About half the natural radiation dose to humans is due to radon and its decay products. It is well known that exposure to high levels of radon causes lung cancer, but as yet, there is still little data on the effect of long-term exposure to low levels of environmental radon. The studies by our radon research group at the NIRS are directed toward determining the health effects of low level exposure to environmental radon. In this paper, our current studies on radon are reviewed.

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In addition to effects on the reproductive system, endocrine disruptors are thought to have brain-disrupting actions based on epidemiological and animal studies. In this review, we discuss the current state of studies on the brain-disrupting actions of diethylstilbestrol (DES), which is a prototype endocrine disruptor that has potent estrogen-like action, focusing on our own findings. The major findings concerning the brain-disrupting action of DES are as follows: Exposure to a minute amount of DES during the late stage of gestation, but not the late stage of the lactation period, caused neurobehavioral changes such as impairment of the passive avoidance response in mice offspring. There is a difference in effect between male and female offspring of DES exposure on dendritic arbolization of hippocampal pyramidal neurons. In 6-week-old male offspring, prenatal DES exposure did not affect the responsiveness of N-methyl-d-aspartate (NMDA)- or GABA_A-receptors in the hippocampal pyramidal neurons, but drastically increased the level of phosphorylated Ca²⁺/calmodulin-dependent protein kinase II in the hippocampus. These findings suggest that, in mice, DES has brain-disrupting actions, with a critical period of exposure. Further studies are needed to clarify the primary site and mechanism of these actions, using various methods and animal models such as estrogen-receptor `knock-out' and/or `knock-in' mice.

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Effect of chemical water pollutants on the growth of two Paramecium species (Paramecium caudatum and Paramecium trichium) were examined. The chemicals used as model chemical pollutants include organic solvents, potential carcinogens, mutagens, metabolic modulators, herbicides, insecticides, fungicides, antimicrobials, heavy metals, and heavy metal-containing chemicals. In this study, the IC₅₀ values indicating the concentration of substances inhibiting the proliferation of Paramecium cells by 50% were used instead of the LD₅₀ value, which indicates the dose of substances killing half the population of organisms, since the former is a more sensitive parameter for assessing the toxicity of substances at lower concentrations. Among 25 chemicals examined, di-(2-ethylhexyl)phthalate, potassium dichromate, 2,4-dichlorophenoxy acetic acid, and paraquat stimulated the growth of paramecia depending on the concentrations used. Dimethyl sulfoxide and formaldehyde were shown to be inert to paramecia in the range of concentrations (up to 1%, v/v) used here. Other chemicals were shown to inhibit the growth of the paramecia and thus the IC₅₀ values for those chemicals were determined. Our data presented here may be a useful reference for assessing the impact of water pollutants on aqueous microecosystems consisting of various microorganisms including protozoa.

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The preventive effect of oolong tea on vigil stress was investigated in 55 Chinese women. The subjects received 4 servings of tea bag daily, the tea bags in each containing 2 g dry weight of oolong tea or barley tea, or else water, for a period of one week. The results of a questionnaire indicated that ingestion of oolong tea improved symptoms of stress such as stiffness of the shoulders, fatigue of the eyes and headaches, as well as ameliorated the stress-induced increase in the number of errors in calculation tasks compared with controls. In addition, plasma cortisol levels were significantly lower in the oolong tea group (17.84 ± 2.46 μg/dl) than in the barley tea group (21.33 ± 6.47 μg /dl) or water group (22.95 ± 6.98 μg/dl). Ingestion of oolong tea significantly alleviated the vigil stress-induced increase in plasma lipid peroxide levels, which may have been related to the stress-relieving effects of caffeine or antioxidant properties of polyphenols contained in the tea. These findings suggested that oolong tea has anti-stress effect, with no adverse effects on appetite or physical fitness.

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The acute and subchronic toxicity studies of Boerhavia diffusa (B. diffusa) leaves in albino mice and rats were investigated. Phytochemical analysis was also carried out. 500, 1000 and 2000 mg/kg of the aqueous leaf extract were administered orally to the test groups while distilled water was given to the control group. The parameters measured include food and fluid intake, body weight, absolute and relative weight of various organs, haematological parameters [total white blood cell (WBC) and packed cell volume (PCV)], and tests for liver function: glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase and total bilirubin. The lethal dose (LD₅₀) was found to be greater than 2000 mg/kg (p.o.) in both mice and rats. Rats treated with the extract had progressive increase in body weight, which was significantly (p < 0.05) different from control. The aqueous extract of B. diffusa leaves increased both food and fluid intake. There were no significant changes in both the absolute and relative organ weights between the control and the test groups. The liver enzymes and haematological parameters were statistically equal in all the groups. B. diffusa aqueous leaf extract is non toxic in albino rats.

