Protective Effect Of Metallothionein Research Articles

Exacerbation of Elastase-Induced Emphysema via Increased Oxidative Stress in Metallothionein-Knockout Mice.

Although the pathogenesis of chronic obstructive pulmonary disease (COPD) is not yet fully understood, recent studies suggest that the disruption of the intracellular balance of oxidative (such as reactive oxygen species (ROS)) and antioxidant molecules plays an important role in COPD development and progression. Metallothionein is an endogenous metal-binding protein with reported ROS scavenging activity. Although there have been many publications on the protective effects of metallothionein in the kidney and liver, its role in COPD models such as elastase- or cigarette smoke (CS)-induced lung injury is unknown. Thus, in the present study, we analyzed the elastase-induced lung injury model using metallothionein-knockout (MT-KO; MT-1 and -2 gene deletion) mice. The expression of MT-1 and MT-2 in the lungs of MT-KO mice was markedly lower compared with that in the lungs of wildtype (WT) mice. Porcine pancreatic elastase (PPE)-induced lung injury (alveolar enlargement and respiratory impairment) was significantly exacerbated in MT-KO mice compared with WT mice. Additionally, PPE-induced increases in the number of inflammatory cells, inflammatory cytokines, and cell death in lung tissue were significantly more pronounced in MT-KO mice compared with WT mice. Finally, using an in vivo imaging system, we also found that PPE-induced ROS production in the lungs was enhanced in MT-KO mice compared with WT mice. These results suggest that metallothionein may act as an inhibitor against elastase-induced lung injury by suppressing ROS production. These results suggest that metallothionein protein, or compounds that can induce metallothionein, could be useful in the treatment of COPD.

Protective effects of metallothionein and vitamin E in the trunk kidney and blood of cadmium poisoned Ctenopharyngodon idellus.

Cadmium (Cd), a substance with one of the most critical health hazard indices, can cause damage to both the blood and kidneys and accumulates in the body at last. The present work studied the toxicological effects of Cd and the therapeutic effects of metallothionein (MT) and vitamin E (VE) on the trunk kidney and blood of freshwater grass carp (Ctenopharyngodon idellus). Grass carp were divided into three groups: Cd + phosphate-buffered saline (PBS) group, Cd + VE group, and the Cd + MT group. Fish were injected with CdCl2 on the first day and then VE, MT, or PBS was administered 4days post-injection. Fish not injected with Cd were used as a negative control. The blood and trunk kidney amassed Cd and suffered severe damage in the forms of organ toxicity cytotoxicity, and immunotoxicity. However, the MT reduced the Cd content in the trunk kidney and blood and partially stabilized the damaged organs. Treatment with VE, however, only demonstrated weaker protection against on Cd-induced toxicity. The results indicate that exogenous MT may play an essential role in restoring homeostasis of the Cd-poisoned urinary and circulatory system and that it may help eliminate Cd in aquatic animals.

Metallothionein Prevents Age-Associated Cardiomyopathy via Inhibiting NF-κB Pathway Activation and Associated Nitrative Damage to 2-OGD.

Cardiac-specific metallothionein (MT) overexpression extends lifespan, but the mechanism underlying the effect of MT protection against age-associated cardiovascular diseases (CVD) remains elusive. To elucidate this, male wild-type and two lines of MT-transgenic (MT-TG) mice, MM and MT-1 (cardiac-specific overexpressing MT about 10- and 80-fold, respectively) at three representative ages (2-3, 9-10, and 18-20 months), were utilized. A stable human MT2A overexpressing cardiomyocytes (H9c2MT7) was also introduced. Histomorphology and echocardiographic analysis revealed that age-associated cardiac hypertrophy, remodeling, and dysfunction were ameliorated in MT-TG mice. Also, aging-accompanied NF-κB activation, characterized by increased nuclear p65 translocation, elevated DNA-binding activity, and upregulation of inflammatory cytokines, was largely attenuated by MT overexpression. Treatment of H9c2 cardiomyocytes with tumor necrosis factor-α (TNF-α), which mimicked an inflammatory environment, significantly increased NF-κB activity, and some age-related phenotypes appeared. The NF-κB activation was further proved to be pivotal for both age-associated and TNF-α-induced nitrative damage to cardiac 2-oxoglutarate dehydrogenase (2-OGD) by virtue of NF-κB p65 gene silencing. MT inhibited NF-κB activation and associated nitrative damage to cardiac 2-OGD in both old MT-TG hearts and TNF-α-treated H9c2MT7 cardiomyocytes; these protective effects were abolished in H9c2MT7 cardiomyocytes by MT-specific gene silencing. Innovation and Conclusion: Together, these findings indicate that the protective effects of MT against age-associated CVD can be attributed mainly to its role in NF-κB inhibition and resultant alleviation of nitrative damage to 2-OGD. Antioxid. Redox Signal. 25, 936-952.

