Beclin-1 expression in normal bladder and in Cd and As exposed and transformed human urothelial cells (UROtsa)

The expression of beclin-1 in normal human bladder and in Cd²⁺ and As³⁺ exposed and transformed urothelial cells (UROtsa) was examined in this study. It was shown using a combination of real-time PCR, Western analysis and immunohistochemistry that beclin-1 was expressed in the urothelial cells of the normal bladder. It was also demonstrated that the parental UROtsa cell line expressed beclin-1 mRNA and protein at levels similar to that of the in situ urothelium. The level of beclin-1 expression underwent only modest alterations when the UROtsa cells were malignantly transformed by Cd²⁺ or As³⁺ or when the parental cells were exposed acutely to Cd²⁺ or As³⁺. While there were instances of significant alterations at individual time points and within cell line-to-cell line comparisons there was no evidence of a dose–response relationship or correlations to the phenotypic properties of the cell lines. Similar results were obtained for the expression of the Atg-5, Atg-7, Atg-12 and LC3B autophagy-related proteins. The findings provide initial evidence for beclin-1 expression in normal bladder and that large alterations in the expression of beclin-1 and associated proteins do not occur when human urothelial cells are malignantly transformed with, or exposed to, either Cd²⁺ or As^3+.     

Cadmium significantly changes major morphometrical points and cardiovascular functional parameters during early development of zebrafish

Living organisms are commonly exposed to cadmium and other toxic metals. A vast body of research has shown the significant effects of these toxic metals on developmental processes. In order to study the role of toxic metals on early developmental stages of eukaryotes, we explored the effect of cadmium (Cd²⁺) contaminant on zebrafish. Thus, zebrafish embryos were exposed to 3 mg/L (16.7 μM) Cd²⁺ for 96 h and imaged every 24 h from the exposure onwards. Hatching rates of the eggs were determined at 72 h, followed by analyses at 96 h for: survival rate, morphometrical factors, and functional parameters of the cardiovascular system. Interestingly enough, significant hatching delays along with smaller cephalic region and some morphological abnormalities were observed in the treatment group. Moreover, substantial changes were noticed in the length of notochord and embryo, absorption of yolk sac with shorter extension, area of swimming bladder, as well as pericardium sac after Cd²⁺ treatment. Cadmium also caused significant abnormalities in heart physiology which could be the leading cause of mentioned morphological deformities. Herein, our results shine light on systematic acute embryological effects of cadmium in the early development of zebrafish for the first time.     

Interaction of metal ions with tRNA: Effects of cadmium,zinc, and magnesium ions on chromatographic behavior of tyrosine and leucine tRNA species

The mechanism by which certain divalent cations may cause toxic effects was studied by examining the in vitro response of certain transfer RNAs to the cations. Two Drosophila tRNAs, those for tyrosine and leucine, were examined by reversed-phase column 5 (RPC-5) chromatography in the presence of each of three metal ions, Zn²⁺, Mg²⁺, and Cd²⁺. As Zn²⁺ concentration increased, both tRNAs eluted at progressively higher ionic strengths and the isoacceptor patterns collapsed and then reappeared. In contrast, when either the Mg²⁺ or the Cd²⁺ concentration increased, both tRNAs eluted at lower ionic strengths and the resolution of each of the isoacceptors was reduced. The resolution of tRNA^Tyr from tRNA^Leu was best with Cd²⁺ and poorest with Mg²⁺. If the observed changes were caused by conformational changes, they should be reversed upon removal of the metal ion; this was found to be so. Recovery of the tRNA after chromatography was reduced with Zn²⁺, presumably through nonspecific hydrolysis. One late-eluting leucyl-tRNA species was labile to incubation in the presence or absence of metal ion, followed by heating to 95°C. This same species was labile to Zn²⁺ exposure in the absence of heating. We suggest that reversed-phase column 5 chromatography is useful for evaluation of the effect of divalent metal ions on tRNA conformation and that the effects of various toxic metal ions could be examined in this manner.     

