In order to evaluate the extent of human exposure to mercury in the Amazon, in particular, in the Tapajos River basin, which is a site of extensive alluvial gold mining, we analyzed samples of human hair, blood and urine, as well as fish, collected from different areas for total levels of mercury and methylmercury. In fishing villages, the inhabitants were found to have accumulated mercury, mostly in the form of methylmercury, at abnormally high levels as the apparent result of consumption of local fish, with very little confounding exposure to inorganic mercury, including mercury vapor in the air. The people living near the main gold-mining areas had accumulated higher levels of methlymercury than people living far downstream from the gold-mining sites. Quite high levels of methylmercury were found in samples of hair from inhabitants of the fishing villages.

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Urinary and biliary metabolites of male Wistar rats dosed orally with 2, 4-dinitrobenzyl glucuronide (2, 4-DNB-G) and 2, 6-dinitrobenzyl glucuronide (2, 6-DNB-G) which are major compounds excreted in bile after administration of carcinogenic 2, 4-dinitrotoluene (2, 4-DNT) and 2, 6-dinitrotoluene (2, 6-DNT) were examined by HPLC. The object of this study is to determine whether mutagenic 2, 4-dinitrobenzaldehyde (2, 4-DNBA1) and genotoxic 2-amino-6-nitrobenzyl alcohol (2A6NB) are produced in the secondary metabolism of 2, 4-DNB-G and 2, 6-DNB-G. Data from HPLC indicated that 2, 4-DNAB1 (about1%), in addition to 2, 4-DNB-G (about 8.6%), 2, 4-dinitrobenzyl alcohol (2, 4-DNB, about 0.1%), two aminonitrotoluenes (about 0.2%), two aminonitrobenzyl alcohols (about 0.1%), 4-acetylamino-2-nitrobenzoic acid (4AA2NBA, about 7.4%) and 4-acetylamino-2-aminobenzoic acid (4AA2ABA, about 1.8%) was excreted in the urine or bile after dosing 2, 4-DNB-G. This result, together with previous findings, indicates that 2, 4-DNBA1 is produced not only by oxidation of 2, 4-DNB formed from 2, 4-DNT, but by oxidation of 2, 4-DNB formed from 2, 4-DNB-G excreted in bile. In addtion, the formation of carcinogenic 2, 4-diaminotoluene (2, 4-DAT) was ascertained from the metabolic pathway of 2, 4-DNB-G based on the metabolites detected. No 2A6NB was found in the urine and bile after dosing 2, 6-DNB-G. However, 2-amino-6-nitrobenzoic acid (2A6NBA, about 0.2%), in addition to 2, 6-dinitrobenzyl alcohol (2, 6-DNB, <0.1%) and 2, 6-DNB-G (about 18%), was detected in the urine or bile after dosing 2, 6-DNB-G. This result, together with previous findings, indicates that 2A6NB is an intermediate in the production of 2A6NBA from 2, 6-DNB, and further suggests that the production of 2A6NB in the metabolism of 2, 6-DNT is coupled to the enterohepatic circulation of 2, 6-DNB. The results of this investigation suggest that the production of 2, 4-DNBA1 and 2, 4-DAT, and 2A6NB from 2, 4-DNB-G and 2, 6-DNB-G may play a role in the hepatocarcinogenicities of 2, 4-DNT and 2, 6-DNT.

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A screening method was established for the determination of 27 pesticides in fresh fruits and vegetables by a super critical fluid extraction (SFE), cleaned up with cartridge columns and HPLC. The multiresidue and semiautomatic analysis was useful for a screening examination, because the determination methods for pesticides under the Japanese Food Sanitation Law are mostly individual determinations. Reported methodologies for multiresidue analysis by HPLC were not adequate to regulated pesticides in Japan. In this report, multiresidue determination of pesticides and their metabolites are discussed using SFE and HPLC. Details of the proposed method are as follows : Wet samples such as fruits and vegetables were not suitable for the SFE instrument, so the water in the samples was removed with an absorptive polymer (Arasorb^[○!R] S-310) prior to SFE. The pesticides were extracted by SFE, the extracts trapped with Extrelut^[○!R] NT + Bond Elut^[○!R] C₁₈ and then eluted with acetonitrile. The eluate was cleaned up with Sep-Pak^[○!R] Florisil+Bond Elut^[○!R] PSA cartridges. After washing with n-hexane, the pesticides were eluted from the cartridges with 15% ether/n-hexane, 15 and 50% acetone/n-hexane. These three fractions were individually determined by HPLC with a photodiode-array detector. The pesticides spiked in samples at 0.5ppm showed satisfactory recoveries except for thiabendazole, imazalil and clofentezine. Detection limits were 0.005-0.01ppm for the 27 pesticides.