Obesity and Metabolomics: Metallothioneins Protect Against High-Fat Diet-Induced Consequences in Metallothionein Knockout Mice

Obesity continues to rise as an alarming global epidemic. System level mechanisms, diagnostics, and therapeutics are sorely needed so as to identify at risk individuals and design appropriate population scale interventions. The present study evaluated the protective role of metallothioneins (MTs) against obesity and high-fat diet-induced effects such as insulin resistance in both male and female MT-1+2 knockout and MT-3 knockout mice. As the metabolome is closest to the functional phenotype, changes in metabolite levels were also evaluated, and the direct or indirect involvement of MTs in metabolism examined. MT-1+2-, MT-3 knockout, and wild-type mice were given a high-fat diet for 2 months. Variation in body weight gain, tissue weight, and response to oral glucose tolerance test and insulin tolerance test were determined and compared to mice that received the control diet. Effect of the high-fat diet on the knockout mice were investigated on the metabolome level in specific tissues using metabolomics. Both knockout mice strains were more susceptible to high-fat diet-induced effects, such as weight gain and moderate insulin resistance, with the MT-3 knockout mice most susceptible. Brain tissue of the knockout mice showed most metabolic variation and pointed to possible impairment of mitochondrial function. The protective effect of MTs against high-fat diet and obesity-induced effects such as insulin resistance was evident from our observations. The putative role MTs play in mitochondrial function is possibly the main contributor to the lack of these effects in wild-type mice. Considering the expression profiles of the MT isoforms and similarity in brain metabolic variation in the knockout strains, it appears that they promote mitochondrial function in the hypothalamus, thereby limiting weight gain and insulin resistance. Furthermore, metabolomics research in preclinical models of obesity and in the clinic is warranted in the near future.

Protective Effects of Metallothionein on Isoniazid and Rifampicin-Induced Hepatotoxicity in Mice

Isoniazid (INH) and Rifampicin (RFP) are widely used in the world for the treatment of tuberculosis, but the hepatotoxicity is a major concern during clinical therapy. Previous studies showed that these drugs induced oxidative stress in liver, and several antioxidants abated this effect. Metallothionein (MT), a member of cysteine-rich protein, has been proposed as a potent antioxidant. This study attempts to determine whether endogenous expression of MT protects against INH and RFP-induced hepatic oxidative stress in mice. Wild type (MT+/+) and MT-null (MT−/−) mice were treated intragastrically with INH (150 mg/kg), RFP (300 mg/kg), or the combination (150 mg/kg INH +300 mg/kg RFP) for 21 days. The results showed that MT−/− mice were more sensitive than MT+/+ mice to INH and RFP-induced hepatic injuries as evidenced by hepatic histopathological alterations, increased serum AST levels and liver index, and hepatic oxidative stress as evidenced by the increase of MDA production and the change of liver antioxidant status. Furthermore, INH increased the protein expression of hepatic CYP2E1 and INH/RFP (alone or in combination) decreased the expression of hepatic CYP1A2. These findings clearly demonstrate that basal MT provides protection against INH and RFP-induced toxicity in hepatocytes. The CYP2E1 and CYP1A2 were involved in the pathogenesis of INH and RFP-induced hepatotoxicity.

