Detection of Extremely Low Level Ciguatoxins through Monitoring of Lithium Adduct Ions by Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry.

Ciguatera poisoning (CP) is the most common type of marine biotoxin food poisoning worldwide, and it is caused by ciguatoxins (CTXs), thermostable polyether toxins produced by dinoflagellate Gambierdiscus and Fukuyoa spp. It is typically caused by the consumption of large fish high on the food chain that have accumulated CTXs in their flesh. CTXs in trace amounts are found in natural samples, and they mainly induce neurotoxic effects in consumers at concentrations as low as 0.2 µg/kg. The U.S. Food and Drug Administration has established CTX maximum permitted levels of 0.01 µg/kg for CTX1B and 0.1 µg/kg for C-CTX1 based on toxicological data. More than 20 variants of the CTX1B and CTX3C series have been identified, and the simultaneous detection of trace amounts of CTX analogs has recently been required. Previously published works using LC-MS/MS achieved the safety levels by monitoring the sodium adduct ions of CTXs ([M+Na]+ > [M+Na]+). In this study, we optimized a highly sensitive method for the detection of CTXs using the sodium or lithium adducts, [M+Na]+ or [M+Li]+, by adding alkali metals such as Na+ or Li+ to the mobile phase. This work demonstrates that CTXs can be successfully detected at the low concentrations recommended by the FDA with good chromatographic separation using LC-MS/MS. It also reports on the method's new analytical conditions and accuracy using [M+Li]+.

Structural Assignment of the Product Ion Generated from a Natural Ciguatoxin-3C Congener, 51-Hydroxyciguatoxin-3C, and Discovery of Distinguishable Signals in Congeners Bearing the 51-Hydroxy Group.

Ciguatoxins (CTXs) stand as the primary toxins causing ciguatera fish poisoning (CFP) and are essential compounds distinguished by their characteristic polycyclic ether structure. In a previous report, we identified the structures of product ions generated via homolytic fragmentation by assuming three charge sites in the mass spectrometry (MS)/MS spectrum of ciguatoxin-3C (CTX3C) using LC-MS. This study aims to elucidate the homolytic fragmentation of a ciguatoxin-3C congener. We assigned detailed structures of the product ions in the MS/MS spectrum of a naturally occurring ciguatoxin-3C congener, 51-hydroxyciguatoxin-3C (51-hydoxyCTX3C), employing liquid chromatography/quadrupole time-of-flight mass spectrometry with an atmospheric pressure chemical ionization (APCI) source. The introduction of a hydroxy substituent on C51 induced different fragmentation pathways, including a novel cleavage mechanism of the M ring involving the elimination of 51-OH and the formation of enol ether. Consequently, new cleavage patterns generated product ions at m/z 979 (C55H79O15), 439 (C24H39O7), 149 (C10H13O), 135 (C9H11O), and 115 (C6H11O2). Additionally, characteristic product ions were observed at m/z 509 (C28H45O8), 491 (C28H43O7), 481 (C26H41O8), 463 (C26H39O7), 439 (C24H39O7), 421 (C24H37O6), 171 (C9H15O3), 153 (C9H13O2), 141 (C8H13O2), and 123 (C8H11O).

[Analysis of Ciguatoxins in the Spotted Knifejaw, Oplegnathus punctatus from the Waters of Japan].

Ciguatera fish poisoning (CFP) is recognized as the most frequent seafood poisoning due to the consumption of fish containing the principal toxins, ciguatoxins (CTXs). In Japan, CFP events have been reported annually from Okinawa and Amami Islands, locating subtropical regions. In addition, there have been reported several outbreaks due to consumption of the fish caught from the Pacific coast of the Mainland and they were often caused by the matured spotted knifejaw, Oplegnathus punctatus. As part of our research on CFP in Japan, we investigated CTXs analysis by LC-MS/MS on 176 individuals of O. punctatus (weight: 100-6,350 g, standard length: 13-60 cm) from the coast of the Mainland (Honshu, Shikoku, and Kyushu), Amami, Okinawa, and Ogasawara (Bonin) Islands. CTXs were detected from only two specimens collected from Okinawa. Total CTXs levels of the two specimens were at 0.014 and 0.040 µg/kg, respectively, exceeding FDA guidance level at 0.01 µg CTX1B equivalent/kg. However, they might be little risk of CFP because consuming over 1.5 kg of flesh is needed to develop intoxication. The toxins consisted of CTX1B analogs including CTX1B, 52-epi-54-deoxyCTX1B, CTX4A, and CTX4B, and no CTX3C analogs, supporting the finding that ciguatoxic fishes in Okinawan Waters containing only CTX1B analogs.

