Uptake of cadmium and zinc by the alga Chlorella vulgaris: II. Multi-ion situation

Many microorganisms are capable of sequestering and concentrating heavy metals from their aqueous environment. While much research has beep carried out on the uptake of single species of metal ions, little attention seems to have been given to the study of multimetal ion systems. A mathematical model has previously been developed to describe the uptake of individual metal species by a microorganism. The model proposes two sequential processes: an initial rapid uptake due to cellular surface adsorption and a subsequent slow uptake due to membrane transport of the metal into the cells. This article extends the treatment by considering the uptake of two metal species together, cadmium and zinc, under different experimental conditions. The results are discussed in terms of possible mechanistic interactions.     

Accumulation of lead by Anabaena cylindrica : mathematical modeling and an energy dispersive X-ray study

The ability of microorganisms to selectively adsorb various heavy metal ions has been recognized for over a decade. We have investigated the biosorption of lead by an active culture of the cyanobacterium Anabaena cylindrica. Energy dispersive X-ray microanalysis was used to evaluate the different uptake mechanisms in the various subcellular regions. Three were identified: a very fast adsorption mechanism in the cell envelope; a time-dependent deposition reaction on the cell surface; and an adsorption mechanism, also time dependent, on the polyphosphate body inside the cell. Atomic absorption spectrometry was then used to quantify the changes with time of bulk fluid concentrations of lead solutions exposed to cyanobacteria. A mass transfer kinetic model was developed which quantitatively predicts the concentration of lead in cells of Anabaena cylindrica as a function of spatial dimensions and time. The model predictions are consistent with a pattern, documented in literature and confirmed by our own experimental evidence, of a very fast uptake in the cell envelope and then a longer uptake period inside the cell. Our experimental evidence also revealed a time-dependent uptake mechanism on the surface of the cells, which is included in the model. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 408-418, 1997.     

Molecular Mechanisms and Genetic Basis of Heavy Metal Tolerance/Hyperaccumulation in Plants

Phytoremediation has gained increased attention as a cost-effective method for the remediation of heavy metal-contaminated sites. Because some plants possess a range of potential mechanisms that may be involved in the detoxification of heavy metals, they manage to survive under metal stresses. High tolerance to heavy metal toxicity could rely either on reduced uptake or increased plant internal sequestration,which is manifested by an interaction between a genotype and its environment. The growing application of molecular genetic technologies has led to increased understanding of mechanisms of heavy metal tolerance/accumulation in plants and, subsequently, many transgenic plants with increased heavy metal resistance,as well as increased uptake of heavy metals, have been developed for the purpose of phytoremediation. In the present review, our major objective is to concisely evaluate the progress made so far in understanding the molecular/cellular mechanisms and genetic basis that control the uptake and detoxification of metals by plants.     

Cadmium uptake and sequestration kinetics in individual leaf cell protoplasts of the Cd/Zn hyperaccumulator Thlaspi caerulescens

Hyperaccumulators store accumulated metals in the vacuoles of large leaf epidermal cells (storage cells). For investigating cadmium uptake, we incubated protoplasts obtained from leaves of Thlaspi caerulescens (Ganges ecotype) with a Cd-specific fluorescent dye. A fluorescence kinetic microscope was used for selectively measuring Cd-uptake and photosynthesis in different cell types, so that physical separation of cell types was not necessary. Few minutes after its addition, cadmium accumulated in the cytoplasm before its transport into the vacuole. This demonstrated that vacuolar sequestration is the rate-limiting step in cadmium uptake into protoplasts of all leaf cell types. During accumulation in the cytoplasm, Cd-rich vesicle-like structures were observed. Cd uptake rates into epidermal storage cells were higher than into standard-sized epidermal cells and mesophyll cells. This shows that the preferential heavy metal accumulation in epidermal storage cells, previously observed for several metals in intact leaves of various hyperaccumulator species, is due to differences in active metal transport and not differences in passive mechanisms like transpiration stream transport or cell wall adhesion. Combining this with previous studies, it seems likely that the transport steps over the plasma and tonoplast membranes of leaf epidermal storage cells are driving forces behind the hyperaccumulation phenotype.     

