Effects of quercetin and rutin on serum and hepatic lipid concentrations, fecal steroid excretion and their antioxidant properties were investigated in rats by oral administration. No toxic symptom was observed even at the dose of 1.0 g/kg of quercetin or rutin. Serum and hepatic lipid concentrations and fecal steroid excretion was not influenced remarkably, but serum thiobarbituric acid reactive substances (TBARS) decreased dose-dependently with the administration of quercetin or rutin. The decrease of serum TBARS was significantly correlated with the increase of serum free flavonoids (p < 0.05-0.001). Serum flavonoid concentrations, especially free quercetin, were higher in rutin-administered rats than in quercetin-administered rats at doses of 1.0g/kg for 10 d (p < 0.05-0.001). When 1.0g/kg of quercetin or rutin was administered in a single dose, they remained in the blood as aglycone or their conjugates of quercetin and isorhamnetin, even three days after administration. Recovered flavonoids were only 0.13% and 0.89% in urine for 3 d and 0.03% and 0.13% in serum on day 3 by administration of quercetin and rutin, respectively. Thus, some part of the administered quercetin or rutin was metabolized and showed antioxidant property, but had no remarkable influence on serum or hepatic lipid concentrations or fecal steroid excretion in rats.

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In-and outpatients with atopic dermatitis and their families were advised to lower the n-6/n-3 ratio of fatty acids of patients' foods throughout one year. Basic nutritional recommendations were to eat traditional Japanese foods, that is, more seafood than meat, and to omit all kinds of high-linoleic acid (n-6) vegetable oils and their products (including fried cookies); use of perilla oil as a cooking oil with a very low n-6/n-3 ratio was advised. Topical steroidal anti-inflammatory drugs were used at the same time but were decreased toward the 3rd month of treatment. The n-6/n-3 ratio of serum lipids decreased significantly, atopic dermatitis area and severity index (ADASI) decreased dramatically and blood eosinophil counts decreased significantly, but the levels of serum IgE, total protein, total cholesterol, hemoglobin, calcium and iron were relatively unchanged. Kampo medicines were also used for some patients with weak constitution, but beneficial effects have been neither proved nor disproved during the year of treatment. Infants were more susceptible to this treatment than adults. Although a longer-term follow-up is necessary, the method was found to be promising and safe for the treatment of atopic dermatitis.

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Chlorinated ethylenes (CEs) such as tetrachloroethylene (PCE), trichloroethylene (TCE), 1, 1-dichloroethylene (1, 1-DCE), cis-1, 2-dichloroethylene (cis-DCE), and trans-1, 2-dichloroethylene (trans-DCE) are members of the class of volatile halogenated hydrocarbons. Each was administered intraperitoneally at 0.5g/kg alone or simultaneously with phenobarbital (PB, 80mg/kg/d) to male Wistar rats weighing about 200g (7-weeks-old). Microsomal fractions of livers and lungs removed from animals sacrificed 24h after treatment were tested for monooxygenase activities and protein content of 4 cytochrome P450 (CYP) forms, i.e., CYP1A1/2, 2B1/2, 2E1 and 3A1/2. In terms of constitutive expression, they were compensatory in liver and lung with CYP1A1/2, 2E1 and 3A1/2 being detected only in liver and CYP2B1 only in lung. All 5 CEs employed in this work suppressed hepatic CYP1A1/2, 2E1 and 3A1/2 in the descending order of 1, 1-DCE > cis-DCE > trans-DCE > TCE > PCE. The magnitude of suppression of pulmonary CYP2B1 by CEs were compared as follows; PCE > trans-DCE > 1, 1-DCE > cis-DCE > TCE. CYP1A1/2, CYP2E1 and CYP3A1/2 in hepatic microsomes from PB-treated animals responded to CEs similarly to those from PB-untreated animals. These 3 enzyme activities could not be detected in pulmonary microsomal fractions, irrespective of the PB-treatment. Hepatic CYP2B1/2 could be detected only when treated with PB in marked contrast to the constitutive expression of pulmonary CYP2B1. The suppression of PB-induced hepatic CYP2B1/2 was observed with all 5 CEs, with special emphasis on complete inhibition with 1, 1-DCE. The adverse effects of TCE and PCE on pulmonary CYP2B1 from PB-treated rats were comparable with those from PB-untreated animals. In contrast, 3 dichloro-isomers were more suppressive to the enzyme in the absence than in the presence of PB. The protein levels of hepatic CYP forms were generally proportional to the enzyme activities in the case of 1, 1-DCE-treatment. The amounts of pulmonary CYP2B1 apoprotein were in good accordance with the enzyme activities when treated with CEs individually.

