Superoxide (O₂^⨪) appears to play an important role in various biological processes, such as inflammation, carcinogenesis, radiation-induced damage, aging, phagocytosis and enzyme reactions. O₂^⨪ has been considered to have diverse but not vigorous reactivities compared with other active oxygen species, but has been reported to exert deleterious effects on living cells. Thus, the mechanism of the biological effects of O₂^⨪ has been of great interest. The review reports that O₂^⨪ reacts with some compounds such as acyl halides, polyhalides, or CO₂ to form peroxy intermediates, which have more vigorous reactivities than O₂^⨪ alone. The peroxy intermediates can oxidize olefins or sulfides to the corresponding epoxides or sulfoxides in good yields. The phosphates, also, enhance the reactivity of O₂^⨪ as shown in the base-release reaction from nucleosides induced by O₂^⨪ and the phosphates. These reactions should be important for consideration of the O₂^⨪ toxicity. The reaction mechanisms have been examined in detail using K¹⁸O₂ and cyclic voltamography. Furthermore, synergism between O₂^⨪ and CCl₄ has been found in the biological system. Thus, CCl₄ toxicity on Escherichia coli is exacerbated by O₂^⨪. The results obtained should contribute not only to the development of O₂^⨪ for use in organic oxidations, but also to the elucidation of the oxidative reactivity of O₂^⨪ in biological systems.

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Contaminant (s) in sodium chondroitin sulfate (SCS) preparations can strongly accelerate formation of nitrosamines from secondary amines and nitrite. About 70% of nitrosation-accelerating activity (NAA) was recovered in the filtrate through a DIAFLO YC 05 ultrafiltration membrane. The results of ultrafiltration and column chromatography using Sephadex G-10 identified that the molecular weight of the main component that showed NAA was several hundreds. It reacted with nitrite and gradually lost its NAA. It did not accelerate formation of nitrosomethylurea. It accelerated formation of nitrosodimethylamine additively with thiocyanate, a well known accelerator of nitrosamine formation.

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A simultaneous method for the determination of fluorescent Whitening agents (FWA), such as disodium 4, 4'-bis [(4-anilino-6-morpholino-1, 3, 5-triazin-2-yl) amino] stilbene-2, 2'-disulfonate (FB 71, trans isomer), disodium 4, 4'-bis [(4-toluidino-6-morpholino-1, 3, 5-triazin-2-yl) amino] stilbene-2, 2'-disulfonate (FB 225, trans isomer and cis isomer) and disodium 4, 4'-bis (2-sulfo-styryl) biphenyl (FB 351, trans isomer and cis isomer), in river waters with high performance liquid chromatography (HPLC) was developed. FWA in river waters were adsorbed on Sep-pak C₁₈ cartridge and eluted with methanol. The eluate was evaporated to dryness, and the residue was dissolved in acetonitrile-water (3 : 7). The solution was filtrated and analysed by HPLC under the following conditions : column, Inertsil ODS 5 (4.6 mm i.d.×150 mm) ; mobile phase, acetonitrile-0.01 M monobasic sodium phosphate (35 : 65) ; flow rate, 1.0 ml/min ; detections, ultraviolet detector (detection wavelength, 270 nm), fluorescent detector (excitation 345 nm and emission 430 nm). The average recoveries of FWA from river waters at 2-15.6 μg/l were 90-102%. This method was applicable to determine the concentration of 0.01μg/l for FB 351, 0.05μg/l for FB 71 (trans), 0.6μg/l for FB 71 (cis), 0.1μg/l for FB 225 (trans) and 1.1μg/l for FB 225 (cis), respectively, using 100 ml samples. On the basis of the present method applied to samples from 6 different rivers located at Kanagawa prefecture, FB 351 (trans), FB 71 (trans, cis) and FB 225 (trans, cis) were determined.

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A convenient method for the determination of small amount of organic pollutants in environmental water has been developed. Five to 101 of water sample was distilled for two hours by using Dean-Stark distillation apparatus and concentrated into 5 ml of n-hexane. As a result of recovery test for 122 kinds of standard pollutants, recoveries of such lipophyllic compounds as chlorobenzens, phthalate esters, organosulfides were above 70% with a few exceptions. But hydrophilic compounds such as organophosphorus or carbamate pesticides and phenols were hardly recovered. By applying the above method, river and tap water were concentrated and analyzed by GC/MS, ECD-GC and HPLC. Phthalate esters, terpenoides etc. were detected from river water and volatile organohalogens were detected from tap water.

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To investigate the relationship between pentachlorobenzene toxicity and the changes of hepatic drug metabolizing enzyme activities, various doses of pentachlorobenzene were administered to rats for 5 days. Both cytochrome P-450 content and UDP-glucuronyltransferase activity increased with an increase in the dose up to 160 mg/kg. However, at the dose of 250 mg/kg, there was no further increase in UDP-glucuronyltransferase activity but a significant decrease in cytochrome P-450 content. In contrast, glutathione S-transferase activity was slightly but significantly increased up to the dose of 250 mg/kg. Glutathione S-transferase activity was inversely correlated with glutathione content in the liver. Content of pentachlorobenzene in the liver linearly increased up to 160 mg/kg, and was markedly enhanced at the dose of 250 mg/kg. Accumulative patterns of pentachlorobenzene in the brain and kidney were similar to that in the liver, while those in adipose tissues were quite different from that in the liver. In adipose tissues, pentachlorobenzene content was 8-27 times higher than that in the liver and the content linearly increased up to 250 mg/kg. A main metabolite of pentachlorobenzene in the liver was pentachlorophenol.

