Marine toxins generally cause two types of human poisoning, one due to ingestion of marine animals, another due to their attack e.g. stinging and biting. Recently, studies on, especially, the former toxins are going to be developed where even their origins as well as properties are pursued. Concerning tetrodotoxin (TTX), a typical marine toxin, the following several interesting facts are elucidated ; its wide distribution in animals and its toxification mechanism mainly through the food web, finally followed by production by marine bacteria. Other typical marine toxins e.g. ciguatera toxin, paralytic shellfish poison, diarrhetic shellfish poison are produced by marine microplankton, dinoflagellates. Consequently, fish or bivalves are toxified by their food chain. The structure of ciguatoxin has recently been elucidated. Many food poisoning incidents due to ingestion of toxic crabs have been known. The causative agent was already elucidated to be PSP. However, their origin remains unknown. Food poisoning incidents due to ingestion of mussel in Canada in 1987 occurred and the causative agent was elucidated to be domoic acid, an excitatory amino acid. Memory loss is featured by the common symptom of serious patients. The causative agent is suspected to be produced by a diatom Nitzschia pungens f. multiseries. Recent topics on studies of marine toxins will mainly be referred herein.

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Lytic characteristics of the blue-green alga, Microcystis aeruginosa by Pseudomonas sp., a strain isolated from the biofilm in a biological treatment facility, were examined in a batch culture experiment. The viable cells of M. aeruginosa were perfectly lysed by the agent for 5 d at 30°C in the dark. Optimum conditions for the lysis of M. aeruginosa were 35°C and pH 7.0. Several important parameters for the estimation of eutrophicated waters, chlorophyll a, turbidity and COD originating from M. aeruginosa were effectively reduced and their removal were 70%, 84% and 41%, respectively, under the condition of 5 d cultivation at 30°C. It was found that M. aeruginosa was efficiently lysed by the agent during a short time.

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A study on the possibility of diarrhea induced by EPA and autoxidized EPA (EPA-Ox ; 61.6% decomposition) was conducted using several assays on rabbits and suckling mice. The result of loop test of both EPA and EPA-Ox in rabbit intestine showed positive diarrheal effect at the dose of more than 12.5 mg per loop though the degree of effect depended on the rabbit used (no sign was observed in 2 out of 6 rabbits). Effect of EPA-Ox was more intense than EPA. In an oral test with suckling mice, however, neither of the acids showed a diarrheal effect at a dose of up to 5 mg/mouse. When a very small amount (1.0μg) of okadaic acid (OA) was given, positive toxicity identified as a typical diarrheal substance derived from phytoplanktons. Significant positive correlation (γ=0.984, p<0.01) was obtained between fluid accumulation ratio (FAR) calculated from the result of the loop test in rabbits and percent of dead mice both animals which are usually viewed as a parameter of diarrhea. A large amount of PUFA such as EPA and its oxides is also considered a potential diarrhea inducer, in addition to substances derived from planktons well known for their diarrheal toxin like OA.

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It is well documented that methylmercury (MeHg) in vivo slowly undergoes demethylation reaction to change to inorganic mercury (Hg-i). The cleavage of the C-Hg bond was suggested to occur via a reactive oxygen-mediated process in vitro. To study the possibility of the involvement of hydroxyl radical (·OH) in the demethylation of MeHg in vivo, the combined effect of Fe-overload and carbon tetrachloride (CCl₄) treatment on the rate of biotransformation of MeHg was examined in rats. The effects of this treatment on H₂O₂-scavenging enzyme activities (catalase and glutathione peroxidase) were also studied. Rats were fed 3.5% Fe (II) fumalate-containing diet for 0, 7 or 21 d to load Fe. Feeding of the Fe-containing diet brought about a time-dependent increase of the hepatic Fe levels by nearly 7-fold after 3 weeks. The serum Fe levels showed a maximum on day 7, while the renal levels increased after day 7. TBA-reactive substance levels in the liver significantly increased along with the Fe feeding, and this increase was drastically accelerated by CCl₄ treatment, suggesting the effective production of ·OH. The CCl₄-induced stimulation of the lipid peroxidation was observed also in serum and kidney, though not as marked as in the liver. The hepatic accumulations of total and inorganic Hg at 72 h after MeHg administration were significantly increased by the Fe-feeding. The proportion of Hg-i to total Hg in this tissue increased markedly by the combined effect of Fe and CCl₄ with the concomitant decrease of catalase activity. On the other hand, the renal accumulation of Hg-i drastically decreased by the Fe-load, while the MeHg levels remained unchanged. Since the renal metallothionein levels were found to be lowered after the Fe-feeding, this might account for the reduced retention of Hg-i in the whole kidney. The present results suggest that reactive oxygen species, probably ·OH, induced by Fe and CCl₄ treatment may play a critical role in the biotransformation of MeHg in the liver.