A smartphone-controlled amperometric immunosensor for the detection of Pacific ciguatoxins in fish.

Ciguatoxins (CTXs) are marine neurotoxins produced by microalgae of the genera Gambierdiscus and Fukuyoa. CTXs may reach humans through food webs and cause ciguatera fish poisoning (CFP). An immunosensor for the detection of Pacific CTXs in fish was developed using multiwalled carbon nanotube (MWCNT)-modified carbon electrodes and a smartphone-controlled potentiostat. The biosensor attained a limit of detection (LOD) and a limit of quantification (LOQ) of 6 and 27 pg/mL of CTX1B, respectively, which were 0.001 and 0.005 µg/kg in fish flesh. In the analysis of fish samples from Japan and Fiji, excellent correlations were found with sandwich enzyme-linked immunosorbent assays (ELISAs), a cell-based assay (CBA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Stability of at least 3 months at -20 °C was predicted. In just over 2 h, the biosensor provides reliable, accurate and precise Pacific CTX contents in fish extracts, being suitable for monitoring and research programs.

[Analysis of Ciguatoxins in Variola louti Captured off the Ogasawara (Bonin) Islands].

Ciguatera poisoning (CP) is one of the most abundant seafood poisonings in the world. CP frequently occurred in the tropical and subtropical Indo-Pacific Ocean and the Caribbean Sea. In Japan, CP cases have been reported annually, from the subtropical regions, including Okinawa Prefecture and Amami Islands, Kagoshima Prefecture. The principal toxins, named ciguatoxins (CTXs), are bio-synthesized by benthic dinoflagellate of genera Gambierdiscus and Fukuyoa. They are bio-transferred herbivorous animals to carnivorous fishes via the food chain.The Ogasawara Islands comprise more than 30 islands, Mukojima Islands, Chichijima (Bonin) Islands, Hahajima Islands, Iwo Islands, Nishinoshima, Minamitorishima, and Okinotorishima, which locate in the tropical to subtropical regions. The Mukojima Islands, Chichijima Islands, and Hahajima Islands locate approximately the same latitude as Okinawa. The distance from Tokyo is approximately 1,000 km for Chichijima, 1,700 km for Okinotorishima (the southernmost tip of Japan), and 1,900 km for Minamitorishima (the easternmost tip of Japan). These islands exist in a wide range of waters, latitudes from 20°25' to 27°44' North and longitudes from 136°04' to 153° 59' East. We collected 65 specimens of a grouper, Variola louti, the most frequent species implicated in CP in Japan, from the waters around the Chichijima, Mukojima, and Hahajima islands. The fish flesh specimens were analyzed CTXs using the liquid chromatography-tandem mass spectrometer (LC-MS/MS). While the peak whose retention time is almost identical to that of CTX1B was detected in all specimens on our routine protocol, no 52-epi-54-deoxyCTX1B nor 54-deoxyCTX1B was detected. The peak retention time was quite different from that of CTX1B when re-analyzing by changing the analytical column. Thus, the CTXs in the specimens in the waters of these islands seemed to be undetectable levels.

Characteristic Distribution of Ciguatoxins in the Edible Parts of a Grouper, Variola louti.

Ciguatera fish poisoning (CFP) is one of the most frequently encountered seafood poisoning syndromes; it is caused by the consumption of marine finfish contaminated with ciguatoxins (CTXs). The majority of CFP cases result from eating fish flesh, but a traditional belief exists among people that the head and viscera are more toxic and should be avoided. Unlike the viscera, scientific data to support the legendary high toxicity of the head is scarce. We prepared tissue samples from the fillet, head, and eyes taken from five yellow-edged lyretail (Variola louti) individuals sourced from Okinawa, Japan, and analyzed the CTXs by LC-MS/MS. Three CTXs, namely, CTX1B, 52-epi-54-deoxyCTX1B, and 54-deoxyCTX1B, were confirmed in similar proportions. The toxins were distributed nearly evenly in the flesh, prepared separately from the fillet and head. Within the same individual specimen, the flesh in the fillet and the flesh from the head, tested separately, had the same level and composition of toxins. We, therefore, conclude that flesh samples for LC-MS/MS analysis can be taken from any part of the body. However, the tissue surrounding the eyeball displayed CTX levels two to four times higher than those of the flesh. The present study is the first to provide scientific data demonstrating the high toxicity of the eyes.

