Identification of Potential Biomarkers From Hepatocellular Carcinoma With MT1 Deletion.

Background: Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Metallothioneins (MTs) are metal-binding proteins involved in multiple biological processes such as metal homeostasis and detoxification, as well as in oncogenesis. Copy number variation (CNV) plays a vital role in pathogenesis and carcinogenesis. Nevertheless, there is no study on the role of MT1 CNV in HCC. Methods: Array-based Comparative Genomic Hybridization (aCGH) analysis was performed to obtain the CNV data of 79 Guangxi HCC patients. The prognostic effect of MT1-deletion was analyzed by univariate and multivariate Cox regression analysis. The differentially expressed genes (DEGs) were screened based on The Gene Expression Omnibus database (GEO) and the Liver Hepatocellular Carcinoma of The Cancer Genome Atlas (TCGA-LIHC). Then function and pathway enrichment analysis, protein-protein interaction (PPI) and hub gene selection were applied on the DEGs. Lastly, the hub genes were validated by immunohistochemistry, tissue expression and prognostic analysis. Results: The MT1-deletion was demonstrated to affect the prognosis of HCC and can act as an independent prognostic factor. 147 common DEGs were screened. The most significant cluster of DEGs identified by Molecular Complex Detection (MCODE) indicated that the expression of four MT1s were down-regulated. MT1X and other five hub genes (TTK, BUB1, CYP3A4, NR1I2, CYP8B1) were associated with the prognosis of HCC. TTK, could affect the prognosis of HCC with MT1-deletion and non-deletion. NR1I2, CYP8B1, and BUB1 were associated with the prognosis of HCC with MT1-deletion. Conclusions: In the current study, we demonstrated that MT1-deletion can be an independent prognostic factor in HCC. We identified TTK, BUB1, NR1I2, CYP8B1 by processing microarray data, for the first time revealed the underlying function of MT1 deletion in HCC, MT1-deletion may influence the gene expression in HCC, which may be the potential biomarkers for HCC with MT1 deletion.

MT2A Promotes Oxaliplatin Resistance in Colorectal Cancer Cells.

We aimed to understand the molecular mechanism underlying the incidence of Oxaliplatin resistance in colorectal cancer. The Oxaliplatin-resistant (OR) HT29 colorectal cell line was established by long-term exposure to Oxaliplatin. Cell viability and proliferation were determined by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide and direct counting assays, respectively. Transcript level of metallothionein 2A (MT2A) was measured by real-time polymerase chain reaction. Protein levels of MT2A, BRCA1-associated RING domain 1 (BARD1), BRCA1, and ß-actin were quantified by immunoblotting. Direct interaction between MT2A with BARD1 and BRCA1 was analyzed by co-immunoprecipitation. Colocalization between of MT2A and BARD1 was determined by immunofluorescence. MT2A was upregulated in OR cells at both transcript and protein levels. Knockdown of MT2A in HT29 OR cells improved sensitivity to Oxaliplatin, while ectopic overexpression of MT2A conferred HT29 cells relative resistance to Oxaliplatin. We further demonstrated that MT2A interacted with and positively regulated BARD1/BRCA1 in colorectal cancer cells. BARD1 overexpression partially restored the compromised Oxaliplatin resistance elicited by MT2A deficiency in terms of both cell proliferation and viability. Our data highlighted the critical contributions of MT2A-BARD1/BRCA1 in Oxaliplatin resistance in colorectal cancer cells.

Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess.

Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concentrations, the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are critical for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.

Zinc protects against cadmium-induced toxicity in neonatal murine engineered cardiac tissues via metallothionein-dependent and independent mechanisms.