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A speciation of heme and non-heme irons in food was carried out with atomic absorption spectrophotometric detection. Both heme and non-heme iron were extracted from an iron-containing protein with methyl isobutyl ketone (MIBK) under acidic conditions. A non-heme iron in the MIBK layer was transferred into the aqueous layer by washing the MIBK layer with water. Irons in each layer was determined by porlarized Zeeman atomic absorption spectrometry using the discrete nebulization technique. The calibration curves were linear between 0.1 and 6.0 μg Fe/ml. Recoveries of heme and non-heme irons from confectionaries fortified at the level of 40-50 μg Fe/g were more than 84%. Amounts of heme iron in commercially available foods determined by this method were in good agreement with the indicated values of iron. This method was recommended for the routine analysis of iron in hemoprotein and non-heme protein because of its simplicity and accuracy.

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The relationships of the concentration of rare earth elements (REEs) and 9 minerals (Ca, Mg, P, Fe, Na, K, Zn, Cu and Mn) in the liver, kidney, spleen, lung, femur and whole blood were investigated in the male rats of Wistar strain. High or low dose of dysprosium chloride (DyCl₃), europium chloride (EuCl₃), ytterbium chloride (YbCl₃) and yttrium chloride (YCl₃) was administered intravenously from the caudal vein. High doses were 20 mg/kg (as REEs) for Dy, Eu and Yb or 10 mg/kg (as REEs) for Y, and low doses were 10 mg/kg (as REEs) for Dy, Eu and Yb or 5 mg/kg (as REEs) for Y. These doses were similar to μmol of each REE per rat. The contents of REEs and 9 minerals in the liver, kidney, spleen, lung, femur and whole blood were estimated by the wet digestionICP-AES method 1 day after the intravenous administration. Results were as follows : 1) The concentrations and distribution rates (% of dose) of REEs in the liver, spleen and lung increased by several times in the rats administered high dose of Dy, Eu and Y [Dy (H), Eu (H) and Y (H)] contrary to the decrease of distribution rate of REEs in the kidney. Distribution rate did not change by dose in the rats administered Yb. 2) Ca concentrations in the liver, spleen and lung increased by several or several decade times in the rats of Dy (H), Eu (H) and Y (H) groups. There were the tendency of Na concentration to be higher and K concentration lower in these rats. Zn concentration in the liver increased slightly by REEs administration. There were no effects of REEs administration otherwise. 3) There were strong positive correlations (p<0.001) between REEs and Ca concentrations in the liver, spleen and lung in the rats administered Dy, Eu or Y ; Ca concentration increased proportionally to REEs concentration. There was no correlation between Ca and Yb concentration. Therefore, it was suggested that there might exist the threshold of REEs dose for the increase of Ca concentrations proportionally to REEs concentrations in the liver, spleen or lung, and the appearence of Yb toxicity might be different from other 3 REEs (Dy, Eu and Y) toxicities.