Studies on the mechanism of zinc uptake by human fibroblasts

The mechanisms of zinc uptake from a complete culture medium by human fibroblasts have been studied. The metal is accumulated in a biphasic pattern; an initial rapid phase followed by a slower linear phase. We suggest that the former represents binding to carriers or receptors on the cell surface followed by uptake to within the cell, or at least to a compartment inaccessible to proteolytic digestion. The uptake correlates well with estimates of the zinc requirement of a growing fibroblast. The process of uptake is saturable, with an apparent association constant of 1.1 X 10(7) M-1. Interestingly, there appears to be a very large number of binding sites, 2 X 10(7) per cell. No explanation for this observation is immediately apparent. The mechanism of uptake is not dependent on metabolic energy, or at least on ATP levels within the cell, but N-ethyl maleimide does block uptake in a dose-dependent manner. Weak bases and ionophores, apart from nigericin, do not affect uptake. The results suggest that zinc is not taken up by a receptor-mediated endocytic pathway as has been described for transferrin and iron.     

Glucose uptake by the cellulolytic ruminal anaerobe Bacteroides succinogenes.

Glucose uptake by Bacteroides succinogenes S85 was measured under conditions that maintained anaerobiosis and osmotic stability. Uptake was inhibited by compounds which interfere with electron transport systems, maintenance of proton or metal ion gradients, or ATP synthesis. The most potent inhibitors were proton and metal ionophores. Oxygen strongly inhibited glucose uptake. Na+ and Li+, but not K+, stimulated glucose uptake. A variety of sugars, including alpha-methylglucoside, did not inhibit glucose uptake. Only cellobiose and 2-deoxy-D-glucose were inhibitory, but neither behaved as a competitive inhibitor. Metabolism of both sugars appeared to be responsible for the inhibition. Cells grown in cellobiose medium transported glucose at one-half the rate of glucose-grown cells. Spheroplasts transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose-binding protein. Differences in glucose uptake patterns were detected in cells harvested during the transition from the lag to the log phase of growth compared with cells obtained during the log phase. These differences were not due to different mechanisms for glucose uptake in the cell types. Based on the results of this study, B. succinogenes contains a highly specific, active transport system for glucose. Evidence of a phosphoenolpyruvate-glucose phosphotransferase system was not found.     

Molecular mechanisms of heavy metal hyperaccumulation and phytoremediation

A relatively small group of hyperaccumulator plants is capable of sequestering heavy metals in their shoot tissues at high concentrations. In recent years, major scientific progress has been made in understanding the physiological mechanisms of metal uptake and transport in these plants. However, relatively little is known about the molecular bases of hyperaccumulation. In this paper, current progresses on understanding cellular/molecular mechanisms of metal tolerance/hyperaccumulation by plants are reviewed. The major processes involved in hyperaccumulation of trace metals from the soil to the shoots by hyperaccumulators include: (a) bioactivation of metals in the rhizosphere through root–microbe interaction; (b) enhanced uptake by metal transporters in the plasma membranes; (c) detoxification of metals by distributing to the apoplasts like binding to cell walls and chelation of metals in the cytoplasm with various ligands, such as phytochelatins, metallothioneins, metal-binding proteins; (d) sequestration of metals into the vacuole by tonoplast-located transporters. The growing application of molecular-genetic technologies led to the well understanding of mechanisms of heavy metal tolerance/accumulation in plants, and subsequently many transgenic plants with increased resistance and uptake of heavy metals were developed for the purpose of phytoremediation. Once the rate-limiting steps for uptake, translocation, and detoxification of metals in hyperaccumulating plants are identified, more informed construction of transgenic plants would result in improved applicability of the phytoremediation technology.     

