Cyclosporin A-sensitive permeability transition pore is involved in Cd(2+)-induced dysfunction of isolated rat liver mitochondria: doubts no more

There is dose-dependent Cd(2+)-evoked swelling of isolated rat liver mitochondria energized by complex I, II, or IV respiratory substrates in sucrose medium in the absence of added Ca(2+) and P(i), which is prevented by Sr(2+). Permeability transition effectors (ADP, CsA, EGTA, RR, DTT, ATR, P(i), and Ca(2+)) affect in a corresponding way Cd(2+)-promoted membrane permeabilization in NH(4)NO(3), KCl, and sucrose media. Maximal depression of Cd(2+)-induced swelling is achieved by simultaneous addition of ADP, Mg(2+), and CsA that produces either synergistic (NH(4)NO(3)) or additive (KCl and sucrose media) action. Sustained activation by low [Cd(2+)] of mitochondrial basal respiration in KCl medium is observed both in the absence and in the presence of rotenone and/or oligomycin but only in the latter case (rotenone+oligomycin) CsA inhibits completely Cd(2+) activation of St 4 respiration and partially reverses DNP-uncoupled respiration depressed by cadmium. Cd(2+) effects are discussed in terms of comparison with those of Zn(2+) and PhAsO.     

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.     

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.     

Lysosomal involvement in hepatocyte cytotoxicity induced by Cu(2+) but not Cd(2+)

Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, "ROS" formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by "ROS" generated by lysosomal Cu(2+) redox cycling.     

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.     

Dimerization of human growth hormone in the presence of metal ions

Zn(2+) and Co(2+) ions are known to promote human growth hormone reversible dimerization. In these studies, dimerization was also shown to be initiated by nine other metal ions: Cd(2+), Hg(2+), Cu(2+), Ag+, Au(3+), Au+, Pd(2+), Ni(2+), and Pt(4+). In some cases (Hg(2+), Ag(+), Au(3+), and Ni(2+)) formation of higher oligomers also took place. In addition further detailed investigation of dimerization in the presence of Zn(2+) ions was carried out.     

Mitochondrial respiratory chain inhibitors modulate the metal-induced inner mitochondrial membrane permeabilization

To elucidate the molecular mechanisms of the protective action of stigmatellin (an inhibitor of complex III of mitochondrial electron transport chain, mtETC) against the heavy metal-induced cytotoxicity, we tested its effectiveness against mitochondrial membrane permeabilization produced by heavy metal ions Cd2(+), Hg2(+), Cu2(+) and Zn2(+), as well as by Ca2(+) (in the presence of P(i)) or Se (in form of Na?SeO?) using isolated rat liver mitochondria. It was shown that stigmatellin modulated mitochondrial swelling produced by these metals/metalloids in the isotonic sucrose medium in the presence of ascorbate plus tetramethyl-p-phenylenediamine (complex IV substrates added for energization of the mitochondria). It was found that stigmatellin and other mtETC inhibitors enhanced the mitochondrial swelling induced by selenite. However, in the same medium, all the mtETC inhibitors tested as well as cyclosporin A and bongkrekic acid did not significantly affect Cu2(+)-induced swelling. In contrast, the high-amplitude swelling produced by Cd2(+), Hg2(+), Zn2(+), or Ca2(+) plus P(i) was significantly depressed by these inhibitors. Significant differences in the action of these metals/metalloids on the redox status of pyridine nucleotides, transmembrane potential and mitochondrial respiration were also observed. In the light of these results as well as the data from the recent literature, our hypothesis on a possible involvement of the respiratory supercomplex, formed by complex I (P-site) and complex III (S-site) in the mitochondrial permeabilization mediated by the mitochondrial transition pore, is updated.     

Screening of heavy metal-tolerant actinomycetes isolated from the Salí River

Fifty-three strains of actinomycetes resistant to heavy metals were isolated from the Salí River in northwest Argentina. Screening procedures that involve solid and liquid synthetic media containing Cd(2+), Cu(2+), or Hg(2+) allowed the selection of six strains. These strains showed a quantitative sorption of Cd(2+) and Cu(2+) by more than 98% of the initial metal concentrations (0.1, 0.5, and 1.0 mM) tested.     

Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection

A novel capacitance biosensor based on synthetic phytochelatins for sensitive detection of heavy metals is described. Synthetic phytochelatin (Glu-Cys)(20)Gly (EC20) fused to the maltose binding domain protein was expressed in Escherichia coli and purified for construction of the biosensor. The new biosensor was able to detect Hg(2+), Cd(2+), Pb(2+), Cu(2+) and Zn(2+) ions in concentration range of 100 fM-10 mM, and the order of sensitivity was S(Zn)>S(Cu)>S(Hg)>S(Cd) congruent with S(Pb). The biological sensing element of the sensor could be regenerated using EDTA and the storage stability of the biosensor was 15 days.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Interaction of metal ions with D-glucobenzothiazoline: isolation and characterization of the resultant products

Six different metal-ion complexes of D-glucobenzothiazoline were synthesized and characterized by analytical and spectral techniques. Formation of different types of species (ML and ML(2)) were observed with Cu(2+), Ag(+), Cd(2+), Hg(2+), and Zn(2+) ions. Existence of an anomeric mixture in the case of the Cu(2+) complex is identified from the EPR spectra, and the results were further supported by the simulated spectra. The structures were proposed based on different studies.     

Preparation and metal-binding behaviour of chitosan functionalized by ester- and amino-terminated hyperbranched polyamidoamine polymers

A series of insoluble chitosan (CTS) derivatives were prepared by grafting ester- and amino-terminated dendrimer-like polyamidoamine (PAMAM) into CTS using a divergent method by repeating two processes: (1) Michael addition of methyl acrylate (MA) to surface amino groups, and (2) amidation of the resulting esters with ethylenediamine (EDA). Their structures were characterized by infrared spectra (IR) and wide-angle X-ray diffraction (WAXD). The adsorption capabilities of the products for Au(3+), Pd(2+), Pt(4+), Ag(+), Cu(2+), Zn(2+), Hg(2+), Ni(2+), and Cd(2+) were studied. The results showed that the products exhibited better adsorption capabilities for Au(3+) and Hg(2+) than for other metal ions, and the adsorption capabilities of amino-terminated products were higher than those of ester-terminated ones. Also it was observed that a high percentage of grafting of PAMAM into CTS does not ensure a high adsorption capacity.     

Membrane potential-controlled inhibition of cytochrome c oxidase by zinc

Like many voltage-sensitive ion pumps, cytochrome c oxidase is inhibited by zinc. Binding of zinc to the outside surface of Rhodobacter sphaeroides cytochrome c oxidase inhibits the enzyme with a K(I) of < or = 5 microm when the enzyme is reconstituted into phospholipid vesicles in the presence of a membrane potential. In the absence of a membrane potential and a pH gradient, millimolar concentrations of zinc are required to inhibit. This differential inhibition causes a dramatic increase in the respiratory control ratio from 6 to 40 for wild-type oxidase. The external zinc inhibition is removed by EDTA and is not competitive with cytochrome c binding but is competitive with protons. Only Cd(2+) of the many metals tested (Mg(2+), Mn(2+), Ca(2+), Ba(2+), Li(2+), Cs(2+), Hg(2+), Ni(2+), Co(2+), Cu(2+) Tb(3+), Tm(3+)) showed inhibitory effects similar to Zn(2+). Proton pumping is slower and less efficient with zinc. The results suggest that zinc inhibits proton movement through a proton exit path, which can allow proton back-leak at high membrane potentials. The physiological and mechanistic significance of proton movement in the exit pathway and its blockage by zinc is discussed in terms of regulation of the efficiency of energy transduction.     

Copper induces permeability transition through its interaction with the adenine nucleotide translocase

In this work we examined the effect of low concentrations of Cu(2+) on the opening of the mitochondrial non-specific pore. The purpose was addressed to further contribute to the knowledge of the mechanisms that regulate the open/closed cycles of the permeability transition pore. Membrane leakage was established by measuring matrix Ca(2+) efflux and mitochondrial swelling. The experimental results indicate that Cu(2+) at very low concentrations promoted the release of accumulated Ca(2+), as well as mitochondrial swelling, provided 1,10-phenanthroline has been added. Carboxyatractyloside and Cu(2+) exhibited additive effects on these parameters. After Cu(2+) titration of membrane thiols, it might be assumed that the blockage of 5.9nmol of SH/mg protein suffices to open the non-specific pore. Taking into account the reinforcing effect of carboxyatractyloside, the increasing ADP concentrations, and that N-ethylmaleimide inhibited the Cu(2+)-induced Ca(2+) efflux, it is proposed that the target site for Cu(2+) is located in the ADP/ATP carrier.     

