Morphological Observation on Euglena gracilis Grown in Zinc-sufficient Media Containing Cadmium Ions

Cadmium ions at 5 to 100 µg/ml induced morphological alterations in Euglena gracilis grown in zinc-sufficient media. The average cell volume was used as a parameter of morphological abnormality. Many types of abnormal-shaped cells were observed when the cadmium-ion concentration was high enough to increase the average cell volume. Most abnormal-shaped cells were starfish-shaped. Scanning electron microscopic observations clearly showed that the structure of the pellicle of starfish-shaped cells was not different from that of normal dividing cells and that these starfish-shaped cells, which were polynucleated and in the process of cytokinesis, resulted from abnormal cytokinesis, but not from cell fusion.     

Cadmium and zinc biosorption byChlorella homosphaera

Summary Cadmium and zinc biosorption byChlorella homosphaera cells were tested under laboratory conditions, in a range of concentrations from 0.5 to 14.0 mg/l. The results indicated two distinct phases for cadmium biosorption: a rapid phase probably associated with metal adsorption around the cell wall and a slower phase associated with the metal transport into the interior of the cells. For zinc biosorption these phases were not well defined probably due to the metabolic use of this metal by the cells.     

BAK and BAX deletion using zinc‐finger nucleases yields apoptosis‐resistant CHO cells

Anoxic and metabolic stresses in large‐scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro‐apoptotic proteins and over‐expression of anti‐apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro‐apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc‐finger nuclease‐mediated gene disruption. Zinc‐finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale‐down systems under conditions that mimic growth in large‐scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX‐ and BAK‐deleted cells produce two‐ to fivefold more IgG than wild‐type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double‐knockout cultures achieve equal or higher cell densities than unmodified wild‐type cultures and reach viable cell densities relevant for large‐scale industrial protein production. Biotechnol. Bioeng. 2010; 105: 330–340. © 2009 Wiley Periodicals, Inc.     

Influence of Cadmium and Zinc on Growth and Photosynthesis of Bacopa monniera Cultivated in vitro

This study evaluates the impact of cadmium and zinc interaction on the amount of soluble proteins, CO₂ fixati stomatal conductance and intercellular CO₂ contents in regenerants of B. monniera. The regenerants were grown 16 weeks on MS medium containing cadmium and zinc in various concentrations. Cadmium decreased the stom conductance, photosynthetic rate and root growth but increased the protein content. Additional supply of zinc in medium reduced the adverse effects of cadmium on these parameters.     

The soluble carbohydrates of Euglena gracilis—their identity and response to increased external solute concentration

Abstract Mannitol and trehalose were the predominant soluble carbohydrates in Euglena gracilis strain z growing heterotrophically in complete darkness or in light. The ratio of trehalose to mannitol was correlated with the water activity of the medium. That is, extracts of Euglena gracilis adapted to grow in media supplemented with either sodium chloride or glucose, thereby reducing the water activity, yielded molar ratios of trehalose to mannitol 10 times greater than extracts of cultures grown under conditions in common usage.     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

Metal-induced expressing of mammal Metallothionein-1 gene in cyanobacteria to promote cadmium-binding preferences

The metal(zinc)-inducible smtA gene promoter (smt O-P) from cyanobacteria was applied for the expression of mouse MT-1 cDNA in the filamentous cyanobacterium Anabaena sp. PCC 7120 to enhance its metal-binding capability and to change its main binding specificity from zinc to cadmium. Shuttle expression vector pKT-MRE transformed the cyanobacterial cells by triparent conjugal transfer. Positive clones were screened and identified by streptomycin, DNA dot blot, SDS-PAGE and Western blot analysis. Photosynthetic oxygen evolution and metal atom absorption indicated that under the cadmium stress the metal-induced expression of foreign mMT-1 doubled their cadmium resistance and developed cells showing a much higher preference to absorb cadmium other than zinc in medium. The cadmium content in cell extract rose from 11% to 36%, and the cadmium cleared from media by transgenic cells rose from 18% to 62%. There was only a slight enhancement for zinc binding in the wild or transgenic type. Received: 1 March 1999 / Received last revision: 9 July 1999 / Accepted: 1 August 1999     

Zinc-, copper- and cadmium-binding protein in Ehrlich ascites tumour cells.

The properties of Ehrlich ascites tumour cells exposed in vivo to cadmium were investigated as a function of the zinc status of the host animals. Tumour-cell growth was inhibited by cadmium in both zinc-sufficient and zinc-deficient animals. However, cells in zinc-sufficient tumours accumulate much less cadmium than those in deficient tumours. The subcellular distributions of cadmium and zinc do not depend on zinc status. Cadmium and zinc are bound to a low-molecular-weight protein with properties similar to metallothionein. Without exposure to cadmium, a zinc- and copper-binding protein is still present that behaves like a metallothionein. This protein can rapidly bind cadmium added to Ehrlich cells in vitro. It is shown that the zinc- and copper-binding protein contains free thiol groups. Ehrlich cells isolated from cadmium-treated animals are viable and show normal incorporation of uridine into RNA, but the cellular uptake of thymidine and its incorporation into DNA are inhibited.     

