Trichothecene mycotoxins are chemically related sesquiterpenoids with 12, 13-epoxy-trichothecene nuclei. T-2 toxin, nivalenol, deoxynivalenol, and satratoxins are isolated as causative toxicants of food-borne diseases such as moldy-corn toxicosis in U.S., Akakabi (red-mold) toxicosis in Japan, vomiting and feed-refusal diseases in U.S., and satratoxicosis in the Central Europe, respectively. Currently, the TLC, HPLC, GC and GC-MS are introduced for chemical analyses of the trichothecene mycotoxins in food and feed, and the accumulated data strongly revealed that, among more than 60 kinds of trichothecenes, deoxynivalenol (=vomitoxin) and nivalenol, both being produced by Fusarium graminearum (Gibberella zeae in perfect stage), are major contaminants in wheat, barley, corn and other cereals and foodstuffs. In 1983, the Canadian goverment set a guideline for deoxynivalenol in wheat, and FDA (US) recommended "level of concern" for this mycotoxin. As for nivalenol, which is also a contaminant in domestic cereals along with deoxynivalenol and possesses more than ten times higher toxicity than deoxynivalenol, neither guideline nor "level of concern" is recommended by the Japanese goverment at present.

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Recent studies on the histochemical staining of a metal in cells and tissues were reviewed mainly on the viewpoint of the mechanisms of metal staining by a chelating agent as follows, i) staining methods for a metal in cells and tissues, ii) molecule designing of a high sensitive chelating agent, iii) mechanisms of staining, iv) masking of interfering metals, v) metal staining in the future. With the modification of a chelating agent to increase the staining sensitivity, a molecular size, the selection of substituent groups and the introducing position of the groups to a chelator molecule, and the hydrolyzing property of metal chelates should be taken into consideration. The most frequently mentioned factors in a staining mechanism are dye-tissue attractive forces, and recent studies have make it clear that weak interactions, hydrogen bond and hydrophobic interaction, would play a significant role in those forces. The Hansch π values were an useful indication for knowing the hydrophobicity of chelating agents for metal staining. The published values of a stability constant were insufficient guide for choosing a masking agent to block interfering metals. The polyphosphates were among the most useful masking agents for metal staining from a practical standpoint. Future prospects about methods for histochemical metal staining developed recently are also referred.

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Reactions of polynuclear aromatic hydrocarbons (PAH) with chlorine were examined under various conditions. The chlorination products of fluoranthene were fractionated by high performance liquid chromatography (HPLC) with a ultraviolet (UV) detecter. The structure of each fraction separated by HPLC was analyzed by gas chromatography (GC)-mass spectrometry (MS), and the mutagenicity of each fraction tested by the Ames Salmonella assay. 1) Eleven products were obtained by the above procedure and extracted with n-hexane. 2) The formation rate of chlorinated substances varied with changes in pH, chlorine concentration and temperature. 3) The fraction of peak No. 5 was found to be a substituted product of fluoranthene by chloro- and hydroxy-group by GC-MS. That of peak No. 7 was mono-chloro-fluoranthene, peak No. 10, di-chloro-fluoranthene and peak No. 11, tri-chloro-fluoranthene. The fractions of peaks 1, 2, 3 and 4 could not be determined precisely by GC-MS, but were considered to be substances produced by the breaking of aromatic carbon rings. 4) The fractions of peaks 1, 2, 3, 4, 5, 6, 8 and 9 were purified by HPLC and found to be mutagenic toward TA 98 and TA 100 strains in the absence of S-9 Mix. But, in the presence of S-9 Mix, fractions of peaks 1, 2, 7, 10 and 11 were all mutagenic toward TA 98 and TA 100 strains. The experimental results indicate that many other hydrocarbons in water may be possibly changed to mutagenic compounds by chlorine reactions.

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The induction of Cd-binding proteins were recognized in liver and kidney of ayu-fish, rainbow trout and catfish injected with CdCl₂. One protein from ayu-fish, two isoproteins from rainbow trout and three isoproteins from catfish were separated as liver Cd-binding proteins by ion exchange chromatography with DEAE Sephadex A-25. These Cd-binding protein of ayu-fish and catfish were purified by rechromatography with Bio gel P-10. All of these purified fractions have much cadmium, a little copper and high absorption at 254 nm. By the analysis of amino acid compositions, each protein has high cysteic contents and absence of aromatic amino acids and histidine. These proteins were identified as (Cd, Cu)-metallothionein, and the isoproteins in catfish named MT-I, MT-II and MT-III. Some researchers presented the theory that the isometallothioneins was induced as more efficient physiological function in the evolution. The authors thought that catfish, α-mesosaprobic organismus, gained duplication of the metallothionein gene, and that the metallothionein acted more reasonable detoxification in polluted water area. Ayu-fish, oligosaprobic organismus, has not duplication of the metallothionein gene. Metal-binding proteins were found in livers of non-injected catfish, rainbow trout and ayu-fish. These metal compositions were zinc and copper and it was suggested that these proteins were (Zn, Cu)-metallothioneins.