Synthesis,characterization and in vivo evaluation of cadmium telluride quantum dots toxicity in mice by toxicometabolomics approach

Quantum dots (QDs) have widespread application in many fields such as medicine and electronics. The need for understanding the potentially harmful side effects of these materials becomes clear. In this study, the toxicity of cadmium telluride quantum dots (CdTe-QDs) and bulk Cd²⁺ has been investigated and compared by applying metabolomics methods. The datasets were ¹H-NMR data from mice plasma which had been taken from four groups of mice in different time intervals. Then, the data were analyzed by applying chemometrics methods and the metabolites were found from Human Metabolome Database (HMDB). The results showed the significant change in the level of some metabolites especially estrogenic steroids in different groups with different amounts of received Cd. The findings also indicated that steroid hormone biosynthesis, lysine biosynthesis and taurine and hypotaurine metabolism are the most affected pathways by CdTe-QDs especially in estrogenic steroids. The over-representation analysis indicated that endoplasmic reticulum, gonads, and hepatocytes are most affected. Since the pattern of metabolite alteration of CdTe-QDs with equivalent Cd²⁺ was similar to those of CdCl₂, it was postulated that beside Cd²⁺ effects, the toxicity of CdTe-QDs is associated with other factors.     

Side-specific effects by cadmium exposure: Apical and basolateral treatment in a coculture model of the blood-air barrier

Cadmium (Cd²⁺) is a widespread environmental pollutant, which is associated with a wide variety of cytotoxic and metabolic effects. Recent studies showed that intoxication with the heavy metal most importantly targets the integrity of the epithelial barrier.In our study, the lung epithelial cell line, NCI H441, was cultured with the endothelial cell line, ISO-HAS-1, as a bilayer on a 24-well HTS-Transwell® filter plate. This coculture model was exposed to various concentrations of CdCl₂.The transepithelial electrical resistance decreased on the apical side only after treatment with high Cd²⁺ concentrations after 48 h. By contrast, a breakdown of TER to less than 5% of baseline could be observed much earlier (after 24 h) when Cd²⁺ was administered from the basal side. Observations of cell layer fragmentation and widening of intercellular spaces confirmed the barrier breakdown only for the basolaterally treated samples. Furthermore, the cytotoxicity and release of proinflammatory markers was enhanced if samples were exposed to Cd²⁺ from the basal side compared to treatment from the apical side. Moreover, we could demonstrate that a high concentration of Ca²⁺ could prevent the barrier-disrupting effect of Cd²⁺.In conclusion, the exposure of Cd²⁺ to cocultures of lung cells caused a decrease in TER, major morphological changes, a reduction of cell viability and an increase of cytokine release, but the effects markedly differed between the two modes of exposure. Therefore, our results suggest that intact epithelial TJs may play a major role in protecting the air-blood barrier from inhaled Cd²⁺.     

Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: exposure to cadmium

The aims of this study were to (1) evaluate the changes in the Cd tolerance of a marine diatom after exposure under different Cd concentrations for various durations and (2) to explore the potential subcellular and biochemical mechanisms underlying these changes. The 72-h toxicity, short-term Cd uptake, subcellular Cd distribution, as well as the synthesis of phytochelatins (PCs) were measured in a marine diatom Thalassiosira nordenskioeldii after exposure to a range of free Cd ion concentrations ([Cd²⁺], 0.01-84 nM) for 1-15 days. Surprisingly, the diatoms did not acquire higher resistance to Cd after exposure; instead their sensitivity to Cd increased with a higher exposed [Cd²⁺] and a longer exposure period. The underlying mechanisms could be traced to the responses of Cd cellular accumulation and the intrinsic detoxification ability of the preconditioned diatoms. Generally, exposure to a higher [Cd²⁺] and for a longer period increased the Cd uptake rate, cellular accumulation, as well as the Cd concentration in metal-sensitive fraction (MSF) in these diatoms. In contrast, although PCs were induced by the environmental Cd stress (with PC₂ being the most affected), the increased intracellular Cd to PC-SH ratio implied that the PCs’ detoxification ability had reduced after Cd exposure. All these responses resulted in an elevated Cd sensitivity as exposed [Cd²⁺] and duration increased. This study shows that the physiological/biochemical and kinetic responses of phytoplankton upon metal exposure deserve further investigation.     

Effect of sodium isopropylxanthate,potassium amylxanthate and sodium diethyldithiocarbamate on the uptake and distribution of cadmium in the brown trout (Salmo trutta)

Brown trout, Salmo trutta, were exposed to water containing 1 μg/l of ¹⁰⁹Cd²⁺, alone or with sodium isopropylxanthate, potassium amylxanthate or sodium diethyldithiocarbamate, respectively. After one week the uptake and distribution of the ¹⁰⁹Cd²⁺ in the fish were examined by whole-body autoradiography and gamma spectrometry. Sodium diethyldithiocarbamate was found to enhance the uptake of the ¹⁰⁹Cd²⁺ in several tissues of the fish and this effect increased with increasing concentration of the carbamate. Potassium amylxanthate induced increase in the levels of ¹⁰⁹Cd²⁺ in several tissues, whereas the brain was the only tissue with increased concentration of ¹⁰⁹Cd²⁺ in the presence of sodium isopropyl-xanthate. A likely mechanism for the enhanced uptake of the ¹⁰⁹Cd²⁺ may be a facilitated penetration over the gill membranes of the lipophilic complexes formed between the studied compounds and the cadmium. A facilitated passage through cellular membranes may also be important for the increased uptake of the metal in other tissues. An elevated uptake of cadmium by the xanthates or the diethyldithiocarbamate may constitute an increased risk for noxious effects of the metal.     

