Selection <Emphasis Type="Italic">in vitro</Emphasis> and accumulation of phytochelatins in cadmium tolerant cell line of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Cucumber (Cucumis sativus L.) cells from suspension culture were selected for their ability to grow and divide rapidly in toxic concentration of cadmium. As a result of selection a cell suspension tolerant to 100 M cadmium chloride (CdCl₂) was initiated. The selected tolerant line exhibited stable and repeatable increase in fresh and dry weight of cells in the presence of cadmium. The accumulated level of phytochelatins in cadmium sensitive (unselected) and tolerant cell line was measured by high performance liquid chromatography (HPLC) after 3, 24 h and 5 days of cadmium treatment. It was shown that in both cell lines Cd induced accumulation of phytochelatins and simultaneous glutathione depletion occurred. No distinct changes were found after 3 and 24 h of cadmium treatment whereas after 5 days of exposure to the metal, the level of phytochelatins was two times higher in the sensitive cell line as compared to the tolerant one. The accumulation of phytochelatins was correlated with cadmium concentration that increased in both cell lines during the course of cell exposure to metal. However, the level of cadmium was always lower in the tolerant cell line. The results showed no direct correlation between the tolerance of cucumber cells to Cd and the accumulated level of phytochelatins. Other mechanisms responsible for the increased tolerance of cucumber cells exposed to Cd are discussed.     

Validation of an in vitro cytotoxicity test for four heavy metals using cell lines derived from a green sea turtle (<Emphasis Type="Italic">Chelonia mydas</Emphasis>)

Ten cell lines established from juvenile green sea turtles were tested and evaluated for their cytotoxic responses to four heavy metals: cadmium (Cd), chromium (Cr), zinc (Zn), and copper (Cu). Following a 24-h exposure to these metal salts at selected concentrations, test cells were comparatively characterized by morphology, viability, and proliferation. Experimental results indicated that all these metal salts were cytotoxic to these turtle cell lines at varied concentrations. Calculated 10% and 50% inhibitory concentration (IC₁₀ and IC₅₀) values revealed that the cytotoxicities of Cd and Cr were significantly more potent than the other two metal salts (p < 0.01). Comparative analysis of IC₁₀ values in these ten cell lines showed that turtle lung cells (GT-LG) are the most sensitive cell line to Cd, Cr, Zn, and Cu. Among these turtle cell lines, turtle liver cells (GT-LV) are more tolerant than other cells to Cd, Cr, and Zn, while GT-EYE cells are more tolerant to Cu, as determined by IC₅₀ values. Overall, GT-LG represents the most sensitive cells to heavy metal contamination and may be used for initial environmental monitoring, while the highly tolerant nature of GT-LV and GT-EYE cells to the tested heavy metals suggest their potential use as an emergency last-resort indicator of potential metal-related adverse effect on human health.     

Effect of Cadmium Stress on the Growth,Physiology, Stress Markers,Antioxidants and Stomatal Behaviour of Two Genotypes of Chickpea ( <i>Cicer arietinum </i>L.)

The current work was performed to know the impact of cadmium (Cd) toxicity on two different genotypes of chickpea (Cicer arietinum L.) namely Pusa-BG1053 and Pusa-BG372. Cadmium was applied in the form of cadmium chloride (CdCl₂), in varying levels, 0, 25, 50, 75, and 100 mg Cd kg^-1 soil. Plant growth as well as physiological attributes were decreased with increasing concentration of Cd. Both genotypes showed the maximum and significant reduction at the maximum dose of Cd (100 mg Cd kg^-1 soil). Results of this study proved that the genotype Pusa-BG1053 was more tolerant and showed a lower decline in growth, photosynthetic and biochemical attributes than Pusa-BG372. This later genotype showed the maximum reduction and was sensitive to Cd stress. A better activity of antioxidants protected Pusa-BG1053 from Cd toxicity; on the other hand, the activity of antioxidants was much lower in Pusa-BG372. Scanning electron microscopic studies showed differences in both genotypes. In Pusa-BG1053, stomatal quantity was higher and stomata were slightly close to the characteristic guard cells. In Pusa-BG372 stomata were lower, slightly open and with highly affected guard cells. Root cell mortality due to the harsh effects of Cd appeared to be more evident in Pusa-BG372 than Pusa-BG1053, which was visible under a confocal microscope. As a result of this study, Pusa-BG1053 was a more tolerant genotype, and exhibited a minimum reduction in terms of all studied parameters than Pusa-BG372, which was a sensitive genotype to Cd toxicity.

