Waterborne cadmium and zinc uptake in a euryhaline teleost Acanthopagrus schlegeli acclimated to different salinities

Metal uptake and toxicity in marine fish are usually much lower than those in freshwater fish, but the underlying mechanisms remain unclear. In this study, we investigated Cd and Zn uptake by the euryhaline black sea bream (Acanthopagrus schlegeli) over a salinity range from 0 to 35 psu. Cd and Zn uptake increased as salinity decreased. The gills were the most sensitive organs in response to salinity change, and played a more important role in Cd and Zn uptake at a lower salinity. Cd and Zn uptake in the viscera contributed to 34-36% of the overall accumulation at full salinity (35 psu), but decreased to 13-16% in freshwater despite the increase of uptake rate. Water permeability, drinking, and major ion uptake (Ca) in the fish at different salinities were also concurrently examined. The overall water uptake was comparable, whereas the drinking rate decreased at lowered salinities. In contrast, the Ca uptake increased significantly with decreasing salinity. The responses of Cd and Zn uptake to salinity challenge were correlated with the Ca uptake, suggesting that they may be taken up through the Ca uptake pathway. At a constant salinity, Cd and Zn uptake increased with reducing Ca concentration, indicating the competitive effect of Ca on metal uptake. Ca channel blockers (verapamil and lanthanum) significantly reduced the uptake of Cd, Zn, and Ca when the fish were acclimated in freshwater, but had no impact on their uptake in marine water. Furthermore, the chloride cell number in the gills could not explain the lower Cd and Zn uptake in seawater. Our results indicated that both ambient physicochemical factors and the physiological responses of fish resulted in difference of metal uptake in marine and freshwater environments.     

A protective effect of dietary calcium against acute waterborne cadmium uptake in rainbow trout

The present study examined the interactions between elevated dietary calcium (as ionic Ca2+ in the form of CaCl2 x 2H2O) and acute waterborne Cd exposure (50 microg/l as CdNO3 for 3 h) on whole body uptake and internal distribution of newly accumulated Cd, Ca2+, and Na+ in juvenile rainbow trout (Oncorhynchus mykiss). Fish were fed with three diets 20 (control), 30 and 60 mg Ca2+/g food: for 7 days before fluxes were measured with radiotracers over a 3h period. The two elevated Ca2+ diets reduced the whole body uptake of both Ca2+ and Cd by >50% and similarly reduced the internalization of both newly accumulated metals in most tissues, effects which reflect the shared branchial uptake route for Ca2+ and Cd. As the Ca2+ concentrations of the fluid phases of the stomach and intestinal contents were greatly elevated by the experimental diets, increased gastrointestinal Ca2+ uptake likely caused the down-regulation of the branchial Ca2+ (and Cd) uptake pathway. Waterborne Na+ uptake and internal distribution were not affected. While plasma Ca2+ surged after the first two feedings of the 60 mg Ca2+/g diet, internal homeostasis was quickly restored. Total Ca2+, Na+, and Cl- levels in tissues were not affected by diets. While dietary Ca2+ protected against waterborne Cd uptake, it did not protect against the relative inhibition of waterborne Ca2+ uptake caused by waterborne Cd. Acute exposure to 50 microg/l Cd reduced the uptake and internalization of newly accumulated Ca2+ (but not Na+) by 70% or more, regardless of diet. Since elevated dietary Ca2+ reduces waterborne Cd uptake, fish eating a Ca(2+)-rich invertebrate diet may be more protected against waterborne Cd toxicity in a field situation.     

Cadmium and zinc kinetics in fish: studies on water-borne 109Cd and 65Zn turnover and intracellular distribution in minnows, Phoxinus phoxinus

This study investigates the short- and long-term turnover of zinc and cadmium in fish. Juvenile minnows, Phoxinus phoxinus, were exposed to water-borne 109Cd and 65Zn for 24 hr. Tracer uptake, redistribution, elimination, and intracellular tracer distribution were studied for 60 days in tracer-free water. The influx of 65Zn from the water to the internal organs was more rapid than the influx of 109Cd, and the 65Zn turnover in the liver and kidney was considerably faster than the turnover of 109Cd. Moreover, in the gills and kidney, intracellular 65Zn was more mobile between intracellular compartments than 109Cd. The liver and kidney accumulated the major part of the 109Cd originating from the gills, whereas the head and skin, and muscle and bone-fractions retained the majority of the 65Zn. This indicates that the metals have different pathways in the fish body, after entering the body from the water. The cadmium levels in the gills, liver and kidney increased drastically because of the cadmium exposure, but the zinc levels in the organs were not markedly affected by a fifteen-fold increase in external zinc concentration, indicating a homeostatic control of zinc in the minnow.     

