Biochemical studies of human (A549) and Simian (CV1) cells labeled with64Cu,67Cu,and125IUdR

CV1 and A549 cells were grown in the presence of 64Cu porphyric complex, 64CuCl2, or 67CuCl2. Radioactive copper determinations were performed on whole cells and on isolated cellular DNA. 125IUdR was used to calibrate the particular extraction and purification procedures we developed because of the half-lives of 64Cu and 67Cu. The results obtained have shown that some radioactive copper atoms remained firmly bound to the DNA molecule. Their amount was of the same order when using two different DNA isolation methods for the two cell lines studied. No significant differences were found when 64Cu was used as CuCl2 or as porphyric complex.     

Physiological responses of Dunaliella salina and Dunaliella tertiolecta to copper toxicity

Species differences in heavy metal tolerance were investigated by comparing the responses of Dunaliella tertiolecta and Dunaliella salina to elevated concentrations of CuCl2. Although both species showed reduced cell number ml(-1) of algal culture, D. salina was more affected by increase in CuCl2. This reflects higher sensitivity of D. salina to CuCl2 compared to D. tertiolecta. Total chlorophyll in terms of microg ml(-1) was higher in D. tertiolecta at all tested CuCl2 levels, but in terms of microg cell(-1) no significant difference was observed between the two species. Total carotenoids in microg cell(-1) increased with increase in CuCl2 in both species and it was about five times higher in D. salina at all CuCl2 concentrations. While both species showed significant increase in lipid peroxidation at elevated CuCl2, the malondialdehyde content of D. salina cells was about three times higher at most CuCl2 concentrations. Although ascorbate peroxidase (APX) activity increased with increase in CuCl2 levels in both species, higher activity was observed in D. tertiolecta at all tested CuCl2 concentrations. Cu content of D. salina cells was higher than D. tertiolecta which may be due to larger volume of D. salina cells. In conclusion, since hydroxyl radical (HO*) produced from H2O2 by Cu2+ (Haber-Weiss cycle) is involved in lipid peroxidation, higher ascorbate peroxidase activity in D. tertiolecta may partly account for lower sensitivity of this species to CuCl2 compared to D. salina.     

Effects of CuCl2 on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart.

CuCl2 non-competitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg2+, 10 muM Ca2+ and phosphodiesterase activator with Ki values of approximately 2 muM for both substrates. CuCl2 inhibition was also non-competitive with Mg2+, Ca2+ and phosphodiesterase activator. Dialysis demonstrated that CuCl2 inhibition is reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl2 was not additive with that of p-hydroxymercuribenzoate, therefore CuCl2 may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl2 also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I50 value of 18 muM. Several effects of Cu2+ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity.     

Induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells is controlled by the level of intracellular copper

A study was carried out on the uptake of copper, zinc, or cadmium ions and their induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells. The main difference between Menkes' and normal cells in the uptake of these metal ions was an increased uptake of copper ions in Menkes' cells at a low concentration of CuCl2 (2.1 microM). The CuCl2 concentration necessary to induce metallothionein synthesis in Menkes' cells was 50 microM, whereas that in normal cells was about 200 microM. The levels of zinc or cadmium ions needed to induce metallothionein in Menkes' cells were similar to those in normal cells. At least four isomers of metallothionein were induced by copper, zinc, and cadmium ions in both types of cells. Metallothionein synthesis in Menkes' and normal cells was induced when the amounts of intracellular copper reached a threshold level of approximately 0.2 nmol/10(6) cells, and the rate of metallothionein synthesis in these cells was increased as a function of the amounts of intracellular copper (0.2-1.7 nmol/10(6) cells). These results indicate that the induction of metallothionein synthesis in lymphoblastoid cells is controlled by the level of intracellular copper, suggesting that the major defect in Menkes' cells is not due to the abnormal regulation of metallothionein synthesis but to an alteration of the copper metabolism in cells by which the levels of intracellular copper become larger than those in normal cells and just lower than the threshold level for induction of metallothionein synthesis.     

Induction of metallothionein mRNA in rat liver and kidney after copper chloride injection.

