Biomedical subjects

M G Cherian

Publications and source records attributed to M G Cherian.

At least 19 recordsLinked to original sources

PubMed2005

Role of p53 and reactive oxygen species in apoptotic response to copper and zinc in epithelial breast cancer cells.

Previous studies revealed that cells may differ in their response to metal stress depending on their p53 status; however, the sequence of events leading to copper-induced apoptosis is still unclear. Exposure of copper (10 and 25 microM) and zinc (10 and 25 microM) caused activation of p53 in ER+/p53+ human epithelial breast cancer MCF7 cells and resulted in up-regulation of p21. Transactivation of p53 in MCF7 cells also led to increase in expression of Bax, proapototic Bcl-2 family member, triggering mitochondrial pore opening, and PIG3 (p53-induced gene 3 product), and also generation of intracellular reactive oxygen species (ROS). The treatment of MCF7 cells with either copper or zinc for 4 h also caused decrease in mitochondrial membrane potential (Delta psi(m)), accompanied by an elevation in the ROS production and redistribution of p53 into mitochondria. The loss of Delta psi(m) was correlated with accumulation of Annexin V positive apoptotic cells. However, the release of apoptosis inducing factor (AIF) and its translocation into nucleus was observed only in MCF7 cells treated with copper. In MDA-MB-231 (ER-/p53-) and MCF7-E6 (ER+/p53-) cells, both p53 and p21 protein levels were not altered in the presence of metals. These cells were resistant to metals, and there was no alteration in Delta psi(m). Copper treatment did not result in accumulation of ROS in these cell lines with an inactive p53 even after exposure to 50 microM of copper for 6 h, indicating a key role for p53 in the ROS generation. Pretreatment of MCF7 cells with p53 inhibitor, pifithrin-alpha, resulted in decrease of copper and zinc induced ROS production to the control level, suppression of both Bax expression and AIF release. Therefore, the activation of p53 seems to play a crucial role in copper and zinc induced generation of ROS in epithelial breast cancer cells, and expression of downstream targets of p53, such as PIG3 and Bax, responsible for increased generation of the intracellular ROS, as well as disruption of mitochondrial integrity. Our data suggest that copper induces apoptosis in MCF-7 cells with no caspases through the depolarization of mitochondrial membrane with release of AIF and its translocation into the nucleus. The results demonstrate that a functional p53 is required for the execution of apoptosis in epithelial cells.

Apoptosis↗

PubMed2004

Differential regulation of signal transduction pathways in wild type and mutated p53 breast cancer epithelial cells by copper and zinc.

Previous studies have suggested that cells may differ in their response to metal stress. This study was undertaken to investigate the role of PI3K/Akt signaling pathway in metal resistance in human breast cancer epithelial cells with different p53 and estrogen receptor status. Exposure to copper and zinc increased Akt phosphorylation with its nuclear localization only in MDA-MB-231 cells with no estrogen receptor and mutated p53. Cyclin D1 expression and cell-cycle progression followed the metal-induced Akt phosphorylation. Treatment with LY294002 abrogated these effects, suggesting the essential role of PI3-kinase. In contrast, in MCF-7 cells with wild type p53 and estrogen receptor, there was no change in Akt activation, while suppression of p53 activity by pifithrin-alpha increased phosphorylation of Akt after the treatment with copper. In MCF-7 cells, the metal treatment increased the phosphorylation of p53 at serine 15, up-regulated p21 expression, and resulted in cell-cycle arrest in G1 phase with apoptosis. These results demonstrate that copper-induced apoptosis in MCF-7 cells is p53 dependent, whereas the metal resistance in MDA-MB-231 cells may be due to activation of Akt in the absence of a functional p53.

Apoptosis↗

PubMed2002

Potential role of p53 on metallothionein induction in human epithelial breast cancer cells.

