Biomedical subjects

A Naganuma

Publications and source records attributed to A Naganuma.

At least 37 records · Page 2Linked to original sources

PubMed1999

Protective effects of triterpene compounds against the cytotoxicity of cadmium in HepG2 cells.

The effects of triterpene compounds on cadmium toxicity were investigated in HepG2 cells. Ten triterpene compounds were examined, namely, betulin, soyasapogenol A, soyasapogenol B, ursolic acid, uvaol, oleanolic acid, friedelin, glycyrrhizin, 18alpha-glycyrrhetinic acid, and 18beta-glycyrrhetinic acid, and betulin, soyasapogenol A, and uvaol were found to reduce the toxicity of CdCl(2). In particular, betulin almost completely abolished the cytotoxicity of CdCl(2) at concentrations as low as 0. 1 microg/ml. The effects of betulin were particularly apparent when added to the culture medium before the addition of CdCl(2). Moreover, when HepG2 cells were incubated with betulin and then incubated in fresh betulin-free medium before the addition of CdCl(2), the toxic effects of cadmium were reduced. Betulin had no significant effect on the intracellular accumulation of cadmium, nor did it bind to cadmium, at least not in a test tube. When HepG2 cells were treated first with cycloheximide or actinomycin D, the subsequent protective effect of betulin against cadmium toxicity was significantly reduced, suggesting that betulin might protect cells against cadmium toxicity by inducing the synthesis of a certain protein or proteins. The synthesis of metallothionein, a protein that is known to reduce the toxicity of heavy metals, was not induced by betulin. However, using the differential display method, we confirmed that betulin promoted the expression of several genes. Our findings suggest that betulin might reduce cadmium toxicity by promoting the synthesis of certain proteins that protect cells against the toxic effects of cadmium.

Cadmium↗

PubMed1998

Determination of metallothionein by high-performance liquid chromatography with fluorescence detection using an isocratic solvent system.

Metallothionein (MT) is a low-molecular-weight protein which plays a role in detoxification of heavy metals and protection against oxidative stress. A sensitive and convenient determination method for MT is necessary to clarify its physiological roles. High-performance liquid chromatography (HPLC) with an isocratic solvent system for MT was, therefore, developed utilizing an unique fluorescence labeling reagent, ammonium 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate (SBD-F). The HPLC system with two separation columns, Shodex RSpak RP18-413 column (a styrene divinylbenzene polymer gel-packed column) and Puresil C18 column (an ODS gel-packed column), connected in tandem successfully separated SBD-labeled MT from biological interference. The SBD-labeled MT was stable and could be stored for at least 1 week without any changes in fluorescence intensity. Although Hg-MT was not detectable, this method is applicable to determination of major MTs such as Zn-MT, Cd-MT, and Cu-MT using commercially available rabbit MT as a standard. Determination of the MT concentration in cells was possible in aliquots of only 1 x 10(4) cultured cells. The present method using a tandem column HPLC system with isocratic elution might be useful for monitoring the concentration of MT in cultured cells as well as in animal tissues.

Animals↗

PubMed1998

Metallothionein-mediated resistance to multiple drugs can be induced by several anticancer drugs in mice.

We examined the role of metallothionein in the chemosensitivity of transplanted tumors in mice. The antitumor activities of cisplatin, adriamycin, bleomycin, peplomycin, cyclophosphamide, and melphalan were significantly suppressed when the concentration of metallothionein in the tumor was increased to only twice the control level. On the other hand, the antitumor activities of mitomycin C, 5-fluorouracil, and vinblastine were hardly affected by increases in the concentration of metallothionein in the tumors in mice. Moreover, all the antitumor drugs examined increased the concentration of metallothionein in transplanted tumors to a level that was high enough to suppress the antitumor activity of these drugs. These observations suggest that treatment of patients with certain antitumor drugs might result in the resistance of their tumors to multiple drugs.

Animals↗

PubMed1998

Determination of gamma-glutamylglutathione and other low-molecular-mass biological thiol compounds by isocratic high-performance liquid chromatography with fluorimetric detection.

