Cadmium accumulation and its effects on intracellular ion pools in a brewing strain of Saccharomyces cerevisiae

The presence of glucose resulted in a two- to three-fold increase in levels of Cd²⁺accumulated by Saccharomyces cerevisiae after 5 h compared with those observed in the absence of glucose. However, time-dependent Cd²⁺ uptake continued in the absence of glucose over 5 h, resulting in an appreciable increase in cellular Cd²⁺levels. Substantial K⁺ efflux but little Mg²⁺ and negligible Ca²⁺release was observed. Cell fractionation revealed that the bulk of intracellular Cd²⁺ was located in the vacuolar (25%) and bound (60%) fractions. Accumulation of Cd²⁺ ions impacted most noticeably on K⁺ rather than Mg²⁺ levels in intracellular compartments. Cytoplasmic and particularly vacuolar K⁺ levels decreased as Cd²⁺ sequestration continued resulting in increased extracellular levels. In contrast, corresponding intracellular Mg²⁺ pools were only modestly affected with a slight increase and decrease observed in the cytoplasmic and vacuolar fractions respectively. However, levels of bound Mg²⁺ decreased in response to continued Cd²⁺ accumulation. Received 07 March 1999/ Accepted in revised form 26 June 1999     

Comparative studies of zinc metabolism in cultured chinese hamster cells with differing metallothionein-induction capacities

Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd^r2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd²⁺ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd²⁺-resistant Cd^r2C10 subline to induce synthesis of the Cd²⁺- and Zn²⁺-binding protein(s), metallothionein(s) (MT). Evidence that Cd²⁺ behaves as an analog of the essential trace metal, Zn²⁺, especially as an inducer of MT synthesis, suggested that the Cd^r and CHO cell types could be employed to investigate cellular Zn²⁺ metabolism. In the present study, measurements were made to compare CHO and Cd^r cell types for (a) growth as a function of the level of ZnCl₂ added to the culture medium, (b) uptake and subcellular distribution of Zn²⁺, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd^r cell types grew normally (T _d≊16–18 h) during exposures to Zn²⁺ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn²⁺ levels, (b) Cd^r cells incorporate fourfold more Zn²⁺ during a 24-h exposure to the maximal subtoxic level of Zn²⁺ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd^r cell is proficient in this response during exposure to the maximal subtoxic Zn²⁺ level. These findings suggest that (a) the CHO and Cd^r cell systems will be useful in further studies of cellular Zn²⁺ metabolism, especially in comparisons of Zn²⁺ metabolism in the presence and absence of induction of the Zn²⁺-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular Zn²⁺ uptake.     

A unique metallothionein-engineered in <Emphasis Type="Italic">Escherichia coli</Emphasis> for biosorption of lead,zinc, and cadmium; absorption or adsorption?

The present study introduced and evaluated modification of E. coli BL21 (DE3) to improve its biosorption capacity by the transfer of the Corynebacterium glutamicum metallothionein gene (C.gMT). The C.gMT sequence was extracted and cloned in pET28a vector and the ligation product was transferred into E. coli BL21 (DE3). It has been also submitted to the GenBank database (accession number KJ638906.1). The performance of the recombinant bacterium was evaluated at different metal ions concentrations, contact times, pH values, and co-ions. The results show that recombinant BL21 (DE3) was able to uptake Pb⁺², and Zn⁺² at greater percentages than could BL21 (DE3). The optimum pH for the removal of each heavy metal was different. As contact time increased, Pb⁺² and Zn⁺² biosorption by the recombinant bacterium increased, while the biosorption of Cd⁺² remained at a nearly steady rate for contact times of more than 1 h. Increasing the concentrations of Pb⁺² and Zn⁺² in solution increased biosorption of these metals by the recombinant BL21 (DE3) over that of Cd⁺². It could be hypothesized that Pb⁺² and Zn⁺² removal by C.gMT-engineered BL21 (DE3) occurred mainly via intracellular biosorption (absorption) and that Cd⁺² was mainly taken up through cell surface biosorption (adsorption).     

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer

Background

Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As⁺³ and Cd⁺² sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants.

Results

It was shown that both As⁺³ and Cd⁺² exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As⁺³ or Cd⁺². Localization studies showed that ENO2 in the MCF-10A cells transformed by As⁺³ or Cd⁺² had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1.

Conclusion

The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As⁺³ or Cd⁺². The findings also suggest a possible link between As⁺³ and Cd⁺² exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As⁺³ and Cd⁺².     

