S.E. Wendelaar Bonga

Cadmium inhibits plasma membrane calcium transport

SummaryThe interaction of Cd2+ with the plasma membrane Ca2+-transporting ATPase of fish gills was studied. ATP-driven Ca2+-transport in basolateral membrane (BLM) vesicles was inhibited by Cd2+ with anI50 value of 3.0nm at 0.25 μm free Ca2+ using EGTA, HEEDTA and NTA to buffer Ca2+ and Cd2+ concentrations. The inhibition was competitive in nature since theK0.5 value for Ca2+ increased linearly with increasing Cd2+ concentrations while theVmax remained unchanged. The Ca2+ pump appeared to be calmodulin dependent, but we conclude that the inhibition by Cd2+ occurs directly on the Ca2+ binding site of the Ca2+-transporting ATPase and not via the Ca2+-binding sites of calmodulin. It is suggested that Cd2+-induced inhibition of Ca2+-transporting enzymes is the primary effect in the Cd2+ toxicity towards cells followed by several secondary effects due to a disturbed cellular Ca2+ metabolism. Our data illustrate that apparent stimulatory effects of low concentrations of Cd2+ on Ca2+-dependent enzymes may derive from increased free-Ca2+ levels when Cd2+ supersedes Ca2+ on the ligands.

Integrated physiological response of tilapia, Oreochromis mossambicus, to sublethal copper exposure

Abstract Juvenile and mature tilapia (Oreochromis mossambicus) were exposed to a range of sublethal copper (Cu) concentrations for 6 days to examine the mechanisms underlying the acclimation to the toxic effects of the metal. The study focuses on the gills, the primary target for waterborne pollutants. To obtain a comprehensive picture of the branchial acclimation processes operating, multiple biochemical and morphological parameters were studied. At all concentrations tested, Cu exposure resulted in the accumulation of the metal in mature fish. At 100 and 200 μg Cu l−1 only, chloride cell proliferation was observed, which was accompanied by an increase in average cell diameter in these groups. Whole body flux measurements in juvenile fish demonstrated a decrease in Na influx in fish exposed to 200 μg l−1 Cu, in the absence of an effect on Ca influx. Gill Na + K + - ATPase activity was also decreased in the crude branchial homogenates of the mature fish exposed to the highest Cu concentration only, but not in the purified branchial vesicle preparations of these fish, which may indicate reactivation of in vivo Cu-inhibited ATPase activity during the isolation process. Plasma pH, Na, Cl, K, glucose and ceruloplasmin concentrations were also affected in the 200 μg Cu l−1 group exclusively. In accordance with the gill accumulation data, plasma Cu levels were clearly elevated in all groups exposed to the metal. The results underscore the integrated response of the gills to Cu, which, however, does not come into play until challenged by relatively high ambient concentrations. These results indicate that, in comparison to the Cu-sensitive rainbow trout, tilapia is more Cu-tolerant. The most sensitive parameters affected by Cu are gill and plasma metal levels, followed by chloride cell number and diameter.

Control of calcium regulating hormones in the vertebrates: parathyroid hormone, calcitonin, prolactin and stanniocalcin

Publisher Summary This chapter discusses the control of calcium regulating hormones in the vertebrates. Control of ionic calcium levels of the extracellular fluids is mandatory for all vertebrates because only minor changes of calcium, in particular the ionized calcium fraction, have pronounced effects on the permeability of cell membranes to ions and, consequently, on important physiological processes, such as muscle contraction, nerve signal transduction, and control of cellular metabolism. Calcium uptake is balanced by renal calcium excretion, with exception of periods of body growth or, in females, periods of reproduction and parental care. Parathyroid hormone (PTH) dominates the control of the Ca2+ concentration of the extracellular fluid by its calcium-mobilizing action on bone and, indirectly, via its control of the synthesis of 1,25(OH)2D3, intestinal calcium absorption. Calcitonin (CT) reduces the extracellular calcium concentration by inhibiting osteoclastic bone resorption and by promoting calcium deposition in the skeleton. In the terrestrial vertebrates, the evidence for the involvement of prolactin (PRL) in calcium metabolism is limited to calcium handling during gestation and lactation in mammals.

