Yeast CUP1 protects HeLa cells against copper-induced stress.

As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. Yeast CUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study, CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.

Metallothionein: an overview on its metal homeostatic regulation in mammals / Metalotioneina: una visión general de su regulación homeostática de metales en mamíferos

Metallothionein (MT) is a ubiquitous protein with a low molecular weight of 6-7 kDa weight and it was first identified in the kidney cortex of equines as a cadmium (Cd)-binding protein responsible for the natural accumulation of Cd in the tissue. The mammalian MT contains 61 to 68 amino acid residues, in which 18 to 23 cysteine residues are present. The expression of MT starts by binding of metal transcription factor-1 (MTF-1) to the regulative region of MT gene called metal responsive elements (MREs). The induction of MT through the MREs region can be initiated by several metal ions such as zinc (Zn), copper (Cu) and Cd. However, Zn is the only heavy metal which can reversibly and directly activate the DNA-binding activity of MTF-1. In mammals four types of MT are expressed and they are termed metallothionein-1 (MT1), metallothionein-2 (MT2), metallothionein-3 (MT3), and metallothionein-4 (MT4). MT1 and MT2 are expressed in almost all tissues while MT3 and MT4 are tissue-specific. MT is a key compound involved in the intracellular handling of a variety of essential and nonessential post-transitional metal ions. In order to the heavy metal binding ability of MT, it is suggested to play roles both in the intracellular fixation of essential trace elements Zn and Cu, in controlling the concentrations, and in neutralizing the harmful influences of exposure to toxic elements...

Metalotioneina (MT) es una proteína, con bajo peso molecular de kDa 6-7 y que fue primero identificada en la corteza renal de equinos como cadmio (Cd)-proteína responsable por la acumulación natural de Cd en los tejidos. La MT en mamíferos contiene 61 a 68 residuos de aminoácidos, de los cuales están presentes 18 a 23 residuos de cisteína. La expresión de MT se inicia por la unión del factor-1 de transcripción de metal (MTF-1) a la región reguladora del gen de la MT llamado elementos metálicos responsable (MREs). La inducción de MT a través de la región MREs puede ser iniciada por varios iones metálicos tales como zinc (Zn), cobre (Cu) y Cd. Sin embargo, el Zn es el único metal pesado que puede revertir y activar directamente la unión ADN de MTF-1. En los mamíferos se expresan cuatro tipos de MT y ellos se denominan metalotioneína-1 (MT1), metalotioneína-2 (MT2), metalotioneína-3 (MT3), y metalotioneína-4 (MT4). MT1 y MT2 se expresan en casi todos los tejidos mientras que MT3 y MT4 son tejido-específico. La MT es un compuesto clave implicado en la manipulación intracelular de una variedad de iones metálicos esenciales y no esenciales post-transicionales. Con el fin de evaluar la capacidad de unión de metales pesados de MT, se sugiere que éste desempeña ambos roles tanto en la fijación intracelular de trazas de elementos de Zn y Cu como en el control de las concentraciones, y neutralizando las influencias perjudiciales a la exposición de elementos tóxicos...

Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines.

The role of metallothioneins (MT) in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-). As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80%) belong to two major categories: 1) metabolism; and 2) cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways.

Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines

The role of metallothioneins (MT) in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-). As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80 percent) belong to two major categories: 1) metabolism; and 2) cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways.

Metallothionein is crucial for safe intracellular copper storage and cell survival at normal and supra-physiological exposure levels.

MTs (metallothioneins) increase the resistance of cells to exposure to high Cu (copper) levels. Characterization of the MT-Cu complex suggests that MT has an important role in the cellular storage and/or delivery of Cu ions to cuproenzymes. In this work we investigate how these properties contribute to Cu homoeostasis by evaluating the uptake, accumulation and efflux of Cu in wild-type and MT I/II null rat fibroblast cell lines. We also assessed changes in the expression of Cu metabolism-related genes in response to Cu exposure. At sub-physiological Cu levels (0.4 microM), the metal content was not dependent on MT; however, when extracellular Cu was increased to physiological levels (10 microM), MTs were required for the cell's ability to accumulate the metal. The subcellular localization of the accumulated metal in the cytoplasm was MT-dependent. Following supra-physiological Cu exposure (>50 microM), MT null cells had a decreased capacity for Cu storage and an elevated sensitivity to a minor increment in intracellular metal levels, suggesting that intracellular Cu toxicity is due not to the metal content but to the interactions of the metal with cellular components. Moreover, MT null cells failed to show increased levels of mRNAs encoding MT I, SOD1 (superoxide dismutase 1) and Ccs1 (Cu chaperone for SOD) in response to Cu exposure. These results support a role for MT in the storage of Cu in a safe compartment and in sequestering an intracellular excess of Cu in response to supra-physiological Cu exposure. Gene expression analysis suggests the necessity of having MT as part of the signalling pathway that induces gene expression in response to Cu.

