Metallothioneins and resistance to cadmium poisoning in Drosophila cells

Toxicity of cadmium on Drosophila cell lines has been studied. Maximal tolerance for cadmium chloride is 10 microM. Metallothioneins are induced in Drosophila cells following cadmium addition. A stable cadmium resistant cell line (Cd R200) has been selected starting from the haploid D clone. The Cd R200 cells are diploid and display metallothionein levels 22 times higher than cells of the original line fully induced with cadmium. The 200 microM CdCl2 tolerance upper limit in Cd R200 line is overcome if L-cysteine is supplemented to the medium. It is thus possible, in the presence of 5 mM L-cysteine, to select cells able to resist 800 microM CdCl2. These cells produce 4 times more metallothioneins than Cd R200 cells.     

Comparison of cadmium-metallothionein synthesis in parenchymal and non-parenchymal rat liver cells.

The time course of cadmium-metallothionein synthesis was studied in non-parenchymal and parenchymal cells, isolated by a cell-separation technique from the livers of rats after the simultaneous injection of CdCl2 (0.05 mg of Cd/kg) and a 10-fold molar excess of 2,3-dimercaptopropanol. Under these conditions of dosing, in contrast with the injection of CdCl2 alone, both cell types accumulate similar concentrations of Cd and synthesize equivalent concentrations of metallothionein. It is concluded that both cell types have a similar capacity to synthesize the metalloprotein, and that the limiting factor under normal cadmium exposure is the relatively inefficient metal uptake into the non-parenchymal cells.     

Metallothionein in the liver of the small lizard Podarcis muralis

A cysteine-rich protein presenting optical and biochemical features typical of metallothionein and a similar amino acid composition was found in the liver of the small lizard Podarcis muralis. Animals were given either CdCl2 (0.8 mg Cd2+/kg body wt) or saline (NaCl 0.9%) by i.p. injection for 3 days. A second group of animals were injected with a single dose of [35S]cysteine plus CdCl2 or saline. Lizard MT contained Zn and Cu when injected with saline and also Cd when injected with CdCl2. Metallothionein induction by cadmium was demonstrated by radioactive labelling.     

Cadmium-bound metallothionein induces apoptosis in rat kidneys, but not in cultured kidney LLC-PK1 cells

The ability of cadmium-bound metallothionein(Cd-MT) to induce apoptosis was investigated in vivo and in vitro. Administration of purified Cd-MT (0.15 mg MT bound Cd per kg body weight) to the rat induces DNA fragmentation, a biochemical characteristic of apoptosis in the kidney at 16 h, which was detectable by ethidium bromide staining on an agarose gel. It was still detected 24 h after administration. Induction of apoptosis by Cd-MT was specific to kidney; it was not observed in cerebrum, cerebellum, heart, lung, liver, testis, dorsolateral prostate, and ventral prostate. In contrast, addition of Cd-MT (0.01-100 microM) to the cultured porcine kidney LLC-PK1 cells failed to induce apoptosis under the condition where cadmium chloride (10 microM) did. There was no additivity of induction of apoptosis by CdCl2 (10 microM) in the presence of Cd-MT (0.01-100 microM). To examine the effect of intracellular MT on cadmium-induced apoptosis in cultured cells, new cell lines were established, which constitutively produce MT, being termed as Cd(r)-LLC-PK1 cells since Cd-MT exogenously added had much less permeability to the cultured cells. Followed by exposure of wild-type LLC-PK1 cells to 50 microM CdCl2 for 24 h, the surviving cells(Cd(r)-LLC-PK1 cells) induce MT at the level of 1.9 microg/2 x 10(6) cells. In Cd(r)-LLC-PK1 cells, 10 microM CdCl2 failed to induce apoptosis, but 60 microM CdCl2 could exert the apoptotic response, indicating that intracellular MT which was induced by CdCl2 did not facilitate CdCl2-elicited apoptosis. Furthermore, chromatin in rat kidneys was condensed by Cd-MT, but not that in LLC-PK1 cells. Thus, Cd-MT induces apoptosis in rat kidneys, but not in the cultured renal cells, suggesting that the ionic form of cadmium was required for programmed cell death.     

