Metallothionein Production Research Articles

Mechanisms for Heavy Metal Detoxification and Tolerance in Algae

重金属是一类具有潜在危害的重要污染物, 越来越多的重金属被排入水体, 对水生生态环境构成严重威胁.藻类在长期响应重金属胁迫过程中, 建立起一系列的适应机制.藻类通过控制重金属的吸收、富集、转运与解毒, 使不同细胞组分中的重金属维持在正常浓度范围内.这些保护机制主要包括藻细胞的某些胞外组分与重金属结合, 从而减少重金属进入胞内;在重金属诱导下藻细胞可合成金属结合蛋白或多肽;重金属诱导藻细胞合成一些代谢物使其免受伤害或修复由重金属胁迫造成的损伤;藻细胞通过液泡区室化作用使重金属远离代谢;藻细胞对重金属具有排斥与排出作用.;With the development of industry and agriculture, more and more heavy metals are released into water bodies.Today, many heavy metals constitute a global environmental hazard.Heavy metals such as copper, zinc, and nickel are essential for many physiological processes yet can be toxic at higher levels.Other metals such as cadmium, mercury and lead are nonessential and potentially highly toxic.Algae possess a range of potential cellular mechanisms that may be involved in the detoxification of heavy metals and thus tolerance to metal stress.These include roles for the following: for sequesteration of metals on extracellular components that reduce metal bioavailability;for chelation of metals in the cytosol by peptides and proteins;for sequestration of metals in polyphosphate bodies;for the com-partmentation of metals away from metabolic process by transporting them into the vacuole;for increasing the efflux or exclusion of metals;for producing stress proteins such as heat shock proteins that repair the stress-damaged proteins;in addition, some heavy metals cause oxidative stress in algae, with the result that metal toxicity can be altered by synthesis of appropriate enzymes or metabolites counteracting metal-induced oxidative stress. In recent years, some attempts to engineer the production of metallothioneins (MTs) and phytochelatins (PCs) in algae to increase metal tolerance and/or accumulation have been reported.To date, however, it is mainly the model plant species that have been genetically engineered.Phytoremediation strategies have been proposed as an attractive alternative owing to their low cost and high efficiency.The concept of phytoremediation of heavy metal contaminated water has been increasingly supported by research.And, algae have been widely used as pollution indicators in water quality determination.Thus, studies on tolerance and detoxification mechanism of heavy metal in algae have numerous ecological and public health implications.

Electrochemical study of heavy metals and metallothionein in yeast Yarrowia lipolytica

The bioaccumulation of heavy metals (cadmium, nickel, cobalt and zinc) and the effect of these metals on the production of metallothionein and metallothionein-like proteins (MT) in Yarrowia lipolytica was studied by electrochemical methods. The concentrations of heavy metals were determined by differential pulse voltammetry (DPV). A combination of the constant current chronopotentiometric stripping analysis (CPSA) and adsorptive transfer stripping technique (AdTS) was used to determine the content of MT in cells. Both the bioaccumulation of heavy metals and the production of MT in different cell compartments of Y. lipolytica exposed to heavy metals were monitored. The LD 50 of each metal was determined from the number of viable cells in yeast cultures: LD 50Cd (37.5 μM), LD 50Ni (570 μM), LD 50Co (700 μM), and LD 50Zn (1800 μM). The highest concentrations of heavy metals were found in the cell wall and membrane debris while the lowest concentrations were detected in the cytoplasm. Cadmium and nickel showed the most significant effect on the production of MT. This study provides new insights into the ecophysiology of microorganisms and demonstrates the potential use of these electrochemical methods in biotechnology.

Dietary cadmium inhibits spontaneous hepatocarcinogenesis in C3H/HeN mice and hepatitis in A/J mice, but not in C57BL/6 mice

Cadmium is known to be a potent carcinogenic and mutagenic metal. However, we demonstrated that dietary supplementation with 50 ppm cadmium inhibits spontaneous carcinogenesis in C3H/HeN and spontaneous hepatitis in A/J mice. We found that the frequencies of spontaneous hepatocarcinogenesis in C3H/HeN mice and of spontaneous hepatitis in A/J mice fed low-dose cadmium for 54 weeks were significantly lower than those in the respective control groups. A cadmium-induced increase in metallothionein production itself and/or metallothionein-associated increases in hepatic zinc concentrations may be involved in the observed preventive effects of cadmium. Our results suggest that low doses of cadmium in the diet or environment may play a beneficial role in the prevention of hepatic disease in humans and animals.