     

Screening Possible Mechanisms Mediating Cadmium Resistance in Rhizobium leguminosarum bv. viciae Isolated from Contaminated Portuguese Soils

Environment heavy-metal contamination is now widespread. Soils may become contaminated from a variety of anthropogenic sources, such as smelters, mining, industry, and application of metal-containing pesticides and fertilizers. Soil microorganisms are very sensitive to moderate heavy-metal concentrations. Therefore, the present work was designed to screen possible mechanisms involved in Rhizobium's Cd resistance; with this purpose, we determined the tolerance levels of several isolates originated from sites with different heavy-metal contamination. Whole-cell-soluble proteins and plasmid profiles were analyzed. We also determined Cd cell concentrations and lipopolysaccharide (LPS) amounts. Results showed different tolerances among Rhizobium isolates; according to their maximum resistance level, isolates were divided in four groups: sensitive (0–125 μM CdCl₂), moderately tolerant (125–210 μM CdCl₂), tolerant (250–500 μM CdCl₂), and extremely tolerant (≥750 μM CdCl₂). Intracellular Cd concentrations were lower when compared to wall-bound Cd. Unexpectedly, extremely tolerant isolates accumulated higher levels of metal, suggesting the presence of intracellular agents that prevent metal interfering with important metabolic pathways. The electrophoretic patterns of whole-cell-soluble proteins evidenced cadmium as an inducer of protein metabolism alterations, which were more evident in some polypeptides. Plasmid profiles also showed differences; most tolerant isolates presented two plasmids with molecular weights of 485 and 415 kb, indicating that extrachromosomal DNA may be involved in cadmium resistance. LPS showed to be a common mechanism of resistance. However, the degree of tolerance conferred by LPS is not enough to support tolerance to the higher levels of stress imposed. Presence of other resistance mechanisms is currently being investigated.     

Involvement of glutathione and enzymatic defense system against cadmium toxicity in Bradyrhizobium sp. strains (peanut symbionts)

In this study, the effects of cadmium (Cd) on cell morphology and antioxidant enzyme activities as well as the distribution of the metal in different cell compartments in Bradyrhizobium sp. strains were investigated. These strains were previously classified as sensitive (Bradyrhizobium sp. SEMIA 6144) and tolerant (Bradyrhizobium sp. NLH25) to Cd. Transmission electron micrographs showed large electron-translucent inclusions in the sensitive strain and electron-dense bodies in the tolerant strain, when exposed to Cd. Analysis of Cd distribution revealed that it was mainly bounded to cell wall in both strains. Antioxidant enzyme activities were significantly different in each strain. Only the tolerant strain was able to maintain a glutathione/oxidized glutathione (GSH/GSSG) ratio by an increase of GSH reductase (GR) and GSH peroxidase (GPX) enzyme activities. GSH S-transferase (GST) and catalase (CAT) activities were drastically inhibited in both strains while superoxide dismutase (SOD) showed a significant decrease only in the sensitive strain. In conclusion, our findings suggest that GSH content and its related enzymes are involved in the Bradyrhizobium sp. tolerance to Cd contributing to the cellular redox balance.     

