Short-term exposure to cadmium modifies phosphorylation of gill proteins in the sea mussel.

1. Sea mussels were exposed to cadmium for short periods of time. The excised gills were incubated with radioactive orthophosphate. The gill proteins were separated by 1- and 2-dimensional gel electrophoresis and the phosphorylation state of the proteins was determined by image analysis of autoradiographs. 2. 1-Dimensional gel electrophoresis revealed that exposure of the animals to cadmium stimulated phosphorylation of the gill proteins in a cadmium concentration-dependent manner. 3. 2-Dimensional gel electrophoresis showed that cadmium differentially affected the phosphorylation of various proteins. Major alterations were observed in the basic, high mol. wt proteins and in the acidic, low mol. wt polypeptides.     

A proteome analysis of the cadmium response in Saccharomyces cerevisiae

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.     

Selection, Isolation, and Characterization of Cadmium-Resistant Datura innoxia Suspension Cultures

Datura innoxia cells from suspension cultures were selected for their ability to grow and divide rapidly in normally lethal concentrations of cadmium. Cells resistant to 12.5, 25, 50, 100, 160, 200, and 250 micromolar cadmium chloride were isolated and utilized to initiate cell suspension cultures resistant to this toxic metal ion. Variant cell lines retained their ability to grow in cadmium after being grown in its absence for more than 400 generations. Resistance to cadmium was correlated with the synthesis of low molecular weight, cysteine-rich, cadium-binding proteins. Synthesis of these proteins was induced rapidly in cadmium-resistant cells in response to a challenge of cadmium. Induction was detectable within one hour after exposure of the cells to the metal ion. Accumulation of protein bound cadmium reached a maximum eight to twelve hours following exposure. Metal-binding proteins were not detectable in the cadmium sensitive D. innoxia cells from which resistant cells were derived.     

Iron regulatory protein 1 is not an early target of cadmium toxicity in mice, but it is sensitive to cadmium stress in a human epithelial cell line

Disruption of iron homeostasis at the levels of intestinal absorption or erythropoiesis contributes to cadmium toxicity. Cellular iron homeostasis in metazoans is maintained by the iron regulatory proteins (IRPs) that regulate the synthesis of proteins involved in the transport, use, and storage of iron. The effect of cadmium intoxication on this regulatory system has been investigated in a cellular model of human epithelium. Cadmium exposure of HeLa cells did not activate the IRPs; rather, the amount of these proteins relative to that of housekeeping proteins decreased. Accordingly, the transferrin receptor mRNA level decreased upon cadmium insult. In a more integrated investigation, separate groups of mice had free access to different doses of cadmium in drinking water for 3 weeks. Cadmium accumulated in all analyzed organs, but its concentration in mouse tissues did not correlate with changes of the activity of the IRPs. The intoxicated mice did not show any sign of anemia, indicating that iron homeostasis was not immediately disrupted after the onset of cadmium accumulation. These data establish that cadmium destabilizes IRPs in mammalian cells, but that iron imbalance is not an early event of cadmium intoxication.     

Seasonal dependence of cadmium molecular effects on Mytilus galloprovincialis (Lamarck, 1819) protamine‐like protein properties

Mussels have a seasonal reproduction and cadmium is a common stressor in estuarine and coastal environments. In previous studies, we have shown that exposure to subtoxic doses of cadmium produced alterations in the properties of winter Mytilus galloprovincialis sperm protamine‐like (PL) proteins. In this study, it was analyzed the possibility that these cadmium effects may be seasonal. Winter and summer mussels were exposed to CdCl₂, and it was tested the PL‐proteins for cadmium bioaccumulation, electrophoretic pattern, DNA binding, and potentiality to induce DNA oxidative damage. It was found that cadmium exposure did not produce the same effects on PL‐proteins of summer mussels that were produced on PL‐proteins of winter mussels, that is: cadmium bioaccumulation, alterations in the acetic acid‐urea polyacrylamide gels (AU‐PAGE) and sodium dodecyl sulfate‐PAGE pattern, a reduced DNA binding affinity and the ability to induce DNA oxidative damage. PL‐proteins from summer mussels, apart from not being affected by all the abovementioned effects of cadmium, also showed a very low DNA binding affinity, independent of cadmium exposure. This study reveals clock‐associated seasonal responses to cadmium in M. galloprovincialis. Understanding the mechanisms through which environmental signals guide biological rhythms is fundamental to understanding the seasonal sensitivity of this bioindicator, to use M. galloprovincialis in appropriate seasonal periods.     