Protective Effect of Zinc against Lipopolysaccharide/D-Galactosamine-Induced Lethality.

Zinc is an essential and multifunctional element for all cells. Metallothionein (MT) is a low molecular weight, cystein-rich, metal-binding protein that is involved in zinc homeostasis. During the acute-phase reaction, hepatic MT production is induced and hepatic zinc accumulation is stimulated. We previously reported that MT-I and II-deficient (MT-null) mice are highly sensitive to the lethal effects of lipopolysaccharide (LPS) plus D-galactosamine (GalN). The sensitization may relate to attenuatoin of α1-acid glycoprotein (AGP) expression in MT-null mice. In the present study, we hypothesized that MT-induced hepatic zinc accumulation promotes AGP expression and prevents LPS/GalN-induced lethality. To determine whether zinc reduces LPS/GalN toxicity, zinc was administered to mice. Simultaneous administration of zinc and LPS/GalN showed no effect on the lethality of LPS/GalN in mice. Zinc administration at 3 hr prior to LPS/GalN challenge reduced LPS/GalN-induced death. However, zinc pre-administration at 24 hr before LPS/GalN challenge did not reduce LPS/GalN-induced death. The expression of AGP mRNA was elevated at 3 hr after zinc administration, 24-hr pretreatment was ineffective. The protective effect of zinc was observed in both wild-type and MT-null mice. These results show that the protective effects of zinc were not caused by MT induction, but by AGP expression. We suggest that MT-induced hepatic zinc accumulation may promote AGP expression and thus prevent LPS/GalN-induced lethality.

Toxicity and Uptake of Zinc in the Planarian Dugesia dorotocephala in Natural Sediments

ABSTRACT Zinc uptake and protein, hemocianine, and metallothionein concentrations were studied in Dugesia dorotocephala exposed to Zn-spiked natural sediments in a recirculation system. Exposures to Zn ranged from 0.129 to 4.43 mg Zn/kg of sediment. Metallothionein and protein concentrations increased after treatment with Zn; however, the hemocianine concentration decreased. Metallothionein production could be an effective defense mechanism in planaria. The 96-h EC50 for Zn against D. dorotocephala was 12.52 mg Zn/kg of sediment.

Prior increase in metallothionein levels is required to prevent doxorubicin cardiotoxicity.

Many studies have shown that metallothionein (MT) can be increased significantly by different oxidative insults in multiple organ systems. However, the increase in MT production often fails to protect against oxidative tissue injury. On the other hand, recent studies using a cardiac-specific, MT-overexpressing, transgenic mouse model have shown that MT protects against oxidative heart injury. Thus, the present study was undertaken to test the hypothesis that prior increase in MT levels is required to prevent oxidative injury. Oxidative heart injury was induced by doxorubicin (DOX), an important anticancer drug that causes severe cardiotoxicity through oxidative stress. Cardiac-specific, MT-overexpressing, transgenic mice and wild-type (WT) FVB mice were treated with DOX at 20 mg/kg. Four days after the treatment, MT concentrations were markedly elevated in the WT mouse heart. The elevated MT concentrations were comparable with those found in the transgenic mouse heart, which did not show further MT elevation in response to DOX challenge. Severe oxidative injury occurred in the heart of WT mice, including myocardial lipid peroxidation, morphological changes as examined by electron microscopy, high levels of serum creatine kinase activity, and decreased total glutathione concentrations in the heart. However, all of these pathological changes were significantly inhibited in the MT-transgenic mice. Therefore, this study demonstrates that there is a correlation between MT induction and oxidative stress in the DOX-treated mouse heart. However, MT can protect the heart from oxidative injury only if it is present prior to induction of oxidative stress.