Poly(gamma-glutamylcysteinyl)glycine Synthesis in Datura innoxia and Binding with Cadmium : Role in Cadmium Tolerance

The effects of Cd on poly(γ-glutamylcysteinyl)glycine [(γEC)_nG] biosynthesis and formation of (γEC)_nG:Cd complexes were measured in two cell lines of Datura innoxia with differing Cd tolerance. In addition, RNA synthesis, protein synthesis, and GSH concentrations were measured during a 48 hour exposure to Cd. Exposure to 250 micromolar CdCl₂ was toxic to the sensitive line, whereas the tolerant line survived and grew in its presence. Cd-sensitive cells synthesized the same amount of (γEC)_nG as tolerant cells during an initial 24 hour exposure to 250 micromolar CdCl₂. However, rates of (γEC)_nG:Cd complex formation differed between the two cell lines with the sensitive cells forming complexes later than tolerant cells. In addition, the complexes formed by sensitive cells were of lower molecular weight than those of tolerant cells and did not bind all of the cellular Cd. Pulse-labeling of cells with l-[³⁵S]cysteine resulted in equivalent rates of incorporation into the (γEC)_nG of both cell lines during the initial 24 hours after Cd. Rates of protein and RNA synthesis were similar for both cell lines during the initial 8 hours after Cd but thereafter declined rapidly in sensitive cells. This was reflected by a decline in viability of sensitive cells. The GSH content of both cell lines declined rapidly upon exposure to Cd but was higher in sensitive cells throughout the experiment. These results show that the biosynthetic pathway for (γEC)_nG synthesis in sensitive cells is operational and that relative overproduction of (γEC)_nG is not the mechanism of Cd-tolerance in a Cd-tolerant cell line of D. innoxia. Rapid formation of (γEC)_nG:Cd complexes that bind all of the cellular Cd within 24 hours appears to correlate with tolerance in these cells.     

Micronucleus frequency and lipid peroxidation in <Emphasis Type="Italic">Allium sativum</Emphasis> root tip cells treated with gibberellic acid and cadmium

Gibberellic acid (GA₃) is a very potent hormone whose natural occurrence in plants controls their development. Cadmium is a particularly dangerous pollutant due to its high toxicity and great solubility in water. In this study, the effect of GA₃ on Allium sativum root tip cells was investigated in the presence of cadmium. A. sativum root tip cells were exposed to CdNO₃ (50, 100, 200 μM), GA₃ (10-3 M), both CdNO₃ and GA₃. Cytogenetic analyses were performed as micronucleus (MN) assay and mitotic index (MI). Lipid peroxidation analysis was also performed in A. sativum root tip cells for determination of membrane damage. MN exhibited a dose-dependent increase in Cd treatments in A. sativum. GA₃ significantly reduced the effect of Cd on the MN frequency. MN was observed in GA₃ and GA₃ + 50 μm Cd treatments at very low frequency. MI slightly decreased in GA₃ and GA₃ + Cd treatments. MI decreased more in high concentrations of Cd than combined GA₃ + Cd treatments. The high concentrations of cadmium induce MN, lipid peroxidation and lead to genotoxicity in A. sativum. Current work reveals that the effect of Cd on genotoxicity can be partially restored with GA₃ application.     

UV-B and cadmium induced changes in pigments,photosynthetic electron transport activity,antioxidant levels and antioxidative enzyme activities of <Emphasis Type="Italic">Riccia</Emphasis> sp.

UV-B and cadmium, alone and together, induced changes in photosynthetic pigment levels, photosynthetic electron transport activity, enzymatic and non-enzymatic (low molecular weight) antioxidants, level of hydrogen peroxide and lipid peroxidation in Riccia sp. were evaluated. Chlorophyll content was found to decrease with the rising concentration of cadmium and UV-B exposure alone and its level further declined when both the stresses were applied together. In contrast to this, carotenoids exhibited varied response, as it showed enhancement with UV-B (15, 30 and 45 min exposure) and low concentration of Cd (1 and 10 μM) treatment alone and in combination. Both the stresses caused strong inhibitory effect on PS II activity (H₂O → p-BQ), while PS I activity (DCPIP/ASC → MV) appeared to be less sensitive. Total peroxide content increased with simultaneous increase in lipid peroxidation. The level of non-enzymatic antioxidant ascorbate and enzymatic antioxidants superoxide dismutase and peroxidase activity were found to increase with simultaneous decrease in catalase activity following UV-B and Cd treatments. These results indicate that 45 min of UV-B exposure and 10, 100 and 1000 μM cadmium alone and together, strongly arrested electron flow through PS II which caused accelerated generation of reactive oxygen species (H₂O₂) and excess accumulation of H₂O₂ due to significant inhibition of catalase activity, led to the oxidative damage in Riccia sp.     