Metallothionein prevention of arsenic trioxide-induced cardiac cell death is associated with its inhibition of mitogen-activated protein kinases activation in vitro and in vivo

Cardiotoxicity induced by arsenic trioxide has become a serious blockade of clinical applications of this effective anticancer agent. The general mechanism responsible for arsenic cardiotoxicity has been attributed to its induction of oxidative stress. Metallothionein (MT) has been extensively proven to be a potent endogenous antioxidant that protects heart against oxidative stress-induced cardiac damage. To investigate whether and how MT protects against arsenic cardiotoxicity, MT-overexpressing H9c2 (MT-H9c2) cardiac cells and transgenic (MT-TG) mice with their corresponding controls were exposed to the clinical relevant dose of arsenic trioxide. Cardiac cell apoptosis was detected by molecular indices, including the cleavage of caspase 3 and caspase 12, Bax/Bcl2 expression ratio, CHOP expression and/or confirmed by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Arsenic trioxide dose- and time-dependently induced cardiac cell death in H9c2 cells with a significant activation of major MAPK subfamily members such as ERK1/2, JNK and p38, but not in MT-H9c2 cells. Importantly, the protective effect of MT on arsenic trioxide-induced apoptotic cell death was completely recaptured in the heart of MT-TG with a significant prevention of MAPKs activation. These results indicate that arsenic trioxide-upregulated MAPKs might play important role in arsenic trioxide-induced apoptotic cell death in cardiac cells both in vivo and in vitro, and MT's suppression of arsenic trioxide apoptotic effect was associated with the inhibition of MAPK activation. Therefore, selective elevation of cardiac MT levels with pharmacological approaches may be a potential strategy for the prevention of arsenic cardiotoxicity.

Protective effects of metallothionein I and II against metal- and ultraviolet radiation-induced damage in cultured lens epithelial cells

Few quantitative analyses of the induction of metallothioneins (MTs) have been conducted, and there are no reports on the contribution of MTs to the protective mechanism against ultraviolet (UV) radiation in the lens. In this study, we quantitatively analyzed the induction of MTs and analyzed the resulting protective effects against both metal- and UV radiation-induced damage in the cultured lens epithelial cell line, alphaTN4-1. The induction profiles of MTs by ZnCl(2) treatment in alphaTN4-1 cells were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The cells in which MTs were induced were either treated with high concentrations of ZnCl(2) or CdCl(2), or irradiated with UV-C, UV-B, or UV-A radiation, followed by analysis of cell viability. The (3)H-thymidine incorporation rate was used as an indicator of cell viability. mRNA expression of MT-I and MT-II, the main MT isoform classes, was induced by ZnCl(2) treatment in a dose-dependent manner. MT induction increased the protective effects against both metaland UV-A radiation-induced cell damage. Our results suggest that MTs play an important role in the protection against damage induced by both toxic metals and UV-A radiation in lens epithelial cells.

Mechanism of protection by metallothionein against acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the US. Metallothionein (MT) expression attenuates APAP-induced liver injury. However, the mechanism of this protection remains incompletely understood. To address this issue, C57BL/6 mice were treated with 100 μmol/kg ZnCl 2 for 3 days to induce MT. Twenty-four hours after the last dose of zinc, the animals received 300 mg/kg APAP. Liver injury (plasma ALT activities, area of necrosis), DNA fragmentation, peroxynitrite formation (nitrotyrosine staining), MT expression, hepatic glutathione (GSH), and glutathione disulfide (GSSG) levels were determined after 6 h. APAP alone caused severe liver injury with oxidant stress (increased GSSG levels), peroxynitrite formation, and DNA fragmentation, all of which were attenuated by zinc-induced MT expression. In contrast, MT knockout mice were not protected by zinc. Hydrogen peroxide-induced cell injury in primary hepatocytes was dependent only on the intracellular GSH levels but not on MT expression. Thus, the protective effect of MT in vivo was not due to the direct scavenging of reactive oxygen species. Zinc treatment had no effect on the early GSH depletion kinetics after APAP administration, which is an indicator of the metabolic activation of APAP to its reactive metabolite N-acetyl- p-benzoquinone imine (NAPQI). However, MT was able to effectively trap NAPQI by covalent binding. We conclude that MT scavenges some of the excess NAPQI after GSH depletion and prevents covalent binding to cellular proteins, which is the trigger for the propagation of the cell injury mechanisms through mitochondrial dysfunction and nuclear DNA damage.