A method was developed for the simultaneous determination of gamma-glutamylglutathione (gamma-GluGSH) and other low-molecular-mass thiol compounds (cysteine, cysteamine, homocysteine, cysteinylglycine, gamma-glutamylcysteine, glutathione and N-acetylcysteine) using high-performance liquid chromatography combined with precolumn fluorescence labeling with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). These SBD-labeled thiol compounds were separated within 35 min on a Cosmosil 5C-18AR column with isocratic elution using 75 mM sodium citrate buffer (pH 2.90)-methanol (98:2) and detected fluorimetrically (ex. 386 nm, em. 516 nm). The calibration graphs using 2-mercaptoethanol as an internal standard showed good linearity in the range from 20 pmol to 10 nmol for all thiol compounds examined. The application of this method for the quantitative determination of thiol compounds in the urine from gamma-glutamyl transpeptidase-deficient mice was also demonstrated. This method is sufficiently simple, rapid and sensitive for the determination of gamma-GluGSH and other low-molecular-mass thiol compounds in biological samples.

Animals↗

PubMed1998

Overexpression of manganese-superoxide dismutase prevents methylmercury toxicity in HeLa cells.

HeLa cells were stably transformed with plasmid constructs that allowed constitutive expression of antioxidant enzymes such as catalase, glutathione peroxidase (GSH-Px), Cu,Zn-superoxide dismutase (Cu,Zn-SOD) or Mn-superoxide dismutase (Mn-SOD) to examine the involvement of reactive oxygen generation in methylmercury toxicity. Overexpression of catalase, GSH-Px or Cu,Zn-SOD did not affect the sensitivity of HeLa cells against methylmercury. However, the sensitivity of HeLa cells against methylmercury was decreased by overexpression of Mn-SOD, an enzyme localized in matrix of mitochondria and which decomposes superoxide anions. These results suggest that formation of superoxide anions in the mitochondria might be involved in the mechanism of the cytotoxicity of methylmercury.

Catalase↗

PubMed1998

Involvement of oxidative stress in paraquat-induced metallothionein synthesis under glutathione depletion.

The inhibition of glutathione (GSH) synthesis by L-buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.

Animals↗

PubMed1998

Epidermal proliferation of the skin in metallothionein-null mice.

Metallothionein (MT) is a low-molecular weight metal-binding protein. Although the physiologic function of MT is not fully known, it is present in various species and various organs including the skin. MT is strongly stained in hyperplastic epidermal tissues in normal skin and in hyperplastic skin lesions, and increased expression of mRNA of the MT gene has been demonstrated in skin stimulated by proliferative agents, suggesting that MT is involved in the proliferation of epidermal keratinocytes. To improve our understanding of the role of MT in epidermal hyperplasia, mice with null mutations in their MT-1 and MT-2 genes were used in this study. We compared the epidermal hyperplasia in MT-null mice and in normal C57BL/6 J mice after treatments with cholera toxin, 12-0-tetradecanoylphorbol-13-acetate, and ultraviolet B irradiation, which stimulate epidermal proliferation. Immunostaining of MT was not detected in the skin of MT-null mice, and these mice developed significantly less epidermal hyperplasia than the normal mice after exposure to each stimulator. We determined the metal contents of skin samples by the proton-induced x-ray emission method. The zinc content of the skin of the MT-null mice was lower than that of the control mice before stimulation. After stimulation of epidermal hyperplasia, MT-null and normal mice showed significantly reduced levels of zinc. These findings indicate that cellular MT is involved in the proliferative process of the epidermis induced by cholera toxin, 12-0-tetradecanoylphorbol-13-acetate, and ultraviolet B light through its regulatory action on the metal metabolism required for cell growth.

Animals↗

PubMed1998

Novel function of metallothionein in photoprotection: metallothionein-null mouse exhibits reduced tolerance against ultraviolet B injury in the skin.

We have shown previously that injection of cadmium chloride (Cd2+) depletes the number of ultraviolet B (UVB)-induced sunburn cells in the mouse skin in vivo, and that Cd2+ treatment enhances UVB resistance in cultured keratinocytes in vitro, indicating the photoprotective role of Cd2+-induced metallothioneins (MT) with antioxidant property against UVB injury; however, there has been no direct evidence for the role of MT in UV protection. To improve our understanding of MT in photoprotection, MT-null mouse deficient in its MT-1 and MT-2 genes was studied. Skin explants were preliminarily exposed to medium alone, Cd2+ and Cd2+ plus buthionine S,R-sulfoximine, an inhibitor of glutathione synthesis. We then compared the number of UVB-induced sunburn cells and apoptotic cells in the epidermis of MT-null mice with that of control mice using organ culture systems. The skin of MT-null mice developed a greater number of sunburn cells and apoptotic cells than did that of normal mice in all experimental conditions. These findings indicate that the skin of MT-null mouse is readily injured by UVB irradiation. MT-null mouse provided direct evidence of the photoprotective effect of cellular MT in the skin.