Cadmium nephrotoxicity in human proximal tubule cell cultures

Summary Human proximal tubule kidney cells grown in a serum-free tissue culture medium were exposed to concentrations of CdCl₂ in a range of 0.5 to 10μg/ml. Cells were observed from 1 to 20 d upon initiation of cadmium in the culture fluid. Both confluent and subconfluent populations of cells were treated and evaluated for cytotoxicity. Both populations exhibited a concentration-dependent toxicity to ionic cadmium. For cells treated with 2.0 to 10 μg/ml Cd, the decreases in cell numbers were largely irreversible. However, cells treated with Cd in a range of 0.5 to 1.0 μg/ml exhibited a partial recovery of cell number and control morphology. In this range, recovery was more efficient in the subconfluent cultures. Fine structural alterations in Cd-treated tubule cells included condensation of nuclear chromatin, loss of microvilli structure, disorganization of lateral membrane interdigitation, as well as decreased uptake of aminoglycoside antibiotics as evidenced by decreased numbers of myeloid bodies in these cells. The results of this study imply that use of a human proximal tubule culture system has potential in discerning structural and functional effects of cadmium as well as other nephrotoxic metals and compounds on the human kidney. This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by the Johns Hopkins Center for Alternatives to Animal Testing.     

An electrophysiological freeze fracture assessment of cadmium nephrotoxicity in vitro

Summary Human proximal tubule cell cultures exposed to doses of cadmium chloride (CdCl₂) between 0.05 μg/ml and 0.5 μg/ml exhibited alterations in cell membrane structure and transport function. At these Cd concentrations, cell numbers were not significantly altered from control values in either nonreplicating confluent, or actively replicating subconfluent cultures. Transmission electron microscopy revealed few alterations in cultures treated with 0.05 μg/ml Cd. Tight junctions were intact; organelles and myeloid body formation appeared normal. Freeze fracture analysis confirmed the integrity of the tight junctions as well as increased numbers of vesicles or pits along the lateral cell membrane, indicating increased endocytotic activity. Cells exposed to 0.1 μg/ml Cd were characterized by decreased numbers of microvilli and inhibited myeloid body formation. Cd doses of 0.5 μg/ml elicited nuclear chromatin condensation, fragmented sealing strands in 5 to 10% of the tight junction profiles, sparse microvilli, and inhibited myeloid body formation. Electrophysiologic assessments of transport function by Ussing chamber analysis revealed decreases in transepithelial potentials for all three concentrations, with significant differences at Cd concentrations of 0.5 to 0.1 μg/ml. Cells treated with 0.5 μg/ml Cd also exhibited slight decreases in electrical resistance, consistent with the minimal fragmentation of sealing strands observed in freeze fracture replicas. Resistance in cultures treated with 0.1 or 0.05 μg/ml Cd remained within control values and indicated that drops in potential difference and short circuit current in these cells reflected true alterations in ion transport. This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association field at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by the Johns Hopkins Center for Alternatives to Animal Testing. The Balzers Freeze Fracture Unit utilized in these studies was provided by equipment grant S10 RR02329 from the National Institutes of Health, Bethesda, MD.     

Subcellular localization of cadmium in the root cells of<Emphasis Type="Italic">Allium cepa</Emphasis> by electron energy loss spectroscopy and cytochemistry

The ultrastructural investigation of the root cells ofAllium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver grains were abundantly localized in the vesicles, which were distributed in the cytoplasm along the cell wall.     

