Haruo Nukaya

BREVETOXIN B1, A NEW POLYETHER MARINE TOXIN FROM THE NEW ZEALAND SHELLFISH, AUSTROVENUS STUTCHBURYI

Abstract A new polyether marine toxin, brevetoxin B 1 , was isolated from a methanol extract of the New Zealand shellfish, Austrovenus stutchburyi , by chromatography on columns of SiO 2 , ODS, and LH-20, followed by reverse-phase HPLC and its structure was elucidated by comparison of its spectral data with those of brevetoxins B and use of NMR techniques.

Furan fatty acid as an anti-inflammatory component from the green-lipped mussel Perna canaliculus

A lipid extract of Perna canaliculus (New Zealand green-lipped mussel) has reportedly displayed anti-inflammatory effects in animal models and in human controlled studies. However, the anti-inflammatory lipid components have not been investigated in detail due to the instability of the lipid extract, which has made the identification of the distinct active components a formidable task. Considering the instability of the active component, we carefully fractionated a lipid extract of Perna canaliculus (Lyprinol) and detected furan fatty acids (F-acids). These naturally but rarely detected fatty acids show potent radical-scavenging ability and are essential constituents of plants and algae. Based on these data, it has been proposed that F-acids could be potential antioxidants, which may contribute to the protective properties of fish and fish oil diets against chronic inflammatory diseases. However, to date, in vivo data to support the hypothesis have not been obtained, presumably due to the limited availability of F-acids. To confirm the in vivo anti-inflammatory effect of F-acids in comparison with that of eicosapentaenoic acid (EPA), we developed a semisynthetic preparation and examined its anti-inflammatory activity in a rat model of adjuvant-induced arthritis. Indeed, the F-acid ethyl ester exhibited more potent anti-inflammatory activity than that of the EPA ethyl ester. We report on the in vivo activity of F-acids, confirming that the lipid extract of the green-lipped mussel includes an unstable fatty acid that is more effective than EPA.

Identification of 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) as a potent mutagen in river water in Kyoto and Aichi prefectures, Japan.

We have previously isolated five mutagens in blue rayon-adsorbed substances from water at a site below sewage plants in the Nishitakase River, in Kyoto, Japan, and identified two of them as 2-phenylbenzotriazole derivatives, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). In the present study, we collected adsorbed materials on blue cotton (3 kg x 9 times) at the same location, and isolated a sufficient amount (97 microg) of one of the remaining three mutagens other than PBTA-1 and PBTA-2, for structural analysis, by multiple column chromatography. The structure of mutagen, accounting for 12% of the total mutagenicity of the blue rayon-adsorbed substances, was determined to be a PBTA-1 analogue, 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4). PBTA-4 is a potent mutagen, inducing 190,000 and 7,800,000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix. In addition to the water of the Nishitakase River, PBTA-4 was detected in water samples from two rivers that flow through other regions where textile-dyeing industries have been developed. Like other PBTA analogues, PBTA-4 might also be produced from azo dyes during industrial processes in dyeing factories and treatment at sewage plants.

Concise Synthesis of Chafurosides A and B

The regioselective synthesis of chafurosides A (1) and B (2) from the same methyl ketone 5 was accomplished using a novel protecting group strategy. Both flavone rings were constructed from beta-diketone intermediate 4, which was readily obtained by condensation of an acyl donor and ketone 5. Construction of the dihydrofuran ring was achieved via an intramolecular Mitsunobu reaction.

Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan

We previously determined the chemical structures of four 2-phenylbenzotriazole mutagens (PBTA-1, -2, -3 and -4) in blue rayon-adsorbed material from the Nishitakase River in Kyoto prefecture and the Nikko River in Aichi prefecture in Japan. On the basis of a synthesis study, these four PBTA derivatives were deduced to have originated from corresponding dinitrophenylazo dyes by reduction and chlorination. 2-[(2-Bromo-4,6-dinitrophenyl)azo]-5-[bis(2-acetoxyethyl) amino]-4-methoxyacetanilide (Color Index Name, Disperse Blue 79:1; CAS Registry Number, 75497-74-4) is a very common dinitrophenylazo dye used in textile dyeing factories. In the present study, we synthesized 2-[4-[bis(2-acetoxyethyl)amino]-2-(acetylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-5) from Disperse Blue 79:1 by reduction with sodium hydrosulfite and subsequent chlorination with sodium hypochlorite. On hydrolysis of PBTA-5 with alkali, 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) was obtained. Both PBTA-5 and -6 were potent mutagens, inducing 723,000 revertants and 485,000 revertants per microgram of Salmonella typhimurium YG1024, respectively, in the presence of S9 mix. To clarify whether PBTA-5 and -6 exist in the environment, water samples were collected from five rivers flowing through regions where textile dyeing industries are developed. PBTA-6 was detected at levels of 3-134 ng/g blue rayon in all water samples that were examined. On the other hand, the amount of PBTA-5 in the samples was less than the detection limit.

Seasonal fluctuation of the mutagenicity of river water in Fukui, Japan, and the contribution of 2-phenylbenzotriazole-type mutagens

To clarify their mutagenic potential, samples of water from the Mawatari, Asuwa and Kitsune rivers, which flow through the central area of Fukui, Japan, were seasonally collected at six sites using blue rayon from July 1998 to August 2000. Forty-five of 52 (87%) of the water samples exhibited mutagenicity toward Salmonella typhimurium YG1024 and YG1029 with and without S9 mix, and the highest potencies were observed in YG1024 with S9 mix. The samples collected in summer and autumn tended to be more mutagenic than those collected in winter and spring. Fractionation using high-performance liquid chromatography (HPLC) suggests that several compounds are responsible for the mutagenicity of river water samples, and some of the major mutagens seem to be common among the samples. Three 2-phenylbenzotriazole (PBTA)-type mutagens, 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-3), 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) and 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6), were quantified in samples collected between July 1998 and April 1999. At least one of these PBTA-type mutagens was detected in 23/24 (96%) of the samples. The amounts of PBTA-3, -4 and -6 were <0.08-58.7, <0.1-15.0 and <0.07-467.9 ng/g of blue rayon, respectively, and high levels of PBTA congeners were detected in the samples collected from each river in July and November 1998. The contributions of these PBTA congeners to the mutagenicity of water samples were also high in July and November 1998. The highest total contribution was observed for samples from the Asuwa river (67.6%). These findings suggest that these three rivers were continually and heavily contaminated with mutagens, and PBTA congeners were some of the major mutagens in these rivers.

Genotoxic activity of 1-nitropyrene in water from the Yodo River, Japan

The identification of mutagenic 1-nitropyrene, one of the direct-acting nitroarenes, in a source of municipal drinking water is described. A diethyl ether extract recovered from river water by the XAD-2 resin column method was separated into neutral, acid and basic fractions. The neutral fraction accounted for 52 and 36%, respectively, of the genotoxicity of the extract in the absence and the presence of the metabolic activation system, for strain NM2009, which is sensitive to nitroarenes and/or aminoarenes. When separated by silica gel column chromatography, the benzene fraction of the neutral fraction, showed the highest genotoxic activity. The genotoxicity of the benzene fraction accounted for 80 and 60%, respectively, of the neutral fraction, in the absence and the presence of S9 mix. These results show that mutagenic nitroarenes might be contained in the benzene fraction of the neutral one. The benzene fraction was further subjected to HPLC and fractionated. A 1-nitropyrene-corresponding fraction was collected and subjected to capillary gas chromatography-mass spectrometry (GC-MS). The mass spectrum showed molecular and fragment ion peaks of 1-nitropyrene: 247, 217, 201, 189. Moreover, the concentration of 1-nitropyrene in municipal river water was 1 ng/l, accounting for only 1% of the total genotoxicity.