Cadmium (Cd) is a nonessential heavy metal and a prevalent environmental toxin that has been shown to induce significant cardiomyocyte apoptosis in neonatal murine engineered cardiac tissues (ECTs). In contrast, zinc (Zn) is a potent metallothionein (MT) inducer, which plays an important role in protection against Cd toxicity. In this study, we investigated the protective effects of Zn against Cd toxicity in ECTs and explore the underlying mechanisms. ECTs were constructed from neonatal ventricular cells of wild-type (WT) mice and mice with global MT gene deletion (MT-KO). In WT-ECTs, Cd (5-20 µM) caused a dose-dependent toxicity that was detected within 8 h evidenced by suppressed beating, apoptosis, and LDH release; Zn (50-200 µM) dose-dependently induced MT expression in ECTs without causing ECT toxicity; co-treatment of ECT with Zn (50 µM) prevented Cd-induced toxicity. In MT-KO ECTs, Cd toxicity was enhanced; but unexpectedly, cotreatment with Zn provided partial protection against Cd toxicity. Furthermore, Cd, but not Zn, significantly activated Nrf2 and its downstream targets, including HO-1; inhibition of HO-1 by a specific HO-1 inhibitor, ZnPP (10 µM), significantly increased Cd-induced toxicity, but did not inhibit Zn protection against Cd injury, suggesting that Nrf2-mediated HO-1 activation was not required for Zn protective effect. Finally, the ability of Zn to reduce Cd uptake provided an additional MT-independent mechanism for reducing Cd toxicity. Thus, Zn exerts protective effects against Cd toxicity for murine ECTs that are partially MT-mediated. Further studies are required to translate these findings towards clinical trials.

On the nature of the Cu-rich aggregates in brain astrocytes.

Fulfilling a bevy of biological roles, copper is an essential metal for healthy brain function. Cu dyshomeostasis has been demonstrated to be involved in some neurological conditions including Menkes and Alzheimer's diseases. We have previously reported localized Cu-rich aggregates in astrocytes of the subventricular zone (SVZ) in rodent brains with Cu concentrations in the hundreds of millimolar. Metallothionein, a cysteine-rich protein critical to metal homeostasis and known to participate in a variety of neuroprotective and neuroregenerative processes, was proposed as a binding protein. Here, we present an analysis of metallothionein(1,2) knockout (MTKO) mice and age-matched controls using X-ray fluorescence microscopy. In large structures such as the corpus callosum, cortex, and striatum, there is no significant difference in Cu, Fe, or Zn concentrations in MTKO mice compared to age-matched controls. In the astrocyte-rich subventricular zone where Cu-rich aggregates reside, approximately 1/3 as many Cu-rich aggregates persist in MTKO mice resulting in a decrease in periventricular Cu concentration. Aggregates in both wild-type and MTKO mice show XANES spectra characteristic of CuxSy multimetallic clusters and have similar [S]/[Cu] ratios. Consistent with assignment as a CuxSy multimetallic cluster, the astrocyte-rich SVZ of both MTKO and wild-type mice exhibit autofluorescent bodies, though MTKO mice exhibit fewer. Furthermore, XRF imaging of Au-labeled lysosomes and ubiquitin demonstrates a lack of co-localization with Cu-rich aggregates suggesting they are not involved in a degradation pathway. Overall, these data suggest that Cu in aggregates is bound by either metallothionein-3 or a yet unknown protein similar to metallothionein.

Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity.

The purpose of this study was to investigate the underlying mechanism of metallothionein (MT) protection from depleted uranium (DU) using a proteomics approach to search for a DU toxicity-differential protein. MT-/- and MT+/+ mice were administrated with a single dose of DU (10 mg/kg, i.p.) or equal volume of saline. After 4 days, protein changes in kidney tissues were evaluated using a proteomics approach. A total of 13 differentially expressed proteins were identified using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The validating results showed that the expression of aminoacylase-3 (ACY-3) and the mitochondrial ethylmalonic encephalopathy 1 (ETHE1) decreased significantly after DU exposure; in addition, the reduction in MT-/- mice was more significant than that in MT+/+ mice. The results also showed that exogenous ETHE1 or ACY-3 could increase the survival rate of human embryonic kidney 293 (HEK293) cells after DU exposure. A specific siRNA of ETHE1 significantly increased cell apoptosis rates after DU exposure, whereas exogenous ETHE1 significantly decreased cell apoptosis rates. In summary, ACY-3 and ETHE1 might involve in protection roles of MT. ETHE1 could be a new sensitive molecular target of DU-induced cell apoptosis.

Persisting neurobehavioral effects of developmental copper exposure in wildtype and metallothionein 1 and 2 knockout mice.

