A comparison of cadmium-induced metallothionein gene expression and Me2+ distribution in the tissues of cadmiumsensitive (rainbow trout; Salmo gairdneri) and tolerant (stone loach; Noemacheilus barbatulus) species of freshwater fish

1. The accumulation of cadmium in the liver, kidney and gills of rainbow trout and stone loach was measured during exposure of the fish to the metal at 3 smg/l in their aquarium water. The pattern of accumulation of the toxic metal in the individual organs was different between the two species.2. The tissue concentrations of metallothionein-specific mRNA and metallothionein protein were also determined in these organs from the same fish. In rainbow trout, the induction of metallothionein gene expression resulted in a gradual increase in metallothionein concentration in gill over the course of the experiment whereas increases in metallothionein in the liver and kidney were detected only at the later time points of analysis (beyond 19 weeks). By contrast, in the same tissues from stone loach, relatively minor changes were quantified in specific mRNA and metallothionein concentrations.3. Throughout the experimental period, tissue concentrations of zinc and copper were determined in the liver, kidney and gills of the rainbow trout and stone loach. Subtle decreases were observed in the zinc concentration of gills in rainbow trout and substantial increases were observed in the hepatic copper concentrations in both species at the later time points of analysis.4. The ability of cadmium to induce metallothionein gene expression and its subsequent ability to compete for the sequestration sites on the newly-synthesized protein is discussed with regard to the relative levels of cadmium, zinc and copper in the organs studied and differing regimes of cadmium administration.     

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss)

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.     

Distribution of calcitonin gene-related peptide and calcitonin-like immunoreactivity in trout

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii. The highest concentrations of the peptide were found in gill (1.68 +/- 0.09 ng/mg protein) and in intestine (1.06 +/- 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 +/- 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species.     

Induction of metallothionein gene expression by cadmium and the retention of the toxic metal in the tissues of rainbow trout (Salmo gairdneri)

1. When rainbow trout were exposed to cadmium by intraperitoneal injection, there was a rapid (within 3hr) and significant (approx. 63%) loss of the metal from the whole bodies of the fish.2. Of the metal retained in the bodies of the fish (approx. 37% of the injected dose), more than 98% was accounted for collectively among the liver, kidney and gills.3. Subsequent maintenance of the rainbow trout in fresh water for up to 98 days post-metal administration, indicated that there was no further loss of the cadmium accumulated in the organs studied and that the distribution of the metal among the liver, kidney and gills remained unchanged over that period.4. During this 98-day period of maintenance of the fish, tissue concentrations of metallothionein-specific mRNA and metallothionein protein were quantified using riboprobe and ELISA systems respectively. Metallothionein-specific mRNA concentrations increased rapidly (within 24 hr) before falling back to levels similar to, or slightly greater than, those found in control animals. The concentration of metallothionein protein also increased significantly (within 3 days) then remained elevated thereafter.5. Throughout the experimental period, the concentrations of zinc and copper were also monitored in the liver, kidney and gills of the rainbow trout. The concentrations of each ion differed between each of the organs but did not change during the experiment.6. The induction of metallothionein gene expression by cadmium in the liver, kidney and gill of rainbow trout and the subsequent sequestration of the toxic metal is discussed with regard to the relative levels of these other essential metal ions.     

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.     

The response of alkaline phosphatase to osmoregulatory changes in the trout,Salmo gairdneri

Summary The relationship between alkaline phosphatase and environmental salinity was examined in the rainbow trout and the migratory rainbow (steelhead),Salmo gairdneri. The enzyme activity in tissues involved in osmoregulation was strongly correlated with the adaptation salinity and thus to the degree of salt and fluid transport in those tissues. After transfer from freshwater to seawater, the specific activity of the enzyme increased over 260% in the intestine, decreased by 50% in kidney, and was unchanged in the liver, an organ not directly involved in osmoregulation. The sea-run steelhead trout response was similar to the nonmigratory rainbow; although, the pre-migratory transformation (smoltification) had no effect on enzyme activity. Amino acid inhibitors of alkaline phosphatase significantly reduced fluid absorption in the isolated intestine of rainbow trout, reaffirming the relationship between the enzyme and fluid movement. Electrophoretic identification of trout alkaline phosphatase isozymes, clearly distinguishes the enzyme from different tissue origins. However, from the analysis of intestinal electrophoretic patterns, osmoregulatory adjustments are not associated with the induction of new alkaline phosphatase isozymes, or in the large scale preferential stimulation of one of the two existing intestinal isozymes over the other.     