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Previously, we found that the cationic cyanine dye tri-S-C₄(5) uncoupled mitochondrial oxidative phosphorylation by acting as an inducer of the mitochondrial permeability transition (PT). In the present study, the actions of cyanine dyes such as tri-S-C₄(5) and tri-S-C₇(5) on the mitochondrial structures and functions were further characterized. In the presence of inorganic phosphate (Pi), cyanine dyes were found to accelerate mitochondrial oxygen consumption that was partially sensitive to the PT inhibitor cyclosporin A (CsA). However, not only the CsA-sensitive but also CsA-insensitive acceleration of mitochondrial respiration was induced by cyanine dyes; and both of them were found to be associated with the release of mitochondrial cytochrome c. On the contrary, in the absence of Pi, moderate acceleration of respiration was induced by cyanine dyes, but this respiratory effect was not associated with the induction of swelling or the release of mitochondrial cytochrome c. Thus, cyanine dyes were concluded to have 3 different effects on the mitochondrial functions depending on the Pi status.

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Ambient carbon monoxide (CO) levels in most of the cities in Japan have not exceeded the National Air Quality Standard. We investigated whether or not this low level of CO pollution has been positively related to the mortality rate. We used computerized data for Tokyo, 1976-1990, obtained from governmental agencies. Subjects were restricted to those 65+ years old, because air-pollution influences this group the most. Although the CO level was higher in earlier years and was correlated with the sulfur dioxide level, the Poisson regression analysis that included chronological year and sulfur dioxide level as covariates showed a positive association between CO level and all-cause mortality rate: The mortality rate ratios for the CO levels 1.1-1.6, 1.6-2.2, 2.2+ ppm compared with the CO level < 1.1 ppm were 1.017 [95% confidence interval (CI) 1.009-1.026], 1.031 (95% CI 1.020-1.041) and 1.051 (95% CI 1.039-1.063), respectively. Although it is possible that the higher CO levels only displace deaths several days earlier, the results warrant further study as to whether or not further reduction of CO level would improve the mortality rate of the elderly.

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The nematode Caenorhabditis elegans (C. elegans) was used for a multiple-generation toxicity bioassay of phenols. We examined the sublethal toxicity (fecundity and reproduction) of bisphenol A (BisA), 4-(1-adamantyl) phenol (Adp), and 4,4′-(1,3-adamantanediyl) diphenol (AdDP) over five generations using a Nematode Growth Medium (NGM) 1.7% agar plate. In the fourth generation, the phenols affected the fecundity rate of C. elegans at doses 100- to 10000-fold lower than the LC₅₀s. In particular, at 1 nM BisA, the number of worms decreased significantly to about 50% of control (p < 0.05). A comparison of the number of viable worms and eggs suggested that the phenols exert hatching toxicity. In addition, individuals with an abnormality in the vulva, which could not ovulate, appeared on the phenol-containing plates. We confirmed that phenols disrupt reproduction in C. elegans and that the method using NGM agar plates facilitates multi-generational toxicity tests of chemicals that are poorly soluble in water.

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Arsenic-contamination of groundwater has long been reported in Mushidabad district of West Bengal, India. We visited 19 arsenic-affected families and 4 non-arsenic -affected families in that area during 20-22 February 2001 and collected 10 tubewell waters used for drinking and cooking and 89 urines from those families. The arsenic concentrations in waters ranged from 0.64 to 75.5 ppb. The average of arsenite, arsenate, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in urine were 23.1, 59.0, 24.6 and 127.4 ng/ml urine, respectively. The average of total arsenic was 234.1 ng/ml urine. On comparison of the ratio of (MMA + DMA) to total arsenic, the average proportion of (MMA + DMA) was 75.7%, but the proportions were from 4.2 to 57.2% for 11 persons. This result suggests that they might be damaged due to the arsenic-methylating capacity. When selecting the members of A to L families because of using the two same tubewell waters except 5 persons due to the luck of enough arsenic-methylating capacity, there were the good relationships (p < 0.01) between As(III) and MMA, As(III) and DMA, MMA and DMA, and DMA and total arsenic. The relationship between the arsenic concentration in tubewell waters and the averages of the total arsenic obtained from each families was not in agreement (p > 0.05). The result suggests the possibility of the contribution of other sources like foodstuffs regarding to the excretion of arsenic species from the urines.