The Dual Nature of Metallothioneins in the Metabolism of Heavy Metals and Reactive Oxygen Species in Aquatic Organisms: Implications of Use as a Biomarker of Heavy-Metal Effects in Field Investigations

The purpose of this study was to examine the function of metallothioneins (MT) in respect to the mobilization of heavy metals and superoxide anion (O2–) scavenging in aquatic organisms. Using an O2– generating system, liberation of free zinc from native and zinc MT (Zn-MT) was measured in vitro. Addition of the O2– generating system and H2O2 readily increased the di- and trimeric forms of MT as determined by gel electrophoresis analysis. To determine whether the proportion of oxidized MT could change in contaminated environments, metal-contaminated Mya arenaria clams were collected from a harbour in the St. Lawrence Estuary. The levels of labile zinc, superoxide dismutase (O2– scavenging enzyme), lipid peroxidation (LPO) and the oxidized/metallic form of MT were determined in the digestive gland. The results revealed that the induction of total MT levels was the result of increased oxidized MT at the expense of the reduced or metallic form of MT. Both superoxide dismutase (SOD) and labile zinc (Zn) levels were induced and they were significantly correlated with the oxidized form of MT, but not the metallic form, in feral clam populations. We concluded that the level of total MT was related to Zn mobility and the activation of antioxidant mechanisms such as SOD, and corresponded to the levels of oxidized MT. The metallic form of MT was negatively associated with Zn mobility but positively associated with oxidative damage such as LPO. Overall, the oxidized fraction of MT appeared to be more closely related to detoxification, while the metallic form of MT was associated with metal mobility and toxicity via oxidative damage. The protective effect of MT during heavy-metal contamination depends on the availability of metals and on its capacity to sequester reactive oxygen species.

Abstract 159: Protective Effect of Metallothionein in Ischemic Hindlimb against Oxidative Stress Induced by Benzo[ a ]pyrene

Background: A wide range of biological responses to and toxic effects of exogenous compounds, such as polycyclic and halogenated aromatic hydrocarbons and dioxins, is mediated by the aryl hydrocarbon receptor (AHR). Given that smoking is an important risk factor for ischemic heart disease and peripheral vascular disease, we hypothesized that benzo-[ a ]pyrene (B[a]P), a prototype carcinogen of tobacco smoke, might impair angiogenesis and AHR might play an important role in the regulation of angiogenesis after exposure to B[a]P. The present study investigated this hypothesis in a surgical model of ischemia. Methods and Results: Ischemia was produced by femoral artery ligature in the AHR gene-deficient (AHR −/ − ) and wild type (WT) mice. B[a]P was administered by oral gavage in corn oil (10 ml/kg) at 125 mg/kg once per week beginning at 12 weeks of age (each group; n = 8). Laser Doppler perfusion analyses showed that the ischemic/nonischemic blood flow ratio of WT mice exposed to B[a]P was significantly reduced compared with that of control WT mice (0.67 ± 0.03 vs. 0.78 ± 0.02, P < 0.05) at 3 weeks after surgery. Furthermore, the blood flow ratio was significantly greater in AHR −/ − mice exposed to B[a]P than in WT mice exposed to B[a]P (P < 0.05). At 3 days after ligature, the abundance of cytochrome P450 1A1 mRNA, determined by quantitative RT-PCR analysis, was markedly greater in both ischemic and nonischemic hindlimbs of WT mice exposed to B[a]P than in those of AHR −/ − mice expose to B[a]P. The abundance of metallothionein I and II mRNA was significantly greater in the ischemic hindlimb than in the nonischemic hindlimb among all groups and was, moreover, significantly greater in the ischemic hindlimb of WT mice exposed to B[a]P than in that of AHR −/ − mice expose to B[a]P. Conclusions: The results show that the deletion of AHR gene reduced the adverse effects of exposure to B[a]P on angiogenesis. Our results also suggest that hypoxia in ischemia tissues activates the expression of metallothionein I and II, which may have protective effects against B[a]P-induced oxidative stress responses.

