In order to regulate contact sensitizers in commercial products, not only is identification of chemical allergens essential but also quantitative evaluation of their allergenicity. We have so far reported threshold values of chemical allergens for both induction and challenge phases in the modified guinea pig maximization test (GPMT). In this study, we examined N-(1-methylheptyl)-N'-phenyl-p-phenylenediamine (MHPPD), an antioxidant for rubber products, and 2-(thiocyanomethylthio) benzothiazole (TCMTB), a biocide for plastic and wood products. MHPPD and TCMTB exhibited distinct profiles of contact allergenicity with the GPMT; that is, both could sensitize guinea pigs by the second induction procedure of topical application alone, even without the first induction procedure of intradermal injection. The calculated challenge threshold values for MHPPD and TCMTB were 0.9 and 0.8 ppm, respectively, which were at least one tenth lower than those of chemical allergens previously examined by us. Octanol-water partition coefficients (log p) used as an index of skin permeability were not necessarily higher for MHPPD and TCMTB than those of other allergens, thus, the skin permeability of these compounds did not seem to be related to their ability to cause sensitization by topical application alone or their low challenge threshold values. The results suggest that the sensitizing potential of both compounds is due to their molecular basis. Cross-reactivities among homologues of both allergens were examined to evaluate their antigenic determinants.

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The adduct formation of 2'-deoxyguanosine (dG) with L-adrenaline under biomimetic conditions (pH 7.5, 37°C) with or without oxidant (MnO₂) was demonstrated in order to clarify the reaction mechanism and the structure. At least two adducts have been observed by liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ion trap MS) and LC-photodiode array detection (LC-PAD) (compound a: more polar than dG, m/z 463(M+H)⁺, λmax: 230, 320 nm; compound b: less polar than dG, m/z 445 (M+H)⁺, λmax: 220, 240, 320, 405 nm). Compound a appeared only in the early stage of the reaction prior to formation of compound b.

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Selenoprotein P is a selenium-rich extracellular protein and is the major selenoprotein in plasma. Although the biological function of selenoprotein P has not been established, we have recently shown that selenoprotein P has phospholipid hydroperoxide glutathione peroxidase-like activity. To study the structure and function of selenoprotein P, we produced monoclonal antibodies against human selenoprotein P. Immunization of rats with purified selenoprotein P was followed by hybridization, cloning, and the establishment of eleven hybridomas producing specific antibodies against human selenoprotein P. With the addition of six kinds of insoluble rat anti-human selenoprotein P monoclonal antibodies to human plasma, the selenium concentration of the supernatant was decreased to 47% of the control. This suggests that selenoprotein P constituted 53% of total plasma selenium. Western blot analysis of the immunoprecipitate from human plasma showed that the antibodies specifically bound to a 69 kDa protein, representing selenoprotein P. Next, we developed an enzyme-linked immunosorbent assay for selenoprotein P using two monoclonal antibodies. The plasma concentration of selenoprotein P in 77 normal individuals was 5.3 ± 1.1 μg/ml. Because preferential depletion of selenoprotein P by low density lipoprotein (LDL)-apheresis has been reported, we next measured selenoprotein P concentration of plasma samples from patients before and after LDL-apheresis. We confirmed that the plasma concentration of selenoprotein P was decreased from 5.7 to 2.3 μg/ml by LDL-apheresis. This result shows that this assay provides a reliable means of measuring the content of selenoprotein P in plasma.

