Alzheimerʼs disease (AD) is the most common form of dementia of the elderly and is a mounting public health problem. There has been steadily growing interest in the involvement of metal ions (especially, zinc and copper)and docosahexaenoic acid (DHA, C22 : 6n-3) in neuronal functions and neurodegenerative disorders including AD. The recent interesting descriptions are in the elucidation of zinc and copper release and flux at the glutamatergic synapse in the cortex and hippocampus, These metals influence the response of the NMDA receptor. AD is also characterized by miscompartmentalization of copper and zinc (e. g. accumulation in amyloid).

DHA, a n-3 polyunsaturated fatty acid, is essential for normal neurological development and vision. DHA deficiency markedly affects neuronal function ; it is thus associated with some neurological dysfunction in aging and AD. Epidemiological studies show a relation between the ingestion of fish oils and AD, suggesting neuroprotective consequences of the oil, especially of DHA. Dietary administration of DHA improves learning ability in rats, and protects against and ameliorates the impairment of learning ability in AD model rats and APP transgenic mouse model of AD. The roles of zinc and copper in pathophysiological of AD and the improvement effect of dietary DHA on AD are reviewed here.

After the introduction of Food for Specified Health Uses system in 1991, circumstances in which functions of foods are evaluated by clinical trials, are improving. Issues of clinical trials of foods in Japan are discussed in comparing with drug cases. Necessity of the addition of systems ensuring the reliability of data is suggested in clinical trial practices of food cases. Some differences of endpoints between drugs and foods are pointed out, and needs of experts who explain medical meanings of such differences to consumers are discussed. In the case of evaluation of disease risk reduction, large scale clinical trials are needed, and then financial supports by government are needed.

The inter-neuronal movement of zinc ions is a key mechanism involved in ischemic neuronal death. Therefore, zinc-induced neuronal cell death is a suitable phenomenon to observe for examining the neurodegenerative damage following ischemia. We have established a convenient and rapid screening system for protective substances against zinc-induced neuronal cell death and isolated carnosine, pyruvate, α-tocopherol, and gadolinium as protective substances. In this study, we modified the screening system to particularly identify substances with weak protective activity. The modifications, earlier administration of the samples and zinc, and the measurement of cell mortality, allowed the present assay to perform the sensitive and reliable detection of protective activity against zinc-induced GT1-7 cell death. Dose-dependency of pyruvate showed that the present assay was improved specially in detecting weak protective activity compared to the previous one. Using the present assay, numerous protective activities could be significantly distinguished from the real samples. The screening method with the present assay will extend the possibilities of screening samples.

We previously prepared a hot water extract and a zinc-rich fraction from oyster and investigated the utilization of zinc from oysters. In the present study, a new oyster extract prepared from a mixture of the hot water extract and the zinc-rich fraction was examined in rats. In experiment 1, changes in the absorption rate and tissue distribution of zinc were examined under different level of sodium phytate administration. Five-week-old male Wistar rats were given diets containing 20 ppm of zinc as zinc sulfate for 29 days ; those diets were replaced and supplemented with graded levels (0 %, 0.2 %, 0.5 %, 1.0 % or 2.0 %) of sodium phytate. The rats fed a diet higher in sodium phytate showed lower tissue zinc concentrations in several tissues. In particular, the zinc concentrations in serum, tibia, ileum and hair responded remarkably to the declination in dietary sodium phytate. There was a negative linear relationship between zinc concentrations in the tissues (serum, tibia, ileum and hair) and dietary sodium phytate level from 0 % to 1.0 %. In experiment 2, effects of differences in zinc source on the absorption and tissue distribution of zinc in rats fed a diet containing sodium phytate were examined. Five-week-old male Wistar rats were given 1.0 % sodium phytate-supplemented diet containing 20 ppm of zinc as zinc sulfate or 3 types of oyster extracts for 29 days. Differences in dietary zinc source (zinc sulfate or oyster extract) did not significantly influence zinc concentrations in serum or various other tissues. The rats fed a diet containing zinc as a new oyster extract showed slightly higher zinc concentrations in several tissues (serum, tibia and ileum) than those fed a diet containing zinc as zinc sulfate or other oyster extracts. These findings suggest that the new oyster extract has higher zinc bioavailability than zinc sulfate or other oyster extracts.