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.     

Energy-dispersive X-ray analysis of the extracellular cadmium sulfide crystallites of Klebsiella aerogenes

Klebsiella aerogenes forms electron-dense partieles on the cell surface in response to the presence of cadmium ions in the growth medium. These particles ranged from 20 to 200 nm in size, and quantitative energy dispersive X-ray analysis established that they comprise cadmium and sulfur in a 1:1 ratio. This observation leads to the conclusion that the particles are cadmium sulfide crystallites. A combination of atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, and acid-labile sulfide analysis revealed that the total intracellular and bound extracellular cadmium:sulfur ratio is also 1:1, which suggests that the bulk of the cadmium is fixed as extracellular cadmium sulfide. The tolerance of K. acrogenes to cadmium ions and the formation of the cadmium sulfide crystallites were dependent on the buffer composition of the growth medium. The addition of cadmium ions to phosphate-buffered media resulted in cadmium phosphate precipitates that remove the potentially toxic cadmium ions from the growth medium. Electrondense particles formed on the surfaces of bacteria grown under these conditions were a combination of cadmium sulfide and cadmium phosphates. The specific bacterial growth rate in the exponential phase of batch cultures was not affected by up to 2mM cadmium in Tricine-buffered medium, but formation of cadmium sulfide crystallites was maximal during the stationary phase of batch culture. Cadmium tolerance was much lower (10 to 150 M) in growth media buffered with Tris, Bistris propane, Bes, Tes, or Hepes. These results illustrate the importance of considering medium composition when comparing levels of bacterial cadmium tolerance.Abbreviations EDXA Energy dispersive X-ray analysis - AAS Atomic absorption spectroscopy - TEM Transmission electron microscopy - SEM Scanning electron microscopy - ICP-MS Inductively coupled plasma mass spectrometry - ALSA Acid-labile sulfide analysis     

Influence of zinc on lotic plants

SUMMARY. The toxicity of zinc to Hormidium rivulare Kütz. in laboratory culture media is decreased by rises in the levels of magnesium, calcium and phosphate, and increased by rises in pH and cadmium. The effects of all these are sufficiently marked that they may be expected to have considerable importance in the field. In contrast, assays with sodium, chloride and sulphate showed no detectable influence of these ions on zinc toxicity.
When applied at higher concentrations, calcium was always more effective than magnesium, but the reverse was sometimes true at lower concentrations. Both magnesium and phosphate were more effective in reducing zinc toxicity with zinc-tolerant populations than with zinc-sensitive ones. Cadmium was highly toxic, either alone or in combination with zinc; however, calcium had a proportionately greater effect in reducing cadmium toxicity than zinc toxicity. The toxic effects of zinc and cadmium were synergistic, and it seems probable that the presence of cadmium at levels of 0.01 mgl⁻¹ and above will usually lead to a significant increase in the toxicity of any zinc also present. Cadmium (in the absence of zinc) was 34 times more toxic than zinc to a zinc + cadmium sensitive population, and 15.5 times more toxic to a zinc + cadmium tolerant population. Because of the synergistic response, cadmium had an even greater effect in the presence of zinc.     

Toxicity and mutual interactions of cadmium and zinc ions in normal and carcinogen-transformed mouse cells

The toxicity of chloride salts of physiological (zinc, manganese, nickel) and non physiological (cadmium) bivalent metal ions was studied in normal or carcinogen-transformed mouse embryo fibroblast cells. The dose response curves for toxicity to both types of cells exhibited similar shapes. The transformed cells, however, were about twice as sensitive to zinc toxicity as normal cells. When normal and transformed cells were grown together and incubated for several hours with an appropriate concentration of zinc, the malignant cells were selectively killed. Cadmium was much more toxic than the three other metal ions in both types of cells. Its toxic effect was reversed by simultaneous addition of zinc at nontoxic concentrations.Abbrevications CFA colony forming ability - MCA 3-methylcholanthrene     

Isolation, culture and characterization of a new strain of Euglena gracilis

A new strain of Euglena gracilis Klebs has been isolated from a highly polluted river; it was named MAT. Strain growth in different culture media was evaluated under heterotrophic and autotrophic conditions. Total lipid, sugar, protein and chlorophyll a production were studied. Results obtained for MAT were compared with data obtained for a UTEX Culture Collection strain. Likewise, cells from both strains were bleached using streptomycin, and grown in the same media used for green samples. Both MAT and UTEX showed clear differences in their biochemical composition and growth rate depending on the media used. They also exhibited different growth patterns. E. gracilis medium proved to be the best culture environment for both strains either in autotrophic or heterotrophic conditions. Results show that basal contents of lipids, sugars, proteins and chlorophyll a vary depending on the strain, and thus values obtained for one strain do not apply to another. Moreover, strain origin may have an influence on the mechanisms of adaptation or defense developed by each sample.     