Physiological and protein profiles alternation of germinating rice seedlings exposed to acute cadmium toxicity

Seed germination is a complex physiological process in plants that can be affected severely by heavy metals. The interference of germination by cadmium stress has not been well documented at the proteomic level. In the present study, in order to investigate the protein profile alternations during the germination stage following exposure to cadmium, a proteomic approach has been adopted in combination with morphological and physiological parameters. Seeds were exposed with a wide range of cadmium between 0.2 and 1.0 mM. Increases of cadmium concentration in the medium resulted in increased cadmium accumulation in seeds and TBARS content, whereas germination rate, shoot elongation, biomass, and water content were decreased significantly. Temporal changes of the total proteins were investigated by two-dimensional electrophoresis (2-DE). Twenty-one proteins were identified using MALDI-TOF mass spectrometry, which were upregulated at least 1.5-fold in response to cadmium stress. The identified proteins are involved in several processes, including defense and detoxification, antioxidant, protein biosynthesis, and germination processes. The identification of these proteins in the cadmium stress response provides new insight that can lead to a better understanding of the molecular basis of heavy metal responses of seeds at the germination stage.     

Comparative proteome analysis of high and low cadmium accumulating soybeans under cadmium stress

A comparative proteomic study was performed to unravel the protein networks involved in cadmium stress response in soybean. Ten-day-old seedlings of contrasting cadmium accumulating soybean cultivars—Harosoy (high cadmium accumulator), Fukuyutaka (low cadmium accumulator), and their recombinant inbred line CDH-80 (high cadmium accumulator) were exposed to 100?μM CdCl₂ treatment for 3?days. Root growth was found to be affected under cadmium stress in all. Varietal differences at root protein level were evaluated. NADP-dependent alkenal double bond reductase P1 was found to be more abundant in low cadmium accumulating Fukuyutaka. Leaf proteome analysis revealed that differentially expressed proteins were primarily involved in metabolism and energy production. The results indicate that both high and low cadmium accumulating cultivars and CDH-80 share some common defense strategies to cope with the cadmium stress. High abundance of enzymes involved in glycolysis and TCA cycle might help cadmium challenged cells to produce more energy necessary to meet the high energy demand. Moreover, enhanced expressions of photosynthesis related proteins indicate quick utilization of photoassimilates in energy generation. Increased abundance of glutamine synthetase in all might be involved in phytochelatin mediated detoxification of cadmium ions. In addition, increased abundance of antioxidant enzymes, namely superoxide dismutase, ascorbate peroxidase, catalase, ensures cellular protection from reactive oxygen species mediated damages under cadmium stress. Enhanced expression of molecular chaperones in high cadmium accumulating cultivar might be another additional defense mechanism for refolding of misfolded proteins and to stabilize protein structure and function, thus maintain cellular homeostasis.     

Proteomic evaluation of cadmium toxicity on the midge Chironomus riparius Meigen larvae

Heavy-metal pollution of aquatic ecosystems is a widespread phenomenon after industrial consumption. Whether aquatic organisms are adapted to the heavy-metal pollutants or not, such environmental stress causes changes in physiological responses. In this study, the aquatic midge, Chironomus riparius Meigen, was used to find changes of expression of proteins in relation to cadmium exposure. Dose-response relationships between cadmium concentrations and mortality of 3rd instar midge larvae were observed and the protein levels were compared using PD-Quest after 2-DE. Comparing the intensity of protein spots, 21 proteins decreased and 18 proteins increased in response to cadmium treatment. With increased proteins, three enzymes such as S-adenosylmethionine decarboxylase, O-methyltransferase, and aspartokinase were involved in the glutathione biosynthesis and a key enzyme regulating fatty acid biosynthesis, oleyl-acyl carrier protein thioesterase was also identified. According to the functional classification of decreased levels of proteins, they were involved in energy production, protein fate, nucleotide biosynthesis, cell division, transport and binding, signal transduction, and fatty acid and phospholipid metabolism in the cell. In addition, phenol hydroxylase, thioesterase, zinc metalloprotease, and aspartate kinase were newly expressed after cadmium exposure at the concentration of the LC(10 )value. Therefore, these proteins seem to be potential biomarkers for cadmium exposure in the aquatic ecosystems.     