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Biological effects of zinc on the lung were examined by biochemical and cytological indicators in the broncho-alveolar lavage fluid. Zinc acetate was instilled intratracheally into male Wistar rats (14 weeks old, mean body weight 371 g) at a dose of 20μg Zn/0.4 ml saline/rat and the animals were killed 1, 3, 6 and 12 h and 1, 2, 3 and 7 d later for the time-dependent experiment. The metal ion was also instilled intratracheally into rats at doses of 0, 5, 10, 20, 50, 100 and 200μg Zn/rat and the animals were killed 2 d later for the dose-dependent experiment. The lung was washed with 5 mM Tris/HCl buffer (pH 7.4, containing 0.25 M glucose, warmed at 37°C) and the broncho-alveolar lavage fluid was centrifuged at 650 g for 5 min, and the supernatant and pellet fractions were used for further examinations. Activities of β-glucuronidase and lactate dehydrogenase and protein content in the lavage fluid were used as biochemical indicators, while numbers of total cells, macrophage and polymorphonuclear leukocytes in the lavage fluid were used as cytological indicators. Concentrations of calcium, magnesium, phosphorus and sulfur were also determined by emission spectrometry. Biological data obtained from time-dependent and dose-dependent experiments indicate that inflammatory responses of the lung can be detected most efficiently two days after intratracheally instillation of zinc acetate. It was suggested that the lung responds to zinc acetate below a dose of 20μg Zn/rat by the native protective mechanism. However, biological indicators were significantly altered at doses more than 20 μg Zn/rat, indicating that the lung was damaged by zinc at a dose of as hight as 20 μg Zn/rat.

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The effects of essential metals, such as Zn, Cu, Mn, Ni, and Co, on the small intestinal absorption of L-histidine and L-leucine in rats were studied. The absorption of L-histidine (0.5 mM) from the small intestine in situ was significantly depressed by the presence of copper and nickel (0.5 mM) in the lumen, and the absorption of L-leucine (0.5 mM) was significantly depressed by the presence of copper. The absorption of L-histidine and L-leucine from the small intestine after pretreatment with copper (0.5 mM) for 1h was more significantly depressed than that of control. However, the pretreatment of the small intestine with nickel did not depress the absorption of L-histidine. The depressed absorption of L-histidine from the small intestine in situ in the presence of copper was significantly restored by the increase in the concentration of L-histidine. These results suggested that the formation of a stable copper-and nickel-L-histidine complex may decrease the amount of free L-histidine available to be absorbed from the intestine, resulting in a decrease in the intestinal absorption of L-histidine. In addition, an in vitro intestinal experiment suggested that copper taken up by the intestinal tissue inhibits the active transport of L-histidine and L-leucine across the small intestine.

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Determination method of alliin in garlic and commercial garlic products was developed by gas chromatography (GC) with a flame photometric detector (FPD). Alliin was extracted by hot water from garlic and commercial garlic products, and the crude extract was purified by using Sep-pak C₁₈ cartridge, followed by Amberlite CG-120 ion exchange column. The eluted alliin was evaporated to dryness, and the residue reacted with trifluoroacetic anhydride (TFAA) to give a volatile compound suitable for GC analysis. An 2% Advance DS+0.5% H₃PO₄ column was employed for GC under the conditions of 130°C as oven temperature, 180°C as injection port temperature, and 30 ml/min as carrier gas flow rate. Calibration curve plotted on logarithmic graph gave a good linearity in the range of 2 to 20μg/ml for alliin. The detection limit of alliin was 5 ng. The average recoveries of alliin added to commercial garlic products at the level of 10 mg/g were obtained from 83.1 to 98.3%.

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The interaction of cannabinoids, tetrahydrocannabinol (THC), cannabidiol (CBD) and some active metabolites with thinner and its components was studied in mice and the following results were obtained. 1) Δ⁹-THC but not CBD (5 mg/kg, i.v.) significantly prolonged thinner-induced anesthesia by 1.8 fold. The latent period to induce the anesthesia was significantly shortened by Δ⁹-THC. 2) 11-Hydroxy-Δ⁸-THC and 11-oxo-Δ⁸-THC (5 mg/kg, i.v.), which are active metabolites of Δ⁸-THC, significantly prolonged thinner-induced anesthesia by 2.0 and 3.2 fold, respectively. 11-Oxo-Δ⁸-THC significantly shortened latent period to induce the anesthesia. 3) Among cannabinoids examined, only 11-hydroxy-Δ⁸-THC significantly prolonged toluene-induced anesthesia by 2.4 fold. Thinner contains about 60% of toluene and a small amount of ethyl acetate, ethylether and ethylalcohol. 4) Δ⁸-THC and its active metabolites, 11-hydroxy-Δ⁸-THC and 11-oxo-Δ⁸-THC significantly prolonged ethyl acetate-induced anesthesia by 4.4, 4.9 and 5.1 fold, respectively. Moreover, Δ⁸-THC, 11-hydroxy-Δ⁸-THC and 11-oxo-Δ⁸-THC significantly shortened latent period to induce anesthesia.