Uptake of six heavy metals by oat as influenced by soil type and additions of cadmium,lead, zinc and copper

Summary The influence of heavy metal additions on availability and uptake of cadmium, lead, zinc, copper, manganese and iron by oat was studied. The experiments were carried out as pot experiments using sandy loam, sandy soil and organic soil. Selective extractants were used to remove metals held in different soil fractions.Lead and copper were preferently bound by organics and oxides, zinc by oxides and inorganics, and cadmium by inorganics and organics.Addition of cadmium to the soils resulted in higher cadmium concentrations in all plant parts but lower concentrations of lead, zinc, copper, manganese and iron, and the accumulation indexes of these metals were also lower when cadmium was added to the soil.Addition of cadmium plus lead, zinc and copper resulted in higher cadmium concentrations in leaves and straw of plants grown in sandy loam and sandy soil, but lower concentrations when plants were grown in organic soil as compared with the results when cadmium was added separately. The transfer of cadmium, lead, zinc and copper from soil to plant was greatest from sandy soil, and zinc and cadmium were more mobile in the plant than were lead and copper.Cadmium concentrations in leaves correlated significantly with CaCl₂ and CH₃COOH extractions in sandy loam and sandy soil and with CH₃COOH extractions in organic soil.Generally, the total metal uptake was lowest from organic soil.     

Implication of distinct proteins in cadmium uptake and transport by intestinal cells HT-29

The mechanisms of intestinal absorption have not been clearly elucidated for cadmium, a toxic metal. In this work, we show the implication of distinct proteins in cadmium transport, and the transport step where these proteins are involved. We first validated the HT-29 model by evaluating nontoxic doses of cadmium (ranging from 1 to 20 μmol/L), and by quantifying metal uptake and transepithelial transport. The time-course of 1 μmol/L cadmium uptake at pH 7.5 showed three steps: a rapid one during the first 4 min, probably due to cadmium binding to the membrane; a slower one, characterized by K _m of 1.65±0.54 μmol/L and V _max of 3.9±0.3 pmol/min per mg protein; and a third, corresponding to slow accumulation that was not equilibrated even after 48 h of cadmium exposure. Intracellular metallothionein content following 1 or 5 μmol/L cadmium exposure showed a significant increase after 6 h of exposure, and was not equilibrated even after 72 h, allowing cadmium accumulation. After 24 h of exposure, metallothionein content was 5-fold, 14-fold, 26-fold, and 50-fold, respectively, for cells grown in the presence of 1, 5, 10, and 20 μmol/L cadmium, compared to control cells. The second step of uptake, characterized by carrier-mediated transport, was markedly increased at pH 5.5, compared to pH 7.5, and strongly inhibited by the metabolic inhibitor dinitrophenol. Moreover Nramp2 transporter cDNA was present in HT-29 cells. These data suggest the involvement of a proton-coupled transporter, which may be the divalent cation transporter Nramp2 (natural resistance-associated macrophage protein 2). Cadmium uptake was also inhibited by copper, zinc, and para-chloromercuribenzenesulfonate (pCMBS), but not by verapamil or ouabain. Taken together, our results indicate that cadmium could enter HT-29 cell by Nramp2 proton-coupled active transport and by diffusion, and accumulates in the cell as long as it binds to metallothionein. Cadmium toxicity could depend partly on the activity of Nramp2, and partly on metallothionein content. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effects of cadmium exposure on the cytology and function of primary cultures from rainbow trout

Cultured epidermal cells from explants of skin of rainbow trout were used to study the cytological and functional changes following sublethal exposure to cadmium stress. The aim was to develop diagnostic markers for ecotoxicology. Cultures were exposed to the pollutant for 48 h. Cell structural and cytological changes were established by light and electron microscopy. Metabolic alterations were detected by immunohistochemistry. The relation between the initiation of cellular alterations and cadmium concentrations was compared in cultures exposed in commercially-available serum-free and serum-containing medium. The expression of stress proteins (metallothionein and heat shock protein) was also studied. Rainbow trout epithelial cells exposed to cadmium showed typical morphological changes indicative of cell death by apoptosis. Sublethal exposure also resulted in cellular metabolic disturbances with increased deposits of glycogen. Increased melanization was also observed. These changes appeared at lower concentrations of cadmium when cells were exposed in serum-free media than in serum-containing media. Cadmium induced the expression of heat shock proteins but not of metallothioneins. The results broadly confirm in vivo findings for cadmium toxicity and suggest that this in vitro technique may have applications in aquatic toxicology. © 1998 John Wiley & Sons, Ltd.     