Distribution of peptidase activity in teleost and rat tissues

Peptides play important roles in cell regulation and signaling in many tissues. The actions of peptides are regulated by peptidases. Although the activity of these enzymes has been thoroughly characterized in mammals, little is known about their presence or function in fish. In the present study, we compared the activity of several peptidases in selected tissues (pituitary gland, different brain areas, kidney and gills) of the gilthead sea bream and rainbow trout with that found in similar rat tissues (lungs studied in place of gills). Soluble puromycin-sensitive aminopeptidase showed the highest values in the pituitary gland of the sea bream, whereas the membrane-bound form was found to be more active in the trout kidney. Very high levels of activity of aminopeptidase N were detected in trout and sea bream plasma. In contrast, the highest levels of activity of aminopeptidase B were found in rat tissues, with the exception of the gills of the trout. Aminopeptidase N levels tended to be higher in sea bream tissues with respect to those of trout. In contrast, the level of activity of aminopeptidase B was found to be consistently much higher in trout tissues than in those of the sea bream. Prolyl endopeptidase activity was principally detected in the pituitary gland and in the brain areas of teleosts. These differences between species could be related to different mechanisms of osmoregulation in saltwater- and in freshwater-adapted fish.     

Selenium-induced autometallographic demonstration of endogenous zinc in organs of the rainbow trout, Salmo gairdneri

Autometallographic (AMG) silver enhancement of endogenous zinc was studied in seven organs of the rainbow trout Salmo gairdneri. Groups of trout were injected intraperitoneally with sodium selenite in doses ranging from 0.08 to 25 ppm, administered 1 h before being killed. The concentration of selenium obtained by each organ was determined by gamma-spectrometry, and compared with the autometallographic deposition of silver grains. The relative accumulation of selenium in the organs was: liver greater than spleen greater than kidney greater than intestine greater than gills greater than brain greater than muscle. In the fish labelled with 10 and 25 ppm Se, AMG-deposits were found (1) within lysosomes of liver cells, (2) within the granules and on the nuclear membrane of melanophores in the spleen, (3) on the microvilli and in the apical cytoplasm of renal proximal tubular cells, (4) within the granules and along the plasma membrane of intestinal eosinophilic granule cells, and in the apical portion of the intestinal epithelium, and (5) in the gills, within granule cells and on the surface of the ionocytes. In the trouts injected with 5 ppm Se, silver grains were still observed in the liver, the intestine, and the gills, whereas, no such grains were found in preparations from fish having received 1 ppm Se. The use of selenium for the histochemical demonstration of endogenous zinc versus exogenous metals is discussed. Also, consideration is given to the question of which part of the total tissue zinc that is histochemically reactive.     

Dietary tissue cadmium accumulation in an amazonian teleost (Tambaqui, Colossoma macropomum Cuvier, 1818).

Understanding the effects of metal contamination in the Amazon basin is important because of the potential impact on this region of high biodiversity. In addition, the significance of fish as the primary source of protein for the local human population (living either alongside the Amazon River or in the city of Manaus) highlights the need for information on the metal transfer through the food chain. Bioaccumulation of metals in fish can occur at significant rates through the dietary route, without necessarily resulting in death of the organism. The goal of this work was to expose an economic relevant species from the Amazon basin (tambaqui, Colossoma macropomum) to dietary cadmium (Cd) at concentrations of 0, 50, 100, 200, and 400 microg.g-1 dry food. Fish were sampled on days 15, 30, and 45 of the feeding trials. Tissues were collected for analysis of Cd concentration using graphite furnace atomic absorption spectrophotometry. Cd accumulation in the tissues occurred in the following order: kidney > liver > gills > muscle. Relative to other freshwater fish (e.g., rainbow trout, tilapia), tambaqui accumulated remarkably high levels of Cd in their tissues. Although Cd is known to affect Ca2+ homeostasis, no mortality or growth impairment occurred during feeding trials.     

