metallothionein and sodium-arsenite

Background Curcumin is extensively used as a therapeutic intervention for treating several ailments. The antioxidant curcumin has an anti-inflammatory and chelating property with arsenic to exhibit a strong therapeutic effect on reproductive organs. This study was undertaken to describe the protective effect of noninvasive administration of curcumin against sodium-arsenite-mediated uterine hazards in female Wistar rats. Methods Twenty-four female Wistar rats were randomly divided into four groups. The treatment was continued for 8 days and given orally sodium arsenite (10 mg/kg body weight) in combination with curcumin (20 mg/kg body weight). Results Our evaluation revealed that 8 days of sodium arsenite (10 mg/kg body weight) treatment reduced the activities of the uterine enzymatic antioxidants superoxide dismutase, catalase, and peroxidase. Blood levels of vitamin B12 and folic acid decreased followed by an increased serum lactate dehydrogenase, homocysteine level, and hepatic metallothionein-1 in arsenic-treated rats. Necrosis of uterine tissue along with the disruption of ovarian steroidogenesis was marked in arsenic-treated rats with an upregulation of uterine NF-κB and IL-6 along with a raised level of serum TNF-α. Oral administration of curcumin (20 mg/kg body weight/day) in arsenic-treated rats significantly reinstated these alterations of the antioxidant system followed by an improvement of ovarian steroidogenesis and the circulating level of B12 and folate along with the downregulation of serum homocysteine, metallothionein-1, and cytokines. Conclusions The findings of this study clearly and strongly elucidated that arsenic-induced oxidative stress in uterus is linked to an alteration of inflammation-signaling biomarkers and these have been protected through the co-administration of curcumin due to its anti-inflammatory, free radical scavenging, and antioxidant activity by the possible regulation of an S-adenosine methionine pool.

Topics: Animals; Antioxidants; Arsenic; Arsenites; Catalase; Curcumin; Cytokines; Female; Glutathione; Glutathione Peroxidase; Inflammation; Metallothionein; NF-kappa B; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Sodium Compounds; Superoxide Dismutase; Uterus

Arsenic (As), a toxic metalloid, is one of the major global concerns. The toxicity resulting from As exposure is linked to the generation of reactive oxygen intermediates during their redox cycling and metabolic activation processes that cause lipid peroxidation (LPO). Zinc (Zn), a redox-inactive metal, helps to maintain cellular functions because of its prominent role in antioxidant network through multiple mechanisms. The present study, therefore, explores the effectiveness of administered Zn to combat against acute As toxicity by analysis of antioxidant defense status, alkaline phosphatase (ALP) activity, histological profile, MT expression, and elemental status in rat liver. To achieve this goal, four experimental groups, one control and three receiving different metal supplementations, were chosen (group 1, control; group 2, Zn supplemented; group 3, As substituted; group 4, Zn + As supplemented). The levels of reduced glutathione (GSH) and activities of glutathione reductase (GR) and ALP were lowered, whereas LPO levels and activity of superoxide dismutase (SOD) were elevated with no significant change in catalase (CAT) activity. Histopathological changes were also observed in the As substituted group in comparison to the control. Particle-induced X-ray emission (PIXE) analysis showed decrease in Fe and S concentration in rat liver after As intoxication, whereas As was below detection limit, i.e., <1 ppm. Zn administration almost restored the antioxidants, ALP activity, histopathological changes, and elemental status. A cumulative increase in MT expression was found with the combined treatment of Zn and As. Also, Zn alone caused no significant change in the antioxidant defense system. It can be concluded that restoration of antioxidant activity and increased MT expression are the two independent protective mechanisms of Zn to reduce acute As toxicity.

Topics: Animals; Antioxidants; Arsenites; Lipid Peroxidation; Liver; Male; Metallothionein; Oxidative Stress; Rats, Sprague-Dawley; RNA; Sodium Compounds; Spectrometry, X-Ray Emission; Zinc Sulfate