Dietary iron lowers the intestinal uptake of cadmium-metallothionein in rats.

It has been shown that addition of extra calcium/phosphorus (Ca/P), zinc (Zn) and iron (Fe2+) to the diet results in a significant protection against cadmium (Cd) accumulation and toxicity in rats fed inorganic Cd salt. However, it is not clear whether the presence of these mineral supplements in the diet also protects against the Cd uptake from cadmium-metallothionein. The present study examines the influence of Ca/P, Zn and Fe2+ on the Cd disposition in rats fed diets containing either 1.5 and 8 mg Cd/kg diet as cadmium-metallothionein (CdMt) or as cadmium chloride (CdCl2) for 4 weeks. The feeding of Cd resulted in a dose-dependent increase of Cd in intestine, liver and kidneys. The total Cd uptake in liver and kidneys after exposure to CdMt was lower than after exposure to CdCl2. At the low dietary Cd level and after addition of the mineral supplement, the kidney/liver concentration ratio increased. However, this ratio was always higher with CdMt than with CdCl2, suggesting a selective renal disposition of dietary CdMt. The uptake of Cd from CdCl2 as well as from CdMt was significantly decreased by the presence of a combined mineral supplement of Ca/P, Zn and Fe2+. The protection which could be achieved was 72 and 75% for CdMt and 85 and 92% for CdCl2 after doses of 1.5 mg/kg and 8 mg/kg respectively. In a following experiment it was shown that the protective effect of the mineral mixture against CdMt was mainly due to the presence of Fe2+. It seems clear that Cd speciation and the mineral status of the diet have a considerable impact on the extent of Cd uptake in rats.     

Characterization of cadmium uptake in human intestinal crypt cells HIEC in relation to inorganic metal speciation

Cadmium (Cd) uptake was studied under inorganic exposure conditions in normal human intestinal crypt cells HIEC. The uptake time course of 0.3 microM Cd in a serum-free chloride medium was analyzed according to a first order equation with rapid initial (U0) and maximal (Umax) accumulation values of 14.1+/-1.4pmol/mgprotein and 41.4+/-2.0 pmol/mgprotein, respectively. The presence of a 300-fold excess of unlabeled Cd dramatically decreased tracer uptake, showing the involvement of specific mechanism(s) of transport. Our speciation studies revealed the preferential uptake of the free ion Cd2+, but also suggested that CdCln(2-n) species may contribute to Cd accumulation. Specific mechanisms of transport of very high and similar affinity (Km approximately 5 microM) have been characterized under both chloride and nitrate exposure conditions, but a two-fold higher capacity (Vmax) was estimated in the nitrate medium used to increase [Cd2+] over chlorocomplex formation. A clear inhibition of 109Cd uptake was observed at external acidic pH under both exposure media. An La-inhibitible 46% increase in 109Cd uptake was obtained in nominally Ca-free nitrate medium, whereas Zn provided additional inhibition. These results show different kinetic parameters for Cd uptake as a function of inorganic metal speciation. Cd2+ uptake would not involve the H+-coupled symport NRAMP2 but would be related instead to the Ca and/or Zn pathways. Because proliferative crypt cells play a critical role in the renewal process of the entire intestinal epithelium, studies on the impact of Cd on HIEC cell functions clearly deserve further investigation.     

Comparative evaluation of the cytotoxicity sensitivity of six fish cell lines to four heavy metals in vitro.

