Reciprocal inhibition of Cd and Ca uptake in human intestinal crypt cells for voltage-independent Zn-activated pathways

Cadmium-Ca-Zn interactions for uptake have been studied in human intestinal crypt cells HIEC. Our results failed to demonstrate any significant cross-inhibition between Cd and Ca uptake under single metal exposure conditions. However, they revealed a strong reciprocal inhibition for a Zn-stimulated mechanism of transport. Optimal stimulation was observed under exposure conditions that favor an inward-directed Zn gradient, suggesting activation by extracellular rather than intracellular Zn. The effect of Zn on the uptake of Ca was concentration-dependent, and zinc-induced stimulation of Cd uptake resulted in a 3- and 5.8-fold increase in the K_m and V_max values, respectively. Neither basal nor Zn-stimulated Ca uptakes were sensitive to membrane depolarization. However, the stimulated component of uptake was inhibited by the trivalent cations Gd³⁺, and La³⁺ and to a lesser extent by Mg²⁺ and Ba²⁺. RT-PCR analysis as well as uptake measurement performed with extracellular ATP and/or suramin do not support the involvement of purinergic P2X receptor channels. Uptake and fluorescence data led to the conclusion that Zn is unlikely to trigger Ca influx in response to Ca release from thapsigargin-sensitive intracellular pools. Our data show that Zn may potentiate Cd accumulation in intestinal crypt cells through mechanism that still needs to be clarified.     

Interaction of Cd and Zn during uptake and loss in the polychaete Capitella capitata: Whole body and subcellular perspectives

The interaction between Cd and Zn in aquatic organisms is known to be highly variable. The purpose of this study was to use a subcellular compartmentalization approach to examine Cd and Zn interactions in the deposit-feeding polychaete Capitella capitata (sp. I). Laboratory-reared C. capitata were co-exposed to Cd (background or 50 μg Cd l^− 1) and Zn (background or 86 μg Zn l^− 1) with ¹⁰⁹Cd and ⁶⁵Zn as radiotracers for 1 week. After the 1-week uptake period, subsets of worms were allowed to depurate accumulated metals for an additional 1 week. Worms from both phases (uptake and loss) were then subjected to subcellular fractionation to determine the compartmentalization of metals as metal-sensitive fractions [MSF — organelles and heat-denaturable proteins (HDP)] and biologically detoxified metals [BDM — heat-stable proteins (HSP) and metal-rich granules (MRG)]. Uptake and loss of Cd and Zn in C. capitata at the whole body level were similar at bkgd-Cd/bkgd-Zn, with worms depurating the majority of accumulated metal (∼ 75% Cd and ∼ 64% Zn). When exposure of Zn or Cd was increased (bkgd-Cd/86-Zn; bkgd-Zn/50-Cd), uptake of background levels of Cd or Zn, respectively, was suppressed by ∼ 50%. These accumulated metals, however, were retained during the loss phase resulting in ∼ 40-50% greater Cd and Zn whole body tissue burdens than those of bkgd-Cd/bkgd-Zn worms. Beyond exhibiting similar patterns of uptake and loss at the whole body level, Cd and Zn behaved similarly at the subcellular level. Under background levels (bkgd-Cd/bkgd-Zn), after uptake, worms partitioned a majority of Cd (∼ 65%) and Zn (∼ 55%) to the HSP and organelles fractions. The HDP and MRG fractions contained less than ∼ 6% of both metals. Following depuration, at bkgd-Cd/bkgd-Zn, Cd and Zn were lost from all subcellular fractions; loss from HSP was the greatest contributor to whole body loss. When exposed to elevated concentrations of Zn or Cd, the suppression in uptake of bkgd-Cd or bkgd-Zn observed in whole body uptake was largely due to suppressions in the storage of Cd and Zn to HSP. These results suggest that Cd-Zn interactions reduce partitioning of both Cd and Zn to HSP, indicating that metal-binding proteins such as metallothioneins play a key role in these interactions.     