An-gong-niu-huang wan (AGNH) is a famous traditional Chinese medicine used for brain trauma, hemorrhage, and coma. AGNH contains 10% realgar (As₄S₄) and 10% cinnabar (HgS). Both As and Hg are well-known for their toxic effects, and the safety of AGNH is of concern. To address this question, the acute toxicity of AGNH, realgar and cinnabar were compared to sodium arsenite (NaAsO₂) and mercuric chloride (HgCl₂). Mice were administrated orally AGNH at 1, 3 and 6g/kg. AGNH at 3g/kg contains 2.8mmol As/kg as realgar and 1.18mmol Hg/kg as cinnabar. Realgar, cinnabar, arsenite (0.28 mmol/kg, 10% of realgar) and HgCl₂ (0.256 mmol/kg, 20% of cinnabar) were orally given to mice for comparison. Blood and tissues were collected 8h later for toxicity evaluation. Serum alanine aminotransferase was increased by arsenite and blood urea nitrogen was increased by HgCl₂. Total As accumulation after arsenite in liver (100-fold) and kidney (13-fold) was much higher than that after realgar. The accumulation of Hg after HgCl₂ in liver was 400-fold higher and kidney 30-fold higher than after cinnabar. Histopathology showed moderate liver and kidney injuries after arsenite and HgCl₂, but injuries were mild or absent after AGNH, realgar, and cinnabar. The expression of metallothionein-1, a biomarker of metal exposure, was increased 4-10-fold by arsenite and HgCl₂, but was unchanged by AGNH, realgar and cinnabar. Thus, AGNH, realgar and cinnabar are much less toxic acutely than arsenite and HgCl₂. The chemical forms of As and Hg are extremely important factors in determining their disposition and toxicity.

Topics: Alanine Transaminase; Animals; Arsenic; Arsenicals; Arsenites; Aspartate Aminotransferases; Blood Urea Nitrogen; Creatinine; Female; Kidney; Liver; Male; Medicine, Chinese Traditional; Mercuric Chloride; Mercury; Mercury Compounds; Metallothionein; Mice; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Compounds; Sulfides

Cilostazol, an antiplatelet drug, exhibits antiatherogenic effects. The purpose of the present study was to determine the effect of cilostazol on the cytotoxicity of cadmium (Cd) and arsenite (iAs(III)), which involved in the pathogenesis of vascular disorders such as atherosclerosis, in cultured vascular endothelial cells. Cytotoxicity was evaluated by the lactate dehydrogenase leakage assay and morphological observation. Cd (10 µM) -induced cytotoxicity was prevented by pretreatment with cilostazol (30 and 100 µM) and simultaneous treatment with cilostazol (100 µM). On the other hand, iAs(III)-induced cytotoxicity was blocked by pretreatment with cilostazol (30 and 100 µM) but not simultaneous treatment with cilostazol. The mRNA level and the protein level of metallothionein (MT) were significantly increased by cilostazol in the cells. These results suggested, therefore, that pretreatment with cilostazol effectively prevents the cytotoxicity of Cd and iAs(III) in cultured vascular endothelial cells, at least in part through the induction of MT synthesis.

Topics: Animals; Aorta; Arsenites; Blotting, Western; Cadmium Chloride; Cattle; Cell Survival; Cells, Cultured; Cilostazol; Endothelium, Vascular; Gene Expression; L-Lactate Dehydrogenase; Metallothionein; RNA, Messenger; Sodium Compounds; Tetrazoles; Vasodilator Agents

There is limited available information on the effects of arsenic on enzymes participating in the folate cycle. Therefore, our aim was to evaluate the effects of sodium arsenite on the protein levels of methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase (DHFR) and its further relationship with the expression MT1/2 and c-myc in MCF-7 cells. Arsenite treatment (0-10 microM) for 4 h decreased MTHFR levels in a concentration-dependent fashion without significant effects on DHFR. The effects on MTHFR were observed at arsenite concentrations not significantly affecting cell viability. We also observed an increase in S-phase recruitment at all concentrations probed. Lower concentrations (<5 microM) induced cell proliferation, showing a high proportion of BrdU-stained cells, indicating a higher DNA synthesis rate. However, higher concentrations (> or =5 microM) or longer treatment periods induced apoptosis. Arsenite also induced dose-dependent increases in MT1/2 and c-Myc protein levels. The levels of MTHFR were inversely correlated to MT1/2 and c-Myc overexpression and increased S-phase recruitment. Our findings indicate that breast epithelial cells are responsive to arsenite and suggest that exposure may pose a risk for breast cancer. The reductions in MTHFR protein levels contribute to understand the mechanisms underlying the induction of genes influencing growth regulation, such as c-myc and MT1/2. However, further research is needed to ascertain if the effects here reported following short-time and high-dose exposure are relevant for human populations chronically exposed to low arsenic concentrations.