Effect of temperature and size on metallothionein synthesis in the gill of Mytilus galloprovincialis exposed to cadmium

Although the degree of metal contamination is expected to be a primary determinant of metallothionein (MT) induction in marine mussels, at least at polluted sites, variability caused by temperature, and biotic factors such as size, may need to be considered when interpreting field data. To test the effects of these variables, mussels, Mytilus galloprovincialis, of different sizes (small: 3.5±0.5 cm and large: 5.2±0.7 cm) were exposed to Cd (100 μg l−1) at different water temperatures (5, 18 and 25 °C) for 34 days. Resultant Cd and MT concentrations in gills were shown to be size dependent and increased significantly with temperature. At the highest temperature tested (25 °C) there appears to be a disproportionate effect on Cd accumulation, which raises MT synthesis to exceptionally high levels. The effect of size on MT concentrations was also temperature-dependent: at 18 and 25 °C, large mussels exhibited higher MT levels than smaller individuals, whilst at 5 °C there were no significant differences between contaminated and control mussels, in either size-group, as a result of the reduced level of MT production at this temperature. When considering the use of MT levels in mussels as a biochemical indicator of metal contamination, the potential influence of factors such as size and temperature on MT-metal relationships needs to be considered. Samples should be of uniform size as far as possible, and collection should be limited to a fixed season (avoiding climatic extremes) to ensure that the effects of these factors on baseline levels of MT is minimised.

Roles of metallothionein in copper homeostasis: responses to Cu-deficient diets in mice

Metallothionein (MT) protects the body from both harmful non-essential and excessive essential metals. Copper (Cu) is an essential metal, and its concentration in the body is regulated at a constant level between excess and deficient ones. Cu accumulating in the livers of Wilson disease patients and its animal model, Long-Evans rats with a cinnamon-like coat color (LEC) rats, is in the form of Cu,Zn–MT, MT being an antioxidant. Contrary to the efficient production of MT in response to excessive accumulation of Cu in LEC rats, Cu-binding to MT only occurs marginally under normal conditions. However, the present study revealed that Cu binds to MT more with a severe Cu-deficiency. Namely, male C57BL/6J mice were fed a Cu-deficient diet (0.037 mg Cu/g) and deionized water containing trientine, and then the concentration and distribution of Cu were determined. It was suggested that the cessation of biliary excretion and limitation of the Cu supply to ceruloplasmin are the first responses on feeding of a Cu-deficient diet, followed by an increase in Cu–MT with maintenance of the Cu concentration in the liver. These results suggest that MT causes the recruitment of Cu in a Cu-deficient environment by sequestering Cu from degraded Cu-enzymes and delivering it to Cu chaperones.

Effect of cadmium on antioxidant enzyme activities and lipid peroxidation in the gills of the clam Ruditapes decussatus

Metals are known to influence the oxidative status of marine organisms, and antioxidant enzymes have been often proposed as biomarkers of effect. The clam Ruditapes decussatus is a well-known metal bioindicator. In this species cadmium (Cd) induces metallothionein (MT) synthesis only after 7 days of exposure. Before MT synthesis is induced, the other mechanisms capable of handling the excess of Cd are unknown. In order to identify some of these mechanisms, variations in antioxidant systems (superoxide dismutase, catalase, selenium-dependent glutathione peroxidase and non-selenium-dependent glutathione peroxidase), malondialdehyde (MDA) and MT were studied in the gills of R. decussatus exposed to different Cd concentrations (4, 40 and 100 gl-1) for 28 days. These parameters, together with total proteins and Cd concentrations, were measured in the gills of the clams over different periods of exposure. Results indicate that Cd accumulation increased linearly in the gills of R. decussatus with the increase in Cd concentration. This increase induces an imbalance in the oxygen metabolism during the first days of Cd exposure. An increase in cytosolic superoxide dismutase (SOD) activity and a decrease in mitochondrial SOD activity was observed at the same time as or after a decrease in cytosolic and mitochondrial catalase activity and of selenium-dependent and non-selenium-dependent glutathione peroxidase activity. After 14 days of exposure, Cd no longer affect these enzymes but there was elevation of other cellular activities, such as MDA and MT production. MT bound excess Cd present in the cell. These variations in these parameters suggest their potential use as biomarkers of effects such as oxidative stress resulting from Cd contamination in molluscs.

