Many metals and metal-containing compounds have been identified to induce various immunological disorders. In the skin, it is well known that metals contained in the dental amalgam and mercury in the clinical thermometer induce dysidrotic eczema and systemic contact dermatitis, respectively, while gold compound used in rheumatic arthritis triggers lichenoid drug eruption. The reason why various metals induce these allergic or immune responses in human is mainly attributed to the potential adjuvant activity of these metals. In general, dendritic cells including epidermal Langerhans cells are established to play a principal role in inducing T cell activation. Therefore, it is supposed that the allergic response induced by various metals is caused by their activation of dendritic cells. In this paper, I will present the effects of metals on dendritic cells.

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Zinc administration prevents bone loss in rats with ovariectomy, skeletal unloading, hydrocortisone treatment, adjuvant arthritics and diabetic condition in rats. Zinc has been shown to play a role in the preservation of bone mass by stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption in rat bone tissues. The cellular mechanism of zinc action in inhibiting osteoclastic bone resorption is reviewed. Zinc inhibits bone resorption which is stimulated by various bone-resorbing factors including parathyroid hormone (PTH), 1, 25-dihydroxyvitamin D₃ [1, 25(OH)₂D₃], and prostaglandin E₂ (PGE₂) in rat bone tissue culture in vitro. Also, zinc has an inhibitory effect on the bone-resorbing factors-induced osteoclast-like cell formation in mouse marrow culture in vitro. The stimulatory effect of bone-resorbing factors on osteoclastogenesis is mediated through receptor activator of NF-κB ligand (RANKL), which plays a key role in development of osteoclasts from preosteoclasts. Zinc inhibits RANKL stimulation in osteoclastogenesis. RANKL is expressed in osteoblasts. In addition, zinc may inhibit RANK expression which is mediated through Ca²⁺ signaling (protein kinase C) by stimulation of PTH, 1, 25(OH)₂D₃ or PGE₂ in osteoblasts. Zinc plays an inhibitory role in the regulation of osteoclastogenesis.

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A metal chelator diethylenetriaminepentaacetic acid (DTPA) has been reported to inhibits replication of human cytomegalovirus (hCMV). In the investigation of the mechanism of DTPA-induced antiviral effect, we found that zinc-regulated expression of metallothionein (MT) enhances promoter activity of hCMV major immediate early gene through the activation of a transcriptional factor NF-κB. Number of endogenous NF-κB-regulated genes have been identified. Modulation of the expression of endogenous NF-κB-regulated gene by zinc and/or MT was investigated in vitro and in vivo. Zinc induces macrophage colony stimulating factor (M-CSF) in L929 fibroblast, MC3T3-E1 osteoblast, and lung fibroblast from MT+/+ wild-type mouse, but not in MT−/− lung fibroblast. Overexpression of MT in L929 cells increases NF-κB-DNA binding and expression of M-CSF. NF-κB is a major signaling protein in lipopolysaccharide (LPS)-induced activation of macrophage. LPS-induced expression of inflammatory cytokines, TNF, IL-1, and IL-6, was lower in MT−/− macrophage than MT+/+ wild-type. Zinc and MT act as an important modulator of cytokines such as M-CSF, TNF, IL-1, and IL-6. The functions of zinc and MT in inflammation and immunoresponse should be further investigated in detail.

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Embryonic stem (ES) cell has attracted the attention of many scientists for application to regenerative medicine. ES cell is a cell line derived from an early embryo which is called blastocyst and is capable of differenciating into all kinds of cell. On the other hand, metallic elements in proteins play important roles as active centers in biological reaction. In this study, we elucidated the differential mechanism of embryonic stem cells by means of elemental analyses, x-ray fluorescence (XRF) and x-ray near edge structure (XANES) analysis. From the results, we found that concentrations of Fe and Zn in the dopaminergic neurons were higher than those in the undifferentiated ES cells. And the results of XANES analysis made it clear that (the number of Fe²⁺)/(the number of Fe atoms) ratio in the dorpaminergic neurons was higher than that in the undifferentiated ES cells. These consequences indicate that tyrosinases (tyrosine hydroxylases) which have Fe²⁺ as active centers during the reaction of dopamine synthesis are actively correlated with the differentiation of ES cells into dopaminergic neurons.

