Dietary metal bioavailability in razor clam Sinonovacula constricta under fluctuating seston environments.

To understand the potential risks of dietary metals to marine bivalves, it is important to study the interaction between dietary metal bioaccumulation and bivalve feeding behavior. Key processes in affecting the dietary metal influx are the selection of different particles during the ingestion process as well as the differential assimilation of metals during the digestion process. In this study, we quantified the influences of seston quality and quantity on the dietary assimilation of Cd and Zn as well as pre-ingestion particle selectivity in a razor clam Sinonovacula constricta following feeding on radiolabeled diatoms and sediments with different mixtures at four food concentrations. Bioavailability of 109Cd and 65Zn from seston was measured by assimilation efficiency (AE) using a pulse-chase feeding technique. The AEs of Cd and Zn were significantly affected by the seston quantity and quality (higher for Zn than they were for Cd and higher for diatoms than for suspended sediments), but were independent of the presence of other particles during the feeding process. Dual gamma radiotracer technique was further employed to study pre-ingestion particle selectivity. Particle selectivity was weak during pre-ingestion in razor clams, although there was evidence that clams might be able to differentiate particles during the process of pseudofeces production. Our study demonstrated that seston composition and quantity substantially affected the bioavailability of Cd and Zn to the razor clams. The results are important to understand the bioaccumulation of metals in clams living in dynamic food environments of estuary.

Effects of ocean acidification on 109Cd, 57Co, and 134Cs bioconcentration by the European oyster (Ostrea edulis): Biokinetics and tissue-to-subcellular partitioning.

The uptake and depuration kinetics of dissolved 109Cd, 57Co and 134Cs were determined experimentally in the European flat oyster Ostrea edulis (Linnaeus, 1758) under different pH conditions (i.e., 8.1, 7.8 and 7.5) for 59 days. Uptake and depuration rates were variable within these elements; no effects were observed under different pH conditions for the uptake biokinetics of 109Cd and 57Co and depuration of 109Cd and 134Cs in oyster. The uptake and depuration rate constants of 134Cs differed during the exposure phase between treatments, while the steady state concentration factors (CFss) were similar. The resulting Cs activity that was purged during short- and long-term depuration phases differed, while the remaining activities after thirty-nine days depuration phase (RA39d) were similar. Co-57 depuration was affected by pCO2 conditions: RA39d were found to be significantly higher in oysters reared in normocapnia (pCO2 = 350 µatm) compared to high pCO2 conditions. Co-57 tissue distribution did not differ among the variable pCO2 conditions, while 109Cd and 134Cs accumulated in soft tissue of oysters were found to be higher under the highest pCO2. Additionally, Cd, Co and Cs were stored differently in various compartments of the oyster cells, i.e. cellular debris, metal-rich granules (MRG) and metallothionein-like proteins (MTLP), respectively. The subcellular sequestration of the elements at the end of the depuration phase did not differ among pH treatments. These results suggest that bioconcentration and tissue/subcellular distribution are element-specific in the oyster, and the effects of higher pCO2 driven acidification and/or coastal acidification variably influence these processes.

Comparing single-feeding and multi-feeding approaches for experimentally assessing trophic transfer of metals in fish.

Diet is an important pathway for metal uptake in marine organisms, and assimilation efficiency is one of the most relevant parameters to quantify trophic transfer of metals along aquatic food webs. The most commonly used method to estimate this parameter is pulse-chase feeding using radiolabeled food. This approach is, however, based on several assumptions that are not always tested in an experimental context. The present study aimed to validate the approach by assessing single-feeding and multiple-feeding approaches, using a model species (the turbot Scophthalmus maximus). Using the kinetic data obtained from the single-feeding experiment, the reconstruction of a multi-feeding experiment was tested for consistency with data provided by an actual multi-feeding performed under the same experimental conditions. The results validated the single-feeding approach. Environ Toxicol Chem 2017;36:1227-1234. © 2016 SETAC.