Phytoaccumulation of cadmium through Azolla from aqueous solution

Azolla, which is an aquatic fern, has proved to be effective in the uptake and accumulation of metals from polluted waters. Azolla spp., namely A. microphylla cv. MH3 and A. caroliniana Willd, were chosen as model plants so that Cd(II) could be accumulated from aqueous solution. An increase in uptake time and the concentration of Cd(II) in aqueous solution resulted in more Cd(II) accumulation in both species. Modified Michaelis-Menten equation was employed to describe the concentration-dependent kinetics of Cd(II) uptake through the roots of A. microphylla cv. MH3, and the values of K_m and V_max were found to be 0.23 mg/L and 16.49 μg/(g.f.wt.h), respectively. Cd(II) uptake by A. microphylla cv. MH3 occurs partly through Ca(II) channels and has the potential to be mediated by Zn(II) transporters.     

Kinetic properties of a micronutrient transporter from<Emphasis Type="Italic"> Pisum sativum</Emphasis> indicate a primary function in Fe uptake from the soil

Fe uptake in dicotyledonous plants is mediated by a root plasma membrane-bound ferric reductase that reduces extracellular Fe(III)-chelates, releasing Fe²⁺ ions, which are then absorbed via a metal ion transporter. We previously showed that Fe deficiency induces an increased capacity to absorb Fe and other micronutrient and heavy metals such as Zn²⁺ and Cd²⁺ into pea (Pisum sativum L.) roots [Cohen et al. (1998) Plant Physiol 116:1063–1072). To investigate the molecular basis for this phenomenon, an Fe-regulated transporter that is a homologue of the Arabidopsis IRT1 micronutrient transporter was isolated from pea seedlings. This cDNA clone, designated RIT1 for root iron transporter, encodes a 348 amino acid polypeptide with eight putative membrane-spanning domains that is induced under Fe deficiency and can functionally complement yeast mutants defective in high- and low-affinity Fe transport. Chelate buffer techniques were used to control Fe²⁺ in the uptake solution at nanomolar activities representative of those found in the rhizosphere, and radiotracer methodologies were employed to show that RIT1 is a very high-affinity ⁵⁹Fe²⁺ uptake system (K _m =54–93 nM). Additionally, radiotracer (⁶⁵Zn, ¹⁰⁹Cd) flux techniques were used to show that RIT can also mediate a lower affinity Zn and Cd influx (K _m of 4 and 100 M, for Zn²⁺ and Cd²⁺, respectively). These findings suggest that, in typical agricultural soils, RIT1 functions primarily as a high-affinity Fe²⁺ transporter that mediates root Fe acquisition. This is consistent with recent findings with Arabidopsis IRT1 knockout mutants that strongly suggest that this transporter plays a key role in root Fe uptake and nutrition. However, the ability of RIT1 to facilitate Zn and Cd uptake when these metals are present at elevated concentrations suggests that RIT1 may be one pathway for the entry of toxic metals into the food chain. Furthermore, the finding that plant Fe deficiency status may promote heavy metal uptake via increased expression of this transporter could have implications both for human nutrition and also for phytoremediation, the use of terrestrial plants to sequester toxic metals from contaminated soil.     

NaCl differently interferes with Cd and Zn toxicities in the wetland halophyte species Kosteletzkya virginica (L.) Presl.

Hydroponic experiments were carried out using seedlings of the wetland halophyte species Kosteletzkya virginica (L.) Presl. exposed to 10???M Cd or 100???M Zn in the absence or presence of 50?mM NaCl. Interaction between salinity and heavy metals was analysed in relation to plant growth, water status and tissue ion contents (Na, K and Ca). Results showed a strong inhibition effect of Cd on leaf emergence, lateral branch development and leaf expansion. Heavy metals induced a significant decrease in plant dry weight, water content, osmotic potential (?? _S) and leaf water potential (?? _w). Cadmium and Zn accumulated to higher extent in the roots than in the shoots. Cadmium increased the leaf K concentration while Zn had an opposite effect. Salinity strongly reduced Cd uptake and translocation from roots to shoots: it mitigated the Cd impact on lateral branch emergence but had no effects on plant dry weight and water status. Cadmium drastically reduced Na translocation in salt-treated plants while Zn increased it. It is concluded that complex interactions exist between heavy metals and monovalent cations in salt conditions and that Cd and Zn display contrasting behaviour in this respect. Stress-induced modification of ion content did not fully explain growth inhibition in Kosteletzkya virginica.     