Topics: Antimetabolites; Arsenites; Blotting, Western; Bromodeoxyuridine; Cell Cycle; Cell Line, Tumor; Cell Survival; Female; Flow Cytometry; Gene Expression; Humans; Metallothionein; Methylenetetrahydrofolate Reductase (NADPH2); Proto-Oncogene Proteins c-myc; S Phase; Sodium Compounds

Arsenite is an important cancer chemotherapeutic. The liver is a major target tissue of arsenic toxicity and hepatotoxicity may limit its chemotherapeutic efficacy. O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO)-producing prodrug metabolized by hepatic P450 enzymes to release NO locally. V-PYRRO/NO protects against various organic or inorganic hepatotoxicants but any role in arsenic hepatotoxicity is undefined. Thus, we studied the effects of V-PYRRO/NO (0-1000muM) pretreatment on inorganic arsenic-induced toxicity in cultured rat liver (TRL 1215) cells. These cells metabolized the prodrug to release NO, producing extracellular nitrite levels to 41.7-fold above control levels (7.50+/-0.38 microM) after 24h V-PYRRO/NO (1000 microM) exposure. The effect of pretreatment with V-PYRRO/NO (24h) on the cytolethality of arsenic (as NaAsO(2)) exposure (24h) was assessed. Arsenic was markedly less toxic in V-PYRRO/NO pretreated cells (LC(50)=30.3 microM) compared to control (LC(50)=20.1 microM) and the increases in LC(50) showed a direct relationship to the level of NO produced (measured as nitrite). Consistent with the cytolethality data, V-PYRRO/NO pretreatment markedly reduced arsenic-induced apoptosis as assessed by DNA fragmentation. Activation of the c-Jun N-terminal kinase (JNK) pathway can be critical to apoptosis and pretreatment with V-PYRRO/NO suppressed arsenic-induced JNK activation. V-PYRRO/NO pretreatment modestly increased metallothionein (MT), a metal-binding protein, but greatly enhanced arsenic induction of MT. Thus, V-PYRRO/NO pretreatment directly mitigates arsenic toxicity in cultured liver cells, reducing cytolethality, apoptosis and related JNK pathway activation, apparently through generation of NO. The role of NO in reducing the hepatotoxicity of arsenical chemotherapeutics in vivo deserves additional study.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arsenites; Cell Line; Cell Survival; Cytochrome P-450 Enzyme System; Cytoprotection; Dose-Response Relationship, Drug; Epithelial Cells; Inhibitory Concentration 50; JNK Mitogen-Activated Protein Kinases; Liver; Metallothionein; Nitric Oxide; Nitric Oxide Donors; Nitrites; Phosphorylation; Protective Agents; Pyrrolidines; Rats; Sodium Compounds; Up-Regulation

Metallothionein (MT) is known to play an important role in the resistance of tumor cells to cis-diamminedichloroplatinum (II) (cisplatin). To identify non-MT factors of cisplatin resistance, we characterized cisplatin-resistant cell lines derived from MT-null cells. All of the cisplatin-resistant MT- null cell lines, namely MKCr-1, -3, -4, -12, and -13, showed strong cisplatin resistance and decreased platinum accumulation. Some multidrug resistance proteins (MRPs) have been reported to contribute to cisplatin resistance. However, no significant difference of the MRPs was observed in any of these cell lines. The MKCrs showed cross-resistance to other metals such as arsenite, arsenate, cadmium and antimony. The arsenate and arsenite sensitivities were highly correlated with sensitivity to cisplatin. In addition, the degree of arsenite accumulation was correlated with the degree of cisplatin accumulation. These results suggest that the cisplatin resistance was strongly correlated with the arsenite transport mechanism in these cells.