Selective uptake, distribution, and redistribution of (109)Cd, (57)Co, (65)Zn, (63)Ni, and (134)Cs via xylem and phloem in the heavy metal hyperaccumulator Solanum nigrum L.

The focus of this article was to explore the translocation of (109)Cd, (57)Co, (65)Zn, (63)Ni, and (134)Cs via xylem and phloem in the newly found hyperaccumulator Solanum nigrum L. Two experiments with the uptake via the roots and transport of (109)Cd, (57)Co, and (65)Zn labeled by roots, and the redistribution of (109)Cd, (65)Zn, (57)Co, (63)Ni, and (134)Cs using flap label in S. nigrum in a hydroponic culture with a standard nutrient solution were conducted. The results showed that (109)Cd added for 24 h to the nutrient medium of young plants was rapidly taken up, transferred to the shoot, and accumulated in the cotyledons and the oldest leaves but was not efficiently redistributed within the shoot afterward leading to a rather low content in the fruits. In contrast, (57)Co was more slowly taken up and released to the shoot, but afterward, this element was redistributed from older leaves to younger leaves and maturing fruits. (65)Zn was rapidly taken up and transferred to the shoot (mainly to the youngest leaves and not to the cotyledons). Afterward, this radionuclide was redistributed within the shoot to the youngest organs and finally accumulated in the maturing fruits. After flap labeling, all five heavy metals tested ((109)Cd, (57)Co, (65)Zn, (63)Ni, (134)Cs) were exported from the labeled leaf and redistributed within the plant. The accumulation in the fruits was most pronounced for (63)Ni and (65)Zn, while a relatively high percentage of (57)Co was finally found in the roots. (134)Cs was roughly in the middle of them. The transport of (109)Cd differed from that previously reported for wheat or lupin and might be important for the potential of S. nigrum to hyperaccumulate cadmium.

Evaluation of in vivo detection properties of 22Na, 65Zn, 86Rb, 109Cd and 137Cs in plant tissues using real-time radioisotope imaging system.

In plant research, radioisotope imaging provides useful information about physiological activities in various tissues and elemental transport between plant organs. To expand the usage of imaging techniques, a new system was developed to visualize beta particles, x-rays and gamma-rays emitted from plant bodies. This real-time radioisotope imaging system (RRIS) visualizes radioactivity after conversion into light with a CsI(Tl) scintillator plate. Herein, the RRIS detection properties of the gamma-ray emitters (22)Na, (65)Zn, (86)Rb, (109)Cd and (137)Cs were evaluated in comparison with those of radioluminography (RLG) using an imaging plate. The lower quantitative detection limit (Bq mm(-2)) during a 15 min period ranged from 0.1 to 4, depending on the nuclide, similar to that of RLG. When the quantitative ability to detect radiation from various Arabidopsis tissues was analyzed, the quantitative capability in silique and the thick internode tended to be low. In an EGS5 simulation, beta particles were the greatest contributors to RRIS imaging of (22)Na, (86)Rb and (137)Cs, and low-energy x-rays contributed significantly to (65)Zn and (109)Cd detection. Thus, both self-absorption and air space between the sample and scintillator surface could impair quantitative RRIS imaging. Despite these issues, RRIS is suggested for quantitative time-course measurements of radionuclide motion within plants.

Characterization of rapid intervascular transport of cadmium in rice stem by radioisotope imaging.

