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In this paper we report two incidents of ciguatera fish poisoning (CFP) that occurred in 2008 on Kakeroma Island in Kagoshima Prefecture, Japan. A family consisting of father (42 y.o.), mother (39 y.o.), daughter (11 y.o.) and son (6 y.o.), as well as a friend of the family (male, 78 y.o.) consumed sliced flesh (sashimi) and developed typical symptoms of CPF 4-5 h later: diarrhea, vomiting, and paresthesia of the extremities. Additionally, the two male adults (42 and 78 y.o.) developed mild hypotention (84/48 and 94/40 mmHg, respectively) and bradycardia (36 and 50 bpm, respectively) and were rushed to the ER of the nearest hospital, located on an adjacent island. The implicated fish were caught together off the west coast of the island and were identified as Variola louti and Lutjanus monostigma based on analysis of 16S rRNA gene coded on mtDNA. Remnants of the implicated fish and other fish caught on the same occasion were examined by the official mouse bioassay method (MBA), which defines the minimum amount of CFP toxin needed to kill a male mouse of ddY strain of 20 g body weight within 24 h as one mouse unit (MU). A significantly high toxicity was detected in the V. louti (0.2 MU/g) eaten by the family and the L. monostigma (0.8 MU/g) eaten by the elderly man. Other specimens of Lethrinus nebulosus, Variola albimarginata, Lutjanus gibbus (2 specimens), Aphareus rutilans, and Sphyraena forsteri (2 specimens) were found to be nontoxic (< 0.025 MU/g). The medical records regarding island inhabitants (ca. 1,500) kept at Kakeroma Tokushukai Clinic, the only medical facility on the Island, and also at Setouchi Tokushukai Hospital, a nearest hospital on an adjacent island (Amami) revealed 6 CFP outbreaks involving 13 cases between 2005 and 2008. The estimated frequency of CFP was 10.0 incidents/10,000 persons/year and the morbidity rate was 21.7 cases/10,000 persons/year. The symptoms and signs observed in the patients and the implicated fish species, L. monostigma and V. louti, were typical of CFP in this region.

Ciguatera fish poisoning is the most extensive and difficult to control of the seafood poisonings. Tofacilitate monitoring of fish toxicity, toxin profiles were investigated by an LC/MS/MS method using 14reference toxins on eight representative species of fish collected in four different areas of the Pacific. Snappers and groupers from Okinawa contained ciguatoxin-1B (CTX1B) and two deoxy congeners at variablebut species-specific ratios, while red snapper, Lutjanus bohar, from Minamitorishima, and amberjack, Seriola dumerili, from Hawaii, contained both CTX1B-type and CTX3C-type toxins. Spotted knifejaw, Oplegnathus punctatus, from Okinawan waters, contained mainly CTX4A and CTX4B, but the same species caught at Miyazaki was contaminated primarily with the CTX3C-type toxins. Otherwise, the toxin profiles were consistently species-specific in fish collected from various locations around Okinawa over 20 years. The LC/MS/MSand mouse bioassay results agreed well, indicating the LC/MS/MS method is a promising alternative to themouse bioassay. Pure CTX1B and CTX3C were prepared for use in future LC/MS/MS analysis.

Ciguatoxins (CTXs) responsible for ciguatera fish poisoning (CFP) in Amami Islands, Kagoshima, Japan in 2008 were determined by LC-MS/MS analysis. Ciguatoxin-1B (CTX1B), 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B were detected in Variola louti and Lutjanus monostigma. The toxin profile distinctly differed from that of a CFP-related fish from Miyazaki, which mainly contained ciguatoxin-3C type toxins. Toxin profiles were species-specific, as observed in fish from Okinawa. The LC-MS/MS and mouse bioassay (MBA) methods produced comparable data, though 54-deoxyCTX1B was not taken into consideration owing to the lack of toxicity data. To improve assessment, toxicity data for this compound are needed. A reef fish caught on the same occasion and judged nontoxic by MBA (