Topics: Animals; Antineoplastic Agents; Arsenites; ATP Binding Cassette Transporter, Subfamily B; Biological Transport; Cell Line, Tumor; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Inhibitory Concentration 50; Metallothionein; Mice; RNA, Messenger; Sodium Compounds

Subcutaneous injection of sodium arsenite (NaAs, 12.5 mg/kg) into BALB/c [wild-type (WT)] mice causes acute renal dysfunction characterized by severe hemorrhages, acute tubular necrosis, and cast formation, with increases in serum blood urea nitrogen and creatinine levels. Concomitant enhancement in intrarenal interferon (IFN)-gamma expression prompted us to examine its roles in this pathology. IFN-gamma-deficient (IFN-gamma-/-) mice exhibited higher serum blood urea nitrogen and creatinine levels and exaggerated histopathological changes, compared with WT mice. Eventually, IFN-gamma-/- mice exhibited a high mortality (87.5%) within 24 hours after NaAs challenge, whereas most WT mice survived. The intrarenal arsenic concentration was significantly higher in IFN-gamma-/- mice later than 10 hours after NaAs treatment, with attenuated intrarenal expression of multidrug resistance-associated protein (MRP) 1, a main transporter for NaAs efflux, compared with WT mice. NF-E2-related factor (Nrf) 2 protein, a transcription factor crucial for MRP1 gene expression, was similarly increased in the kidneys of both strains of mice after NaAs treatment. In contrast, the absence of IFN-gamma augmented transforming growth factor-beta-Smad3 signal pathway and eventually enhanced the expression of activating transcription factor 3, which is presumed to repress Nrf2-mediated MRP1 gene expression. Thus, IFN-gamma can protect against NaAs-induced acute renal injury, probably by maintaining Nrf2-mediated intrarenal MRP1 gene expression.

Topics: Acute Kidney Injury; Animals; Antibodies; Arsenites; ATP Binding Cassette Transporter, Subfamily B, Member 1; Gene Expression Regulation; Interferon-gamma; Kidney Tubules; Male; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Multidrug Resistance-Associated Proteins; NF-E2-Related Factor 2; RNA, Messenger; Signal Transduction; Sodium Compounds; Transforming Growth Factor beta; Up-Regulation

Studies have shown that metallothionein (MT) is overexpressed in some human bladder cancers and that overexpression can predict treatment response to neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy. In the present study the UROtsa cell line, a model of normal human urothelium, was used to determine the expression of the human MT-1 and MT-2 genes and MT protein following exposure to CdCl(2) or NaAsO(2) at lethal and sublethal levels. Acute exposure was modeled by treating confluent cultures with 100 microM NaAsO(2) or 53.4 microM CdCl(2) for 4 h followed by a 48-h recovery period. Extended exposure was modeled by treating confluent cells with 1, 4, and 8 microM As(3+) or 1, 5, and 9 microM Cd(2+) for 16 d, with the highest concentrations producing cell lethality. The expression of MT mRNAs and protein were determined by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblot analysis. Cell viability was determined by the MTT assay. It was shown that acute exposure to either As(3+) or Cd(2+) increased the levels of mRNAs for the MT-1E, MT-1X, and MT-2A genes, whereas extended exposure only increased these mRNAs following exposure to Cd(2+). It was shown that both acute and extended exposure to either As(3+) or Cd(2+) increased the levels of MT protein, reaching a maximal value of 8 ng MT protein/microg total protein for acute exposure to Cd(2+). This is in contrast to previous studies using cultured human proximal tubule cells, where similar extended treatment with Cd(2+) resulted in over 20-fold higher MT protein levels. These studies demonstrate that human urothelial cells accumulate only modest amounts of MT protein when exposed to either Cd(2+) or As(2+) for a 16-d time period.

Topics: Arsenites; Cadmium Chloride; Cell Line; Enzyme Inhibitors; Gene Expression Regulation; Humans; Metallothionein; Sodium Compounds; Urothelium

Arsenic exposure is clearly linked to human cancer. In rodent cells, arsenic has been reported to induce aberrant gene expression, including activation of the proto-oncogene c-myc. Abnormal or altered expression of such oncogenes can be involved in the acquisition of a malignant phenotype. Although its mechanism of action is unclear, arsenic is known to exert at least some of its toxic effects through interaction with sulfhydryl groups, and the non-protein sulfhydryl glutathione (GSH) appears to play an important role in detoxication of arsenic. Similarly, metallothionein (MT), a metal-binding protein with high sulfhydryl content, often functions in defense against metal-induced or oxidative cellular injury. Therefore, we examined the relationship among GSH, MT gene expression, and arsenic-induced toxicity or c-myc expression in cultured rat myoblast (L6) cells. In initial toxicity studies, arsenic was used in both the trivalent (arsenite) and pentavalent (arsenate) forms. The role of GSH was studied by pretreating cells with L-buthionine sulfoximine (BSO), which induces a marked depletion of GSH. In vitro exposure of L6 cells to BSO (1 to 25 microM) resulted in dose-dependent decreases in GSH. GSH depletion sensitized cells to both arsenite and arsenate. Zinc pretreatment, at levels which highly activated MT expression, had no effect on arsenite-induced cytotoxicity. Arsenite (1 microM) alone modestly increased c-myc expression from 1 to 4 h after treatment (maximum of 2.0-fold over control). After GSH depletion cells responded to arsenite exposure with much larger increases in c-myc transcription (3.2-fold over control). Zinc pretreatment had no reductive effect on arsenite-induced c-myc expression despite markedly activating the MT gene. Thus, it appears that the cellular levels of GSH, but not MT gene expression, play an important role in resistance to arsenic toxicity and aberrant gene activation. Moreover, depletion of GSH enhances arsenic-induced proto-oncogene activation, which might contribute to subsequent transformation.