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The bio-activity of the brownish scale of onion (Allium cepa) was studied together with identifying the active components and addressing the mode of action. A crude MeOH extract (0.5-1.0 mg/ml) showed the inhibitory effects on human platelet aggregation induced by collagen, adenosine 5′-diphosphate (ADP), thrombin and epinephrine. The anti-platelet extract (1.0 μg/ml) rigidified liposomal membranes by acting on the hydrocarbon core more intensively than the surface of membrane lipid bilayers. Serial solvent extractions and chromatographic purifications provided four isolates which were structurally identified as different quercetin dimers (1 and 2), quercetin (3) and quercetin-4′-glucoside (4). The flavonoidal components 1, 3, 2 and 4 (0.5-2 mM) inhibited collagen-induced platelet aggregation in increasing order of intensity. More active 1 and 3 (2 mM) also dissociated the aggregates produced by ADP. The anti-platelet flavonoids (0.25-10 μM) acted on liposomes of the lipid composition resembling human platelets to cause membrane rigidification which was greatest in the order of 1, 2, 3 and 4. The interaction with membrane lipids to modify membrane fluidity appears to be partly responsible for the anti-aggregatory and disaggregatory effects on human platelets. Although the inedible scale of onion is usually regarded as waste, it has the possibility to be a medicinal resource.

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Phytoestrogens, such as daidzein and genistein in plants are suspected as endocrine-disrupting chemicals (EDCs), because their chemical structures are similar to natural or synthetic estrogens, and have estrogenic activity in vitro and in vivo. An EDC study was carried out on the diets of various animals in vivo. However, many of these diets include phytoestrogens and may already possess estrogenic activity. In this study, we evaluated the estrogenic activity of phytoestrogens (such as daidzin, genistin, daidzein, genistein, coumestrol and equol) and the feed diets for experimental animals, such as fish, amphibians and reptiles, towards human estrogen receptors α (hER-α) and β (hER-β), and the genistein and daidzein content in these diets from HPLC analysis. Coumestrol showed the highest estrogenic activity for hER-α and -β in the -S9 test. Equol showed the highest estrogenic activity for hER-α in the +S9 test. The estrogenic activities of coumestrol, equol and genistein were approximately one hundred to two thousand times higher than that of daidzein. Many of these compounds showed higher compatibility with hER-β than with hER-α. A diet for fish from soybean was indicated to contain the highest amounts of genistein and daidzein. Moreover, this fish diet had the highest estrogenic activity for hER-α and -β. The estrogenic activity was found with hydrolysis by β-glucuronidase, showing higher compatibility with hER-β than with hER-α. In addition, correlation between the contents of genistein and estrogenic activities in the diets was found, with the exception of part of the diet. Therefore, this indicates that the genistein content contributes to the estrogenic activity of the diets. These results suggest that in vivo estrogenic activity might be caused by the diet provided to an experimental animal, indicating the necessity for more careful selection of the feeding diet and measurement of estrogenic substances when performing an in vivo screening assay for EDCs.

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A method was developed for the determination of 6-chloro-2,3-dihydrobenzoxazol-2-one (CDHB) generated by the acid decomposition of fenoxaprop-ethyl and fenoxaprop in agricultural products. Fenoxaprop-ethyl and fenoxaprop were extracted from agricultural products using acetonitrile, and the extract was acidified by 0.5 mol/l hydrochloric acid to make CDHB. The CDHB was extracted again into ethyl acetate and cleaned up using Sep-Pak^® Plus Diol and Bond Elut^® AccuCAT cartridge columns. The recoveries from brown rice, wheat, cotton seed, onion, carrot, sweet potato and cabbage exceeded 70% by HPLC (UV). However, soybean, green soybeans and kidney beans showed many interference peaks in the UV spectra, and Florisil column chromatography was necessary for additional purification. The fortified peaks were confirmed by liquid chromatograph/mass spectrometry (LC/MS) with electrospray ionization (ESI), and the CDHB peak was quantitatively determined. Almost the same result was obtained by HPLC (UV) and LC/MS selected ion monitoring (SIM). Consequently, for agricultural products which included many interfering peaks during UV detection, using LC/MS (SIM) significantly improved the quantitative and qualitative analyses and the number of interfering peaks was fewer than by UV detection.