Modulation of acute cadmium toxicity by Emblica officinalis fruit in rat

The efficacy of Emblica officinalis in modifying the acute cytotoxicity of cadmium in male rats was evaluated. Oral administration of Emblica fruit juice (500 mg/kg, b.w.) for 8 days followed by a single toxic dose of Cd as CdCl2 (3 mg/kg,b.w. ip), considerably reduced the mortality in rats as well as prevented to some extent the cadmium induced histopathological damage in testis, liver and kidneys. Biochemical investigation also revealed reduced levels of Cd induced serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and gamma glutamyltranspeptidase. The enhanced levels of Cd and lipid peroxidation in liver, kidney, and testes and metallothionein and total sulphydryl in liver and kidney by Cd were significantly reduced by Emblica pretreatment. These results suggest cytoprotective potential of Emblica fruit in acute cadmium toxicity which could be due to its multiple role in biological system.     

Growth and Heavy Metal Binding Properties of Transgenic Chlamydomonas Expressing a Foreign Metallothionein Gene

We have compared the growth rates and cadmium binding capacity of wild-type and transgenic Chlamydomonas reinhardtii cells expressing a foreign class-II metallothionein. We observed that cells expressing metallothionein grew to significantly higher cell densities than wild-type cells in the presence of a toxic cadmium concentration (40 μM). When grown at a low (5 μM) cadmium concentration, cells expressing metallothionein bound twofold more cadmium (0.43 μg Cd)mg Ch1) than wild-type. At cadmium concentrations (40 μM), which induce phytochelatin synthesis in wild-type cells the cadmium binding capacity of both wild-type (79.6 μg Cd)mg Ch1) and transformed cells (86.4 μg Cd)mg Ch1) was similar; however, the transformed cells grew to higher densities than the wild type. These results suggest that under conditions that apparently induce phytochelatin expression, the presence of metallothionein in the cytoplasm reduces heavy metal toxicity. Furthermore, because cells expressing metallothionein grow to higher densities than wild-type cells at a toxic cadmium concentration (40 μM), the transgenic cells sequester more total cadmium (9% of total Cd) from the medium than the wild type (5.5% of total Cd). These results indicate that the trace-metal binding properties of Chlamydomonas can be enhanced through the expression of trace-metal-specific binding proteins.     

Protection against cadmium toxicity and enzyme inhibition by dithiothreitol

In the present in vivo studies the alterations in cation transporting enzymes of the brain, kidney and liver tissues were assessed at intervals between 0 to 48 h after a single, acute (10 mg kg-1, i.p.) dose of cadmium (Cd). The inhibition of Na+-K+-ATPase during the first 24 h does not parallel the changes in K+-PNPPase suggesting differential effects on phosphorylation and dephosphorylation steps of the overall ATPase reaction. Between 30 min to 2 h the inhibition in enzyme activity was steep (27 per cent in brain, 54 per cent in liver) followed by a rapid reversal between 2-6 h. This critical period may correspond to the time of induction of metallothionein. This enzyme reversal was followed by a significant decrease in Na+-K+ ATPase (40-68 per cent) and K+-PNPPase (44-60 per cent) between 24 to 48 h. A similar pattern was observed in Ca2+-ATPase in all the three tissues. A 33 per cent mortality was observed in rats after 48 h of cadmium challenge. Administration of dithiothreitol (DTT, 20 mg kg-1, i.p.) to CdCl2 pretreated rats at 24 h resulted in mortality reduced from 33 per cent to 0 and reversal in the inhibition of Na+-K+-ATPase in brain and kidney and Ca2+-ATPase in brain. Since protection of brain and kidney enzymes by DTT paralleled its protection against Cd toxicity, their inhibition by Cd may, in part, constitute the biochemical basis of Cd toxicity.     

Glutathione, a first line of defense against cadmium toxicity

Experimental modulation of cellular glutathione levels has been used to explore the role of glutathione in cadmium toxicity. Mice treated with buthionine sulfoximine [an effective irreversible inhibitor of gamma-glutamylcysteine synthetase (EC 6.3.2.2) that decreases cellular levels of glutathione markedly] were sensitized to the toxic effects of CdCl2. Mice pretreated with a sublethal dose of Cd2+ to induce metallothionein synthesis were not sensitized to Cd2+ by buthionine sulfoximine. Mice sensitized to Cd2+ by buthionine sulfoximine were protected against a lethal dose of Cd2+ by glutathione mono isopropyl ester (L-gamma-glutamyl-L-cysteinylglycylisopropyl ester), but not by glutathione. These results are in accord with studies that showed that glutathione mono esters (in contrast to glutathione) are efficiently transported into cells and converted intracellularly to glutathione. The findings indicate that intracellular glutathione functions in protection against Cd2+ toxicity, and that this tripeptide provides a first line of defense against Cd2+ before induction of metallothionein synthesis occurs. The experimental approach used here in which cellular levels of glutathione are decreased or increased seems applicable to investigation of other types of metal toxicity and of other glutathione-dependent biological phenomena.     

Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum.

Using a yeast expression vector system, we have expressed both wild type and six mutated Chinese hamster metallothionein coding sequences in a metal-sensitive yeast strain in which the endogenous metallothionein gene has been deleted. The mutant proteins have single or double cysteine to tyrosine replacements (C13Y, C50Y, and C13,50Y), single cysteine to serine replacements (C13S and C50S), or a single cysteine to alanine replacement (C50A). These proteins function in their yeast host in cadmium detoxification to differing extents. Metallothioneins which contain a cysteine mutation at position 50 (C50Y, C50S, C50A, and C13,50Y) conferred markedly less cadmium resistance than wild type metallothionein, or metallothionein with a single cysteine mutation at position 13 (C13Y and C13S). Wild type and three of the mutant Chinese hamster metallothioneins (C13Y, C50Y, and C13,50Y) were purified from yeast grown in subtoxic levels of either CdCl2 or 113CdCl2. All three of the mutant proteins bound less cadmium than the wild type protein when metal-binding stoichiometries were determined. The one-dimensional 113Cd NMR spectrum of the recombinant wild type Chinese hamster metallothionein was compared to the spectra of native rat and rabbit liver metallothioneins. The close correspondence between the 113Cd chemical shifts in these metallothioneins is consistent with the presence of two separate metal clusters, A and B, corresponding, respectively, to the alpha- and beta-domains, in the recombinant metallothionein. The one-dimensional 113Cd NMR spectra recorded on each of the three mutant metallothioneins, on the other hand, provide some indication as to the structural basis for the reduced, by one, metal stoichiometry of each of the mutant metallothioneins. For the C13Y mutant, it appears that the beta-domain now binds a total of two metal ions whereas with the C50Y mutant, the alpha-domain appears metal-deficient. For the double mutant, C13,50Y, the 113Cd resonances are indicative of major structural reorganizations in both domains.     

Cadmium uptake and toxicity via voltage-sensitive calcium channels

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.     

Uptake of cadmium is diminished in transfected mouse NIH/3T3 cells enriched for metallothionein.

To determine the relationship between cellular uptake of cadmium and content of metallothionein, we measured uptake of 109Cd in cells that differed in content of metallothionein (MT). MT cells were derived from NIH/3T3 cells by transfection with a plasmid containing the genome of bovine papilloma virus and the mouse metallothionein-I gene, driven by the promotor for the glucose-regulated protein of 78 kDa. Control cells were similarly transfected with bovine papilloma virus-based plasmids with the gene for metallothionein inverted and thus separated from the promoter (TM), or deleted, along with the promoter (BPA). The number of copies of bovine papilloma virus-based plasmids was similar in MT, TM, and BPA cells, approximately 100 per cell. MT cells were more than 10 times more resistant to the lethal effect of cadmium than were the control cells. Synthesis of metallothionein was 15-fold greater in the MT cells than in the TM or BPA cells. The uptake of 109Cd by the cells enriched in metallothionein was 4-fold less than by the control cells. These data suggest that an increased content of metallothionein may protect some cells from the toxic effects of cadmium, in part, by diminishing uptake of the metal.     

Taurine protects the antioxidant defense system in the erythrocytes of cadmium treated mice

The present study was undertaken to investigate the protective role of taurine (2-aminoethanesulfonic acid) against cadmium (Cd) induced oxidative stress in murine erythrocytes. Cadmium chloride (CdCl(2)) was chosen as the source of Cd. Experimental animals were treated with either CdCl(2) alone or taurine, followed by Cd exposure. Cd intoxication reduced hemoglobin content and the intracellular Ferric Reducing/Antioxidant Power of erythrocytes, along with the activities of antioxidant enzymes, glutathione content, and total thiols. Conversely, intracellular Cd content, lipid peroxidation, protein carbonylation, and glutathione disulphides were significantly enhanced in these cells. Treatment with taurine before Cd intoxication prevented the toxin-induced oxidative impairments in the erythrocytes of the experimental animals. Overall, the results suggest that Cd could cause oxidative damage in murine erythrocytes and that taurine may play a protective role in reducing the toxic effects of this particular metal.     