The expression and induction of metallothionein has been associated with protection against oxidative stress and apoptosis. This study examines the effect of tumour suppressor protein p53 on metallothionein expression following CdCl2 treatment in eight human epithelial breast cancer cell lines differing in p53 and oestrogen-receptor status. Cells were treated with 10 microM CdCl2 for 24 h and metallothionein protein levels were measured by cadmium binding assay. MCF7 cells which are p53-positive (p53+) and oestrogen-receptor-positive showed a large induction in metallothionein synthesis by 10.79+/-1.36-fold. Other breast cancer cell lines which are p53-negative (p53-) and oestrogen-receptor-negative or weakly oestrogen-receptor-positive showed a small induction ranging from 1.40+/-0.10 to 3.65+/-0.30-fold. RT-PCR analysis showed an induction of metallothionein mRNA in MCF7 cells by about 1.61+/-0.08-fold, while in HCC1806 cells (p53-, oestrogen-receptor-negative) by 1.11+/-0.13-fold, and in MDA-MB-231 (p53-, oestrogen-receptor-negative) by 1.25+/-0.06-fold. Metallothionein localisation was determined by immunohistochemical staining. Prior to metal treatment, metallothionein was localised mainly in the cytoplasm of MCF7 and MDA-MB-231 cells. After treatment with 10 microM CdCl2 for 24 h, MCF7 cells showed intense nuclear and cytoplasmic staining for metallothionein, while MDA-MB-231 cells showed staining in the cytoplasm with weak nuclear staining. Apoptosis induced by 10-40 microM CdCl2 at time points between 4 and 48 h was examined with TUNEL assay. In MCF7 cells, apoptosis increased with higher concentrations of CdCl2, it peaked at 6-8 h and appeared again at 48 h for all concentrations of CdCl2 tested. In MDA-MB-231 cells, apoptosis remained at low levels for 10-40 microM CdCl2 at all time points. Studies on cadmium uptake showed similar uptake and accumulation of cadmium at 8 and 24 h in all the cell lines. The data demonstrate that treatment of epithelial breast cancer cells with 10 microM CdCl2 for 24 h caused a greater induction of metallothionein protein and mRNA expression in p53+ and oestrogen-receptor-positive cells as compared to p53- and oestrogen-receptor-negative or weakly oestrogen-receptor-positive cells. This effect may be associated with the occurrence of apoptosis and suggests a role for p53 and oestrogen-receptor on the expression and induction of metallothionein in epithelial cells.

Apoptosis↗

PubMed2001

Metallothionein expression in patients with small cell carcinoma of the lung: correlation with other molecular markers and clinical outcome.

BACKGROUND: Patients with small cell carcinoma of the lung (SCLC) are known to have an extremely poor prognosis, with a 5-year survivor rate of only 5%. Chemotherapeutic drug resistance is a major obstacle to curative therapy in patients with SCLC. METHODS: The authors evaluated retrospectively the expression of metallothionen (MT), proliferating cell nuclear antigen (PCNA), p53, and retinoblastoma gene product (RBGP) in biopsy samples from 58 patients with SCLC prior to standard chemotherapy. The objective was to study the correlation between MT and other molecular markers in SCLC and correlate these data with the clinical outcome of patients. The authors studied 28 short-term survivors (STS; survival < 24 months) and 30 long-term survivors (LTS; survival > 24 months). RESULTS: In line with expectations, the authors found a strong inverse association between stage and survival. Of 58 patients with SCLC, 26 patients (45%; 17 STS and 9 LTS) showed MT expression, 55 patients (94%; 28 STS and 27 LTS) were positive for PCNA, 28 patients (48%; 16 STS and 12 LTS) were positive for p53, and only 6 patients (10%; 1 STS and 5 LTS) showed positivity for RBGP. On comparing the percent positivity of various markers in the two survivor groups, there was greater frequency of expression of MT, PCNA, and p53 and lower RBGP expression in the STS group compared with the LTS group. However, only the difference in expression of MT between the two survivor groups was statistically significant (Fisher exact test; P = 0.034). Multivariable analysis using a logistic regression model showed a significant association between MT expression and patient survival after adjusting for disease stage (chi-square test; P = 0.022). There was also a statistically significant association between MT expression and p53 expression (chi-square test; P = 0.001). CONCLUSIONS: In this study, of the molecular markers studied, the authors demonstrated that only MT overexpression was independently predictive of short-term survival in patients with SCLC undergoing chemotherapy.

Adult↗

PubMed2001

Roles of vitamin C in radiation-induced DNA damage in presence and absence of copper.