Emerging mechanisms for heavy metal transport in plants

Heavy metal ions such as Cu(2+), Zn(2+), Mn(2+), Fe(2+), Ni(2+) and Co(2+) are essential micronutrients for plant metabolism but when present in excess, these, and non-essential metals such as Cd(2+), Hg(2+) and Pb(2+), can become extremely toxic. Thus mechanisms must exist to satisfy the requirements of cellular metabolism but also to protect cells from toxic effects. The mechanisms deployed in the acquisition of essential heavy metal micronutrients have not been clearly defined although a number of genes have now been identified which encode potential transporters. This review concentrates on three classes of membrane transporters that have been implicated in the transport of heavy metals in a variety of organisms and could serve such a role in plants: the heavy metal (CPx-type) ATPases, the natural resistance-associated macrophage protein (Nramp) family and members of the cation diffusion facilitator (CDF) family. We aim to give an overview of the main features of these transporters in plants in terms of structure, function and regulation drawing on information from studies in a wide variety of organisms.     

Metal cofactor requirement of β-lactamase II

1. The apoenzyme obtained on removal of Zn(2+) from beta-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn(2+)-containing enzyme. 2. Removal of Zn(2+) led to a conformational change in the enzyme and partial unmasking of a thiol group. 3. Replacement of Zn(2+) by Co(2+), Cd(2+), Mn(2+) or Hg(2+) gave enzymes with significant, but lower, beta-lactamase activity. No activity was detected in the presence of Cu(2+), Ni(2+), Mg(2+) or Ca(2+). 4. Equilibrium dialysis indicated that the enzyme had at least two Zn(2+) binding sites. With benzylpenicillin as substrate the variation in activity with concentration of Zn(2+) indicated that activity paralleled binding of Zn(2+) to the site of highest affinity. 5. Replacement of Zn(2+) by Co(2+) and Cd(2+) gave enzymes with absorption bands at 340 and 245nm respectively, and raised the question of whether the thiol group in the enzyme is a metal-ion ligand. 6. Reduction of the product obtained by reaction of denatured beta-lactamase II with Ellman's reagent [5,5'-dithiobis-(2-nitrobenzoic acid)] gave a protein which could refold to produce beta-lactamase II activity in high yield.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Effects of Ce3+, Cd2+, and Hg2+ on activities and secondary structure of trypsin

The different effects of Ce³⁺, Cd²⁺, and Hg²⁺ on the activities and secondary structure of trypsin were studied. The results showed that trypsin activity was increased substantially by Ce³⁺ in 0.5–5 μmol/L concentration, but the activity was decreased significantly by Cd²⁺ or Hg²⁺ in 0.5–5 μmol/L concentration. The ultraviolet-visible spectrum of trypsin with 4 μmol/L Ce³⁺ treatment was the same as that of the control, but the 232-nm characteristic peak of trypsin with 4 μmol/L Cd²⁺ or Hg²⁺ treatment was blue-shifted and the peak intensity weakened. The circular dichroism (CD) spectrum of trypsin with 4 μmol/L Ce³⁺ treatment was similar to that of the control. The secondary structure of trypsin did not change with Ce³⁺ treatment. However, the CD spectrum of trypsin with 4 μmol/L Cd²⁺ or Hg²⁺ treatment was different from that of the control and Ce³⁺ treatment. The secondary structure of trypsin with Cd²⁺ or Hg²⁺ treatment changed greatly; for example, the α-helix and β-sheet contents were reduced significantly, the β-turn was enhanced greatly, and the random coil contents increased or decreased.     

Copper induces apoptosis in Aedes C6/36 cells

The Aedes albopictus C6/36 cell clone is used as a model system to study the effects of heavy metals on insect cells. Here we report on the effects of Cu(2+) on these cells. Similar to Cd(2+) and Hg(2+), Cu(2+) induces hyperpolymerization of the microtubules; moreover, with Cu(2+) this is followed by cell aggregation and massive apoptosis. This process, which is cell density dependent, is maximal between 0.75 and 1 mM; this is just under the LC(50) as determined by a membrane integrity test. At higher Cu(2+) concentrations, cell death occurs by necrosis. Apoptosis was ascertained by fluorescence and electron microscopy and by agarose gel electrophoresis. At 0.75 mM, apoptosis started at 18-hr exposure time and the amount of apoptotic cells increased almost linearly until 42 hr; then a plateau was reached with 70-80% apoptotic cells. This is the first report on Cu(2+)-induced apoptosis in insect cells. Possible induction mechanisms are discussed in the light of existing literature on vertebrate cells.