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Analysis of Trp-P-2 in municipal sewage water and investigation on its behavior during activated sludge treatment of Trp-P-2 was carried out. Prior to HPLC, we established the most suitable pretreatment-condition for soluble and adsorptive Trp-P-2 in the sewage water. Determination of soluble and adsorptive Trp-P-2 in the sewage water showed that Trp-P-2 exists in the sewage water and adsorptive form is too much more than soluble one. Activated sludge treatment of Trp-P-2 demonstrated that the most of Trp-P-2 adsorbs to activated sludge at an earlier period after the treatment of Trp-P-2. It is considered that the release of Trp-P-2 from activated sludge was recognized by sonication, alkalization or ethyl acetate extraction. Indirect frameshift mutagenicity of treated water during activated sludge treatment of Trp-P-2 showed a decrease with treatment time.

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The mutagenicity of airborne particulates in Tokyo was evaluated by the Salmonella microsuspension procedure. Airborne particles were collected in 9 collection stages according to their aerodynamic diameter and tested for the mutagenicity with S. typhimurium strain TA 98. Because the microsuspension procedure is highly sensitive, it was possible to detect the mutagenic activity from only 10 μg of the particles of less than 2.5 μm in diameter. Moreover we could detect the mutagenic activity of the particles ranging from 2.5 to 10 μm, which had been difficult to be detected by the original Ames test. The particles collected in winter showed higher activity than those in summer. The mutagenic activity was high in the particles of less than 0.4 μm and of about 1 μm in winter. In the same season, the specific activity for the mutagenicity of the particulates did not differ in the sampling area. The concentration of polyaromatic hydrocarbons in airborne particulates correlated with the mutagenicity of those in every collection stage, and it was suggested that mutagens in particulate matter behave as polyaromatic hydrocarbons.

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Blackfoot disease (BFD) is a peripheral vascular disease resulting in gangrene of the lower extremities. In the present work, the objective was to examine the amount of arsenic and mercury in urine of BFD patients. The urine specimens were acidified with nitric acid and digested in a microwave oven. A solid phase extraction (SPE) cartridge was used for sample purification and preconcentration. The analytical technique for the determination of arsenic was by hydride atomic absorption spectrophotometry (HAAS) and for mercury by cold vapor atomic absorption spectrophotometry (CVAAS). The sensitivity and accuracy of the analytical techniques were checked with Lanornorm control urine. Arsenic and mercury concentrations in the urine of normal controls and BFD patients were found to be 11.3±4.7 μg/1 and 33.6±23.1 μg/1 for As ; 5.0±1.8 μg/1 and 11.6±5.9 μg/1 for Hg, respectively. The life background of the BFD patients is also briefly mentioned.

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The determination and confirmation of trans-10-hydroxydecenoic acid (10-HDA) in royal jelly (RJ) were developed by capillary gas chromatography (GC) and GC-chemical ionization mass spectrometry (CI-MS) equipped with an ion trap detector (ITD) as a mass spectrometer. It was found that 10-HDA extracted with dichloromethane at pH 2.5 was converted into trimethylsilyl (TMS) derivative in a constant yield by the reaction with N, O-bis (trimethylsilyl) acetamide at room temperature for 15 min. The recovery of 10-HDA (50 μg) was 99.5%. The TMS derivatives of raw RJ tested showed a satisfactory result of separation and sensitivity by GC using a DB-1 capillary column with a flame ionization detector. The linear dynamic detection range was approximately from 50 ng to 500 μg (16.7-167000 ppm) determined using n-eicosane as an internal standard. The minimum detectable amount of 10-HDA was found to be 50 ng (16.7 ppm) at the split ratio of 40 : 1 of the split injection. The amount of 10-HDA in the raw RJ sample was 2.28±0.04% determined by the GC method and 2.25±0.01% by the HPLC method as described previously, respectively. The TMS derivatives of 10-HDA and its related compounds in the raw RJ were confirmed by ITD-GC. The diagnostic ions of TMS-HDA were presented at m/z 331 (M+1), 315 (M-Me), and 241 (M-OTMS) in isobutane CI mode. The present GC method is an easier and more convenient method for the analysis of 10-HDA in RJ and is applicable for the determination and confirmation of 10-HDA in forensic and food samples.

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[in Japanese]

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[in Japanese]

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