Metallothionein-bound cadmium in the gut of the insect Orchesella cincta (Collembola) in relation to dietary cadmium exposure

Metallothionein is considered to be a potential biomarker for heavy metal exposure in the terrestrial environment. However, limited information is available on metallothioneins from insects, a major class of terrestrial invertebrates. In this study we have quantified metallothioneins in the springtail Orchesella cincta by determining metallothionein-bound cadmium after separation of these proteins using gel filtration and reversed phase chromatography from total body homogenates of animals dietary exposed to different concentrations of cadmium. Furthermore, we have studied in more detail where cadmium and metallothionein-bound cadmium is located within this animal. The concentration of metallothionein-bound cadmium increases with the exposure concentration in the same way as the total internal concentration. Both reach a plateau at an exposure concentration of approximately 1.0 μmol Cd/dry food. Cadmium is primarily located within the gut of O. cincta and isolation of metallothionein from this organ gives results identical to isolations from total bodies. Based on this results an estimation of the metallothionein level at the highest exposure concentration results in a concentration of about 115 μg metallothionein/g fresh gut. The O. cincta metallothionein gives the possibility of using this protein as a biomarker for heavy metal exposure in soil insects.     

Effects of redox agents on protein tyrosine phosphorylation in pea roots

Posttranslational protein modifications and their interaction are an important way for the regulation of activities of proteins and their supramolecular complexes. More than 50 soluble proteins phosphorylated on tyrosine were detected in pea (Pisum sativum L., cv. Truzhenik) roots by one- and two-dimensional electrophoresis and immunoblotting with a highly specific antibody (PY20). The level of tyrosine phosphorylation of these proteins was changed under in situ action of redox and alkylating agents.     

Effect of tungstate on pea root growth and protein tyrosine phosphorylation

Tungsten belong to heavy metal group, which physiological, biochemical, and molecular action mechanisms are essentially unstudied despite metal wide application in light, heavy, and military industries and the gradual accumulation in the environment. Protein phosphorylation/dephosphorylation (one of the most important posttranslational modifications) is a highly conserved mechanism of intracellular signaling and regulation of many processes of cell activity. Protein tyrosine phosphorylation/dephosphorylation is required for the cell cycle processing, plant growth and differentiation. In this work, the effects of sodium tungstate on pea (Pisum sativum L. cv. Truzhenik) root growth, protein tyrosine phosphorylation, and phosphatase activity in the roots were studied. It was shown that sodium tungstate suppressed growth, changed the mitotic index in the root meristem, and delayed cells at some mitosis phases. Under the influence of tungstate, hydrogen peroxide accumulated in the roots and phosphatase activity was inhibited. It was established by two-dimension electrophoresis and immunoblotting with the highly specific to phosphotyrosine antibody (PY20) that tungstate treatment increased both the number of such proteins and their specific phosphorylation. It is supposed that the inhibition of protein tyrosine phosphatases was one of the reasons for tungstateinduced pea root growth inhibition.     

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.     

Effects of cadmium acetate contaminated drinking water on vital organs: A histopathological and biochemical study

Cadmium (Cd) is a heavy metal with various human exposure sources. It accumulates in the liver, forming a complex with metallothionein protein and progresses to other organs. As a heavy metal, cadmium can replace calcium and other divalent ions and disturb their cascades, ultimately affecting the vital organs. Since cadmium acetate (CA) is considered more lethal than other Cd compounds, the current study examines the effect of different concentrations of CA doses in drinking water for different exposure times in murine models (Mus musculus). After the exposure period, the murine models were then examined histopathologically and biochemically. The histopathological examination of the heart, liver, and kidneys of the experimental group showed extensive degenerative effects. Atomic absorption spectroscopy was used to determine the quantity of cadmium in serum, kidney, and hepatic tissues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hepatic proteins, especially metallothionein, directly related to Cd administration. The biochemical parameters, including creatine kinase, alanine aminotransferase, aspartate aminotransferase, total proteins, glucose, urea, uric acid, and creatinine, were also analyzed. After thorough histochemical and biochemical analysis, it was concluded that even low dose exposure of CA is hazardous to murine models with damaging effects.     