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The phylogenetic composition and physiological activity of bacterioplankton communities in two different rivers in Southeast Asian countries, the Kelang River basin in Malaysia and Chao Phraya River in Thailand, which are polluted by untreated or incompletely treated sewage, were analyzed by fluorescent in situ hybridization (FISH) and FISH combined with the direct viable count technique (DVC-FISH). The results were also compared with those from temperate zone habitats in eutrophic rivers in Osaka, Japan. FISH detected 56% to 78% of total cells with the probe EUB338 targeted for the domain Bacteria in samples from the Kelang River basin, compared with 14% to 33% in samples from the Chao Phraya River. DVC-FISH with an antibiotic cocktail increased the fraction of bacteria detectable with EUB338 in the Chao Phraya River (72% to 75% of total bacteria), while no appreciable change was found in samples from the Kelang River basin. These results show that in situ physiologic activity of resident bacteria was generally high in the Kelang River basin and low but present in the Chao Phraya River. Bacterial community structures in both rivers were dominated by the beta (5% to 39%) and gamma (4% to 41%) subclasses of Proteobacteria. In river water samples from Osaka, bacterial community structures determined by FISH were dominated by the beta subclass, but those determined by DVC-FISH were dominated by both beta (26% to 39%) and gamma (17% to 47%) subclasses. This result implies that in situ physiological activity of the gamma subclass is low in the eutrophic river in Osaka, but those bacteria have the potential for cell division.

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Absorbed dose rates in air (dose rate) from terrestrial gamma radiations obtained using a scintillation survey meter ranged from 13.8 to 187 nGy/h depending on the geological features in Kochi Prefecture. Mean dose rates of four geological belts were found in the following order; Alluvial Deposits < Sambagawa Belt = Chichibu Belt < Shimanto Belt. The mean dose rate of the prefecture was about 60 nGy/h, which was 20% higher than that of Japan as a whole. The mean effective dose rate per person in this prefecture was about 0.37 mSv/y, which was the same as the Japanese mean. The ratio of the mean dose rates of ⁴⁰K, nuclides of the thorium series and the uranium series in the soil determined using a germanium detector were 2 : 2 : 1. The relationship between ⁴⁰K, ²⁰⁸Tl and ²¹⁴Bi was positive, and therefore ⁴⁰K, nuclides of the thorium series and the uranium series were considered to have the same behavior in the soil. ¹³⁷Cs in the soil ranged from 1.4 to 150 Bq/kg with a mean of 24.5 ± 23.1 (S.D.) Bq/kg, suggesting that we continue to be under the influence of nuclear tests in the atmosphere.

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Three polychlorinated ethylenes (CEs), tetrachloroethylene (PCE), trichloroethylene (TCE) and 1, 1-dichloroethylene (DCE), were comparatively studied for their effects on the expression of cytochrome P450 (CYP) mRNA forms in the liver and lung using 7-weeks-old male Wistar rats. The animals were i.p. inoculated with 0.5 g/kg of the individual CEs and sacrificed at 6-hr intervals for 30 hr for the determination of the mRNA levels of hepatic and pulmonary CYP2B and hepatic CYP2E1. A 3.5-fold increase in the expression of hepatic CYP2B mRNA was noted transiently at 6 hr in the presence of PCE. In contrast, 1, 1-DCE was suppressive and started within 6 hr and lasted for more than 30 hr, with a trough value of 15% of the control being observed around 12 to 18 hr, while there was no marked effect observed in the case of TCE-treatment. The expression of pulmonary CYP2B mRNA was severely suppressed in the presence of 1, 1-DCE during the entire observation period as was the case with its hepatic counterpart, while the temporarily enhanced expression of mRNA at 6 hr by PCE and TCE was followed by a moderate suppression, with the trough values of ca. 80 and 65% of the control, respectively, at 18 hr. Concerning the hepatic CYP2E1, the expression of mRNA was adversely affected by all the CEs with a descending order of magnitude of 1, 1-DCE, TCE and PCE with peak inhibition values of 85%, 50% and 35%, respectively. The nonselective suppressive effect of 1, 1-DCE on the expression of divergent CYP mRNAs was well correlated with the IL-1β-dependent suppression of various types of CYP mRNA in primary cultured hepatocytes previously reported. Although PCE and TCE are under stringent legislative restriction in Japan as environmental pollutants owing to their relatively stable natures, they are less toxic in terms of the inhibition of the mRNA expression of CYP forms in acute phases than short-lived 1, 1-DCE, which is inflammatory to the host animals; the shorter the environmental life span (1, 1-DCE > TCE > PCE), the more severe the acute toxicity.

