R.A.C. Lock

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.

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.

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.

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.

Involvement of cortisol and metallothionein-like proteins in the physiological responses of tilapia (Oreochromis mossambicus) to sublethal cadmium stress

Abstract Exposure of freshwater tilapia to a sublethal cadmium concentration in the water (10 μg Cd/l) induced a rapid elevation (within 1 h) of plasma cortisol levels. Concentrations remained increased for at least 4 days during which time hypocalcemia, hyponatremia and hyperglycemia were observed; concurrently the opercular chloride cell density doubled. After this time, cortisol levels gradually decreased, plasma cadmium, natrium and glucose returned to (near) normal levels while a concomitant rise in the metallothionein-like protein content of gill, kidney and liver was observed. We conclude that, in tilapia, cortisol, by initiating physiological responses and via possible synergism with other hormones like prolactin, enables the fish to counter-act the adverse effects of Cd. This hormonal response apparently forms an intrinsic part of a general adaptation response of fish to sublethal Cd-stress.

Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine

SummaryThe inhibition of Ca2−-ATPase, (Na++K+)-ATPase and Na+/Ca2+ exchange by Cd2+ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven 45Ca2+ uptake into inside-out membrane vesicles displayed a Km for Ca2+ of 88±17 nm, and was extremely sensitive to Cd2+ with an IC50 of 8.2±3.0 pM Cd2+, indicating an inhibition via the Ca2+ site. (Na++K+)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd2+, displaying an IC50 of 2.6±0.6 μm Cd2+. Cd2+ ions apparently compete for the Mg2+ site of the (Na− +K+)-ATPase. The Na+/Ca2+ exchanger was inhibited by Cd2+ with an IC50 of 73±11 nm. Cd2+ is a competitive inhibitor of the exchanger via an interaction with the Ca2+ site (Ki = 11 nm). Bepridil, a Na+ site specific inhibitor of Na+/Ca2+ exchange, induced an additional inhibition, but did not change the Ki of Cd2+. Also, Cd2+ is exchanged against Ca2+, albeit to a lesser extent than Ca2+. The exchanger is only partly blocked by the binding of Cd2+. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na+/Ca2+ exchanger. We conclude that intracellular Cd2+ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.

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.

Metallothionein response in gills of Oreochromis mossambicus exposed to copper in fresh water.

Freshwater Oreochromis mossambicus (tilapia) were exposed to 3.2 micromol/l Cu(NO(3))(2) in the water for up to 80 days, and copper (Cu) and immunoreactive metallothionein (irMT) were localized in the branchial epithelium. Cu was demonstrated in mucous cells (MC), chloride cells (CC), pavement cells (PC), respiratory cells (RC), and basal layer cells (BLC) via autometallography combined with alcian blue staining for MC and Na(+)-K(+)-ATPase immunostaining for CC and, on the basis of their location in the epithelium of PC, RC, and BLC. In control fish (water with Cu concentration </=90 nmol/l) incidentally irMT was observed in the area where progenitor cells of the branchial epithelia reside, as demonstrated by proliferating cell nuclear antigen staining. This was also the area where the first increase irMT expression of the Cu exposure was observed. After 2 days of exposure to Cu, irMT was found in CC and PC. From 5 days on, a pronounced irMT staining was observed in BLC of branchial epithelium, which then appeared to migrate and differentiate into mature CC, PC, and RC. We conclude that MT expression in mature CC, PC, and RC requires exposure to Cu in a earlier stage of development of these cells. Once expression is initiated in undifferentiated cells, MT remains expressed throughout the life cycle of the cell.

Interactions between copper and cadmium modify metal organ distribution in mature tilapia, Oreochromis mossambicus.

Sexually mature female tilapia were exposed to sublethal concentrations of waterborne Cu and/or Cd over 6 days, and subsequent body concentrations of these metals were determined in several organs. The results show that the distribution of Cu and Cd was metal and organ specific. This is demonstrated, for example, by the observation that in tilapia, Cu exposure did not result in Cu accumulation in the liver, whereas in the intestinal wall, notably high concentrations of Cu and Cd were measured in metal exposed fish. In addition to single metal exposed fish, we also determined Cu and Cd body distribution in Cu?Cd co-exposed fish. The observed interactions in metal accumulation were most pronounced in the organs of fish exposed to low, environmentally realistic, metal concentrations.