Influence of zinc on cadmium-induced toxicity in nodules of pigeonpea (Cajanus cajan L. Millsp.) inoculated with arbuscular mycorrhizal (AM) fungi

Arbuscular mycorrhizal (AM) fungi are known to alleviate heavy-metal stress in plants. The intent of the present work was to analyze accumulation of heavy metals (Cd and Zn) in nodules of two Cajanus cajan (L.) Millsp. genotypes and their subsequent impact on nitrogen fixation, oxidative stress, and non-protein thiols (glutathione and phytochelatins) with and without AM fungus Glomus mosseae. Accumulation of Cd and Zn in nodules resulted in sharp reduction in nodule number, nodule dry mass as well as nitrogen fixation (leghemoglobin and nitrogenase (N₂ase)), although Cd had more pronounced effects than Zn. Cd-induced lipid peroxidation, H₂O₂ accumulation, and electrolyte leakage were largely reversed by Zn supplementation. Zn application significantly altered the negative effects of Cd on the synthesis of non-protein thiols, suggesting antagonistic behaviour of Zn. Higher concentration of Zn was more effective in lessening the negative effects of Cd than its lower concentration. Remarkable genotypic variation was found, with more severe effects of both the metals in P792 than Sel 85N. Glomus mosseae attenuated the phytotoxic effects of metals in nodules by decreasing metal uptake, oxidative stress, and by enhancing defense system ultimately leading to better nitrogen-fixing potential of pigeonpea nodules.     

Acute dietary pre-exposure and trace metal bioavailability to the barnacle Balanus amphitrite

Rates of uptake from solution and assimilation efficiencies of the trace metals Ag, Cd and Zn were investigated in the barnacle Balanus amphitrite after exposure in the laboratory for 19 days to low and high doses of added Ag and Cd in a diatom (Thalassiosira weissflogii) diet, the major route of metal uptake in barnacles. The hypothesis under test was that acute metal pre-exposure would affect the assimilation efficiency (AE) of that and other metals and their rate of uptake from solution. It was found that pre-exposure of the barnacles to atypically high dietary challenges of Cd and Ag did not cause changes in the rates of uptake of Cd, Ag or Zn from solution. Similarly, there was no clear consistent effect of dietary pre-exposure to Cd or Ag on the assimilation efficiency of Cd, Ag or Zn. The efflux rates of the metals were also comparable following the acute dietary exposure. Subcellular fractionation data indicated that the majority of the three metals were partitioned in the insoluble fraction, with very little in the soluble fraction consisting of metallothionein-like proteins and other (heat-sensitive) proteins. The lack of induction of increased Cd or Ag AE after pre-exposure in barnacles contrasts with results for mussels; this inconsistency is interpreted to result from differences in physiological accumulation patterns, the barnacles relying to an extreme extent on insoluble detoxification.     

Biotechnological potential of Microcystis sp. in Cu, Zn and Cd biosorption from single and multimetallic systems

This paper provides information on biosorption of Cu, Zn and Cd by Microcystis sp. in single, bi and trimetallic combination. Highest biosorption of Cu followed by Zn and Cd in single as well as in mixtures containing two or three metals was noticed. The order of inhibition of Cu, Zn and Cd biosorption in bi and trimetallic combinations was suggestive of screening or competition for the binding sites on the cell surface. This observation was reconfirmed by Freundlich adsorption isotherm. K_f values were maximum for Cu (K_f=45.18), followed by Zn (K_f=16.71), and Cd (K_f=15.63) in single metallic system. The K_f values for each test metal was reduced in solution containing more than one metal. Further, the reduction in biosorption of each metal ion due to presence of other metal ion was of greater magnitude at relatively higher concentrations of interfering metal ion. The biosorption of Cu at saturation was less affected when secondary metal (Cd or Zn) was added in the medium. Above results suggest that Microcystis holds great potential for metal biosorption from mixture.     