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Metallothionein (MT) is known to play an important role in the resistance of tumor cells to cisdiamminedichloroplatinum (II) (CDDP, cisplatin). To identify non-MT factors that play important roles in CDDP resistance, we established CDDP-resistant cell lines from simian virus 40-transformed MT null cells. Subclones of CDDP-resistant MT null cells, designated as MKCr-3, -12, and -18, exhibited 24- to 62-fold resistance to CDDP compared with parental cells. The contents of glutathione (GSH) and the activities of GSH-related enzymes and antioxidant enzymes in MKCr-12 and -18 were higher than those in parental cells. However, MKCr-3 cells did not show any significant increase in the levels of GSH nor in enzyme activities except for superoxide dismutase. Accumulation of platinum in CDDP-resistant subclones was 15-26% of that in parental cells 24 h after administration of CDDP. Eleven other subclones of CDDP-resistant MT null cells also exhibited low accumulation of platinum. These results suggest that the decreased accumulation of CDDP may be a predominant factor in the resistance to CDDP in the absence of MT, an intracellular metal- and free radical-scavenger.

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Airborne particulates were collected during the same periods in downtown Kanazawa, Sapporo and Tokyo and polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs) were determined together with several heavy metal elements and water-soluble inorganic ions. The mean concentrations of metal elements and inorganic ions were the highest in Tokyo, followed by Sapporo and the lowest in Kanazawa both in winter and summer, and were strongly dependent on the amount of airborne particulates. These tendencies were different from the cases of PAHs and NPAHs. Correlations between the organic compounds (PAHs and NPAHs) and several inorganic pollutants were strong only in winter samples of Sapporo, but not so strong in Kanazawa and Tokyo. Because PAHs and NPAHs and several combustion-origin inorganic substances are, in general, detected mainly in the fine airborne particulates, it was reasonable that the significant correlations were found only in the winter samples of Sapporo where the generation of the coarse and crustal particulates were suppressed by snow or ice. Though the atmospheric concentrations of PAHs and NPAHs in Kanazawa were much lower than in Sapporo, the correlation among the NPAH compounds and between PAHs and NPAHs were surprisingly similar in these two cities. The strong correlations indicated that the atmospheric behaviors of the PAHs and NPAHs studied were similar in the two cities and that the source of PAHs and NPAHs might be the same. On the contrary, strong correlations were not observed in Tokyo, suggesting the possibility of multiple sources of formations/degradations of PAHs and NPAHs.

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The content of 9 types of isoflavonoids (daidzein, glycitein, genistein, formononetin, biochanin A, coumestrol, daidzin, glycitin and genistin) in 34 domestic or imported raw beans including soybeans, 7 immature beans and 5 bean sprouts consumed in Japan were systematically analyzed. Each isoflavonoid was analyzed in total after acid hydrolysis to the aglycone, and intact individual isoflavonoids were also analyzed without hydrolysis. After the sample clean up, daidzein, glycitein, genistein, formononetin, biochanin A, daidzin, glycitin and genistin were determined by HPLC with a diode array detector and coumestrol was determined by spectrofluorimetry. The content and composition of isoflavonoids varied greatly between soybean sprouts, immature soybeans and mature beans of the same type but of different source. Isoflavonoid content was highest in mature soybeans. The composition of isoflavonoids differed in each growth stage of soybeans. In other beans, the largest content of isoflavonoids was found in mature chickpeas, but this value was less than 1/27 of the isoflavonoid content in mature soybeans. Thus, the contribution of beans other than soybeans to the daily intake of isoflavonoids in a Japanese diet is negligible.