Development and Validation of an LC-MS/MS Method for the Ultra-Trace Analysis of Pacific Ciguatoxins in Fish.

BACKGROUND: Ciguatera fish poisoning (CFP) poses a serious threat to both public health and the use of aquatic resources from the various warm-water regions of the world. Hence, a process for the efficient determination of the relevant toxins is required. OBJECTIVE: We sought to develop and validate the first LC-MS/MS method to quantify the major toxins prevalent in fish from the Pacific Ocean. METHOD: Toxins were extracted from fish flesh (2 g) using a methanol-water mixture (9:1, v/v). The extract was heated at 80°C, and low-polarity lipids were eliminated using hexane, initially from the basic solution and later from the acidic solution. The cleanup was performed using solid-phase extraction, Florisil, silica, reversed-phase C18, and primary secondary amine columns. A validation study was conducted by spiking fish flesh with two representative toxins having different skeletal structures and polarities and was calibrated by NMR (qNMR) spectroscopy. RESULTS: The validation parameters for the ciguatera toxins CTX1B and CTX3C at spiked levels of 0.1 µg/kg were as follows: repeatabilities of 2.3-3.5% and 3.2-5.3%; intermediate precisions of 6.3-9.8% and 6.0-7.4%; recoveries of 80-107% and 95-120%, respectively. The lowest detection levels were 0.004 µg/kg for CTX1B, 0.005 µg/kg for 51-hydroxyCTX3C, and 0.009 µg/kg for CTX3C. CONCLUSIONS: The described method practically clears the international action level of 0.01 µg/kg CTX1B equivalents set by the U.S. Food and Drug Administration and the European Food Safety Authority and satisfies the global standards set by Codex and AOAC INTERNATIONAL. HIGHLIGHTS: A validation study for an LC-MS/MS method for ciguatoxin detection was completed for the first time using calibrated toxin standards.

[Detection of Ciguatoxins from Fish Introduced into a Wholesale Market in Japan].

Ciguatera fish poisoning (CFP), one of the most frequently occurring seafood poisonings due to marine finfish consumption, mainly affects the tropical and subtropical Indo-Pacific region and the Caribbean Sea. The principal class of toxins, ciguatoxins (CTXs) from the Pacific, includes more than 20 derivatives and are classified into two groups, CTX1B and CTX3C congeners, based on their skeletal structures. As part of risk management of CFP by the Japanese government, the import of certain species of fish into Japan is prohibited. Additionally, local governments recommend rejecting certain fish species caught in Japan. In this study, we used LC-MS/MS to analyze CTXs from 18 fish specimens belonging to 7 species that had been brought to a wholesale market but were disapproved for sale because of their potential danger of CFP. CTXs were detected in four specimens of Lutjanus bohar and one specimen of Variola louti. It was estimated that the two most poisonous specimens (no. 5: 0.348 µg/kg, no. 8: 0.362 µg/kg) had a toxicity of 0.05 MU/g. Consumption of 200 g of flesh from these fish could cause CFP. Thus, the guidance of the local government to disallow the sale of these fish species in the market contributed to the prevention of CFP.Only CTX1B congeners were detected in L. bohar (specimen no. 5), which had no record of the area where it captured from. It is presumed that the origin of specimen no. 5 was the same as that of the Okinawan L. bohar because the CTX compositions were similar. In two specimens (nos. 6 and 8) from Wakayama, both CTX1B and CTX3C congeners were detected. This is the first report to reveal the CTX profile in fish collected off the Honshu island in Japan.

First Report of Microcystis Strains Producing MC-FR and -WR Toxins in Japan.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography-mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan.

Biooxidation of Ciguatoxins Leads to Species-Specific Toxin Profiles.

