Inverse correlation between resistance towards copper and towards the redox-cycling compound paraquat: a study in copper-tolerant hepatocytes in tissue culture

The essential mediatory role of copper or iron in the manifestation of paraquat toxicity has been demonstrated (Kohen and Chevion (1985) Free Rad. Res. Commun. 1, 79-88; Korbashi, P. et al. (1986) J. Biol. Chem. 261, 12472-12476). Several liver cell lines, characterized by their resistance to copper, were challenged with paraquat and their cross-resistance to paraquat and copper was studied. Cell growth and survival data showed that copper-resistant cells, containing elevated copper, are more sensitive towards paraquat than wild type cells. Copper-deprived resistant cells did not have this sensitivity. Paraquat was also shown to cause a marked degradation of cellular glutathione in all cell lines. Albeit the fact that the basal glutathione levels are higher in copper-resistant than in wild type cells, there is more paraquat-induced degradation of cellular glutathione (GSH + GSSG) in resistant cells. It is suggested that in copper-resistant cells which contain elevated levels of copper, paraquat-induced cellular injury is potentiated even where glutathione levels are elevated. Additionally, in vitro experiments are presented that support the in vivo findings demonstrating a role for copper in glutathione degradation.     

Resistance of cultured hepatoma cells to copper toxicity. Purification and characterization of the hepatoma metallothionein

The mechanism of resistance to the toxic effects of copper was investigated using a series of copper-resistant hepatoma cell lines maintained in copper-enriched medium. Gel electrophoresis of carboxyamidated cell extracts demonstrated the presence of a pair of low molecular mass cysteine-rich proteins in wild-type and resistant cell lines. These proteins were purified to homogeneity and contained approx. 60% of the total cellular copper. Comparisons of molecular masses, pI values and amino-acid compositions for the purified hepatoma proteins with authentic rat liver metallothionein, as well as cross-reactivity with anti-rat metallothionein antibody, confirmed that the cysteine-rich hepatoma proteins were metallothioneins. The cellular concentration of these hepatoma copper-metallothioneins was proportional to both the level of metal resistance and the amount of copper accumulated by individual cell lines. Further, resistant cells removed from copper-enriched medium for 6-12 months, yet maintaining their level of resistance, showed only a slight decrease in metallothionein concentration. Thus it is proposed that the level of resistance to metal toxicity is mediated by the concentration of copper-metallothionein. It is also suggested that the steady-state level of copper metallothionein is controlled by the degree of metal exposure.     

Intracellular copper transport in cultured hepatoma cells

The distribution of copper in lysates prepared anaerobically from copper-resistant hepatoma cells radiolabeled with 67Cu was examined in pulse-chase experiments. Initially, the majority of the radioactivity (greater than 85%) coeluted with copper-metallothionein. As the chase time increased there was a gradual loss of 67Cu from metallothionein, with a concomitant increase in the level of 67Cu-labeled glutathione. There was also an increase in 67Cu incorporation into superoxide dismutase. These results suggest that the chelation of copper by metallothionein from a copper-glutathione complex (Freedman, J. H., Ciriolo, M. R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605) is a reversible process. Further, they demonstrate that the copper bound to metallothionein is not permanently sequestered, but can be incorporated into other copper proteins.     

Neurotoxicity from glutathione depletion is mediated by Cu-dependent p53 activation

Loss of intracellular neuronal glutathione (GSH) is an important feature of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The consequences of GSH depletion include increased oxidative damage to proteins, lipids, and DNA and subsequent cytotoxic effects. GSH is also an important modulator of cellular copper (Cu) homeostasis and altered Cu metabolism is central to the pathology of several neurodegenerative diseases. The cytotoxic effects of Cu in cells depleted of GSH are not well understood. We have previously reported that depletion of neuronal GSH levels results in cell death from trace levels of extracellular Cu due to elevated Cu(I)-mediated free radical production. In this study we further examined the molecular pathway of trace Cu toxicity in neurons and fibroblasts depleted of GSH. Treatment of primary cortical neurons or 3T3 fibroblasts with the glutathione synthetase inhibitor buthionine sulfoximine resulted in substantial loss of intracellular GSH and increased cytotoxicity. We found that both neurons and fibroblasts revealed increased expression and activation of p53 after depletion of GSH. The increased p53 activity was induced by extracellular trace Cu. Furthermore, we showed that in GSH-depleted cells, Cu induced an increase in oxidative stress resulting in DNA damage and activation of p53-dependent cell death. These findings may have important implications for neurodegenerative disorders that involve GSH depletion and aberrant Cu metabolism.     