Nitric oxide induces metallothionein (MT) gene expression apparently by displacing zinc bound to MT

The metal binding protein metallothionein (MT) is involved in zinc homeostasis since it typically binds large amounts of zinc. Free zinc can control MT gene expression by interacting with metal-sensitive transcription factors. However, the precise factors governing intracellular release of metal ions from MT remain unknown. Aerobic nitric oxide (NO) can nitrosate thiol groups in proteins, and MT-bound cadmium is released by NO exposure. Thus, we hypothesized that NO may also be effective at displacing zinc from MT in cultured cells and that this could be an important physiological control mechanism in zinc homeostasis and utilization. In this study, DETA/NO, an agent that spontaneously generates NO with a 20-h half life in physiological media, was used to study the release of zinc from MT and the induction of MT in TRL1215 cells (a normal rat liver cell line). Zinc or cadmium was given at levels inducing MT production, followed by DETA/NO (20–200 μM) to produce controlled NO exposure in both cell lines. Although both metals activated MT gene expression, MT-I mRNA and MT protein were further increased when DETA/NO was given after zinc or cadmium treatment. Additionally, NO from DETA/NO clearly displaced MT-bound zinc, as evidenced by G-75 gel-filtration chromatography. The released zinc or cadmium probably then stimulates further MT gene expression. These results suggest that NO may play an important role in regulation of cellular zinc homeostasis by providing a controlled release mechanism for metal ions stored in MT, and NO-mediated release of MT-bound zinc could in turn activate gene expression, such as with the MT gene.

Metallothionein gene expression and secretion in white adipose tissue.

White adipose tissue (WAT) has been examined to determine whether the gene encoding metallothionein (MT), a low-molecular-weight stress response protein, is expressed in the tissue and whether MT may be a secretory product of adipocytes. The MT-1 gene was expressed in epididymal WAT, with MT-1 mRNA levels being similar in lean and obese (ob/ob) mice. MT-1 mRNA was found in each of the main adipose tissue sites (epididymal, perirenal, omental, subcutaneous), and there was no major difference between depots. Separation of adipocytes from the stromal-vascular fraction of WAT indicated that the MT gene (MT-1 and MT-2) was expressed in adipocytes themselves. Treatment of mice with zinc had no effect on MT-1 mRNA levels in WAT, despite strong induction of MT-1 expression in the liver. MT-1 gene expression in WAT was also unaltered by fasting or norepinephrine. However, administration of a beta(3)-adrenoceptor agonist, BRL-35153A, led to a significant increase in MT-1 mRNA. On differentiation of fibroblastic preadipocytes to adipocytes in primary culture, MT was detected in the medium, suggesting that the protein may be secreted from WAT. It is concluded that WAT may be a significant site of MT production; within adipocytes, MT could play an antioxidant role in protecting fatty acids from damage.

Zinc sulphate induces metallothionein in pancreatic islets of mice and protects against diabetes induced by multiple low doses of streptozotocin

Diabetes is induced by multiple low doses of streptozotocin (MLD-STZ) in male mice of susceptible strains. In this model beta-cell injury and T-cell-mediated inflammatory reactions are induced. Probably, reactive oxygen species (ROS) participate in the destruction of beta cells. The effects of ROS can be counterbalanced by several antioxidant systems. One of these is metallothionein (MT), cytosolic proteins that are induced by zinc ions (Zn2+) and scavenge hydroxyl radicals (OH). The effect of Zn2+ on MLD-STZ-diabetes was studied. We gave C57BL/6 and (C57BL/6 x SJL)F1 hybrid mice either MLD-STZ or in addition Zn2+-enriched drinking water. We analysed metallothionein ex vivo in pancreatic islets for protein and mRNA concentration for the isoforms 1 and 2. Pancreatic sections were examined by immunohistochemistry for metallothionein and histologically for insulitis. In both strains, Zn2+-enriched drinking water significantly up-regulated metallothionein and prevented MLD-STZ-diabetes and loss of beta-cell function. In the F1 hybrid mice a variant of MLD-STZ-diabetes was observed. These mice developed hyperglycaemia 10 weeks after the first injection of STZ (in contrast to 2 weeks observed in other mouse strains) and pronounced insulitis. The mRNA of the metallothionein isoforms 1 and 2 were constitutively expressed and slightly up-regulated by Zn2+-enriched drinking water. All islets cells stained for metallothionein. Drinking water enriched with Zn2+ significantly up-regulated metallothionein production in pancreatic islets of mice and prevented diabetes induced with MLD-STZ.