Ciguatoxins (CTXs) contaminate fish worldwide and cause the foodborne illness ciguatera. In the Pacific, these toxins are produced by the dinoflagellate Gambierdiscus toxicus, which accumulates in fish through the food chain and undergoes oxidative modification, giving rise to numerous analogs. In this study, we examined the oxidation of CTXs in vitro with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using reference toxins, and found that CTX4A, CTX4B, and CTX3C, which are produced by the alga, are oxidized to the analogs found in fish, namely CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, 2-hydroxyCTX3C, and 2,3-dihydroxyCTX3C. This oxidation was catalyzed by human CYP3A4, fish liver S9 fractions, and microsomal fractions prepared from representative ciguateric fishes (Lutjanus bohar, L. monostigumus, and Oplegnathus punctatus). In addition, fish liver S9 fractions prepared from non-ciguateric fishes (L. gibbus and L. fulviflamma) in Okinawa also converted CTX4A and CTX4B to CTX1B, 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B in vitro. This is the first study to demonstrate the enzymatic oxidation of these toxins, and provides insight into the mechanism underlying the development of species-specific toxin profiles and the fate of these toxins in humans and fish.

[Accumulation of tetrodotoxin from diet in two species of scavenging marine snails].

A feeding experiment of TTX-containing diet was conducted using the small scavenging marine snails Pliarcularia globosa and Reticunassa festiva. Seventy-five specimens of each species were divided into 15 groups of 5 individuals, of which 3 groups were directly submitted, without feeding, to toxin quantification as described below. TTX was not detected. Each of the remaining 12 groups was accommodated in a plastic case (80×70×40 mm) filled with seawater, and fed for 24 hours with ovary tissue (0.1 g) of the pufferfish Takifugu vermicularis, whose TTX content had previously been determined. Then the seawater was exchanged for fresh seawater, the snails were reared for 4 days without feeding, and then the seawater was changed again. This feeding/rearing cycle (5 days) was repeated 8 times, and 3 groups were sampled every 2 cycles. The combined viscera and combined muscle of each group were each extracted with 0.1% aqueous acetic acid, and then TTX was quantified by liquid chromatography-mass spectrometry. The estimated amount of ingested TTX was calculated by multiplying the difference between the amounts of ovary tissue supplied and remaining by the toxin content (122-126 MU/g). Similar mean values of 5.1 MU/group/cycle in P. globosa and 5.3 MU/group/cycle in R. festiva were obtained. Toxin content (TTX amount per gram of tissue) and toxin amount (TTX amount per group) during the experimental period were 0.23-2.85 MU/g and 0.05-0.96 MU/group, respectively, in P. globosa viscera. Both values increased markedly from the 2nd cycle to the 6th cycle. In contrast, no such increase in toxin content/amount was observed throughout the experimental period in P. globosa muscle (<0.05-0.86 MU/g, <0.02-0.27 MU/group), R. festiva viscera (<0.05-0.8 MU/g, <0.02-0.33 MU/group), and R. festiva muscle (<0.05-0.81 MU/g, <0.02-0.23 MU/group). The remaining ratio of TTX (percentage of total toxin amount [sum of the toxin amount of viscera and muscle] to estimated TTX ingestion amount) was less than 4% in P. globosa, and less than 2% in R. festiva after the 4th cycle, suggesting that the possibility that these two species would accumulate TTX at levels high enough to raise food hygiene issues is low.

Determination of toxins involved in ciguatera fish poisoning in the Pacific by LC/MS.

Ciguatera fish poisoning is the most extensive and difficult to control of the seafood poisonings. To facilitate monitoring of fish toxicity, toxin profiles were investigated by an LC/MS/MS method using 14 reference 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 variable but 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/MS and mouse bioassay results agreed well, indicating the LC/MS/MS method is a promising alternative to the mouse bioassay. Pure CTX1B and CTX3C were prepared for use in future LC/MS/MS analysis.

[Toxicity and toxin profile of scavenging and carnivorous gastropods from the coastal waters of Okinawa Prefecture, Japan].