Role of Nerve Growth Factor in Oxidanl Homeostasis: Glutathione Metabolism

Abstract— Free radicals are generated in the CNS by ongoing oxygen metabolism and biological events associated with injury and inflammation. Increased free radical levels may also persist in some chronic neurological diseases and in the aged. Nerve growth factor (NGF) is a member of the neurotrophin family of proteins that can regulate neuronal development, maintenance, and recovery from injury. NGF protected rat pheochromocytoma PC12 cells, an adrenal chromaffin-like NGF-responsive cell line, from the oxidant stress accompanying hydrogen peroxide treatment by stimulating GSH levels and enzymes in the GSH metabolism cycle and in the GSH/GSH peroxidase antioxidant redox system, a ubiquitous cellular antioxidant system. Specifically, NGF increased γ-glutamylcysteine synthetase (GCS) activity, the rate-limiting enzyme for GSH synthesis, by 50% after 9h and GSH levels by 100% after 24 h of treatment. NGF stimulated GSH peroxidase by 30% after 3 days and glucose 6-phosphate dehydroge-nase by 50% after 2 days. Treatment with NGF and cyclo-heximide, or actinomycin D, which inhibit protein and RNA synthesis, respectively, blocked the NGF stimulation of GCS and glucose 6-phosphate dehydrogenase. Increased GSH levels due to NGF treatment were responsible for the significant protection of PC12 cells from hydrogen peroxide-induced stress. Pretreatment of PC12 cells with NGF for 24 h rescued cells from the toxic effects of the extracellular hydrogen peroxide generated by the glucose/glucose oxidase system but did not rescue cells that were subjected to GSH deprivation due to treatment with 10 μMl -buthionine-(S,R)-sulfoximine, an inhibitor of GCS. However, treatment with 10 μMl -buthionine-(S,R)-sulfoximine alone did not affect PC12 cell viability, NGF stimulation of neurite extension, and NGF induction of GCS, GSH peroxidase, and glucose 6-phosphate dehydrogenase activity. When GSH levels were measured in PC12 cells that were treated for 24 h with other neurotrophins and growth factors, such as brain-derived neurotrophic factor, neurotro-phin-3, epidermal growth factor, insulin-like growth factor-I, and basic fibroblast growth factor, only epidermal growth factor was found to increase GSH levels by 30%. Whereas NGF increased GSH levels in the human neuro-blastoma SK-N-SH-SY5Y and the human melanoma A-875 in serum-free medium, addition of fetal calf serum to the medium abolished the NGF effects on GSH levels in the NGF-responsive cell lines, SK-N-SH-SY5Y, A-875, and the CNS C6 rat glioma subclone 2BD.     

Tolerance and stress response of sclerotiogenic <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> G15 to copper and lead

Aspergillus oryzae G15 was cultured on Czapek yeast extract agar medium containing different concentrations of copper and lead to investigate the mechanisms sustaining metal tolerance. The effects of heavy metals on biomass, metal accumulation, metallothionein (MT), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were evaluated. Cu and Pb treatment remarkably delayed sclerotial maturation and inhibited mycelial growth, indicating the toxic effects of the metals. Cu decreased sclerotial biomass, whereas Pb led to an increase in sclerotial biomass. G15 bioadsorbed most Cu and Pb ions on the cell surface, revealing the involvement of the extracellular mechanism. Cu treatment significantly elevated MT level in mycelia, and Pb treatment at concentrations of 50–100 mg/L also caused an increase in MT content in mycelia. Both metals significantly increased MDA level in sclerotia. The variations in MT and MDA levels revealed the appearance of heavy metal-induced oxidative stress. The activities of SOD, CAT, and POD varied with heavy metal concentrations, which demonstrated that tolerance of G15 to Cu and Pb was associated with an efficient antioxidant defense system. In sum, the santioxidative detoxification system allowed the strain to survive in high concentrations of Cu and Pb. G15 depended mostly on sclerotial differentiation to defend against Pb stress.     

