Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.     

Interactions of bismuth complexes with metallothionein(II).

Bismuth complexes are widely used as anti-ulcer drugs and can significantly reduce the side effects of platinum anti-cancer drugs. Bismuth is known to induce the synthesis of metallothionein (MT) in the kidney, but there are few chemical studies on the interactions of bismuth complexes with metallothionein. Here we show that Bi(3+) binds strongly to metallothionein with a stoichiometry bismuth:MT = 7:1 (Bi(7)MT) and can readily displace Zn(2+) and Cd(2+). Bismuth is still bound to the protein even in strongly acidic solutions (pH 1). Reactions of bismuth citrate with MT are faster than those of [Bi(EDTA)](-), and both exhibit biphasic kinetics. (1)H NMR data show that Zn(2+) is displaced faster than Cd(2+), and that both Zn(2+) and Cd(2+) in the beta-domain (three metal cluster) of MT are displaced by Bi(3+) much faster than from the alpha-domain (four metal cluster). The extended x-ray absorption fine structure spectrum of Bi(7)MT is very similar to that for the glutathione and N-acetyl-L-cysteine complexes [Bi(GS)(3)] and [Bi(NAC)(3)] with an inner coordination sphere of three sulfur atoms and average Bi-S distances of 2.55 A. Some sites appear to contain additional short Bi-O bonds of 2.2 A and longer Bi-S bonds of 3.1 A. The Bi(3+) sites in Bi(7)MT are therefore highly distorted in comparison with those of Zn(2+) and Cd(2+).     

Renal function in the freshwater rainbow trout after dietary cadmium acclimation and waterborne cadmium challenge

Renal function was examined in adult rainbow trout (Oncorhynchus mykiss) after chronic exposure to a sublethal level of dietary Cd (500 mg/kg diet) for 52 d and during a subsequent challenge to waterborne Cd (10 microg/L) for 72 h. Dietary Cd had no major effects on UFR (urine flow rate) and GFR (glomerular filtration rate) but caused increased renal excretion of glucose, protein, and major ions (Mg(2+), Zn(2+), K(+), Na(+), Cl(-) but Ca(2+)). However, dietary Cd did not affect any plasma ions except Na(+) which was significantly elevated in the Cd-acclimated trout. Plasma glucose and ammonia levels fell by 25% and 36% respectively, but neither plasma nor urine urea was affected in Cd-acclimated fish. Dietary Cd exposure resulted in a remarkable increase of Cd load in the plasma (48-fold, approximately 22 ng/mL) and urine (60-fold, 8.9 ng/mL), but Cd excretion via the kidney was negligible on a mass-balance basis. Clearance ratio analysis indicates that all ions, Cd, and metabolites were reabsorbed strongly (58-100%) in both na?ve and dietary Cd exposed fish, except ammonia which was secreted in both groups. Mg(2+), Na(+), Cl(-) and K(+) reabsorption decreased significantly (3-15%) in the Cd-exposed fish relative to the control. Following waterborne Cd challenge, GFR and UFR were affected transiently, and only Mg(2+) and protein excretion remained elevated with no recovery with time in Cd-acclimated trout. Urinary Ca(2+) and Zn(2+) excretion rates dropped with an indication of renal compensation towards plasma declines of both ions. Cadmium challenge did not cause any notable effects on urinary excretion rates of metabolites. However, a significant decrease in Mg(2+) reabsorption but an increase in total ammonia secretion was observed in the Cd-acclimated fish. The study suggests that dietary Cd acclimation involves physiological costs in terms of renal dysfunction and elevated urinary losses.     

Evidence for cadmium uptake through Nramp2: metal speciation studies with Caco-2 cells.