Zinc and cadmium specifically interfere with RNA-binding activity of human iron regulatory protein 1

The cellular pro-oxidative stress induced by high zinc concentrations or cadmium is most likely mediated by disruption of redox (mainly thiol) homeostasis or by mishandling of redox-active transition metals. The impact of zinc and cadmium on the main regulators of iron homeostasis in metazoans, the iron regulatory proteins (IRP) 1 and 2, has been probed with the human recombinant proteins. Using purified proteins or extracts of yeast producing human IRP, zinc and cadmium were shown to interfere with the IRE-binding activity of IRP1, but not with that of IRP2 or the aconitase activity of IRP1. The IRP1 active site cysteines in positions 437, 503 and 506 were not directly involved in the effects of zinc and cadmium. The loss of RNA-binding activity is due to the reversible and specific aggregation of the IRP1 apoprotein with zinc and cadmium, since precipitation did not occur with other divalent metals such as manganese, cobalt or magnesium. The reported data suggest a new mechanism for the biological toxicity of cadmium and high zinc concentrations by interference with iron metabolism.     

Induction of metal binding proteins in striped bass, Morone saxatilis, following cadmium treatment

1. The time course of induction of cytosolic metal binding proteins (MBP) was observed follow up to three daily intramuscular injections of cadmium chloride (0.2 mg cadmium/injection). 2. Low molecular weight binding proteins were resolved by gel permeation chromatography on Sephadex G-75. Based on total metal binding capacity, the concentration of crude MBP increased 2.6 fold. This level of induction of MBP was confirmed by polarographic analysis. 3. Initial binding of cadmium to MBP resulted in displacement of zinc, while at later times, zinc associated with MBP increased above control levels. 4. Using 35S-cysteine incorporation, it was shown that the rate of hepatic MBP synthesis was significantly greater than controls and sham injected fish 18 hr after the third cadmium injection. 5. Due to interfering proteins of molecular weights similar to the metal binding proteins one dimensional PAGE was not capable of verifying induction. However, the metal binding proteins were resolved using two dimensional gel electrophoresis.     

Effects of cadmium on structure and enzymatic activity of Cu,Zn-SOD and oxidative status in neural cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder disease. Ten percent of the ALS patients are congenital (familial ALS), and the other 90% are sporadic ALS (SALS). It has been shown that mutations found in the Cu,Zn-SOD cause 20% of the familial ALS due to its low enzyme activity. We hypothesized that heavy metals may interfere the structure of Cu,Zn-SOD protein to suppress its activity in some of the SALS. In this study, we expressed and characterized the recombinant human Cu,Zn-SOD under various concentrations of Cu(2+), Zn(2+), and Cd(2+). By atomic absorption spectrophotometry, we demonstrated that adding of cadmium significantly increased the content of cadmium ion, but reduced its Zn(2+) content and enzyme activity of the Cu,Zn-SOD protein. The data of circular dichroism spectra demonstrated that the secondary structure of Cu,Zn-SOD/Cd is different from Cu,Zn-SOD, but close to apo-SOD. In addition to the effect of cadmium on Cu,Zn-SOD, cadmium was also shown to induce neural cell apoptosis. To further investigate the mechanism of neural cell apoptosis induced by cadmium, we used proteomics to analyze the altered protein expressions in neural cells treated with cadmium. The altered proteins include cellular structural proteins, stress-related and chaperone proteins, proteins involved in reactive oxygen species (ROS), enzyme proteins, and proteins that mediated cell death and survival signaling. Taken together, in this paper, we demonstrate that cadmium decreases the content of Zn(2+), changes the conformation of Cu,Zn-SOD protein to decrease its enzyme activity, and causes oxidative stress-induced neural cell apoptosis.     