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Wilson’s disease (WD), an autosomal recessive disorder of copper transport, usually presents with symptoms of the liver or central nervous system. The known disease-caused mutations identified in the responsible gene, ATP7B, exceed 200 variations. The H1069Q mutation is the most common mutation in European and North American patients with WD, while the R778L mutation is found in most Asian patients. In the present study we identified 12 homozygotes and 22 heterozygotes for R778L. We summarize our results here along with those of previous studies. There were a total of 46 homozygotes and 66 heterozygotes for R778L. The phenotypes of R778L homozygotes and heterozygotes were grouped as hepatic presentation, neurologic presentation, or other presentation according to the most recent standards. No significant differences were found in three clinical features (mean age of onset, number of patients with hepatic presentation, and number of patients with neurologic presentation) between R778L homozygote and heterozygote groups, suggesting that the phenotype of WD is influenced by a series of factors, rather than only by the ATP7B gene.

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Speciation of arsenic in the extracts from salmon egg cell cytoplasm and cell membrane was carried out by an HPLC/ICP-MS hyphenated system. The extraction of arsenic species from salmon egg cells was performed by using methanol/water (1:1, v/v) with the aid of sonication. The extracts were evaporated to dryness, and the residues were dissolved with the HPLC mobile phase solution. In the ⁷⁵As-detected chromatograms, arsenobetaine (AB), trimethylarsine oxide (TMAO), dimethylarsinic acid (DMA), and arsenate were found for egg cell cytoplasm, while arsenate, AB, DMA, and monomethyl-arsonic acid (MMA) were for egg cell membrane. The concentration of each arsenic species was determined by using the arsenic standard compounds. As a result, it was found that the total concentrations of arsenic species in the extracts from cell cytoplasm and cell membrane were 22.7 ng g^-1 and 44.2 ng g^-1, respectively, which corresponded to ca. 12% and 35% of the total amounts of arsenic in cell cytoplasm and cell membrane, respectively. The percentage of methylated arsenic species (AB, DMA, and TMAO) in the extract from cell cytoplasm was 93% of the total extracted arsenic species, and that of inorganic arsenic (arsenate) was only 7%, in which AB was relatively the most abundant (ca. 80%). On the contrary, the abundance of arsenate in the extract from egg cell membrane was 35%, which was more abundant than that in egg cell cytoplasm.

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We examined a removal effect and a removal mechanism of residual chlorine by copper fiber made from copper sticks. A copper fiber of 1.0 g could remove residual chlorine of about 1.0 mg/L (100 mL) for 60 min. However, the removal effects by copper board, copper line, copper eluate, copper sulfate solution and copper nitrate solution were low. Furthermore, because the removal effects were depressed by addition of bathocuproine which is a chelating agent of copper (I) ion or DMPO (5, 5-dimethyl-1-pyrroline-N-oxide) which is a spin trapping agent of radical species, it was presumed that an active substance, such as radical species which might be formed from dissolved oxygen in case of changing from copper (I) ion to copper (II) ion, decomposed residual chlorine. Practically, when chemiluminescence by luminol was measured, the chemiluminescence from a test of copper fiber was detected more than from the test of copper sulfate solution. Furthermore, when the removal effect in sodium hypochlorite solution prepared with degassed purified water was tested, the removal effect was depressed. Therefore, it was presumed that radical species were formed from copper and dissolved oxygen in the water. With regard to the mechanism of the removal effect by copper fiber, it was shown that active radical species, which formed from copper and dissolved oxygen in for copper (I) ion changing to copper (II) ion, partially decomposed residual chlorine.

