metallothionein and Carcinoma--Ehrlich-Tumor

To investigate the chemopreventive efficacy of the Indian medicinal plant Acanthus ilicifolius L Acanthaceae in a transplantable Ehrlich ascites carcinoma (EAC)-bearing murine model.. Male Swiss albino mice were divided into four groups: Group A was the untreated normal control; Group B was the EAC control mice group that received serial, intraperitoneal (ip) inoculations of rapidly proliferating 2 x 10(5) viable EAC cells in 0.2 mL of sterile phosphate buffered saline; Group C was the plant extract-treated group that received the aqueous leaf extract (ALE) of the plant at a dose of 2.5 mg/kg body weight by single ip injections, once daily for 10, 20 and 30 consecutive days following tumour inoculation (ALE control); and Group D was the EAC + ALE-treatment group. The chemopreventive potential of the ALE was evaluated in a murine model by studying various biological parameters and genotoxic markers, such as tumour cell count, mean survival of the animals, haematological indices, hepatocellular histology, immunohistochemical expression of liver metallothionein (MT) protein, sister-chromatid exchanges (SCEs), and DNA alterations.. Treatment of the EAC-bearing mice with the ALE significantly (P < 0.001) reduced viable tumour cell count by 68.34% (228.7 x 10(6) +/- 0.53) when compared to EAC control mice (72.4 x 10(6) +/- 0.49), and restored body and organ weights almost to the normal values. ALE administration also increased (P < 0.001) mean survival of the hosts from 35 +/- 3.46 d in EAC control mice to 83 +/- 2.69 d in EAC + ALE-treated mice. Haematological indices also showed marked improvement with administration of ALE in EAC-bearing animals. There was a significant increase in RBC count (P < 0.001), hemoglobin percent (P < 0.001), and haematocrit value (P < 0.001) from 4.3 +/- 0.12, 6.4 +/- 0.93, and 17.63 +/- 0.72 respectively in EAC control mice to 7.1 +/- 0.13, 12.1 +/- 0.77, and 30.23 +/- 0.57 respectively in EAC + ALE-treated group, along with concurrent decrement (P < 0.001) in WBC count from 18.8 +/- 0.54 in EAC control to 8.4 +/- 0.71 in EAC + ALE. Furthermore, treatment with ALE substantially improved hepatocellular architecture and no noticeable neoplastic lesions or foci of cellular alteration were observed. Daily administration of the ALE was found to limit liver MT expression, an important marker of cell proliferation with concomitant reduction in MT immunoreactivity (62.25 +/- 2.58 vs 86.24 +/- 5.69, P < 0.01). ALE was also potentially effective in reducing (P < 0.001) the frequency of SCEs from 14.94 +/- 2.14 in EAC control to 5.12 +/- 1.16 in EAC + ALE-treated group. Finally, in comparison to the EAC control, ALE was able to suppress in vivo DNA damage by abating the generations of 'tailed' DNA by 53.59% (98.65 +/- 2.31 vs 45.06 +/- 1.14, P < 0.001), and DNA single-strand breaks (SSBs) by 38.53% (3.14 +/- 0.31 vs 1.93 +/- 0.23, P < 0.01) in EAC-bearing murine liver.. Our data indicate that, ALE is beneficial in restoring haematological and hepatic histological profiles and in lengthening the survival of the animals against the proliferation of ascites tumour in vivo. Finally, the chemopreventive efficacy of the ALE is manifested in limiting MT expression and in preventing DNA alterations in murine liver. The promising results of this study suggest further investigation into the chemopreventive mechanisms of the medicinal plant A. ilicifolius in vivo and in vitro.

Topics: Acanthaceae; Animals; Carcinoma, Ehrlich Tumor; Cell Proliferation; Cell Transplantation; Disease Models, Animal; DNA Breaks, Single-Stranded; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Liver; Male; Metallothionein; Mice; Plant Extracts; Sister Chromatid Exchange