Cadmium induced oxidative damage and apoptosis in the hepatopancreas of <Emphasis Type="Italic">Meretrix meretrix</Emphasis>

Even trace amounts of cadmium (Cd), a non-essential metal, are known to be toxic to aquatic organisms. Here we investigated the relationship between cadmium ion (Cd²⁺) exposure and oxidative damage and apoptosis in the hepatopancreas of the clam Meretrix meretrix. Clams were exposed to different concentrations of Cd²⁺ (0, 1.5, 3, 6 and 12 mg L^?1) for 5 days. We monitored both antioxidant enzyme activity, including that of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx), and levels of malondialdehyde (MDA), glutathione (GSH) and glutathione disulfide (GSSG). Apoptosis of hepatopancreatic cells was detected by DNA laddering and AO/EB double fluorescent staining. The results show that the rate of apoptotis, MDA levels, and caspase-3 activity, increased with Cd²⁺ concentration, whereas GPx activity and the ratio of GSH/GSSG, decreased. SOD and CAT enzyme activity first increased, then decreased, with increasing Cd²⁺ concentration; peak activity of these enzymes was recorded in the 3 mg L^?1 Cd²⁺-treatment group. These results show that Cd-induced oxidative damage can both induce, and aggravate, apoptosis in the hepatopancreatic cells of clams, even at Cd²⁺ concentrations far below the semi-lethal dose for adult clams. The observed changes in caspase-3 activity enhanced significantly at lower Cd²⁺ concentrations, indicating that caspase-3 is a suitable biomarker for heavy metal pollution, especially cadmium pollution, in marine organisms.     

Mechanism of primary Cd2+-induced rat liver mitochondria dysfunction: discrete modes of Cd2+ action on calcium and thiol-dependent domains

We attempted to discern discrete sites of Cd²⁺ deleterious action on rat liver mitochondrial function. In particular, EGTA, ADP, and cyclosporin A (potent mitochondrial permeability transition antagonists) affected mainly Cd²⁺ -induced changes in resting state respiration, eliminating its stimulation in KCl medium, while dithiothreitol (DTT, a dithiol reductant) produced its effect both on Cd²⁺ activation of the basal respiration and Cd²⁺ depression of uncoupler-stimulated respiration, evoking its restoration. Substantial differences in DTT influence on mitochondrial respiration at low and high [Cd²⁺] were revealed, namely, an enhanced mitochondrial permeabilization in the presence of saturated [DTT] at high [Cd²⁺] took place. Besides, DTT only partially reversed Cd²⁺ -induced swelling in NH₄NO₃ medium when glutamate plus malate or succinate without rotenone was used. Contrarily, DTT produced complete reversal of the swelling of succinate-energized mitochondria when rotenone was present in the medium. In addition, in the presence of rotenone both Cd²⁺ -produced activation of the resting state respiration in KCl medium and Cd²⁺-induced swelling in sucrose medium of succinate-energized mitochondria were more sensitive to cyclosporin A than the same Cd²⁺ effects obtained on mitochondria oxidizing succinate (without rotenone) or glutamate plus malate. We have concluded that Cd²⁺, producing primary mitochondrial dysfunction, acts both as a thiol and Me²⁺ binding site reagent. Suppositions about possible localization of separate sites of direct Cd²⁺ effects on mitochondrial function were made.     