The kinetics of the increase of metallothionein mRNA in rat liver and kidney after CuCl2 injection was determined by cell-free translation and dot-blot hybridization of total RNA isolated at various times after the injection. Both assay procedures gave essentially the same result: a 16-fold increase in hepatic metallothionein mRNA was observed 7h after CuCl2 injection, with a decline to basal values by 15 h. The response in the kidney was less dramatic, with a 6-fold increase in metallothionein mRNA 5 h after injection, and basal values were attained by 12h. The rise in Cu2+ concentration in both organs was closely correlated with the increase in metallothionein mRNA; hepatic Cu2+ was increased 5.9-fold by 5h after injection and renal Cu2+ was increased 4.3-fold 5h after injection. The Zn2+ concentration in the liver had not risen significantly within 5h of Cu2+ injection. Renal Zn2+ concentrations did not alter appreciably in the Cu2+-treated animals. These results support the conclusion that Cu2+ is acting as a primary inducer of metallothionein mRNA in the rat.     

Copper inhibits activated protein C: protective effect of human albumin and an analogue of its high-affinity copper-binding site, d-DAHK

Activated protein C (APC) is useful in the treatment of sepsis. Ischemia and acidosis, which often accompany sepsis, cause the release of copper from loosely bound sites. We investigated (i) whether physiological concentrations of copper inhibit APC anticoagulant activity and (ii) if any copper-induced APC inhibition is reversible by human serum albumin (HSA) or a high-affinity copper-binding analogue of the human albumin N-terminus, d-Asp-d-Ala-d-His-d-Lys (d-DAHK). APC activity after 30 min of incubation with CuCl2 (10 microM) was decreased 26% below baseline. HSA, both alone and when combined with various ratios of CuCl2, increased APC activity significantly above baseline. d-DAHK alone and 2:1 and 4:1 ratios of d-DAHK:CuCl2 also increased APC activity. APC contained 1.4 microM copper, which helps explain the increased APC activity with HSA and d-DAHK alone. These in vitro results indicate that copper inhibits APC activity and that albumin and d-DAHK reverse the copper-induced APC deactivation.     

Effect of Intracellular Glutathione Level on the Production of 6-Methoxymellein in Cultured Carrot (Daucus carota) Cells

To produce phytoalexin, 6-methoxymellein (6-MM) was induced in suspension cultures of carrot (Daucus carota) by buthionine sulfoximine (BSO) and CuCl2. Addition of BSO (a specific inhibitor of glutathione [GSH] synthesis) to the cultures lowered the cellular GSH levels. This depletion of GSH was BSO-concentration dependent, and the extent of 6-MM accumulation was dependent on the GSH depletion. The accumulation of 6-MM induced by BSO was suppressed by exogenous GSH. Exogenous H2O2 stimulated the production of 6-MM when added 1 d after BSO treatment, whereas H2O2 added at time zero or on the 4th d of BSO treatment did not. Moreover, a synergistic effect of simultaneous addition of BSO and CuCl2 was observed. These results suggest that active oxygen species may be involved in the triggering of 6-MM synthesis.     

Glutathione level of Desulfovibrio desulfuricans IMV K-6 under the influence of heavy metal salts

Glutathione is the metal stress protector and changes of its level in the sulfate-reducing bacteria cells under the influence of heavy metal salts have not been studied yet. CdCl2, Pb(NO3)2, CuCl2, and ZnCl2 influence on the total glutathione level in cell-free extracts of sulfate-reducing bacteria Desulfovibrio desulfuricans IMV K-6 was studied. The research has been carried out using Ellman, Lowry methods, statistical processing of the results. It was shown that the glutathione level depends on the heavy metal salts concentration in the medium. The total glutathione level was the highest under the influence of Pb(NO3)2. Other salts were also toxic to bacteria because glutathione level increased in bacterial cells after addition of these salts to the medium. On the basis of the results of our work the range of heavy metal salts influence on D. desulfuricans IMV K-6 cells glutathione level has been formed for the first time: Pb(NO3)2 > CuCl2 > CdCl2 > ZnCl2.     

CU(II)-dependent inactivation of Mn-catalase by hydroxylamine

Hydroxylamine is a strong inhibitor of the Mn-catalase of Lactobacillus plantarum in the presence of hydrogen peroxide [Kono, Y., and Fridovich, I. (1983) J. Biol. Chem. 258, 13646-13648]. In the presence of CuCl2 the Mn-catalase was rapidly inactivated by hydroxylamine without the addition of hydrogen peroxide. FeSO4 and MnCl2 were approximately 10% and 4% as effective as was CuCl2. Under anaerobic conditions, the inactivation did not occur. The chelating agents such as EDTA and histidine completely prevent the inactivation. These results indicate that the hydrogen peroxide produced during the autooxidation of hydroxylamine catalyzed by CuCl2 participates in the CuCl2-dependent inactivation by hydroxylamine.     