Participation of the intervascular transport system within the rice stem during cadmium (Cd) partitioning was investigated by characterizing (109)Cd behaviour in the shoot. In addition, (45)Ca, (32)P, and (35)S partitioning patterns were analysed for comparison with that of (109)Cd. Each tracer was applied to the seedling roots for 15 min, and the shoots were harvested either at 15 min (i.e. immediately after tracer application) or at 48 h. Distribution patterns of each element at 15 min were studied to identify the primary transport pathway before remobilization was initiated. (32)P was preferentially transported to completely expanded leaf blades having the highest transpiration rate. The newest leaf received minimal amounts of (32)P. In contrast, the amount of (35)S transported to the newest leaf was similar to that transported to the other mature leaf blades. Preferential movement towards the newest leaf was evident for (109)Cd and (45)Ca. These results directly indicate that elemental transport is differentially regulated in the vegetative stem as early as 15 min before the elements are transported to leaves. Cd behaviour in the stem was investigated in detail by obtaining serial section images from the bottom part of shoots after (109)Cd was applied to a single crown root. At 30 min, the maximum amount of (109)Cd was distributed in the peripheral cylinder of the longitudinal vascular bundles (PV) and, interestingly, some amount of (109)Cd was transported downwards along the PV. This transport manner of (109)Cd provides evidence that Cd can be loaded on the phloem at the stem immediately after Cd is transported from the root.

In situ analysis of cadmium uptake in four sections of the gastro-intestinal tract of rainbow trout (Oncorhynchus mykiss).

This study links results from past in vitro and in vivo experiments, by implementing an in situ experiment in order to determine the relative importance for cadmium (Cd) uptake of different sections of the gastro-intestinal tract (GIT) of rainbow trout. Transport of Cd from four sections of the GIT of adult rainbow trout (~220 g) was individually examined by infusing ligated sections of the GIT in live, free-swimming fish with 50 µM Cd spiked with radiolabelled (109)Cd (0.5 µCi ml(-1)). Fish were exposed for an 8-h period. The percentage of the total injected (109)Cd which was internalized from the different segments was only between ~0.1 and ~7%, indicating low uptake efficiency. The stomach is the most important GIT segment for Cd transport into the internal compartment of the animal, while the posterior intestine also plays a significant role. The majority of (109)Cd recovered at the end of the flux period was detected within gut material (ranging from 28 to 95%); the portion of Cd which was internalized was largely found in the carcass (32 to 60%). Distribution between the measured organs varied with uptake from the various GIT sections. Our results also confirm that the GIT acts as a protective barrier against Cd uptake from dietary exposure.

Characterization of basolateral-to-apical transepithelial transport of cadmium in intestinal TC7 cell monolayers.

Cadmium (Cd) is a toxic metal with an extremely long half-life in humans. The intestinal absorption of Cd has been extensively studied but the role the intestinal epithelium may play in metal excretion has never been considered. The basolateral (BL)-to-apical (AP) transepithelial transport of Cd was characterized in TC7 human intestinal cells. Both AP and BL uptakes varied with days in culture, and BL uptake was twofold higher compared to AP in differentiated cultures. A 50% increase in the BL uptake of 0.5 µM (109)Cd was observed at pH 8.5 in a chloride but not nitrate medium, suggesting the involvement of a pH-sensitive mechanism of transport for chloro-complexes. Fe and Zn inhibited the BL uptake of Cd whereas complexation by albumin had no effect, but the stimulatory effect of pH 8.5 was lost in the presence of albumin. The BL uptake of [(3)H]-MPP(+) and (109)Cd were both inhibited by decynium22 without reciprocal inhibition. MRP2 and MDR1 mRNA levels increased as a function of days in culture. A 25 and 20% decrease in the cellular AP efflux of Cd was observed in the presence of verapamil and probenecid, respectively. In cells treated with BSO, which lowered by 26% the total cellular thiol content, the inhibitory effect of verapamil increased, whereas that of probenecid decreased. These results reveal the existence of a decynium22-sensitive mechanism of transport for Cd at the BL membrane, and suggest the involvement of MDR1 and MRP2 in cellular Cd efflux at the AP membrane. It is conceivable that the intestinal epithelium may contribute to Cd blood excretion.

Assimilation and subcellular partitioning of elements by grass shrimp collected along an impact gradient.