Exposure to either ionizing radiation or certain transition metals results in generation of reactive oxygen species that induce DNA damage, mutation, and cancer. Vitamin C (a reactive oxygen scavenger) is considered to be a dietary radioprotective agent. However, it has been reported to be genotoxic in the presence of certain transition metals, including copper. In order to explore the capacity of vitamin C to protect DNA from radiation-induced damage, and the influence of the presence of copper on this protection, we investigated vitamin C-mediated protection against radiation-induced damage to calf thymus DNA in vitro in the presence or absence of copper(II). Vitamin C (0.08-8.00 mM, pH 7.0) significantly reduced DNA damage induced by gamma-irradiation (30-150 Gy) by 30-50%, similar to the protective effect of glutathione. However, vitamin C plus copper (50 microM) significantly enhanced gamma-radiation-induced DNA damage. Low levels of added copper (5 microM), or chelation of copper with 1-N-benzyltriethylenetetraine tetrahydrochloride (BzTrien) and bathocuprinedisulfonic acid (BCSA), abolished the enhanced damage without diminishing the protective effect of vitamin C. These results indicate that vitamin C can act as: (1) an antioxidant to protect DNA damage from ionizing radiation; and (2) a reducing agent in the presence of copper to induce DNA damage. These effects are important in assessing the role of vitamin C, in the presence of mineral supplements or radioprotective therapeutic agents, particularly in patients with abnormally high tissue copper levels.

Animals↗

PubMed2001

Metallothionein in human gingival amalgam tattoos.

Amalgam tattoos occur when small particles of dental amalgam, composed largely of silver (Ag) and mercury (Hg), are inadvertently implanted into oral soft tissues during dental procedures. Metallothioneins (MTs) are ubiquitous, low molecular weight, cysteine-rich, metal-binding proteins that are inducible by many agents including metals and may be involved in the detoxification of toxic metals such as Hg. In this study, the correlation between MT expression and amalgam tattoos in human gingiva was investigated using energy-dispersive X-ray microanalysis (EDX) and immunohistochemical techniques. Light microscopically, amalgam tattoos presented as either fine granular particles or larger discrete opaque globular particles in connective tissues. EDX revealed the smaller particles to be silver sulphide (Ag(2)S), while the larger particles exhibited a shell of Ag(2)S that contained irregularly distributed masses of Ag and Hg. Particles of tin (Sn) were also found. No MT staining was observed in collagen, fibroblasts or blood vessels in areas exhibiting abundant amounts of embedded fine granular Ag(2)S particles. Blood vessels exhibiting relatively few amalgam particles stained positively for MT. Cells with the morphological features of histiocytes located directly adjacent to larger pieces of amalgam showed intense MT staining. These results indicate that amalgam tattoos contain no Hg or free Ag except in large globular pieces of amalgam, which still contain Hg and which induce MT expression in adjacent histiocytes. This suggests that Hg leaching from impacted dental amalgam particles induces MT, while residual Ag(2)S and Sn particles do not. MT may therefore act to reduce Hg exposure in patients with amalgam tattoos.

Blood Vessels↗

PubMed2001

High-glucose-induced metallothionein expression in endothelial cells: an endothelin-mediated mechanism.

Vascular endothelial cells are constantly exposed to oxidative stress and must be protected by physiological responses. In diabetes mellitus, endothelial cell permeability is impaired and may be increased by high extracellular glucose concentrations. It has been postulated that metallothionein (MT) can protect endothelial cells from oxidative stress with its increased expression by cytokines, thrombin, and endothelin (ET)-1. In this study, we demonstrate that high glucose concentration can induce MT expression in endothelial cells through a distinct ET-dependent pathway. Exposure of human umbilical vein endothelial cells (HUVEC) to increasing concentrations of glucose resulted in a rapid dose-dependent increase in MT-2 and ET-1 mRNA expression. MT expression may be further augmented with addition of ET-1. Preincubation of the cells with the specific ET(B) antagonist BQ-788 blocked MT-2 mRNA expression more effectively than the ET(A) inhibitor TBC-11251. High glucose also increased immunoreactive MT protein expression and induced translocation of MT into the perinuclear area. Perinuclear localization of MT was related to high-glucose-induced reorganization of F-actin filaments. These results demonstrate that an increase in extracellular glucose in HUVEC can lead to a rapid dose-dependent increase in MT-2 mRNA expression and to perinuclear localization of MT protein with changes to the cytoskeleton. These effects are mediated via the ET receptor-dependent pathway.