Imbalance in zinc homeostasis enhances lung Tissue Loss following cigarette smoke exposure

Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease. Cadmium is a leading toxic component of cigarette smoke. Cadmium and zinc are highly related metals. Whereas, zinc is an essential metal required for normal health, cadmium is highly toxic. Zrt- and Irt-like protein 8 (ZIP8) is an avid transporter of both zinc and cadmium into cells and is abundantly expressed in the lung of smokers compared to nonsmokers. Our objective was to determine whether disturbed zinc homeostasis through diet or the zinc transporter ZIP8 increase susceptibility to lung damage following prolonged cigarette smoke exposure.MethodsCigarette smoke exposure was evaluated in the lungs of mice subject to insufficient and sufficient zinc intakes, in transgenic ZIP8 overexpressing mice, and a novel myeloid-specific ZIP8 knockout strain.ResultsModerate depletion of zinc intakes in adult mice resulted in a significant increase in lung cadmium burden and permanent lung tissue loss following prolonged smoke exposure. Overexpression of ZIP8 resulted in increased lung cadmium burden and more extensive lung damage, whereas cigarette smoke exposure in ZIP8 knockout mice resulted in increased lung tissue loss without a change in lung cadmium content, but a decrease in zinc.ConclusionsOverall, findings were consistent with past human studies. Imbalance in Zn homeostasis increases susceptibility to permanent lung injury following prolonged cigarette smoke exposure. Based on animal studies, both increased and decreased ZIP8 expression enhanced irreversible tissue damage in response to prolonged tobacco smoke exposure. We believe these findings represent an important advancement in our understanding of how imbalance in zinc homeostasis and cadmium exposure via tobacco smoke may increase susceptibility to smoking-induced lung disease.     

EFFECTS OF CADMIUM ON GROWTH AND SUPEROXIDE DISMUTASE ACTIVITY OF THE MARINE MICROALGA TETRASELMIS GRACILIS (PRASINOPHYCEAE)1

Marine planktonic algae are frequently exposed to metallic contaminants. Because heavy metals can be assimilated and accumulated by algal cells, they can then be transferred to higher trophic levels of food chains. We studied the effects of cadmium on protein production and the growth of the marine prasinophyte Tetraselmis gracilis (Kylin) Butcher. By means of toxicological assays, we estimated the LC₅₀ of cadmium as 3.2 ppm and 1.8 ppm after 48 h and 96 h of exposure to this heavy metal, respectively. The growth curves and survival percentages of cell cultures in the presence of cadmium were determined, and a proportional reduction of both parameters with increasing metal concentrations was found. When chronically exposed to sublethal concentrations of cadmium, T. gracilis contained high levels of superoxide dismutase (SOD) activity, one of the main enzymes of the cell's antioxidant defense mechanism. Under these growth conditions, total SOD activity in crude extracts was increased by 41% (at 1.5 ppm) and 107% (at 3.0 ppm). Assays of SOD activity in nondenaturing polyacrylamide gels also showed a similar induction by cadmium. These results show that cadmium has potentially toxic properties since it significantly inhibited the growth of T. gracilis at low concentrations and promoted the induction of SOD activity, suggestive of an oxidative stress state. Besides being the first report of SOD in T. gracilis, this work describes experimental evidence of SOD induction by cadmium in this species.     

Effects of Iron-, Manganese-, or Magnesium-Deficiency on the Growth and Morphology of Euglena gracilis1

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.     

Cadmium-induced abnormality in strains of Euglena gracilis: morphological alteration and its prevention by zinc and cyanocobalamin

Euglena gracilis is susceptible to cadmium (Cd) at high concentrations. There are no comparative data on cytotoxicity or abnormality of CdCl₂ to E. gracilis Z and its achlorophyllous mutant SMZ. The present study examined the cytotoxicity of CdCl₂ under continual exposure at levels ranging from sub-ppm to ppm, and assessed the effects of zinc (Zn) or cyanocobalamin (VB₁₂) supplementation on the suppression of Cd-induced abnormal cell proliferation and hypertrophy. With Zn levels restricted to 1 ppm [as Zn⁺⁺], cell growth of both E. gracilis strains was reduced in proportion to Cd concentration. More abnormal cells (hypertrophied, V-shape and starfish-shape) were observed in both strains at sub-ppm levels of Cd. ZnSO₄ supplementation from 2 to 63 ppm significantly suppressed the incidence of Cd-induced abnormality. However, a significant increase in abnormal cells was observed following Zn supplementation at levels of 125 and 250 ppm, which produced remarkable differences in cell morphology. The incidence of abnormal cells varied with supplemented VB₁₂ levels ranging from 4 to 250 ppb in both E. gracilis strains.