Monthly changes in macro mineral concentrations of feeds in dairy farms were investigated to evaluate mineral status of dairy cattle and buffalo. Fifteen dairy farms in three villages located around Rampur in Chitwan District were chosen, and feed amounts and its concentrations fed to mature dairy cattle and buffalo were quantified in every month from April 2007 to March 2008. The animals were fed green fodders mainly from May to July and straws from August to April. The total DM amounts of feeds were decreased in July, which was reflected from the highest amount of green fodders. The concentrations of magnesium (Mg) and potassium (K) in feedstuffs were as much as the requirements for dry and pregnant cows. However, the concentrations of phosphorus (P) and calcium (Ca) were lower than the requirements for dry and pregnant cows in some months. In particular, the concentration of sodium (Na) was considerably lower than the requirement for dry and pregnant cows. It was suggested that attention should be paid for P, Ca and Na status of dairy cattle and buffalo in the region because the concentrations of the minerals in feedstuff might be insufficient in the particular months for P and Ca and in year-round for Na.

Magnesium deficiency enhances insulin sensitivity in rats whereas it reduces insulin sensitivity in humans. Magnesium deficiency causes hypocalcemia in humans while the hypocalcemia is not observed in magnesium-deficient rats. In the present study, we examined the effect of pair-feeding or magnesium deficiency with calcium deficiency on insulin sensitivity in rats. Rats were pair-fed control diets or magnesium and/or calcium deficient diets for three weeks before intravenous insulin tolerance test (IVITT).

Magnesium deficiency significantly (P<0.05) increased glucose disappearance rate (Kitt) from plasma and decreased area under the curve (AUC) of plasma glucose in IVITT. Pair-feeding did not affect Kitt and AUC. Magnesium and calcium deficiency enhanced insulin sensitivity as well as magnesium deficiency alone. These results suggest that magnesium deficiency enhances insulin sensitivity not via the reduction of feed intake and the disturbance of calcium metabolism.

Adipocyte differentiation is primainly regulated by a cascade of transcription factors, including CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor γ (PPARγ). Ascorbic acid was reported to promote the adipocyte differentiation. However, its detail mechanism has not been clarified. In the present experiment, we investigated the molecular mechanism of ascorbate phosphate on differentiation in 3T3-L1 preadipocytes. Treatment with ascorbate phosphate enhanced the induction of some markers of adipocyte differentiation such as glycerol-3-phosphate dehydrogenase activity and triglyceride accumulation. Gene expression of PPARγ and C/EBPα were increased by ascorbate phosphate treatment. Moreover, gene expression of C/EBPβ, expressing at early stage in adipogenesis, was also stimulated by ascorbate phosphate. These results suggest that ascorbic acid promotes adipocyte differentiation at least via affecting C/EBPβ expression level at the early phase of differentiation.

In Japan, number of people who have taste abnormality due to zinc deficiency is increasing. It was reported that chronic intake of alcohol induces the zinc deficiency. In this paper, mice were caused severe zinc deficiency through zinc deficiency diet and alcohol intake and we studied the influence of zinc deficiency and alcohol intake on immune functions by flow cytometory. Then mice were given recovery diet and the recovery of immune function was examined. Zinc introduced into yeast or Zn-CO₃ was used as a recovery diet in this study. It was observed that the immune function of thymus from zinc deficiency mice was lower than that of the control mice. When mice were given Zn-CO₃ or the zinc introduced into yeast diet, the immune function of the mice was recovered. Previously, we have shown that the absorption of the zinc from the zinc introduced into yeast is slow compared with Zn-CO₃.Though the absorption of the zinc introduced into yeast is slow, it has recovered function of the immune as well as Zn-CO₃.

The serine racemase is a bifunctional enzyme that catalyzes a racemization and a dehydratation (α, β-elimination) of serine, and it has been found in various eukaryotes, such as human, mouse, fission yeast, cellular slime mold and plants. The serine racemase belongs to the fold-type II of the pyridoxal 5ʼ-phosphate (PLP) enzymes. We reported the clonig and expression of the serine racemase from Oryza sativa L. (SerR) and discovered the regulation mechanism of two enzyme reactions by Magnesium (II) ion (Mg²⁺). To examine the regulation mechanism of Mg²⁺, we tried to analyze the fluorescence quenching of tryptophan (Trp) residues in SerR by acrylamide. The result showed that the structure of SerR is distorted by the addition of Mg²⁺, and this structural change probably regulates the two enzyme activities.