Cadmium‐induced cell death in BY‐2 cell culture starts with vacuolization of cytoplasm and terminates with necrosis

Cadmium is a potent inducer of programmed cell death (PCD) in plants but the morphological changes in cells exposed to cadmium are poorly characterized. Using light and transmission electron microscopy (TEM) we have investigated the changes in ultrastructure of tobacco BY‐2 cells treated with 50 µM CdSO₄. The cadmium‐induced alterations in cell morphology occurred gradually over a period of 3–4 days and the first stages of the response resembled vacuolar type of cell death. The initial formation of numerous small cytoplasmic vacuoles and dilation of endoplasmic reticulum was followed first by fusion of smaller vacuoles with each other and with big vacuoles, and then by the appearance of autophagic vacuoles containing autophagic bodies. The final stages of cell death were accompanied by necrotic features including loss of plasmalemma integrity, shrinkage of the protoplast and unprocessed cellular components. In addition, we observed a gradual degradation of nuclear material. Our results demonstrate that cadmium‐induced plant cell death is a slow process featuring elements of vacuolar cell death and terminating with necrosis.     

Changes in phytochelatins and their biosynthetic intermediates in red spruce (<Emphasis Type="Italic">Picea rubens </Emphasis>Sarg.) cell suspension cultures under cadmium and zinc stress

Cell suspension cultures of red spruce (Picea rubens Sarg.) were selected to study the effects of cadmium (Cd) and zinc (Zn) on phytochelatins (PCs) and related metabolites after 24 h exposure. The PC₂ and its precursor, γ-glutamylcysteine (γ-EC) increased two to fourfold with Cd concentrations ranging from 12.5 to 200 μM as compared to the control. However, Zn-treated cells showed a less than twofold increase in γ-EC and PC₂ levels as compared to the control even at the highest concentration of 800 μM. In addition, unidentified higher chain PCs were also found in both the Cd and Zn treated cells and they increased significantly with increasing concentrations of Cd and Zn. The cellular ratio of PC₂ : Cd or Zn content clearly indicated that Cd (with ratios ranging from 0.131 to 0.546) is a more effective inducer of PC₂ synthesis/accumulation than Zn (with ratios ranging from 0.032 to 0.102) in red spruce cells. A marginal decrease in glutathione (GSH) was observed in both Cd and Zn treated cells. However, the GSH precursor, cysteine, declined twofold with all Cd concentrations while the decrease with Zn was 1.5–2-fold only at the higher treatment concentrations of Zn as compared to control. In addition, changes in other free amino acids, polyamines, and inorganic ions were also studied. These results suggest that PCs and their biosynthetic intermediates play a significant role in red spruce cells protecting against Cd and Zn toxicity.     

Accumulation and translocation of Cd metal and the Cd-induced production of glutathione and phytochelatins in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

Translocation of cadmium (Cd) in the tissues of Vicia faba, the water content in biomass, the biomass production, and the glutathione and phytochelatin tissue concentrations were studied and correlated with the plant sensitivity and/or tolerance to Cd. The total concentrations of Cd were determined by inductively coupled plasma/mass spectrometry (ICP-MS), the concentrations of glutathione (GSH) and phytochelatins 2 and 3 (PC2 and PC3) were determined by on-line high performance liquid chromatography/electrospray-ionization tandem mass spectrometry (HPLC–ESI–MS–MS) in the roots and leaves of the sensitive and the tolerant cultivars of V. faba grown in Cd containing nutrient solutions (NS, 0–100 μmol l⁻¹ Cd²⁺). Both the cultivars of V. faba accumulate a major portion of Cd in the roots and only a minor part of ca. 4% in the leaves. The differences between the cultivars concerning Cd accumulation in leaves were apparent from higher Cd concentrations in NS and the Cd amount in the sensitive cultivar was approximately twice as high. In the roots, the differences between the cultivars in the Cd accumulation were only statistically significant with the highest Cd concentrations in NS, with the tolerant cultivar accumulating about 16% more of Cd compared to the sensitive one. The biomass production of the sensitive cultivar decreased approximately twice as fast with increasing Cd concentration in NS. The biomass water content decreased with increasing Cd concentration in NS in both the cultivars. In general, the GSH concentration did not linearly correlate with Cd accumulation, except for the roots of the sensitive cultivar where it was independent, and was higher in the sensitive cultivar than in the tolerant one in both the leaves and roots. The GSH concentration in leaves was approximately one order of magnitude higher than that in the roots for both the cultivars. The relationships between the PC and Cd concentrations in tissues were found nonlinear. At lower Cd accumulation levels, the PC concentrations followed an increase in the Cd accumulation in both the roots and leaves, whereas at higher Cd accumulations the relations differed between roots and leaves. In the roots, the PC concentrations decreased with increasing Cd accumulation, whereas the PC concentration in the leaves followed the decrease in the Cd accumulation.     