Characterization of oat vicilin-like polypeptides

The 7S and 3S globulin fractions were extracted and characterized from Avena sativa L. seeds which showed similar solubility characteristics and holoprotein size to those of the vicilin fraction in legumes. These holoproteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectrofocussing, and two-dimensional electrophoresis. Among the components of the oat reserve globulins, the 7S fraction was capable of binding to concanavalin A-Sepharose, thus indicating it to be a glycoprotein. This pattern of the glycosylation of the reserve proteins in oat resembles that observed in certain legume seeds such as Pisum sativum and Glycine max. The results support the notion that the protein components of cereal and legume globulins may be homologous.     

Assessment of heavy metal induced stress responses in pea (Pisum sativum L.)

Exposure of plants to heavy metals severely affects their growth and physiological processes. Nevertheless, different plants show variable responses to different heavy metals, generally in a concentration-dependent manner. In this study, phytotoxic effects of cadmium (Cd), cobalt (Co) and lead (Pb) applied as chlorides at concentration 500, 750, 1000 and 1250 ppm were evaluated on seed germination, early seedling growth and dry biomass of pea (Pisum sativum L.). A lower concentration (500 ppm) of Pb promoted seed germination but declined other growth parameters. Higher concentration had a phytotoxic influence on the pea. Cd and Co severely affected germination and seedling growth of pea resulting in complete failure of germination and seedling growth at higher metal concentration. Tolerance index (TI) calculated for seed germination and dry biomass indicated that tested plant had zero tolerance to 1250 ppm of Cd as well as 750 ppm and higher concentrations of Co. The order of heavy metals for their phytotoxic effects was Co > Cd > Pb. The study suggests that P. sativum is relatively tolerant to Pb but highly sensitive to Co and Cd.     

Embryogenic and callus-specific proteins in somatic embryogenesis of the grass,Dactylis glomerata L.

Somatic embryogenesis occurs spontaneously in some monocotyledoneous callus and cell suspension cultures maintained in suitable culture conditions. Nevertherless, the processes involved in somatic embryo development, and factors inducing this differentiation, are poorly understood. In order to study the changes in protein composition accompanying embryogenesis in cell suspension cultures of Dactylis glomerata L., embryos of various sizes and “undifferentiated” callus cells were separated and their total cellular protein extracts analyzed by two-dimensional polyacrylamide gel electrophoresis. Several proteins could be identified that are specific for embryos or callus under various culture conditions. Three independent detection methods were employed: silver-staining of proteins, in vivo labeling of proteins with [³⁵S]methionine, and in vitro translation of poly(A)⁺ RNA. All culture conditions tested, including those that induce embryonic proteins in carrot, fail to induce embryonic proteins in D. glomerata callus cells.     

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.     

Traffic and industrial activities around Riyadh cause the accumulation of heavy metals in legumes: A case study

The objective of this study was to analyse the effect of the continuously increasing anthropogenic activities around Riyadh, Saudi Arabia on the accumulation of heavy metals in leguminous crops. This study determined whether four legume crops, Pisum sativum L., Vicia faba L., Glycine max and Vigna sinensis, could accumulate the heavy metals Cu, Mn, Pb and Zn in their leaves, pods and grains during the summer when grown under conditions with ambient air pollution from heavy traffic and industrial activities in Riyadh, Saudi Arabia. The effect of the air pollution was examined by quantifying the protein and trace element Cu, Mn, Pb and Zn concentrations in the leaves, pods and grains of the four plant species. Analysis of the results indicated that air pollution significantly increased the heavy metal concentrations in the leaves, pods and grains. Toxic concentrations of the heavy metals were found in the plants grown at L3, L4 and L5. In conclusion, the air pollution increases as the traffic, industrial activities and population density increase.     

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.     

Differential proteomic analysis of cadmium-responsive proteins in wheat leaves

To gain a comprehensive understanding of plant response to Cd, physiological and proteomic changes in wheat (Triticum aestivum L.) leaves exposed to a range of Cd concentrations (10, 100 and 200 μM) were investigated. Leaf elongation was decreased, whereas the H₂O₂ and malondialdehyde content increased significantly at higher Cd concentrations. Changes in protein profiles were analyzed by two-dimensional electrophoresis. Twenty-one proteins which showed 1.5-fold change in protein abundance in response to Cd were identified. These proteins can be functionally grouped into three groups: 1) oxidative stress response, 2) photosynthesis and sugar metabolism and 3) protein metabolism and others. These results provide a new insight into our understanding of the molecular basis of heavy metal response in plants.     