Protective effects of metallothionein against dopamine quinone-induced dopaminergic neurotoxicity

Dopamine (DA) quinone as DA neuron-specific oxidative stress conjugates with cysteine residues in functional proteins to form quinoproteins. Here, we examined the effects of cysteine-rich metal-binding proteins, metallothionein (MT)-1 and -2, on DA quinone-induced neurotoxicity. MT quenched DA semiquinones in vitro. In dopaminergic cells, DA exposure increased quinoproteins and decreased cell viability; these were ameliorated by pretreatment with MT-inducer zinc. Repeated L-DOPA administration markedly elevated striatal quinoprotein levels and reduced the DA nerve terminals specifically on the lesioned side in MT-knockout parkinsonian mice, but not in wild-type mice. Our results suggested that intrinsic MT protects against L-DOPA-induced DA quinone neurotoxicity in parkinsonian mice by its quinone-quenching property.

Diabetes and Metallothionein

Diabetes is a widespread disease, and its development and toxic effects on various organs have been attributed to increased oxidative stress. Metallothionein (MT) is a group of intracellular metal-binding and cysteine-rich proteins, being highly inducible in many tissues. Although it mainly acts as a regulator of metal homeostasis such as zinc and copper in tissues, MT was found to be a potent antioxidant and adaptive (or stress) protein to protect cells and tissues from oxidative stress. Studies showed that zinc-induced or genetically enhanced MT synthesis in the pancreas prevented the development of spontaneous or chemically-induced diabetes. Genetically or pharmacologically enhanced MT expression in various organs including heart and kidney provided significant protection from diabetes-induced organ dysfunction such as cardiomyopathy and nephropathy. These studies suggest that MT as an adaptive protein can prevent both diabetes development and diabetic complications. This mini-review will thus briefly describe MT's biochemical features and then summarize the data on the protective effect of MT against diabetes and diabetic complications. In addition, the coordinative role of MT with zinc in the prevention of diabetes and its complications will also be discussed.

Attenuation of AngII‐induced early cell death by MT results in a prevention of the late development of cardiomyopathy

Angiotensin II (Ang II) plays a critical role in the diabetic cardiovascular diseases. We have demonstrated that cardiomyocytes with high levels of metallothionein (MT) are significantly resistant to diabetes- and Ang II-induced apoptotic cell death mediated by NOX activation. To investigate the effects of MT on Ang II – induced cardiac injury, we administrated the cardiac MT transgenic (MT-TG) and WT mice with a single injection of Ang II at 1 mg/kg body weight. Ang II caused an increase in apoptosis, examined by TUNEL and Western blot for the caspase-3 activation along with an increased 3-NT formation and an increased expression of NOX isoform p47phox in the membrane proteins 7h after treatment. All these effects of Ang II were significantly prevented in the heart of Ang II-treated MT-TG mice, indicating the protective effect of MT on Ang II-induced apoptosis via suppression of NOX activation. To further evaluate antiapoptotic effect of MT, MT-TG and WT mice were chronically infused with Ang II at 0.5 mg/kg body weight for two weeks and then cardiac tissues were collected at 3 and 6 months. Ang II treatment caused a significant increase in cardiac fibrosis, shown by increased collagen content and up-regulation of connective tissue growth factor in the heart of WT but not in MT-TG mice. These results suggest that Ang II induces cardiac apoptosis through NOX activation and generation of peroxynitrite. MT prevents Ang II-induced cardiac cell death through suppression of NOX activation, peroxynitrite formation and nitrosative damage. The protection of Ang II-induced early cell death by MT leads to a prevention of the late cardiomyopathy.