Growth inhibition of the filamentous fungus Aspergillus nidulans by cadmium: an antioxidant enzyme approach

The heavy metal cadmium is very toxic to biological systems. Although its effect on the growth of microorganisms and plants has been investigated, the response of antioxidant enzymes of Aspergillus nidulans to cadmium is not well documented. We have studied the effect of cadmium (supplied as CdCl(2)) on catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR). 0.005 mM CdCl(2) had a very slight stimulatory effect on the growth rate of A. nidulans, but at concentrations above 0.025 mM, growth was totally inhibited. The accumulation of Cd within the mycelium was directly correlated with the increase in the concentration of CdC(2) used in the treatments. Although a cadmium-stimulated increase in SOD activity was observed, there was no change in the relative proportions of the individual Mn-SOD isoenzymes. Higher concentrations of CdCl(2) induced a small increase in total CAT activity, but there was a major increase in one isoenzymic form, that could be separated by gel electrophoresis. GR activity increased significantly following treatment with the highest concentration (0.05 mM) of CdCl(2). The increases in SOD, CAT, and GR activities suggest that CdCl(2) induces the formation of reactive oxygen species inside the mycelia of A. nidulans.     

Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress

Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 microM CdCl(2) stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 microM CdCl(2). Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels.     

Prevention by copper of cadmium sequestration by metallothionein in liver.

Following chronic CdCl2 administration to rats, more than 98% of the metal in liver supernatant is bound to the low molecular weight binding protein, metallothionein. Simultaneous administration of high doses of Cd and copper salts result in an increase in toxicity which is accompanied by a failure of Cd sequestration by metallothionein in vivo. This may be due to an aggregation of metallothionein which has been observed in the presence of copper in vitro.     

Properties of cadmium-binding proteins in rat testes. Characteristics unlike metallothionein.

Since the exposure of rats to cadmium causes zinc to accumulate in metallothionein in liver and kidney but not in a similar protein in the testes, the properties of the low-Mr cadmium-binding proteins were investigated in rat testes. Weanling rats that had been given dietary cadmium for 6 weeks were injected with 109CdCl2 and subsequently killed, and the 109Cd-labelled low-Mr proteins from testes were purified. The pooled low-Mr cadmium-containing fractions from the gel-filtration (Sephadex G-75) columns were eluted through DEAE-Sephacel columns, yielding two peaks. Each of the individual peaks from this Sephacel column was further purified by rechromatography on DEAE-Sephacel and on Bio-Gel P-10 columns. Amino acid analysis of the two purified proteins revealed a low cysteine (about 3%) content, with aspartate, glutamate and glycine as the predominant amino acids. Thus these low-Mr cadmium-binding proteins induced by cadmium in rat testes do not appear to be metallothionein.     

Combination effects of chronic cadmium exposure and gamma-irradiation on the genotoxicity and cytotoxicity of peripheral blood lymphocytes and bone marrow cells in rats

This work investigated the effects of chronic cadmium (Cd) exposure combined with γ-ray irradiation on the cytotoxicity and genotoxicity of peripheral blood cells and bone marrow cells in rats. Results showed that when the rats were exposed to low dose (LD) Cd of 0.1mg CdCl?/(kgd) for 8 and 12 weeks, the Cd concentration in blood reached to 135-140 μg/L and no toxic effects on peripheral blood lymphocytes, white blood cells (WBC) and granulocyte-monocyte (GM) progenitor cells were observed except polychromatic erythrocytes (PCE) of bone marrow. Moreover, this chronic LD Cd exposure significantly decreased irradiation-induced micronucleus (MN) formation and hypoxanthine-guanine phosphoribosyl transferase (hprt) mutation in lymphocytes and PCE, while the combination of LD Cd exposure and irradiation induced the additive metallothionein (MT) protein expression in bone marrow cells. When the rats were exposed to a high dose (HD) Cd of 0.5mg CdCl/?(kgd) for 8 and 12 weeks, the blood Cd level approached to 458-613 μg/L and an inflammatory response was induced, meanwhile, MN formation and hprt mutation were markedly increased, and the ratio of PCE/NCE (normochromatic erythrocyte) was significantly decreased. Furthermore, when the rats were exposed to HD Cd plus 2 Gy irradiation, additive toxic effects on MN formation, hprt mutation, PCE damage and GM progenitor cell proliferation were observed, while this combination treatment resulted in an obvious reduction of MT protein compared to HD Cd group. In conclusion, chronic exposure to LD Cd induced the adaptive response to irradiation in the genotoxicity of peripheral blood lymphocytes and PCE of bone marrow by the up-regulation of Cd-induced MT protein, but the combination of HD Cd exposure and irradiation generated the additive effects on the cytotoxicity and genotoxicity in peripheral blood lymphocytes and bone marrow cells.     