Animals↗

PubMed1998

Strain difference in sensitivity of mice to renal toxicity of inorganic mercury.

Inorganic mercury has a high affinity for the kidneys and causes acute renal failure. The present investigation was designed to determine the cause of the strain difference in sensitivity of mice to the renal toxicity of inorganic mercury. Renal damage caused by HgCl2 was estimated by histopathological and biochemical assessment, such as increase in blood urea nitrogen and plasma creatinine levels, and was found to be more remarkable in C3H/He than in C57BL/6 mice. Increase in renal lipid peroxidation in C3H/He was greater than that in C57BL/6 mice. However, no strain difference was observed in renal activities of glutathione (GSH) peroxidase, superoxide dismutase and GSH S-transferase in HgCl2-untreated mice. The GSH content and activities of catalase and GSSG reductase in kidney of HgCl2-untreated mice were higher in C3H/He than in C57BL/6. Background level of renal metallothionein content and the extent of metallothionein induction by HgCl2 showed no strain difference. On the other hand, renal mercury accumulation was higher and urinary mercury excretion was lower in C3H/He than in C57BL/6. The activity of renal gamma-glutamyltranspeptidase (gamma-GTP), which plays a key role in renal mercury accumulation, was higher in C3H/He than in C57BL/6. Furthermore, the increase in blood urea nitrogen by HgCl2, renal mercury accumulation and renal gamma-GTP activity in B6C3F1 mice were intermediate between those of the parent strains. These results suggest that the strain difference in renal toxicity of inorganic mercury seems to be caused by the discrepancy in renal mercury accumulation, and therefore, renal gamma-GTP may be an important factor determining the susceptibility of mice to the toxic action of inorganic mercury.

Animals↗

PubMed1998

Protective effect of bismuth nitrate against injury to the bone marrow by gamma-irradiation in mice: possible involvement of induction of metallothionein synthesis.

The effects of bismuth nitrate (BN) on the lethal effect of and injury to bone marrow by gamma-irradiation were examined. Mice were given daily s.c. injections of BN for 2 days and were exposed to whole-body irradiation (137Cs; 8 grays) 24 hr after the second injection of BN. All mice exposed to gamma-irradiation without treatment with BN died within 30 days, but the lethal effect of gamma-irradiation was markedly reduced in mice given BN before irradiation. Irradiation (3 grays) significantly reduced the total number of leukocytes 1 day after irradiation but the number of leukocytes subsequently increased in both nontreated and BN-treated irradiated mice. However, the rate of recovery of the total number of leukocytes, as monitored from 5 days after irradiation, was significantly higher in BN-treated mice than in the nontreated mice. Reductions in the viability of hematopoietic stem cells (determined by monitoring the number of colony-forming units in the spleen) that were induced by gamma-irradiation (3 grays) were considerably diminished by the treatment of mice with BN before irradiation. BN significantly increased the concentration of metallothionein in the bone marrow cells of mice, but levels of other cellular antioxidants, such as catalase, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase and glutathione, were unchanged. These results suggest that BN protects bone marrow cells against the toxic effects of gamma-irradiation by inducing the synthesis of metallothionein in the bone marrow. Metallothionein might play an important role in determining the sensitivity of animals to gamma-irradiation.

Animals↗

PubMed1997

Inhibitory effect of selenium on biliary secretion of methyl mercury in rats.

The inhibitory effect of sodium selenite on biliary secretion of methyl mercury was examined in rats. The biliary secretion of methyl mercury in rat treated with 1 mumol/kg of methyl mercury was significantly decreased by administration of selenite at doses of 0.05 mumol/kg or higher. In rats given 10 mumol/kg of methyl mercury, marked depression of biliary secretion of mercury was observed when selenite was injected at a dose of 0.2 mumol/kg. On the other hand, secretion of substantial amounts of selenium was observed when biliary secretion of mercury was depressed. When the concentration of selenium in the bile was higher than 5 nmol/ml, biliary secretion of mercury was markedly depressed independently of the dose of methyl mercury administered (1 mumol/kg or 10 mumol/kg). These results suggest that the degree of inhibitory effect of selenite may be determined by the selenium concentration in the liver or the bile after treatment with selenite rather than the molar ratio of the dose of methyl mercury and selenite. We concluded that the decrease in biliary secretion of methyl mercury induced by selenite may result from inhibition of pathway for secretion of methyl mercury from liver to bile rather than the direct formation of a complex between methyl mercury and selenium. Methyl mercury has been considered to be secreted from liver to bile as a complex with glutathione (GSH). However, administration of selenite did not affect biliary secretion of GSH or hepatic glutathione S-transferase activity. Moreover, gel filtration of liver cytosol demonstrated that the distribution pattern of hepatic methyl mercury between macromolecules and GSH was not significantly changed by administration of selenite. These results suggest that selenite does not affect complex formation of methyl mercury with GSH at least in the liver. Selenite might specifically inhibit the activity of the canalicular transporter(s) which transport complexes of methyl mercury and GSH from the liver to bile.