To establish the potential use of cell cultures as a simple and sensitive biological tool to detect environmental pollutants, six cell lines established from several fish species including GCF (grass carp fins), CIK (Ctenopharyngodon idellus kidney), EPC (epithelioma papulosum cyprini), CCO (channel catfish ovary), BB (brown bullhead caudal trunk) and FHM (fathead minnow muscle) were tested and compared for their cytotoxic sensitivity to four heavy metals: cadmium (Cd), chromium (Cr), zinc (Zn), and copper (Cu). Following a 24-h exposure to these metal salts at selected concentrations, test cells were characterized by morphology, viability and proliferation. Our results indicate that all these metal salts are cytotoxic to these fish cell lines, but at varied levels. Calculated inhibitory concentration (IC(50)) values revealed that the cytotoxicity of Cr and Cd was significantly more pronounced than that of the other two metal salts. Comparative analysis of these fish cell lines showed that C. idellus kidney (CIK) cells are the most sensitive cell line to copper, epithelioma papulosum cyprini (EPC) cells are more sensitive than other cells to Cr and Zn, while channel catfish ovary (CCO) cell line is the most sensitive one to Cd. In conclusion, CIK, EPC and CCO could potentially be sensitive bio-indicators for the initial monitoring and assessment of acute cytotoxicity of heavy metals in the aquatic environment.     

In vitro characterization of cadmium and zinc uptake via the gastro-intestinal tract of the rainbow trout (Oncorhynchus mykiss): Interactive effects and the influence of calcium

An in vitro gut sac technique was employed to study whether Cd and Zn uptake mechanisms in the gastro-intestinal tract of the rainbow trout are similar to those at the gills, where both metals are taken up via the Ca transport pathway. Metal accumulation in surface mucus, in the mucosal epithelium, and transport into the blood space were assayed using radiolabelled Cd or Zn concentrations of 50micromolL(-1) in the luminal (internal) saline. Elevated luminal Ca (10 or 100mmolL(-1)versus 1mmolL(-1)) reduced Cd uptake into all three phases by approximately 60% in the stomach, but had no effect in the anterior, mid, or posterior intestine. This finding is in accordance with recent in vivo evidence that Ca is taken up mainly via the stomach, and that high [Ca] diets inhibit Cd accumulation from the food specifically in this section of the tract. In contrast, 10mmolL(-1) luminal Ca had no effect on Zn transport in any section, whereas 100mmolL(-1) Ca stimulated Zn uptake, by approximately threefold, into all three phases in the stomach only. There was no influence of elevated luminal Zn (10mmolL(-1)) on Cd uptake in the stomach or anterior intestine, or of high Cd (10mmolL(-1)) on Zn uptake in these sections. However, high [Zn] stimulated Cd transport into the blood space but inhibited accumulation in the mucosal epithelium and/or mucus-binding in the mid and posterior intestine, whereas high [Cd] exerted a reciprocal effect in the mid-intestine only. We conclude that Cd uptake occurs via an important Ca-sensitive mechanism in the stomach which is different from that at the gills, while Cd transport mechanisms in the intestine are not directly Ca-sensitive. Zn uptake does not appear to involve Ca uptake pathways, in contrast to the gills. These results are discussed in the context of other possible Cd and Zn transport pathways, and the emerging role of the stomach as an organ of divalent metal uptake.     

Fe- and Zn-induced inhibition of Cd uptake in human lung cell lines: Speciation studies with H441 and A549 cells

Cadmium transport was studied in human lung cell lines A549 and H441 as a function of inorganic metal speciation. A 2-fold higher equilibrium accumulation was obtained in the A549 cells, but a specific system of transport of high affinity and low capacity was characterized in both these cells. Exposure conditions optimizing [Cd²⁺] increased Cd uptake, but species are also taken up. Studies on Cd-Ca reciprocal inhibition do not support a role for Ca pathways in Cd uptake. Nramp2 and Zip8 mRNAs were detected in both cell lines. Fe inhibited Cd uptake in the nitrate medium with an apparent at pH 5.5. NRAMP2 may contribute to Cd uptake but a major role is precluded since the overall process of accumulation was inhibited by acidic conditions. Zn and Mn inhibited Cd uptake with the following observations: (1) Similar apparent K_i values for Zn-induced inhibition in the chloride and nitrate media. (2) Whatever the medium, a higher Mn-induced inhibition at pH 5.5 compared to pH 7.4, with much lower K_i values under acidic conditions. (3) Elimination of the stimulatory effect of by Zn but not by Mn. Zn may inhibit a ZIP8-mediated Cd uptake, whereas the Mn-sensitive component of uptake would be related to other transport processes.     