The specific uptake of 0.3 microM (109)Cd by the TC7 clone of the human enterocytic-like Caco-2 cells increased 4-fold as the pH(out) was lowered from 7.5 to 5.5; the stimulatory effect of acidic media being more pronounced when the level of the free ion (109)Cd(2+), relative to total (109)Cd, was increased. The initial uptake rate was 12-fold higher under conditions, optimizing (109)Cd(2+) accumulation over that of (109)CdCl(2-n)(n) (NO(-)(3)/pH(out) 5.5); a saturable system of transport has been characterized (K(m) = 1.1 +/- 0.1 microM, V(max) = 87 +/- 3 pmol/3 min/mg protein). An excess of Fe(2+) failed to affect (109)Cd uptake when the pH(out) was 7.4, whereas a strong inhibition was observed under NO(-)(3)/pH(out) 5.5 conditions. In contrast, the maximal inhibitory effect of Zn(2+) was observed under Cl(-)/pH(out) 7.4 conditions. This results strongly suggest that Fe(2+) may compete with Cd(2+) for Nramp2, whereas Zn and CdCl(2-n)(n) compete for another system of transport that has yet to be identified.     

Antiserum specific for the intact isoform-3 of metallothionein

The recombinant form of isoform-3 of mouse brain metallothionein (MT3) was used as an antigen to immunize rabbits and raise MT3-selective antiserum. The antiserum was essentially specific for MT3 with 100-fold greater sensitivity for MT3 compared to MT1 or MT2. Immunonblot analysis of whole mouse brain homogenates showed that MT3 was present only in the fraction retained by a 30,000-Da cut-off filter. The antiserum was used to immunoprecipitate MT3 from mouse brain extracts of Swiss Webster mice and provided evidence that MT3 was a member of a macromolecular complex of greater than 30,000 Da mass in brain. An ELISA was developed using purified, recombinant mouse brain Cd(7)-MT3 as the antigen and used to quantify MT3 in mouse brain extracts. The concentration of MT3 was found to be 3.0+/-0.8 microg/ml or approximately 3.5 microg/g mouse brain (wet weight).     

Ni2+ Transport and Accumulation in Rhodospirillum rubrum

The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni(2+) transport assay was used to monitor uptake and accumulation of (63)Ni(2+) into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on (63)Ni(2+) transport and accumulation. Cells grown either in the presence or absence of CO transported Ni(2+) with a K(m) of 19 +/- 4 microM and a V(max) of 310 +/- 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni(2+) the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn(2+), Mg(2+), Ca(2+), and Zn(2+) showed no inhibition of (63)Ni(2+) transport but Co(2+), Cd(2+), and Cu(2+) inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4 degrees C), by protonophores (carbonyl cyanide m-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N, N'-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni(2+) transport. (63)Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated (63)Ni(2+) accumulation coincided with the appearance of CODH activity in the culture, suggesting that the (63)Ni(2+) was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased (63)Ni(2+) accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated (63)Ni(2+) like cells unexposed to CO.     

Maternal cadmium exposure induces mt2 and smtB mRNA expression in zebrafish (Danio rerio) females and their offspring

The present study aimed to identify the effects of maternal cadmium (Cd(2+)) exposure on the mRNA expression of mt2 (metallothionein-2) and smtB (similar to metallothionein-B) in female zebrafish (Danio rerio) and their offspring (F1 larvae). Zebrafish females were exposed to 0, 8.9, 17.8, and 35.6 μM Cd(2+) for 72 h, and their ovaries and F1 larvae were collected to measure their Cd(2+) contents and their smtB and mt2 mRNA expression. Cd(2+) contents and the mRNA expression of smtB and mt2 in F1 larvae all showed positive correlations with the maternal Cd(2+) treatment dose. The mt2 was 1.9- to 3.4-fold higher than smtB in F1 larvae. Furthermore, F1 larvae had noticeably enhanced Cd(2+) tolerance after maternal Cd(2+) treatment. These results demonstrate that maternal Cd(2+) was transferred to larval fish and induced mt2 and smtB mRNA expression to protect larva against the impacts of Cd(2+). In female ovaries, mt2 expression showed a noticeable increase after exposure to a metal environment, while smtB did not show exactly the same effect. The study can only conclude that smtB might have a much different role other than just protecting against the impacts of metals.     