Axonal Transport of Cadmium in the Olfactory Nerve of the Pike

¹⁰⁹Cd²⁺ was applied in the olfactory chambers of pikes (Esox lucius) and the dynamics of the axoplasmic flow of the metal was determined in the olfactory nerves by gamma spectrometry and autoradiography. The results showed that the ¹⁰⁹Cd²⁺ is transported at a constant rate along the olfactory nerves. The profile of the ¹⁰⁹Cd²⁺ in the nerves showed a wave front of transported metal followed by a saddle region. When the nasal chambers were washed 2 hr after application of the ¹⁰⁹Cd²⁺ well-defined transport peaks for the metal were seen in the olfactory axons. The maximal velocity for the transport of ¹⁰⁹Cd²⁺, which corresponds to the movement of the wave front, was 2.38±0.10 mm hr (mean±S.E.) at the experimental temperature (10 C). The average velocity for the transport of the ¹⁰⁹Cd²⁺, which corresponds to the peak apex movement of the wave, was 2.18±0.05 mm/hr (mean±S.E.) at 10 C. The transported ¹⁰⁹Cd²⁺ was strongly accumulated in the anterior parts of the olfactory bulbs, whereas in other brain areas the levels of the metal remained low. Autoradiography of a pike exposed to ¹⁰⁹Cd²⁺ via the water showed a strong labelling in the receptor-cell-containing olfactory rosettes, whereas other structures in the olfactory chambers were only weakly labelled. The accumulation and axonal transport in the olfactory neurons may be noxious and constitute an important component in the toxicology of cadmium in fish, and this may apply also to some other heavy metals.     

Leucocytes and leucopoietic capacity in goldfish,Carassius auratus,exposed to sublethal levels of cadmium

By comparison with animals in essentially cadmium-free water (< 2 μg Cd²⁺/l) goldfish exposed for 3 wk to 90, 270 and 445 μg Cd²⁺/l (5, 15 and 25% 240-h LC₅₀) exhibited significant reductions in total leucocyte counts. These were the result of decreases in lymphocyte and thrombocyte numbers. Mitogenic response to administered PHA as well as sharp decreases in blast cell numbers suggested that lympho- and thrombopenia reflect, in part at least, decreased proliferative capacity. By contrast, neutrophil, cosinophil and basophil numbers increased in cadmium-intoxicated fish. Cadmium apparently reduced PHA-related changes in granulocyte abundances.     

Effect of cadmium on the extracellular Na+, K+, and Ca2+ in the gill and small intestine of Goldfish Carassius auratus

In this study, the toxic effect of cadmium on extracellular Na⁺, K⁺, and Ca²⁺ in the gill and small intestine of goldfish Carassius auratus was determined with the technique of ion chromatograph. Two-way ANOVA indicated that the two factors (Cd²⁺ treatment and time) and the interaction factor had significant effect on the level of Na⁺, K⁺, and Ca²⁺ in the small intestine and gill. 1.0 mg/L Cd²⁺ significantly increased Ca²⁺ level in the small intestine, but Ca²⁺ level in the gill was significantly decreased by 1.0 and 5.0 mg/L Cd²⁺ at 24, 48, and 72 h. Na⁺ and K⁺ level in the small intestine and gill was increased by 1.0 mg/L Cd²⁺ at three time points, but increased by 5.0 mg/L Cd²⁺ at a certain different time. In addition, Na⁺ level was significantly decreased by 5.0 mg/L Cd²⁺ at 24 or 48 h in the small intestine and gill. The results indicated that Cd²⁺ played an important role in regulating the level of Na⁺, K⁺, and Ca²⁺ in the small intestine and gill of goldfish C. auratus. A method was constructed to investigate the extracellular Na⁺, K⁺ and Ca²⁺ in the tissues of gold fish with ion chromatography.     

Cadmium stimulates mouse skin fibroblast apoptosis by affecting intracellular homeostasis

Context: Cadmium (Cd²⁺) is an important industrial and environmental pollutant and has been shown to induce apoptosis in a variety of cell types and tissues. Objective: To assess the specific effects of low-dose Cd^2+?on the skin. This organ is easily exposed to Cd²⁺, but how it damages cells is not fully understood. Materials and methods: Mouse skin fibroblasts were treated with low doses of Cd^2+?(0.4, 0.8 or 1.6?μM) for 12–48?h, and we observed cell morphological alterations, measured DNA damage and quantified cell viability changes. Results: Cd²⁺-treated fibroblasts exhibited morphological changes and evidence of DNA damage, as well as higher numbers of apoptotic and necrotic cells. There were increased caspase ?3, ?8 and ?9 activities when fibroblasts were treated with 0.4, 0.8 and 1.6?μM CdCl₂ for 24?h. Higher intracellular calcium (Ca²⁺) and reactive oxygen species (ROS) levels, and enhanced efflux of extracellular Ca^2+?and potassium (K⁺). The mitochondrial membrane potential was lowered in treated cells, and the cell cycle arrested in the G0/G1 phase. Bax and Fas gene expression increased and Bcl-2 gene expression decreased. Discussion: The results demonstrate that Cd^2+?exerts typical apoptotic effects in mouse skin fibroblasts. It strongly inhibited proliferation and induced apoptosis in a dose- and duration-dependent manner. Ca^2+?homeostasis was disturbed by oxidative stress, mitochondrial dysfunction and caspase-mediated apoptosis. Conclusion: K^+?efflux and Bax, Bcl-2 and Fas gene expression regulation play important roles in Cd²⁺-induced dysfunction by disrupting intracellular homeostasis in mouse skin fibroblasts.     