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Cs uptake in Streptomyces lividans TK24 and Streptomyces sp. TOHO-2, as representatives of actinomycetes, was investigated. Growth of S. lividans TK24 was inhibited above 50 mM CsCl in yeast extract-malt extract agar, while that of Streptomyces sp. TOHO-2, which was isolated from soil as a Cs tolerant strain, was inhibited above 200 mM CsCl. The amount of Cs incorporated into the mycelia of these strains increased with the concentration of Cs in the medium; S. lividans TK24, grown in the presence of 25 mM Cs, accumulated 61.5 mgCs/g dry weight of mycelia, and Streptomyces sp. TOHO-2, grown in the presence of 150 mM Cs, accumulated 142.2 mgCs/g dry weight of mycelia. Scanning electron microscopy (SEM) of the substrate mycelia grown on the YM agar plate containing CsCl showed white spots with a similar distribution of mycelia. Elemental analysis using SEM-energy dispersive X-ray analysis showed that there were larger amounts of Cs, P and O in the brilliant spots than in other regions of the cell. Streptomyces lividans TK24 and Streptomyces sp. TOHO-2 could grow even in the presence of high concentrations of Cs, and accumulated high concentration of Cs in the cells.

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The effect of experimental diets with fermented soybean (natto) containing isoflavone and zinc on ovariectomy (OVX)-induced bone loss was investigated. Sham-operated rats or OVX rats were given experimental diets containing soybean protein for 3 months, and other OVX rats were fed dietary natto, with or without calcium, calcium plus zinc, calcium plus isoflavone or calcium plus zinc and isoflavone for 3 months. Experimental diets contained 2.1 to 9.7mg zinc per 100g and 44.6 to 92.4mg isoflavone (including genistin, genistein, daidzin, and daidzein) per 100g. OVX caused a significant reduction in the dry weight and mineral density of the femur. Also, the calcium content, zinc content and alkaline phosphatase activity in femoral-diaphyseal and metaphyseal tissues was significantly reduced by OVX. These reductions were largely prevented by feeding natto diets. This prevention was significantly enhanced in OVX rats fed natto diets supplemented with isoflavone and zinc. This study demonstrates that the prolonged intake of dietary natto supplemented with isoflavone and zinc has a preventive effect on OVX-induced bone loss, suggesting that it may have a role in the prevention of osteoporosis.

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We showed previously that orally administered bisphenol A (BPA) easily crosses the placental barrier and enters the fetus. However, BPA glucuronide transport and metabolism in the fetus was not studied. We examined the transport of orally administered BPA and BPA glucuronide into mature rat testis and fetus of pregnant rats. After administration of an oral dose of 10mg BPA per kg body weight to pregnant female rats, BPA glucuronide in the fetus was not detected. BPA glucuronide does not easily pass through the placental barrier. One hour after oral administration of 10mg BPA per kg body weight to mature male rats, approximately 90% of the BPA was present as BPA glucuronide in both blood plasma and testis. Although the concentration of free BPA in blood plasma decreased gradually, free BPA in the testis had increased slightly 8 h after administration. Eight hours after oral administration of BPA, BPA glucuronide gradually decreased in rat testis. In contrast, following oral BPA administration, blood plasma BPA glucuronide decreased to 55% of the maximum observed concentration after 3h, but then increased to 100% of the maximum observed concentration after 8h. These results suggest that BPA easily passes through the testicular barrier, is converted by UDP-glucuronosyltransferase to BPA glucuronide, and gradually breaks down to BPA by β-glucuronidase.

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Stimulated phospholipid degradation was observed in liver homogenates harvested from rats who had received an injection of carbon tetrachloride (CCl₄). When the liver homogenates obtained from CCl₄-treated rats were incubated for 1 h at 37°C, approximately half of the phosphatidylethanolamine (PE) was hydrolyzed, producing a stoichiometrical amount of lysophosphatidylethanolamine (LysoPE). The homogenates obtained from control rats showed only poor hydrolytic activity toward endogenous PE. The fatty acid composition of lysoPE produced was essentially identical to that at the sn-1 position of PE, suggesting that A₂ type of phospholipase (PLase A₂) was involved in the degradation during incubation. The hydrolysis of endogenous PE was dependent on calcium ion. The hydrolytic activity was almost exclusively associated with the membrane fraction which was precipitated by centrifugation at 10000 × g, and did not require the cytosol fraction. Para-bromophenacylbromide, rabbit antibody against rat 14 kDa type II PLase A₂, and thielocin A₁, a specific inhibitor of type II PLase A₂, suppressed the hydrolysis almost completely. These results indicate that the accelerated degradation of endogenous PE observed in liver homogenates of CCl₄-treated rat may be mainly due to the activity of the enzyme closely related to type II PLase A₂.

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One of the urgent tasks in understanding endocrine disruptors (EDs) is to compile a list of suspected substances among the huge number of chemicals by using the screening test method. We developed a simple and rapid screening method using the yeast two-hybrid system based on the ligand-dependent interaction of nuclear hormone receptors with coactivators. To date, we have tested the estrogenic activity of more than 500 chemicals including natural substances, medicines, pesticides, and industrial chemicals. 64 compounds were evaluated as positive, and most of these demonstrated a common structure; phenol with a hydrophobic moiety at the para-position without bulky groups at the ortho-position. These results are expected to facilitate further risk assessment of chemicals.