Chronic exposure to polluted field conditions can impact metal bioavailability in prey and may influence metal transfer to predators. The present study investigated the assimilation of Cd, Hg and organic carbon by grass shrimp Palaemonetes pugio, collected along an impact gradient within the New York/New Jersey Harbor Estuary. Adult shrimp were collected from five Staten Island, New York study sites, fed (109)Cd- or (203)Hg-labeled amphipods or (14)C-labeled meals and analyzed for assimilation efficiencies (AE). Subsamples of amphipods and shrimp were subjected to subcellular fractionation to isolate metal associated with a compartment presumed to contain trophically available metal (TAM) (metal associated with heat-stable proteins [HSP - e.g., metallothionein-like proteins], heat-denatured proteins [HDP - e.g., enzymes] and organelles [ORG]). TAM-(109)Cd% and TAM-(203)Hg% in radiolabeled amphipods were approximately 64% and approximately 73%, respectively. Gradients in AE-(109)Cd% ( approximately 54% to approximately 75%) and AE-(203)Hg% ( approximately 61% to approximately 78%) were observed for grass shrimp, with the highest values exhibited by shrimp collected from sites within the heavily polluted Arthur Kill complex. Population differences in AE-(14)C% were not observed. Assimilated (109)Cd% partitioned to the TAM compartment in grass shrimp varied between approximately 67% and approximately 75%. (109)Cd bound to HSP in shrimp varied between approximately 15% and approximately 47%, while (109)Cd associated with metal-sensitive HDP was approximately 17% to approximately 44%. Percentages of assimilated (109)Cd bound to ORG were constant at approximately 10%. Assimilated (203)Hg% associated with TAM in grass shrimp did not exhibit significant variation. Percentages of assimilated (203)Hg bound to HDP ( approximately 47%) and ORG ( approximately 11%) did not vary among populations and partitioning of (203)Hg to HSP was not observed. Using a simplified biokinetic model of metal accumulation from the diet, it is estimated that site-specific variability in Cd AE by shrimp and tissue Cd burdens in field-collected prey (polychaetes Nereis spp.) could potentially result in up to approximately 3.2-fold differences in the dose of Cd assimilated by shrimp from a meal in the field. The results of this study also suggest that chronic field exposure can impact mechanisms of metal transport across the gut epithelium that do not influence carbon assimilation. Differences in the assimilation and subcellular partitioning of metal may have important implications for metal toxicity in impacted shrimp populations.

Uptake and loss of dissolved 109Cd and 75Se in estuarine macroinvertebrates.

Semaphore crabs (Heloecius cordiformis), soldier crabs (Mictyris platycheles), ghost shrimps (Trypaea australiensis), pygmy mussels (Xenostrobus securis), and polychaetes (Eunice sp.), key benthic prey items of predatory fish commonly found in estuaries throughout southeastern Australia, were exposed to dissolved (109)Cd and (75)Se for 385 h at 30 k Bq/l (uptake phase), followed by exposure to radionuclide-free water for 189 h (loss phase). The whole body uptake rates of (75)Se by pygmy mussels, semaphore crabs and soldier crabs were 1.9, 2.4 and 4.1 times higher than (109)Cd, respectively. There were no significant (P>0.05) differences between the uptake rates of (75)Se and (109)Cd for ghost shrimps and polychaetes. The uptake rates of (109)Cd and (75)Se were highest in pygmy mussels; about six times higher than in soldier crabs for (109)Cd and in polychaetes for (75)Se - the organisms with the lowest uptake rates. The loss rates of (109)Cd and (75)Se were highest in semaphore crabs; about four times higher than in polychaetes for (109)Cd and nine times higher than in ghost shrimps for (75)Se - the organisms with the lowest loss rates. The loss of (109)Cd and (75)Se in all organisms was best described by a two (i.e. short and a longer-lived) compartment model. In the short-lived, or rapidly exchanging, compartment, the biological half-lives of (75)Se (16-39 h) were about three times greater than those of (109)Cd (5-12h). In contrast, the biological half-lives of (109)Cd in the longer-lived, or slowly exchanging compartment(s), were typically greater (1370-5950 h) than those of (75)Se (161-1500 h). Semaphore crabs had the shortest biological half-lives of both radionuclides in the long-lived compartment, whereas polychaetes had the greatest biological half-life for (109)Cd (5950 h), and ghost shrimps had the greatest biological half-life for (75)Se (1500 h). This study provides the first reported data for the biological half-lives of Se in estuarine decapod crustaceans. Moreover, it emphasises the importance of determining metal(loid) accumulation and loss kinetics in keystone prey items, which consequently influences their trophic transfer potential to higher-order predators.