A proteome analysis of the cadmium and mercury response in Corynebacterium glutamicum

Cadmium and mercury are well-known toxic heavy metals, but the basis of their toxicity is not well understood. In this study, we analyzed the cellular response of Corynebacterium glutamicum to sublethal concentrations of cadmium and mercury ions using 2-DE and MS. Mercury induced the over-expression of 13 C. glutamicum proteins, whereas 35 proteins were induced, and 8 proteins were repressed, respectively, under cadmium stress. The principal response to these metals was protection against oxidative stress, as demonstrated by upregulation of, e.g., Mn/Zn superoxide dismutase. Thioredoxin and oxidoreductase responded most strongly to cadmium and mercury. The increased level of heat-shock proteins, enzymes involved in energy metabolism, as well as in lipoic acid and terpenoid biosynthesis after the treatment of cells with cadmium was also registered. Identification of these proteins and their mapping into specific cellular processes enable a global understanding of the way in which C. glutamicum adapts to heavy-metal stress and may help to gain deeper insight into the toxic mechanism of these metals.     

Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 100 microM verapamil. Quantification of intracellular cadmium concentration by atomic absorption spectrometry showed a reduced cadmium accumulation in cells expressing the MDR genes. Inside-out membrane vesicles of L. lactis overexpressing lmrA displayed an ATP-dependent (109)Cd(2+) uptake that was stimulated by glutathione. An evolutionary model is discussed in which MDR proteins have evolved independently from an ancestor protein displaying both organic xenobiotic- and divalent metal-extrusion abilities.     

Protein lysine methylation contributes to modulating the response of sensitive and tolerant Arabidopsis species to cadmium stress

The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analysed the importance of protein lysine methylation for plants to cope with cadmium. We analysed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species, Arabidopsis thaliana and A. lyrata, and in three populations of A. halleri with contrasting cadmium accumulation and tolerance traits. We showed that some proteins are differentially methylated at lysine residues in response to Cd and that a few genes coding KMTs are regulated by cadmium. Also, we showed that 9 out of 23 A. thaliana mutants disrupted in KMT genes have a tolerance to cadmium that is significantly different from that of wild-type seedlings. We further characterized two of these mutants, one was knocked out in the calmodulin lysine methyltransferase gene and displayed increased tolerance to cadmium, and the other was interrupted in a KMT gene of unknown function and showed a decreased capacity to cope with cadmium. Together, our results showed that lysine methylation of non-histone proteins is impacted by cadmium and that several methylation events are important for modulating the response of Arabidopsis plants to cadmium stress.     

Proteome analysis associated with cadmium adaptation in U937 cells: identification of calbindin-D28k as a secondary cadmium-responsive protein that confers resistance to cadmium-induced apoptosis

Cadmium is a well known environmental toxicant and carcinogen. To identify proteins involved in cellular adaptive responses to cadmium, we established cadmium-adapted U937 cells that exhibit resistance to cadmium-induced apoptosis, and we performed comparative proteome analysis of these cells with parental cells that were either untreated or treated with cadmium. Newly identified proteins that were changed in expression level in both adapted cells and cadmium-treated parental cells included proteins implicated in cell proliferation and malignant transformation. Most interesting, a calcium-binding protein calbindin-D(28k) was increased only in the adapted cells but not in cadmium-exposed parental cells. The level of calbindin-D(28k) increased by the degree of cadmium adaptation and was stably maintained without selective pressure of cadmium. Cadmium-adapted U937 cells were resistant to the toxic effects of cytosolic calcium rise by cadmium treatment and by depletion of intracellular calcium stores, suggesting that enhanced calcium buffering by up-regulated calbindin-D(28k) may be responsible for acquiring resistance to cadmium-induced apoptosis. We demonstrated that overexpression of calbindin-D(28k) in MN9D neuronal cells resulted in reduced cadmium-induced apoptosis. Our study documents for the first time that cells respond to long term cadmium exposure by increasing calbindin-D(28k) expression, thereby attenuating cadmium-induced apoptosis.     