Cadmium accumulation and interactions with zinc, copper, and manganese, analysed by ICP-MS in a long-term Caco-2 TC7 cell model

The influence of long-term exposure to cadmium (Cd) on essential minerals was investigated using a Caco-2 TC7 cells and a multi-analytical tool: microwave digestion and inductively coupled plasma mass spectrometry. Intracellular levels, effects on cadmium accumulation, distribution, and reference concentration ranges of the following elements were determined: Na, Mg, Ca, Cr, Fe, Mn, Co, Ni, Cu, Zn, Mo, and Cd. Results showed that Caco-2 TC7 cells incubated long-term with cadmium concentrations ranging from 0 to 10 μmol Cd/l for 5 weeks exhibited a significant increase in cadmium accumulation. Furthermore, this accumulation was more marked in cells exposed long-term to cadmium compared with controls, and that this exposure resulted in a significant accumulation of copper and zinc but not of the other elements measured. Interactions of Cd with three elements: zinc, copper, and manganese were particularly studied. Exposed to 30 μmol/l of the element, manganese showed the highest inhibition and copper the lowest on cadmium intracellular accumulation but Zn, Cu, and Mn behave differently in terms of their mutual competition with Cd. Indeed, increasing cadmium in the culture medium resulted in a gradual and significant increase in the accumulation of zinc. There was a significant decrease in manganese from 5 μmol Cd/l exposure, and no variation was observed with copper.     

Subcellular localization of Cd in the root cells of<Emphasis Type="Italic">Allium sativum</Emphasis> by electron energy loss spectroscopy

The ultrastructural investigation of the root cells ofAllium sativum L. exposed to three different concentrations of Cd (100 (AM, 1 μM and 10 mM) for 9 days was carried out. The results showed that Cd induced several significant ultrastructural changes — high vacuolization in cytoplasm, deposition of electron-dense material in vacuoles and nucleoli and increment of disintegrated organelles. Data from electron energy loss spectroscopy (EELS) revealed that Cd was localized in the electron-dense precipitates in the root cells treated with 10 mM Cd. High amounts of Cd were mainly accumulated in the vacuoles and nucleoli of cortical cells in differentiating and mature root tissues. The mechanisms of detoxification and tolerance of Cd are briefly explained.     

Cd-induced system of defence in the garlic root meristematic cells

Studies on cadmium effects in the root meristematic cells of Allium sativum L. were carried out using electron microscopy in order to explain the possible mechanisms of garlic seedlings’ tolerance to Cd stress. Seedlings were treated with 0.01, 0.10 and 1.00 mM CdCl₂ solutions for 0.5, 1, 2, 4, 8, 10, 12, 24 and 48 h, respectively. The results indicated that cell walls, plasma membrane and main organelles actively participated in Cd detoxification and tolerance at low Cd concentrations. Once excessive Cd ions entered the cytosol, a defence mechanism becomes activated, protecting the cells against cadmium toxicity. However, under high Cd content in cells, the cell structure was damaged, even leading to cells death.     