The ultrastructure of chloride cells in the gills of the teleost Oreochromis mossambicus during exposure to acidified water

SummaryBranchial chloride cells, which actively take up ions in the gills of freshwater fish, were studied in tilapia (Oreochromis mossambicus) exposed to sublethally acidified freshwater. Structural damage of cells, resulting in cell death by necrosis, only occurred transiently, when the reduction of water pH was acute rather than gradual. The most prominent effects of water acidification were the rapid increase in the number of chloride cells and the changes in frequency of the different stages of the chloride cell cycle. In the opercular inner epithelium, a twofold increase in cells occurred 48 h after gradual acidification. Cell density stabilized after 4 weeks at a level 5 times that of control fish. Four transitory stages were distinguished in the chloride cell cycle: accessory or replacement cells, immature, mature, and degenerating (apoptotic) cells. In control fish, mature chloride cells dominated (over 50%) with immature and apoptotic cells totalling about 40%. After 4 weeks in acid water, only 13% of the cells were mature. Immature and apoptotic cells dominated, each representing about 40% of the total number of chloride cells. Mature cells apparently age rapidly under these conditions. Thus, chloride cells turn over quickly in acid water, with a minor increase in ion transport capacity of the gills. This conclusion is supported by the observation that opercular and branchial Na+/K+ ATPase activities in treated fish are only 40%–50% higher than in controls.

Degeneration and death, by apoptosis and necrosis, of the pavement and chloride cells in the gills of the teleost Oreochromis mossambicus

SummaryDegeneration and death of branchial epithelial cells were studied in an African cichlid fish. In both freshwater and seawater fish the superficially located pavement cells are sloughed off at the end of their lifecycle. This process is preceded by degeneration via a process of cytoplasmic shrinkage and condensation related to apoptotic (physiologically controlled) cell death. The chloride cells are pleomorphic, i.e., accessory, mature, and degenerating cells. Degeneration of chloride cells mainly occurs by apoptosis. Degenerating cells show shrinkage and densification of cytoplasm and nuclei, and swelling of the tubular system; these cells are then separated from the ambient water by pavement cells. They are finally phagocytosed and digested by macrophages. Apoptosis of chloride cells, but not of pavement cells, is greatly stimulated when the fish are in seawater; this reflects an increase in cellular turnover of the chloride cells. Accidental cell death (necrosis) of pavement cells or chloride cells is rarely observed in fully adapted freshwater and seawater fish. Its incidence increases in the first few days following transfer of fish from fresh water to seawater.

Effect of waterborne and dietary cadmium on plasma ions of the teleost Oreochromis mossambicus in relation to water calcium levels