Animals↗

PubMed1997

[Metallothionein].

Metallothionein is a low-molecular weight protein involved in resistance to toxicity of heavy metals. Recent experimental evidences indicate that metallothionein appears to be a factor in determining the responsiveness of tumor cells to anticancer drugs, such as cisplatin and adriamycin. The resistance of tumor cells having high concentration of metallothionein to cisplatin has been observed in cultured cells and transplanted tumors. On the other hand, increase in metallothionein concentration in normal tissues of tumor-bearing animals prevents side effects of anticancer drugs. Metallothionein may be an important endogenous factor in cancer chemotherapy.

Animals↗

PubMed1997

Enhanced renal toxicity by inorganic mercury in metallothionein-null mice.

To elucidate a protective role of metallothionein (MT) in the manifestation of inorganic mercury toxicity, we studied the susceptibility of MT-null mice to the renal toxicity of mercuric chloride. Because the MT-null (J) mice are a genetic background of 129/Sv strain, the 129/Sv mice were used as wild-type controls. Nine-week-old male MT-null (J) and 129/Sv mice were given subcutaneous injections of mercuric chloride at doses of 10 to 40 micromol/kg. The basal MT level in the kidney of MT-null (J) mice was undetectable (<0.2 microg/g of tissue) and approximately 2.5 microg/g of tissue in 129/Sv mice. The sensitivity to the renal toxicity of mercuric chloride was markedly enhanced in the MT-null (J) mice compared with the 129/Sv mice. The renal mercury level was similar for the MT-null (J) and 129/Sv mice at 4 hr after the injection of mercuric chloride (20 micromol/kg) but became significantly lower in MT-null (J) mice than in 129/Sv mice at 24 and 72 hr. Based on the present results, we conclude that MT is an important protective factor against the renal toxicity caused by inorganic mercury and that it may play a major role in the retention of mercury in the kidney.

Animals↗

PubMed1995

Effect of preinduction of metallothionein synthesis on clastogenicity of anticancer drugs in mice.

The effect of pretreatment with metallothionein (MT) inducers (bismuth nitrate or zinc chloride) on clastogenicity of anticancer drugs was investigated. Bismuth nitrate (50 mumol/kg) or zinc chloride (400 mumol/kg) was administered s.c. to mice once a day for two days prior to treatment with 3.3 mumol/kg of cis-diamminedichloroplatinum(II) (cis-DDP), 3.4 mumol/kg of adriamycin (ADR), 72 mumol/kg of cyclophosphamide (CPA) or 0.41 mumol/kg of L-phenylalanine mustard (L-PAM). The frequency of occurrence of erythrocytes with micronuclei in bone marrow was increased by each anticancer drug at 24 h after treatment. Micronucleus formation was significantly prevented by pretreatment with either bismuth nitrate or zinc chloride. MT concentration in bone marrow cells of mice at the time of treatment with anticancer drugs increased to 2- and 3.5-fold by pretreatment with bismuth nitrate and zinc chloride, respectively. These results indicate that MT induction in bone marrow cells effectively prevents micronucleus induction of anticancer drugs.

Animals↗

PubMed1995

Effect of glutathione depletion on metallothionein synthesis induced by paraquat in mice.