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Cyanide and thiocyanate levels were determined by head space gas chromatography and the spectrophotometric Konig method respectively, for blood and salivary samples collected from 40 healthy adult volunteers. Blood cyanide (mean±standard deviation : 0.27±0.07μм), plasma thiocyanate (111.2±92.1μм), salivary cyanide (0.66±0.52μм) and salivary thiocyanate (1655±841μм) levels were significantly higher in the group of 20 tobacco smokers than in the group of 20 nonsmokers (0.17±0.04μм, 33.5±25.4μм, 0.38±0.26μм, 542±406μм). Statistical correlations were observed between the blood cyanide and the salivary thiocyanate levels (γ=0.64), between the blood cyanide and the plasma thiocyanate levels (γ=0.54), and between the plasma thiocyanate and the salivary thiocyanate levels (γ=0.46). It is concluded that not only plasma and salivary thiocyanate levels but also blood cyanide level can be suitable indices for distinguishing smokers from nonsmokers.

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Norephedrine and tyramine are familiar as sympathetic nerve-stimulating chemicals. Analysis of norephedrine in urine sample is necessary for the evaluation of intake. Analysis of tyramine in food samples is also necessary for evaluation of its daily intake. However, both are difficult to extract by conventional liquid-liquid extraction. The present work proposed to monitor both using a small amount of a mixture of two solvents. For norephedrine, a filtrate (100ml) of human urine sample (pH11) was mixed with 5% sodium dodecyl sulfate solution (100μl) and acetone (2.5ml), then shaken with chloroform (2.5ml). This method recovered 5-10% of norephedrine added. For tyramine, a filtrate (5ml) of intact or concentrated sample solution (pH11) was salted out with NaCl (1g), then shaken with a mixture of acetone (3ml) and ethyl ether (2ml). This method recovered 50-60% of tyramine added.

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The effects of heavy metal exposure on the immune system were determined by measuring cytokine production of human peripheral blood mononuclear cells (h-PBMCs) from a healthy female. The h-PBMCs were exposed for 3d to CdSO₄, K₂Cr₂O₇, and HgCl₂ at 1, 5, 10μм and to (CH₃COO)Pb at 10, 50, and 100μм. Concentrations of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, IL-6, IL-8, and IL-10 were quantified using the ELISA method. The results showed that cytokines assayed were differentially affected by heavy metal exposure. Chromium significantly increased the production of IL-1β while decreasing the production of IFN-γ, IL-6, IL-8 and IL-10. Mercury at low concentrations increased the levels of TNF-α and IL-1β. At higher concentrations, an opposite effect was seen. Cytokines may function as biomarkers in elucidating the mechanisms of the immunotoxic effects of heavy metals.

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The effect of the supplemental intake of isoflavone and a zinc-containing mineral mixture on bone components in the femoral tissue of rats was investigated. Rats (5 weeks old) were orally administered either vehicle, isoflavone glycoside (5mg/100g body weight), aglycone isofavone (5mg/100g, including 3.772mg daidzein, 0.395mg genistein, and 0.833mg glycitein), zinc-containing mineral mixture (0.833mg zinc, 50mg calcium, 25mg magnesium, and 0.208μg vitamin D₃ per 100g body weight, respectively), or both aglycone isoflavone (5mg/100g) and zinc-containing mineral mixture for 7d. Administrations resulted in a significant increase in femoral dry weight, calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal and metaphyseal tissues. Femoral zinc content was significantly increased by the administration of zinc-containing mineral mixtures with or without aglycone isoflavone. The effect of aglycone isoflavone in elevating bone components was significantly enhanced in combination with the zinc-containing mineral mixture. Moreover, the oral administration of both aglycone isoflavones and zinc-containing mineral mixture to aged rats (50 weeks old) for 21d resulted in a significant increase in femoral dry weight, zinc content, and bone components. This study demonstrates that supplemental intake of both aglycone isoflavone and a zinc-containing mineral mixture has an anabolic effect on bone components in aged rats, suggesting the role of nutrients in the prevention of osteoporosis with increasing age.

