Organic toxic substances, consisting of natural and synthetic toxins, are diverse in structure and toxicity. In poisoning cases, such toxins can be determined using a variety of analytical methods. Mass spectrometry is superior for obtaining structural information. Low molecular weight (MW) lipophilic toxins can be analyzed by gas chromatography-mass spectrometry (GC-MS), as exemplified by chemical warfare agents. Low MW polar toxins and metabolites of lipophilic toxins can be also determined by GC-MS after conversion to volatile derivatives. Liquid chromatography (LC)-MS with electrospray ionization (ESI) or atmospheric pressure chemical ionization can be used in the direct analysis for polar low MW toxins, even though the chromatographic peak resolution is low, as exemplified by saxitoxin. Intermediate MW (around 1000) polar toxins can be analyzed by matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-MS and LC-ESI/MS, and the associated MS/MS technique, which permits structural information, such as the peptide sequence to be obtained. High MW proteinous toxins can be also analyzed by LC-MS and MALDI-TOF-MS, as exemplified by staphylococcal enterotoxin B. However, only an approximate MW estimation can be obtained for ricin, because of the difficulty of obtaining accurate MW values, due to the presence of multiple monoisotopic molecular ion peaks and heterogeneity as the result of posttranslational modification. Instead, LC-MS/MS analysis after proteolytic digestion is typically used in the identification of digested intermediate MW peptides and the subsequent sequencing of the ricin protein.

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A simple and rapid method for the simultaneous determination of seven golf course pesticides in aqueous samples was developed by using in-tube solid-phase microextraction (in-tube SPME) coupled with liquid chromatography. In-tube SPME, in which the analytes were extracted from the sample directly into an open capillary column, is an extraction technique for organic compounds in aqueous samples. Although an open tubular capillary column with a thick film of polymer was used for the conventional in-tube SPME, we used a porous-layer open-tabular (PLOT) column in which there was a porous layer on the inner wall and has a larger surface area. A microsyringe pump equipped with a gastight syringe was employed to sequentially pump the sample solution through the capillary. The detection limits were from 0.9 to 4.1 ng ml^-1. The calibration curves were linear in the range from 1 to 50 ng ml^-1. We took a survey of seven golf course pesticides in several water samples by using the developed method.

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The effects of three inhibitors of mitochondrial respiratory functions on the induction of metallothionein (MT) in the liver were investigated in mice. C57BL/6 mice were injected subcutaneously with sodium azide, atractyloside, or malonic acid and were analyzed for the hepatic levels of thiobarbituric acid-reactive substances (TBARS) and MT. Sodium azide and atractyloside increased the hepatic MT level transiently around 6 and 12 hr, respectively, after the injection. Malonic acid significantly increased the MT level even at 3 hr and the increase continued until at least 24 hr after the injection. In all three cases, the changes in hepatic MT level showed almost the same patterns as those of TBARS with respect to both kinetics and magnitude. The increases in both TBARS and MT in the liver due to malonic acid were inhibited by about 90% by preinjection of succinic acid. These results suggest that the inhibitors of mitochondrial respiratory functions directly affect the mitochondria of hepatic cells, causing the production of reactive oxygen species, and consequently resulting in the induction of MT in mice.

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The chemical structures of individual isomers present in a mixture of 4-nonylphenol have recently been clarified, and differences in estrogen activity due to differences in the structure have been reported. Considering the importance of investigations of the chemical structure of 4-nonylphenol contained in commonly used synthetic resin products, we investigated an analytical method of individual isomers of 4-nonylphenol using positive chemical ionization-gas chromatography/mass spectrometry (PCI-GC/MS). Furthermore, we established a highly sensitive simultaneous analysis method using PCI-GC/MS [selected ion monitoring (SIM)], in which 4-alkylphenol and bisphenol A were extracted using a simple dissolution method without pretreatment such as solid-phase extraction and derivatized by trimethylsilylation.

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Rice bran was evaluated for the prevention of bulking in the activated sludge process. After the addition of rice bran to the aeration tank of a food plant, a decrease in the sludge volume index was observed. Both biochemical oxygen demand and chemical oxygen demand showed significant decreases relative to that before the addition of rice bran. Rice bran accelerated the growth of the effective microorganism and thus inhibited the growth of filamentous organisms that cause bulking. The results suggest that phytic acid in rice bran is useful in controlling filamentous bulking in laboratory-scale investigations.