Cd2+ versus Zn2+ uptake by the ZIP8 HCO3--dependent symporter: kinetics, electrogenicity and trafficking

The mouse Slc39a8 gene encodes the ZIP8 transporter, which has been shown to be a divalent cation/HCO3- symporter. Using ZIP8 cRNA-injected Xenopus oocyte cultures, we show herein that: [a] ZIP8-mediated cadmium (Cd(2+)) and zinc (Zn(2+)) uptake have V(max) values of 1.8+/-0.08 and 1.0+/-0.08 pmol/oocyte/h, and K(m) values of 0.48+/-0.08 and 0.26+/-0.09 microM, respectively; [b] ZIP8-mediated Cd(2+) uptake is most inhibited by Zn(2+), second-best inhibited by Cu(2+), Pb(2+) and Hg(2+), and not inhibited by Mn(2+) or Fe(2+); and [c] electrogenicity studies demonstrate an influx of two HCO3- anions per one Cd(2+) (or one Zn(2+)) cation, i.e. electroneutral complexes. Using Madin-Darby canine kidney (MDCK) polarized epithelial cells retrovirally infected with ZIP8 cDNA and tagged with hemagglutinin at the C-terminus, we show that-similar to ZIP4-the ZIP8 eight-transmembrane protein is largely internalized during Zn(2+) homeostasis, but moves predominantly to the cell surface membrane (trafficking) under conditions of Zn(2+) depletion.     

Identification and characterization of heavy metal-resistant unicellular alga isolated from soil and its potential for phytoremediation

A unicellular alga displaying a high growth rate under heterotrophic growth conditions was isolated from soil and identified as Chlorella sorokiniana. The optimal temperature for growth was 35 degrees C and the optimal pH was 6.0-7.0. Glucose, sucrose, galactose, maltose, and soluble starch served as carbon sources supporting growth under dark conditions. The cell yield was 50 g/l (wet weight) in a heterotrophic medium containing 3% glucose. Isolated unicellular algae were highly resistant to heavy metals such as Cd(2+), of which the minimal inhibitory concentration was 4 mM. Algae were capable of taking up the heavy metal ions Cd(2+), Zn(2+) and Cu(2+) at 43.0, 42.0 and 46.4 microg/mg dry weight, respectively. Growth inhibition of Oryza sative shoots by 5 ppm Cd(2+) in hydroponic medium was completely prevented by the addition of 0.25 mg of wet Chlorella cells. These results indicated that this isolate was potentially useful for phytoremediation by preventing environmental dispersion of heavy metals.     

The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd(2+), Mn(2+), Mg(2+), Zn(2+), Co(2+) and Ni(2+) were approximately a linear function of heats of hydration of the corresponding ions. Ba(2+), Sr(2+), Ca(2+), Cu(2+) and Be(2+) did not activate the enzyme; Be(2+) inhibited the reaction in the presence of Mg(2+) and, to a greater extent, in the presence of Ni(2+). 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8.0 and 25 degrees , in the presence of 3mm concentrations of the unvaried reactants, were 88+/-7mum-ATP, 42+/-4mum-NMN and 85+/-4mum-Mg(2+). The results at this pH and at pH7.5 were consistent with mechanisms in which Mg(2+)-ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide-hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni(2+) and Co(2+) than with Mg(2+).     

Maternal transfer of cadmium tolerance in larval Oreochromis mossambicus

Exposing female tilapia to Cd did not affect reproduction and egg quality, nor the survival of their offspring. The LT ₅₀ of Cd-exposed larvae (within 24 h post hatching) from Cd-treated female Oreochromis mossambicus (mean ±S.E.=10·9±0·5 days, n =4) was greater than that of Cd-exposed larvae from control females (6·9± 1·2 days, n =4). A substantial amount of metallothionein mRNA (MT mRNA) was found in both oocytes and newly hatched larvae from Cd-treated females, but MT mRNA levels were low in newly fertilized eggs. The amount of MT mRNA was very low in all these stages in the control group. Cadmium could not be detected at any stage for either control or Cd-treated group, and metallothionein protein levels were also very low in both groups. The early appearance of MT mRNA in oocytes and newly hatched larvae from Cd-treated females may result in early translation after hatching and explain their tolerance to Cd.     

Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants.