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The concentrations of 48 kinds of major and trace elements in dry milk were determined by microwave-induced plasma mass spectrometry, atomic absorption spectrometry, or colorimetry. The order of the element concentrations in dry milk was K > Ca, P > Na > Mg >> Zn > Rb > Fe > Sr > Cu, Ba, Ni, Mo, Mn, As > Se, Co, Cr. Other elements determined were much lower or under detection limit. Element concentrations in fractions of milk (skim milk, butter milk, acidic casein, whey, whey protein concentrate (WPC), and milk mineral) were also determined. The concentrations of each element in these powdered samples were dependent on the fractions. Concentrations of Ca and Mg in skim milk, butter milk, whey and WPC were similar to those in dry milk, but those in acidic casein fraction were very low and those in milk mineral fraction were extremely high. The concentrations of P in milk mineral fraction were about twice of dry milk, and Na was almost the same, and K was lower than those in dry milk. The present study indicates that the milk mineral fraction is a good source of Ca and Mg.

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In order to understand the behaviors of inorganic elements at the confluence of the river water and water supply process, the Watarase, Tone, and Edo River System was studied in regard to inorganic contamination. This river system starts from the base of the Ashio copper mine and ends at Tokyo Bay. Along the rivers, there are 14 local municipalities in Gunma, Saitama, Ibaragi and Chiba Prefectures, as well as Tokyo. This area is in the center of the Kanto Plain and includes the main source of water pollution from human activities. Moreover, the water of the river system is the source water for human activities in the Kanto area. We analyzed some inorganic elements to clarify water environmental status and outline the water environment problems in the research area. Water samples from 18 river sites and 42 water faucets at public facilities from the Watarase, Tone, and Edo basins which include 14 local municipalities were collected, and the degree of contamination was analyzed. Inorganic elements were analyzed by an inductively coupled plasma atomic emission spectrometer (ICP) and inductively coupled plasma mass spectrometer (ICP-MS). In river water, the concentrations of inorganic elements showed characteristic changes for each element as functions of location. As and Cu were detected in river water near the base of the Ashio copper mine. In tap water, we detected some samples containing inorganic elements exceeding the limits recommended by Japan Drinking Water Quality Standards. The current findings suggest that present water filtration plant procedures are not sufficient to remove certain inorganic elements from source water. Moreover, it is possible that the water works may have caused inorganic contaminations. These results may be important in the understanding of distribution patterns of inorganic elements.

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The effects of zinc deficiency on protein utilization in rats during gastric tube feeding were investigated. Rats fed a zinc-deficient diet ad libitum ceased growing on the 4th day. Thereafter, rats were fed equal amounts of the zinc-deficient diet or a control diet via gastric tube. Apparent absorption of diet and protein were not reduced, but urinary excretion of nitrogen and urea were increased in zinc-deficient rats. These results show that utilization of absorbed amino acids was reduced in zinc-deficient rats.

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The form by which Al enters brain cells as well as the intracellular consequences of Al in relation to neurodegenerative diseases remains unresolved. In this report, Al was differentially taken up from Al amino acid complexes by primary culture of cortical astrocytes. Aluminum uptake from different amino acid complexes in the presence and absence of the respective amino acid transporter blockers were compared. The results indicate that none of the amino acid transporter blockers, as well as ouabain, employed in the present study apparently inhibited the uptake of Al. There is a possibility that passive diffusion, influenced by concentration gradient and exposure time, is a major mechanism involved in the Al transport in the forms employed here. The apoptotic effect of Al amino acid complex on astrocytes was also confirmed in the present study with evidence of nuclear shrinkage and chromatin condensation that occurred in more than 20% of the cells, as early as 3 days and also at concentrations as low as 0.0125 mM.

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It has been suggested that metallothioneins (MTs) play a role to protect cells from toxic effects of oxidative stress and heavy metals. On the other hand, the ability of Cu-containing MT to protect cells from oxidative damage is controversial. In the present study, we have constructed HeLa cell lines overexpressing metallothioneins-1E and -2A for the first time, and have examined cytoprotective effects of MT against oxidative damage induced by hydrogen peroxide (H₂O₂) in these cell lines pretreated with or without Cu. As a result, the HeLa cells overexpressing MT have suppressed oxidative damage induced by H₂O₂ at low concentrations of Cu, suggesting that Cu-containing MT has cytoprotective effects against oxidative damage induced by H₂O₂ if the amount of MT is sufficient to contain Cu in the protein and not to liberate Cu bound to it.