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An androgen receptor (AR)-reporter gene assay for some chemicals was examined using two different types of stably transfected CHO-K1 cell lines (AR-EcoScreen cells for androgenic activity and c-luc cells for cell toxicity evaluation). One stably expresses luciferase with androgen induction. The other stably expresses it without the need for androgen induction. We studied the responsiveness of the luciferase of AR-EcoScreen to androgen agonists and androgen antagonists. The luciferase activity of AR-EcoScreen was highly induced by androgens such as 5a-dihydroxytestosterone and testosterone. Furthermore, the luciferase activity of AR-EcoScreen was very susceptible to androgen antagonists such as hydroxyflutamide and cyproproterone acetate compared with that of MDA-kb2, which stably expresses androgen and glucocorticoid-responsive luciferase and represents a tool for screening for androgenicity. We examined the AR-reporter gene assay of some chemicals containing alkylphenols and parabens using AR-EcoScreen and c-luc cells (AR-EcoScreen system). None of the tested chemicals had any androgenic activity. However, 4-t-octylphenol, 4-n-dodecylphenol, 4-n-nonylphenol, 4-n-hexylphenol, 4-n-pentylphenol, 4-n-octylphenol, and 4-n-propylphenol exerted antiandrogenicity. Isobutylparaben, n-butylparaben, isopropylparaben, and n-propylparaben also had antiandrogenic activity. The level at which these parabens effects were observed is a value that is lower than the upper limit of the acceptable daily intake (ADI, 10 mg/kg/day). 4-Ethylphenol, 4-methylphenol, ethylparaben, and methylparaben had no effect on the androgen-responsive luciferase activity. We propose that the AR-EcoScreen system is suitable for a battery of titer-1 tests to identify chemicals that affect the endocrine system, and that the ADI value of total parabens may have to be lowered in Japan.

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3-Nitrobenzanthrone (3-NBA) is an extremely potent direct-acting bacterial mutagen that has been detected in diesel exhaust particles, airborne particles and so forth. Recently, 3-aminobenzanthrone (3-ABA) and 3-acetylaminobenzanthrone (3-AABA) were identified as metabolites of 3-NBA in mammalian cells. In this study, to clarify the genotoxicity of 3-ABA, 3-AABA and 3-NBA in vitro and in vivo, the mutagenicity and DNA-damaging activity of these chemicals were investigated by the Ames assay and by alkaline single gel electrophoresis (Comet assay), respectively. 3-ABA and 3-AABA were mutagenic toward four Salmonella typhimurium strains, i.e. TA98, TA100, YG1024 and YG1029, in the presence of a mammalian metabolic system (S9 mix). Both chemicals showed the highest mutagenicity toward YG1024, and induced 2180000 revertants/nmol of 3-ABA and 131000 revertants/nmol of 3-AABA. 3-NBA also showed mutagenicity toward these four strains with and without S9 mix, but the potencies of 3-NBA in these strains were decreased by the addition of S9 mix. In the presence of S9 mix, the mutagenic activities of 3-NBA in four strains were comparable to or lower than those on 3-ABA. 3-ABA, 3-AABA and 3-NBA produced statistically significant DNA damage, which was detected by an increase in the DNA tail moment, in vivo 3 hr after intraperitoneal injection at 160 mg/kg body weight. 3-ABA, 3-AABA and 3-NBA induced significant DNA damage in the liver, kidney, spleen and lung, spleen only, and liver, kidney, lung and bone marrow, respectively. At a lower dose (40 mg/kg), 3-NBA produced significant DNA damage in the lung. These results indicate that 3-ABA, 3-AABA and 3-NBA are not only mutagenic in vitro in bacteria but also genotoxic in vivo in mouse.

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It has been suggested that the properties of vascular endothelial cells differ depending on the type of blood vessel. Proteoglycans are macromolecules that contribute to vascular properties through regulation of endothelial cell functions. In this study, we characterized proteoglycans synthesized by cultured human brain microvascular endothelial cells by biochemical techniques. The experiments indicated that the cells synthesize and secrete two types of proteoglycans: large heparan sulfate proteoglycans bearing heparan sulfate chains of approximately M_r∼68000 and chondroitin/dermatan sulfate proteoglycans bearing chondroitin/dermatan sulfate chains of approximately M_r∼48000. The heparan and chondroitin/dermatan sulfate proteoglycans were identified as perlecan and biglycan, respectively, by Western blot analysis. A part of perlecan was associated with the cell layer, while most of biglycan was secreted into the medium. These results suggest that the characteristics of proteoglycans synthesized by brain microvascular endothelial cells are similar to the characteristics of those synthesized by previously reported arterial endothelial cells. Regulation of endothelial cell functions by proteoglycans may be independent of blood vessel types.

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Circulating immune complexes (CICs) in sera from patients with liver cirrhosis (LC), primary biliary cirrhosis (PBC), hepatitis B type (BCH) and hepatitis not including BCH (nonBCH) were analyzed using F(ab′)₂anti-C3 ELISA, since CICs' functions are intimately associated with the composition and their sizes. Positive percentages for IgG-, IgA- and IgM-CIC were significantly increased in LC, PBC, BCH and nonBCH as compared to healthy specimens. The combination of F(ab′)₂anti-C3 ELISA and sucrose density gradient fractionation allowed us to detect the classes of antibodies and hepatitis B surface antigens in the fractionated CICs from sera of patients with liver diseases. Properties of the fractionized CICs are discussed in the text.