Two different Cd(2+) uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn(2+) uptake system which also takes up Cd(2+) and is induced by Mn(2+) starvation. The calculated K(m) and V(max) are 0.26 microM and 3.6 micromol g of dry cell(-1) min(-1), respectively. Unlike Mn(2+) uptake, which is facilitated by citrate and related tricarboxylic acids, Cd(2+) uptake is weakly inhibited by citrate. Cd(2+) and Mn(2+) are competitive inhibitors of each other, and the affinity of the system for Cd(2+) is higher than that for Mn(2+). The other Cd(2+) uptake system is expressed in Mn(2+)-sufficient cells, and no K(m) can be calculated for it because uptake is nonsaturable. Mn(2+) does not compete for transport through this system, nor does any other tested cation, i.e., Zn(2+), Cu(2+), Co(2+), Mg(2+), Ca(2+), Fe(2+), or Ni(2+). Both systems require energy, since uncouplers completely inhibit their activities. Two Mn(2+)-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn(2+) for growth as the parental strain. Mn(2+) starvation-induced Cd(2+) uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn(2+) or Cd(2+) accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn(2+) and Cd(2+) uptake system.     

Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line)

The rapid generation of H(2)O(2) by Cd(2+)-treated plant cells was investigated in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. The starting point for the generation of H(2)O(2) has been located at the cell plasma membrane using cytochemical methods. Treatment of the cells with diphenyleneiodonium (DPI) and imidazol, both inhibitors of the neutrophil NADPH oxidase, prevented the generation of H(2)O(2) induced by Cd(2+). These data suggest the involvement of an NADPH oxidase-like enzyme leading to H(2)O(2) production through O(2)(*-) dismutation by superoxide dismutase enzymes. To investigate the implication of Ca(2+) channels in a Cd(2+)-induced oxidative burst, different inhibitors of Ca(2+) channels were used. Only La(3+) totally inhibited the generation of H(2)O(2) induced by Cd(2+). However, verapamil and nifedipine, inhibitors of Ca(2+) channels, were not effective. Calmodulin or a Ca(2+)-dependent protein kinase is also implicated in the signal transduction sequence, based on the results obtained with two types of calmodulin antagonists, fluphenazine and N-(-6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7) and staurosporine, an inhibitor of protein kinases. However, neomycin, an inhibitor of the phosphoinositide cycle, did not inhibit the generation of H(2)O(2) induced by Cd(2+), suggesting mainly an induction of the oxidative burst mediated by calmodulin and/or calmodulin-dependent proteins.     

Purification and characterization of specific 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Escherichia coli B

A phosphatase specific for the hydrolysis of 3-deoxy-d-manno-octulosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of Escherichia coli B. The hydrolysis of KDO-8-phosphate to KDO and inorganic phosphate in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated three- to fourfold by the addition of 1.0 mM Co(+) or Mg(2+) and to a lesser extent by 1.0 mM Ba(2+), Zn(2+), and Mn(2+). The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu(2+), Ca(2+), Cd(2+), Hg(2+), and chloride ions (50% at 0.1 M). The pH optimum was determined to be 5.5 to 6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7 to 4.8 and a molecular weight of 80,000 +/- 6,000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of two identical subunits of 40,000 to 43,000 molecular weight. The apparent K(m) for KDO-8-phosphate was determined to be 5.8 +/- 0.9 x 10(-5) M in the presence of 1.0 mM Co(2+), 9.1 +/- 1 x 10(-5) M in the presence of 1.0 mM Mg(2+), and 1.0 +/- 0.2 x 10(-4) M in the absence of added Co(2+) or Mg(2+).     

Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages

The sub-chronic (28–56 days) effects of exposure to low concentrations of cadmium (Cd; 0·05, 0·25, 0·50 and 2·50 μg l⁻¹) shortly following fertilization on embryos, larvae and juvenile rainbow trout Oncorhynchus mykiss were examined. Premature hatching occurred at lower concentrations (0·05 and 0·25 μg l⁻¹ Cd), however, delayed hatching was seen in the 2·50 μg l⁻¹ Cd group, with >90% of hatching occurring on the last day of the hatching period. Larval growth was negatively affected by Cd exposure in a concentration-dependent manner. Larvae exposed to 2·50 μg l⁻¹ Cd were 13·9 ± 0·8% shorter in total length ( L _T) and weighed 22·4 ± 3·5% (mean ± s . e .) less than controls at the end of the exposure period. Plasma sex steroid concentrations (oestradiol in juvenile females and 11-ketotestosterone in juvenile males) were elevated (four- to 10-fold over controls) in exposed fish in both males and females, following 28 days of exposure to 0·05, 0·25 and 0·50 μg l⁻¹ Cd, respectively. These results suggest that environmentally realistic concentrations (in the μg l⁻¹ range) of Cd can affect the development of O. mykiss impacting embryos, larvae and juvenile fish.     

