquisition by the branchial and epidermal epithelia. The potential for Pacific hagfish, Eptatretus stoutii, to absorb amino acids from the environment across the skin and gill was thus investigated. l-alanine and glycine were absorbed via specific transport pathways across both gill and skin surfaces, the first such documentation of direct organic nutrient acquisition in a vertebrate animal. Uptake occurred via distinct mechanisms with respect to concentration dependence, sodium dependence and effects of putative transport inhibitors across each epithelium. Significant differences in the absorbed amino acid distribution between the skin of juveniles and adults were noted. The ability to absorb dissolved organic matter across the skin and gill may be an adaptation to a scavenging lifestyle, allowing hagfish to maximize sporadic opportunities for organic nutrient acquisition. From an evolutionary perspective, hagfish represent a transitory state between the generalized nutrient absorption pathways of aquatic invertebrates and the more specialized digestive systems of aquatic vertebrates.