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This study was conducted to establish a practical method to cultivate selenium-enriched vegetables in a large scale. Due to the toxicity of inorganic selenium compounds used as a source of selenium, concerns are the safety of agricultural workers operating the system and also effects on environments, especially the pollution of the ground water.
Barium selenate and barium selenite were selected as the selenium source for the cultivation because of the easiness of handlings, a slow diffusion into environments and the constant release of selenium in soil for an extended period of time. The cultivation was carried out in a concrete 10m×15m×1m frame equipped with a water tank beneath the frame. The frame was filled with a soil. The water tank was used to store the permeated water from the soil. The system was set in the ground. Barium selenate (500mg/m² as selenium) and barium selenite (500mg/m² as selenium) were applied on the soil and then the soil was plowed.
Garlic and onion were planted in fall and harvested in the next autumn. Cabbage was planted in early winter and harvested in the next spring. Peanut, pepper, ginger, tomato, and eggplant were planted in spring and harvested in the late summer. Through the study period, selenium contents in the water permeated into the tank from the soil (Soil Water) were monitored. The selenium content in the soil in the frame was also determined. The selenium content in Soil Water reached the maximum value (1.4ppm) 14 months after the application of the selenium salts. On the other hand, the total selenium in the soil was slightly decreased during this period. Selenium contents in fresh edible portions of harvested vegetables as follow:
Garlic 130ppm, onion 20ppm, peanut 57ppm, pepper 7.1ppm, ginger 2.7ppm, cabbage 1.3ppm, tomato 2.9ppm, and eggplant 3.7ppm.
Major selenium compounds in these vegetables are selenoamino acids and their derivatives. Organic selenium components in the soil would participate with the selenium absorption by the vegetables.

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Recently, it has been reported that vanadium exists in a relatively high concentration in the natural water around Mt. Fuji in Japan. Vanadium is known to have a blood glucose lowering effect and improve the diabetic state in human and rodents. Therefore, many researchers have interested in the relationship between diabetes mellitus and vanadium in the undercurrent water from Mt. Fuji. In this study, we examined whether or not 3- or 5-fold concentrated undercurrent water from Mt. Fuji improves hyperglycemia and diabetic state in type 2 diabetic KK-A^y mice with obesity. The concentrated undercurrent water given as drinking water did not reduce high blood glucose level, however, suppressed the progress of obesity, in terms of body weight gain, by the treatment for 12 weeks. Moreover, vanadium was found to accumulate in the tissues such as bone, spleen and liver by the treatment of 5-fold concentrated undercurrent water from Mt. Fuji for 12 weeks.

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Urinary selenium concentrations of 400 healthy adults in Aichi Prefecture were measured between 1998-2002 to assess the normal range of selenium concentrations in urine. In analyzing the data, we have evaluated the appropriate way of correction using urinary creatinine concentration and the specific gravity. Results and conclusions:
1) The urinary selenium concentration was 40.3±21.4 μg/l (mean±SD, n=400; median=36.5), and 43.4±20.1 for males (n=200), 37.3±22.2 for females (n=200). The concentration for males was significantly higher (P<0.01) than that for females. The selenium concentrations were found to decrease significantly with age.
2) Correction using either with urinary creatinine or the specific gravity has made the standard deviation of the corrected concentrations smaller, the results of which may suggest that the correction was successfully achieved. It should be noted, however, that the corrected concentrations by the creatinine and by the specific gravity showed opposite tendency in the relationships between aging and gender. The opposite tendency in relation to these factors was considered to derive from the fact that urinary creatinine levels reflect the amount of each individual’s muscle volume. Accordingly, the specific gravity correction is considered to be more suitable for the correction of the measured selenium in spot urinary samples.