Metallothionein provides zinc-mediated protective effects against methamphetamine toxicity in SK-N-SH cells

Methamphetamine (METH) is a drug of abuse and neurotoxin that induces Parkinson's-like pathology after chronic usage by targeting dopaminergic neurons. Elucidation of the intracellular mechanisms that underlie METH-induced dopaminergic neuron toxicity may help in understanding the mechanism by which neurons die in Parkinson's disease. In the present study, we examined the role of reactive oxygen species (ROS) in the METH-induced death of human dopaminergic SK-N-SH cells and further assessed the neuroprotective effects of zinc and metallothionein (MT) against METH-induced toxicity in culture. METH significantly increased the production of reactive oxygen species, decreased intracellular ATP levels and reduced the cell viability. Pre-treatment with zinc markedly prevented the loss of cell viability caused by METH treatment. Zinc pre-treatment mainly increased the expression of metallothionein and prevented the generation of reactive oxygen species and ATP depletion caused by METH. Chelation of zinc by CaEDTA caused a significant decrease in MT expression and loss of protective effects of MT against METH toxicity. These results suggest that zinc-induced MT expression protects dopaminergic neurons via preventing the accumulation of toxic reactive oxygen species and halting the decrease in ATP levels. Furthermore, MT may prevent the loss of mitochondrial functions caused by neurotoxins. In conclusion, our study suggests that MT, a potent scavenger of free radicals is neuroprotective against dopaminergic toxicity in conditions such as drug of abuse and in Parkinson's disease.

Metallothionein induction in the liver, kidney, heart and aorta of cadmium and isoproterenol treated rats

Metallothionein (MT), induced in different organs in response to heavy metals and oxidative conditions, exerts antioxidant properties and thus could be implicated in cardiovascular physiopathology. The aim of this study was to investigate the capacity of cadmium (Cd) and isoproterenol to induce in vivo MT not only in rat liver and kidneys but also in heart and aorta. Tissue MT levels, catalase (CAT) and glutathione peroxidase (GPX) activities were assayed at different times after Cd or isoproterenol injection. Cd induced a dose-dependent induction of MT with a higher response in the liver than in the kidney, aorta and heart. The hepatic increase was early (12 h) and maintained (72 h), whereas the elevation was maximal around 48 h for the other organs. Isoproterenol induced a transient (12 h) hepatic and a biphasic (12 and 36 h) renal and cardiac increase. CAT activity was decreased in the liver and increased in the heart with the higher Cd doses. Isoproterenol increased the cardiac GPX activity. In conclusion, the results demonstrate that MT can be induced in rat liver and kidneys but also in heart after a Cd or isoproterenol injection. This enhancement of cardiac and vascular MT levels could be used to study the potential protective effect of MT in cardiovascular diseases.

Protective effect of metallothioneins against oxidative stress evaluated on wild type and MT-null cell lines by means of flow cytometry.

It is generally accepted that metallothioneins (MTs) are devoted to the regulation of the metabolism of essential trace metals and to chelation of toxic metals. Nowadays, there is increasing evidence that MTs also act as free radical scavengers. We employed wild type mouse embryo fibroblast cell line, GKA1, and its MT-null variant, GKA2, in order to correlate the presence of MTs to the sensitivity of cells to reactive oxygen species (ROS), spontaneously generated by the aerobic cellular metabolism, or chemically induced by hydrogen peroxide. The absence of MTs in GKA2 cells was unambiguously correlated to higher sensitivity to ROS attack, as evaluated by detection and quantification of 8-oxo-2'-deoxyguanosine (8-oxo-G), the first product of oxidative attack to DNA, using Fluorescence-Activated Cell Sorter (FACS). When compared to MT-null cell line, the wild type cells (GKA1) were less sensitive to ROS attack. In GKA1 cells, MT biosynthesis is readily induced by Cd2+ treatment, and such an induction caused a further decrease in sensitivity to ROS injury. On the contrary, the MT-null cells (GKA2) expressed no detectable metallothioneins either constitutively, or after heavy metal pretreatment. Indeed, in GKA2 cell line, pretreatment with Cd2+ did not reduce but even enhanced the oxidative stress.