Mechanism of cadmium-induced cytotoxicity in rat hepatocytes

Exposure of rat hepatocytes to cadmium below 50 μM for a short period (10 min) resulted in cellular acidification. Conversely, exposure to Cd more than 50 μM for a long period (60 min) caused cellular alkalinization accompanied by membrane damage as reflected by decrease in cellular K content and loss of intracellular lactic dehydrogenase. In hepatocytes exposed to 5 μM Cd, a concentration sufficient to induce acidification without cytotoxicity, the metal was preferentially associated with the crude nuclei and cell debris fractions, suggesting an interaction between Cd and cell membranes to cause acidification. Omission of bicarbonate from the incubation medium induced cellular acidification. The presence of Cd in this medium did not potentiate the medium-induced acidification. Mg-ATP (25 μM) induced cellular acidification in relation to an increase in the concentration of cytosolic free Ca. The coexistence of Mg-ATP and Cd at the concentrations which had no effect on cellular pH in the presence of either agants induced cellular acidification. These observations suggest that Cd induced cellular acidification by modulating the process connected with the rise in cytosolic free Ca via interaction with plasma membranes. This acidification had no strong immediate cytotoxic actions but led to subsequent cellular alkalinization accompanied with severe cytotoxicity and membrane breakage.     

Cadmium induced oxidative stress and biochemical responses in cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis>

The present study deals with the growth, photosynthesis, oxidative stress and heavy metal accumulation ability of Nostoc muscorum exposed to different levels (2, 4, 8, 16, 20 μM) of cadmium (Cd) concentrations. Growth and photosynthetic pigments i.e., chlorophyll a, carotenoids and phycocyanin were significantly affected by cadmium exposure and inhibition was found to be dose dependent. ¹⁴C-fixation appeared to be more sensitive to Cd than whole cell oxygen evolution. Significant accumulation of Cd in the cells of N. muscorum was noticed after 1 and 2 h of exposure and the accumulation rate was dose and time dependent. Furthermore, the levels of superoxide radicals and hydrogen peroxide (H₂O₂) were found significantly increased by cadmium exposure which in turn accelerated the formation of malondialdehyde (MDA) content, and protein and DNA damage. The selected dose of Cd (20 μM) showed the induction of new polypeptide of ~23.24 kD and the loss of ~37.84 kD and ~69.63 kD whereas the remaining bands were inhibited as compared to control. Significant DNA fragmentation which is a hallmark of programmed cell death (PCD) was also observed in the cells treated with 20 μM of Cd for 48 h. The decrease in proline and total phenol content at 8 and 16 μM suggest that the cells of N. muscorum were not able to mitigate the oxidative stress induced by cadmium exposure. Similarly, the decreased activities of antioxidant enzymes i.e., superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) also indicates the failure of the antioxidant defense system of N. muscorum to survive at higher concentration (8 and 16 μM) of cadmium.     

Responses of <Emphasis Type="Italic">Rhodotorula</Emphasis> sp. Y11 to cadmium

Some aspects of the cellular responses to cadmium were extensively investigated in the yeast Rhodotorula sp. Y11. Scanning electron microscopy indicated that accumulation of cadmium in the Y11 did not cause any visible effects on cell morphology. More than 20% yeast cells still showed viability after 15 h of cadmium accumulation under 100 mg l⁻¹ cadmium concentration, and transmission electron microscopy analysis showed that plasmolysis and thickened cell wall were not observed in all of the cells. In the presence of cadmium, the activities of superoxide dismutase (SOD) and catalase (CAT) were all greater than the control, but the increase was in a dose-independent manner. Changes in SOD and CAT activities were also dependent on the time of exposure. Therefore, it suggests that antioxidative defenses play an important role in cadmium tolerance in Rhodotorula sp. Y11. Nondenaturing polyacrylamide gels revealed only one SOD isoforms in Y11 even under exposure to cadmium.