BACKGROUND: Metallothioneins (MT) are small proteins, which are crucial for the distribution of heavy and transition metals. Previously, we found in mice that knockout of MT 1 and 2 genes (MTKO) impaired spatial learning and potentiated the learning impairment caused by developmental mercury exposure. The current study examined the neurocognitive and neurochemical effects of MTKO with the developmental copper (Cu) supplementation. METHODS: Wildtype (WT) and MTKO mice were given supplemental Cu (0, 10 or 50 mg/l) in their drinking water during gestation and until weaning. When the mice were young adults they were trained on the win-shift 8-arm radial maze test of spatial learning and memory. After cognitive testing, their brains were analyzed for norepinepherine, dopamine and serotonin levels. RESULTS: In the spatial learning test, wildtype mice showed the normal sex difference with males performing more accurately than the females. This effect was eliminated by MTKO and restored by moderate Cu supplementation during development. In neurochemical studies, MTKO caused a significant overall increase in serotonin in all of the regions studied: the frontal cortex, posterior cortex, hippocampus, striatum, midbrain, and brainstem. MTKO also caused a significant increase in norepinepherine in the brainstem and hippocampus. In wildtype mice, Cu supplementation during development caused a significant decline in dopamine and norepinepherine in the midbrain and dopamine in the frontal cortex. These effects were blocked by MTKO. CONCLUSIONS: The normal sex difference in spatial working memory accuracy, which was eliminated by MTKO, was restored by moderate copper supplementation. MTKO increased serotonin across all brain areas studied and increased norepinepherine only in the hippocampus and brainstem. MTKO blocked copper-induced decreases in dopamine and norepinepherine in the midbrain and dopamine in the frontal cortex.

A Distinct Gene Module for Dysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells.

Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8(+) tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8(+) TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact.

Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity.

Depleted uranium (DU) has been widely used in both civilian and military activities, and the kidney is the main target organ of DU during acute high-dose exposures. In this study, the nephrotoxicity caused by DU in metallothionein-1/2-null mice (MT-/-) and corresponding wild-type (MT+/+) mice was investigated to determine any associations with MT. Each MT-/- or MT+/+ mouse was pretreated with a single dose of DU (10mg/kg, intraperitoneal injection) or an equivalent volume of saline. After 4days of DU administration, kidney changes were assessed. After DU exposure, serum creatinine and serum urea nitrogen in MT-/- mice significantly increased than in MT+/+ mice, with more severe kidney pathological damage. Moreover, catalase and superoxide dismutase (SOD) decreased, and generation of reactive oxygen species and malondialdehyde increased in MT-/- mice. The apoptosis rate in MT-/- mice significantly increased, with a significant increase in both Bax and caspase 3 and a decrease in Bcl-2. Furthermore, sodium-glucose cotransporter (SGLT) and sodium-phosphate cotransporter (NaPi-II) were significantly reduced after DU exposure, and the change of SGLT was more evident in MT-/- mice. Finally, exogenous MT was used to evaluate the correlation between kidney changes induced by DU and MT doses in MT-/- mice. The results showed that, the pathological damage and cell apoptosis decreased, and SOD and SGLT levels increased with increasing dose of MT. In conclusion, MT deficiency aggravated DU-induced nephrotoxicity, and the molecular mechanisms appeared to be related to the increased oxidative stress and apoptosis, and decreased SGLT expression.

Metallothionein-I/II Knockout Mice Aggravate Mitochondrial Superoxide Production and Peroxiredoxin 3 Expression in Thyroid after Excessive Iodide Exposure.

PURPOSE: We aim to figure out the effect of metallothioneins on iodide excess induced oxidative stress in the thyroid. METHODS: Eight-week-old MT-I/II knockout (MT-I/II KO) mice and background-matched wild-type (WT) mice were used. Mitochondrial superoxide production and peroxiredoxin (Prx) 3 expression were measured. RESULTS: In in vitro study, more significant increases in mitochondrial superoxide production and Prx 3 expression were detected in the MT-I/II KO groups. In in vivo study, significantly higher concentrations of urinary iodine level were detected in MT-I/II KO mice in 100 HI group. Compared to the NI group, there was no significant difference existing in serum thyroid hormones level in either groups (P > 0.05), while the mitochondrial superoxide production was significantly increased in 100 HI groups with significantly increased LDH activity and decreased relative cell viability. Compared to WT mice, more significant changes were detected in MT-I/II KO mice in 100 HI groups. No significant differences were detected between the NI group and 10 HI group in both the MT-I/II KO and WT mice groups (P > 0.05). CONCLUSIONS: Iodide excess in a thyroid without MT I/II protection may result in strong mitochondrial oxidative stress, which further leads to the damage of thyrocytes.

Zinc-binding metallothioneins are key modulators of IL-4 production by basophils.