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We present a new selenium (Se) map of Japan in more detail than in our previous report (J. Health Sci., 42, 360-366). It contains 150 measured points, especially in the northeast of Japan. In the new sampling points, we did not find high Se levels and most of the measurements were generally at values below 1 mg/kg. In the Se map of Japan, there are two particularly high measurements, at Mt.Zao (10 mg/kg) and Tateyama Murodo Jigokudani (148 mg/kg). These two areas are the origin of a minor branch of the Abukuma River and the Jouganji River, respectively. We also analyzed the Se levels near two major copper mines, Asio and Besshi. We detected high Se content in rocks at both copper mines and an average Se level in soil from the area near the Asio copper mine. The Se level in soil at Besshi was slightly higher than that at the area near the Asio copper mine. We also measured the Se levels of some pyrites in Japan. The Se levels of most pyrites were some ten mg/kg higher than the general Se level in soil. This study provides information about the fundamental background values of Se levels in soil and rocks to correlate with Se pollution if it occurs in the future

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Both calcined shell calcium, which was recently reported to have bactericidal effects on Escherichia coli O157 : H7, Pseudomonas aeruginosa, and Staphylococcus aureus, and calcium oxide, which is the main component in calcined shell calcium, were examined as a bactericide against Legionella species. A calcined shell calcium solution of 0.025% or above reduced L. pneumophila counts from the original 7.0 × 10⁶ CFU/ml to less than 300 CFU/ml after an incubation period of 1 hr. In water samples taken from the cooling tower of a prefabricated house, a calcined shell calcium solution of 0.05% or above reduced L. pneumophila counts to less than 10 CFU/ml after an incubation period of 1 hr. There was no difference between the bactericidal effects of the calcined shell calcium manufactured from the shells of surf clams and that manufactured from oysters; surf clams and oysters are equally effective. Calcium oxide also showed similar bactericidal effects against L. pneumophila, and thus we believe that the effect is not specific to calcined shell calcium, but rather to effects caused by the alkalinity of calcium oxide. The use of calcium oxide or calcined shell calcium as a bactericidal agent against Legionella species in the cooling tower water of hotels or other buildings is therefore expected.

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A rapid enzyme immunoassay (EIA) for cocaine and its main metabolite, benzoylecgonine (BZE), using glucose oxidase (GOD) was established. Drug concentrations in samples were determined by the inhibition test. BZE-conjugated GOD and samples were added into the wells of a 96-well microtest plate coated with anti-BZE monoclonal antibody. After washings, color development was performed by additions of glucose, horse radish peroxidase (HRP) and 3, 3', 5, 5'-tetramethylbenzidine (TMB), which was a non-mutagenic and non-carcinogenic chromogen. The time required for the immune reaction and the subsequent color development was only 12 min in this EIA. The EIA specifically detected cocaine and BZE at the low detection limit, that is, about 45.4 pg/well for cocaine, and 433 pg/well for BZE, respectively. The values were hardly interfered by urine diluted to 10%. The present assay method was rapid, sensitive, simple, and safe, and was easily carried out anyplace a micro-plate reader was available. Therefore, it might be suitable to screen cocaine and BZE in urine at the scene of abuse.

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Biotin-binding human immunoglobulin G (B-IgG) was detected by two different methods in the avidin-coated multiwell format for the first time. In the first method, B-IgG was caught by avidin supported on the solid phase and was detected by alkaline phosphatase (ALP)-labeled anti-human IgG (method A). This method, however, was not able to distinguish B-IgG-positive and -negative human sera. The second method included peroxidase-labeled avidin instead of ALP-labeled anti-human IgG (method B). The sensitivity of method B was 28 times higher than that of method A. Method B in the multiwell-microplate format could detect B-IgG in the IgG fraction purified from human sera for the first time. This suggests that the level of B-IgG in human sera is very low.

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Adsorption isotherms of 10 pesticides and their biodegradation intermediates on clay minerals and soils were investigated to predict the fate of pesticides in the environment. The adsorption isotherms were expressed by the Freundlich isotherm equation. Pentachloronitrobenzene, 2,4,6-trichlorophenyl-4'-nitro-phenylether, and various intermediates were highly adsorbed on soils, although isoprothiolane was only slightly adsorbed. The adsorbabilities of pesticides on ando soil, gray lowland soil, and montmorillonite were higher than those on allophene and kaolinite. These results can be used to study the fate of pesticides.

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