Topics: Animals; Arsenates; Arsenic; Arsenites; Cell Line; Gene Expression; Glutathione; Metallothionein; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Rats; Sodium Compounds

Arsenic exerts its toxicity by the generation of reactive oxygen intermediates which caused lipid peroxidation and cellular damage. Metallothioneins (MTs) have been shown to be induced by oxidative stress and act as scavengers of reactive oxygen intermediates. Thus, hepatic MT was examined in channel catfish treated with the herbicide monosodium methyl arsonate (MSMA) and compared to equal doses of trivalent and pentavalent arsenic. Fish were exposed to 0.01, 0.1, and 1.0 mg/L of each compound for 1 week by static renewal. Hepatic MT was measured by the cadmium/hemoglobin (Cd/Hb) saturation assay, ELISA using antibodies raised against the first 10 amino acids of piscine MT, and Northern blot analysis using a cDNA encoding winter flounder hepatic MT. Cd/Hb and ELISA measurements of low molecular weight fractions from the hepatic cytosolic component of fish exposed to MSMA revealed a dose dependent increase in MT. MTs and MT mRNA of fish receiving the 1.0 mg/L dose were significantly induced vs control. Responses to arsenate exposure were more variable, but showed a trend toward a dose-dependent induction of MT and MT mRNA. MT mRNA and protein also showed a dose-dependent increase with arsenite exposure with no significant differences with untreated animals. Hepatic lipid peroxidation (as determined by TBARS) and glutathione was unaltered by any of the arsenical treatments. Thus, the lack of correlation between oxidative stress and MT expression suggest MT may not be a reliable indicator of oxidative stress. In addition, the induction of hepatic MT by various forms of As does not appear to be mediated through an oxidative stress mechanism in the liver.

Topics: Animals; Arsenates; Arsenic Poisoning; Arsenicals; Arsenites; Cadmium Chloride; Copper Sulfate; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Glutathione; Herbicides; Ictaluridae; Lipid Peroxidation; Liver; Male; Metallothionein; Sodium Compounds; Zinc Sulfate

A glutathione S-transferase (GST) was purified from an arsenic-resistant Chinese hamster ovary cell line, SA7. The SA7 GST was shown to catalyse the conjugation of glutathione and ethacrynic acid, a specific substrate for Pi class GST. Its N-terminal amino-acid sequence has 80% identical residues to that of rat GST P and human GST pi. Thus, the GST purified from SA7 cells belongs to the Pi family. Treatment with Cibacron Blue or ethacrynic acid, which are GST inhibitors, significantly decreased the resistance of SA7 cells to sodium arsenite. On the other hand, pretreatment of SA7N cells, a partial revertant of SA7 cells, with sublethal doses of sodium arsenite, cadmium acetate or zinc sulphate resulted in re-elevation of GST activities and the cells regained the arsenic resistance. The regained arsenic resistance was well correlated with the levels of GST pi which were induced dose-dependently by zinc sulphate. Heat-shock treatment (45 degrees C for 10 min) did not increase GST pi expression or arsenic resistance of SA7N cells. The results indicate that GST pi is possibly involved in the mechanism of arsenic detoxification.

Topics: Amino Acid Sequence; Animals; Arsenic; Arsenites; CHO Cells; Cricetinae; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Glutathione Transferase; Heat-Shock Proteins; Humans; Isoenzymes; Metallothionein; Molecular Sequence Data; Rats; Sodium Compounds; Substrate Specificity; Sulfates; Zinc; Zinc Sulfate