Actins↗

PubMed2000

Astrocyte cultures from transgenic mice to study the role of metallothionein in cytotoxicity of tert-butyl hydroperoxide.

The cell viability, lipid peroxidation (LPO) and hydrogen peroxide (H(2)O(2)) generation were measured in cultured primary astrocytes, from metallothionein (MT)-I isoform overexpressing transgenic (MT-I*), MT-I/MT-II null and control mice after exposure to tert-butylhydroperoxide (tBH). Astrocytes from MT-I* mice have high basal levels of both MT-I mRNA and MT protein, whereas there is only MT-III isoform in astrocytes from MT-I/MT-II null mice. The results showed that (1) cultured astrocytes from MT-I* mice were most resistant to the cytotoxicity of tBH and those from MT-I/MT-II null mice were most sensitive to the cytotoxicity of tBH; (2) LPO after exposure to tBH were increased in all cells, but the levels were the highest in astrocytes from MT-I/MT-II null mice, while those in MT-I* mice were the lowest; (3) the levels of H(2)O(2) in cultured astrocytes from MT-I* mice were the lowest, while those in astrocytes from MT-I/MT-II null mice were the highest. These results support the hypothesis that MT can scavenge free radicals and protect astrocytes from oxidative stress.

Animals↗

PubMed2000

Delay of M-phase onset by aphidicolin can retain the nuclear localization of zinc and metallothionein in 3T3-L1 fibroblasts.

The transient nuclear localization of metallothionein during cell growth and differentiation may be related to the increased requirement of zinc for DNA synthesis, activation of metalloenzymes, and transcription factors. Treatment of 3T3-L1 fibroblasts with aphidicolin, an inhibitor of nuclear DNA synthesis, caused a cell-cycle block at G1/S phase and a delay in the onset of M phase. This also resulted in the accumulation of both zinc and metallothionein in the nucleus. After removal of aphidicolin, the cells rapidly reentered S phase, and during the G2/M phase of cell cycle both zinc and metallothionein began to relocate to the cytoplasm. Delaying the onset of M phase in 3T3-L1 cells could prevent the cytoplasmic relocation of metallothionein. The nuclear translocation of both zinc and metallothionein during the cell cycle can be considered as a normal process and this may be a general mechanism in response to mitogenic signals.

3T3 Cells↗

PubMed2000

Induction of metallothionein synthesis with preservation of testicular function in rats following long term renal transplantation.

Metallothionein (MT), as an acute phase or stress-response protein and free radical scavenger, is related to inflammation and cellular protection from oxidative damage. In order to evaluate long-term testicular damage and the role of MT following renal transplant, nine allogenic (Fisher 344 --> Lewis) and seven isogenic (Lewis --> Lewis) renal transplants were performed and the recipient rats were followed for 140 days when allografts develop chronic transplant rejection. Testicular weight, light microscopic morphology, and lactate dehydrogenase-X enzyme activity were assessed. Testicular MT was determined by Cd-heme assay, and was localized immunocytochemically using a polyclonal rabbit antibody. No differences in testis weight, morphology, or LDH-X enzyme activity were found between allograft and isograft recipients. Testicular MT level was significantly increased in the testis of allograft recipients. Testicular zinc (Zn) and copper (Cu) levels, but not iron (Fe) level, were significantly higher in testis with allograft kidney than that with isograft kidney. In addition, Cu/Zn ratio was also significantly high in the allograft group. However, the MT level did not show any significant correlation either with Cu and Zn alone or with Cu/Zn and Fe/Zn ratios. These data suggest that allogenic stimuli may induce MT synthesis in the recipient testis. The increased MT level in an allograft may offer a protective action from oxidative damage in the testis.

Animals↗

PubMed2000

Interaction of endothelin-1 with vasoactive factors in mediating glucose-induced increased permeability in endothelial cells.