Characterization of stress responses of heavy metal and metalloid inducible promoters in synechocystis PCC6803

In several biotechnological applications of living bacterial cells with inducible gene expression systems, the extent of overexpression and the specificity to the inducer are key elements. In the present study, we established the concentration ranges of Zn(2+), Ni(2+), Co(2+), AsO(2)(-), and Cd(2+) ions that caused significant activation of the respective promoters of Synechocystis sp. without concomitant unspecific stress responses. The low expression levels can be increased up to 10-100-fold upon treatments with Cd(2+), AsO(2)(-), Zn(2+), and Co(2+) ions and up to 800-fold upon Ni(2+) treatment. These results facilitate the development of conditional gene expression systems in cyanobacteria.     

The effects of copper and other ions on the ribonucleic acid polymerase activity of isolated rat liver nuclei

1. The effects of various ions on the Mg(2+)- and Mn(2+)/ammonium sulphate-activated RNA polymerase activities of isolated liver nuclei were studied. 2. The Mg(2+)-activated RNA polymerase reaction was inhibited by more than 60% by Cd(2+), SeO(3) (2-), Be(2+), Cu(2+), Co(2+), Ca(2+) and La(3+), all at 1mm concentrations. 3. The Mn(2+)/ammonium sulphate-activated RNA polymerase reaction was strongly inhibited by Hg(2+), Cd(2+), Cu(2+) and Ag(+). The effect of Hg(2+), Cd(2+) and Ag(+) was relieved by cysteine or mercaptoethanol. 4. Inhibition by Cu(2+) was not affected by addition of DNA, and was relieved only partially by EDTA or histidine. 5. No changes of RNA polymerase activities were observed in nuclei isolated from the liver of rats treated with copper albuminate.     

Multiple characterizations of Listeria monocytogenes sensitive and insensitive variants to divergicin M35, a new pediocin-like bacteriocin

AIMS: Divergicin M35 is a new class IIa bacteriocin produced by Carnobacterium divergicin M35. The bactericidal activity of this antimicrobial peptide was tested against a set of 11 strains of Listeria monocytogenes isolated from food. METHODS AND RESULTS: The minimal inhibitory concentration (MIC) was determined by the microdilution method. The strains tested displayed a different level of sensitivity to divergicin M35. L. monocytogenes LSD530, referred to as DivS strain, was the most sensitive and appeared to be inhibited by concentration of divergicin M35 below 0.13 microg ml(-1). The mutant resistant to divergicin M35, called DivM, was obtained from L. monocytogenes LSD530 (DivS) by gradually increasing the amounts of divergicin M35 until 1.3 microg ml(-1). Notably, DivM was stable after 50 generations. DivS parental strain was inhibited by a concentration of 4 microg ml(-1). L. monocytogenes LSD530 was shown to be resistant to divergicin M35 at 1.3 microg ml(-1). Remarkably, in the presence of divalent cations such as Ca(2+), Mg(2+) and Mn(2+), the lethality caused by divergicin M35 was reduced by 0.48, 0.54 and 0.63 log CFU per ml (after 18 h at 30 degrees C), respectively. The total DNA profiles of DivS and DivM were similar. DivS and DivM showed variable sensitivity to antibiotics. The two-dimensional (2-D) electrophoresis of cell wall proteins did not show any significant difference between DivS and DivM strains but their fatty acid composition showed a significant difference in C(16:0) content. CONCLUSIONS: Resistance to divergicin M35 is likely ascribed to modification in cell wall fatty acid composition rather than protein modification. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides original results contributing to understanding of the resistance of L. monocytogenes to divergicin M35, a new class IIa bacteriocin.