This study was concerned with the role of copper (Cu) and Cu-metallothionein (Cu-MT) in oxidative stress. Hydrogen peroxide (H(2)O(2))-induced oxidative injury was examined in Ehrlich ascites tumour cells isolated from host mice pretreated with 0, 1 or 2mg of CuSO(4) (ip) 24h earlier. Control Ehrlich cells contained low levels of Cu and Cu treatment produced dose-related increases in cellular Cu and Cu-MT levels and corresponding increases in sensitivity to oxidative toxicity of H(2)O(2) (LC(50), cell blebbing, lipid peroxidation, GSH depletion, and increase in intracellular free [Ca(2+)](i)). Hydrogen peroxide treatment also resulted in the oxidation of MT thiolates, reduction in the binding of Cu to MT resulting in translocation of Cu to other subcellular sites. d-penicillamine, a Cu-chelating agent, obliterated the sensitization effect of Cu-pretreatment and reduced the redistribution of MT-bound Cu, suggesting the participation of Cu ions derived from MT in promoting oxidant stress. Additional experiments with desferoxamine and mannitol have revealed the involvement of a Cu-dependent Fenton reaction in the mediation of the prooxidative effect of Cu-MT. These data suggest that cells with high levels of Cu-MT may be particularly susceptible to oxidative stress.

Topics: Animals; Calcium; Carcinoma, Ehrlich Tumor; Cell Survival; Copper; Copper Sulfate; Deferoxamine; Dose-Response Relationship, Drug; Glutathione Disulfide; Hydrogen Peroxide; Injections, Intraperitoneal; Iron; Lipid Peroxidation; Male; Mannitol; Metallothionein; Mice; Oxidative Stress; Penicillamine; Pinocytosis; Time Factors; Tumor Cells, Cultured; Zinc

This study was concerned with the role of zinc (Zn) and zinc-metallothionein (Zn-MT) in oxidative stress. Hydrogen peroxide-induced oxidative injury was examined in Ehrlich ascites tumour cells isolated from control host mice, mice pretreated with 10 mg/kg ZnSO4 (i.p.) to increase cellular Zn/Zn-MT levels, and mice exposed to Zn-deficient diet to reduce the cellular Zn/Zn-MT levels. The results of the present study showed that Ehrlich cells with seven-fold differences in Zn-MT concentrations could be obtained by manipulating the Zn status of host mice and that high Zn and Zn-MT levels can make Ehrlich cells more resistant to H2O2-induced oxidative injury (cell viability, lipid peroxidation, [Ca2+]i) while cells with reduced Zn/Zn-MT levels were more susceptible to this treatment. H2O2 treatment resulted in oxidation of MT thiolate groups and loss of its metal binding capacity with translocation of Zn released from oxidized MT to other cellular sites. Preincubation of Ehrlich cells with ZnSO4 in vitro also conferred some degree of resistance to H2O2 toxicity, suggesting the inherent antioxidative property of Zn ions. These data suggested that Zn-MT can be considered as an antioxidant by virtue of its thiolate groups and its Zn ions that are released in the presence of oxidative stress.

Topics: Animals; Antioxidants; Calcium; Carcinoma, Ehrlich Tumor; Cell Line, Tumor; Cell Survival; Copper; Cytosol; Glutathione; Hydrogen Peroxide; Lipid Peroxidation; Male; Metallothionein; Mice; Neoplasm Transplantation; Protein Binding; Time Factors; Tumor Cells, Cultured; Zinc

Based on previous findings that liver zinc and metallothionein (MT) levels increase after tumor transplantation, zinc metabolism in tumor-bearing mice was studied to clarify the role of zinc-MT in host defense systems. Zinc in the hepatic cytosolic MT fraction did not increase in tumor-bearing mice fed a zinc-deficient diet, suggesting that dietary zinc is necessary for apo-MT induction in the liver after tumor transplantation and is then incorporated into the apo-MT. When (65)ZnCl(2) was intravenously injected, liver (65)Zn levels in the tumor-bearing mice were higher than those in control mice for 72 h after the injection. Pancreatic and blood (65)Zn levels in tumor-bearing mice were lower than those in controls for 24 h (pancreas) and 6 h (blood) after the injection. These findings indicate that the hepatic zinc response via MT induction influences zinc metabolism in the body after tumor transplantation. Moreover, (65)Zn uptake in the liver of MT-deficient tumor-bearing mice was lower than that in control tumor-bearing mice 1 h after injection. (65)Zn uptake in the tumor and blood (65)Zn levels in the MT-deficient tumor-bearing mice were higher than those in the control tumor-bearing mice. Tumor weight increased more in MT-deficient mice than in control mice. The formation of zinc-MT in the liver of tumor-bearing mice might decrease blood zinc availability for tumors and other tissues, such as the pancreas.