Regulation of metallothionein gene expression and secretion in rat adipocytes differentiated from preadipocytes in primary culture.

The gene encoding metallothionein, a low mol. wt. metal binding and stress response protein, is expressed in white adipose tissue. In the present study, metallothionein (MT-1) gene expression and factors regulating metallothionein production have been examined in adipocytes induced to differentiate from fibroblastic preadipocytes in primary cell culture. On the induction of differentiation, the metallothionein-1 gene was strongly expressed in the cells and metallothionein released into the medium. A peak in metallothionein-1 mRNA level and metallothionein secretion occurred at 2 and 10 days post-differentiation, respectively, with a decrease in protein release after this time. The metallothionein-1 gene was expressed in the adipocytes prior to the adipsin and lipoprotein lipase genes, suggesting that it is an early marker of adipocyte differentiation. The addition of the glucocorticoid, dexamethasone, led to a substantial increase in metallothionein-1 mRNA in the cells and metallothionein secretion. Insulin and leptin also stimulated metallothionein production, although the effect was small. Neither noradrenaline nor the beta3-adrenoceptor agonist, BRL 37 344, altered metallothionein release but forskolin and bromo-cAMP were stimulatory, markedly increasing both metallothionein-1 level and metallothionein secretion. It is suggested that metallothionein is a novel secretory product of the differentiated white adipocyte and that its production is regulated particularly by glucocorticoids and through a cAMP-dependent pathway.

GENETIC BASIS OF A BETWEEN-ENVIRONMENT TRADE-OFF INVOLVING RESISTANCE TO CADMIUM IN DROSOPHILA MELANOGASTER.

In a replicated, laboratory, natural selection experiment Drosophila melanogaster populations were maintained for 20 generations either on unpolluted medium or on polluted medium containing cadmium chloride at a concentration of 80 μg/ml. Lines maintained on polluted medium evolved resistance. In comparison with unpolluted lines, their juvenile survivorship increased from 35% to 46%, developmental period decreased from 13.7 days to 13.0 days, and fecundity increased from 3 to 29 eggs per two-day period. Emergence weights, however, did not change. By contrast the "environmental" effect of moving susceptible flies onto polluted medium was that after two generations survivorship fell 62%, developmental period increased 40%, and fecundity fell 97%. Emergence weights fell 31% in females and 28% in males. Resistant lines paid a fitness cost in unpolluted environments, with fecundity being reduced by 44% and emergence weights being reduced by 4% in females and 6% in males. Developmental period, however, was unaffected. Analyses of crosses and backcrosses between the lines suggested that the evolved cadmium resistance was due to a single sex-linked gene. Levels of dominance were calculated, and in each life-history character the resistant allele was found to be completely dominant. Because the life-history effects appear to be produced by a single gene, it is probable that they all depend on the same metabolic pathway. Metallothionein production is a likely candidate because this is known to be controlled by genes on the X-chromosome. The study adds to a small number of examples of single or closely linked genes with large antagonistic pleiotropic effects on life histories. The result here is a between-environment trade-off, allowing animals increased fitness in polluted environments, but only at the cost of reduced growth and reproduction in unpolluted environments.