Surface binding and uptake of cadmium (Cd2+) by LLC-PK1 cells on permeable membrane supports

Recent studies have shown that Cd²⁺ has relatively specific damaging effects on cell-cell junctions in the renal epithelial cell line, LLC-PK₁. The objective of the present studies was to examine the surface binding and uptake of Cd²⁺ by LLC-PK₁ cells in relation to the disruption of cell-cell junctions. LLC-PK₁ cells on Falcon Cell Culture Inserts were exposed to CdCl₂ containing trace amounts of¹⁰⁹Cd²⁺ from either the apical or the basolateral compartments, and the accumulation of¹⁰⁹Cd²⁺ was monitored for up to 8 h. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that the cells accumulated 3–4 times more Cd²⁺ from the basolateral compartment than from the apical compartment. The accumulation of Cd²⁺ from the basolateral compartment occurred in two phases: a rapid, exponential phase that occurred in 1–2 h and coincided with a decrease in transepithelial resistance, and a slower, linear phase that continued for 6–8 h. The Cd²⁺ that accumulated during the rapid phase was easily removed by washing the cells in EGTA, indicating that most of it was bound to sites on the cell surface. By contrast, most of the Cd²⁺ that accumulated during the slower phase could not be removed by EGTA, indicating that it had been taken up by the cells. Additional studies showed that the rapid phase of Cd²⁺ accumulation was enhanced when Ca²⁺ was present at low concentrations (0.1 mM), and was greatly reduced when Ca²⁺ was present at high concentrations (10 mM). These results suggest that Cd²⁺ damages the junctions between LLC-PK₁ cells by interacting with Ca²⁺-sensitive sites on the basolateral cell surface.     

Effects of cadmium on Drosophila: toxicity, proteins, and transfer RNAs

An animal model with well-defined genetic and biochemical characteristics is needed for a detailed understanding of the mechanism of toxicity by metal ions. Drosophila melanogaster was used in the present study to demonstrate a number of responses of Cd²⁺, including lethality, age-related changes in resistance, alterations of the normal developmental changes in proteins, and alterations in specific transfer RNAs. Genotype-specific differences in resistance to Cd²⁺ were found: the ν; bw strain was 5–10 times more resistant than su(s)²ν; bw for developmental exposure; upon treatment of the young adults the differences were in the same direction, but the sensitivities differed by only two- to three-fold. The adult fly became more sensitive to Cd²⁺ as it aged through 2 weeks, but changed little thereafter. The electrophoretic patterns of proteins of adult flies underwent changes during aging from 1 to 8 days; these changes were markedly altered by 0.55 mm CdCl₂ but not by 0.74 mm ZnCl₂ in the medium on which the flies were maintained. The appearance of queuosine-containing tRNA was stimulated by CdCl₂ (0.05–0.8 mm) in the growth medium, but not by ZnCl₂ (0.07–1.1 mm). Further studies involving D. melanogaster should be useful in defining specific interactions of toxic metal ions with macromolecules to enhance the understanding of the toxic effects of these and similar pollutants.     

Metallothionein accumulation in response to cadmium in a clonal rat liver cell line

RLC cells, a clonal line of cells derived from rat liver, accumulate ¹⁰⁹Cd in a unique bound form in response to exposure to Cd²⁺. Cadmium administration also causes increased incorporation of [³⁵S]cystine into a chromatographically distinct macromolecule. The ¹⁰⁹Cd-binding activity and the macromolecule preferentially labeled by [³⁵S]cystine cochromatograph both on gel filtration and ion exchange columns. In addition, the Cd²⁺ responsive [³H]cystine-labeled moiety of RLC cells cochromatographs in both systems with rat liver [³⁵S]cystine metallothionein labeled in response to Cd²⁺ challenge in vivo. These data suggest that RLC cells are able to accumulate authentic metallothionein in culture in response to Cd²⁺ challenge. Time course and concentration data for metallothionein accumulation and labeling with ¹⁰⁹Cd and [³⁵S]cystine are presented. RLC cells in culture appear to be able to accumulate Cd²⁺ into metallothionein at a rate comparable to rat liver tissue in vivo when both systems are maximally activated.