Stress proteins induced by arsenic.

The elevated expression of stress proteins is considered to be a universal response to adverse conditions, representing a potential mechanism of cellular defense against disease and a potential target for novel therapeutics. Exposure to arsenicals either in vitro or in vivo in a variety of model systems has been shown to cause the induction of a number of the major stress protein families such as heat shock proteins (Hsp). Among them are members with low molecular weight, such as metallotionein and ubiquitin, as well as ones with masses of 27, 32, 60, 70, 90, and 110 kDa. In most of the cases, the induction of stress proteins depends on the capacity of the arsenical to reach the target, its valence, and the type of exposure, arsenite being the biggest inducer of most Hsp in several organs and systems. Hsp induction is a rapid dose-dependent response (1-8 h) to the acute exposure to arsenite. Thus, the stress response appears to be useful to monitor the sublethal toxicity resulting from a single exposure to arsenite. The present paper offers a critical review of the capacity of arsenicals to modulate the expression and/or accumulation of stress proteins. The physiological consequences of the arsenic-induced stress and its usefulness in monitoring effects resulting from arsenic exposure in humans and other organisms are discussed.

Toxicología ambiental: metalotioneínas y metabolismo celular / Environmental toxicology: metallothioneins and cellular metabolism

Las metalotioneínas (MT) constituyen una familia de proteínas de bajo peso molecular (6-8 kDa), presentes en procariotes y eucariotes, con elevado contenido en cisteína (30-35 por ciento) y marcada capacidad para combinarse con iones metálicos. dentro del metabolismo celular, se le han asignado múltiples funciones, desde un papel central en la homeostasis celular de ciertos metales pesados esenciales ("pool" citosólico de Zn y/o Cu), la detoxificación de metales pesados no esenciales, hasta su participación en ciertos procesos inflamatorios y la inactivación de radicales libres. La variación en el nivel de MT es indicador de exposición a xenobióticos, de situaciones ambientales adversas así como de situaciones de estrés fisiológico. Como tal, esta respuesta puede utilizarse tanto a nivel de la toxicología laboral, clínica y en estudios ecotoxicológicos. A este respecto, se encuentra en fase experimental su utilización con fines de diagnóstico ambiental. Esta revisión tiene como objeto constituir una breve reseña sobre los principales conocimientos reunidos hasta la fecha. Se ha avanzado significativamente en los aspectos básicos y metodológicos de la inducción de metalotioneínas en situaciones de estrés. Sin embargo, y a pesar de contarse con un número importante de estudios específicos, y a diferencia de otras proteínas menos estudiadas, queda aún sin precisar su función básica dentro del metabolismo celular

Toxicología ambiental: metalotioneínas y metabolismo celular / Environmental toxicology: metallothioneins and cellular metabolism

Las metalotioneínas (MT) constituyen una familia de proteínas de bajo peso molecular (6-8 kDa), presentes en procariotes y eucariotes, con elevado contenido en cisteína (30-35 por ciento) y marcada capacidad para combinarse con iones metálicos. dentro del metabolismo celular, se le han asignado múltiples funciones, desde un papel central en la homeostasis celular de ciertos metales pesados esenciales ("pool" citosólico de Zn y/o Cu), la detoxificación de metales pesados no esenciales, hasta su participación en ciertos procesos inflamatorios y la inactivación de radicales libres. La variación en el nivel de MT es indicador de exposición a xenobióticos, de situaciones ambientales adversas así como de situaciones de estrés fisiológico. Como tal, esta respuesta puede utilizarse tanto a nivel de la toxicología laboral, clínica y en estudios ecotoxicológicos. A este respecto, se encuentra en fase experimental su utilización con fines de diagnóstico ambiental. Esta revisión tiene como objeto constituir una breve reseña sobre los principales conocimientos reunidos hasta la fecha. Se ha avanzado significativamente en los aspectos básicos y metodológicos de la inducción de metalotioneínas en situaciones de estrés. Sin embargo, y a pesar de contarse con un número importante de estudios específicos, y a diferencia de otras proteínas menos estudiadas, queda aún sin precisar su función básica dentro del metabolismo celular (AU)

Thermogenic activity of growth hormone transgenic mice.