Cadmium uptake, translocation and tolerance in the hyperaccumulator Arabidopsis halleri.

Arabidopsis halleri is a well-known zinc (Zn) hyperaccumulator, but its status as a cadmium (Cd) hyperaccumulator is less certain. Here, we investigated whether A. halleri can hyperaccumulate Cd and whether Cd is transported via the Zn pathway. Growth and Cd and Zn uptake were determined in hydroponic experiments with different Cd and Zn concentrations. Short-term uptake and root-to-shoot transport were measured with radioactive 109Cd and 65Zn labelling. A. halleri accumulated > 1000 mg Cd kg(-1) in shoot dry weight at external Cd concentrations >or= 5 microm, but the short-term uptake rate of 109Cd was much lower than that of 65Zn. Zinc inhibited short-term 109Cd uptake kinetics and root-to-shoot translocation, as well as long-term Cd accumulation in shoots. Uptake of 109Cd and 65Zn were up-regulated, respectively, by low iron (Fe) or Zn status. A. halleri was much less tolerant to Cd than to Zn. We conclude that A. halleri is able to hyperaccumulate Cd partly, at least, through the Zn pathway, but the mechanisms responsible for cellular Zn tolerance cannot detoxify Cd effectively.

Iron and cadmium uptake by duodenum of hypotransferrinaemic mice.

Absorption from food is an important route for entry of the toxic metal, cadmium, into the body. Both cadmium and iron are believed to be taken up by duodenal enterocytes via the iron regulated, proton-coupled transporter, DMT1. This means that cadmium uptake could be enhanced in conditions where iron absorption is increased. We measured pH dependent uptake of (109)Cd and (59)Fe by duodenum from mice with an in vitro method. Mice with experimental (hypoxia, iron deficiency) or hereditary (hypotransferrinaemia) increased iron absorption were studied. All three groups of mice showed increased (59)Fe uptake (p<0.05) compared to their respective controls. Hypotransferrinaemic and iron deficient mice exhibited an increase in (109)Cd uptake (p<0.05). Cadmium uptake was not, however, increased by lowering the medium pH from 7.4 to 6. In contrast, (59)Fe uptake (from (59)FeNTA(2)) and ferric reductase activity was increased by lowering medium pH in control and iron deficient mice (p<0.05). The data show that duodenal cadmium uptake can be increased by hereditary iron overload conditions. The uptake is not, however, altered by lowering medium pH suggesting that DMT1-independent uptake pathways may operate.

Cadmium in the shore crab Carcinus maenas: seasonal variation in cadmium content and uptake and elimination of cadmium after administration via food.