The molecular characteristics of two metallothioneins in Ostrinia furnacalis and expression under conditions of heavy metal stress

Metallothioneins are ubiquitously-expressed metal-binding proteins. Despite their potential ecological relevance, no prior reports have identified any metallothioneins in Ostrinia furnacalis or other Lepidoptera species. A better understanding of the molecular characteristics and regulatory dynamics of metallothionein genes in O. furnacalis under heavy metal stress conditions would enable future studies of the roles played by these proteins in the context of heavy metal detoxification. Herein, we identified and characterized two metallothionein (OfMT) genes in O. furnacalis, including the 147 bp OfMT1 gene encoding a 48 amino acid protein containing 10 cysteine residues, and the 141 bp OfMT2 gene encoding a 46 amino acid protein containing 12 cysteine residues. The expression of OfMT2 was found to be related to Cu and Cd concentrations in a dose-dependent manner but was unaffected by Zn exposure. Overall, these results indicate that OfMT genes likely encode metal-binding proteins consistent with their potential role in the maintenance of heavy metal homeostasis.     

Response of Pteris vittata to different cadmium treatments

Pteris vittata is known as an arsenic hyperaccumulator, but there is little information about its tolerance to cadmium and on its ability to accumulate this heavy metal. Our aim was to analyse the accumulation capacity, oxidative stress and antioxidant response of this fern after cadmium treatments. Cadmium content, main markers of oxidative stress and antioxidant response were detected in leaves of plants grown in hydroponics for both short- (5 days) and long- (15 days) term exposure to 0 (control) 60 and 100 μM CdCl₂. In leaves, the concentration of cadmium and oxidative stress were parallel with the increase of cadmium exposure. In the short-term exposure, antioxidant response was sufficient to contrast cadmium phytotoxicity only in 60 μM cadmium-treated plants. In the long-term exposure all treated plants, in spite of the increase in activity of some peroxide-scavenging enzymes, showed a significant increase in oxidative damage. As in the long-term stress markers were comparable in all treated plants, with no clear correlation with hydrogen peroxide content, at least part of cadmium-induced oxidative injury seems not mediated by H₂O₂. Based on our studies, P. vittata, able to uptake relatively high concentrations of cadmium, is only partially tolerant to this heavy metal.     

Effect of heavy metal ions on growth and biochemical characteristics of photosynthesis of barley and maize seedlings

The effects of Cu²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ on growth and the biochemical characteristics of photosynthesis were more expressed in barley (Hordeum vulgare L.) than in maize (Zea mays L.) seedlings. The barley and maize seedlings exhibited retardation in shoot and root growth after exposure of Cu²⁺, Cd²⁺ and Pb²⁺. The Zn²⁺ions practically did not influence these characteristics. The total protein content of barley and maize roots declined with an increase in heavy metal ion concentrations. The protein content of barley shoots was only slighly decreased with an increase in heavy metal ion concentrations, but the protein content in maize shoots was increased under the same conditions. The chlorophyll content was decreased in barley shoots and increased in maize. The ribulose-l,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activities were decreased drastically by Cu²⁺, Cd²⁺ and Pb²⁺ in thein vivo experiments. The tested heavy metal ions affect photosynthesis probably mainly by inhibition of these key carboxylating enzymes: this mechanism was studied in thein vitro experiments.     

Metallothionein induction in human peripheral blood lymphocytes by heavy metals

Human peripheral blood lymphocytes have the capacity to produce metallothioneins (MTs) as a protective response to cadmium exposure. To define the range of metal species inducing lymphocyte MTs, cellular proteins synthesized after exposure to each of 11 heavy metals were analyzed by gel electrophoresis. Toxic metals such as cadmium, mercury and silver were found to induce thioneins (apoproteins of MTs) at relatively low concentrations (maximum at approximately 10 microM), whereas less toxic metals such as zinc, copper and nickel were inductive at relatively high concentrations (maximum at approximately 200 microM). Tin, lead, iron, cobalt, and manganese did not induce thioneins. The heavy metal specificity of MT induction in the lymphocyte resembles that in the liver, and the regulatory mechanism of MT production seems to be similar in both of these tissues. In the cells exposed to highly toxic metals such as cadmium and mercury, expression of cytotoxicity (represented by decline of cysteine uptake) was remarkable at the metal concentrations higher than those saturating thionein induction, supporting the protective role of MTs against heavy metals.     