Understanding cellular responses to toxic agents: a model for mechanism-choice in bacterial metal resistance

Summary Bacterial resistances to metals are heterogeneous in both their genetic and biochemical bases. Metal resistance may be chromosomally-, plasmid- or transposonencoded, and one or more genes may be involved; at the biochemical level at least six different mechanisms are responsible for resistance. Various types of resistance mechanisms can occur singly or in combination and for a particular metal different mechanisms of resistance can occur in the same species. To understand better the diverse responses of bacteria to metal ion challenge we have constructed a qualitative model for the selection of metal resistance in bacteria. How a bacterium becomes resistant to a particular metal depends on the number and location of cellular components sensitive to the specific metal ion. Other important selective factors include the nature of the uptake systems for the metal, the role and interactions of the metal in the normal metabolism of the cell and the availability of plasmid (or transposon) encoded resistance mechanisms. The selection model presented is based on the interaction of these factors and allows predictions to be made about the evolution of metal resistance in bacterial populations. It also allows prediction of the genetic basis and of mechanisms of resistance which are in substantial agreement with those in well-documented populations. The interaction of, and selection for resistance to, toxic substances in addition to metals, such as antibiotics and toxic analogues, involve similar principles to those concerning metals. Potentially, models for selection of resistance to any substance can be derived using this approach.     

Silicon Alleviation of Cadmium Toxicity in Mangrove (Avicennia marina) in Relation to Cadmium Compartmentation

Mangrove plants seem to be highly tolerant of high levels of heavy-metal pollution. Recently, some researchers have focused on the mechanisms involved in their metal uptake and tolerance. However, the important mechanisms involved are still only partly understood. This investigation studied whether silicon (Si) affected cadmium (Cd) subcellular distribution in the leaves and root tips of Avicennia marina (Forsk.) Vierh seedlings, resulting in the amelioration of the toxicity of Cd. The results showed that Si partly overcame the reduction in growth due to Cd. This amelioration was correlated with a reduction in Cd uptake and alteration of Cd subcellular distribution. The mechanisms of Si amelioration of Cd stress were tissue dependent. In the leaves and root tips, Si reduced Cd concentration in subcellular fractions, Cd mobility, and the concentration of biologically active Cd in the cell wall active space. Si did not change the distribution of Cd between compartments in the leaves, but it increased the proportion of Cd in the cell walls and reduced the proportion of Cd in the symplast of the root tips.     

High sensitivity of RBL-2H3 cells to cadmium and manganese: an implication of the role of ZIP8

Cellular incorporation of Cd involves multiple transport systems for other metals such as Fe, Zn, Mn, and Ca. Metal transporters including divalent metal transporter 1, Zrt/Irt-related protein (ZIP) 8, and ZIP14, and certain types of voltage-dependent Ca channels have been shown to be involved in cellular Cd uptake. However, tissue- or cell-specific roles of these metal transporters in the accumulation and toxicity of Cd remains unclear. In the present study, we compared the sensitivity to and accumulation of Cd, Mn, and Zn among four types of rat cell lines. Rat basophilic leukemia RBL-2H3 cells showed the highest sensitivity to Cd and Mn due to the highest accumulation of Cd and Mn among the four cell lines. The high accumulation of Cd and Mn was caused by high uptake rates of Cd and Mn. Since relatively high expression of ZIP8 and ZIP14 was found in RBL-2H3 cells, siRNAs of ZIP8 and ZIP14 were transfected into RBL-2H3 cells. The knockdown of ZIP8, but not of ZIP14, significantly reduced the uptake rates of Cd and Mn in RBL-2H3 cells, especially in the presence of bicarbonate. These results suggest that the high expression of ZIP8, which is known to have affinities for both Cd and Mn, resulted in high accumulation of Cd and Mn, leading to high sensitivity to these metals in RBL-2H3 cells. Thus, RBL-2H3 cells may serve as a good model for clarifying the mechanisms of Cd and Mn transport via ZIP8.     

The role of glutathione in copper metabolism and toxicity

Cellular copper metabolism and the mechanism of resistance to copper toxicity were investigated using a wild type hepatoma cell line (HAC) and a copper-resistant cell line (HAC600) that accumulates copper and has a highly elevated level of metallothionein (MT). Of the enzymes involved in reactive oxygen metabolism, only glutathionine peroxidase was elevated (3-4-fold) in resistant cells, suggestive of an increase in the cellular flux of hydrogen peroxide. A majority of the cytoplasmic copper (greater than 60%) was isolated from both cell lines as a GSH complex. Kinetic studies of 67Cu uptake showed that GSH bound 67Cu before the metal was complexed by MT. Depletion of cellular GSH with buthionine sulfoximine inhibited the incorporation of 67Cu into MT by greater than 50%. These results support a model of copper metabolism in which the metal is complexed by GSH soon after entering the cell. The complexed metal is then transferred to MT where it is stored. This study also indicates that resistance to metal toxicity in copper-resistant hepatoma cells is due to increases in both cellular GSH and MT. Furthermore, it is suggested that elevated levels of GSH peroxidase allows cells to more efficiently accommodate an increased cellular hydrogen peroxide flux that may occur as a consequence of elevated levels of cytoplasmic copper.     