Expression of HvHMA2 in tobacco modifies Zn–Fe–Cd homeostasis

HvHMA2 is a plasma membrane P_1B-ATPase from barley that functions in Zn/Cd root-to-shoot transport. To assess the usefulness of HvHMA2 for modifying the metal content in aerial plant parts, it was expressed in tobacco under the CaMV35S promoter. Transformation with HvHMA2 did not produce one unique pattern of Zn and Cd accumulation; instead it depended on external metal supply. Thus Zn and Cd root-to-shoot translocation was facilitated, but not at all applied Zn/Cd concentrations. Metal uptake was restricted in HvHMA2-transformed plants and the level in the shoot was not enhanced. It was shown that HvHMA2 localizes to the plasma membrane of tobacco cells, and overloads the apoplast with Zn, which could explain the overall decrease in metal uptake observed. Despite the lower levels in the shoot, HvHMA2 transformants showed increased Zn sensitivity. Moreover, introduction of HvHMA2 into tobacco interfered with Fe metabolism and Fe accumulation was modified in HvHMA2-transformants in a Zn- and Cd-concentration dependent manner. The results indicate that ectopic expression of the export protein HvHMA2 in tobacco interferes with tobacco metal Zn–Cd–Fe cross-homeostasis, inducing internal mechanisms regulating metal uptake and tolerance.     

Particle sources and trace element reactivity in the Humber plume

Samples of suspended particulate matter (SPM) collected from the Humber Estuary had higher concentrations of particulate metals than SPM from Holderness coastal waters (U.K.). Characterised SPM from both sources was used in laboratory experiments involving the uptake of radiotracer¹⁰⁹Cd,¹³⁷Cs,⁵⁴Mn and⁶⁵Zn. Kinetic experiments, over five days, showed that the rate and extent of uptake was highly dependent on particle type, with¹⁰⁹Cd,⁵⁴Mn and⁶⁵Zn being more reactive with Humber Estuary particles than those from Holderness and¹³⁷Cs having the opposite trend. Adsorption experiments were also carried out on suspensions in which SPM from the Humber Estuary and Holderness coastal water were mixed in various proportions. These experiments revealed that K_d for⁶⁵Zn increased linearly with the proportion of Humber SPM, K_d for¹³⁷Cs decreased linearly with increase in Humber SPM and K_d for⁵⁴Mn and¹⁰⁹Cd displayed non-linear behaviour. The results of the study were used to develop an algorithm for predicting the partition coefficients in the Humber Plume based on the extent of particle mixing from the two source regions. The use of^206/207Pb ratios in determining the extent of particle mixing is discussed, along with the application of the algorithm to the modelling of particulate trace metal behaviour in the Humber-Wash coastal zone.     

Relationships between Cadmium, Zinc, Cd-Peptide, and Organic Acid in Tobacco Suspension Cells

Responses of tobacco (Nicotiana tabacum) suspension cells to Cd and Zn were studied in the presence and absence of ligand of Cd-peptide in order to understand the role of this peptide versus other mechanisms in Cd and Zn accumulation and accommodation in plants. With 45 micromolar Cd and 300 micromolar Zn (non-growth-inhibiting levels), metals appeared rapidly within cells, and intracellular Cd and Zn reached medium concentrations after 6 to 10 hours. Cd-peptide was observed in response to Cd after 2 hours, but this form only accounted for ~30% of soluble Cd after 24 hours. Peptide was not observed in cells exposed to 300 micromolar Zn for up to 7 days. Organic acid-to-metal stoichiometry indicated that endogenous organic acid content of cells was more than sufficient to complex absorbed metals and no evidence was found for stimulation of organic acid biosynthesis by Cd or Zn. Metal-complexing potential of organic acids for Cd and Zn versus endogenous cations is discussed as is vacuolar-extravacuolar distribution of metals. The absence of Cd-peptide does not limit Cd-accumulation in the system studied. Results suggest that tobacco suspension cells accommodate the presence of non-growth-inhibiting and growth-inhibiting levels of Cd and Zn by sequestration in the vacuole as complexes with endogenous organic acids and that this may be a principal means for accommodation of Cd as well as Zn in the presence and absence of Cd-peptide.     

