Structural effects of cobalt-amine compounds on DNA condensation.

Light scattering and electron microscopy have been used to investigate the structural effects of the trivalent complexes hexaammine cobalt (III) chloride (Cohex), tris(ethylenediamine) cobalt(III) chloride (Coen), and cobalt(III) sepulchrate chloride (Cosep) on DNA condensation. These cobalt-amine compounds have similar ligand coordination geometries but differ slightly in size. Their hydrophobicity is in the order Cosep > Coen > Cohex, according to the numbers of methylene groups in these ligands. All of these compounds effectively precipitate DNA at high concentrations; but despite a lower surface charge density, Cosep condenses DNA twice as effectively as Coen or Cohex. UV and CD measurements of the supernatants of cobalt-amine/DNA solutions reveal a preferential binding of Delta-Coen over Lambda-Coen to the precipitated DNA, but there is no chiral selectivity for Cosep. Competition experiments show that the binding strengths of these three cobalt-amine compounds to aggregated DNA are comparable. A charge neutralization of 88-90% is required for DNA condensation. Our data indicate that 1) electrostatic interaction is the main driving force for binding of multivalent cations to DNA; 2) DNA condensation is dependent on the structure of the condensing agent; and 3) the hydration pattern or polarization of water molecules on the surface of condensing agents plays an important role in DNA condensation and chiral recognition.     

Cobalt chloride-induced apoptosis and extracellular signal-regulated protein kinase 1/2 activation in rat C6 glioma cells

Brain ischemia brings about hypoxic insults. Hypoxia is one of the major pathological factors inducing neuronal injury and central nervous system infection. We studied the involvement of mitogen-activated protein (MAP) kinase in hypoxia-induced apoptosis using cobalt chloride in C6 glioma cells. In vitro cytotoxicity of cobalt chloride was tested by MTT assay. Its IC(50) value was 400 microM. The DNA fragment became evident after incubation of the cells with 300 microM cobalt chloride for 24 h. We also evidenced nuclear cleavage with morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signal pathway of cobalt chloride-induced apoptosis in C6 cells. The activation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) started to increase at 1 h and was activated further at 6 h after treatment of 400 M cobalt chloride. In addition, pretreatment of PD98059 inhibited cobalt chloride-induced apoptotic cell morphology in Electron Microscopy. These results suggest that cobalt chloride is able to induce the apoptotic activity in C6 glioma cells, and its apoptotic mechanism may be associated with signal transduction via MAP kinase (ERK 1/2).     

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100?kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved.     

Methylation and restriction endonuclease cleavage of linear Z-DNA in the presence of hexamminecobalt (III) ions.

These studies employed the synthetic linear DNA, poly dGdC, in the B and cobalt hexammine chloride (Co)-induced Z form to determine the effect of conformation on protein-DNA interactions. The rate of the reaction of the restriction endonucleases, Hha I and Cfo I, are reduced with Z DNA as compared to B DNA. The ability of both restriction endonucleases to react with an aggregate form of Z DNA (Z* DNA) is found to depend upon how the Z* DNA is formed. When Z* DNA is induced by low concentrations of Co (50 microM), the endonucleases remain active. In the presence of 100 microM Co, which causes increased aggregation, the endonucleases are inactive. The Hha I DNA methyltransferase reacts at equal rates with the B, Z and low cobalt Z* forms and at a greatly reduced rate with the high cobalt Z* form. These results are significantly different than those observed with Z form dGdC tracts inserted into circular DNA molecules.     

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

 Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100 kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved. Received: 21 December 1995/Accepted: 10 June 1996     

Inhibitory effects of cobalt chloride and cinnamaldehyde on 5-azacytidine-induced digital malformations in rats.

The effect of two DNA repair inhibitors in bacteria, cobalt chloride and cinnamaldehyde, on 5-azacytidine (5-AC)-induced digital malformations was studied. Both agents inhibited the induced digital malformations. The effect of cobalt chloride was significant 3 hr before to 1 hr after the 5-AC treatment, and the effect of cinnamaldehyde was significant 3 hr before to 24 hr after the treatment. However, an increase in fetal mortality was observed with the latter agent. The mechanisms underlying the suppressive effects of both agents may be different, but their natures require elucidation.     

