Arsenic (As) is a ubiquitous element in the environment. Chronic As poisoning from natural and anthropogenic sources has become one of the most significant environmental health problems affecting over 100 million people worldwide¹⁾. As leads human carcinogen. It causes increased cancer risk of skin, lung, bladder and other internal organ systems²⁾. There had been many failed attempts, as review by Wang et al.³⁾, to demonstrate As carcinogenicity in- vivo animal systems until recent years. Chronic exposure of sodium arsenate caused multiple tumours in C57Bl/6J mice⁴⁾. Waalkes et al.⁵⁾ were able to demonstrate transplacental carcinogenic effects of arsenite dosed to the pregnant C3H mice resulting in a higher incidence of cancers in the offspring at their late stage of life.

Solar ultraviolet radiation (UVR) is responsible for the induction of more than 90% of non-melanoma skin cancers¹¹⁾. This is of particular concern in view of the consequences of stratospheric ozone depletion. It has been estimated that every 1% depletion of ozone concentration results in a 2% increase in the amount of UVR the earth⁶⁾.

It has been demonstrated that As enhanced the number of skin cancers in mice induced by UV exposure⁷⁾. Since people who live in As-endemic areas are also exposing to UV radiation. The significance of the combined effect of arsenic and UV radiation need further investigation.

Euglena gracilis SMZ strain.has been shown as a useful model for the study of oxidative damage induced by a number of metals including mercury, lead and cadmium^8,9), In this study, we investigate the oxidative effect of arsenic and/or UVB exposure in E. gracilus SMZ strain.

Cytochrome P450 is a group of enzymes that are responsible for the biotransformation of numerous endogenous and exogenous compounds. However, the precise process of dioxygen activation involved in P450-dependent substrate oxygenations has not been established. Previously, we reported that singlet oxygen (¹O₂) played an important role in microsomal P450-dependent oxygenation. In order to confirm the ¹O₂ involvement, we investigated further the relationship between the formation of reactive oxygen species (ROS) and a substrate oxygenation in the ethanol-induced rat liver microsomal P450.

VP-16 is a clinical anticancer drug for the treatment of a number of human cancers. Anticancer activity of VP-16 has been suggested to be due to a covalent enzyme-DNA complex between the drug and type II topoisomerase or to involvement of free radical species, which in turn cause double-strand DNA breaks in cancer. Previously, we have found that hydroxyl radical (・OH)is generated during a redox-dependent complex formation between Cu(II) and VP-16 in the presence of molecular dioxygen (O₂), suggesting the ・OH participation in the single-and double-strand breaks of DNA. On the basis of results, we extended our investigation to look into the mechanism in detail, and VP-16 and its related compounds were found to form both organic free radical species and ・OH in the presence of Cu(II) as detected by ESR and spin-trapping methods, respectively, their structures being analyzed by computer simulation and molecular orbital calculation. Then, we examined the relationship between the free radical formations and DNA-cleaving activities, and proposed a new mechanism of the free radicals-dependent DNA cleavage.

Antioxidant action of eugenol compounds was analyzed in relation to the role of transition metal. Eugenol and isoeugenol inhibited iron-mediated lipid peroxidation, and autooxidation of Fe²⁺ ion. Inhibitory effects of isoeugenol on lipid peroxidation and iron oxidation were more potent than those of eugenol. Eugenol and isoeugenol protected low density lipoprotein (LDL) from copper-dependent oxidation, and showed a potent copper-reducing activity to the same extent. Both compounds effectively scavenged a stable radical, 1,1’-diphenyl-2-picrylhydrazyl(DPPH). Antioxidant properties of eugenol compounds can be explained by forming complexes with reduced metals, and the potent preventive effect of isoeugenol on lipid peroxidation may be related to the decreased formation of perferryl ion or the iron-oxygen chelate complex as the initiating factor of lipid peroxidation by keeping iron at a reduced state.