Dietary iron lowers the intestinal uptake of cadmium-metallothionein in rats

It has been shown that addition of extra calcium/phosphorus (Ca/P), zinc (Zn) and iron (Fe²⁺) to the diet results in a significant protection against cadmium (Cd) accumulation and toxicity in rats fed inorganic Cd salt. However, it is not clear whether the presence of these miniral supplements in the diet also protects against the Cd uptake from cadmium-metallothionein. The present study examines the influence of Ca/P, Zn and Fe²⁺ on the Cd disposition in rats fed diets containing either 1.5 and 8 mg Cd/kg diet as cadmium-metallothionein (CdMt) or as cadmium chloride (CdCl₂) for 4 weeks. The feeding of Cd resulted in a dose-dependent increase of Cd in intestine, liver and kidneys. The total Cd uptake in liver and kidneys after exposure to CdMt was lower than after exposure to CdCl₂. At the low dietary Cd level and after addition of the mineral supplement, the kidney/liver concentration ratio increased. However, this ratio was always higher with CdMt than with CdCl₂, suggesting a selective renal disposition of dietary CdMt. The uptake of Cd from CdCl₂ as well as from CdMt was significantly decreased by the presence of a combined mineral supplement of Ca/P, Zn and Fe²⁺. The protection which could be achieved was 72 and 75% for CdMt and 85 and 92% for CdCl₂ after doses of 1.5 mg/kg and 8 mg/kg respectively. In a following experiment it was shown that the protective effect of the mineral mixture against CdMt was mainly due to the presence of Fe²⁺. It seems clear that Cd speciation and the mineral status of the diet have a considerable impact on the extent of Cd uptake in rats.     

Effects of Zn pre-exposure on Cd and Zn bioaccumulation and metallothionein levels in two species of marine fish

Zinc is an essential trace metal but also a potential toxicant to aquatic organisms. In this study, two juvenile marine fish species, the black sea bream Acanthopagrus schlegeli and the grunt Teraponjarbua, were pre-exposed to Zn either from waterborne (0.74-170 microg L-1) or dietary (39-5926 microg g-1) Zn for 1 or 3 weeks. The concentrations of Zn and metallothionein (MT) in the whole body of the black sea bream and in the gills, viscera and carcass of the grunt were then measured during this pre-exposure. Following the pre-exposure, both fish species were then exposed to 109Cd and 65Zn labeled food or water to quantify the dietary assimilation efficiency (AE) and the uptake rate of dissolved Cd and Zn. Zn concentrations in both fish species were enhanced after pre-exposure, but the increases were much less than the increase of ambient Zn pre-exposure concentration. Following Zn pre-exposure, MT concentrations in the viscera and carcass were significantly elevated, whereas the MT levels were not significantly elevated in the gills. Waterborne and dietary Zn exposure enhanced the uptake rates of dissolved Cd and Zn in both fish. The maximum increases of uptake rate constants of dissolved Cd and Zn were up to 1.9-2.8 and 2.1-2.6 times, respectively, in the seabream and grunt. In contrast, dietary assimilation efficiency of Cd and Zn was not significantly enhanced following Zn pre-exposure. A positive linear relationship was found between the uptake rate constants of dissolved metals and Zn or MT concentrations in the fish. The results suggested that Zn pre-exposure increased the potential of metal uptake from ambient water, but had little effect on dietary metal uptake. Furthermore, the Zn body concentration and metal uptake from the dissolved phase were significantly dependent on the fish body size.     

Branchial cadmium and copper binding and intestinal cadmium uptake in wild yellow perch (Perca flavescens) from clean and metal-contaminated lakes