Distributions of α-1,4 unit chains of the endosperm starch molecules of so-called ancient-type, pigmented rice grains were precisely analyzed by HPAEC, after enzymic debranching of their amylopectin fractions, and compared with those of cultivated, ordinary Japanese rice, and also of various non-kernel cereals. The results show that the unit chain of color pigmented rice starches are distributed DP 6 to 30 with high proportions of DP 10-12 peak, similar to those of other cereal starches, suggesting that the rice endosperm starches, used as main energy-supplied source would not significantly different during human long history. In the present studies we noted that, in the case of rice starch, the unit chain of amylopectin of the endosperm starch, might be shifted significantly to longer, as milling degrees are increased, as in the case of 10 %, 40 %, and 60 % milled Yamadanishiki rice, which would affect to sake-fermentation. The antioxidant activity in the bran fraction of red color pigmented rice grain was also confirmed.

Mimosine (3-hydroxy-4-oxo-1 (4H)-pyridinealanine), a tyrosine analogue is produced by plant Leucaena leucocephala. Cytotoxic effects of mimosine were analyzed in relation to the generation of reactive oxygen species. 1. Treatment of glioma C6 cells with mimosine induced cell death with DNA damage and repair processes. 2. Mimosine/iron complex inactivated aconitase, the most sensitive enzyme to oxidative stress. The inactivation was dependent on sodium azide, an inhibitor of catalase, indicating that mimosine/iron complex can generate hydrogen peroxide as a principal product. 3. Mimosine stimulated the atuooxidation of Fe²⁺ suggesting that mimosine promotes the activation of dioxygen molecule by reduced iron. 4. Mimosine enhanced the Fe/ascorbate-dependent formation of 8-hydroxy-2ʼ-deoxyguanosine in DNA, indicating generation of hydroxyl radical. Prooxidant action of mimosine may explain the cytotoxic effects of this compound including alopecia and growth retardation reported previously.

Humic substances (HS) have physical, chemical and biological activities. One of the main elements of HS is humic acid (HA) which is constituted of the mixture of heterogeneous large molecule electrolytes. In order to reuse HA effectively, the antioxidative activities of partially decomposed HAs were evaluated since HA had reported to show some antioxidative activities. Native HA (HA-a) from peat was treated with high temperature (132°C) and high pressure (190 kPa) for 20 min (HA-b), 60 min (HA-c) and 120 min (HA-d). The peak top time of HA-a-d by high-performance size exclusion chromatography were 15.4, 15.8, 16.0 and 16.0 min, respectively. All of HA-b-d showed the antioxidative activities, such as scavenging activities for 1,1-diphenyl-2-picrylhydrazyl radical and inhibitory effect on 2,2ʼ-azobis (2-amidinopropane) dihydro chloride-induced hemolysis of rabbit erythrocytes in vitro as much as HA-a. HA-a and HA-c were tested on the inhibitory effect of the damages by UVA irradiation (100 mJ/cm²) using human skin fibroblast cells. Both of them showed the inhibitory effect with significant differences.

This study was conducted to examine the effects of excessive biotin intake during gestation on the reproductive performance in mice. There were no changes in the weight and relative weight of organs in the biotin-excessive dams, which did not exhibit any overt signs of skin. However, the fetal body weight and length were significantly decreased by excessive biotin intake. The biotin concentration in the liver and amniotic fluid in fetuses was higher, compared to that in their dams with an excessive biotin intake. This indicates that much more biotin was easily transported to the fetuses from the dams. In addition, many kinds of external malformations were observed in these fetuses. The predominant malformations were micrognathia, micromelia and edema. However, cleft palate which is induced by biotin deficiency was not induced. These findings demonstrate that in mice the fetuses are more sensitive to excessive biotin intake during gestation than their dams.

Recently, chronic kidney disease (CKD) has been estimated to affect about 20 million people in Japan. CKD is associated with a several fold increased risk of arteriosclerosis related disease. Oysters (Crassostrea gigas) contain many nutrients and are used widely in Japan. Oyster extract contains major components including minerals, carbohydrates, protein, and amino acids. Various effects on health maintenance and life-style related diseases of oyster extracts have been reported so far. The present study evaluated the preventive effects of oyster extract on the formation of chemically induced nephrotoxicity in rats. Rats fed an AIN-93G modified diet containing oyster extract (1.0 %) After 6 weeks, the rats were injected intraperitoneally with p-aminophenol (PAP, 0.5 mmol/kg body weight). Urine was collected before and after injection, and the kidney was removed at 48 h after injection.