Topics: Animals; Carcinoma, Ehrlich Tumor; Chlorides; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; Mice; Mice, Inbred Strains; Mice, Knockout; Pancreas; Rats; Rats, Inbred Strains; Zinc; Zinc Compounds; Zinc Radioisotopes

Interleukin-6 (IL-6) has been thought to play a key role in the induction of hepatic metallothionein (MT) synthesis in tumor-bearing animals. In order to clarify the role of IL-6 and to distinguish its effect from those of other cytokines, we inoculated IL-6-null and B6J129Sv (wild-type control) mice SC with 1 x 10(7) Ehrlich carcinoma cells and examined tumor growth, hepatic MT, and serum cytokines. We have found that tumor growth was followed by an increase of hepatic MT in both IL-6-null and wild-type mice and that the two strains of mice had a similar hepatic MT induction followed by zinc accumulation in the liver. These results could be explained by our finding that tumor-bearing IL-6-null mice had a high level of tumor-secreted IL-6 in the blood. In conclusion, by utilizing tumor-bearing IL-6-null mice, we have demonstrated that IL-6 secreted from the tumor cells is responsible for the tumor-evoked increase of hepatic MT level.

Topics: Animals; Carcinoma, Ehrlich Tumor; Copper; Cytokines; Interleukin-6; Liver; Metallothionein; Mice; Mice, Knockout; Neoplasm Transplantation; Neoplasms, Experimental; Time Factors; Zinc

Based on the previous finding that hepatic metallothionein (MT) level was tumor-growth dependently elevated in tumor-transplanted mice, the mode of induction of hepatic MT in tumor-bearing mice was comparatively studied with inflammation-induced and stress-subjected mice. The prefeeding with zinc (Zn)-deficient diet for 1 wk suppressed both the growth of tumor and the increase of hepatic MT and Zn in tumor-bearing mice. The postfeeding with Zn-deficient diet also suppressed hepatic MT induction in the course of tumor growth. On the other hand, in the other two experimental model mice, the prefeeding with Zn-deficient diet did not suppress the increase of hepatic MT and Zn. Further, the dexamethasone treatment stimulated hepatic MT induction in tumor-bearing mice, but rather reduced that in inflammation-induced mice. These results suggest that hepatic MT was induced uniquely in tumor-bearing mice and that Zn may play a key role for the induction of hepatic MT by tumor transplantation.

Topics: Animals; Carcinoma, Ehrlich Tumor; Dexamethasone; Diet; Liver; Male; Metallothionein; Mice; Neoplasm Transplantation; Restraint, Physical; Sarcoma 180; Stress, Physiological; Turpentine; Zinc

In order to determine if the radioimaging of the level of hepatic metallothionein (MT) is able to detect malignancy, we performed, using 45Zn, a trial to measure MT levels in the liver of experimental tumor-bearing mice and rats. The distribution of 65Zn in the livers of the mice and rats increased with s.c. transplantation of Ehrlich carcinoma and ascites hepatoma 7974F, respectively. A Sephadex G-75 elution profile of the cytosol fraction of the liver showed that the elevation of 65Zn level was due to elevation of the MT level. At 2 days after tumor transplantation in mice, the 65Zn-image demonstrated elevation of the hepatic MT level, while 67Ga-citrate, which is currently utilized, could not image the tumor at such an early period of growth. These results suggest that hepatic MT is a useful marker for the detection of malignancy at the early stage.

Topics: Animals; Carcinoma, Ehrlich Tumor; Gamma Cameras; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; Mice; Radionuclide Imaging; Rats; Zinc Radioisotopes

A tumor growth-dependent elevation in the hepatic levels of Zn and metallothionein (MT), without a change in the level of Cu, was found in mice and rats bearing solid tumors in the inguinal region. The levels of Zn and MT thus elevated gave a significant correlation (r = 0.95) between them. Nevertheless, when tumor-bearing mice and rats were fed a Zn-deficient diet, the hepatic levels of Zn and MT did not increase. In mice in which inflammation was induced at the same region, on the other hand, hepatic levels of Zn and MT increased transiently after the injection of turpentine or carrageenan even when they were fed the Zn-deficient diet. These results suggest that the elevation of MT and Zn levels can be a helpful marker for detecting malignancy.