X-ray microanalysis (EDXMA) of cadmium-exposed eggs of Bothriocephalus acheilognathi (Cestoda: Bothriocephalidea) and the influence of this heavy metal on coracidial hatching and activity

Over recent years it has been established that pollutants can have a significant impact on host-parasite systems in the aquatic environment, so much so that it has been proposed that parasite fauna may be a useful parameter to monitor water quality. Surprisingly, with perhaps the exception of trematodes and bioaccumulation in adult acanthocephalans, detailed observations on the interaction between helminths, particularly cestodes, and pollutants such as heavy metals, are lacking. In this study, eggs of the carp tapeworm, Bothriocephalus acheilognathi were exposed to a range of cadmium concentrations (0.1, 10, 100 and 10,000 μg/L) and coracidial hatching and survival assessed. Results indicated that the egg is highly resistant to heavy metal pollution and hatching occurs even at 10,000 μg/L. In contrast, the activity of the liberated coracidium significantly decreased after 1 h exposure to cadmium at 10 and 100 μg/L. Electron microscopic X-ray microanalysis of parasite eggs exposed to 1000 and 10,000 μg/L cadmium revealed that cadmium accumulates on the surface of the egg and does not penetrate detectably into the enclosed coracidium. This means that the parasite eggs may be able to withstand a heavy metal pollutant incident.     

Synthesis of the low molecular weight heat shock proteins in plants

Heat shock of living tissue induces the synthesis of a unique group of proteins, the heat shock proteins. In plants, the major group of heat shock proteins has a molecular mass of 15 to 25 kilodaltons. Accumulation of these proteins to stainable levels has been reported in only a few species. To examine accumulation of the low molecular weight heat shock proteins in a broader range of species, two-dimensional electrophoresis was used to resolve total protein from the following species: soybean (Glycine max L. Merr., var Wayne), pea (Pisum sativum L., var Early Alaska), sunflower (Helianthus annuus L.), wheat (Triticum aestivum L.), rice (Oryza sativa L., cv IR-36), maize (Zea mays L.), pearl millet (Pennisetum americanum L. Leeke, line 23DB), and Panicum miliaceum L. When identified by both silver staining and incorporation of radiolabel, a diverse array of low molecular weight heat shock proteins was synthesized in each of these species. These proteins accumulated to significant levels after three hours of heat shock but exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation. Although most appeared to be synthesized only during heat shock, some were detectable at low levels in control tissue. Compared to the monocots, a higher proportion of low molecular weight heat shock proteins was detectable in control tissues from dicots.     

Effects of heavy metal stress on free amino acids in the haemolymph and proteins in haemolymph and total body tissue of Lymantria dispar larvae parasitized by Glyptapanteles liparidis

Heavy metal contamination of the forest pest insect Lymantria dispar (L.) (Lepidoptera; Lymantriidae), the gypsy moth, can alter its haemolymph composition, as has already been shown for carbohydrates and lipids in recent studies. L. dispar larvae are frequently parasitized by Glyptapanteles liparidis (Bouché) (Hymenoptera; Braconidae) larvae, which can—to some extent—regulate the population size of the pest insect. The parasitoids feed on the haemolymph of L. dispar larvae; hence, a different haemolymph composition of the host alters the trophic situation of the parasitoids. The aim of the present study was to investigate whether metal contamination also affects the concentrations of free amino acids in L. dispar haemolymph, and protein concentrations in their haemolymph and tissue. L. dispar larvae were parasitized on the first day of the second instar and then reared on diets contaminated with Cd, Pb, Cu or Zn at two concentrations each. Haemolymph and total body tissue of the larvae (fourth instar/third day) were analyzed. The concentrations of the free amino acids were elevated in five out of the eight contamination groups (Cd6, Pb4, Cu6, Cu10, Zn60), whereas haemolymph protein concentrations were significantly reduced in all contaminated individuals. The haemolymph protein concentration was 18 mg/ml in the control group and decreased to less than 10 mg/ml due to cadmium and zinc contamination at both concentrations and in the low copper contamination group. In contrast, total body proteins (136 g/mg dry weight in the control group) were elevated due to heavy metal stress. Analyses of haemolymph protein concentrations during the fourth instar demonstrated an increase of the proteins from day one to day four (followed by a decrease on the fifth day) in the control group and the cadmium contamination group. A steady increase of proteins from the first to the fifth day in the copper and zinc contaminated larvae indicated a retarded development in these groups. Thus, the present study along with other recent studies demonstrated, that heavy metal stress changes the concentrations of all main haemolymph compounds of L. dispar larvae.