Zinc (Zn) is an essential nutrient, and Zn deficiency causes immunodeficiency and skin disorders. Basophils express FcɛRI on their surface and release multiple mediators after receptor cross-linking, including large amounts of IL-4. However, the mechanisms involved in the FcɛRI-mediated regulation of basophil IL-4 production are currently unclear. Here, we show that the Zn-binding metallothionein (MT) proteins are essential for the FcɛRI-induced basophil production of IL-4. Basophils from MT-I/II(-/-) mice produced significantly less FcɛRI-induced IL-4 than did wild-type basophils. The MTs were involved in maintaining intracellular Zn levels, thereby regulated the calcineurin activity and nuclear factor of activated T-cell (NFAT)-mediated IL-4 production. These results suggest that the MT-dependent control of Zn homeostasis is a novel mechanism for regulating basophil IL-4 production.

Deletion of metallothionein exacerbates intermittent hypoxia-induced oxidative and inflammatory injury in aorta.

The present study was to explore the effect of metallothionein (MT) on intermittent hypoxia (IH) induced aortic pathogenic changes. Markers of oxidative damages, inflammation, and vascular remodeling were observed by immunohistochemical staining after 3 days and 1, 3, and 8 weeks after IH exposures. Endogenous MT was induced after 3 days of IH but was significantly decreased after 8 weeks of IH. Compared with the wild-type mice, MT knock-out mice exhibited earlier and more severe pathogenic changes of oxidative damages, inflammatory responses, and cellular apoptosis, as indicated by the significant accumulation of collagen, increased levels of connective tissue growth factor, transforming growth factor ß1, tumor necrosis factor-alpha, vascular cell adhesion molecule 1,3-nitrotyrosine, and 4-hydroxy-2-nonenal in the aorta. These findings suggested that chronic IH may lead to aortic damages characterized by oxidative stress and inflammation, and MT may play a pivotal role in the above pathogenesis process.

Cardioprotection against doxorubicin by metallothionein Is associated with preservation of mitochondrial biogenesis involving PGC-1α pathway.

Metallothionein (MT) has been shown to inhibit cardiac oxidative stress and protect against the cardiotoxicity induced by doxorubicin (DOX), a potent and widely used chemotherapeutic agent. However, the mechanism of MT׳s protective action against DOX still remains obscure. Mitochondrial biogenesis impairment has been implicated to play an important role in the etiology and progression of DOX-induced cardiotoxicity. Increasing evidence indicates an intimate link between MT-mediated cardioprotection and mitochondrial biogenesis. This study was aimed to explore the possible contribution of mitochondrial biogenesis in MT׳s cardioprotective action against DOX. Adult male MT-I/II-null (MT(-/-)) and wild-type (MT(+/+)) mice were given a single dose of DOX intraperitoneally. Our results revealed that MT deficiency significantly sensitized mice to DOX-induced cardiac dysfunction, ultrastructural alterations, and mortality. DOX disrupted cardiac mitochondrial biogenesis indicated by mitochondrial DNA copy number and decreased mitochondrial number, and these effects were greater in MT(-/-) mice. Basal MT effectively protected against DOX-induced inhibition on the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a key regulator of mitochondrial biogenesis, and its downstream factors including mitochondrial transcription factor A. Moreover, MT was found to preserve the protein expression of manganese superoxide dismutase, a transcriptional target of PGC-1α. in vitro study showed that MT absence augmented DOX-induced increase of mitochondrial superoxide production in primary cultured cardiomyocytes. These findings suggest that MT׳s cardioprotection against DOX is mediated, at least in part, by preservation of mitochondrial biogenesis involving PGC-1α pathway.

Metallothionein deficiency impacts copper accumulation and redistribution in leaves and seeds of Arabidopsis.

Most angiosperm genomes contain several genes encoding metallothionein (MT) proteins that can bind metals including copper (Cu) and zinc (Zn). Metallothionein genes are highly expressed under various conditions but there is limited information about their function. We have studied Arabidopsis mutants that are deficient in multiple MTs to learn about the functions of MTs in plants. T-DNA insertions were identified in four of the five Arabidopsis MT genes expressed in vegetative tissues. These were crossed to produce plants deficient in four MTs (mt1a/mt2a/mt2b/mt3). The concentration of Cu was lower in seeds but higher in old leaves of the quad-MT mutant compared to wild-type plants. Experiments with stable isotopes showed that Cu in seeds came from two sources: directly from roots and via remobilization from other organs. Mobilization of Cu out of senescing leaves was disrupted in MT-deficient plants. Tolerance to Cu, Zn and paraquat was unaffected by MT deficiency but these plants were slightly more sensitive to cadmium (Cd). The quad-MT mutant showed no change in resistance to a number of microbial pathogens, or in the progression of leaf senescence. Although these MTs are not required to complete the plant's life cycle, MTs are important for Cu homeostasis and distribution in Arabidopsis.