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The arylhydrocarbon receptor (AhR)-dependent induction of 7-ethoxycoumarin O-deethylase (ECOD) activity in HepG2 cells was employed in a longitudinal study of pollution in the Sagami River flowing into Sagami Bay using river sediment extracts. The six sampling points for river sediment were Ogurabashi, Shimomizo, Atsugi, Kurami, Shinomiya, and Hiratsuka (in descending order of distance from the estuary). In samples obtained at the four midpoints along the river, ECOD activity was induced almost to the same extent. Ogurabashi, the point farthest from the estuary, seemed less polluted than downstream areas based on ECOD activity, while a marginal increase in the activity above the baseline was found at the highest concentration in the sample from Hiratsuka. The points where the river sediment caused a marked induction of ECOD activity in HepG2 cells were surrounded by inland industrial areas scattered along the river.

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The deacylation of N-formylanilines and N-acetylanilines to aniline derivatives was examined in rat liver preparations. When N⁴-formylsulfanilamide or N⁴-acetylsulfanilamide was incubated with rat liver cytosol, the deacylated metabolite, sulfanilamide, was formed. These deacylating activities were inhibited by paraoxone and diisopropyl fluorophosphate, inhibitors of formamidase. N⁴-Formylsulfanilamide and N⁴-acetylsulfanilamide were deacylated by formamidase purified from rat liver cytosol. N-Acetyl- or N-formylanilines bearing an electron-withdrawing group at the para position were deacylated to anilines by formamidase. In contrast, anilines bearing an electron-donating group at the para position were formylated to N-formylanilines in the presence of N-formyl-L-kynurenine. Formamidase catalyzed both N-formylation of aniline derivatives, and deacylation of N-formylanilines and N-acetylanilines.

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Rice bran, wheat bran, rapeseed, linseed, okara, and sakekasu were evaluated for correlations between their effectiveness in adsorbing organochlorine compounds such as chloroform, dichloromethane or benzene and the number of spherosomes. Wheat bran contained the highest concentration of spherosomes, with a mean level of 4.37 × 10¹⁰ spherosomes/g. The lowest was sakekasu, with a mean level of 2.28 × 10¹⁰ spherosomes/g. There was a high correlation between removal efficiency and the number of spherosomes.

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The in vitro effects of exposure to tributyltin chloride (TBT), dibutyltin dichloride (DBT), and triphenyltin chloride (TPT) on testosterone production in isolated Leydig cells from neonatal pig testes were examined. These organotin compounds strongly suppressed human chorionic gonadotropin (10 IU/ml) or 8-bromo-cAMP (0.1 mM) induced testosterone production when the cells were exposed to nontoxic concentrations. The suppression of testosterone production was dose-dependent over the concentration range of 0.03-0.3 μM for TBT and DBT, and 0.01-0.3 μM for TPT. These effective concentrations for the suppression of testosterone production were lower than that of the cytotoxic concentrations of the organotin compounds in Leydig cells and were equivalent to the accumulated concentration levels that were previously reported in marine organisms.

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From the viewpoints of decreasing environmental burdens and recycling of materials, the adsorption ability of charcoal from coffee grounds for acidic dye (acid orange 7) removal was investigated by the batch method. Differences in the removal ratio and removal rate of acid orange 7 could be explained by differences in the properties of charcoal such as specific surface area and pore volume. In the relationship between the amount of acid orange 7 adsorbed at each elapsed time and the square root of elapsed time, a good linearity was recognized. Since the good linearity between the acid orange 7 adsorption and the square root of elapsed time was recognized, the intraparticle diffusion of acid orange 7 onto pores of adsorbents was identified as the rate-limiting step in the adsorption process.

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To investigate whether multidrug resistance protein 5 (MRP5) functions as a xenobiotic detoxification factor, we measured MRP mRNA expression levels in metallothionein (MT)-I, II knockout mouse, and the results showed that MRP5b/SMRP, a splicing variant of MRP5, was highly expressed in the liver of MT-I, -II null mice.

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For the accurate quantitation of glutathione (GSH), cysteine (Cys) and cysteinylglycine (CysGly) in human plasma by detection of fluorescence after derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F), a thiol-specific fluorogenic agent and high-performance liquid chromatography (HPLC) , we attempted to identify optimum conditions for the derivatization and treatment of samples. The extent of derivatization with SBD-F of endogenous thiol compounds in human plasma was affected by several factors, such as the concentration of SBD-F and the reaction time. Acidification and deproteinization of plasma immediately after the separation of plasma from whole blood were essential and prevented the determination of inappropriately low values. The method developed in the present study allowed recovery that was closed to 100% for each compound. The mean values determined for 61 healthy individuals were as follows: Cys, 50.1 μM; CysGly, 12.2 μM; and GSH, 5.3 μM.