Branchial binding kinetics and gastro-intestinal uptake of copper and cadmium where examined in yellow perch (Perca flavescens) from a metal-contaminated lake (Hannah Lake, Sudbury, Ontario, Canada) and an uncontaminated lake (James Lake, North Bay, Ontario, Canada). An in vivo approach was taken for gill binding comparisons while an in vitro gut binding assay was employed for gastro-intestinal tract (GIT) uptake analysis. By investigating metal uptake at the gill and the gut we cover the two main routes of metal entry into fish. Comparisons of water and sediment chemistries, metal burdens in benthic invertebrate, and metal burdens in the livers of perch from the two study lakes clearly show that yellow perch from Hannah L. are chronically exposed to a highly metal-contaminated environment compared to a reference lake. We found that metal-contaminated yellow perch showed no significant difference in gill Cd binding compared to reference fish, but they did show significant decreases in new Cd binding and absorption in their GITs. The results show that gill Cd binding may involve low-capacity, high-affinity binding sites, while gastro-intestinal Cd uptake involves binding sites that are high-capacity, low-affinity. From this we infer that Cd may be more critically controlled at the gut rather than gills. Significant differences in branchial Cu binding (increased binding) were observed in metal-contaminated yellow perch. We suggest that chronic waterborne exposure to Cu (and/or other metals) may be the dominant influence in gill Cu binding rather than chronic exposure to high Cu diets. We give supporting evidence that Cd is taken up in the GIT, at least in part, by a similar pathway as Ca(2+), principally that elevated dietary Ca(2+) reduces Cd binding and uptake. Overall our study reveals that metal pre-exposure via water and diet can alter uptake kinetics of Cu and Cd at the gill and/or the gut.     

Influence of acclimation and cross-acclimation of metals on acute Cd toxicity and Cd uptake and distribution in rainbow trout (Oncorhynchus mykiss)

The development of chronic metal toxicity models for fresh water fish is complicated by the physiological adjustments made by the animal during exposure which results in acclimation. This study examines the influence of a pre-exposure to a chronic sublethal waterborne metal on acclimation responses as well as the uptake and distribution of new metal into juvenile rainbow trout. In one series of tests, trout were exposed to either 20 or 60 microg/L Cu, or 150 microg/L Zn for a month in moderately hard water and then cross-acclimation responses to Cd were measured in 96 h LC(50) tests. Cu exposed trout showed a cross-acclimation response but Zn exposed trout did not. Using these results, a detailed examination of Cd uptake and tissue distribution in metal-acclimated trout was done. Trout were exposed to either 75 microg/L Cu or 3 microg/L Cd for 1 month to induce acclimation and subsequently, the uptake and distribution of new Cd was assessed in both Cd- and Cu-acclimated fish using (109)Cd. The pattern of accumulation of new metal was dramatically altered in acclimated fish. For example, in 3 h gill Cd binding experiments, Cd- and Cu-acclimated trout both had a higher capacity to accumulate new Cd but only Cu-acclimated fish showed a higher affinity for Cd compared to unexposed controls. Experiments measuring Cd uptake over 72 h at 3 microgCd/L showed that the Cd uptake rate was lower for Cd-acclimated fish compared to both Cu-acclimated fish and unexposed controls. The results demonstrate the phenomenon of cross-acclimation to Cd and that chronic sublethal exposure to one metal can alter the uptake and tissue distribution of another. Understanding how acclimation influences toxicity and bioaccumulation is important in the context of risk assessment. This study illustrates that knowledge of previous exposure conditions is essential, not only for the metal of concern, but also for other metals as well.     

Plasma clearance of cadmium and zinc in non-acclimated and metal-acclimated trout

Adult rainbow trout were pre-exposed to a sublethal concentration of waterborne cadmium (Cd, 26.7 nmol/l) or waterborne zinc (Zn, 2294 nmol/l) for 30 days to induce acclimation. A single dose of radiolabeled Cd (64.4 nmol/kg) or Zn (183.8 nmol/kg) was injected into the vascular system of non-acclimated and Cd- or Zn-acclimated trout through indwelling arterial catheters. Subsequently, repetitive blood samples over 10 h and terminal tissue samples (liver, heart, bile, stomach, intestine, kidney, gills, muscle, and spleen) were taken to characterize the effect of metal acclimation on clearance kinetics in vivo. Plasma clearance of Cd in Cd-acclimated fish (0.726+/-0.015 and 0.477+/-0.012 ml/min per kg for total and newly accumulated Cd, respectively), was faster than that in non-acclimated trout (0.493+/-0.013 and 0.394+/-0.009 ml/min per kg). Unlike plasma Cd, the levels of Cd in red blood cells (RBCs) were 1.2-2.2 times higher in Cd-acclimated fish than in non-acclimated fish. At 10 h post-injection, the liver accumulated the highest proportion ( approximately 22%) of the injected Cd dose in both non-acclimated and Cd-acclimated fish but did not account for the difference in plasma levels of Cd between two groups. Plasma clearance of Zn ( approximately 0.23 ml/min per kg for new Zn) was substantially lower than Cd clearance. Pre-acclimation to waterborne Zn reduced the new Zn levels in RBCs, but did not affect the clearance of Zn from blood plasma or tissue burdens of Zn in fish. Bile concentrations of both Cd and Zn were elevated in acclimated fish, but total bile burden accounted for <1% of the injected metal dose. The results suggest that the detoxification process of injected plasma Cd is stimulated by pre-acclimation to waterborne Cd, and that Zn levels are homeostatically controlled in both non-acclimated and acclimated trout.     

