Nicolas R. Bury

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.

Corticosteroid receptors involved in stress regulation in common carp, Cyprinus carpio

In higher vertebrates, mineralo- (aldosterone) and glucocorticoids (cortisol/corticosterone) exert their multiple actions via specific transcription factors, glucocorticoid (GR) and mineralocorticoid (MR) receptors. Teleostean fishes lack aldosterone and mineral regulatory processes seem under dominant control by cortisol. Despite the absence of the classical mineralocorticoid aldosterone, teleostean fishes do have an MR with cortisol and possibly 11-deoxycorticosterone (DOC) (as alternative for aldosterone) as predominant ligands. We studied corticoid receptors in common carp (Cyprinus carpio L). Through homology cloning and bioinformatic analysis, we found duplicated GR genes and a single MR gene. The GR genes likely result from a major genomic duplication event in the teleostean lineage; we propose that the gene for a second MR was lost. Transactivation studies show that the carp GRs and MR have comparable affinity for cortisol; the MR has significantly higher sensitivity to DOC, and this favours a role for DOC as MR ligand in fish physiology. mRNA of the GRs and the MR is expressed in forebrain (in pallial areas homologous to mammalian hippocampus), corticotrophin-releasing hormone (CRH) cells in the pre-optic nucleus (NPO) and pituitary pars distalis ACTH cells, three key neural/endocrine components of the stress axis. After exposure to prolonged and strong (not to mild acute) stressors, mRNA levels of both GRs and MR become down-regulated in the brain, but not in the NPO CRH cells or pituitary ACTH cells. Our data predicts a function in stress physiology for all CRs and suggest telencephalon as a first line cortisol target in stress.

Cortisol protects against copper induced necrosis and promotes apoptosis in fish gill chloride cells in vitro

Abstract In order to distinguish between toxic actions of copper (Cu) and the indirect actions of the metal mediated via the stress hormone cortisol, a 24 h in vitro gill filament culture was used to investigate the effects of this heavy metal and hormone, singly and in combination, on apoptosis and necrosis of chloride cells in the cichlid fish, tilapia (Oreochromis mossambicus). Cell death was identified after fluorescent double-labelling using a confocal laser scanning microscope. Incubation of filaments with 50 μM and 100 μM CuSO1 caused an approximate 5- and 16-fold increase, respectively, in chloride cell necrosis when compared to control, but had no significant effect on apoptosis. A 12 h incubation with 0.28 μM cortisol prior to exposure to 100 μM CuSO1 reduced necrosis by about 75%. The apparent protection provided by cortisol against copper toxicity could be blocked by the glucocorticoid receptor blocker RU 486. Incubation with 0.83 μM cortisol induced apoptosis to the same extent as that of camptothecin, a topoisomerase I inhibitor. We conclude that Cu directly causes necrosis of chloride cells, whilst cortisol protects against copper toxicity at lower concentrations, and induces apoptosis at higher concentrations, typical for severely stressed fish.

Stress and innate immunity in carp: corticosteroid receptors and pro-inflammatory cytokines.

The stress hormone cortisol is deeply involved in immune regulation in all vertebrates. Common carp (Cyprinus carpio L.) express four corticoid receptors that may modulate immune responses: three glucocorticoid receptors (GR); GR1, with two splice variants (GR1a and GR1b), GR2 and a single mineralocorticoid receptor (MR). All receptors are expressed as of 4 days post-fertilization and may thus play a critical role in development and functioning of the adult immune system. Immune tissues and cells predominantly express mRNA for GRs compared to mRNA for the MR. Three-dimensional protein structure modeling predicts, and transfection assays confirm that alternative splicing of GR1 does not influence the capacity to induce transcription of effector genes. When tested for cortisol activation, GR2 is the most sensitive corticoid receptor in carp, followed by the MR and GR1a and GR1b. Lipopolysacharide (LPS) treatment of head kidney phagocytes quickly induces GR1 expression and inhibits GR2 expression. Cortisol treatment in vivo enhances GR1a and MR mRNA expression, but only mildly, and cortisol treatment in vitro does not affect receptor expression of phagocytes. Cortisol has no direct effect on the LPS-induced receptor profile. Therefore, an immune rather than a stress stimulus regulates GR expression. Cortisol administered at stress levels to phagocytes in vitro significantly inhibits LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-12 (IL-12) (subunit p35) and of inducible nitric oxide synthase (iNOS) expression. A physiologically differential function for GR1 and GR2 in the immune response of fish to infection is indicated.

Iron acquisition by teleost fish

Iron is a vital micronutrient for teleost fish, being an integral component of proteins involved in cellular respiration and oxygen transfer. However, in excess iron is toxic, and fish need to balance uptake to prevent deficiency vs. potential toxicity. This review assesses the current physiological and molecular knowledge of the mechanisms of iron acquisition in the teleost fish. It focuses on freshwater teleost fish when assessing the gill as a possible site for iron acquisition, and includes a summary of geochemical processes that govern aquatic iron bioavailability. It focuses on marine teleost fish for assessing the mechanism of intestinal iron uptake. Physiological evidence indicates that iron preferentially crosses the apical membrane of both the gills and intestine in the ferrous (Fe2+) state. Molecular evidence supports this, demonstrating the presence of homologues in fish to the large Slc 11a family of evolutionary conserved proteins linked to Fe2+ transport. This symporter is probably linked to a reductase, which reduces either ferric (Fe3+) or organic complexed iron to Fe2+ prior to uptake.