Silver aids healing in the sterile skin wound: experimental studies in the laboratory rat

Incisional wounds 15 mm long were induced surgically in the back skin of young adult Wistar rats. They were sutured and used as an experimental model in the therapeutic evaluation of daily applications of 0.5 mL of silver nitrate (SN) at 0.01, 0.1 or 1.0% w/v aqueous solution, or 0.5 g silver sulphadiazine (SSD) over a 10-day period. Control wounds received deionized water only. The silver preparations were not toxic but SN did stain the hair and superficial layers of the stratum corneum. The wounds remained microbiologically clean. Wounds exposed to SN (0.1 or 1.0%) or SSD healed more rapidly than controls. From about the fourth day of treatment, we noted a more rapid exteriorization of sutures, improved wound closure and an earlier loss of scabs and wound debris. Silver treatment appeared to reduce the inflammatory and granulation tissue phases of healing and enhance epidermal repair. Silver from SN was deposited as silver sulphide in extrafollicular hair shafts and superficial aspects of the skin and wound debris but not at deeper levels. Silver uptake was four-fold higher in damaged skin than in intact tissue. SSD was absorbed by intact and wounded skin but the silver did not precipitate as silver sulphide and its localization in the tissue is not known. Uptake of silver from SN or SSD was associated with changes in the concentrations of zinc and calcium in the skin. Zinc levels were depressed during the inflammatory and proliferative phases of healing and then increased. Zinc concentrations had normalized by 10 days when wound healing was achieved. Calcium levels remained higher than normal throughout the observation period. The mechanism of action of silver in advancing wound healing in the rat is unclear. Its ability to reduce the inflammatory and granulation phases of healing, and to invoke metallothionein production and influence metal ion binding are possibly important.

Quantification of metallothioneins in the common asteroid Asterias rubens (Echinodermata) exposed experimentally or naturally to cadmium

Uptake and intracellular fate of Cd was assessed in the pyloric caeca of the common asteroid Asterias rubens exposed experimentally or naturally to Cd. Cd partitioning among intracellular metal-binding pools was studied by graphite furnace atomic absorption spectrometry of heat-treated cytosolic fractions which had been separated by gel filtration chromatography. Low molecular mass proteins bound > 80% of cytosolic Cd and appeared to become saturated with the metal after experimental exposure to 20 μg Cd 1 −1 for 10–20 days. These proteins presented several features of metallothioneins (MTs), namely molecular mass (13–22 kDa), heat-stability (90 °C, 10 min), high Cd content, and high thiolic content as determined by differential pulse polarography. Analysis of Chromatographie fractions indicated that the majority of thiolic groups (66–73%) present in heat-treated cytosol was associated with the MT pool. Subsequent determinations of MT concentrations were made using whole cytosol extracts after correcting for interference from thiolic groups present in residual, heat-stable, high molecular mass proteins. Asteroids collected from unpolluted sites in SW England, SW Netherlands and SW Norway contained basal levels of 2.5 – 4.5 mg MT g −1 dw. Asteroids experimentally exposed to Cd (20 μg Cd 1 −1) responded by a 2-fold increase in the production of MT after 30 days. Turnover of MTs was rapid in A. rubens and probably accounted for the low net increase in protein and characteristic rapid loss of Cd from the pyloric caeca. Alkaline phosphatase (AP) activity was measured as a marker of the functional state of the pyloric caeca. It was not significantly affected during Cd exposure in the laboratory. Asteroids collected in the heavy metal polluted Sørfjord (SW Norway) contained significantly higher MT concentrations (5 – 5.6 mg MT g −1 dw) and significantly lower AP activity in the most heavily contaminated region, though these two parameters were not correlated.

The effect of cold stress on UVB injury in mouse skin and cultured keratinocytes.

The effect of cold stress on skin damage caused by UVB irradiation was investigated both in vivo and in vitro. Ear skin of mice that had been exposed to cold stress at 0 degree C for 20 min and at 5 degrees C for 24 h was exposed to UVB radiation. Sunburn cell production was less in mice exposed to the lower temperature. In addition, the effect of cold stress on the survival rate of UVB-irradiated rat keratinocytes was examined in a cytotoxicity test, with the results showing that keratinocytes exposed to cold stress of 0 degree C had a higher survival rate than control cells. To pursue a promising clue for explaining the result, we examined metallothionein (MT) production in rat keratinocytes that had been exposed to cold stress at 0 degree C. Microfluorometric quantification showed a positive correlation between the time course and the intensity of immunofluorescence for MT, indicating that the molecule is inducible by exposure to cold stress in our experimental system. These results suggest that epidermal cells that have been exposed to cold stress maintain a higher resistance to UV radiation than nonexposed controls in vivo and in vitro, and that MT with radical-scavenging activity might contribute, at least in part, to photoprotection against UVB-induced oxidative damage in mammalian skin.