Evidence of Microbial Translocation Associated with Perturbations in T Cell and Antigen-Presenting Cell Homeostasis in Hookworm Infections

Background

Microbial translocation (MT) is the process by which microbes or microbial products translocate from the intestine to the systemic circulation. MT is a common cause of systemic immune activation in HIV infection and is associated with reduced frequencies of CD4⁺ T cells; no data exist, however, on the role of MT in intestinal helminth infections.

Methods

We measured the plasma levels of MT markers, acute-phase proteins, and pro- and anti - inflammatory cytokines in individuals with or without hookworm infections. We also estimated the absolute counts of CD4⁺ and CD8⁺ T cells as well as the frequencies of memory T cell and dendritic cell subsets. Finally, we also measured the levels of all of these parameters in a subset of individuals following treatment of hookworm infection.

Results

Our data suggest that hookworm infection is characterized by increased levels of markers associated with MT but not acute-phase proteins nor pro-inflammatory cytokines. Hookworm infections were also associated with increased levels of the anti – inflammatory cytokine – IL-10, which was positively correlated with levels of lipopolysaccharide (LPS). In addition, MT was associated with decreased numbers of CD8⁺ T cells and diminished frequencies of particular dendritic cell subsets. Antihelmintic treatment of hookworm infection resulted in reversal of some of the hematologic and microbiologic alterations.

Conclusions

Our data provide compelling evidence for MT in a human intestinal helminth infection and its association with perturbations in the T cell and antigen-presenting cell compartments of the immune system. Our data also reveal that at least one dominant counter-regulatory mechanism i.e. increased IL-10 production might potentially protect against systemic immune activation in hookworm infections.     

Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

重金属Cd2+对拟水狼蛛体内金属硫蛋白含量及其生长发育的影响

为了明确重金属Cd²⁺对拟水狼蛛Pirata subpiraticus体内金属硫蛋白(metallothionein, MT)含量及其生长发育的影响, 在室内条件下用5个不同浓度（0, 10, 20, 40和80 μg/g）的Cd²⁺培养的黑腹果蝇Drosophila melanogaster饲喂从5种不同生境下（S1, S2, S3, S4和S5）采集的拟水狼蛛性成熟雌蛛产卵孵化的幼蛛,待幼蛛性成熟后取所得雌蛛成蛛采用原子吸收光谱法测定了Cd²⁺ 诱导下拟水狼蛛体内金属硫蛋白含量及其存活率和生长率。结果表明： 食物中过量的Cd²⁺能够通过食物链进行传递并在拟水狼蛛体内积累, 积累量随黑腹果蝇培养基中Cd²⁺浓度的增加而增加,存在显著的浓度-效应关系。不同浓度的Cd²⁺能够诱导拟水狼蛛体内MT不同的表达,表达量与Cd²⁺浓度显著正相关(P<0.05)。当浓度低于20 μg/g时,污染点（S1, S2, S3和S4）拟水狼蛛体内MT表达量显著高于参照组S5（P<0.05）; 当高于20 μg/g时, 所有样点拟水狼蛛体内MT表达量差异不显著（P＞0.05）。拟水狼蛛存活率和成长率随着Cd²⁺浓度的升高呈下降趋势。据此认为,金属硫蛋白可能是蜘蛛耐受重金属污染的重要机制,与重金属具有一定浓度-效应关系。     

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

Background

This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As⁺³) and cadmium (Cd⁺²)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd⁺²-and As⁺³-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.

Methods

Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As⁺³-and Cd⁺²-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.

Results

It was shown that the As⁺³-and Cd⁺²-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.

Conclusions

Tumor initiating cells isolated from SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.     

Analysis of Metal-Regulated Metallothionein and Heat Shock Gene Expression in HeLa-Derived Cadmium-Resistant Cells

The expression of metallothionein (MT) and heat shock protein gene families was investigated in normal and in HeLa-derived cadmium-resistant cells, named H454. In the absence of amplification of MT genes H454 cells accumulated elevated concentrations of cadmium ions and synthesized higher levels of MT proteins than unselected HeLa cells. Northern blot analyses revealed higher levels of MT mRNAs in the resistant cells than in wild-type cells after Cd²⁺and Zn²⁺exposure. Evaluation of the cytotoxic potential of the different metals confirmed the high resistance to cadmium of the H454 cells. Two proteins of the heat shock family, hsp70 and GRP78, were synthesized in Cd²⁺-exposed H454 cells at levels comparable to the ones present in Cd²⁺-treated normal cells. Northern blot analyses of the mRNA levels corresponding to these proteins revealed elevated expression of both hsp70 and GRP78 mRNAs in H454 cells upon exposure to cadmium ions and no response to zinc induction. These data suggest the existence in the H454 cells of a cadmium-specific pathway of regulation of MT and heat shock genes.     