Cu~(2 )作用下辣椒膜脂过氧化及倍半萜环化酶基因转录

分析了在ＣｕＣｌ２作用下辣椒叶片倍半萜环化酶活性、倍半萜环化酶ｍＲＮＡ表达、细胞ＧＳ Ｈ和ＧＳＳＧ代谢及膜脂过氧化。结果表明,Ｃｕ２＋能诱导辣椒叶片表达倍半萜环化酶活性,酶活性的表达与相应的基因转录有关。辣椒叶片在ＣｕＣｌ２作用下合成了含－ＳＨ的螯合肽,相应地ＧＳＨ含量下降,ＧＳＳＧ的含量有所上升,细胞膜脂过氧化作用加剧。推测细胞膜脂过氧化产物或ＧＳＨ氧化产物可能参与了ＣｕＣｌ２诱导辣椒倍半萜环化酶基因表达的信号传递作用。     

CuCl2胁迫对烟草BY-2悬浮细胞死亡的诱导

本文以不含叶绿体的烟草BY-2悬浮细胞系为实验材料,研究了CuCl2胁迫对BY-2细胞呼吸速率、呼吸途径以及细胞内H2O2和ATP含量的影响。结果表明：0.5mmol·L-1CuCl2胁迫明显导致了烟草BY-2细胞的死亡,引起了胞内H2O2爆发和ATP含量下降,严重抑制了BY-2细胞的总呼吸和交替氧化酶途径（alternative oxidase pathway,AOX）。此外,CuCl2对BY-2细胞的线粒体电子传递具有即时的抑制作用。加入外源腺苷（ATP合成的底物）显著抑制了CuCl2胁迫引起的ATP损耗,并阻止了细胞死亡。上述结果表明CuCl2胁迫导致的ATP损耗在CuCl2诱导烟草BY-2细胞死亡过程中起重要的作用。     

Involvement of prostaglandins in the effect of cupric ions on the human uterus

The effect of cupric ions on the human uterus and the involvement of prostaglandins (PGs) in mediating this effect was studied by recording of isometric contractions of isolated myometrial strips and pieces of uterine arteries, and by intrauterine pressure recordings in women before the onset of menstruation. In vitro, CuCl2 in concentrations of 10(-4) M and higher caused a significant inhibition of myometrial contractile activity, but no effect on the artery preparations was seen. Furthermore, the contractile response of myometrial strips to PGF2 alpha and PGE2 (10 ng/ml) decreased in the presence of CuCl2 in concentrations of 5 and 50 mumol. In vivo, instillations of 0.3, 1.0 and 2.0 mM of CuCl2 in 0.7 ml of saline solution into the uterine cavity caused a dose-dependent stimulation of uterine activity, but after pretreatment with naproxen, 500 mg orally, the effect of these substances was abolished. After naproxen treatment, but during infusion of PGF2 alpha (5 micrograms/min), the response to the CuCl2 solutions was partially restored. It is suggested that cupric ions, at high concentrations, have an inhibiting effect on myometrial activity. The stimulatory effect of low doses of CuCl2 seen after instillation into the uterine cavity is largely exerted via initiation of synthesis and release of endometrial PGs.     

Degradation of high-molecular-weight hyaluronan by hydrogen peroxide in the presence of cupric ions

Dynamic viscosity (eta) of the high-molecular-weight hyaluronan (HA) solution was measured by a Brookfield rotational viscometer equipped with a Teflon cup and spindle of coaxial cylindrical geometry. The decrease of eta of the HA solution, indicating degradation of the biopolymer, was induced by a system containing H2O2 alone or H2O2 plus CuCl2. The reaction system H2O2 plus CuCl2 as investigated by EPR spin-trapping technique revealed the formation of a four-line EPR signal characteristic of a *DMPO-OH spin adduct. Thus, hydroxyl radicals are implicated in degradation of high-molecular-weight HA by the system containing H2O2 and CuCl2.     

Chemometric optimisation of parameters for biocatalytic reduction of copper ion by a crude enzyme lysate of Saccharomyces cerevisiae grown under catabolic repression conditions

The capacity of the microbes to reduce the metal has been demonstrated. The immobilised induced microbes with toxic chemical CuCl(2) was used to reduce the Cu ions as elemental metal and by using the response surface methodology the parameters such as the inducer concentration, the time of inducer addition which are concerned with the growth and formation of specific enzymes and the initial substrate concentration, initial pH of the substrate solution and the time of reaction which are concerned with the biocatalytic reduction of the metal ions were optimised for maximum reduction. The elemental copper reduced and removed experimentally from its ionic state at the optimum conditions was 54.82ppm.     