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In order to analyze metals in deposit rapidly, the ash of deposit was fused with sodium hydroxide and suspended with water. The mixture was filtrated and the filtrate was used for the estimation of aluminium, silicon, vanadium, and zinc. The residue after filtration was dissolved with sulfuric acid and titanium, iron, manganese, copper and cobalt were assayed from the solution The measurement of metals in the filtrate or the sulfuric acid solution was performed with colorimetry, titration, and atomic-absorption spectrometry. The complete analysis of the metals in deposit at Kumamoto city could be rapidly determined by this method.

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Hydrolyses of cyanogen glycosides, amygdalin and linamarin, by various β-glucosidases were examined. The β-glucosidases from almond emulsin and apricot stone hydrolyzed amygdalin, but little hydrolyzed linamarin. On the other hand, the enzymes from lima bean and butter bean hydrolyzed linamarin, but did not hydrolyze amygdalin. These results show that the ability to hydrolyze amygdalin or linamarin is restricted to certain β-glucosidase species. An application of β-glucosidase is useful for the analysis of cyanogenic glycosides in food. However, it should be noted that the substrate specificities of the enzymes are rather complex.

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In order to clarify a mechanism of chloropicrin formation, chloropicrin and chloroform formation potential by aqueous chlorination of organic nitrogen compounds were investigated from viewpoints of structural correlation and stoichiometry. The results obtained were as follws. 1) Chloropicrin formation potential by aqueous chlorination of alanine related compounds, phenols and resorcinols containing nitrogen depended on oxidation state of nitrogen in functional group, the potential being in the decreasing order of -NO₂>-NO>-NH₂. 2) By the reaction of chloropicrin formation by aqueous chlorination of alanine, it was considered that oxidation of amino group to nitro group was rate-determining step and this reaction was antagonized reaction of chloroform formation. 3) Mechanism of chloropicrin formation by aqueons chlorination of aminophenol was proposed as follows ; Amino group was oxidized (-NH₂→-NO→-NO₂) and consequently chlorinated to chloropicrin by way of cleavage of aromatic ring. It was recognized that oxidation of nitrosophenol to nitrophenol was rate-determining step. 4) By the reaction of chloropicrin formation by aqueous chlorination of aminoresorcinol, it was recognized that two steps of oxidation (-NH₂→NO→-NO₂) were rate-determining steps and this reaction was not antagonize reaction of chloroform formation.

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The methods for analyzing residual methylene blue and malachite green in the muscle and liver of rainbow trout and eel were established. Since methylene blue and malachite green in the muscle and liver homogenates were effectively extracted by n-butanol in the presence of ZnSO₄, the recoveries of methylene blue and malachite green were determined by measuring the optical density (665 nm for methylene blue, 620 nm for malachite green) of butanol solution extracted from the liver and muscle homogenates containing the dyes. However, the optical density of malachite green in butanol solution extracted from the liver homogenates was measured at 500 nm, 620 nm and 700 nm, and was then calculated from the following equation ; Net OD₆₂₀=(MOD₆₂₀-OD₇₀₀)-[(OD₅₀₀-OD₇₀₀)×0.4815-0.0033] MOD₆₂₀ : OD observed at 620 nm : malachite green + pigments of liver homogenates The recoveries of methylene blue and malachite green added to the muscle homogenates were 80-87% and 80-88%, respectively. The limits of detection in the muscle were 0.1 μg/g for both dyes. Also, the recoveries of methylene blue and malachite green added to the liver homogenates were approximately 85-87% and 88-95%, respectively. As little as 0.5 μg/g of both dyes in the liver was detectable. On the other hand, when rainbow trout were exposed to 0.5, 1.0 and 2.0 ppm of malachite green solution at 16°C for 1 h, residual amounts of malachite green in the muscle and liver were 1.41±0.28 μg/g (muscle) and 5.9±1.2 g/g (liver), 2.41±0.11 μg/g (muscle) and 10.1±1.4 μg/g (liver), and 5.90±1.61 μg/g (muscle) and 19.1±2.8 μg/g (liver), respectively. Also, when eel were exposed to 3.0 ppm of methylene blue solution at 16°C for 1, 2 and 3 h, residual amounts of methylene blue in the liver was 1.20±0.3 μg/h, 1.10±0.5 μg/2 h and 0.90±0.2 μg/3 h, respectively, and residual methylene blue in the muscle could not be detected.