The effect of Glutathione (GSH) depletion on the induction of metallothionein (MT) synthesis by paraquat (PQ) was examined in ICR mice. An increase in hepatic MT level in mice was observed after a single PQ administration. Pretreatment of mice with L-buthionine-SR-sulfoximine (BSO), an inhibitor of GSH synthesis, enhanced the induction of hepatic and renal MT synthesis by PQ depending on the decreased tissue GSH level. A similar result was obtained by pretreatment with diethylmaleate, a GSH depleting agent. The ratio of hepatic MT-I to MT-II induced by PQ was not changed by BSO pretreatment. An increase in the hepatic MT level in GSH depleted mice was observed from 3 hr to 24 hr after PQ administration. An increase in the hepatic MT-I mRNA level after treatment with PQ was observed prior to hepatic MT induction in BSO pretreated mice. Pretreatment with actinomycin D, an inhibitor of mRNA synthesis, inhibits the PQ-induced increase in hepatic MT and MT-I mRNA levels in BSO pretreated mice. Pretreatment with BSO did not affect the induction of MT synthesis by zinc, cadmium or dexamethasone. Pretreatment with dexamethasone, an anti-inflammatory agent, enhanced the hepatic MT induction by PQ treatment in GSH depleted mice, while dexamethasone reduced the MT induction by turpentine oil, which is known to induce inflammation and hepatic MT synthesis. These findings suggest that GSH depletion enhances the induction of MT synthesis by PQ because of an increase in the transcription rate, and this enhancement of MT synthesis is not due to an inflammatory response caused by PQ.

Animals↗

PubMed1995

Enhancement of paraquat toxicity by glutathione depletion in mice in vivo and in vitro.

Effect of glutathione (GSH) depletion on paraquat (PQ) toxicity in the liver and kidneys of mice was examined. Glutamic-pyruvate transaminase (GPT) and blood urea nitrogen (BUN) levels in plasma of mice were hardly changed by treatment with 150 micro mol/kg of PQ. However, significant increases in the plasma GPT and BUN levels after PQ injection were observed in mice which were pretreated with L-buthionine-SR-sulfoximine (BSO), an inhibitor of GSH synthesis, at 4 hr prior to PQ administration. This result supports the previous observation that hepatotoxicity of PQ was enhanced in diethyl maleate-pretreated mice (Cagen and Gibson, 1977). In the present study, lipid peroxidation evaluated by thiobarbituric acid-reactive substances (TBA-RS) level in the liver of mice given PQ was elevated by pretreatment with BSO. Moreover, enhancement of PQ cytotoxicity by BSO pretreatment was also observed in cultured mouse hepatoma cell line (NCTC clone 1469). Vitamin E, an antioxidant, and Desferal, an iron chelator, significantly prevented mice from the BSO-enhanced hepato- and nephrotoxicity of PQ. These findings suggest that the tissues or cells of low GSH concentration are highly vulnerable to PQ toxicity and GSH may play a major role in diminishing the toxic action of PQ exerted through oxidative stress.

Animals↗

PubMed1994

Establishment and characterization of methylmercury-resistant PC12 cell line.

Methylmercury (MeHg)-resistant sublines of rat pheochromocytoma (PC12) cells were isolated by repeated exposure to stepwise increased concentrations of MeHg. One of the sublines (PC12/TM) showed an 8- to 10-fold increase in resistance to MeHg compared with parent PC12 cells on the basis of the concentration required for 50% inhibition (IC50) of growth. PC12/TM cells accumulated smaller amounts of MeHg than parent PC12 cells. This reduction in MeHg accumulation in PC12/TM cells resulted from slow uptake and rapid efflux. The intracellular glutathione (GSH) level in PC12/TM cells was four times higher than that of PC12 cells. Pretreatment of PC12/TM cells with buthionine sulfoximine, which decreased the GSH level to that of the parent PC12 cells, increased the sensitivity of PC12/TM cells to MeHg. A close correlation between the MeHg accumulation and MeHg sensitivity was found among seven sublines of PC12 cells and parent PC12 cell line. The GSH level in PC12 sublines was also correlated with their sensitivity to MeHg.

Animals↗

PubMed1994

[Role of metallothionein in cancer chemotherapy].

Metallothionein (MT) shows a protective effect against toxic actions of some antitumor drugs and gamma-irradiation in animals. Preadministration of bismuth subnitrate, an MT inducing drug, significantly reduced side effects of antitumor drugs without affecting antitumor activity of the drugs. This specific effect of bismuth on the toxicity of antitumor drugs appears to be attributable to its specific induction of MT in normal tissues, but not in tumor tissue. Preinduction of MT synthesis in the lung also prevented mice from carcinogenesis caused by cisplatin and melphalan in the lung. On the other hand, zinc compound induced MT synthesis in the tumor, and significantly suppressed the antitumor activity of some antitumor drugs. Propargylglycine (PPG), an inhibitor of cystathionine pathway, significantly inhibited MT induction by zinc in the tumor inoculated in mice, and, consequently, PPG could diminish cisplatin resistance acquired by an increase in the tumor MT levels.

Alkynes↗