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Punica granatum (P. granatum) Linn. pericarp has been commonly employed as a crude drug in Thai traditional medicines for the treatment of diarrhea. The antibacterial activity of extracts from P. granatum pericarp against different strains of enterohemorrhagic Escherichia coli (E. coli) O157 : H7 and other strains of enterohemorrhagic E. coli were investigated. Successive chloroform, 95% ethanol, and water extracts of the plant were examined. The ethanolic extract was found to be the most effective against all the strains examined. Both the ethyl acetate and n-butanol fractions of P. granatum pericarp were demonstrated to have high activity with the highest minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of 0.05 and 0.05 and 0.39 and 0.19 mg/ml, respectively. The inhibitory effects of both fractions on the production of verocytotoxin (VT)1 and VT2 by enterohemorrhagic E. coli O157 : H7 was observed at very low concentrations of 1/10 of the MIC value (0.05-0.09 mg/ml, respectively). Phytochemical screening of the ethanolic extracts demonstrated that they contain flavonoids, sterols, triterpenes, phenols, and tannins. Our findings suggest that an appropriate bioactive compound may be developed from P. granatum pericarp as alternative treatment for this E. coli O157 : H7 infection. The crude plant may also be administered to prevent VT production in the human intestine to solve the problem of subinhibitory effects from the use of antibiotics.

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The amount of benzo[a]pyrene (BaP), benz[a]anthracene (BaA), and dibenz[a,h]anthracene (DBA) has been restricted to a concentration of 10 μg/g each in creosotes, and 3 μg/g each in creosote-treated woods, respectively, because of the possibility of the risk of skin cancer in consumers, and creosotes can otherwise contain high concentrations of each chemical. We already reported the content of 16 polycyclic aromatic hydrocarbons (PAHs) and phenols in creosotes and creosote-treated wood as determined by gas chromatography-mass spectrometry (GC-MS) and absorptiometry [Chemosphere, 60, 1279-1287 (2005)]. However, the limit of determination of each PAH per sample was > 40 μg/g according to that method, the sensitivity of which was insufficient for determining the allowable levels of these 3 compounds. Moreover, a substantial amount of time was needed for GC-MS analysis. In the present study, we improved upon our previous analytical method in order to increase the sensitivity of the test and to reduce the duration of GC-MS analysis. Creosote was extracted from treated wood samples by dichloromethane-soak incubation, and was placed on a Sep-Pak silica cartridge and eluted with dichloromethane. The eluates were evaporated and dissolved in dichloromethane. The sample solution spiked with the internal standard solution was injected into the GC-MS system. The limit of determination of each chemical in the test solution was approximately 0.2 μg/ml, which corresponded to 1-2 μg/g in each sample. The duration of GC-MS analysis was approximately 17 min. A collaborative study was also carried out in order to evaluate the reproducibility of the method for determining low levels of BaP and related compounds in creosotes. The present method was applied for the analysis of certain commercially available creosotes and creosote-treated wood samples in Japan. It was confirmed that some creosotes and railway sleepers contained these compounds in high concentrations, thus exceeding the allowed control value.

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We examined the mineral contents in Chinese traditional medicines from Japanese or Chinese markets by X-ray fluorescence spectrometry, and found extremely high levels of arsenic (N.D.-11.8%) and/or mercury (N.D.-18.8%) in medicines from Chinese markets but not Japanese markets. Such traditional medicines also contained sulfur in high levels, suggesting that these toxic metals present as their respective sulfide. Arsenic was partially eluted with hot water, artificial gastric juice, or artificial intestinal juice from rokushingan (Liushenwan), one of the most famous Chinese medicines. These results suggest that high arsenic-Chinese medicines are harmful to human beings and special attention should be given to traditional medicines in Chinese markets.

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Dried thyroid added to dietary supplements was analyzed using liquid chromatographyl mass spectroscopy (LC/MS). However, rapid analysis of many dietary supplements on the market is difficult because the analysis uses expensive instruments (LC/MS) and requires a long time for pretreatment. The enzyme-linked immunosorbent assay (ELISA) is rapid, simple, and lowcost and thus suitable for processing many samples. In this study, we investigated rapid analysis of dried thyroid contained in dietary supplements with ELISA using commercial anti-thyroglobulin antibody. The analytical method using ELISA established in this study may be useful for the screening of dried thyroid added to dietary supplements.

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Validation of multiresidue screening methods for the determination of 186 pesticides in 11 agricultural products: broccoli, asparagus, carrot, spinach, burdock, matsutake mushroom, cauliflower, orange, soybean, sesame and millet was done by gas chromatography (GC). The investigated pesticides were selected on the based of such compounds that are commonly used around the world. Although the recovery of 58 of the pesticides was low (< 50%) in some crops, the 128 pesticides that spiked in samples at 0.1 mg/kg showed satisfactory recoveries (≥ 50%) in all crops with relative standard deviation of 4-21%. These validated 128 pesticides were therefore newly acceptable for the pesticide-monitoring programme at the Quarantine Station in Japan; the quantitative limits ranged from 0.005 to 0.1 ppm by GC on a crop basis. The screening methods were applied to monitor the residue from a total of 200 pesticides including 72 previously validated in imported foods at the Station in Japan. Pesticide residue from 188 (12.4%) was found in 1516 samples. Of these, 4 (0.26%) were in violation of Japanese maximum residue limits (MRLs). No detectable residue was found in 1328 (87.6%) samples.

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