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A molybdenum (Mo) thiocyanate complex, Mo(SCN)₅ had been used previously for the determination of Mo in seawater using electron spin resonance (ESR) method. In the present ESR study, a reduced Mo complex with diethyldithiocarbamate (DDC), Mo-DDC, was extracted with cycohexanol and used for the determination of Mo. Mo-DDC shows a peak at g = 1.980 with peak-to-peak width of 0.3 mT, whereas Mo(SCN)₅ shows a peak at g = 1.940 with peak-to-peak width of 1.2 mT. DDC reacts with Mo more specifically than SCN^-: The peak height of Mo-DDC is nearly 30 times that of Mo(SCN)₅ produced from the solution containing both DDC and SCN^- with the molar ratio of DDC: SCN^- = 1 : 30. The limit of detection and the time required for measurement are 0.1 ng and 5 min, respectively in the present method, whereas those are 5 ng and 60 min, respectively in the previous SCN method.

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We prepared six new zinc(II) complexes of halogenated picolinic acids with a Zn(N₂O₂) coordination mode and evaluated for their insulinomimetic activities by in vitro study. By introducing an electron withdrawing halogen groups into the picolinic acid, we prepared bis(4- or 6-chloro picolinato), bis(4-, 5- or 6-iodo picolinato), and bis(6-bromo picolinato)/zinc(II) complexes (Zn(4cpa)₂, Zn(6cpa)₂, Zn(4ipa)₂, Zn(5ipa)₂, Zn(6ipa)₂, and Zn(6bpa)₂, respectively). By in vitro evaluation of the inhibition of free fatty acid (FFA) release from isolated rat adipocytes in the presence of epinephrine, the insulinomimetic activities of Zn(4cpa)₂, Zn(6cpa)₂, Zn(4ipa)₂, Zn(6ipa)₂, and Zn(6bpa)₂ (IC₅₀ = 0.64, 0.60, 0.77, 0.85, and 0.50 mM, respectvely) were found to be higher than that of bis(picolinato)/zinc(II) complex (Zn(pic)₂) (IC₅₀ = 1.00 mM) in terms of IC₅₀ value, the 50% inhibition concentrations for the FFA release from rat adipocytes.

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A 5-year-old girl presented with chronic liver dysfunction. Her serum ceruloplasmin level was within normal range. Urinary copper excretion was slightly elevated. We performed ATP7B gene analysis. And mutations were found intron 4 and exon 18. One splicing donor site mutation, 1708-5 t to g, and missense mutation, N1270S, were detected. We thus diagnosed her to have Wilson disease with normo-ceruloplasminine-
mia. And we confirmed her diagnosis by measurement of hepatic copper content. Then, familial analysis was performed by gene analysis. Three carriers (parents and older brother) were detected. We conclude that molecular diagnosis is very useful for atypical cases of Wilson disease.

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Serum nonceruloplasmin - bound Cu was calculated as the formula : serum Cu - serum ceruloplasmin × 2.89, when one molecule of ceruloplasmin (CEP) was combined with 6 atoms of Cu. In this study 55 inpatients (25men, 30 women) with hospital diets, 49 (24, 25) with conventional liquid foods of enteral feeding, 22 (9, 13) with Cu - rich “L-6PM plus” in order to substitute for conventional liquid foods and the other inpatients were recruited, who were aged 50 to 100 years. There were found significantly positive correlations (r=0.981, n=245) between serum Cu and CEP levels, and (r=0.956, n=245) between serum Cu and nonceruloplasmin - bound Cu in almost elderly inpatients, who were suffered from dysphagia due to cerebrovascular accidents.

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The almost elderly inpatients with enteral liquid foods of tube feeding were divided into 5 groups of I to V. In this study 60 inpatients (28 men, 32 women) aged mean 78.8 years were given with Cu - poor conventional liquid foods such as MA-8, PN-Hi and Isocal plus for mean 145 days in I, 4 (2, 2) aged 84 years with slightly Cu - rich ISO-CZ for 93 days in II, 18 (5, 13) aged 80.9 years with one pack of Cu - rich L-6PM plus (Asahi-Kasei Pharma) substituted for one pack of conventionals for 103 days in III, 13 (7, 6) aged 76.3 years with two packs of L-6PM plus substituted for two packs of conventionals for 106 days in IV, and 6 (1, 5) aged 74 years only with 5 - 6 packs of L-6PM plus for 52 days in V. Then in III and IV serum Cu levels were measured in the periods for 120 to 340 days. The decreasing rates were shown in I and the increasing rates were shown in II to V. The grades of the increasing rates were II < III < IV significantly. In III 50 % of cases were improved to normal serum Cu from hypocupremia, showing daily Cu intake of 0.35 mg as an estimated average requirements. In IV all cases were improved to normal serum Cu from hypocupremia, showing 0.63 mg of Cu intake as an adequate intake. Recommended allowance was calculated from average requirement plus 2 standard deviations, indicating 0.4 mg of Cu intake.