Since an antimicrobial ingradient of hiba oil (Japanese cypress oil), hinokitiol, has a wide antimicrobial spectrum, it has long been added to toothpaste and hair cosmetics. Hinokitiolis approved as a food additive for preservation. Currently, hinokitiol is chemically synthesized. Recently, addition of hinokitiol and hinoki oil, as a safe natural antimicrobial agent, to antimicrobial-processed textiles has rapidly increased. However, patients with allergic disorders caused by hinokitiol and its teratogenicity have been reported, indicating that the safety of hinokitiol has not been sufficiently established. First, we developed an analytical method for measuring the concentration of hinokitiol used in commercially available antimicrobial products. There had been no reproducible methods. for analysis of hinokitiol due to its characteristics, such as photodegradability, sublimation ability, and high metal complex formation ability. We established a method for the detection of hinokitiol at a level of μg/ml by HPLC using a photodiode array detector. We also established a method for the extraction of hinokitiol with methanol from fabric samples prepared by treating with hiba oil, and a purification method using sodium dihydrogen phosphate buffer/cyclohexane partition and a C₁₈ cartridge column. We examined commercially available textiles with which processing with hiba oil or hinokitiol was indicated by the analytic method, but did not detect hinokitiol in any sample.

To clarify the causes, we produced standard fabric samples by fixing microcapsules filled with synthesized hinokitiol, hinoki oil, or hinoki leaf oil, performed the JIS light fastness test of the fabric samples, and examined the amount of residual hinokitiol at intervals by HPLC. As a result, the major cause of the disappearance of hinokitiol was photolysis. We also examined the antimicrobial activity of hinokitiol by the new agar plate method using gram-positive bacteria Staphylococcus aureus and gram-negative bacteria Klebsiella pneumoniae, and found that the hinokitiol treated fabric samples after photoirradiation showed higher antimicrobial activity, suggesting that degradation products of hinokitiol by photoirradiation had higher antimicrobial activity. It will be necessary to evaluate the safety of degradation products of hinokitiol by photoirradiation.

To prepare selenium (Se) -enriched sprouts, accumulation of Se in seeds and sprouts of kaiware daikon (A type of Raphanus sativus, the sprouts of which are eaten in Japan) was examined. The seeds were kept in a graded concentration (1 to 100 μg Se/ml) of selenite solution at room temperature for 48 h and cultivated to sprouts on absorbent cotton holding deionized water. After being kept in the selenite solution for 48h, the seeds accumulated Se dose-dependently. The seeds kept in a 100 μg Se/ml of selenite solution accumulated Se at a level of more than 250 μg/g dry weight. Abilities of germination of the seeds were significantly inhibited by the selenite solution at a level of more than 20 μg Se/ml. However, almost all the seeds kept in 20 μg Se/ml of selenite solution had abilities of germination and could grow to the sprouts normally. After cultivation of the sprouts for 7 days, Se concentration of the sprouts derived from the seeds kept in the 20 μg Se/ml solution was about 40 μg/ g dry weight. Analysis using high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP MS) showed that chemical species of most Se in the sprouts was Se-methylseleocysteine.

Dietary lectins have been established to lead to depressed activities of some intestinal enzymes, growth retardation and even death for experimental animals, particularly when consumed in large quantities. ln this study, we examined the effect of dietary lectin (concanavalin A) on serum and hepatic levels of cholesterol in rats fed a 0.5% cholesterol containing diet at doses that did not affect food intake and body weight. Dietary lectin inhibited the increase in the hepatic and serum levels of cholesterol in the cholesterol-feeding rats. Lectin-mediated decrease in the serum cholesterol was mainly due to the reduction of very low-density lipoprotein and low-density lipoprotein fractions. but a high-density lipoprotein fraction was not at all affected by dietary lectin.These results suggest that the dietary lectin might be an effective hypocholesterolemic factor.