Zinc-metallothionein protects from DNA damage induced by radiation better than glutathione and copper- or cadmium-metallothioneins

Protection of radiation-induced DNA damage by metallothionein (MT) has been documented, but there is no detailed information about its efficiency compared to other antioxidants or the effect of metals which bind to MT on the protective effect of MT in radiation-induced DNA damage. In this study, we used a cell-free system to investigate the effect of MT with other antioxidants, such as albumin and glutathione and we compared the efficiency of MT bound to different metals on radiation-induced DNA damage. DNA damage was measured by loss in ethidium bromide/DNA fluorescence and increased mobility of DNA on gel electrophoresis. Gamma rays at 30 Gy induced significant DNA damage and zinc-MT showed a significant higher protection from radiation-induced DNA damage than both glutathione and albumin. Metallothionein bound to other metals, such as copper and cadmium, also showed protection of radiation-induced DNA damage, but the protective effect by zinc-MT was the highest. These results suggest that MT, in particular bound to zinc, is a high-capacity antioxidant to protect radiation-induced DNA damage.

Protective effect of metallothionein against the toxicity of cadmium and other metals

Metallothionein (MT) is a small-molecular weight, cysteine-rich protein that binds metals. The protective role of MT in Cd toxicity is well established but its ability to protect against toxicity of other metals remains unclear. In this study, wild-type and MT-I and -II null mice (MT-null mice) were used to determine whether MT is protective against the lethality of not only Cd but also Zn, Cu, Fe, Pb, Hg and As. Following daily subcutaneous administration of an increasing dose of each metal, starting with a low, non-toxic dose, we compared the cumulative median lethal dose (LD 50) of each metal between wild-type and MT-null mice. The LD 50 of Cd for wild-type mice was 6.9-fold higher than for MT-null mice. The LD 50 of Zn was 2.4-fold higher for wild-type mice than for MT-null mice, and 1.4-fold higher for Cu and As. The LD 50 of Hg was 1.3-fold higher for wild-type mice than for MT-null mice, but this was not statistically significant. No difference in LD 50 values was observed between wild-type and MT-null mice following Pb and Fe administration. These results suggest that MT is an important protein in the cellular defense against Cd toxicity and lethality, but it provides much less protection against the lethality of the other metals.

Levels of cellular glutathione and metallothionein affect the toxicity of oxidative stressors in an established carp cell line

The comparative toxicity of a variety of oxidative stressors was studied in the epithelioma papulosum cyprini line from carp using the neutral red cytoxicity assay. LC 50's decreased in the order t-butylhydroperoxide>hydrogen peroxide>diquat>paraquat. The cytoxicity of hydrogen peroxide was significantly reduced when the cells were grown in L-15 medium rather than MEM and this could be attributed to elevated cellular glutathione and metallothionein levels and higher activities of GSH-dependent detoxification systems. The protective effect of metallothionein in radical scavenging was demonstrated by decreased toxicity of the redox-cycling toxicants, diquat and menadione after metallothionein levels had been pre-induced by Cd-exposure. This study demonstrates the relationship between toxic effects of oxidative stressors and expression of detoxification systems in fish.

Protective role of zinc-metallothionein on DNA damage in vitro by ferric nitrilotriacetate (Fe–NTA) and ferric salts

Oxidative DNA damage can be caused by radicals generated by transitional metals like iron in Fenton reaction. Metallothionein (MT) may play an important role in preventing oxidative DNA damage. Therefore, after comparing the effects of ferric salts (Fe), and complexes of ferric salts with nitrilotriacetic acid (Fe–NTA) on DNA damage, the protective effects of zinc-MT (Zn-MT) on DNA damage of Fe salts or Fe–NTA were investigated in vitro. DNA damage was measured by loss of fluorescence of DNA binding to ethidium bromide, and also by increased DNA mobility in agarose gel electrophoresis. Both Fe salts and Fe–NTA could induce calf thymus DNA damage in presence of hydrogen peroxide and ascorbate. However, the degree of DNA damage was lower with Fe salts than that with Fe–NTA complex. Addition of 50 μM Zn-MT could only protect DNA from Fe–NTA, but not from Fe salt induced damage. The protective effect of MT was about five times better than that of glutathione (GSH). These results suggest a potential role for MT in protection from Fe–NTA-induced DNA damage.