The effects of cadmium administered via ambient water or food on plasma ions of the African freshwater cichlidOreochromis mossambicus were studied for 2, 4, 14, and 35 days, in low calcium (0.2 mM) and high calcium (0.8 mM) water. In low calcium water, an environmentally relevant concentration of 10 μg/L water-borne cadmium induced a significant and dramatic hypocalcemia on days 2 and 4. Recovery of plasma calcium was observed on days 14 and 35. Hypermagnesemia was observed on day 2, but normal levels were already found on day 4. In high calcium water adapted fish, the extent of hypocalcemia and hypermagnesemia was less pronounced than in fish from low calcium water. Water-borne cadmium caused no significant changes in plasma phosphate, sodium, potassium, or osmolality. On days 2 and 4, dietary cadmium (averaging 10 μg Cd/fish/day) caused hypermagnesemia and hypocalcemia in low calcium wateradapted fish. Recovery was observed on days 4 and 14, respectively. In fish from high calcium water, dietary cadmium caused a significant reduction in plasma calcium on day 4 only; plasma magnesium was unaffected. Hyperphosphatemia was apparent on day 14, irrespective of the water calcium concentration. No changes in plasma sodium, potassium, or osmolality were found.The results show that sublethal concentrations of cadmium, administered via the water as well as via the food, affect calcium and magnesium metabolism in tilapia. High water calcium ameliorates the effects of both water and dietary cadmium on plasma calcium and magnesium levels.Among the various heavy metal pollutants, cadmium is frequently present in natural water bodies as a result of discharges from industrial processes or other anthropogenic contamination. The harmful effects of cadmium on mammals and other terrestrial animals have been widely studied and reviewed (Flicket al. 1971; Vallee and Ulmer 1972; Webb 1979; Korte 1983; Foulkes 1986). Aquatic vertebrates such as fish, live in very intimate contact with the environment through their gills. This makes them very susceptible to aquatic pollutants.Since it is well established that freshwater fish take up most of the ions necessary for homeostasis from the water via the gills (Eddy 1982), cadmiuminduced plasma ionic disturbances are apparently caused by impaired uptake and diffusional losses of ions via these organs (Larssonet al. 1981; Giles 1984). Ionic disturbances have also been reported after exposure of fish to sublethal concentrations of heavy metals. For example, changes in the plasma ionic composition have been observed in fish exposed to copper and zinc (Lewis and Lewis 1971; Spry and Wood 1985), mercury (Locket al. 1981), and chromium (Van der Putteet al. 1983). With respect to cadmium, exposure of rainbow trout to sublethal levels induced hypocalcemia, with reduced plasma sodium, potassium, chloride and increased plasma magnesium (Giles 1984). In European flounder, cadmium-induced hypocalcemia and elevated levels of plasma phosphate, magnesium and potassium were observed (Larssonet al. 1981).In addition to water, food could also be a source of cadmium for fish, since it accumulates in aquatic organisms through trophic transfers (Anonymous 1971; Williams and Giesy 1978; Coombs 1979). Indeed, Bryan (1976) concluded that food as a source of Zn, Mn, Co, and Fe for molluscs, crustaceans and fish was more important than water. From various studies on both water-borne and food-containing metals, reviewed by Dallingeret al. (1987), there is evidence that uptake of heavy metals such as Cd, Cu, Co, Pb, Hg, and Zn from food is also the predominant pathway in freshwater fish. Koyama and Itazawa (1977) reported significant hypocalcemia and elevated plasma phosphate levels in cadmium-fed carps. Similarly, plaice and thornback ray both accumulated more cadmium from food than from seawater (Pentreath 1977). In general, cadmium concentrations in natural waters are extremely low and a more important route of cadmium uptake by fish may be represented via the gut. Experiments with dietary cadmium may therefore yield more representative information for field situations.In this investigation, we have compared the effects of a sublethal concentration of cadmium administered via the water or via the food in the African cichlid fishOreochromis mossambicus (tilapia). Plasma ions and osmolality were determined. Cadmium was administered at sublethal concentrations, in the order of magnitude that may occur in natural waters (⩽10 μg Cd/L). In many studies aimed at evaluating the effects of cadmium on fishes, high concentrations (>1 mg Cd/L) of cadmium have been used. Hence severe physiological, behavioral and detrimental effects have been reported. Such high concentrations are rarely found in nature, except in cases of spillage or heavily polluted waters. The Working Group on Cadmium Toxicity (EIFAC 1977) has suggested that chronic exposure to low cadmium concentrations is more relevant to understanding the mechanisms involved in the intoxication process in teleost fish.We further studied the influence of relatively low and high calcium concentration of the water on the toxic effects of cadmium. The effects of water hardness (mainly Ca2+ and Mg2+ ions) on heavy metal toxicity have been demonstrated in various species of teleosts (Pärtet al. 1985). Increased toxicity of cadmium to fish in soft water as compared to hard water has been demonstrated in catfish and guppies (Kinkade and Erdman 1975), goldfish (McCartyet al. 1978), striped bass (Palawskiet al. 1985), brook trout (Carrollet al. 1979) and rainbow trout (Calamariet al. 1980; Pascoet al. 1986). Similar observations on teleosts exposed to zinc, copper and lead (Sinleyet al. 1974; Zitko and Carson 1976; Judy and Davies 1979; Laurén and McDonald 1986) indicate a protective role of calcium against the toxic effects of heavy metals. It was also investigated whether the protective effect of the water-calcium concentration is limited to water-borne cadmium only, or also applies to dietary cadmium.