Dietary oyster extract caused a decrease in blood urea nitrogen (BUN), return to the normal levels of urine volume, urinary N-acetyl-β-D-glucosaminidase (NAG) activity, and creatinine concentration, and reduction in the severity of tubular basophilic change and nuclear division in tubular epithelium compared with control diets. However, the severity of tubular necrosis and tubular basophilic change in the midzone area and vacuolar degeneration in renal and arcuate arteries were not significantly different. Dietary oyster extract recovered proximal tubular epithelial cell function in PAP-induced nephrotoxicity in rats.

Pheophorbide a is a catabolite of chlorophyll a and often found in food such as abalones, pickles and herb tea. Upon ingestion of food contained pheophorbide a and subsequent exposure to sunlight, humans and animals will develop cutaneous photosensitivity. The molecular mechanism of this photosensitivity is that pheophorbide a, sensitized by sunlight, produces singlet oxygen (reactive oxygen) and this in turn oxidizes lipids and proteins in the cell membrane causing damage to the cells. In search for photoprotective agents, we have screened herbs and vitamin-like substances using photo-oxidized hemolysis as an in vitro model for cutaneous photosensitivity. Red blood cell suspensions from Wistar rats were exposed to visible light in the presence of pheophorbide a with or without test substances. At the end of light exposure, absorbance of the supernatants at 570 nm was measured and hemolysis ratios were calculated. Among ten herbs studied, the Indian herb Bacopa monnieri (Otomeazena) inhibited photo-oxidized hemolysis most effectively. Among vitamin-like substances, carnosine inhibited photo-oxidized hemolysis more effectively than histidine, which is a well known singlet oxygen scavenger. The photoprotective effects of these substances will be further investigated in an animal model.

Focusing on the changes on eating behavior and sleep pattern in Muslim, their adaptation to fasting in the daytime were examined any possibility to lead to adapt in our irregular life style.

Using self-administration questionnaires about body measurements and daily practices (life events and food habit), their changes were compared with before, during, and after Ramadan for Malaysian students who were divided into two, Muslim group and Non-Muslim group. <Research 1> Fasting started, calorie intake and amount of diet such as grain dishes, fish and meat and vegetables of Muslim male (MM) were decreased significantly during Ramadan, however in Muslim female (MF) only grain dishes was decreased significantly. Calorie intake from soft drinks by MM and MF was significantly increased. <Research 2> Sleeping hours of both of MM and MF was shortened significantly during fasting. 90 % of Muslim spent their fasting time with taking naps, 20 % of them did light exercise.

From their changes above and the way of spending the fasting time, it was suggested that effect on eating behavior and sleeps was different by sex. More analyzed and discussed, relations with timing of food intake and quality and quantity of food, alternation of sleepiness.

A vegetarian diet is considered better for prevention and treatment of lifestyle-related diseases. However, zinc and selenium, currently recognized as antioxidants, are abundant in animal food, and so it is necessary to assess the nutritional status of vegetarians with respect to these nutrients. We evaluated the serum levels of zinc, selenium, superoxide dismutase (SOD), dietary intake, and other elements in 13 non-vegetarian and 11 vegetarian individuals in 2007. In addition, we put the data in our 2002-2003 study on 58 non-vegetarian and 31 vegetarian individuals together. So we analyzed the dietary intake and constituents of serum in 113 subjects. We found that the intake and serum levels of zinc and selenium were lower in the vegetarian than in the non-vegetarian individuals. Although the difference was not significant, the serum SOD was also lower in the vegetarians. Accordingly, the deficient intake of some nutrients by the vegetable diet was considered to negatively affect the health. However, there were more intake of antioxidants, such as β-carotene and manganese, in the vegetarian than in non-vegetarian diet, and the positive effect of these elements was noticed.

Although the vegetable diet is reportedly an effective method from the viewpoint of obesity control and lifestyle-related disease prevention, we think that it is necessary to consider well the trace nutrients intake in this vegetarian diet.

It has been about ten years since drinking water was developed originally from Deep-Sea Water which was made for the first time in the world. However, different methods of making drinking water from clean and rich in minerals Deep-Sea Water that is very valuable for use cause to make water including different mineral composition and relations to health for each. The authors succeeded development of refining sea water from Deep-Sea Water using trehalose firstly in the world, which was made from Muroto Deep-Sea Water with the original method. This study are reported about the use of trehalose/refining sea water from Deep-Sea Water complex as follows ; 1) response of magnesium readministration and effect on blood glucose level to animal and human 2) the possibility of application to food for individuals with mastication and swallowing difficulty, which is demanding better food in clinical business.