Toxin profiles of representative ciguatera species caught at different locations of Japan were investigated in fish flesh by high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Identification and quantification of 16 toxins were facilitated by the use of 14 reference toxins prepared by either synthesis or isolation from natural sources and the previous LC-MS data thereof. Sodium adduct ions [M + Na](+) were used as parent and product ions. Distinct regional differences were unveiled: ciguatoxin-1B type toxins were found in snappers and groupers from Okinawa, ciguatoxin-3C type toxins were found in a spotted knifejaw, Oplegnathus punctatus, from Miyazaki located 730 km north of Okinawa, and both types of toxins were found in a red snapper, Lutjanus bohar, from Minamitorishima (Marcus) Island. Twelve toxins were identified in a dinoflagellate, Gambierdiscus toxicus, collected as the primary toxin source in French Polynesia. Occurrence of M-seco-toxins in fish and oxidized toxins in the dinoflagellate was confirmed for the first time. The present LC-MS/MS method is rapid, specific, and accurate. It not only outperforms the currently employed mouse bioassays but also enables the study of the toxin dynamics during the food chain transmission.

Okinawa being located in the subtropical region has the highest incidence of ciguatera in Japan. Officially, 33 outbreaks involving 103 patients have been reported between 1997 and 2006. The implicated species were Variola louti, Lutjanus bohar, Lutjanus monostigma, Epinephelus fuscoguttatus, unidentified Lutjanus sp., Plectropomus areolatus, Oplegnathus punctatus, Epinephelus polyphekadion, Caranx ignobilis and moray eel. Toxicities of the leftover meals, as determined by mouse bioassays, ranged from 0.025 to 0.8 MU/g or above (equivalent to 0.175–5.6 ng CTX1B/g). We collected 612 specimens of fish belonging to L. monostigma, L. bohar, Lutjanus argentimaculatus, Lutjanus russellii, V. louti, Variola albimarginata, and E. fuscoguttatus from the coasts around Okinawa and examined the toxicity of the flesh by the mouse bioassay. The rate of toxic fish was as follows: L. monostigma: 32.3%, L. bohar: 11.9%, V. louti: 14.3%, E. fuscoguttatus: 20.8%. Only one out of 36 samples of V. albimarginata and two of 74 samples of L. russellii were found toxic. None of the 35 samples of L. argentimaculatus was toxic. Nor the L. bohar samples weighing less than 4 kg were toxic. In all toxic samples, CTX1B was detected by LC/MS analysis but CTX3C and 51-hydroxyCTX3C were not.

We previously found and isolated a novel natural product, designated kohamaic acid A (KA-A), which inhibited the first cleavage of fertilized sea urchin eggs. In this paper, we report that this compound could selectively inhibit the activities of DNA polymerases (pol. alpha, beta, gamma, delta and epsilon ) only from species in the deuterostome branch in the animal kingdom, like sea urchin, fish and mammals, but not from protostomes including insects (fruit fly, Drosophila melanogaster) and mollusks (octopus and oyster). Inhibition of deuterostome DNA polymerases was dose dependent. IC(50) values for DNA polymerases of mammals and fish occurred at approximately 5.8-14.9 microM and those of sea urchin at 6.1-30.3 microM. In the sea urchin DNA polymerases, the activities of the replicative DNA polymerases such as alpha, delta and epsilon were more strongly inhibited than that of the repair-related pol. beta. KA-A is an inhibitor of replicative DNA polymerases from the deuterostome species, and subsequently, the inhibition of the first cleavage of fertilized sea urchin eggs might occur as a result of the suppression of DNA replication.

Two new sesterterpenic acids, which exhibited moderate cytotoxicity, were isolated from the marine sponge Ircinia sp. Their absolute stereochemistries were determined by spectroscopic and chemical analyses. Their configurations at C15 were different from those of previously isolated sesterterpenes. This result suggests the existence of a (14Z)-geranylfarnesyl pyrophosphate, which is yet unknown in nature.