Assimilation efficiency of zinc and cadmium in the decapod crustacean Penaeus indicus

The assimilation of trace metals from food can be the main route of metal uptake into aquatic invertebrates. The assimilation efficiencies of zinc and cadmium from muscle tissue of a cephalopod mollusc (40% to 70% Zn, 64% to 83% Cd) and from a macrophytic alga (50% to 69% Zn, 39% to 50% Cd) were measured in juvenile penaeid prawns Penaeus indicus. Assimilated Zn and Cd were retained mostly in the hepatopancreas of the prawns, some metal being excreted (Zn k_e 0.10 and 0.11 and Cd k_e 0.004 and 0.009, from cephalopod muscle and alga, respectively). There were no significant differences between k_es (efflux rate constants) of one metal from either diet. Given the high trace metal assimilation efficiencies measured here, it is highly probable that metal assimilation from food plays an important role in Zn and Cd accumulation in the body of P. indicus, particularly in estuarine stages of the life cycle, for estuaries are particularly prone to metal pollution and are likely to offer the prawns abundant metal-rich diets such as detrital material derived from local macrophytes.     

Cadmium-Sensitive Mutants of Arabidopsis thaliana

A screening procedure for identifying Cd-sensitive mutants of Arabidopsis thaliana is described. With this procedure, two Cd-sensitive mutants were isolated. These represent independent mutations in the same locus, referred to as CAD1. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed that the mutation is closely linked to the tt3 locus on chromosome 5. In addition to Cd, the mutants are also significantly more sensitive to mercuric ions and only slightly more sensitive to Cu and Zn, while being no more sensitive than the wild type to Mn, thus indicating a degree of specificity in the mechanism affected by the mutation. Undifferentiated callus tissue is also Cd sensitive, suggesting that the mutant phenotype is expressed at the cellular level. Both wild-type and mutant plants showed increased sensitivity to Cd in the presence of buthionine sulfoximine, an inhibitor of the biosynthesis of the cadmium-binding (γ-glutamylcysteine)_n-glycine peptides, suggesting that the mutant is still able to synthesize these peptides. However, the effects of a cad1 mutation and buthionine sulfoximine together on cadmium sensitivity are essentially nonadditive, indicating that they may affect different aspects of the same detoxification mechanism. Assays of Cd uptake by intact plants indicate that the mutant is deficient in its ability to sequester Cd.     

Mycorrhizal symbiosis and phosphorus fertilization effects on Zea mays growth and heavy metals uptake

Arbuscular mycorrhizal fungi (AMF) can promote plant growth and reduce plant uptake of heavy metals. Phosphorus (P) fertilization can affect this relationship. We investigated maize (Zea mays L.) uptake of heavy metals after soil AMF inoculation and P fertilization. Maize biomass, glomaline and chlorophyll contents and uptake of Fe, Mn, Zn, Cu, Cd and Pb have been determined in a soil inoculated with AMF (Glomus aggregatum, or Glomus intraradices) and treated with 30 or 60 µg P-K₂HPO₄ g^?1 soil. Consistent variations were found between the two mycorrhizal species with respect to the colonization and glomalin content. Shoot dry weight and chlorophyll content were higher with G. intraradices than with G. aggregatum inoculation. The biomass was highest with 30 µg P g^?1 soil. Shoot concentrations of Cd, Pb and Zn decreased with G. aggregatum inoculation, but that of Cd and Pb increased with G. intraradices inoculation. Addition of P fertilizers decreased Cd and Zn concentrations in the shoot. AMF with P fertilization greatly reduced maize content of heavy metals. The results provide that native AMF with a moderate application rate of P fertilizers can be exploited in polluted soils to minimize the heavy metals uptake and to increase maize growth.     

Testosterone metabolism by homogenates of human prostates with benign hyperplasia: Effects of zinc,cadmium and other bivalent cations

The effects of various quantities of Ba, Be, Ca, Cd, Co, Cu, Mg, Mn, Sr, Zn and EDTA on the formation of 5α-reduced metabolites of testosterone (T) substrate and of 3α-/3β -reduced metabolites of 5α-dihydrotestosterone substrates by homogenates of 6 human hyperplastic prostate glands were studied in incubations at pH 7.4 with NADPH-generating system. Effects of these cations and EDTA on the V_M and K_M of the 5α-reductase and 3α-/3β-hydroxysteroid dehydrogenases (-HSD) were also measured. Quantities of 5α-reduced T metabolites were significantly increased by Cd, Cu and Zn supplementations. These increments were shown to result from significant augmentations of the V_M but no change in K_M of the NADPH-dependent 5α -reductase. Quantities of 3α -reduced DHT metabolites were significantly decreased by Cd and Cu supplementations and resulted from an increase of the K_M of the NADPH-dependent 3α-HSD by Cd and both an increase of K_M and a decrease of V_M by Cu. Quantities of β-reduced DHT metabolites were significantly decreased by Cd and Cu supplementations. Increase of the K_M of the NADPH-dependent 3β-HSD by Cd was found significant while Cu both increased the Am and decreased the V_M of the enzyme. EDTA-related changes in 5α-reductase activity were shown to result from the EDTA-induced decrease of the pH of the medium. No effect of EDTA was observed on the activities of both 3α/3β-HSD.     