Plant cells have two different mechanisms for the assembly of iron-sulfur clusters: the mitochondrial mechanism and the chloroplastic mechanism, which is less well characterized. Cysteine desulfurase catalyzes the desulfurization of L-cysteine and is supposed to deliver the sulfur atom for the synthesis of iron-sulfur clusters in both organelles. However, it remains unclear what proteins cooperate with cysteine desulfurase for cluster assembly in chloroplasts. The protein encoded by slr0077 of Synechocystis sp. PCC6803, named SsCsd3, shows a high sequence similarity to chloroplastic cysteine desulfurase from Arabidopsis thaliana (AtCpNifS) (60% identity). Thus, the mechanism for cluster assembly involving SsCsd3 can be regarded as a model of the mechanism operating in chloroplasts. In this study, SsCsd3 was overproduced, purified, and characterized. SsCsd3 acted not only on L-cysteine but also on L-selenocysteine, although the physiological significance of its activity toward L-selenocysteine is unknown. The specific activity of purified SsCsd3 toward 10 mM L-selenocysteine (5.4 units/mg) was comparable to that of AtCpNifS (3.7 units/mg), and the activities of these enzymes toward レselenocysteine were much higher (over 100 times) than those toward L-cysteine. Thus, SsCsd3 is similar to AtCpNifS not only in its primary structure but also in its catalytic properties. The iron-sulfur cluster of ferredoxin was reconstituted in vitro by using SsCsd3 as the sulfur delivery protein.

To evaluate the molybdenum (Mo) status of infants in Japan, the Mo contents in human milk and formula milk were determined using inductively coupled plasma mass spectrometry (ICP-MS) and the average Mo intake of infants in Japan was estimated. Seventy-eight human milk samples were randomly collected from all over the country. The median, arithmetical mean, geometrical mean and range of Mo in these human milk samples were 2.9 ng/ml, 4.5 ng/ml, 3.4 ng/ml and 0.8 to 34.7 ng/ml, respectively. Based on these results, a reference range of Mo concentration of human milk in Japan was estimated to be 0.8 to 14.0 ng/ml. Analysis of Mo in seventeen human milk samples collected from 3 healthy volunteer showed that intra-individual variation was comparable to inter-individual variation in the Mo concentration. Mo concentration in Japanese formula milk was ranged to 1.1 to 2.4 ng/ml. Based on the Mo levels in human milk and formula milk, the Mo intake of Japanese infants was estimated to be 1 to 4 μg/day/capita.

In this study, we examined the effects of chronic low-level exposure to methylated arsenicals on tolerance to inorganic arsenite in vitro using the normal rat liver cell line TRL 1215. For 20 weeks, cells were exposed to methylated mammalian metabolites of inorganic arsenicals; monomethylarsonic acid (MMAs^v; 1.3 mM), dimethylarsinic acid (DMAs^v; 700 μM) or trimethylarsine oxide (TMAs^vO; 10 mM). These cells acquired tolerance to the acute cytolethality of inorganic arsenite. Cellular arsenic accumulation was less in chronic methylated arsenical-exposed cells than passage-matched control cells. Long-term exposure to methylated arsenicals increased cellular glutathione S-transferase (GST) activity and cellular reduced glutathione (GSH) levels, although only chronic exposure to DMAs^v significantly decreased cellular GSH levels. Cellular GSH depletion increased arsenic accumulation and decreased tolerance to the acute cytolethality of arsenite in these chronic methylated arsenical-exposed cells. These results indicate that chronic exposure to methylated arsenicals induces tolerance to inorganic arsenite, and that cellular GSH may play an important role in the induction of this tolerance.

Since the finding of insulin-mimetic effect of Zn(II) ion, several insulin-mimetic Zn(II) complexes have been proposed. Previous studies reported an extremely high insulin-mimetic activity of Zn(II) complex with maltol (Zn(ma)₂) in in vitro and in vivo evaluations. To develop more effective insulin-mimetic Zn(II) complexes than Zn(ma)₂, we examined the structure-activity relationships for the Zn(ma)₂ and its related complexes. From the results, a new Zn(II) complex (Zn(alx)₂) with allixin, isolated from garlic as a bioactive product induced by continuous environmental stress, was found to exhibit the highest in vitro insulin-mimetic activity among these complexes. Then, we examined the anti-diabetic effects of Zn(alx)₂ complex in type 2 diabetic animals. The blood glucose lowering effects of Zn(alx)₂ and Zn(ma)₂ were compared, and both complexes were found to lower the high blood glucose level in type 2 diabetic KKA^y mice after a 14-day course of daily i.p. injections. However, Zn(alx)₂ improved glucose tolerance in KKA^y mice much more than Zn(ma)₂, indicating that Zn(alx)₂ possesses a higher in vivo anti-diabetic activity than Zn(ma)₂. In addition, Zn(alx)₂ improved the leptin resistance, suppressing the progress of obesity in KKA^y mice. On the basis of these observations, we propose that Zn(alx)₂ complex is a novel potent candidate for the treatment of type 2 diabetes mellitus.