Topics: Animals; Carcinoma, Ehrlich Tumor; Copper; Inflammation; Kinetics; Leukemia L1210; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; Mice; Mice, Inbred Strains; Organ Size; Rats; Rats, Inbred Strains; Zinc

Topics: Animals; Apoproteins; Binding Sites; Binding, Competitive; Cadmium; Carcinoma, Ehrlich Tumor; Cell Death; Cell Division; Chromatography, Gel; Copper; DNA Replication; Kinetics; Metallothionein; Metals; Mice; Tumor Cells, Cultured; Zinc

Host zinc deficiency halts the proliferation of the mouse Ehrlich ascites tumor. The major site of measurable cellular zinc depletion is a cytosolic zinc binding protein. This protein is characterized as a metallothionein on the basis of its presence as two isoproteins which behave on DEAE-Sephadex and in polyacrylamide gel electrophoresis like metallothioneins, the lack of protein absorbance at 280 nm, its sulfhydryl/zinc ratio of 3.5, and its reactivity in zinc transfer to apocarbonic anhydrase. Finally, the protein exhibits cross-reactivity with a known rat metallothionein in a radioimmunoassay. Coupled with the similarity in structure and antigenicity of rat and mouse metallothioneins, this adds strong support to the identification of the zinc-binding protein as a metallothionein. In zinc-deficient cells this metallothionein-like protein appears to exist as an apoprotein. When small amounts of dietary zinc stimulate the deficient cells to divide, zinc is not observed in metallothionein. Larger concentrations of dietary zinc support both proliferation and the steady state presence of zinc in this protein. It is demonstrated that metallothionein is the principal donor of zinc to apocarbonic anhydrase added to Ehrlich cytosol. These results are used to construct a model of zinc metabolism in which zinc metallothionein is a labile depot of zinc in the Ehrlich cell which can be mobilized under zinc-deficient conditions.

Topics: Animals; Apoenzymes; Carbonic Anhydrases; Carcinoma, Ehrlich Tumor; Carrier Proteins; Female; Metallothionein; Mice; Mice, Inbred ICR; Zinc

The effect of preinduction of metallothionein (MT) by bismuth (Bi) compounds on the toxic side effects and antitumor activity of adriamycin (ADR) was investigated in mice. Preinduction of MT by oral administration of bismuth subnitrate (BSN) significantly decreased the lethal toxicity, cardiotoxicity and bone marrow toxicity observed with a single subcutaneous injection of ADR. A significant increase in the concentration of cardiac MT was observed in mice treated with BSN. The MT level in the heart was significantly correlated with the protective effect of BSN against the cardiotoxicity of ADR. In tumor-bearing mice, pretreatment with BSN did not affect the antitumor activity of ADR, although its cardiotoxicity was significantly depressed. The ability of BSN to reduce specifically the toxicity of ADR may be ascribed to the fact that Bi induces MT in the target tissue of ADR toxicity but not in a tumor. The protective effect of MT against the toxicity of ADR, which is believed to act as an anticancer agent by generating active oxygen, can be assumed to be due to its ability to scavenge free radicals or inhibit their formation.

Topics: Animals; Bismuth; Bone Marrow; Carcinoma, Ehrlich Tumor; Diarrhea; Doxorubicin; Drug Administration Schedule; Heart; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Inbred ICR

Ehrlich ascites-tumour cells normally contain a large concentration of Zn-metallothionein. When cells are placed in culture media, containing or pretreated with the metal-ion-chelating resin Chelex-100, they stop growing, remain viable and lose zinc specifically from the metallothionein (MT) pool. The kinetics of loss of zinc are first-order and are very rapid, having a rate constant of greater than or equal to 0.6 h-1. MT protein labelled with 35S is biodegraded with a rate constant of 0.07-0.014 h-1 in control cells, 0.08 h-1 in cells exposed to the zinc-deficient medium and 0.12-0.18 h-1 in cells treated directly with Chelex. Over the 6 h period in which zinc is totally lost from Zn-MT there is relatively little decrease in MT-like protein as measured by cadmium-binding to the 10,000-Mr protein fraction. Other pools of zinc and 35S-labelled protein turn over more slowly. There is no loss of zinc from rat liver Zn-MT that is dialysed against Chelex to model the possible reaction of the resin with Ehrlich-cell Zn-MT. However, Chelex does compete slowly for MT-bound zinc when resin and MT are directly mixed. Analysis of the known and possible pathways of zinc metabolism in cells in relationship to these rate constants shows that biodegradation of MT protein cannot account for the rate of loss of zinc from Zn-MT.