The uptake and assimilation efficiency of cadmium administered via the food in the shore crab Carcinus maenas were investigated together with elimination kinetics and seasonal variations in cadmium content. The majority of shore crabs assimilated between 41 and 86% of the cadmium administered in their food. More than 90% of the cadmium taken up from food was retained in midgut gland. Elimination of cadmium after uptake from one meal of radioactively labelled soft parts of blue mussels could be described by a three-compartment model (percent 109Cd-retained = 64 x e(-0.001107 x t) + 25 x e(-0.0385 x t)+11 x e(-0.888 x t)). The biological half-life for cadmium in the most slowly exchanging compartment (containing 64% of the body burden) was 626 days. Groups of male and female shore crabs were collected from an uncontaminated site in the period May till October and the concentrations of cadmium in midgut gland and gills were determined. Male crabs had higher cadmium concentrations in the midgut gland in June and August (mean 2.7 microg Cd g(-1) dry weight) than they had in May, September and October (mean 1.7 microg Cd g(-1) dry weight). Females generally had slightly lower cadmium concentrations in the midgut gland than the males, except for a relatively high concentration in May. The cadmium concentrations in gills generally ranged between 0.3 and 0.5 microg Cd g(-1) dry weight) except for male values in October (mean 1 microg Cd g(-1) dry weight). Some of the seasonal changes in cadmium content of the crabs might plausibly be explained by changes in cadmium uptake from water, i.e. changes during the moult cycle and changes in cadmium uptake rates from water brought about by changes in ambient factors such as salinity and temperature. However, uptake of cadmium from water and transfer to the midgut gland take place at a rate that is two orders of magnitude too low to account for the increase in the cadmium concentrations in midgut gland in male crabs between May and June. The distribution of cadmium among tissues in crabs collected at uncontaminated sites also corresponds better with results obtained after administration of cadmium via the food than via water, and the exposure of the crabs to cadmium via the food is large enough to explain the increase in concentration between May and June.

In vivo and in vitro cadmium accumulation during the moult cycle of the male shore crab Carcinus maenas--interaction with calcium metabolism.

The effect of moult stage on cadmium accumulation and distribution was investigated in vivo in male shore crabs Carcinus maenas exposed to 1 mg Cd l(-1) for 7 days. The accumulation of cadmium in all tissues examined was markedly higher in postmoult (A(1-2) and B(1-2)) compared to intermoult (C1, C3 and C4) and premoult (D(0-3)). In addition, elevated levels of cadmium were found in gills of late premoult (D(2-3)) animals. The total amount of cadmium accumulated in the tissues (haemolymph, gills, midgut gland and muscle) increased from 43 microg Cd in early premoult (D(0-1)) to 391 microg Cd in late postmoult (B(1-2)). Gills and midgut gland were the primary cadmium accumulating tissues in C4-intermoult and premoult (D(0-3)); in early postmoult (A(1-2)) haemolymph and midgut gland were the main cadmium containing tissues, while midgut gland dominated in late postmoult (B(1-2)) and early intermoult (C1 and C3). A detailed account of calcium distribution in haemolymph, gills, midgut gland, muscle and exoskeleton during the moult cycle is presented. Mechanistic links between cadmium and calcium uptake in posterior gills of C4-intermoult and early postmoult (A(1-2)) crabs were explored using an in vitro gill perfusion technique. Calcium and cadmium influxes were markedly higher in postmoult compared to intermoult. No differences between intermoult and postmoult effluxes were found for either calcium or cadmium. From intermoult to postmoult net influx increased from 2.4 to 29 micromol Ca2+ g(-1) ww(gill) h(-1) and from 0.24 to 25 nmol Cd2+ g(-1) ww(gill) h(-1). The results indicate that the postmoult increase in cadmium influx is due to increased active transport of cadmium, at least partly, by accidental uptake via calcium transporting proteins. The in vitro net influx rates corresponded accurately to the observed in vivo accumulation of both cadmium and calcium. Although cadmium accumulation and distribution are clearly linked to changes in calcium requirements, cadmium did not interfere with calcium accumulation or distribution at any stage during the moult cycle.

Effects of dietary calcium and cadmium on cadmium accumulation, calcium and cadmium uptake from the water, and their interactions in juvenile rainbow trout.