Ultrastructural and autoradiographic analysis of the cellular reactivity of the exo- and endocrine epithelium of the frog pancreas under the action of cobalt chloride]

The frog pancreatic epithelium was studied using morphometry, autoradiography and electron microscopy during the treatment with cobalt chloride (10 mg/100 g body wieght). In response to this treatment, generative changes in some exocrine cells were revealed. At the same time, those of cells, whose nuclei were in the state of DNA synthesis, increased in number. Cobalt chloride resulted in endocrine A-cells damage. A compensation of the broken endocrine A-cell function was maintained in a transformation of the exocrine epithelium into endocrine A-cells and in the appearance of intermediate acinar-islet cells, as well as in islet A-cell hyperplasia.     

The essential role of cobalt in the inhibition of the cytosolic isozyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Nicotiana silvestris by glyphosate

The prime molecular target of glyphosate (N-[phosphonomethyl]glycine), a potent herbicidal and antimicrobial agent, is known to be the shikimate-pathway enzyme, 5-enol-pyruvylshikimate-3-phosphate synthase. Inhibition by glyphosate of an earlier pathway enzyme that is located in the cytosol of higher plants, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DS-Co), has raised the possibility of dual enzyme targets in vivo. With the recent appreciation that magnesium (and manganese) can replace cobalt as the divalent-metal activator of DS-Co, it has now been possible to show that sensitivity of DS-Co to inhibition by glyphosate is obligately dependent upon the presence of cobalt. Evidence for a cobalt(II):glyphosate complex with octahedral coordination was obtained through examination of the effect of glyphosate upon the visible electronic spectrum of aqueous solutions of cobalt(II) chloride. The presence of glyphosate increased the concentration of cobalt(II) chloride required for enzyme activity, and the concentration of cobalt(II) chloride markedly affected the concentration of glyphosate required for inhibition of DS-Co activity. The extent to which DS-Co is vulnerable to inhibition by glyphosate in vivo depends, therefore, upon the unknown extent to which DS-Co molecules in the cytosol might be associated with cobalt.     

Effects of Cobalt Nanoparticles on Human T Cells In Vitro

Limited information is available on the potential risk of degradation products of metal-on-metal bearings in joint arthroplasty. The aim of this study was to investigate the cytotoxicity and genotoxicity of orthopedic-related cobalt nanoparticles on human T cells in vitro. T cells were collected using magnetic CD3 microbeads and exposed to different concentrations of cobalt nanoparticles and cobalt chloride. Cytotoxicity was evaluated by methyl thiazolyl tetrazolium and lactate dehydrogenase release assay. Cobalt nanoparticles dissolution in culture medium was determined by inductively coupled plasma-mass spectrometry. To study the probable mechanism of cobalt nanoparticles effects on T cells, superoxide dismutase, catalase, and glutathione peroxidase level was measured. Cobalt nanoparticles and cobalt ions could inhibit cell viability and enhance lactate dehydrogenase release in a concentration- and time-dependent manner (P < 0.05). The levels of cobalt ion released from cobalt nanoparticles in the culture medium were less than 40% and increased with cobalt nanoparticles concentration. Cobalt nanoparticles could induce primary DNA damage in a concentration-dependent manner, and the DNA damage caused by cobalt nanoparticles was heavier than that caused by cobalt ions. Cobalt nanoparticles exposure could significantly decrease superoxide dismutase, catalase, and glutathione peroxidase activities at subtoxic concentrations (6 μM, <CC₅₀). These findings suggested that cobalt nanoparticles could generate potential risks to the T cells of patients suffer from metal-on-metal total hip arthroplasty, and the inhibition of antioxidant capacity may play important role in cobalt nanoparticles effects on T cells.     

Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase.

Hexamine cobalt chloride (HCC) increases the efficiency of blunt end ligation by T4 DNA ligase about 50 fold. Maximum stimulation occurs when standard buffers for ligation are supplemented with 1 mM HCC. All the ligation events are intermolecular regardless of the initial DNA concentration. In the presence of monovalent cations (eg. 25 mM KCl) HCC still increases the extent of T4 catalyzed ligation but intramolecular ligation products are also formed. Therefore, intermolecular ligation can be performed rapidly and at low DNA concentrations.     