Inhibitory Effects of Whisky Congeners on IgE-Mediated Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells and Passive Cutaneous Anaphylaxis Reaction in Mice

Whisky is matured in oak casks. Many nonvolatile substances (whisky congeners, WC) seep from the oak cask during the maturing process. In this study, three antiallergic agents (syringaldehyde, SA; lyoniresinol, Lyo; and ellagic acid, EA) were isolated from WC. Treatment with SA, Lyo, and EA reduced the elevation of intracellular free Ca(2+) concentration ([Ca(2+)]i) and intracellular ROS production caused by FcepsilonRI activation. The inhibitions of the elevation of [Ca(2+)]i and intracellular ROS production by SA and Lyo were mainly due to the suppression of the NADPH oxidase activity and scavenging of the produced radical, respectively. On the other hand, EA inactivated spleen tyrosine kinase and led to the inhibition of the elevation of [Ca(2+)]i and intracellular ROS production. Furthermore, it was found that WC strongly inhibited IgE binding to the FcepsilonRIalpha chain, whereas SA, Lyo, and EA did not indicate this inhibitory effect. These results suggest that WC inhibits allergic reactions through multiple mechanisms. To disclose the in vivo effects of WC, SA, Lyo, and EA, these compounds were administered to type I allergic model mice, and the passive cutaneous anaphylaxis (PCA) reaction was measured. These compounds remarkably suppressed the PCA reaction. Taken together, these findings suggest that WC seemed to be beneficial to ameliorate allergic reactions.

Blue Chitin columns for the extraction of heterocyclic amines from cooked meat

Mutagenic/carcinogenic heterocyclic amines (HAs) are formed at low levels (ng/g) during heat processing of protein-rich food such as meat and fish. The complex matrix requires effective extraction and purification methods. Blue Chitin columns were used for the extraction of HAs from fried chicken fillets and the samples were analysed with LC-MS. Several HAs were identified at levels ranging from 0.04 to 0.10 ng/g. The use of Blue Chitin columns provides a simple and fast method for the extraction of HAs from meat samples.

Chromosomal effects of newly identified water pollutants PBTA-1 and PBTA-2 and their possible mother compounds (azo dyes) and intermediates (non-ClPBTAs) in two Chinese hamster cell lines.

We performed the in vitro micronucleus (MN) test on 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)-ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), which are newly identified water pollutants from the Nishitakase river in Kyoto, Japan, and on their possible mother compounds (AZO DYE) and intermediates (non-ClPBTAs). We tested these compounds in the absence and presence of S9 mix in two Chinese hamster cell lines CHL and V79-MZ and scored MN, polynuclear and karyorrhectic (PN), and mitotic (M) cells. PBTA-2 in the absence of S9 mix induced the strongest responses in both cell lines. It was also a strong inducer of binucleate cells in PN cells in both cell lines, which suggested that it induced polyploidy. PBTA-1 showed clear positive results only in the absence of S9 mix and only in V79-MZ cells, inducing aneuploidy. In CHL cells AZO DYE-1 significantly induced MN cells in the presence of S9 mix, and AZO DYE-2 induced MN and PN cells, including binucleate cells and cells with a multilobed nucleus, in the absence of S9 mix. In V79-MZ cells, AZO DYE-1 and -2 induced primarily M cells in the presence of S9 mix. 9% of the M cells treated with 50 microg/ml AZO DYE-1 showed endoreduplication. AZO DYE-2 at 200 microg/ml condensed the chromatin in 100% of the cells. The non-ClPBTAs were a bit more cytotoxic than the other compounds and induced a slight increase in MN cells in both cell lines. Some of the chemicals tested induced a characteristic karyomorphology that might reflect abnormal cell division. Abnormalities of cell division could be detected in PN and M cells as well as in MN cells. Structure-activity relationships have also been discussed.