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In order to obtain information on the relationship between calculi and urine-crystal constituents, component analyses of the biphenyl sulfate conjugates in urine and urine crystals in rats fed a diet containing biphenyl and KHCO₃ were performed by LC-MS/MS and FT-IR. LC-MS/MS analysis revealed the presence of biphenyl metabolites, i.e., three isomers of monohydroxy-biphenyl-O-sulfate (HBPOS) and five isomers of dihydroxy-biphenyl-O-sulfate (DHBPOS), in rat urine. The same results were obtained for the crystals in rat urine. These findings suggested that the metabolism of biphenyl resulting in the formation of sulfate conjugates follows the process : biphenyl → monoor dihydroxylation → sulfate conjugation. FT-IR analysis of the urine crystals indicated that the major constituent was the potassium salt of 4-hydroxy-biphenyl-O-sulfate (4-HBPOS), which is known to be the main constituent of urinary bladder calculi. Consequently, in view of the similarity of the major constituent, the potassium salt of 4-HBPOS, in both the urine crystals and calculi, it is thought that the formation of the calculi can be attributed to the lower solubility of the potassium salt of 4-HBPOS, as compared to the other sulfate conjugates.

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An o-Tyrosine method for detection of irradiation of foods was studied by HPLC using a novel light amplification by stimulated emission of radiation (LASER) fluorometric detection system with pre-column reaction. Sample was prepared and purified by eliminating fat and sugars using a mixture of acetone and chloroform, and then the purified protein was hydrolyzed using hydrochloric acid at 110°C for 24h in a vacuum. The sample was reacted with 4-fluoro-7-nitrobenzofurazan (NBD-F) reagent by an automatic pipetting system and was introduced into the HPLC system. Irradiated chicken, pork, beef, and tuna were examined by irradiating at 0, 1, 5, 10 kGy. Irradiation of chicken and pork irradiated at or over 10 kGy was successfully detected, but that of beef and tuna were more difficult to detect. After 3 months storage at-20°C, the irradiation was still detectable in chicken irradiated at 10 kGy. Thus this detection procedure can be used to detect irradiation in some chilled meats irradiated at 10 kGy. Non-irradiated o-tyrosine formation and reduction of o-tyrosine by hydroxylation are also discussed.

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A monoclonal antibody (MAb-4A4) against Δ⁹-tetrahydrocannabinolic acid (THCA) was prepared and its cross-reactivity analyzed with various cannabinoids including tetrahydrocannabinol metabolites by competitive enzyme-linked immunoassay (ELISA). Δ⁹-THC (1-100μg/ml) could be determined by the ELISA. Many metabolites of Δ⁸-THC and Δ⁹-THC reacted with the antibody, and their cross-reactivities were 44-157% of Δ⁸-THC. However, the antibody did not recognize the lipophilic compounds cholesterol, testosterone, β-carotene, androstene-3, 17-dione or an endogenous cannabinoid, anandamide.

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Rare earth metals (REMs) are widely used in several emerging technologies, however, little is known about their biological effects. In this study we examined the effects of REMs, including lanthanum (La), neodymium (Nd), gadolinium (Gd), terbium (Tb) and ytterbium (Yb) on the testes of mice. Mice were treated i.v. with a single dose of 20 or 200μmol REM/kg as chloride. The testicular weights, lipid peroxidation, and alterations such as hemorrhagic inflammation, were examined 5 d after the administration. The concentration of Ca in the testes was also determined by atomic absorption spectrometer. No significant changes in testicular weights, lipid peroxidation, or alterations were observed by the administration of these REMs at either dose. However, the concentration of Ca in the testes was markedly increased at both doses. There was no significant difference in the Ca concentration between dose levels or among the REMs. These results suggest that the REMs induce increased Ca concentration in mice testes, and this might be a significant aspect of their toxic mechanisms.

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The change in circulating vitamin K₂ (menaquinone7; MK-7) and γ-carboxylated osteocalcin (Gla osteocalcin) concentrations in normal individuals with the intake of fermented soybean (natto) was investeigated. Forty eight volunteers (forty five males and three females) were divided into three groups of sixteen volunteers each (fifteen males and one female), and each group was given sequentially the fermented soybean (natto; 50g) containing three different contents of MK-7 once a day for 14 d as follows : either regular natto with 865μg MK-7/100g of natto, reinforced natto containing 1295μg MK-7/100g, or 1730μg MK-7/100g. Serum MK-7 was not found in normal individuals who had not had natto intake. Serum MK-7 and γ-carboxylated osteocalcin concentrations were significantly raised 7, 10, and 14 d after the start of the intake of reinforced natto containing 1295 or 1730μg MK-7/100g. However, serum γ-carboxylated osteocalcin levels were not significantly elevated by the intake of regular natto, although serum-MK-7 levels were significantly raised. Moreover, serum γ-carboxylated osteocalcin concentration was significantly elevated 14 d after the intake of natto containing either 1295 or 1730μg MK-7/100g, as compared with that of regular natto intake. The present study suggests that the intake of dietary MK-7 in the reinforced natto can stimulate γ-carboxylation of osteocalcin, which plays an important role in bone formation in normal individuals.

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