Role of metallothioneins as danger signals in the pathogenesis of colitis.

Inflammatory bowel diseases (IBDs) are recurrent intestinal pathologies characterized by a compromised epithelial barrier and an exaggerated immune activation. Mediators of immune cell infiltration may represent new therapeutic opportunities. Metallothioneins (MTs) are stress-responsive proteins with immune-modulating functions. Metallothioneins have been linked to IBDs, but their role in intestinal inflammation is inconclusive. We investigated MT expression in colonic biopsies from IBDs and acute infectious colitis patients and healthy controls and evaluated MT's role in experimental colitis using MT knockout mice and anti-MT antibodies. Antibody potential to target extracellular MT and its mechanism was tested in vitro. Biopsies of patients with active colitis showed infiltration of MT-positive cells in a pattern that correlated with the grade of inflammation. MT knockout mice displayed less severe acute dextran sulphate sodium (DSS)-induced colitis compared to congenic wild-type mice based on survival, weight loss, colon length, histological inflammation and leukocyte infiltration. Chronic DSS-colitis confirmed that Mt1 and Mt2 gene disruption enhances clinical outcome. Blockade of extracellular MT with antibodies reduced F4/80-positive macrophage infiltration in DSS- and trinitrobenzene sulphonic acid-colitis, with a tendency towards a better outcome. Whole-body single-photon emission computer tomography of mice injected with radioactive anti-MT antibodies showed antibody accumulation in the colon during colitis and clearance during recovery. Necrotic and not apoptotic cell death resulted in western blot MT detection in HT29 cell supernatant. In a Boyden chamber migration assay, leukocyte attraction towards the necrotic cell supernatant could be abolished with anti-MT antibody, indicating the chemotactic potential of endogenous released MT. Our results show that human colitis is associated with infiltration of MT-positive inflammatory cells. Since antibody blockade of extracellular MT can reduce colitis in mice, MT may act as a danger signal and may represent a novel target for reducing leukocyte infiltration and inflammation in IBD patients.

Cooperative functions of ZnT1, metallothionein and ZnT4 in the cytoplasm are required for full activation of TNAP in the early secretory pathway.

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1(-/-) MT(-/-) ZnT4(-/-) cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1(-/-) MT(-/-) ZnT4(-/-) cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1(-/-) MT(-/-) ZnT4(-/-) cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.

Preventive effects of metallothionein against DNA and lipid metabolic damages in dyslipidemic mice under repeated mild stress.

The effects of repeated mild stress on DNA and lipid metabolic damages in multiple organs of dyslipidemic mice, and the preventive role of metallothionein (MT) were investigated. Female adult wild-type and MT-null mice fed high-fat diet (HFD) or standard diet (STD) were repeatedly subjected to fasting or restraint for three weeks. The liver, pancreas, spleen, bone marrow and serum samples were taken for evaluating DNA damage, MT, glutathione (GSH), corticosterone, carnitine and adiponectin. Body weights of restraint groups were reduced with the intensity of stress increased, even if the energy intakes were higher than those of STD group. Hepatic GSH levels were reduced in HFD control group and were further reduced in stress groups, especially in restraint groups, while the hepatic MT and serum corticosterone levels were increased in concert with the intensity of stress. Cellular DNA damages were generally increased by the restraint stress, especially in MT-null mice. Hepatic carnitine levels of MT-null mice were markedly lower than those of wild-type mice. The data suggest that MT plays a preventive role by acting as an antioxidant in corporation with GSH decreased by repeated stress and that MT may be an essential factor for inducing carnitine under the stress.

Cadmium modulates adipocyte functions in metallothionein-null mice.