Calcineurin (CN) is a Ca²⁺/calmodulin-binding protein serine/threonine phosphatase that plays a pivotal role in a variety of cellular functions, such as immune and nerve systems in our body. It is well known that the phosphatase activity is stimulated by Ni²⁺ and Mn²⁺ ions in vitro . Previously, we examined the effect of three types of vanadium ion, orthovanadate ions (VO₄^3-), metavanadate ions (VO₃^-) and vanadyl ions (VO²⁺) on Ni²⁺-stimulated phosphatase activity of CN. We have found that Ni²⁺-stimulated CN activity was inhibited by VO₄^3-, VO₃^- or VO²⁺. In the present study, we further examined effect of vanadium ions (VO₄^3- or VO₃^-) inhibition on the kinetics of Ni²⁺-stimulated CN activity. It has been shown that VO₄^3- and VO₃^- inhibitions of Ni²⁺-stimulated phosphatase were competitive inhibition using Lineweaver-Burk plot. Inhibition constants (Ki) for VO₄^3- and for VO₃^- were seen at 4.76 μmol/L and 7.16 μmol/L, respectively.

Chemical composition in various domestic and foreign commercially grown oysters were examined on 2005 to 2007. Specimens of Crassostrea gigas (Japan, U.S.A., New Zealand and Australia), Crassostrea nippona (Japan), Crassostrea sikamea (U.S.A.), Crassostrea virginica (U.S.A.), Ostrea edulis (U.S.A.) and Ostrea lurida (U.S.A.) were used, and similar shell sizes of them were selected. After stripping the shell, the samples were weighed and homogenized, and then the moisture, zinc (Zn), copper (Cu), iron (Fe), cadmium (Cd), calcium (Ca), magnesium (Mg), (atomic absorption analysis), taurine (HPLC) and glycogen (phenol-sulfuric acid colorimetric method) were determined. In the samples of Crassostrea gigas, differences in the chemical composition were recognized based on the year collected. In specimens from Japan and New Zealand, Zn and Cu concentrations were higher than those in specimens from Australia and U.S.A.. The Cd concentration in oysters from U.S.A. was higher than that in those from Japan and New Zealand. Moisture showed an inverse correlation with taurine. There was a strong correlation between Zn and Cu, but not between Cd and Cu or between Cd and Zn.

Arsenic accumulation in Akamoku, Sargassum horneri, a member of the Phaeophyta Family, was determined throughout the whole plant by thermal neutron activation analysis. We collected Akamoku plants at a sea coast along Ise Bay, where Hijiki plants have also been harvested. The arsenic distribution in the plant body was not uniform along the stem as found in Hijiki plants ; however, the arsenic concentration was higher than the level in the Hijiki plants harvested at the same coast. The arsenic concentrations were discussed comparing the characteristic distributions in the two species.

Commercial dried Hijiki, Sargassum fusiforme¹⁾, was soaked in 30 volumes of water for 20 min at various temperatures (0-75°C). The total amount of arsenic was determined by thermal neutron activation analysis and the types of the arsenic compounds released were determined by an ICP-MS instrument equipped with HPLC.

The ratios of the total arsenic amounts retained in the swollen Hijiki tissues to those released into water indicated validity of the water-soaking process as a method to diminish arsenic levels in Hijiki. The higher the temperature, the more arsenic was extracted from the Hijiki tissues within a short time. Out of the arsenic compounds extracted at 30°C, 60 % was arsenate and the rest (40 %) was an organic arsenic compound, X1, having a chromatographically corresponding retention time to arsenobetaine. Other components were less than a few percentage of the total arsenic.

Those arsenic compounds seem to exist in a dispersed form in the tissues, because the observation under a scanning electron microscope did not show any peak of arsenic by line analysis, selected area analysis and/or particle analysis.