From January to June 2009, a total of 64 gastropod specimens of 15 species were collected from the coastal waters of Okinawa Prefecture, Japan, and examined for toxicity by means of mouse bioassay. Among the specimens tested, 5 species, Nassarius glans, Nassariuscoronatus, Olivaannulata, Oliva concavospira and Zeuxis sp., were toxic. The toxicity scores of N. glans were very high; 39.6-461 MU/g in muscle, and 98.6-189 MU/g in viscera including digestive gland, followed by Zeuxis sp. (12.7 MU/g in whole body), N. coronatus (5.64-11.1 MU/g in whole body), O. annulata (10.8 MU/g in the whole body), O. concavospira (6.65 MU/g in the muscle). Liquid chromatography/mass spectrometry (LC-MS) revealed that the major toxic component was tetrodotoxin (TTX), which accounting for 13-82% of the total toxicity. As for the remaining toxicity in the case of N. glans, 4,9-anhydroTTX, 4-epiTTX and 11-oxoTTX were contributors. Moreover, Niotha albescens showed no toxicity (less than 10 MU/g) in mouse bioassay, but TTX (5.08 MU/g) was detected by LC-MS. Paralytic shellfish poison was not detected in any of the specimens by high-performance liquid chromatography with fluorometric detection (HPLC-FLD).

[Toxin profiles in fish implicated in ciguatera fish poisoning in Amami and Kakeroma Islands, Kagoshima Prefecture, Japan].

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 (<0.025 MU/g) was found to contain low levels of CTX, indicating a potential risk for CFP.

Detailed LC-MS/MS analysis of ciguatoxins revealing distinct regional and species characteristics in fish and causative alga from the Pacific.

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.

The first F-ring modified ciguatoxin analogue showing significant toxicity.

Ciguatoxins, the principal causative toxins of ciguatera seafood poisoning, are potent neurotoxic polycyclic ethers. We report herein the total synthesis of a 10-membered F-ring analogue of 51-hydroxyCTX3C, which constitutes the first example of an F-ring modified ciguatoxin that exhibits potent cytotoxicity as well as mouse acute toxicity.

Ciguatera incidence and fish toxicity in Okinawa, Japan.

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 ngCTX1B/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.

The effect of structural variation in 21 microcystins on their inhibition of PP2A and the effect of replacing cys269 with glycine.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. The representative MC, MC-LR, strongly inhibits protein phosphatase 2A (PP2A), while the inhibitory potencies of at least 60MC analogs characterized from bloom samples and cultured strains have not been fully elucidated. In this study, we determined the IC(50) values for 21MC analogs for inhibiting the recombinant PP2A catalytic subunit (rPP2Ac). Of the 21MC analogs, MC-LR was the strongest inhibitor of rPP2Ac. Comparison of the IC(50) values indicates that demethylation of the amino acids at positions 3 or 7 leads to a greater reduction in activity than the substitution of l-amino acids at positions 2 and 4. To obtain further insight into the MC-PP2A interaction, we substituted cysteine at position 269 in PP2Ac with glycine. The mutant PP2Ac (C269G) was comparable to the wild-type PP2Ac in the hydrolysis of p-NPP, but was more resistant to MCs as indicated by the greater IC(50) values. Our results indicate that cys269 in PP2Ac and N-methyldehydroalanine (Mdha) at position 7 in MCs play important roles in the enzyme-inhibitor interaction. We also determined the LC(50) values of the MCs for cytotoxicity assay. Our results indicate that there is a weak correlation between the cytotoxicity and PP2A inhibiting activities of the MCs. The MCs and rPP2Ac used in this study were of high purity and the IC(50) values were determined under the same experimental conditions, ensuring the quality of the data. The IC(50) values are of practical importance because they enable the precise conversion of the amounts of various MCs detected using instrumental methods to MC-LR equivalents.

[A case of food poisoning due to ingestion of eggplant, Solanum melongena, grafted on Devil's trumpet, Datura metel].

A husband and wife successively visited an emergency room with symptoms of staggering, slurred speech, mydriasis, and drowsiness, three hours after separately eating spaghetti with meat sauce. The sauce contained eggplant that had been grafted onto a Devil's trumpet, Datura metel. Scopolamine and atropine were detected in the leftover sauce and in the sera of the patients. This is the first case of food poisoning related to Datura in Okinawa, Japan and also might be the first report of food poisoning caused by intake of a vegetable grafted onto Datura in Japan.