Alteration of endothelins (ET) and/or their receptors may be important in mediating vascular dysfunction in diabetes. We investigated mechanisms regulating ET-1 expression in human umbilical vein endothelial cells (HUVEC) in response to glucose and the functional significance of these mechanisms. Permeability across HUVEC, grown in medium containing either low (5 mmol/l) or high (25 mmol/l) D-glucose were investigated. L-glucose was used as a control. ET-1, ET(A), and ET(B) mRNA were assessed by semiquantitative RT-PCR. ET-1 immunoreactivity and F-actin microfilament assembly were investigated using confocal microscopy. Increased transendothelial permeability was noted in cells cultured in high glucose or when the cells grown in low (physiologic) glucose were incubated with ET-1, vascular endothelial growth factor (VEGF), or N (G) -nitro-L-arginine methyl ester but not when they were incubated with ET-3, N(G)-nitro-D-arginine methyl ester, or L-glucose. Increased permeability was associated with increased ET-1, ET(A), and ET(B) mRNA expression and augmented ET-1 immunoreactivity. High glucose induced increased permeability, increased ET-1, ET(A), and ET(B) mRNA expression. ET-1 immunoreactivity was blocked by the protein kinase C (PKC) inhibitor chelerythrine, the specific PKC isoform inhibitor 379196, VEGF-neutralizing antibody, or the ET(A) blocker TBC11251, but was not blocked by the specific ET(B) blocker BQ788 or by a VEGF-non-neutralizing antibody. Increased permeability was also associated with deranged F-actin assembly in the endothelial cells and by derangement of endothelial cell junctions as assessed by electron microscopy. Data from this study suggest that high glucose-induced increased permeability may be induced through increased ET-1 expression and disorganization of F-actin assembly. ET-1 expression and increased permeability may occur secondary to PKC isoform activation and may be modulated by VEGF and nitric oxide.

Base Sequence↗

PubMed2000

Metallothionein induction in human CNS in vitro: neuroprotection from ionizing radiation.

PURPOSE: There have been extensive studies on the regulation of metallothionein (MT) synthesis, and its biological role in liver and kidney. Although there are few reports on brain MT, there is a growing interest in the role of MT in brain. There have been no publications to date on MT synthesis in the human central nervous system (CNS) following exposure to ionizing radiation. In the present study, primary human CNS cultures were used to examine the effect of ionizing radiation on MT mRNA and protein synthesis. In the same cultures, the neuroprotective effects of zinc (Zn) and cadmium (Cd)-induced MT synthesis from high-dose radiation were also examined. MATERIALS AND METHODS: Primary, serum-free, human CNS cultures were exposed to 30 or 60 Gy gamma-rays. The total MT protein was then measured by a Cd-heme assay, and mRNA for MT-II and MT-III was detected by reverse transcription polymerase chain reaction (RT-PCR). Cytotoxicity was measured by LDH release and apoptotic cell death by DNA fragmentation analysis. Sublethal neuroglial injury was assessed morphologically using specific astrocytic (glial fibrillary acidic protein--GFAP) and neuronal (microtubule-associated protein 2--MAP2) immunohistochemical markers. RESULTS: The total MT protein content was increased 12h after exposure to 30Gy. The increase in MT content in response to 60Gy was not statistically significant. MT-II mRNA levels increased at 3 and 6h after exposure to 30Gy gamma-rays, with a maximum expression at 12-24 h. MT-III mRNA was not significantly affected. Exposure to 60 Gy, but not 30 Gy, caused a marked increase in LDH release. Cells exposed to 30 Gy or less showed some apoptotic cell death by DNA fragmentation analysis, while exposure to 60 Gy resulted in a DNA smear confirmed by LDH assays. Preinduction of MT by 5 microM Cd or 100 microM Zn resulted in a significant reduction in radiation-induced LDH release. Morphological evaluations revealed that Cd or Zn preincubation led to relative preservation of MAP2 staining and GFAP. CONCLUSION: Both MT protein and MT-II mRNA can be induced in human CNS cells by ionizing radiation. Furthermore, induction of MT synthesis with Zn and Cd can protect human CNS cells from radiation-induced cytocidal and sublethal injuries. Both findings have implications in the development of strategies to protect human CNS tissue from damage during radiotherapy.