Zinc is an essential trace element and crucial for the normal development of the central nervous system. Zinc is abundantly present in the brain and is secreted to synaptic clefts with neurotransmitters. However, its precise role is still under investigation. Meanwhile, excess zinc is reported to contribute to neuronal death in the global ischemia. We have investigated the effects of zinc in immortalized hypothalamic neurons (GT1-7 cells). Zinc chelator caused marked death of GT1-7 cells, while zinc itself caused degeneration of GT1-7 cells. Neurotoxicity of zinc was prevented by pyruvate and calcium. However, zinc inhibited death of GT1-7 cells induced by excessive extracellular calcium. Our results suggest that zinc homeostasis is important for neuronal survival and that calcium homeostasis is implicated in zinc-induced neurotoxicity.

A relationship between the inhibition of protein kinase C activation by phospholipid -interacting compounds and the associated change in the physical properties of phospholipid vesicle membranes was studied using physically characterizable phosphatidylserine -phosphatidylcholine-diacylglycerol (3:6:1) mixture vesicle membranes. The vesicles supported protein kinase C activation as well as phosphatidylserine-diacylglycerol (9:1) vesicles. Under the same enzyme assay conditions, trifluoperazine and to a lesser degree chlorpromazine inhibited the enzyme activation and increased the fluorescence polarization of 1,6-diphynylhexatriene in the vesicle membranes in a dose-related fashion. At the concentration of 10^-4M, n-dibutyltin dichloride also showed a slight tendency to inhibit the enzyme activation and to increase the polarization whereas dibucaine scarcely showed any significant effect. At least DBC of more than 5×10^-4M was required for manifestation of the detectable effect.There was a good correlation between the relative order of potency of these compounds for the inhibition of the enzyme activation and for the ordering of the membranes. Moreover, the compounds inhibited the enzyme and substrate binding to the vesicle membranes in a dose-related fashion. These results shows that the phospholipids -interacting compounds such as trifluoperazine and chlorpromazine may exert their inhibitory action on the enzyme activation by ordering the vesicle membranes or by the change in phase separation.

A vegetarian diet is thought to be better for the prevention and treatment of lifestyle related diseases. However, selenium and zinc are abundant in animal products, so it is necessary to assess the nutritional status of vegetarians with respect to these substances. We evaluated the serum levels of selenium and zinc in 45 women who were on a 45 day vegetarian diet and 61 women who were normal diet (normal diet group). As a result, the intake of selenium was lower in the vegetarian group (29.8 ± 30.2μg) than in the normal diet group (125.9 ± 41.9μg). Zinc intake was also lower in the vegetarian group (6.2 ± 2.2 mg) than in the normal diet group (7.9 ± 1.8 mg). In addition, serum levels of selenium and zinc were lower in the vegetarian group than in the normal diet group. Furthermore, a lower intake of selenium was related to a lower serum level, and reduced intake of zinc from animal products was related to a lower zinc level. These results suggest that reduced selenium and zinc intake due to a vegetarian diet may potentially have adverse effects.

Inductively coupled plasma-mass spectrometry (ICP-MS) was applied to determination of selenium (Se) in food samples. When the concentration of nitric acid in the sample solution was adjusted to 1.0 to 1.5 M, a suitable analytical value of Se was obtained without using any internal substances. The concentration detection limit was 25 ng/g in the dried food samples. In quadruplicate assays of 7 biological reference materials using the proposed ICP-MS method, measured Se concentrations were not significantly different from their certified values. Using the proposed method, Se concentrations in domestic or imported buckwheat and barley were determined. Imported cereals grown in Manitoba (Canada) or North Dakota (USA), known to be high Se areas, showed high Se values at a level of more than 300 ng/g, while most domestic cereals showed low Se values at a level of less than 100 ng/g. These results indicate that the proposed method is reliable and suitable for the determination of trace levels of Se in foods.