The objective was to determine the effect of a mouse metallothionein/bovine growth hormone transgene on resting metabolic rate (RMR), cold-induced thermogenesis, and beta-agonist stimulated nonshivering thermogenesis in mice. Non-transgenic littermates were used as controls. Open-circuit indirect calorimetry was used to assess RMR and cold-induced thermogenesis in 64 mice. Air temperature in the chamber was set at 31 degrees C for RMR and was decreased to 28, 25, 21, or 17 degrees C to determine cold-induced thermogenesis. Response to the beta-agonist isoproterenol was evaluated by monitoring changes in colonic temperature of 34 mice upon injection of the drug or saline. Despite the fact that RMR tended to be lower in transgenics than in nontransgenics, at 31 degrees C transgenic mice were able to regulate colonic temperature at the same level as nontransgenics, but colonic temperature decreased in transgenics relative to nontransgenics as air temperature was reduced. For each degree decrease in air temperature between 31 and 17 degrees C, nontransgenic mice increased heat production by 1.03 +/- .10 watt/kg, whereas transgenic mice increased it by only .56 +/- .08 watt/kg, indicating that the thermogenic response of transgenics to cold was inferior. The magnitude of the maximal increase in colonic temperature after isoproterenol injection was similar for both groups, but the response was slower in transgenics. We suggest that lean body mass and substrate availability for shivering thermogenesis are reduced in transgenics relative to total body weight, and that they allow colonic temperature to decrease to conserve energy.

Increase of striatal dopamine release by cadmium in nursing rats and its prevention by dexamethasone-induced metallothionein.

Repeated daily intraperitoneal (i.p.) administrations of cadmium (CdCl2, 1 mg/kg per day for 5 days) increased striatal dopamine (DA) release (180% of controls) and turnover (150% of controls) in 13-day-old rats. Cd treatment also increased striatal metallothionein (MT) content (161%), Cd (127%) and lipid peroxidation (LPO, 190%). In addition, Cd treatment decreased striatal tyrosine hydroxylase (TH) activity (-28%), and such an effect may result from D-2 receptor blockade as a consequence of excessive dopamine release, since sulpiride (a specific D-2 receptor antagonist) administration to Cd-treated rats abolished the effect of Cd on TH. No effect was observed on striatal monoamine oxidase (MAO) activity. Dexamethasone (Dx) treatment increased striatal MT content and caused no effect on either DA release or turnover. However, Dx administration prevented the effects caused by Cd, including the increased DA release and enhanced striatal lipid peroxidation. These results indicate that toxic effects on the brain are to be expected as a result of Cd exposure and that Dx administration can attenuate them.

Effects of bovine growth hormone (GH) expression in transgenic mice on serum and pituitary immunoreactive mouse GH levels and pituitary GH-releasing factor binding sites.

Pituitary and serum levels of homologous growth hormone (GH) and characteristics of specific GH-releasing factor (GHRF) binding to pituitary homogenates were examined in transgenic mice expressing bovine GH (bGH) gene regulated by different promoters [mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase (PEPCK)] and in their normal littermates. Pituitary GH concentration and GHRF binding were reduced by approximately 50% in transgenic MT-bGH mice in which serum bGH levels were about 20 micrograms/l and by approximately 95% in transgenic PEPCK-bGH mice in which serum bGH levels were tenfold higher. Suppression of plasma immunoreactive mouse GH (mGH) levels was detected in MT-bGH but not in PEPCK-bGH animals, presumably due to cross-reaction of the antiserum employed with bGH. Scatchard plots of GHRF binding to washed homogenates of pituitary glands from normal and young adult MT-bGH transgenic mice were curvilinear, indicating the presence of two types of binding sites, with low and high affinities. Both types of binding sites were reduced in number in MT-bGH transgenic mice without changes in their affinity. In 5-7-month-old MT-bGH transgenic mice there were changes in pituitary GH levels, in GHRF binding levels and in characteristics of GHRF binding that closely resembled the alterations described previously in aging rats. We conclude that over-expression of heterologous GH genes in transgenic mice can lead to partial or virtually complete suppression of somatotroph function, depending on the levels of heterologous GH in the circulation, and that transgenic MT-bGH mice exhibit symptoms of remarkably early onset of neuroendocrine aging.