Quercetin and Vitamin C Mitigate Cobalt Chloride-Induced Hypertension through Reduction in Oxidative Stress and Nuclear Factor Kappa Beta (NF-Kb) Expression in Experimental Rat Model

The objective of the present work was to evaluate the toxic effects of cobalt chloride, a potent oxidative stress-inducing chemical, at 650 ppm in rats and the protective effect of quercetin and/or vitamin C against the cobalt chloride-induced toxicity. Thirty rats were randomly selected, and assigned to one of five groups: control, cobalt chloride, cobalt chloride + quercetin, cobalt chloride + vitamin C and cobalt chloride + quercetin + vitamin C. The exposure of rats to cobalt chloride led to a significant increase (p < 0.05) in malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) generated, but decreased nitric oxide (NO) bioavailability. Also, significant (p < 0.05) reductions were observed in the activity of glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the cardiac and renal tissues. Treatment with quercetin and vitamin C reversed the effect of cobalt chloride on MDA, H₂O₂ and NO, more potently than with either of the two antioxidants, and increased the antioxidant defence system. Further, treatment of rats with quercetin and vitamin C in combination resulted in significant (p < 0.05) decreases in the systolic, diastolic, and mean arterial blood pressure of rats, relative to those exposed to cobalt chloride alone. Immunohistochemical studies revealed a greater expression of nuclear factor kappa beta (NF-kB) in the cobalt chloride group compared with the control- and antioxidants-treated rats. The results of this study suggest a protective role for quercetin and vitamin C in the amelioration of the toxic mechanisms leading to cobalt chloride-induced hypertension and its associated cardiac and renal complications in rats.     

Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages

Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl(2)) and examined their apoptosis by DNA fragmentation assay, 4',6-diamidino-2'-phenylindol dihydrochloride staining, and Western blot analysis. CoCl(2) induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl(2) inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl(2) released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.     

Upstream regulatory elements in chick heme oxygenase-1 promoter: A study in primary cultures of chick embryo liver cells

Previously, chick heme oxygenase-1 (cHO-1) gene was cloned by us and two regions important for induction by sodium arsenite were identified. These two regions were found to contain consensus sequences of an AP-1 (-1580 to -1573) and a MRE/cMyc complex (-52 to -41). In the current study, the roles of these two elements in mediating the sodium arsenite or cobalt chloride dependent induction of cHO-1 were investigated further. DNA binding studies and site-directed mutagenesis studies indicated that both the AP-1 and MRE/cMyc elements are important for the sodium arsenite induction, while cobalt chloride induction involves only the AP-1 element. Electrophoretic mobility shift assays showed that nuclear proteins binding to the AP-1 element was increased by both sodium arsenite or cobalt chloride treatment, whereas the binding of proteins to the MRE/cMyc element showed a high basal expression in untreated cells and the binding activity was only slightly increased by sodium arsenite treatment. Site-directed mutagenesis studies showed that, to completely abolish sodium arsenite induction, both the AP-1 and MRE/cMyc elements must be mutated; mutation of either element alone resulted in only a partial effect. In contrast, a single mutation at AP-1 element was sufficient to reduce the cobalt chloride induction almost completely. The MRE/cMyc complex plays a major role in the basal level expression, and shares some similarities to the upstream stimulatory factor element (USF) identified in the promoter regions of mammalian HO-1 genes and other stress regulated genes. Because sodium arsenite is known to cause oxidative stress and because activation of AP-1 proteins has been shown to be a key step in the oxidative stress response pathway, we also explored the possibility that the induction of the cHO-1 gene by sodium arsenite is mediated through oxidative stress pathway(s) by activation of AP-1 proteins. We found that pretreatment with antioxidants (N-acetyl cysteine or quercetin) reduced the induction of the endogenous cHO-1 message or cHO-1 reporter construct activities induced by sodium arsenite or cobalt chloride. These antioxidants also reduced the protein binding activities to the AP-1 element in the electrophoretic mobility shift assays. In summary, induction of the cHO-1 gene by sodium arsenite or cobalt chloride is mediated by activation of the AP-1 element located at -1,573 to -1,580 of the 5 UTR.     