Biosorption of heavy metals by Saccharomyces cerevisiae: a review

Heavy metal pollution has become one of the most serious environmental problems today. Biosorption, using biomaterials such as bacteria, fungi, yeast and algae, is regarded as a cost-effective biotechnology for the treatment of high volume and low concentration complex wastewaters containing heavy metal(s) in the order of 1 to 100 mg/L. Among the promising biosorbents for heavy metal removal which have been researched during the past decades, Saccharomyces cerevisiae has received increasing attention due to the unique nature in spite of its mediocre capacity for metal uptake compared with other fungi. S. cerevisiae is widely used in food and beverage production, is easily cultivated using cheap media, is also a by-product in large quantity as a waste of the fermentation industry, and is easily manipulated at molecular level. The state of the art in the field of biosorption of heavy metals by S. cerevisiae not only in China, but also worldwide, is reviewed in this paper, based on a substantial number of relevant references published recently on the background of biosorption achievements and development. Characteristics of S. cerevisiae in heavy metal biosorption are extensively discussed. The yeast can be studied in various forms for different purposes. Metal-binding capacity for various heavy metals by S. cerevisiae under different conditions is compared. Lead and uranium, for instances, could be removed from dilute solutions more effectively in comparison with other metals. The yeast biosorption largely depends on parameters such as pH, the ratio of the initial metal ion and initial biomass concentration, culture conditions, presence of various ligands and competitive metal ions in solution and to a limited extent on temperature. An assessment of the isotherm equilibrium model, as well as kinetics was performed. The mechanisms of biosorption are understood only to a limited extent. Elucidation of the mechanism of metal uptake is a real challenge in the field of biosorption. Various mechanism assumptions of metal uptake by S. cerevisiae are summarized.     

Fluorimetric Analysis of Copper Transport Mechanisms in the B104 Neuroblastoma Cell Model: A Contribution from Cellular Prion Protein to Copper Supplying

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrP^C) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrP^C proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrP^C-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrP^C in mediating copper uptake by clathrin-dependent endocytosis.     

Copper resistance mechanisms in bacteria and fungi

Abstract: Copper is both an essential micronutrient and a toxic heavy metal for most living cells. The presence of high concentrations of cupric ions in the environment promotes the selection of microorganisms possessing genetic determinants for copper resistance. Several examples of chromosomal and plasmid copper-resistance systems in bacteria have been reported, and the mechanisms of resistance have started to be understood at the molecular level. Bacterial mechanisms of copper resistance are related to reduced copper transport, enhanced effiux of cupric ions, or copper complexation by cell components. Copper tolerance in fungi has also been ascribed to diverse mechanisms involving trapping of the metal by cell-wall components, altered uptake of copper, extracellular chelation or precipitation by secreted metabolites, and intracellular complexing by metallothioneins and phytochelatins; only the metallothionein chelation mechanism has been approached with molecular detail.     

Differential modulation of Alzheimer's disease amyloid beta-peptide accumulation by diverse classes of metal ligands