The objective of this study was to examine the effects of chronically elevated dietary Ca2+ (as CaCO3), alone and in combination with elevated dietary Cd, on survival, growth, and Cd and Ca2+ accumulation in several internal compartments in juvenile rainbow trout (Oncorhynchus mykiss). In addition, effects on short-term branchial uptake and internal distribution of newly accumulated waterborne Ca2+ and Cd during acute waterborne Cd exposure (50 microg/L as CdNO3 for 3 h) were monitored using radiotracers (45Ca, 65Cd). Fish were fed with four diets: 20 mg Ca2+/g food (control), 50 mg Ca2+/g food, 300 microg Cd/g food, and 50 mg Ca2+/g + 300 microg Cd/g food for 30 days. There were no significant effects on growth, mortality, or total body Ca2+ accumulation. The presence of elevated Ca2+, Cd, or Ca2+ + Cd in the diet all reduced waterborne Ca2+ uptake in a short-term experiment (3 h), though the inhibitory mechanisms appeared to differ. The effects were marked after 15 days of feeding, but attenuated by 30 days, except when the diet was elevated in both Ca2+ and Cd. The presence of elevated Ca2+ in the diet had only modest influence on Cd uptake from the water during acute Cd challenges but greatly depressed Cd uptake from the diet and accumulation in most internal tissues. None of the treatment diets prevented the decreases in waterborne Ca2+ uptake and new Ca2+ accumulation in internal tissues caused by acute exposure to waterborne Cd. In conclusion, there are complex interactions between waterborne and dietary effects of Ca2+ and Cd. Elevated dietary Ca2+ protects against both dietary and waterborne Cd uptake, whereas both waterborne and dietary Cd elevations cause reduced waterborne Ca2+ uptake.

Hyperaccumulation of cadmium and zinc in Thlaspi caerulescens and Arabidopsis halleri at the leaf cellular level.

Vacuolar compartmentalization or cell wall binding in leaves could play a major role in hyperaccumulation of heavy metals. However, little is known about the physiology of intracellular cadmium (Cd) sequestration in plants. We investigated the role of the leaf cells in allocating metal in hyperaccumulating plants by measuring short-term (109)Cd and (65)Zn uptake in mesophyll protoplasts of Thlaspi caerulescens "Ganges" and Arabidopsis halleri, both hyperaccumulators of zinc (Zn) and Cd, and T. caerulescens "Prayon," accumulating Cd at a lower degree. The effects of low temperature, several divalent cations, and pre-exposure of the plants to metals were investigated. There was no significant difference between the Michaelis-Menten kinetic constants of the three plants. It indicates that differences in metal uptake cannot be explained by different constitutive transport capacities at the leaf protoplast level and that plasma and vacuole membranes of mesophyll cells are not responsible for the differences observed in heavy metal allocation. This suggests the existence of regulation mechanisms before the plasma membrane of leaf mesophyll protoplasts. However, pre-exposure of the plants to Cd induced an increase in Cd accumulation in protoplasts of "Ganges," whereas it decreased Cd accumulation in A. halleri protoplasts, indicating that Cd-permeable transport proteins are differentially regulated. The experiment with competitors has shown that probably more than one single transport system is carrying Cd in parallel into the cell and that in T. caerulescens "Prayon," Cd could be transported by a Zn and Ca pathway, whereas in "Ganges," Cd could be transported mainly by other pathways.

Cadmium transport by human Nramp 2 expressed in Xenopus laevis oocytes.

Using the Xenopus oocyte expression system, human Nramp2, a human intestinal iron transporter, was shown to work as a cadmium transporter. An 1824-bp human Nramp2 cDNA was constructed by PCR cloning from reverse transcription products of human kidney mRNA. When the pH of the extracellular solution was 6.0, human Nramp2 transported (109)Cd(2+). Substitution of external Cl(-) with NO3- had no effect on human Nramp2-dependent cadmium uptake. The concentration-dependent Cd(2+) transport of human Nramp2 indicated Michaelis-Menten type transport with an average K(m) value of 1.04 +/- 0.13 microM and an average V(max) of 14.7 +/- 1.9 pmol/oocyte/h (n = 3). Cd(2+) transport via human Nramp2 was inhibited significantly by Cd(2+), Fe(2+), Pb(2+), Mn(2+), Cu(2+), and Ni(2+), while it was not inhibited by Hg(2+) and Zn(2+). Transport of 0.1 microM Cd(2+) by human Nramp2 was inhibited by metallothionein (IC50 = 0.14 microM). Therefore, human Nramp2 is suggested to function as a pH-dependent cadmium absorption transporter on the luminal membrane of human intestinal cells.