Comparison of dietary and waterborne exposure to benzo a pyrene: bioavailability,tissue disposition and CYP1A1 induction in rainbow trout Oncorhynchus mykiss

Absorption and tissue distribution of benzo\[ a ]pyrene (BaP)-derived radioactivity were studied in juvenile rainbow trout following dietary or waterborne exposure. In order to compare the bioavailability of BaP, the fish were exposed to 1.5 mCi 3H-BaP kg-1 fish, either in the diet or in the water as a 2 days static exposure. Furthermore, tissue levels of BaP-derived radioactivity bound to macromolecules in different tissues were studied in non-induced fish, and in fish induced by additional treatment with unlabelled BaP (corresponding to 5 mg kg-1 fish) in the water. Absorption and tissue distribution of 3H BaP were studied by liquid scintillation counting and whole-body autoradiography. BaPderived radioactivity bound to macromolecules in different tissues was studied by autoradiography of solvent-extracted whole-body sections. The hepatic CYP1A induction was measured as EROD activity. Exposure to unlabelled BaP resulted in a marked induction of hepatic EROD activity in rainbow trout 2 days after the start of the exposure. Significant higher concentrations of radiolabelled compound were observed in waterborne-exposed fish, in contrast to dietary-exposed fish. High concentrations of radiolabelling were observed in the gills, liver, bile, intestines, olfactory organ, kidney and the skin of the waterborne-exposed fish. In the dietary-exposed fish, high levels of radioactivity were observed in the intestines and the bile, whereas lower concentrations were present in the liver. Only traces of radioactive compound were observed in the gills. In contrast to waterborne-exposed fish, no radioactivity was detected in the olfactory organ or skin. In autoradiograms of sections extracted with a series of polar and non-polar solvents, a large fraction of radioactivity was still present in the gills, olfactory organ, liver, kidney, skin and intestinal mucosa of the waterborne-exposed fish, indicating that reactive BaP intermediates formed by CYP1A-mediated metabolism were bound to macromolecules in these tissues.     

Distribution and characteristics of D-amino acid and D-aspartate oxidases in fish tissues

The distributions of D-amino acid oxidase (D-AAO, EC 1.4.3.3) and D-aspartate oxidase (D-AspO, EC 1.4.3.1) activities were examined on several tissues of various fish species. Both enzyme activities were commonly high in kidney and liver and low in intestine with some exceptions. After oral administration of D-alanine at 5 micromol /g body weight(-1)day(-1) to carp for 30 days, D-AAO activity increased by about 8-, 3-, and 1.5-fold in intestine, hepatopancreas, and kidney, respectively, whereas no increase was found in brain. In contrast, oral administration of D-glutamate or D-aspartate did not show any increase of D-AspO activity in any tissues. D-AAO and D-AspO of common carp kidney and hepatopancreas were subcellularly localized in peroxisomes, as clarified in mammals. D-proline was the best substrate for D-AAO in rainbow trout kidney, common carp kidney, and hepatopancreas, followed by D-alanine and D-phenylalanine. N-methyl-D-aspartate was the best substrate for D-AspO in rainbow trout kidney and common carp hepatopancreas. The optimal pH for D-AAO in rainbow trout kidney was broad, from 7.4 to 8.2, and that for D-AspO was around 10. D-AAO was inhibited by benzoate known as D-AAO inhibitor and D-AspO was strongly inhibited by meso-tartarate as D-AspO inhibitor. From these results, at least D-AAO in fish is considered to work as a metabolizing agent of exogenous and endogenous free D-alanine that is abundant in aquatic invertebrates such as crustaceans and bivalve mollusks, which are potential food sources of these fishes.