Inhibition of zinc-induced metallothionein mRNA accumulation by gonadotropin-releasing hormone in human hepatocarcinoma cell line HepG2.

Recently we have demonstrated that the human hepatocellular carcinoma-derived cell lines, HepG2 and HuH7, contain gonadotropin-releasing hormone (gonadoliberin) receptors and respond to various molecular forms of gonadoliberin in terms of suppressed proliferation in vitro. This study provides the first demonstration that gonadoliberin inhibits the zinc-induced production of metallothionein mRNA in HepG2 and HuH7 cells. Administration of gonadoliberin agonist (gonadoliberin-A) inhibited the Zn-induced metallothionein mRNA level in a time-related and dose-related manner. The effect of gonadoliberin-A was found to be specific, because concomitant treatment with a gonadoliberin antagonist (gonadoliberin-ANT) blocked gonadoliberin-A inhibition of metallothionein mRNA accumulation. Furthermore, the gonadoliberin-A-induced inhibition of Zn-mediated metallothionein accumulation was found to correlate closely with suppresion of cell proliferation and [3H]thymidine uptake in these cells. It is known that the metal-binding protein metallothionein plays an important role in tumor cell pathobiology and resistance to chemotherapeutic drugs. The present findings may have important implications in the development of an effective chemotherapy for treatment of human liver cancer, in part, by improving the sensitivity of tumor cells through suppression of metallothionein production by gonadoliberin peptides.

Induction and characterization of metallothionein in different organs ofOstrea edulis L.

In this study, the induction of metallothionein (MT) in one species of oyster Ostrea edulis exposed to copper (Cu) and zinc (Zn) was examined. The quantity of total protein (mg protein/mg dry wt) in each sample and the measurement of MT were determined by the Lowry and silver (Ag)-saturation methods. Our results show that the gonads are the organs producing the greatest quantity of MT in the controls and in the groups induced by Cu and Zn, followed by the mantle, gut, and by muscle and plasma. Competition by Zn with respect to Cu for the production of MT has been established.

Studies on the Mechanism of Early Onset Macular Degeneration in Cynomolgus Monkeys. II. Suppression of Metallothionein Synthesis in the Retina in Oxidative Stress

Initial investigations done in this laboratory detected increased albumin and decreased glyceraldehyde 3-phosphate dehydrogenase concentrations in the retina of an animal model manifesting early onset macular degeneration. Both glyceraldehyde 3-phosphate dehydrogenase and albumin are markers of oxidative stress in cells. In this study, we used the same animal model to study further biochemical and physiological processes which may be involved in the pathogenesis of early onset macular degeneration in monkeys. We detected 60% lower catalase and glutathione peroxidase activities in the affected retinas suggesting lower antioxidant activities and oxidative stress. One of the consequences of oxidative stress is the production of metallothionein, a low molecular weight protein also induced by high concentrations of heavy metals such as zinc. Metallothionein was detected by RT-PCR in these monkey retinas. However initial quantitative PCR studies on this protein showed that the synthesis of metallothionein in affected retinas appears to be less than in normal controls. The affected retinas also showed a fourfold lower zinc concentration compared with the normal controls. No significant difference, however, could be detected in the zinc concentrations in plasma samples. Since induction of metallothionein synthesis is mediated by transcription factors which require heavy metals such as zinc for binding to specific sites in the DNA, the lowered zinc concentration may, thus, correlate with the lowered metallothionein expression. And since metallothionein is suggested to function as a free radical scavenger, the lowered metallothionein synthesis may consequently contribute to increased peroxidation reactions in the affected retinas. It appears therefore, that oxidative stress and the decreased metallothionein synthesis may be involved in the pathogenesis of early onset macular degeneration in this animal model.