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When cadmium (CdCl₂) was administered subcutaneously in a high single dose of 5.0 mg/kg to rats, their liver and testis tissues showed a statistically significant increase in alkaline phosphatase (Al-P) activity at 24 h or more after the administration while the reverse was the case in the serum of the rats. Lactate dehydrogenase (LDH) activity in the serum showed a statistically significant decrease at 15 to 24 h after the administration of CdCl₂. An increase in LDH activity for the serum coincided with that for the testis. During the study period there was no change in the calcium level in the serum, but the level of zinc in the serum decreased in a similar fashion to serum Al-P activity. As for the level of cadmium in the serum, it showed a very high value (ca. 0.03 mM) at 15 and 24 h after CdCl₂ administration. When this concentration of cadmium was added in the form of CdCl₂ to rat serum in vitro, it produced an inhibitory effect on serum Al-P activity. These facts suggest that the decrease in serum Al-P activity is not due to a change in tissue levels of Al-P, but reflects a direct inhibition of Al-P activity in the serum by cadmium.

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A method for the quantitative determination of estradiol benzoate (EB) in cosmetics by highperformance liquid chromatography (HPLC) was developed. The EB was extracted from samples with methylene chloride, the extract was evaporated, and the residue was subjected to silica gel column chromatography. The column was washed with benzene to remove impurities, and EB was eluted with ethanol-benzene (1 : 9, v/v) and determined by HPLC on an octadecylsilylated silica column using a mobile phase of acetonitrile-water, with UV detection (230 nm). Recoveries of EB obtained from a variety of cosmetic samples spiked at 0.001 and 0.002% levels were 91.8-99.8%, and the coefficients of variation were between 0.41-2.5%. When this method was applied to the analysis of EB in commercial samples, EB could be determined without interference.

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In river sediments, heavy metals are adsorbed to the components, such as, clay and humic substances. The adsorbing abilities of clay and humic substance are compared. The adsorbing ability of humic acid is stronger than that of clay for all heavy metals at pH 4. The ability of humic acid reduced under the co-existence with clay.

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Extraction method for sorbic acid (SOA), dehydroacetic acid (DHA), benzoic acid (BA) and p-hydroxy benzoic acid-esters (PHBAs) with 0.5 ml of n-butyl acetate (AcoBu), compared with the official extraction procedure, was cheep, fast and simple. Proper quantity (3-15 μg of SOA, 5-25 μg of DHA, BA and PHBAs determined by UV absorption of the distillate) of the distillate of food or 1 g of soft drinks was added to the test tube containing 0.1 ml of 10% of HCl and 3 g of NaCl. The sample was made up to 10 ml with water, 0.5 ml of 90 ppm n-tridecanoic acid-AcoBu solution (in the case of PHBAs, AcoBu was used) was added to the tube, and the tube was shaken vigorously. Upper layer was applied to GC. The recovery rates of the preservatives from the distillates of foods and from soft drinks were 95-102%. The results of marketed foods by our method were almost the same as the results by the official method.

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The components of paralytic shellfish poisons produced by toxic plankton Protogonyaulax tamarensis from Funka Bay, Hokkaido, were investigated. The cells of P. tamarensis cultured in BSW-4 medium were extracted with 80% ethanol (pH 2.0). The extract was defatted with chloroform, and then purified by two types of column chromatography on Bio-Gel P-2 and Bio-Rex 70 (H⁺). The toxins thus obtained were analyzed by using cellulose acetate membrane electrophoresis and thin layer chromatography on silica-gel. From the toxic fractions unbound on Bio-Rex 70 column, four components coded tentatively as PTX₁, PTX₂, PTX₃ and PTX₄ were detected. Judging from Rm values, Rf values and the result of acid-hydrolysis, PTX₁ and PTX₂ were demonstrated to be gonyautoxin-8 and its epimer, respectively. On the other hand, from the toxic fractions bound on Bio-Rex 70 column, gonyautoxins 1-4, neosaxitoxin and saxitoxin were detected.

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