In this study, for the development of a new way of using crushed fish bones (which contain many minerals) in a recycling society, mineral distribution in the body of bonito and calcium extraction from bone were examined. A large amount of calcium (15,000 mg/100 g) in crushed bone, and, also, much potassium in a blood-colored fish known as "chiai" (710 mg/100 g) were contained in bonito. Furthermore, iron and calcium were abundantly contained in the head of mackerel (28.8 mg and 7,200 mg/100 g, respectively). The iron concentration of the "chiai" was as same as in the head of mackerel. The calcium of the bone was not extracted in hot water under high pressure, but easily extracted in a solution of brewing vinegar (4.8 % acetic acid) and 30 % acetic acid. A difference was not seen in the amount of calcium extraction between the concentrations of acidity. From these findings, we demonstrated that crushed fish bones are an important nourishment source because a lot of minerals such as calcium and magnesium are contained in bonito bone and mackerel head. Further studies will be needed to develop new foods using crushed bone.

Trace elements including magnesium (Mg), aluminium (Al), calcium (Ca), manganese (Mn), iron (Fe) and zinc (Zn) in coffee samples were measured by ICP-Atomic Emission Spectrometry (ICPS1000IV). Twenty four coffee bean samples were collected from 12 different areas in Indonesia and Guatemala.

Coffee bean samples were classified into two geographic origins with 91.67 % correction rate by discriminant analysis.

Selenoamino acids in selenium (Se)-enriched foods were identified by gas chromatography-mass spectrometry (GC-MS) after derivatization using a commercial amino acid analysis kit (EZ : faast^TM). After the derivatization, a compound eluted at the same retention time as derivatized Se -methylselenocysteine (MeSec) in GC was detected in Se-enriched Kaiware radish sprouts and Se-enriched garlic bulb. Mass spectrum of this derivatized compound was coincident with that of derivatized MeSec ; MeSec was identified in these Se-enriched foods. Similarly, selenomethionine was identified in Se-enriched yeast using EZ : faast^TM and GC-MS. Analysis by high performance liquid chromatography-inductively coupled plasma mass spectrometry showed a presence of selenohomolanthionine (SeHL) in Se-enriched mung bean sprouts. However, SeHL could not be analyzed by GC-MS after the derivatization by EZ : faast^TM.

Molybdenum cofactors contain molybdenum and sulfur atoms as functionally essential components. These cofactors are required for the activities of the molybdoenzymes. It has been shown that CsdA, one of 3 cysteine desulfurases in Escherichia coli, can efficiently transfer sulfur from l-cysteine to the C-terminal thiocarboxylate of the smaller subunit of molybdopterin synthase in a defined in vitro system to generate the dithiolene group of molybdopterin from precursor Z. Precursor Z is the immediate precursor of molybdopterin in molybdenum cofactor biosynthesis. In this study, we found that a csdA-deletion strain of E. coli does not accumulate compound Z, a direct oxidation product of precursor Z, unlike a ΔmoaD strain. Although a two-dimensional PAGE analysis suggests that csdA is involved in l-cysteine metabolism, an assay of a molybdenum-dependent enzyme, namely, sulfite oxidase, revealed that the enzyme activity was not markedly different among the ΔcsdA, ΔsufS, and wild-type strains. In contrast, the activity of sulfite oxidase was greatly decreased in the ΔiscS strain. These results suggest that CsdA is not essential for the biosynthesis of molybdenum cofactor and IscS is a possible candidate for the physiological sulfur-donating enzyme in molybdopterin biosynthesis.

AIM : To analyze the dynamic state of intracellular calcium and the mechanism for induction of apoptosis in olfactory neurons and astrocyte-like RCR-1 cells induced by the exposure to tributyltin (TBTC).

METHODS : Astrocyte-like RCR-1 cells and primary cultures of olfactory neurons were cultured in a serum-free DMEM medium and exposed to TBTC. The intracellular calcium of olfactory neurons or astrocyte-like RCR-1 cells was labeled using the calcium specific fluorescent dye Fluo3-AM, and examined before and after administration of TBTC, utilizing confocal laser scanning microscope (CLSM). Apoptotic cells were further analyzed by flow cytometry.

RESULTS: Intracellular concentration of calcium increased within 5 min (as noted by fluorescent intensity) of exposure to TBTC and cytosolic calcium was transported to the nucleus in olfactory neurons or RCR-1 cells. Finally, caspase-dependent apoptosis occurred.

CONCLUSION : The TBTC-induced significant increase in calcium may be associated with caspase-dependent cell death. This calcium is a critical messenger in the apoptotic pathway. Further investigation is warranted to examine the TBTC-induced calcium-dependent apoptotic pathway and the apoptotic signaling messenger for olfactory neurons and astrocyte cells.