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Fungal identification methods based on DNA sequence analysis were recently published in Methods of Analysis in the Health Sciences (Eisei-shiken-ho in Japanese) issued by the Pharmaceutical Society of Japan (PSJ) and in the draft second supplement to the 14th edition of the Japanese Pharmacopoeia (JP). We carefully compared and assessed the effectiveness of the two methods of fungal identification. The PSJ method analyzes D1/D2 26S rDNA, while the JP method analyzes the internal transcribed spacer (ITS) 1 region. The former method is superior to the latter in that the identification criterion described in the PSJ method is rationally defined based on the species concept, whereas distinct species can be misidentified as being of the same species using the identification criterion described in the JP method; and there are more sequence data for the D1/D2 26S rDNA regions in the DNA data libraries (DDBJ/GenBank/EMBL) than for ITS1 data. Based on our assessment, we conclude that the PSJ method is superior to the JP method for fungal identification.

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Chondroitin/dermatan sulfate chains consist of a repeating disaccharide unit of glucuronic acid (GlcA)/iduronic acid (IdoA) and N-acetylgalactosamine (GalNAc) with or without O-sulfation at the C-4 and C-6 position of GalNAc and at the C-2 position of IdoA. Lead and cadmium influence the synthesis of chondroitin/dermatan sulfate proteoglycan core proteins in vascular smooth muscle cells when the cell density is high and low, respectively. However, it has been unclear whether the metals influence the synthesis of chondroitin/dermatan sulfate chains. In the present study, it was shown that lead inhibits the formation of GlcAβ1-3GalNAc, GlcAβ1-3GalNAc(4S) and GlcAβ1-3GalNAc(6S) in dense cells, whereas cadmium inhibits the formation of GlcAβ1-3GalNAc(4S) and GlcAβ1-3GalNAc(6S) but increases that of IdoAβ1-3GalNAc(4S) in the sparse cells. The present data support the hypothesis that lead and cadmium may influence the composition of chondroitin/dermatan sulfate in atherosclerotic vascular wall depending on the density of vascular smooth muscle cells.

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The mammalian constitutively active receptor (CAR) is a ligand-activated transcription factor that participates in controlling the expression of cytochrome P450 2B (CYP2B) genes in response to xenobiotics in an organ-specific manner. In the presence of phenobarbital (PB) or PB-type agents, hepatic CYP2B forms are highly inducible. In contrast, PB-dependent increases in the expression of CYP2B activities are rarely observed in the lung. Mature CAR mRNA could be detected in the liver of 7-week-old Wistar rats by RT-PCR, while lung CAR mRNA had a 91 bp extra nucleotide sequence inserted in a coding region of hepatic CAR mRNA. By comparing the full- length sequence of hepatic CAR mRNA, including 5′- and 3′-untranslated region (3′-UTR), with the genomic sequence completed in the present study, the genomic structure was clarified to consist of 9 exons and 8 introns, in which the coding region expanded from exon 2 in close to its 5′-end to the first one-third of exon 9 after 159 bp of 5′-UTR in the most frequently obtained cDNA clones. In pulmonary CAR mRNA, intron 6 was not spliced out, implying that the lack of CAR in the lung might in part result from the incomplete splicing of precursor mRNA during its maturation.

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Thyroxine (T4), one of the thyroid hormones, in adulterated dietary supplements was analyzed using two different methods; enzyme-linked immunosorbent assay (ELISA) and LC/MS. To release T4 from thyroglobulin, samples were first hydrolyzed with proteolytic enzyme, and then the supernatant was diluted and directly analyzed using a commercial free T4 ELISA kit for diagnostic discrimination. In contrast, T4 was extracted with ethyl acetate from the supernatant, and then ethyl acetate layer was evaporated. The residue was dissolved in the mobile phase and analyzed by LC/MS with electrospray ionization (ESI) interface under positive ion mode. These methods were applied to the analyses of 13 dietary supplements advertised as weight reducers. T4 was detected in four of the samples and the analytical results by ELISA agreed well with those obtained by LC/MS. The ELISA technology described here is available for the screening of T4 in adulterated supplements.

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