Our previous study has demonstrated that exposure to cadmium (Cd), a toxic heavy metal, causes a reduction of adipocyte size and the modulation of adipokine expression. To further investigate the significance of the Cd action, we studied the effect of Cd on the white adipose tissue (WAT) of metallothionein null (MT(-/-)) mice, which cannot form atoxic Cd-MT complexes and are used for evaluating Cd as free ions, and wild type (MT(+/+)) mice. Cd administration more significantly reduced the adipocyte size of MT(-/-) mice than that of MT(+/+) mice. Cd exposure also induced macrophage recruitment to WAT with an increase in the expression level of Ccl2 (MCP-1) in the MT(-/-) mice. The in vitro exposure of Cd to adipocytes induce triglyceride release into culture medium, decrease in the expression levels of genes involved in fatty acid synthesis and lipid hydrolysis at 24 h, and at 48 h increase in phosphorylation of the lipid-droplet-associated protein perilipin, which facilitates the degradation of stored lipids in adipocytes. Therefore, the reduction in adipocyte size by Cd may arise from an imbalance between lipid synthesis and lipolysis. In addition, the expression levels of leptin, adiponectin and resistin decreased in adipocytes. Taken together, exposure to Cd may induce unusually small adipocytes and modulate the expression of adipokines differently from the case of physiologically small adipocytes, and may accelerate the risk of developing insulin resistance and type 2 diabetes.

Metallothionein blocks oxidative DNA damage in vitro.

The role of metallothionein (MT) in mitigation of oxidative DNA damage (ODD) induced by either cadmium (Cd) or the direct oxidant hydrogen peroxide (H(2)O(2)) was systematically examined using MT-I/II double knockout (MT-null) or MT-competent wild-type (WT) cells. Both toxicants were much more lethal to MT-null cells (Cd LC(50) = 6.6 µM; H(2)O(2) LC(50) = 550 µM) than to WT cells (Cd LC(50) = 16.5 µM; H(2)O(2) LC(50) = 930 µM). Cd induced concentration-related MT increases in WT cells, while the basal levels were undetectable and not increased by Cd in MT-null cells. ODD, measured by the immuno-spin trapping method, was minimally induced by sub-toxic Cd levels (1 or 5 µM; 24 h) in WT cells, but markedly increased in MT-null cells (>430 %). Similarly, ODD was induced to higher levels by lower concentrations of H(2)O(2) in MT-null cells than WT cells. Transfection of MT-I into MT-null cells reduced both Cd- and H(2)O(2)-induced cytolethality and ODD. Cd increased the expression of the oxidant defense genes, HO-1, and GSTa2 to a much greater extent in MT-null cells than in WT. Cd or H(2)O(2) exposure increased the expression of key transport genes, Mrp1 and Mrp2, in WT cells but not in MT-null cells. MT protects against Cd- and H(2)O(2)-induced ODD in MT-competent cells possibly by multiple mechanisms, potentially including direct metal ion sequestration and sequestration of oxidant radicals by MT. MT-deficient cells appear to adapt to Cd primarily by turning on oxidant response systems, while MT-competent cells activate MT and transport systems.

Protective effect of naringenin against acetaminophen-induced acute liver injury in metallothionein (MT)-null mice.

Naringenin is a natural flavonoid aglycone of naringin that has been reported to have a wide range of pharmacological properties, such as antioxidant activity and free radical scavenging capacity. This study was designed to examine the hepatoprotective effect of naringenin against acetaminophen (250 mg kg(-1), sc) in metallothionein (MT)-null mice. 42 SPF MT-knockout mice were used. Naringenin (200, 400, and 800 mg kg(-1), ig) was administered for 4 days before exposure to acetaminophen (250 mg kg(-1), sc). Liver injury was measured by serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), as well as liver malondialdehyde (MDA). The glutathione-to-oxidized glutathione ratio (GSH/GSSG) was also assessed. The evidence of liver injury induced by acetaminophen included not only a significant increase in the levels of serum ALT, AST, LDH and liver MDA, and also a significant decrease in GSH/GSSG. Pretreatment of mice with naringenin at 400 and 800 mg kg(-1) reversed the altered parameters. Such reversal effects were dose-dependent: ALT decreased 78.62% and 98.03%, AST decreased 88.35% and 92.64%, LDH decreased 76.54% and 81.63%, MDA decreased 48.59% and 66.27% at a dose of 400 and 800 mg kg(-1) respectively; GSH/GSSG increased 22.57% and 16.93% at a dose of 400 and 800 mg kg(-1) respectively. Histopathological observation findings were also consistent with these effects. Together, this study suggests that naringenin can potentially reverse the hepatotoxic damage of acetaminophen intoxication in MT-null mice.