Topics: Animals; Cadmium; Carcinoma, Ehrlich Tumor; Chelating Agents; Chromatography, Gel; Cysteine; Kinetics; Metallothionein; Models, Biological; Resins, Synthetic; Sulfur Radioisotopes; Trichloroacetic Acid; Tumor Cells, Cultured; Zinc

The protective effects of metallothionein (MT) preinduction by bismuth subnitrate (BSN) against the renal toxicity of cis-diamminedichloroplatinum (cis-DDP), the cardiotoxicity of adriamycin (ADR), and the lethal and bone marrow toxicities of these drugs were observed in mice receiving cis-DDP and ADR simultaneously. Preinduction of MT biosynthesis by BSN, which is currently used as an antidiarrheal drug, did not affect the antitumor activities of the two drugs, suggesting that the beneficial effects of the preinduction of MT biosynthesis by BSN may be applicable for therapy involving cis-DDP and ADR, either alone or in combination.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Bismuth; Carcinoma, Ehrlich Tumor; Cisplatin; Colonic Neoplasms; Doxorubicin; Heart; Kidney; Male; Metallothionein; Mice; Mice, Inbred ICR

This summary examines some of the known and hypothesized roles of metallothionein and related proteins in mediating the metal metabolism and toxicity from a chemical perspective. It attempts to examine in kinetic terms how such molecules may exert homeostatic control over the intracellular bioavailability of metal ions to essential enzymatic or other molecular systems. The concept of ongoing competition between metallothionein and related proteins with other intracellular metal-binding sites for various metals is also examined in relation to the thermodynamic stability of these proteins. Comparisons between mammalian metallothionein and analogous nonmammalian proteins demonstrate both similarities and great differences in types of metal-binding sites, metal-binding constants, amino acid composition, and secondary structures such that apparent diversity of these low molecular weight metal-binding molecules in nature appears to be growing ever wider. The potential value of these data rests both in delineating new hypotheses for metallothionein evolution and in suggesting new model systems for discovering the normal function of metallothionein and related proteins in cells.

Topics: Animals; Cadmium; Carcinoma, Ehrlich Tumor; Cells, Cultured; Copper; Dose-Response Relationship, Drug; Metallothionein; Metals; Tissue Distribution; Zinc

The antitumor agent, cis-dichlorodiammine Pt(II), is cytotoxic to Ehrlich cells in culture. These cells contain a substantial amount of metallothionein in the absence of inducers of the protein. At concentrations of drug which cause 60% inhibition of cell proliferation, most of the platinum is found in the cytosol. Of this about 30% is bound in the metallothionein fraction. Isolated rat liver metallothionein reacts slowly with hydrolyzed cis-dichlorodiammine Pt(II). Thus, metallothionein is a major cellular site of binding of the platinum complex at concentrations which inhibit tumor growth.

Topics: Animals; Cadmium; Carcinoma, Ehrlich Tumor; Cell Compartmentation; Cell Division; Cells, Cultured; Cisplatin; Dose-Response Relationship, Drug; Metallothionein; Organometallic Compounds; Rats; Subcellular Fractions; Thiosemicarbazones; Zinc

The copper complex of 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazone) or CuKTS is reduced and dissociated upon reaction with Ehrlich cells. Titration of the cells with the complex leads to the specific binding of copper to metallothionein with 1 to 1 displacement of its complement of zinc. Under conditions of complete titration of metallothionein, 1.25-2.5 nmol CuKTS/10(7) cells, cellular DNA synthesis is rapidly inhibited but no long term effects on cell proliferation are observed. The kinetics of redistribution of Cu and Zn in Ehrlich cells in culture and in animals were studied after pulse reaction of CuKTS with cells. After exposure of cells to the noncytotoxic concentration of 2.5 nmol of CuKTS/10(7) cells, nonmetallothionein bound copper is lost rapidly from the cells, after which copper in metallothionein decays. New zinc metallothionein is made as soon as exposed cells are placed in culture. New synthesis stops when the level of zinc in metallothionein reaches control levels. A second pulse treatment of cells with CuKTS to displace zinc from metallothionein again stimulates new synthesis of the protein to restore its normal concentration. The kinetics of metal metabolism in Ehrlich cells exposed to 5.5 nmol of CuKTS/10(7) cells, which inhibits cell proliferation, are qualitatively similar except there is a pronounced lag before new zinc metallothionein is synthesized. The Ehrlich ascites tumor in mice responds to CuKTS similarly to cells in culture. It is also shown that cultured Ehrlich cells do not make extra zinc metallothionein in the presence of high levels of ZnCl2, and fail to accumulate copper in the presence of large concentrations of CuCl2.

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Cell Division; Cells, Cultured; Chlorides; Copper; Female; Kinetics; Metallothionein; Mice; Mice, Inbred ICR; Organometallic Compounds; Thiosemicarbazones; Time Factors; Zinc; Zinc Compounds