Effects of Amendments on Growth and Uptake of Cd and Zn by Wetland Plants,Typha angustifolia and Colocasia esculenta from Contaminated Sediments

A pot study was conducted to compare the effects of amendments (CaHPO₄ and cow manure) on growth and uptake of Cd and Zn from contaminated sediments by two wetland plant species, Typha angustifolia and Colocasia esculenta. Contaminated sediments (Cd 33.2 mg kg^–1 and Zn 363 mg kg^–1) were collected from Mae Tao basin, Mae Sot district, Tak province, Thailand. The experiment consisted of 4 treatments: control (uncontaminated sediment), Cd/Zn, Cd/Zn + 5% CaHPO₄, and Cd/Zn + 10% cow manure. Plants were grown for 3 months in the greenhouse. The addition of CaHPO₄ resulted in the highest relative growth rate (RGR) and highest Cd accumulation in both T. angustifolia and C. esculenta while the lowest RGR was found in C. esculenta grown in the cow manure treatment. Both plant species had higher concentrations of metals (Cd, Zn) in their belowground parts. None of the amendments affected Zn accumulation. C. esculenta exhibited the highest uptake of both Cd and Zn. The results clearly demonstrated the phytoremediation potential of C. esculenta and the enhancement of this potential by CaHPO₄ amendment.     

The role of heavy-metal ATPases,HMAs, in zinc and cadmium transport in rice

The P_1B-type heavy metal ATPases (HMAs) are diverse in terms of tissue distribution, subcellular localization, and metal specificity. Functional studies of HMAs have shown that these transporters can be divided into two subgroups based on their metal-substrate specificity: a copper (Cu)/silver (Ag) group and a zinc (Zn)/cobalt (Co)/cadmium (Cd)/lead (Pb) group. Studies on Arabidopsis thaliana and metal hyperaccumulator plants indicate that HMAs play an important role in the translocation or detoxification of Zn and Cd in plants. Rice possesses nine HMA genes, of which OsHMA1–OsHMA3 belong to the Zn/Co/Cd/Pb subgroup. OsHMA2 plays an important role in root-to-shoot translocation of Zn and Cd, and participates in Zn and Cd transport to developing seeds in rice. OsHMA3 transports Cd and plays a role in the sequestration of Cd into vacuoles in root cells. Modification of the expression of these genes might be an effective approach for reducing the Cd concentration in rice grains.     

Dynamics of plant metal uptake and metal changes in whole soil and soil particle fractions during repeated phytoextraction

Aims

Phytoextration of metal polluted soils using hyperaccumulators is a promising technology but requires long term successive cropping. This study investigated the dynamics of plant metal uptake and changes in soil metals over a long remediation time.

Methods

A soil slightly polluted with metals (S1) was mixed with highly polluted soil (S4) to give two intermediate pollution levels (S2, S3). The four resulting soils were repeatedly phyto-extracted using nine successive crops of Cd/Zn-hyperaccumulator Sedum plumbizincicola over a period of 4 years.

Results

Shoot Cd concentration decreased with harvest time in all soils but shoot Zn declined in S1 only. Similar shoot Zn concentrations were found in S2, S3 and S4 although these soils differed markedly in metal availability, and their available metals decreased during phytoextraction. A possible explanation is that plant active acquisition ability served to maintain plant metal uptake. Plant uptake resulted in the largest decrease in the acid-soluble metal fraction followed by reducible metals. Oxidisable and residual fractions were less available to plants. The coarse soil particle fractions made the major contribution to metal decline overall than the fine fractions.

Conclusion

Sedum plumbizincicola maintained long term metal uptake and the coarse soil particles played the most important role in phytoextraction.