Intestinal administration of copper and its transient release into venous rat blood serum concomitantly with metallothionein

The molecular side of copper transport in biological systems is unknown. It was attempted to examine the copper and metallothionein (MT) release into the portal blood in rats in vivo. After direct administration of Cu(II) into the jejunum the copper and MT levels were distinctively higher in the portal venous serum compared with that of the vena cava inferior. MT in gel filtrated serum samples was analyzed immunologically employing ELISA and a monoclonal antibody to rat MT-I. Affinity chromatography on Protein A-Sepharose resulted in a higher immunoreactivity in the portal compartment as deduced from an elevated MT-antibody complex. It is assumed that MT serves as a genuine transport system for cuprous copper during the mucosal-to-serosal flux of this biologically important transition metal.     

Transfer of copper from metallothionein to nonmetallothionein proteins in cultured cells

Copper uptake and distribution with time among cytoplasmic proteins were followed in cultured cells under several conditions: (1) CHO cells, which cannot synthesize metallothioneins, were labeled with⁶⁷Cu in the presence of 100 μM ZnCl₂; (2) Cd^r30F9 cells, which contain some constitutive metallothionein (MT), were labeled in the absence of additional ZnCl₂ and; (3) Cd^r30F9 cells were labeled in the presence of ZnCl₂, under which conditions they synthesized additional metallothioneins. The exogenous⁶⁷Cu and ZnCl₂, where present, were then removed, and the distributions of⁶⁷Cu among size fractions of the cellular proteins were observed at intervals for 16 h. In addition, a culture identical to condition (3) above was also treated with 100 μM ZnCl₂ during the redistribution period. The⁶⁷Cu was initially resolved into three peaks by Sephadex G-75 chromatography: high molecular weight, intermediate molecular weight, and MT. The⁶⁷Cu in the MT fraction decreased with at _1/2 of 10–12 h. In contrast to this, generally, in cells with a higher initial⁶⁷Cu bound to metallothionein, there was a progressive increase in the amount of⁶⁷Cu eluting with the high- and intermediate-molecular-weight fractions. Since no other source of⁶⁷Cu was available, these experiments suggest that copper stored in MT can be transferred to other proteins in these cells.     

Growth-modulating tripeptide (glycylhistidyllysine): association with copper and iron in plasma, and stimulation of adhesiveness and growth of hepatoma cells in culture by tripeptide-metal ion complexes

The tripeptide H-Gly-His-Lys-OH (GHL) is a human plasma constituent which has been previously shown to modulate the growth and viability of a variety of cell types and organisms. Experimental observations presented herein indicate that GHL is complexed with the transition metal ions Cu++ and Fe++ in vivo and may exert its biological effects as a peptide-metal chelate. At physiological pH in vitro, GHL associates with ionic copper, cobalt, iron, molybdenum, manganese, nickel, and zinc, but has no affinity for calcium, manganese, potassium, and sodium. GHL acts synergistically with copper, iron, cobalt, and zinc to alter patterns of cell growth in monolayer cultures of a tumorigenic hepatoma cell line (HTC4). These transition metals induce cellular flattening and adhesion to support surfaces, and inhibit DNA synthesis and lactic acid production when growth is limited by reduction of serum concentrations in medium. These inhibitory effects are neutralized, and intercellular adhesion and growth are stimulated by GHL in medium at nanomolar concentrations. Cu and Fe are the most active metals when combined with GHL. The results suggest that the inability of HTC4 cultures to replicate without adequate concentrations of serum in medium may reflect deficiency of GHL and transition metals, which appear to form complexes prior to interaction with cells. Chelation of transition metals with GHL and, potentially, with other growth-modulating peptide factors in plasma or medium, may provide a mechanism for expression and regulation of biological activities influenced by transition metals and polypeptide growth factors. The observed effects of GHL-metal complexes, including stimulation of cellular adhesiveness to substratum (flattening) and intercellular attachment (monolayer formation), appear to satisfy requirements for growth of hepatoma cells in monolayer culture.     

Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus.

Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes.     

Effects of cadmium, copper and metallothionein synthesis inhibiting and stimulating compounds on zinc uptake and accumulation in rat hepatoma HTC cells.