Biometals have an important role in AD (Alzheimer's disease) and metal ligands have been investigated as potential therapeutic agents for treatment of AD. In recent studies the 8HQ (8-hydroxyquinoline) derivative CQ (clioquinol) has shown promising results in animal models and small clinical trials; however, the actual mode of action in vivo is still being investigated. We previously reported that CQ-metal complexes up-regulated MMP (matrix metalloprotease) activity in vitro by activating PI3K (phosphoinositide 3-kinase) and JNK (c-jun N-terminal kinase), and that the increased MMP activity resulted in enhanced degradation of secreted Abeta (amyloid beta) peptide. In the present study, we have further investigated the biochemical mechanisms by which metal ligands affect Abeta metabolism. To achieve this, we measured the effects of diverse metal ligands on cellular metal uptake and secreted Abeta levels in cell culture. We report that different classes of metal ligands including 8HQ and phenanthroline derivatives and the sulfur compound PDTC (pyrrolidine dithiocarbamate) elevated cellular metal levels (copper and zinc), and resulted in substantial loss of secreted Abeta. Generally, the ability to inhibit Abeta levels correlated with a higher lipid solubility of the ligands and their capacity to increase metal uptake. However, we also identified several ligands that potently inhibited Abeta levels while only inducing minimal change to cellular metal levels. Metal ligands that inhibited Abeta levels [e.g. CQ, 8HQ, NC (neocuproine), 1,10-phenanthroline and PDTC] induced metal-dependent activation of PI3K and JNK, resulting in JNK-mediated up-regulation of metalloprotease activity and subsequent loss of secreted Abeta. The findings in the present study show that diverse metal ligands with high lipid solubility can elevate cellular metal levels resulting in metalloprotease-dependent inhibition of Abeta. Given that a structurally diverse array of ligands was assessed, the results are consistent with the effects being due to metal transport rather than the chelating ligand interacting directly with a receptor.     

Cadmium uptake by Caco-2 cells. Effect of some milk components

The effect of some milk components on the cellular uptake of cadmium has been studied using a human intestinal cell line (Caco-2). Cadmium uptake by Caco-2 cells increased with the concentration of this metal in the culture medium, in a saturable way. These cells were exposed to different concentrations of cadmium and the synthesis of metallothionein was studied by a cadmium-saturation method. The levels of metallothionein increased with the cadmium concentration in the medium up to 20 μM of metal. Supplementation of the culture medium with 10% bovine milk caused a 25% decrease in the uptake of cadmium with respect to that internalized by the cells maintained in the culture medium alone. However, the uptake of cadmium from the medium supplemented with 10% human milk was similar to that with serum-free medium. β-Lactoglobulin interacted with cadmium when studied by equilibrium dialysis, showing a stoichiometric binding constant of 5 × 10⁴l/mol. Interaction of lactoferrin with cadmium, however, was negligible. When Caco-2 cells were incubated in culture medium containing lactoferrin, cadmium uptake decreased with respect to that observed incubating the cells in a medium containing β-lactoglobulin or in the free-protein medium. The inhibitory effect of lactoferrin on the uptake of cadmium might be due to a reduction of the cell surface charge, through its binding to the membrane.     

The PvdRT-OpmQ efflux pump controls the metal selectivity of the iron uptake pathway mediated by the siderophore pyoverdine in Pseudomonas aeruginosa

Pyoverdine (PVD) is the major siderophore produced by Pseudomonas aeruginosa for iron acquisition. PvdRT-OpmQ is an ATP-dependent efflux pump involved in the secretion of newly synthesized pyoverdine (PVD) and of PVD that has transported and released its iron into the bacterium from the periplasm into the extracellular medium. This iron uptake pathway also involves an outer membrane transporter, FpvA, for PVD-Fe uptake from the extracellular medium into the periplasm. In binding assays, FpvA bound PVD in complex with many different metals, with affinities from 2.9?nM for PVD-Fe to 13?μM for PVD-Al. Uptake assays with various FpvA and PvdRT-OpmQ mutants, monitored by inductively coupled plasma-atomic emission spectrometry (ICP-AES) for metal detection, and by fluorescence for PVD detection, showed that both metals and PVD accumulated in P.?aeruginosa, due to the uptake of these compounds via the FpvA/PVD pathway. Higher levels of accumulation were observed in the absence of PvdRT-OpmQ expression. Thus, FpvA has a broad metal specificity for both the binding and uptake of PVD-metal complexes, and the PvdRT-OpmQ efflux pump exports unwanted metals complexed with PVD from the bacterium. This study provides the first evidence of efflux pump involvement in the export of unwanted siderophore-metal complexes and insight into the molecular mechanisms involved controlling the metal selectivity of siderophore-mediated iron uptake pathways.