Alterations in metal toxicity and metal-induced metallothionein gene expression elicited by growth medium calcium concentration

The calcium content of the growth medium has been shown to influence the growth and differentiation of primary epithelial cells in culture. The goal of the present study was to determine if growth medium calcium concentration could influence the susceptibility to metal toxicity and metallothionein gene expression of an immortalized human prostate-derived epithelial cell line (RWPE-1). The RWPE-1 cell line was grown in medium containing either 0.1 or 1.4 mM calcium. Confluent cells were exposed to either Zn⁺² (50, 100, or 150 μM) or Cd⁺² (3, 6, or 12 μM) for 13 days, and cell toxicity and MT gene expression were determined along the time course of exposure. It was demonstrated that the calcium content of the growth medium had a marked influence on Zn⁺² toxicity and a lesser but significant effect on Cd⁺² toxicity to the RWPE-1 cells. Calcium concentration of the growth medium was also shown to alter the accumulation of MT-1/2 protein and MT-1E, MT-1X, and MT-2A mRNAs. It was shown that MT-1/2 protein was markedly increased for metal-exposed cells grown in medium containing 0.1 mM calcium; however, the increased expression did not cause an increase in the resistance of the cells to Zn⁺² or Cd⁺² exposure. These observations show that growth medium calcium concentration can influence metal toxicity and the pattern of expression of the MT mRNAs and protein for RWPE-1 cells. The results suggest that caution should be exercised when comparing toxicological responses between cell lines that may be grown in growth formulations differing in calcium concentration.     

Effects of cadmium and lead on the accumulation of Ca2+ and K+ and on the influx and translocation of K+ in wheat of low and high K+ status

The effects of cadmium and lead on the internal concentrations of Ca²⁺ and K⁺, as well as on the uptake and translocation of K(⁸⁶Rb⁺) were studied in winter wheat (Triticum aestivum L. a. MV-8) grown hydroponically at 2 levels of K⁺ (100 uM and 10 mM). Cd²⁺ and Pb²⁺ were applied in the nutrient solution in the range of 0.3 to 1000 u.M. Growth was more severely inhibited by Cd²⁺ and in the high-K⁺ plants as compared to Pb^z+ and low-K⁺ plants. Ions of both heavy metals accumulated in the roots and shoots, but the K⁺ status influenced their levels. Ca²⁺ accumulation was increased by low concentrations of Cd²⁺ mainly in low-K⁺ shoots, whereas it was less influenced by Pb²⁺. The distribution of Cd²⁺ and Ca²⁺ in the plant and in the growth media indicated high selectivity for Cd²⁺ in the root uptake, while Ca²⁺ was preferred in the radial and/or xylem transport. Cd²⁺ strongly inhibited net K⁺ accumulation in high-K⁺ plants but caused stimulation at low K⁺ supply. In contrast, the metabolis-dependent influx of K⁺(⁸⁶Rb⁺) was inhibited in low-K⁺ plants, while the passive influx in high-K⁺ plants was stimulated. Translocation of K⁺ from the roots to the shoots was inhibited by Cd²⁺ but less influenced in Pb²⁺-treated plants. It is concluded that the effects of heavy metals depend upon the K⁺-status of the plants.     

Structural and functional analysis of the human metallothionein-IA gene: Differential induction by metal ions and glucocorticoids

We describe a region of human DNA containing four metallothionein (hMT) genes. One of these genes, hMT-I_A, was found to encode a functional protein that confers heavy metal resistance to NIH 3T3 cells after transfer on a bovine papilloma virus-derived vector. This gene is expressed in cultured human cell lines, but at a lower basal level than the hMT-II_A gene; it shows a different induction response to heavy metals and glucocorticoids than the hMT-II_A gene. Induction of the human MT family therefore does not represent an equivalent elevation in the level of expression of individual genes, but is the sum of the differential responses of active members. The differential response is due to functional differences of the respective promoter/regulatory regions of the genes as shown by gene-fusion experiments. While the hMT-II_A promoter is responsive to Cd⁺⁺, Zn⁺⁺, and glucocorticoids, the hMT-I_A promoter mediates response only to Cd⁺⁺.     

Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium

Due to its exemplary resistance to ionising radiation, oxidative stress, desiccation and several DNA damaging agents, Deinococcus radiodurans R1 (DR1) is considered as one of the most appropriate candidates for the bioremediation of the nuclear waste sites. However, the high sensitivity of this bacterium to heavy metals, which are usually preponderant at nuclear waste dump sites, precludes its application for bioremediation. This study deals with the expression two metal binding peptides in DR1 as an attractive strategy for developing metal tolerance in this bacterium. A synthetic gene (EC20) encoding a phytochelatin analogue with twenty repeating units of glutamate and cysteine was constructed by overlap extension and expressed in DR1. The cyanobacterial metallothionein (MT) gene, smtA was cloned for intracellular expression in DR1. Both the genes were expressed under the native groESL promoter. DR1 strain carrying the recombinant EC20 demonstrated 2.5-fold higher tolerance to Cd²⁺ and accumulated 1.21-fold greater Cd²⁺ as opposed to the control while the heterologous expression of MT SmtA in DR1 imparted the transformant superior tolerance to Cd²⁺ amassing 2.5-fold greater Cd²⁺ than DR1 expressing EC20.     