Calcium effects on cadmium uptake,redistribution, and elimination in minnows,Phoxinus phoxinus,acclimated to different calcium concentrations

A short ‘pulse’ of ¹⁰⁹Cd (0.01 μg Cd/l) was given in the synthetic experimental water to four groups of minnows, Phoxinus phoxinus, acclimated to different calcium concentrations (0.2, 0.5, 2, and 5 mM Ca). The fate of the radioactive metal within the fish was followed during an elimination period. The study was designed as an uptake-release study and the main effect of calcium was observed on the ¹⁰⁹Cd uptake in the gills which decreased with increasing Ca levels. This caused a lower metal accumulation in the liver and kidney. Increased Ca levels resulted in a slower transfer of ¹⁰⁹Cd from the gills to the blood indicated both by the slower ¹⁰⁹Cd elimination rates in the gills and slower accumulation rates in liver and kidney. This effect may not have been caused by Ca directly since the ¹⁰⁹Cd transfer rate could have been dependent on the amount of ¹⁰⁹Cd retained in the gill. Liming of acidified fresh waters increases the calcium concentrations in the water. One of the positive effects of this increase might be a lower heavy-metal uptake in fish and a subsequent decreased metal toxicity to fish.     

Cadmium-metallothionein nephrotoxicity in the rat: transient calcuria and proteinuria

After a s.c. injection of 0.4 mg Cd/kg as cadmium-metallothionein (CdMT) in rats, a marked increase in urinary protein concentration appeared at 16-40 h. There was a peak of urinary Cd content during the first 4 h after the treatment. Urinary Ca was increased at 8 h after the CdMT injection and returned to normal level at 32 h. Luminal and basolateral renal membrane vesicles were isolated from both control group and CdMT (0.4 mg Cd/kg) group at 24 h after the injection. Calcium uptake and binding of both fractions were decreased in the group treated with CdMT. Cd, Zn and MT concentrations in the kidney cortex were increased, but Ca concentration was not significantly changed. Since injected CdMT is probably only partly reabsorbed by tubular cells at the dose level of 0.4 mg Cd/kg as CdMT, excessive plasma CdMT is rapidly excreted in urine, explaining the increased Cd excretion during the first few hours observed in the present experiment. Decreased Ca binding in the luminal membranes as observed in vitro could be one of the mechanisms of production of calcuria if occurring in vivo. Another possible explanation of calcuria is that Cd ions released from CdMT into the cytoplasm of the tubular cell, may exert ionic interference with Ca transport across the luminal membranes and produce decreased Ca reabsorption. It is known that a disturbance of Ca metabolism could influence the membrane stability and such a change may contribute to explaining the proteinuria characteristic of CdMT nephrotoxicity. The reversibility of the proteinuria observed after a single dose of CdMT may be related to the induction of metallothionein synthesis in the renal cells.     

Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependent manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of ¹⁰⁹Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd²⁺ species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive, suggesting that each ion inhibits the same uptake mechanism. Our results indicate that Cd uptake in human osteoblastic cells occurs, at least in part, through Ca- and Mg-inhibitable transport mechanisms, which may involve channels of the TRPM family. The effect of Cd on bone metabolism may be enhanced under low Ca and/or Mg levels.     