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Mushrooms collected from a sub-alpine forest of Mt. Fuji and some other locations in Japan in 1996 were analyzed for radiocesium. The ¹³⁷Cs concentrations in 37 mushrooms varied widely from 1.6 to 783 Bq kg ^-1 fresh wt. The characteristics of Cs accumulation were analyzed by culturing fruiting bodies of the edible mushroom Pleurotus ostreatus (Fr.) Kummer Y-1 (P. ostreatus Y-1). The ¹³⁷Cs and stable Cs accumulation expressed as the concentration ratio (CR, ¹³⁷Cs or Cs concentration in the dried fruiting body / ¹³⁷Cs or Cs concentration in the fresh medium) were in good agreement, indicating similar migration. The CR of Cs grown on medium containing both 0.1% Cs and 0.1% K, 10.2, showed a decrease of about 30 percent as compared with that containing 0.1% Cs only. These CR values suggested that Cs accumulation by the fruiting bodies of P. ostreatus Y-1 is affected by the presence of K similarly to previous observations in the mycelia. The ¹³³Cs-NMR spectra from the fruiting bodies of P.ostreatus Y-1 showed two resonance signals, whereas those from the media after harvesting of fruiting bodies showed only one signal. Just before growth of the fruiting bodies, bunches consisting of many mycelia were observed by scanning electron microscopy (SEM). No significant differences in the elemental distribution (Cs, K, P and C) were detected in the mycelium surface by SEM equipped with an energy dispersive X-ray microanalyzer.

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Tributylin (TBT)-induced imposex phenomena have been reported only in mollusks. Our findings reveal that imposex is not a phenomenon exclusive to mollusks. The imposex phenomenon has been found to occur in a common Japanese freshwater crab, Geothelphusa dehaani, which inhabits TBT-uncontaminated mountain streams in Japan. In addition, the imposex of G. dehaani occurred not only in females but also in males (dual-gender imposex) : either female genital opening (gonopore) or a single ovary occured in male crabs. However, the definitive causal factors and mechanics relating to the occurrence of imposex are still unclear.

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Soil samples from 30 sites in Tokushima, Japan, were analyzed to obtain contemporary normal (or typical) levels of polycyclic aromatic hydrocarbons (PAHs) in local soils. A very wide range of PAH concentration has been observed in these soils. The ΣPAH values ranged over 2orders of magnitude, from 1 to 147μg/kg. Fluoranthene was consistently found to be the most abundant compound. Other major constituents were pyrene, benzo[b]fluoranthene, and benzo[a]anthracene. Biomass burning and vehicular exhaust contribute PAH to soils. Higher PAH contents in rural soils principally came from biomass burning. Mobile sources (diesel-and gasoline-powered vehicles) raise PAH contents in urban soils. In addition, soil properties play some role in controlling/influencing the PAHs contents of soils.

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Nitrate was determined using nitrate reductase (NR) in a flow system. A merging zone method was applied in the system in which a zone of NR and that of nitrate in separate streams were merged to react. The NADPH decreased by the enzymatic reaction was detected at 340nm. The length of the reaction coil used for the enzymatic reaction was 250cm. Of the concentrations from 0 to 0.6mм NADPH in a carrier, 0.02mм gave the maximum peak area due to decreased NADPH, suggesting that NR may be inhibited by a coenzyme, NADPH. The buffer of pH 7.5 was found to be optimum in the pH range from 6.5 to 8.0 of the buffer used as a carrier medium. Of the various buffer types(pH 7.5) used as the medium of carriers, piperazine-1, 4-bis(2-ethanesulfonic acid) (PIPES) buffer afforded the maximum peak area. The elevated temperatures of the water bath for enzymatic reaction gave reduced peak areas and the maximum peak area was observed at 32°C. Under the optimum conditions, a linear calibration curve (r=0.996) was obtained in the nitrate concentration range from 5 to 100μм and detection limit (S/N=3) was 1.8μM. The relative standard deviation of the peak area at 20μM nitrate was 4.2% (n=7). The method was applied to the determination of nitrate in samples of natural water. Nitrate content obtained by the present method agreed well with that determined by the JIS method.

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The addition of phenolic antioxidants, cysteine, N-acetylcysteine, unsaturated fatty acids, ascorbate and erythorbate has been evaluated in the reduction of free radical Maillard intermediates, the pyrazine cation radicals, and also the mutagenicity of a heated model system composed of glucose/glycine/creatinine. The aim of the present study was to determine whether these components were useful to reduce the mutagenicity of cooked hambuger. The effect of these components, added at low concentrations to ground beef, on the generation of the mutagenicity of cooked hambuger was examined. Mutagenicity of hamburger was assayed by the Ames test using Salmonella typhimurium TA98 strain with metabolic activation after the mutagens were purified by the use of blue rayon. Mutagenicity of hamburger was reduced to a half by the addition of ascorbate or erythorbate at 0.33%, whereas the mutagenicity was not reduced by epigallocatechin gallate(EGCG), cysteine, N-acetylcysteine, soybean oil or lard at the low concentrations.

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