Cd2+-induced damage to yeast plasma membrane and its alleviation by Zn2+: studies on Schizosaccharomyces pombe cells and reconstituted plasma membrane vesicles

In Schizosaccharomyces pombe, Cd²⁺ shares the same uphill uptake system with Zn²⁺. Both heavy metals inhibited growth, respiration, H⁺/glucose uptake, and glucose-induced proton extrusion, Cd²⁺ being a 10–15-fold stronger inhibitor. In contrast, both had a similar effect on the plasma membrane H⁺-ATPase, enhancing its affinity for ATP and reducing the rate of ATP splitting. Cd²⁺ caused protracted strong fluidization of the plasma membrane of energized cells, whereas deenergized cells, phosphatidylcholine liposomes, and plasma membrane fragments, either purified or incorporated into the liposomes, exhibited only a short initial fluidization. Zn²⁺, which caused only a marginal membrane fluidization, suppressed the fluidizing action of Cd²⁺. The fluidizing effect of both heavy metals on liposomes was reduced by the presence of plasma membrane fragments in the liposome membrane. At 50 μM, Cd²⁺ brought about loss K⁺ (18 K⁺/1 Cd²⁺) from energized, but not from deenergized cells since Cd²⁺ must first accumulate in the cells before causing a detectable effect. A simple membrane disruption by external Cd²⁺ is, therefore, unlikely to be the main mechanism of cadmium-induced potassium loss in intact cells. Zn²⁺ had virtually no effect below 1 mM concentration, and it again weakened the K⁺-releasing effect of Cd²⁺. Cd²⁺ caused a strong loss of K⁺ also from K⁺-containing liposomes, probably because of a direct interaction with liposome phospholipids. Incorporation of plasma membrane fragments into the liposomes reduced the K⁺ loss sixfold. Received: 13 November 1995 / Accepted: 31 January 1996     

Energy metabolism and ATP turnover time during the cell cycle of Ehrlich ascites tumour cells

The energy production in different parts of the cell cycle due to aerobic and aerobic glycolytic metabolism and ATP turnover time was estimated by measuring the oxygen consumption, lactate-pyruvate and ATP content of Ehrlich ascites tumour cells growing in vivo. Cell fractions of high purity from the various parts of the cell cycle were obtained by means of elutriator centrifuging. The total energy production for one cell cycle was estimated to be 19 × 10^?12 mol ATP, 60% of which was due to the aerobic metabolism. Whereas the total ATP production is unchanged during G1 a fairly exponential increase is found during the S and G2 + M phases. The total cellular ATP content increases from 12 fmol ATP at early G1 to 28 fmol ATP at G2 + M; this increase, however, is discontinuous and is most pronounced during G1 and during late S phase S phase/G2 + M. The ATP turnover time, as defined as the ratio between ATP content and ATP production, was found to increase significantly from 75 sec in early G1 to 120 sec in late G1 but was constantly 100 sec during the early, middle and late S phase as well as G2 + M. These variations indicate maximum energy-requiring processes during early G1 period of the cell cycle and are discussed in relation to K⁺Na⁺ flux and macromolecule synthesis.     

Cultivation of thermophilic methanogen KN-15 on H2-CO2 under pressurized conditions

Cultivation of the thermophilic methanogen KN-15 was carried out under pressurized batch and continuous conditions. In pressurized batch culture, both the turning point at which cell growth changed from exponential to linear and the growth rate during the linear growth phase increased with the rise of total pressure of the gas phase in the fermentor. The cell concentration reached 18.5 g dry cell/l after 10 h of batch cultivation under 3.0 × 10⁵ Pa pressurized conditions. Under pressurized continuous conditions, it was also observed that the cell concentration and cell productivities increased with the rise of total pressure of the gas phase. Cell and methane productivities of 3.0 g dry cell/l/h and 1.28 mol/l/h, respectively, were achieved in 3.0 × 10⁵ Pa pressurized continuous culture. According to the results from the Monod model application, the achievable cell productivities (V_max) and the Monod type saturation constant for cell productivities (K_s^′) were 12.8 g dry cell/l/h and 9.7 × 10⁵ Pa, respectively.