Incorporation of cadmium into proteins in a cadmium tolerant fungi

Aspergillus carbonarius and a strain of Penicillium, a cadmium tolerant fungi, are able to metabolize cadmium chloride up to 2% (w/v). Their amino acids analysis on cadmium free and cadmium chloride containing media indicated certain disorders in their metabolic activities. Cystathionine was only detected in both fungi in the presence of cadmium chloride. However, cadmium was incorporated into several types of low and high molecular weight proteins. The amino acids hydrolyzates of a cadmium containing protein are characterized by the presence of high levels of sulfur amino acids; cysteine and methionine. Ethylasparagine was detected in the hydrolyzate of that cadmium containing protein as well.     

A comparison of the sequestration of cadmium and zinc in the tissues of rainbow trout (Salmo gairdneri) following exposure to the metals singly or in combination

Rainbow trout were exposed to either cadmium (9 micrograms/l) or zinc (100 micrograms/l) in their aquarium water. They were then transferred to water containing concentrations of cadmium (54 micrograms/l) that would have otherwise proved fatal to the majority of the fish without the pretreatment. Most of the fish survived under both sets of conditions. However, two different mechanisms seem to be involved in the protection of the animals against the toxic manifestations of cadmium. In both cases, more than 99% of the total body load of cadmium was found in the liver, kidney and gills of the animals. Analysis of the metal-binding proteins in these organs was carried out. In the fish exposed to the two concentrations of cadmium, the toxic metal was found only in association with two low mol. wt specific binding proteins despite the presence of zinc- (and copper)-containing isometallothioneins in all three organs. On the other hand, cadmium was distributed between these binding-proteins and metallothioneins in the liver, kidney and gill of the trout pretreated with zinc before their exposure to cadmium.     

Postinductive actinomycin D effects on the concentrations of cadmium thionein, zinc thionein, and copper chelatin in rat liver

The time courses of induction in rat liver of copper chelatin by copper, cadmium thionein by cadmium, and zinc thionein by copper, cadmium, and zinc were monitorg metal were used in order to avoid toxic effects, being 5 mg zinc, 0.5 mg copper, and 0.25 mg cadmium per kg body weight. Peak times of induction and half times of decay observed were: copper chelatin (9 h, 8.6 h), cadmium thionein (18 h, 6.80 days), and zinc thionein (zinc rats, 18 h, 10.1 h; copper rats, 9 h, 18.2 h; cadmium rats, 24 h, 4.53 days). Administration of actinomycin D (1 mg per kg body weight) at the peak times of induction of the various proteins had no effect on the concentrations of chelatin or cadmium thionein observed up to 24 hours later, but in the case of zinc thionein, induced by zinc, copper, or cadmium, elevated concentrations were observed up to 23 h after administration of the drug. Such behavior is reminiscent of superinduction previously seen with other proteins and enzymes. We postulate that the intracellular concentration of free zinc in liver is of fundamental importance in the induction of zinc thionein, and this can be distributed by exogenous copper or cadmium resulting in the induction of synthesis of zinc thionein.     

Cadmium-binding to transferrin in the plasma of the common carp Cyprinus carpio

In this study, it was investigated by autoradiography with radioactive cadmium after Western blotting of two-dimensional electrophoresis gels, to which proteins cadmium is mainly bound in plasma of common carp Cyprinus carpio. The obtained results demonstrate that in carp plasma, cadmium is primarily bound to two high molecular weight proteins. Relative small amounts are bound to a protein with M(r) approximately 60000. The other metal-binding protein, with M(r) approximately 70000 and pI approximately 6.7 was identified as transferrin. The conditional equilibrium constants for the binding of cadmium ions to the two metal-binding sites of this protein were calculated as logK(1)=5.40+/-0.12 and logK(2)=4.66+/-0.21, which are comparable to those of human transferrin under the same experimental conditions. Transport of cadmium in plasma of carp was found to be different from that of brown trout Salmo trutta and man, where cadmium is mainly bound to albumin and transferrin. The prominent binding of cadmium to transferrin can be explained by the absence or at least the very low concentrations in which albumin is present in carp plasma.