Brain↗

PubMed2000

Repeated cadmium exposures enhance the malignant progression of ensuing tumors in rats.

Prior studies show that a single subcutaneous (sc) exposure to cadmium (Cd) will induce injection site sarcomas (ISS) in rats. These tumors, thought clearly malignant, do not often metastasize or invade subdermal muscle layers because of their location. Recent evidence indicates that when tumorigenic cells chronically exposed to Cd in vitro are inoculated into mice, tumor progression and invasiveness in the mice are enhanced. Thus, we studied the effects of repeated Cd exposures on tumor incidence, progression, and metastatic potential in rats. Wistar (WF) and Fischer (F344) rats (30 per group) were injected sc in the dorsal thoracic midline with CdCl2 once weekly for 18 weeks with doses of 0, 10, 20, or 30 micromol Cd/kg. This resulted in total doses of 0, 180, 360, or 540 micromol/kg. One other group of each strain received a low, loading dose of Cd (3 micromol/kg) prior to 17 weekly injections of 30 micromol/kg (total dose 513 micromol/kg). Rats were observed for 2 years. Many F344 rats (57%) died within one week after the first injection of the highest dose, but WF rats were not affected. The low loading dose prevented acute lethality of the high dose in F344 rats. Surprisingly, latency (time to death by tumor) of ISS was the shortest in the groups given the low loading dose in both strains. ISS in these groups also showed the highest rate of metastasis and subdermal muscle layer invasion. Based on ISS incidence in the groups given the lowest total dose of Cd (180 micromoles/kg), F344 rats were more sensitive to tumor induction, showing an incidence of 37% compared to 3% in WF rats. On the other hand, Cd-induced ISS showed a higher overall metastatic rate in WF rats (18 metastatic ISS/68 total tumors in all treated groups; 27%) compared to F344 rats (6%). Immunohistochemically, the primary ISS showed high levels of metallothionein (MT), a cadmium-binding protein, while metastases were essentially devoid of MT. These results indicate that repeated Cd exposures more rapidly induce ISS. An initial low exposure to Cd further accelerates the appearance and enhances the metastatic potential and invasiveness of these tumors. The primary and metastatic ISS appear to have a differing phenotype, at least with regard to MT production. The association between multiple Cd exposures and enhanced metastatic potential of the ensuing tumors may have important implications in chronic exposures to Cd, or in cases of co-exposure of Cd with organic carcinogens, as in tobacco smoking.

Animals↗

PubMed2000

Radiation exposure does not alter metallothionein III isoform expression in mouse brain.

The level of metallothionein III mRNA and protein, a brain-specific isoform of metallothionein (MT), was investigated in the brain of MT-I and -II gene knockout (MT-null) mice exposed to 20 Gy whole-body gamma-irradiation. Because MT-null mice did not express MT-I or MT-II isoforms, the total brain MT content in these mice represented the isoform MT-III only. MT-III protein content was determined by a cadmium-binding assay, and the MT mRNA level was measured by reverse transcription followed by polymerase chain reaction (RT-PCR). Both MT-III protein content and mRNA expression in the brains of MT-null mice were not affected by exposure to whole-body irradiation. These results indicate that mouse brain MT-III expression is not induced by ionizing radiation.

Animals↗

PubMed2000

Signal transduction pathways, and nuclear translocation of zinc and metallothionein during differentiation of myoblasts.

The changes in subcellular localization of metallothionein during differentiation were studied in two myoblast cell lines, L6 and H9C2. Addition of insulin like growth factor-I or lowering foetal bovine serum to 1% can induce differentiation of myoblasts to myotubes. Metallothionein and zinc were localized mainly in the cytoplasm in myoblasts but were translocated into the nucleus of newly formed myotubes during early differentiation. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. Addition of an inhibitor of mitogen-activated protein kinase, PD 98059, did not affect differentiation but blocked nuclear translocation of metallothionein. LY 294092, an inhibitor of PI3 kinase, and rapamycin, an inhibitor of p70S6 serine/threonine kinase, abolished insulin-like growth factor-I induced differentiation of myoblasts, retained metallothionein in the cytoplasm, and decreased metallothionein content. These results demonstrate that the cytoplasmic-nuclear translocation of metallothionein occurs during the early stage of differentiation of myoblasts to myotubes and can be blocked by inhibition of certain signal transduction pathways. The transient nuclear localization of metallothionein and zinc may be related to a high requirement for zinc for metabolic activities during the early stage of differentiation.