Effect of ambient and dietary cadmium on pavement cells, chloride cells, and Na+/K+-ATPase activity in the gills of the freshwater teleost Oreochromis mossambicus at normal and high calcium levels in the ambient water

The effects of cadmium on the gills of the African freshwater cichlid Oreochromis mossambicus in water with normal and relatively high calcium concentrations were studied for periods up to 35 days. The exposure was either through the ambient water or via the diet. Changes in the ultrastructure of the gill epithelium upon exposure to cadmium in the ambient water indicated degeneration of pavement cells and chloride cells, and acceleration in the turnover of the chloride cells. Studies of the Na+/K+-ATPase activity of the gills indicated that a transient increase in the total number of chloride cells was not associated with an increase, but rather a decrease of the total ion-exchange capacity of the chloride cells. Macrophages, lymphocytes, rodlet cells and neutrophilic granulocytes infiltrated the filament epithelium. Recovery of the gills was observed after 35 days. Dietary cadmium caused similar, although delayed, effects. High water calcium concentration reduced the impact of water-borne cadmium, but had no ameliorating effect on dietary cadmium. The data indicate that the disturbance of ion regulation in fish caused by sublethal levels of dietary cadmium is the result of effects of the metals on the gills rather than on the kidney.

Toxicants and osmoregulation in fish

Fish are extremely sensitive to many water-borne toxicants, because these affect the gills by increasing the permeability to water and ions of the gill epithelium and by inhibition of the ion exchange activity of the chloride cells. The compensatory responses of the fish will significantly increase the energy required for maintenance of water and ion homeostasis, and this will result in reduced growth and reproduction. The effects of toxicants are to a great extent comparable to those of stressors such as confinement, transport, and handling, not only where the endocrine and metabolic responses are concerned, but also with respect to the osmoregulatory disturbances produced. Stressors may affect osmoregulation indirectly through the action of catecholamines on the gills. Furthermore, stressors induce immunosuppression and this may result in gill damage by infectious agents. Many toxicants evoke a stress response, and thus it is difficult to determine the mechanism of action of toxicants on the gills, because the specific effects of the toxicants are hard to distinguish from the effects of non-specific stress responses on the gills. This further implies that the negative effects of many toxicants and non-toxicant stressors on gill structure and hydromineral balance are additive. This aspect needs more attention in aquaculture.

Background colour influence on the stress response in cultured red porgy Pagrus pagrus

Red porgy Pagrus pagrus were placed and maintained in white, grey and black background fibreglass tanks for 2 weeks. Additionally, fish kept in white and black background tanks were then subjected to crowding stress. After 2, 9, 16 and 23 days, blood samples were taken and plasma cortisol, alpha melanocyte stimulating hormone (α-MSH) and glucose values were analysed and compared with values from uncrowded fish from white or black tanks. Measurements of plasma cortisol and α-MSH in unstressed red porgy from white, grey and black tanks revealed no significant differences among the three groups. However, the results show that background colour markedly affects the in vitro interrenal sensitivity to both α-MSH and ACTH, as interrenal cells from black adapted fish had become virtually unresponsive to both secretagogues. Crowded fish on a black background showed a prominent increase in plasma cortisol after 2 days, which was followed by a recovery. In fish crowded on a white background however, the increase of cortisol was lower but was maintained through the entire experiment. Plasma α-MSH levels increased at 23 days as a consequence of crowding; this increase was also dependent on the background, being more prominent in fish placed in white background tanks. Thus, in the red porgy, a white background appears to modify the stress response, particularly in the long term.

Effects of water-borne copper on branchial chloride cells and Na+/K+-ATPase activities in Mozambique tilapia (Oreochromis mossambicus)

Abstract Freshwater tilapia (Oreochromis mossambicus) were exposed for different periods up to 28 days to 3.2 μM of water-borne Cu. Electron microscopical analysis of the gills demonstrated significant changes in the structure and number of chloride cells (CCs) from Cu-exposed fish when compared to controls. These cells, which are the main location of the Na+/K+-ATPase of the gills and which play a crucial role in transepithlial Na+ transport, showed a time-related increase of degeneration by apoptosis and necrosis in the Cu-exposed fish. After 28 days of Cu exposure, apoptotic CCs had doubled in number while necrotic CCs had even increased by a factor of ten. The activity of the gill Na+/K+-ATPase and the plasma Na+ concentration decreased in time and in parallel. An inverse relationship between the Na+/K+-ATPase specific activity and the branchial Cu content further supports the notion that this enzyme is very sensitive to Cu2+ inhibition. In contrast to controls, no significant correlation was found in the Cu-exposed fish between the opercular CC number and the gill Na+/K+-ATPase total activities, despite the large increase in number of these cells. This study provides further evidence that not only the number but also the quality of the CCs, may determine to a large extent the branchial capacity of a freshwater fish to absorb Na+ from the surrounding water.