Induction of chromosomal aberrations in cultured Chinese hamster cells by short-term treatment with cadmium chloride

Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride (CdCl2) was investigated under 3 different treatment conditions: (i) 2-h treatment in MEM medium supplemented with 10% fetal bovine serum (MEM + 10% FBS) or (ii) in HEPES-buffered Hanks' solution (HEPES-Hanks), and (iii) continuous treatment for 24 h in MEM + 10% FBS. Two-h treatment with CdCl2 in HEPES-Hanks or continuous treatment for 24 h in MEM + 10% FBS was respectively 2 or 3 times more cytotoxic than 2-h treatment with the metal in MEM + 10% FBS. Continuous treatment for 24 h with a CdCl2 concentration in excess of 5 X 10(-6) M was too toxic to the cells to allow chromosomal analysis, and moreover, only a slight increase in incidence of chromosomal aberrations was observed at a concentration of 5 X 10(-6) M CdCl2. In contrast, a marked and concentration-dependent increase in incidence of chromosomal aberrations was observed after post-treatment culture for 22 h follows 2-h treatment with 1 X 10(-6) M to 5 X 10(-5) M of CdCl2 in both MEM + 10% FBS and HEPES-Hanks. Two-h treatment with cadmium in HEPES-Hanks was approximately 3 times more potent for the induction of chromosomal aberrations than that in MEM + 10% FBS. Types of aberrations induced by CdCl2 mainly consisted of chromatid gaps and breaks, although a few exchanges, dicentrics and fragmentations were observed at high concentrations of cadmium. Increase in incidence of tetraploidy was also observed with a concentration dependency after 2-h treatment with CdCl2. Potency of CdCl2 to induce chromosomal aberrations after 2-h exposure was comparable to that of benzo[a]pyrene activated with S9 at equitoxic concentrations. Two-h treatment with cadmium markedly inhibited incorporation of [3H]thymidine, even at concentrations at which incorporation of [3H]uridine or [3H]leucine was less inhibited. However, the inhibition of [3H]thymidine incorporation by cadmium was reversible and the incorporation restored to the control level during 2-6 h of post-treatment incubation. These findings suggest that restoration of DNA synthesis after cadmium exposure is required for the efficient detection of chromosomal aberrations induced by the metal.     

土壤镉污染与作物

通过土壤镉污染对作物影响的盆栽模拟试验,以揭示重金属镉在土壤—植物系统中的转移、分布规律及其对植物生长、发育的影响。试验证明不同化合形态的镉施入土壤(砂壤质褐土,pH值8.2),水稻对镉吸收的多寡依次为CdCl2>CdSO4>CdO>CdS>CdCO3。白菜的镉吸收表现为CdSO4>CdCl2>CdO>CdCO3。土壤的不同镉浓度(施加CdCl2,以纯镉计)对作物影响的试验结果表明,可食部位达到食品污染标准(谷物含镉量0.4ppm、蔬菜0.2ppm为暂定标准)时的土壤镉污染临界值分别为：小麦、莴苣、白菜<1ppm,茄子、萝卜<2.5ppm,番茄、菜豆<20ppm。土壤因子的处理影响镉的活动性;降低土壤pH值,水稻的镉吸收增加。增施有机肥、ZnSO4、S、CaO、CaSO4可降低糙米含镉量13.4%一30%。白菜的镉吸收,由于增施有机肥、FeSO4、Fe2O3、CaO或S而降低菜叶含镉量28%一61%。以Cd,Zn比1:100或1:200施入土壤,叶内含镉量分别下降61%和76.4%,但白菜产量减少61%和76%。