A crystallization inhibitory material of calcium carbonate was purified from the crude extract of a scallop shell. The inhibition pattern of this inhibition material was the same as that of lithostathine, could inhibit the growth of calcium carbonate crystal. This inhibitory material was purified by ethanol fractionation and column chromatographies on both of DEAE-TOYOPEARL 650M and DEAE-TOYOPEARL HW-55F. The molecular weight of purified crystallization inhibitory material of calcium carbonate was estimated to be approximately 2.7×10³ according to gel filtration chromatography.

Recently, the increase of morbidity and mortality of cardiovascular disease in Japanese caused by high calcium/magnesium ratio in dietary intake are focused. In this report, calcium, magnesium and phosphorus intake, and these concentrations in blood and plasma of Japanese adult men are examined.

Subjects in this survey were 20 healthy adult men (42 ± 6.6 years) and the intakes of calcium (Ca), magnesium (Mg), phosphorus (P), Ca and Mg ratio (Ca/Mg ratio) and P and Ca ratio (P /Ca ratio) were calculated from the daily food intakes. Ca, P and Mg concentrations in plasma and blood were measured by using inductively coupled plasma- atomic emission spectrometry (ICP-AES: Perkin Elmer Co. Ltd) after wet digestion by nitric acid, and biochemical parameters in plasma were monitored. No significant correlation was found between Ca, P and Mg concentrations in plasma or blood and Ca, P, Mg, P /Ca ratio and Ca/Mg ratio in dietary intakes. Ca/Mg ratio in food intake correlated positively with phospholipid and triglyceride values in plasma (p < 0.05).

These results indicate that Ca, Mg and P concentrations in plasma are not so much change for the most part by the homeostasis mechanism, so that not always reflected in the nutritional status. However, they support that increase of Ca/Mg ratio in daily food intakes induces arteriosclerosis and ischemic heart disease, and showed high correlation with plasma index for these diseases. It is suggested that anew not only individual mineral intake but also mineral balance is important.

To evaluate a contribution of tea to manganese intake of Japanese, manganese contents in several types of tea leaves and tea infusions were determined and chemical species of this element in the tea infusions were examined. Thirty-five samples of green tea, 15 samples of black tea and 13 samples of oolong tea were collected. Their manganese contents were ranged in 200 to 1000 μg/g and were similar values to those listed in Standard Tables of Food Composition in Japan. Infusions prepared from these tea samples also showed similar manganese concentrations (2 to 5 μg/ml) to those listed in the Tables. However, manganese concentrations in tea infusions and commercial tea drinks consumed by healthy 108 university students were 0.5 to 2 μg/ml and were significantly less than those of tea infusions prepared in the laboratory or those listed in the Tables. Based on the present analytical results, manganese intake from tea is estimated to be less than half of total dietary manganese intake in Japanese. Most of manganese in the tea infusions prepared in the laboratory was filterable to an ultrafilter with molecular mass cut-off of 5kDa. In analysis of high-performance liquid chromatography using a column of molecular exclusive resin, manganese in the tea infusion was eluted at the same retention time of bivalent manganese ion.

Pressure ulcer is a disease seen in the elderly and bedridden. Once a pressure ulcers is created, it takes several months to heal. Nutrition management is indispensable to heal chronic wounds such as pressure ulcers. In this research, we examined whether changes were observed in pressure ulcer wound healing conditions, according to the kind of foods they ingested and whether adding zinc, one of the micronutrients reported to have effect on wound healing, was a factor. We have made a comparative study based on these results.