Experiments were carried out to investigate the uptake and accumulation of Zn in rat hepatoma HTC cells, as affected by interfering metals (Cd, Cu), metallothionein synthesis inhibiting compounds (Actinomycin D, cycloheximide) and metallothionein synthesis stimulating compounds (dexamethasone, dibu-cAMP). Cell viability was tested under all experimental conditions by the measurement of LDH leakage, K+ uptake and total cell protein. Determinations of Zn were performed by AAS (total Zn) or by gamma-ray spectrometry (65Zn). Metallothionein analysis was carried out by Cd-saturation tests. The results indicate that cellular responses in rat hepatoma HTC cells with respect to the uptake and accumulation of 65Zn are fully comparable with literature data existing for 65Zn accumulation in rat hepatocytes, under all experimental conditions applied. Cu2+ and dibutyryl-cAMP did not significantly affect rates of 65Zn accumulation. Cd2+, Actinomycin D and cycloheximide reduced 65Zn uptake, but dexamethasone additions resulted in increased 65Zn accumulation in the cells. Effects on 65Zn were shown both in cytosolic and in the membranes/organelles cell fractions. HPLC chromatography in control cells suggested that newly accumulated cytosolic 65Zn was predominantly MT-associated. Dexamethasone-induced 65Zn accumulation could not be related to elevated cellular MT levels, nor were the total cytosolic Zn levels significantly affected. Non-specific attenuations in MT levels (Actinomycin D, cycloheximide and dibu-cAMP) yielded linear relations between cytosolic 65Zn and MT levels, without any change in cytosolic Zn (AAS). Combined addition of Cd and dexamethasone yielded elevated MT levels, but severely reduced total cytosolic Zn and 65Zn concentrations. The results further indicate the non-Zn-specific nature of dexamethasone-action and suggest the relatively easy Zn-complexing and Zn-release of MT. The simultaneous determinations of total cytosolic zinc and cytosolic 65Zn levels showed that the application and sole measurement of radiotracers may yield only one-sided views of what is actually present or occurring in the cells.     

Glutathione and transition-metal homeostasis in Escherichia coli

Glutathione (GSH) and its derivative phytochelatin are important binding factors in transition-metal homeostasis in many eukaryotes. Here, we demonstrate that GSH is also involved in chromate, Zn(II), Cd(II), and Cu(II) homeostasis and resistance in Escherichia coli. While the loss of the ability to synthesize GSH influenced metal tolerance in wild-type cells only slightly, GSH was important for residual metal resistance in cells without metal efflux systems. In mutant cells without the P-type ATPase ZntA, the additional deletion of the GSH biosynthesis system led to a strong decrease in resistance to Cd(II) and Zn(II). Likewise, in mutant cells without the P-type ATPase CopA, the removal of GSH led to a strong decrease of Cu(II) resistance. The precursor of GSH, gamma-glutamylcysteine (gammaEC), was not able to compensate for a lack of GSH. On the contrary, gammaEC-containing cells were less copper and cadmium tolerant than cells that contained neither gammaEC nor GSH. Thus, GSH may play an important role in trace-element metabolism not only in higher organisms but also in bacteria.     

An investigation into copper catalyzed D-penicillamine oxidation and subsequent hydrogen peroxide generation

D-Penicillamine is a potent copper (Cu) chelating agent. D-Pen reduces Cu(II) to Cu(I) in the process of chelation while at the same time being oxidized to D-penicillamine disulfide. It has been proposed that hydrogen peroxide is generated during this process. However, definitive experimental proof that hydrogen peroxide is generated remains lacking. Thus, the major aims of these studies were to confirm and quantitatively assess the in vitro production of hydrogen peroxide during copper catalyzed D-penicillamine oxidation. The potential cytotoxic effect of hydrogen peroxide generation was also investigated in vitro against MCF-7 human breast cancer cells. Cell cytotoxicity resulting from the incubation of D-penicillamine with copper was compared to that of D-penicillamine, copper and hydrogen peroxide. The mechanism of copper catalyzed D-penicillamine oxidation and simultaneous hydrogen peroxide production was investigated as a function of time, concentration of cupric sulfate or ferric chloride, temperature, pH, anaerobic condition and chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid. A simple, sensitive and rapid HPLC assay was developed to simultaneously detect D-penicillamine, its major oxidation product D-penicillamine disulfide, and hydrogen peroxide in a single run. Hydrogen peroxide was shown to be generated in a concentration dependent manner as a result of D-penicillamine oxidation in the presence of cupric sulfate. Chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid were able to inhibit D-penicillamine oxidation. The incubation of MCF-7 human breast cancer cells with D-penicillamine plus cupric sulfate resulted in the production of reactive oxygen species within the cell and cytotoxicity that was comparable to free hydrogen peroxide.     