Phosphodiesterase Found in Young Wheat Roots

Phosphodiesterase has been found in the particulate and soluble fractions from young wheat roots. The intracellular distribution of this enzyme was studied by using RNA, oligo DNA and DNPP as the substrates. When oligo DNA was used, 50 to 60 per cent of PPDase activity was found in the soluble fraction and 30 to 40 per cent in the microsomal fraction. Besides magnesium ion, calcium, cobalt, manganese and nickel ions were effective for its activity. The pH optimum of the enzyme was found at 6.0. This PPDase produced 5′-nucleotides from RNA at pH 6.9 on addition of magnesium chloride.     

Inhibition of proteasomal degradation of Mcl-1 by cobalt chloride suppresses cobalt chloride-induced apoptosis in HCT116 colorectal cancer cells

Cobalt promotes apoptosis in multiple cell systems, however, the molecular mechanisms that influence cobalt-induced apoptosis are not fully understood. We investigated mechanisms of cobalt chloride induced apoptosis in HCT116 colorectal cancer cells. Cobalt chloride induced dose dependent apoptosis in HCT116 cells (250–750 μM) which, at higher concentrations (500–750 μM), was associated with an increase in the expression of the Bcl-2-related Mcl-1 survival protein. Cobalt chloride caused the accumulation of higher molecular weight ubiquitin-conjugates of Mcl-1 in intact HCT116 cells and inhibited the activity of the trypsin-like site of the 20S proteasome in an in vitro assay. Although siRNA-mediated knockdown of Mcl-1 increased apoptosis in HCT116 cells, the combination of Mcl-1 siRNA and cobalt chloride induced very high levels of cell killing. Therefore, inhibition of the proteasome by cobalt chloride leads to the accumulation of Mcl-1 which acts to limit cobalt chloride induced apoptosis.     

Selective and effective bactericidal activity of the cobalt (II) cation against Helicobacter pylori

BACKGROUND: Although the anti-Helicobacter pylori activity of bismuth is well established, the therapeutic potential of other metal ions against the organism is not known. MATERIALS AND METHODS: We measured the minimum inhibitory concentrations of a series of metal ions, including several cobalt (II) compounds against four type strains and seven clinical isolates of H. pylori using three standard broth culture media and a defined medium. Other intestinal bacteria were also investigated for specificity of action. RESULTS: Cobalt chloride had marked activity against H. pylori (minimum inhibitory concentration range was 0.03-1.0 mg/l). The effect was specific because other transition metals had no effect and other intestinal bacteria were not affected by cobalt chloride. Activity was attributable to free cobalt ions as ligands inhibited activity in proportion to their affinity for the ions. Inhibition of cobalt activity was also observed in the presence of nickel, in a dose dependent fashion. However, cobalt activity was not directed towards the nickel-dependent urease enzyme because its effect was similar in wild-type and urease negative mutant strains of H. pylori. Finally, the viability of H. pylori was reduced at the same rate with 2 mg/l cobalt as with 1 mg/l amoxicillin. CONCLUSIONS: Cobalt competes for nickel in its acquisition by H. pylori, but mediates toxicity in a nonurease dependent fashion. As cobalt MIC is similar to some antibiotics and 10 to a hundred times lower than for bismuth, cobalt may represent an effective form of therapy for H. pylori infection.     

Cobalt mapping of the nervous system: how to avoid artifacts

Infusion of cobalt ions into cut axons is an established method for tracing neuron projections in the central nervous system. Artifacts, where unintended neurons are stained, however, have been reported, leading to difficulties in interpretation. Experiments in the locust Schistocerca gregaria Forsk?l show that such artifacts can be induced through damage to axons caused by cutting peripheral nerves and by using high cobalt chloride concentrations (0.4M and above). Mixtures of cobalt and nickel chlorides and nickel chloride alone were introduced into different branches of the same nerve and developed with rubeanic acid to give precipitates of different colors in the two sets of axons. Preparations were examined with the light microscope, where mixing of ions would appear as intermediate colors, and by x-ray probe microanalysis. No evidence for leakage of metal ions from the filled axons or for ion uptake by other axons could be detected, provided that low concentrations of cobalt and nickel chlorides were used and nerve cutting was reduced to a minimum by making preparations in vivo. If extreme conditions are avoided when making the preparation, the risk of producing artifacts is minimized, thus enabling the cobalt method to be used with greater confidence for describing neuronal projections.