Changes in the absorption rate and tissue distribution of zinc under different nutritional status of this element were examined. Five-week-old male Wistar rats were given a zinc-deficient basal diet (zinc content, 0.6 ppm) or the basal diet supplemented with graded levels (5.5 ppm, 10 ppm, 15 ppm, 20 ppm, 30 ppm or 40 ppm) of zinc as zinc sulfate for 4 weeks. Feeding of zinc-deficient diet for 4 weeks induced poor growth and a significant decrease in zinc concentrations in many tissues. Rats fed a diet containing zinc at a level of 5.5 ppm showed lower growth and tissue zinc concentrations than those fed diet containing zinc at level of 10 ppm or more. In particular, the zinc concentration in tibia responded remarkably to the elevation of dietary zinc level. Apparent absorption of zinc was inversely correlated to the dietary zinc level; apparent absorption in rats fed diet containing 5.5 ppm zinc was 92%, while that in rats fed diet con.taining 30 ppm zinc was 27%. These results indicate that the minimum dietary requirement for zinc is 5.5 to 10 ppm in growing rats.

We determined the concentrations of metallothionein-1 (MT-1) and MT-2 in the liver cytosol of Long-Evans Cinnamon (LEC) rats, an animal model of Wilson disease, which develop hepatic disorders spontaneously, by using capillary zone electrophoresis (CZE) with polyacrylamid-coated capillary. In addition, by comparing the values of MT by ELISA (total concentrations of MT-1 and MT-2),we confirmed that each concentration of MT-1 and MT-2 can be determined by CZE. Total MTs in the liver cytosol of LEC rats increased age-dependently with accumulation of Cu. MT-1/MT-2 ratio decreased age-dependently (1.73, 1.38 and 0.76), indicating that MT-2 was induced more than MT-1 with development of hepatic disorders.

Transcription of mammalian metallothionein (MT) genes is activated by a various metals including Zn, Cd, Cu, Hg, Ag and so on. The control sequences located in the 5’-flanking region of MT genes revealed that the short DNA elements, which are called metal responsive elements (MREs) and contain less conserved GC-rich region, mediate metal responsiveness. MREs were initially shown to mediate transcriptional response of MT gene to Zn and Cd, and more recently to oxidative stress. However, the other metals have not shown to be essential for metal-induced transcriptional regulation of MT. In this study, the effect of Cu²⁺ ion to binding of MREs sequences to the nuclear protein from HeLa cells stimulated with TPA (12-O-tetradecanoyl phorbol-13-acetate) was investigated by the gel shift assay, using the plasmid pKB8 which was constructed the human metallothionein (MT)-IIA promoter gene in pUC19.

The microbiological assay is one of the most common methods for biotin determination, which generally uses biotin-dependent L. plantarum ATCC 8014. The growth of microorganisms in the presence of biotin was evaluated by turbidity optical density of the assay solution at 610 nm, which is known as a turbidimetric assay. We developed a new improved bioassay for biotin levels using ATP production in L. plantarum. The standard curve was linear in the range from 0.01 to 800 pg/ml and the detection levels of this assay as low as 50 pg/ml could be realized. The intra-assay precision test revealed that the reproducibility of duplicates was within 8%. These findings suggest that this ATP assay is more sensitive and simple for the determination of biotin than the classic turbidimetric assay, thus making it appropriate for clinical laboratory use.

A plant-based diet is thought to be better for the prevention and treatment of lifestyle-related diseases. However, zinc, copper and selenium, which are essential trace elements, are abundant in animal products, so it is necessary to assess the nutritional status of vegetarians with respect to these nutrients. We evaluated the serum levels of selenium, zinc, and copper in 19 middle-aged and elderly women who were on a 45-day plant-based diet therapy and 27 healthy middle-aged and elderly women. Selenium content in the meal was found to be lower than the daily requirement in the women on a plant-based diet, while the contents of the zinc and copper did not satisfy daily requirements in both the healthy women and plant-based diet groups. Serum selenium and zinc levels were lower trend in the group after eating the plant-based diet than in the healthy women group. Analysis of correlations between the component in the meal and the serum levels of zinc, copper, and selenium only showed a correlation between the component in the meal and the serum level of selenium. These results suggest that reduced selenium intake due to a plant-based diet may have the potential adverse effects.