Oxidative Stress Induced by Cadmium in the C6 Cell Line: Role of Copper and Zinc

In this report, we have investigated the role of copper (Cu) and zinc (Zn) in oxidative stress induced by cadmium (Cd) in C6 cells. Cells were exposed to 20 μM Cd, 500 μM Cu, and 450 μM Zn for 24 h. Then, toxic effects, cellular metals levels, oxidative stress parameters, cell death, as well as DNA damage were evaluated. Cd induced an increase in cellular Cd, Cu, and Zn levels. This results not only in the inhibition of GSH-Px, GRase, CAT, and SOD activities but also in ROS overproduction, oxidative damage, and apoptotic cell death not related to Cu and Zn mechanisms. The thiol groups and GSH levels decreased, whereas the lipid peroxidation and DNA damage increased. The toxicity of Zn results from the imbalance between the inhibition of antioxidant activities and the induction of MT synthesis. The increase in Cu and Zn levels could be explained by the disruption of specific transporter activities, Cd interference with signaling pathways, and metal displacement. Our results suggest that the alteration of Cu and Zn homeostasis is involved in the oxidative stress induced by Cd.     

高温胁迫下外源褪黑素对黄瓜幼苗活性氧代谢的影响

以黄瓜品种‘津春4号’为试材,用叶面喷施的方法,研究了高温胁迫条件下外源褪黑素(melatonin,MT)对黄瓜幼苗活性氧（ROS）代谢的影响.结果表明：外源MT能显著降低高温胁迫下黄瓜叶片超氧阴离子自由基（O₂^-.）产生速率、过氧化氢（H₂O₂）含量、电解质漏渗率（relative electric conductivity, REC）及丙二醛(MDA)含量,增强黄瓜幼苗叶片中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性,提高抗坏血酸（AsA）、谷胱甘肽（GSH）及可溶性蛋白质含量.说明MT预处理能抑制高温胁迫条件下黄瓜幼苗体内ROS的产生,提高抗氧化酶系的活性及抗氧化物质的含量,降低膜质过氧化水平,保护脂膜的完整性,减少电解质的外渗,减轻高温胁迫对幼苗造成的伤害,提高幼苗抗高温胁迫的能力.     

Metallothionein accumulation may account for intracellular copper retention in Menkes' disease

Cultured lymphoblasts derived from infants with Menkes' disease exhibit the same increased avidity for copper as do fibroblasts and most extrahepatic tissues from these patients. The Menkes' cells preferentially take up not only copper but also, on exposure to elevated metal concentrations, the other metallothionein-binding metals, zinc and cadmium. Menkes' lymphoblasts contain larger amounts of metallothionein than normal cells following exposure to each of these metals; the amount bound to this protein quantitatively accounted for the total cellular increment in metal in Menkes' cells. Induction of metallothionein synthesis caused both normal and Menkes' cells to subsequently take up increased amounts of 67Cu. These observations suggest that an enhanced capacity of Menkes' cells to accumulate metallothionein may be responsible for their increased uptake and retention of copper.     

Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I).glutathione complex

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I).GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (b) NMR spectroscopy following the broadening of the resonances of the Cu(I).GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I), Co enzyme, Cu(I).GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(II) complexes. In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I).GSH.protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper.GSH complexes in copper-over-loaded hepatoma cells (Freedman, J.H., Ciriolo, M.R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605), Cu(I).GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in vivo.     

Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking.

The Menkes P-type ATPase (MNK), encoded by the Menkes gene (MNK; ATP7A), is a transmembrane copper-translocating pump which is defective in the human disorder of copper metabolism, Menkes disease. Recent evidence that the MNK P-type ATPase has a role in copper efflux has come from studies using copper-resistant variants of cultured Chinese hamster ovary (CHO) cells. These variants have MNK gene amplification and consequently overexpress MNK, the extents of which correlate with the degree of elevated copper efflux. Here, we report on the localization of MNK in these copper-resistant CHO cells when cultured in different levels of copper. Immunofluorescence studies demonstrated that MNK is predominantly localized to the Golgi apparatus of cells in basal medium. In elevated copper conditions there was a rapid trafficking of MNK from the Golgi to the plasma membrane. This shift in steady-state distribution of MNK was reversible and not dependent on new protein synthesis. In media containing basal copper, MNK accumulated in cytoplasmic vesicles after treatment of cells with a variety of agents that inhibit endosomal recycling. We suggest that MNK continuously recycles between the Golgi and the plasma membrane and elevated copper shifts the steady-state distribution from the Golgi to the plasma membrane. These data reveal a novel system of regulated protein trafficking which ultimately leads to the efflux of an essential yet potentially toxic ligand, where the ligand itself appears directly and specifically to stimulate the trafficking of its own transporter.