Stimulation of cadmium uptake by estradiol in the kidney of male rats treated with cadmium

The present study was carried out to analyze the sex differences in the retention of Cd in rats treated with a small amount of Cd, and its mechanisms. Cd and Zn concentrations in the kidney and liver of female rats treated with 28 nmol Cd or 1 nmole Zn were significantly higher than those in male rats. Pretreatment with estradiol (1.8 mumol/kg of b.w., twice a day, 6 consecutive days) increased the Cd and Zn concentrations in the kidney of male rats treated with Cd or Zn. Incubation of MDCK cells with 10(-5) M estradiol, 10(-5) M stilboestrol and 10(-5) M progesterone caused a significant increase in Cd uptake. These results suggest that endogenous female sex hormones may play a role in a higher concentration of Cd and Zn in the kidney of female rats than that in male rats. The basal level of metallothionein (MT) in the liver and kidney of control female rats was within the same range as that in the control male rats. Cd and Zn accumulations caused by pretreatment with estradiol in the kidney of male rats treated with Cd or Zn were so low (Cd: 38 ppb, Zn: 1.0 ppb) as to be probably unable to induce the synthesis of MT. An increase in the concentration of Cd in the cultured renal cells occurred 1 hr after treatment with estradiol and Cd. Pretreatment with estradiol alone also resulted in a modification of the concentration of Na and K, which cannot be bound to MT. Together, all of the above findings suggest that estradiol directly increases the accumulation of Cd into the renal cells without inducing the synthesis of MT.     

Effects of copper and cadmium on ion transport and gill metal binding in the Amazonian teleost tambaqui (Colossoma macropomum) in extremely soft water

Metal toxicity in fish is expected to be most severe in soft waters because of the low availability of cations (particularly Ca(2+)) to out-compete the metal forms for binding sites on the gills. Natural waters in the Amazon basin are typically soft due to regional geochemistry, but few studies have focused on metal toxicity in fish native to the basin. We assessed the ionoregulatory effects of waterborne copper (Cu) and cadmium (Cd) on tambaqui (Colossoma macropomum) in extremely soft water (10 micromoll(-1) Ca(2+)). Tambaqui had a very high tolerance to Cu (50-400 microgl(-1)), as indicated by a complete lack of inhibition of Na(+) uptake and an ability to gradually recover over 6h from elevated diffusive Na(+) losses caused by Cu. The insensitivity of active Na(+) influx to Cu further supports the notion that Amazonian fish may have a unique Na(+) transport system. Addition of 5-10 mgCl(-1) of dissolved organic matter (DOM) did not prevent initial (0-3h) negative Na(+) balance in tambaqui exposed to Cu. Exposure to 40 mgCl(-1) DOM prevented Na(+) losses in tambaqui even at 400 microgl(-1) Cu, probably because most Cu was complexed to DOM. Tambaqui exposed to waterborne Cd (10-80 microgl(-1)) experienced an average of 42% inhibition in whole body Ca(2+) uptake relative to controls within 3h of exposure to the metal. Inhibition of Ca(2+) uptake increased over time and, at 24h, Ca(2+) uptake was suppressed by 51% and 91% in fish exposed to 10 and 80 microgl(-1) Cd, respectively. Previous acclimation of fish to either elevated [Ca(2+)] or elevated [DOM] proved to be very effective in protecting against acute short-term metal accumulation at the gills of tambaqui in soft water (in the absence of the protective agent during metal exposure), suggesting a conditioning effect on gill metal binding physiology.     

Modulatory effect of calcium on the influx and binding of cadmium in primary cultures of neonatal rat hepatocytes

Primary cultures of rat hepatocytes were exposed to 3 or 30 microM stable cadmium (Cd) in the presence or absence of 1.8 or 3.6 mM calcium (Ca). The presence of Ca significantly reduced the efflux of lactate dehydrogenase (LDH) from cells regardless of Cd concentration or exposure time. During a 3-h time interval, the influx of Cd into hepatocytes increased from about 5 to 14% of the total extracellular Cd present. The presence of Ca during 30 microM Cd exposures resulted in an 18% reduction (P less than 0.01 or 0.001) in Cd influx during a 3-h exposure. About 11% more Cd was bound to those cells exposed in the absence of Ca following 2-h, but not 0.5-h, exposures. Therefore, binding of Cd to hepatocytes was not related directly to Cd uptake since Cd uptake (but not binding) was elevated at the 0.5-h time interval. Although the presence of Cd did not affect the influx of Ca, the presence of Cd increased the binding of Ca by 557% (P less than 0.001). Interaction between these cations at the same binding and/or entry sites on (or adjacent to) phospholipid head groups could account for the modulatory effect of Ca on Cd-challenged hepatocytes.