Maitotoxin stimulates Cd influx in Madin-Darby kidney cells by activating Ca-permeable cation channels.

This study examined the role of calcium channels for the uptake of cadmium (Cd) into Madin-Darby canine kidney (MDCK) cells. Maitotoxin, an activator of different types of calcium channels, increased accumulation of 109Cd and 45Ca in MDCK cells. We found that maitotoxin increased accumulation by stimulating 109Cd influx because it did not affect efflux. An inhibitor of store-operated Ca channels, SKF96365, partially blocked 45Ca influx but did not affect 109Cd influx. Ni and Mn, and loperamide and proadifen (SKF 525a), inhibited 45Ca and 109Cd influx in cells stimulated with maitotoxin, but La and nifedipine did not. Overnight treatment with phorbol 12, 13-ibutyrate (PDBu) to activate protein kinase C resulted in a decrease in the concentration of maitotoxin needed to stimulate 45Ca and 109Cd influx. The effect of PDBu was blocked by treating cells with the protein kinase C inhibitor GF109203X. Additionally, the effect of PDBu was lost in cells treated with an inhibitor of RNA synthesis actinomycin D. These results suggest that a Ca permeable cation channel different from voltage-dependent and store-operated Ca channels mediates the uptake of Cd in MDCK cells. The expression of this channel is regulated by protein kinase C.

Loss of the metal binding properties of metallothionein induced by hydrogen peroxide and free radicals.

The relationship between the metal-binding properties of metallothionein (MT) and its ability to interact with peroxides and free radicals was explored in vitro. The binding of 109Cd to MT and the thiol density of the protein were determined after incubation of a purified Zn/Cd-metallothionein preparation with either hydrogen peroxide alone, or with a number of free radical generating systems. Exposure of MT to H2O2, whether in the presence or absence of Fe2+, resulted in the progressive loss of the thiol residues of the protein and led to a parallel decrease of its 109Cd-binding capacity. These changes correlated with r values of 0.999 (P = 0.001) and 0.998 (P = 0.001), in the absence and presence of iron, respectively. The effects of H2O2, alone or plus Fe2+, on MT were completely prevented by catalase, but totally unaffected by superoxide dismutase or desferrioxamine. Exposure of MT to xanthine/xanthine oxidase also led to thiol oxidation and to a concomitant loss of the Cd-binding properties. In this system, both changes correlated with an r of 0.993 (P = 0.001) and were completely inhibited by superoxide dismutase. Exposure of MT to the peroxyl radical generator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), resulted in the progressive loss of its the metal-binding properties and its thiol residues, both changes correlating with an r of 0.986 (P = 0.002). The ability of MT to bind 109Cd, lost as a result of its prior exposure to either H2O2 alone, H2O2 plus Fe2+, xanthine/xanthine oxidase, or to AAPH was, in all cases, completely recovered after incubation of the modified protein with dithiothreitol. These results indicate that H2O2 alone, and/or the oxygen-derived species, superoxide anion and peroxyl radicals, can all directly interact in vitro with MT to modify the protein oxidatively, and suggest that, under in vivo conditions, these species may be implicated as modifying factors of the metal-binding capacity of metallothionein.

Cadmium binding by bacteria: screening and characterization of new isolates and mutants.

A fast and simple methodology was developed that enables screening of microbial strains for their ability to bind cadmium. It is based on the use of a radioisotope of cadmium (109Cd) for screening colonies and for evaluation of cadmium binding. The methods described here can be used to screen new environmental isolates or to obtain mutants with altered ability to bind cadmium. Examples for the two uses are described in the paper.