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In order to obtain basic information of trace element distribution in the liver of the Formosan squirrel that accumulates Cu at high level, subcellular and molecular weight distributions in this species were examined.
Mean Cu concentrations in the whole liver of the Formosan squirrel were 610±360 (μ g/g wet wt.). And Cu concentrations in the fraction containing nuclei, mitochondria and lysosome were the largest in the liver of the Formosan squirrel. Mean Cu burden in the fraction containing nuclei, mitochondria and lysosome in the hepatocytes was 73% of the total copper contain in the hepatocytes. On the other hand, the average Cu burden was 3.5% in the microsome and 23% in the cytosol of the hepatocytes.
The results of the gel filtration showed that most of the Cu in the cytosol was contained in the void volume fraction, the molecular weight of which is larger than that of metallothionein. About only 6.5% of Cu was associated with metallothionein fractions in the liver of the Formosan squirrel. In the metallothineine fraction, Cu was contained while zinc was not detected, suggesting that Cu accumulates beyond the capacity of metallothinein synthesis. These results suggest that the fraction containing nuclei, mitochondria and lysosome contributes the high level of Cu accumulation in the liver of this species.

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Our object of this study was to investigate the effects of metallothionein, metal binding protein, on the change of the tissue distribution of essential metals by restraint stress. Wild-type and MT-null mice were restrained in wire net frame for 18 hours. After the stress load, 10 organs of brain, thymus gland, testis, spleen, kidney, lung, heart, small intestine, stomach, and liver were removed. Each organ measured the 9 metals (Mg, Cr, Mn, Fe, Co, Cu, Zn, Se, Mo) concentration by inductively coupled plasma mass spectrometer (ICP-MS). In conclusion, Zn, Mn and Mo in liver, stomach, and small intestine were easy to respond to the restraint stress load. Though MT is indispensable for liver Zn concentration increase by the restraint stress, the factor except for MT seems to be concerned in the part in the increase of zinc in stomach and small intestine.

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It is reported that metabolism in hyperthyroidism case are abnormal. The trend of iodine conc. in them are problem. We investigated the trend of iodine conc. in hair of hyperthyroidism and remission. Therefore, these hairs were analyzed by instrumental neutron activation analysis. Iodine concentrations in hair were measured. Except for I, elements were measured Al, Au, Br, Ca, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, S, Se, V and Zn. It was recognized that iodine conc. in hair of hyperthroidism case and remission case were lower than normal hair specimen.

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The effect of 50% dietary restriction on the mineral content of the liver in spontaneously obese rat with abnormal lipid metabolism selected among Wistar rat (Minko rat) was studied. Twenty-four male Minko rats and eighteen female rats (8 weeks old) were separated each into two groups. One group was fed 50% dietary restriction and other group was fed normal diet. These dietary regiments were performed for 13 weeks. The concentrations of calcium, phosphorus, sodium, magnesium, iron, zinc, potassium, sulfur, manganese, copper and molybdenum in liver of Minko rats were determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES: Perkin Elmer Co. Ltd) after wet digestion by nitric acid.
The significant changes seen in 50% dietary restricted rats to compared with normal group rats were as follows: In male rats, phosphorus, zinc, sulfur, copper and molybdenum levels in liver decreased, and potassium, iron and manganese levels in liver increased. In female rats, calcium, zinc, copper and molybdenum levels in liver decreased, and potassium, iron, and manganese levels in liver increased. Plasma iron level was higher, and total iron binding capacity was lower in male 50% restriction rats. These results suggest that 50% dietary restriction induced iron accumulation and the mineral imbalances in liver, and abnormal metabolism losing homeostatic regulation of plasma iron.

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