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The root cytoplasmic fraction of Salix bakko, a metal tolerant plant collected from the Ashio mine where soil was polluted with copper, were separated into three fractions by a chromatography on Bio-Gel P-30. The fraction with the highest affinity for copper was eluted with an apparent molecular weight of 9200 by the gel filtration. The fraction was purified by chromatography on a column of DEAE-Cellulofine and subsequently by high performance liquid chromatography. The purified copper-binding protein was characterized by an animal metallothionein-like amino acid composition of high contents of cysteine (11.5%) and acidic amino acids (25.3%), and of a slight amount of aromatic amino acids.

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Simultaneous analysis of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) in oily foods and dried fish was investigated by using high-performance liquid chromatography (HPLC). Authentic BHA and BHT (each 100μg/g) were added to antioxidant-free oily foods and dried fish (0.1g, 0.5 g), followed by distillation with a modified continuous extractor for 2 h (oily foods) or 3 h (dried fish). HPLC was carried out on a normal-phase partition column (TSK gel silica-60, 4.6 mm i.d.×25 cm) with butyl acetate-heptane (1 : 9) solution as the mobile phase and with ultraviolet detection at 285 nm. Less time was required for distillation as compared with previous distillation methods, and recovery of BHA and BHT was higher. Moreover, only one column was required in HPLC, and BHA and BHT could thus be recovered more easily and accurately. The determination limit of BHA and BHT by this method was much improved.

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A simple and rapid enzymatic analysis was developed for the determination of formaldehyde in foods. The liquid food stuffs were diluted with 0.2 M triethanolamine hydrochloride buffer solution (pH 8.1). After the solid food stuffs were steam distilled, the distillate were diluted with the buffer solution (pH 8.1). The test solution was incubated for 15 min at 25±5°C with formaldehyde dehydrogenase (FADH) in the presence of β-nicotinamide adenine dinucleotide (NAD) and reduced glutathione, and then the content of formaldehyde were determined quantitatively from the increasing absorbance at 340 nm by reduced NAD (NADH) formed. The calibration curve for formaldehyde in the range of 0.1μg-10μg was linear. The recoveries of formaldehyde added to various kinds of foods at levels of 2-100μg/g were in the range of 75-105%, and the detection limit was 1μg/g of sample. When the proposed method was applied to 9 kinds of commercial foods, formaldehyde was detected in the range of 1.6-732μg/g. From the results described this enzymatic method was concluded to be a simple and precise analysis of formaldehyde in foods.

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Zinc acetate was instilled intratracheally into rats and the metabolic fate of zinc ions in the lung was examined. Male Wistar rats (14 weeks old, mean body weight 371 g) were instilled at a dose of 20μg Zn/0.4 ml saline/rat and killed 1/6, 1/2, 1, 3, 6 and 12 h and 1, 2, 3 and 7 d for the time-course experiment, and the animals were instilled at doses of 5, 10, 20, 50, 100 and 200 μg/rat and killed 2 d later for the dose-dependent experiment. Concentrations of zinc and other elements in the lungs, livers, pancreases, kidneys, supernatants of lungs and broncho-alveolar lavage fluids were determined by inductively coupled argon plasma-atomic emission spectrometry (ICP). Zinc instilled in the lung was shown to be taken up more than 90% by the lung within 3 h. However, the zinc taken up by the lung was transferred immediately to the liver, pancreas and other organs. A portion of the zinc taken up by the lung was present in the supernatant fraction of the lung and the zinc in this fraction disappeared more slowly than that in the whole lung. Distribution profiles of zinc and other elements in the supernatant of the lung were determined by the HPLC-ICP method. Only copper profiles showed a significant change. Zinc was not found in the metallothionein fraction, while copper increased in this fraction with doses of zinc instilled into the lung.

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In order to research the origin of taste of so-called "delicious natural water", chemical components of tap and natural water samples were measured and compared to each other. The values of pH, carbonate ion, KMnO₄-consumed values and ion levels in the samples were determined by the officially established methods for drinking water. Twenty-one kinds of tap water samples were collected from the domestic water supply of Hyogo, Osaka and Nara prefectures. On the other hand, 10 kinds of natural water samples were collected from Hyogo, Osaka and Nara prefectures, while the other 11 samples purchased at markets were originated from several parts of Japan. The tap water samples gave significantly higher values on the KMnO₄-consumed values and the concentrations of chloride and sulfate ions and iron (Fe²⁺+Fe³⁺) than the respective data in the natural water samples. In contrast, the concentrations of carbonate ion in the natural water samples were significantly higher than those in the tap water samples.

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