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.

Global Transcriptome Profiling Reveals Molecular Mechanisms of Metal Tolerance in a Chronically Exposed Wild Population of Brown Trout

Worldwide, a number of viable populations of fish are found in environments heavily contaminated with metals, including brown trout (Salmo trutta) inhabiting the River Hayle in South-West of England. This population is chronically exposed to a water-borne mixture of metals, including copper and zinc, at concentrations lethal to naïve fish. We aimed to investigate the molecular mechanisms employed by the River Hayle brown trout to tolerate high metal concentrations. To achieve this, we combined tissue metal analysis with whole-transcriptome profiling using RNA-seq on an Illumina platform. Metal concentrations in the Hayle trout, compared to fish from a relatively unimpacted river, were significantly increased in the gills, liver and kidney (63-, 34- and 19-fold respectively), but not the gut. This confirms that these fish can tolerate considerable metal accumulation, highlighting the importance of these tissues in metal uptake (gill), storage and detoxification (liver, kidney). We sequenced, assembled and annotated the brown trout transcriptome using a de novo approach. Subsequent gene expression analysis identified 998 differentially expressed transcripts and functional analysis revealed that metal- and ion-homeostasis pathways are likely to be the most important mechanisms contributing to the metal tolerance exhibited by this population.

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.

Waterborne iron acquisition by a freshwater teleost fish, zebrafish Danio rerio

SUMMARY Waterborne iron accumulation by the gills of the zebrafish Danio rerio was assessed in ion-poor water. Branchial iron uptake, which comprises both the iron that has entered the gill cells and iron that is strongly bound to the epithelia, has high- and low-affinity components. At low nominal [Fe] (<40 nmol l-1) the high-affinity component demonstrated saturation kinetics, with an apparent Km of 5.9 nmol l-1 Fe and Vmax of 2.1 pmol g-1 h-1. Over a range of higher nominal [Fe] (40-200 nmol l-1), branchial uptake was linear. In the presence of 2μ mol l-1 of the reducing agent dithiothreitol (DTT), branchial iron accumulation was significantly enhanced at [Fe]>15 nmol l-1. The proton pump inhibitor bafilomycin A significantly reduced iron uptake in the presence of DTT. On the basis of these observations we conclude that branchial iron uptake at low [Fe] shows characteristics similar to those of other iron-transporting epithelia, coupling an apical membrane ferric reductase to a Fe2+/H+ symporter. Zebrafish branchial iron transport at 18.6 nmol l-1 was inhibited by 200 nmol l-1 Cd2+. But, unlike other Fe2+/H+ symporters, iron uptake was not affected by other divalent metals (Co2+, Ni2+, Pb2+, Cu2+, Zn2+ and Mn2+). Zebrafish loaded with 59Fe from the water showed a loss of 7.9 pmol Fe g-1 body mass over the first day and a further loss of 5.7 pmol Fe g-1 body mass over the following 28 days. The depuration kinetics followed a two-component exponential model; for the short-lived component, t1/2=0.31 days, and for the long-lived component, t1/2=13.2 days. The daily iron loss by zebrafish can be compensated by iron uptake at exceedingly low water iron concentrations (uptake rate at 1.625 nmol l-1 Fe=0.425 pmol g-1 h-1), demonstrating that uptake of iron from the water is potentially an important source of this nutritive metal in freshwater teleost fish.

Differential tolerance of two Gammarus pulex populations transplanted from different metallogenic regions to a polymetal gradient

The River Hayle, Cornwall, UK exhibits pronounced Cu and Zn concentration gradients which were used to compare the metal handling abilities of two populations of Gammarus pulex (Crustacea: Amphipoda). One population was native to the Hayle region (Drym) and presumably has been historically impacted by elevated Cu and Zn levels, whilst naïve gammarids were collected from the River Cray, Kent, UK. Both populations were subject to a 32 day in situ exposure at four R. Hayle sites (Drym, Godolphin, Relubbus and St. Erth). Mortality (LT50), Cu and Zn accumulation and sub-cellular distribution, and oxidative stress (malondialdehyde production) increased with the expected Cu and Zn bioavailabilities at the four sites (i.e. Godolphin>Relubbus>St. Erth>Drym). The naïve population experienced greater metal induced effects in terms of Cu and Zn accumulation, oxidative stress responses and lower LT50s. Analysis of Cu and Zn sub-cellular distribution, however, revealed no significant differences in metal handling. In both populations each metal was localised predominantly to the sub-cellular fraction containing metal bound to metallothionein-like proteins (MTLP) or that holding both metal-rich granules (MRG) and exoskeleton, MTLP and MRG binding being indicative of metal detoxification. However, a greater capacity for detoxified metal storage is not a mechanism implicated in the perceived tolerance of the historically impacted gammarids. Instead our results suggest that the historically impacted population was adapted for lower uptake of Cu and Zn leading to lower bioaccumulation, stress response and ultimately mortality. These results demonstrate not only the usefulness of the in situ methodology, but also that differences in population exposure history can cause significant differences in metal responses during exposure at higher concentrations.