The effect of zinc ions on fertilization of mouse ova in vitro

The fertilization rate of mouse gametes in a Krebs-Ringer bicarbonate medium was 60%. The rate fell to 0% when 250 micron-ZnCl2 was added, but MgCl2, CaCl2, MnCl2, FeCl2, and CuCl2 (each at 250 micron) had no effect. The inhibitory effect of Zn2+ was not evident if the spermatozoa or eggs were preincubated with Zn2+ or if previously capacitated spermatozoa were used. The Zn2+ is thought to act by interference with capacitation.     

The Effects of Heavy Metals and Root Immersion on Isoflavonoid Metabolism in Alfalfa (Medicago sativa L.)

Modest increases in the concentration of medicarpin, 6-fold in leaves and 4-fold in roots, were observed in alfalfa (Medicago sativa L.) seedlings treated with 1 mM metal salts for 72 h. However, medicarpin-3-O-glucoside-6"-O-malonate (MGM) and formononetin-7-O-glucoside-6"-O-malonate (FGM) levels were up to 50-fold lower in metal-treated compared to control roots. Approximately 10% of the "missing" conjugates could be accounted for in the root treatment solution, where FGM and MGM transiently accumulated prior to their hydrolysis. Time-course studies revealed that total isoflavonoid content (roots plus solution) increased slightly after CuCl2 treatment, whereas the levels of FGM and MGM increased rapidly in alfalfa roots immersed in water. This increase was reduced by aeration. The phenylalanine ammonia-lyase inhibitor L-[alpha]-aminooxy-[beta]-phenylpropionic acid was used to show that immersion of the roots reduced conjugate rates of degradation, which explains their accumulation. In contrast, conjugate rates of degradation were elevated in CuCl2-treated roots, with 50% of the increase being due to hydrolysis. Up to 90% of formononetin and medicarpin produced in response to CuCl2 treatment arose via conjugate hydrolysis. Our results demonstrate that both immersion/anaerobiosis and abiotic elicitation modify isoflavonoid metabolism in alfalfa, and that metal-stimulated accumulation of phytoalexins may arise through the release from preformed stores rather than de novo synthesis.     

The interaction of copper chloride with erythrocyte membrane as a source of activated oxygen species. A chemiluminescent study

The possibility for the generation of activated oxygen species during the interaction between copper chloride and erythrocyte membranes was investigated. A chemiluminescent method for detecting superoxide radicals and hydrogen peroxide was used. It was found that the interaction of CuCl2 with erythrocyte membrane is accompanied with O-2 and H2O2 generation. On the base of this result it is proposed that the activated oxygen species generated by CuCl2-membrane interaction may be able to initiate peroxidative breakdown processes in erythrocytes eventually leading to haemolysis.     

Interaction and conformational changes of chromatin with divalent ions.

We have investigated the interaction of divalent ions with chromatin towards a closer understanding of the role of metal ions in the cell nucleus. The first row transition metal ion chlorides MnCl2, CoCl2, NiCl2 and CuCl2 lead to precipitation of chicken erythrocyte chromatin at a significantly lower concentration than the alkali earth metal chlorides MgCl2, CaCl2 and BaCl2. A similar distinction can be made for the compaction of chromatin to the "30 nm" solenoid higher order structure which occurs at lower MeCl2 concentration in the first group but at the same MeCl2 concentration within each group. In other experiments in which mixed solutions of NaCl and of MgCl2 were examined, it is shown that increasing NaCl concentration leads to increasing solubility in the presence of MgCl2. Best compaction of chromatin was obtained at 40 mM NaCl and 0.8 mM MgCl2 at a value A260 approximately 0.8. Similar experiments were undertaken with mixtures of NaCl and MnCl2.     

Killing of Bacillus subtilis spores by a modified Fenton reagent containing CuCl2 and ascorbic acid

Bacillus subtilis spores were killed by CuCl(2)-ascorbic acid, chloride ions were essential for killing of spores, and spores with defective coats were killed more rapidly. CuCl(2)-ascorbic acid did not damage spore DNA, and spores killed by this reagent initiated germination. However, spores killed by CuCl(2)-ascorbic acid may have damage to their inner membrane.