Animals↗

PubMed2000

Nuclear localization of metallothionein during cell proliferation and differentiation.

Although MT is detected as a cytoplasmic protein in hepatocytes of adult liver, it can be localized in the hepatocyte nuclei in human fetal liver bound to zinc and copper. Both nuclear and cytoplasmic localization of MT have been observed in several human tumours, especially in regions of high proliferation. Transient co-localization of zinc and MT has been shown in differentiating myoblast and 3T3-L1 fibroblasts, and during the G1-/S-phase progression in cell cycle. Several mechanisms have been proposed for the import and retention of MT in the nucleus, including signal transduction pathways. The high levels of MT in the nucleus of cells under certain conditions may be related to the increased requirement for zinc for several metallo-enzymes and transcription factors during rapid growth. The function of nuclear MT may be to protect the cell from DNA damage and apoptosis, and also to regulate gene expression during certain stages of cell cycle.

Animals↗

PubMed2000

Effect of ethanol on brain metallothionein in transgenic mice.

BACKGROUND: Three isoforms of metallothionein (MT-I, -II, and -III) have been detected in mice brain but little is known on their inducibility. Therefore, changes in MT levels in brain were investigated in MT-I overexpressing transgenic mice (MT-I*) and wild type mice of the same strain (C57BL/6J) after oral administration of ethanol. METHODS: All experimental mice were given 4 g/kg of ethanol by gastric intubation. In addition, to account for the stress of animal handling, both groups of mice (MT-I* and C57) were given distilled water by gastric intubation and used as controls. At different times after ethanol/water treatment, the mice were killed and the brains were dissected into six regions. The total amount of MT in the brain and different regions were measured by a cadmium-binding assay. Zinc and copper were measured by atomic absorption spectrometry. The results were compared with untreated mice and corresponding control mice. RESULTS: In C57BL/6J mice, the whole brain MT was increased by approximately 1.29-fold at 12 hr and approximately 1.27-fold at 18 hr after ethanol administration compared with basal levels. In MT-I* male mice, the whole brain MT was increased by approximately 1.64-fold at 12 hr and approximately 1.62-fold at 18 hr after ethanol administration, whereas in MT-I* female mice, it was increased by approximately 1.57-fold at 12 hr and approximately 1.61-fold at 18 hr after ethanol administration. Although the zinc and copper levels in the whole brain were not altered, the zinc level in the cerebellum at 12 and 18 hr in male and female MT-I* mice were increased, and zinc levels in the hippocampus of male MT-I* mice were increased at 12 and 18 hr after ethanol treatment. CONCLUSIONS: Acute or chronic ethanol administration can cause generation of oxygen free radical and oxidative stress to the brain. The generation of free radicals is thought to be one of the causes of cell injury after ethanol administration. The mechanism of induction of MT in brain after oral ethanol administration may be associated with oxidative stress caused by ethanol or its metabolites, and can be considered as a protective mechanism against ethanol toxicity.

Animals↗

PubMed1999

Metallothionein and apoptosis during differentiation of myoblasts to myotubes: protection against free radical toxicity.

The changes in subcellular localization of metallothionein (MT) during differentiation were studied in two muscle cell lines, L6 and H9C2, myoblasts in order to understand the nuclear presence of MT and its antiapoptotic property. In myoblasts, MT and zinc were localized mainly in the cytoplasm but were translocated into the nucleus of newly formed myotubes during early stage of differentiation, which was initiated by lowering FBS from 10% to 1%. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. These changes in subcellular localization of MT and Zn were accompanied by increased apoptosis in myotubes. The changes in the apoptosis at different stages of differentiation were measured by both DNA ladder formation and TUNEL technique. The results also show that the apoptosis may be initiated by free radical generation and may be accompanied by p53 expression. The H9C2 cells contained high levels of MT, differentiated slowly, and had low incidence of apoptotic bodies compared to L6 cell line.

Animals↗