MT test Immunochromato-PSP had been developed in a collaborative research project. In this kit, the previously developed mouse monoclonal antibody GT-13A designed against GTX2/3 is used. Since STX and its analogs (STXs) are small molecules, a competitive inhibition format with modified-STX is applied. The formation of Avidin Biotin complexes to trap modified-STX on the test line showed interference by the bivalve matrix, so we improved the kit with oligonucleotides trapping complementary strands. The affinity of the GT-13 antibody differs depending on the STX analogs present and does not correspond to relative toxicity. Therefore, it is necessary to accumulate data in advance on paralytic shellfish toxins (PSTs) toxin profiles for the local target species and area. Since this kit is intended to be used in screening, it is necessary to consider a dilution factor that will never lead to a false negative against the regulatory value. Although this kit is qualitative, it can be recorded and compared objectively as semi-quantitative data by imaging and quantifying. It can also be used to determine PSTs presence in seawater samples. In recent years, the problem of PSTs has become more serious in the east and north of Japan. We are considering using the kit for monitoring scallops in one prefecture and have confirmed that some of the samples could be assessed with the kit and applied to screening. However, we also observed transformation of PSTs after the shellfish became highly toxic, limiting the utility of the kit in these cases.

Paralytic shellfish toxins (PSTs) consist of saxitoxin (STX) and approximately 60 structural analogues. We report here a new STX analogue, 12β-deoxyM2, detected in bivalve molluscs. Because 12β-deoxyM2 and STX are detected on the same selected reaction monitoring (SRM) transition in LC-MS/MS and exhibit very close retention times, detailed mass spectrometric and chromatographic characterization was carried out to enable identification of 12β-deoxyM2. In our present study, presence of 12β-deoxyM2 in bivalves was unambiguously demonstrated.

This study aimed to investigate if imidazole dipeptides (IDPs) undergo heat-induced denaturation and to identify denatured IDPs in food products. Herein, IDPs were heated at 120 °C for 4 h in both model systems and various foods for denaturation and subjected to mass spectrometry and nuclear magnetic resonance analyses. Notably, denatured carnosine, anserine, and balenine were identified as acryloyl-histidine, acryloyl-Nπ-methyl-histidine, and acryloyl-Nτ-methyl-histidine, respectively. These acryloyl-histidine-related components were not detected in raw meat samples, but their levels increased significantly after heat treatment. Additionally, they were found in commercially available processed meat products and IDP-containing supplements. Further, acrylamide was not detected post-heating at 120 °C, indicating that IDPs do not serve as a precursor for acrylamide under typical cooking conditions below 150 °C. Overall, these findings link the IDP denaturation pathway to the Maillard reaction and its influence on the bioavailability of acryloyl-histidine-related components and potential health effects, considering their regular consumption in daily diets.

Okadaic acid (OA) is a lipophilic phycotoxin that causes acute diarrhoea when ingested. OA is an inhibitor of protein phosphatase 2 A, but the mechanism of toxicity behind the diarrhoea remains unclear. OA modulated inflammatory markers in epithelial cells, however, the effect on endothelial cells, with a key role in the inflammatory cascade, has not been previously addressed. Therefore, the aim of the present work was to test the effect of OA in human (HMEC-1) and mouse (MS1) endothelial cells. After 3, 6 and 24 h of incubation in the presence of OA (10-1000 nM) cell viability was significantly reduced, showing a higher effect on human cells with half inhibitory concentrations (IC50) in HMEC-1 cells five times lower than in mouse cells. Furthermore, when cells were treated with OA, significant amounts of the proinflammatory mediators ROS, CD147, IL-6 and monocyte chemoattractant protein 1 (MCP-1) were detected. Some of these effects were observed only in HMEC-1 cells and around three hours earlier, pointing again to a higher sensitivity in human models. Finally, OA triggered phosphorylation of NFκB at 100 nM after 3 and 6 h of treatment, while the signal transducer and activator of transcription 3 (STAT3) was increased after 3 h but decreased after 6 h in both cell lines. Altogether, these data suggest that the toxic effect of OA in endothelial cells could be related with the activation of the inflammatory cascade.

Tetrodotoxin (TTX), the pufferfish toxin, has the potential to cause fatal food poisoning because of its potent voltage-gated sodium channel (Nav) blocking activity. 4-epiTTX, 11-norTTX-6(S)-ol, and 11-oxoTTX are the major TTX analogues found in marine animals; thus, their chemical properties and biological activities should be determined. In this study, these three TTX analogues were purified to a high level (purity >97%) from pufferfish and newts. The ratios of the hemilactal to the 10,7-lactone forms were determined using 1H NMR, and 13C NMR data were also obtained. 4-epiTTX underwent considerable transformation to TTX in physiological conditions. The Nav blocking activity of these analogues was evaluated by whole-cell patch-clamp recording using a human Nav1.2, colorimetric cell-based assay and mouse bioassay. The toxicity equivalency factors (TEFs setting TTX 1) of the three analogues were estimated; 4-epiTTX (0.06), 11-norTTX-6(S)-ol (0.02), and 11-oxoTTX (1.2) using patch-clamp recording, and 11-norTTX-6(S)-ol (0.50) and 11-oxoTTX (0.42) using the mouse bioassay. These data confirmed the low Nav blocking activity of 4-epiTTX and high activity of 11-oxoTTX.

Ascidian Halocynthia roretzi is a very important aquacultured species on the coast of Miyagi prefecture. In Miyagi prefecture, the harvest season of this species is from late spring to early summer, which is also the season when Alexandrium species as the causative plankton of paralytic shellfish poisoning (PSP) have been observed in the coastal waters. There have been reports on the detection and tissue distribution of paralytic shellfish toxins (PSTs) in H. roretzi, but there are few reports on such tissue distribution throughout the PSP season and on the metabolism of PSTs. In this study, we detected PSTs from H. roretzi throughout the PSP season. The results indicated that most of the PST was localized in the hepatopancreas of H. roretzi throughout the PSP season and there is a possibility that the PST amounts of individuals could be reduced to about 10% by removing the hepatopancreas of the contaminated H. roretzi.

Saxitoxin (STX) and its analogues produced by toxic dinoflagellates in marine environments are known as specific voltage-gated sodium channel blockers and can be detected by the mouse bioassay (MBA) that has been used as the official testing method for paralytic shellfish toxin monitoring systems worldwide. Considering animal welfare issues and improved performance of analytical instruments, it would be better to replace MBA with non-animal testing methods such as LC-MS/MS which have been recently reported for the detection of STXs in shellfish. However, STX itself is regulated by the Act on the Prohibition of Chemical Weapons, so the domestic use of the reference material is required with permission from the relevant government department. Therein, nontoxic enantiomeric STX (ent-STX) was developed as an alternative reference material for STX. In this study, the LC-MS/MS method using ent-STX-fortified scallops homogenate was validated in accordance with a previous report and CODEX-STAN 292-2008. The spiked concentrations of ent-STX were set to the regulatory limits of CODEX (0.8 mg of STX-2HCl equivalents/kg) and approximately half of the limits. The calibration standard solutions were prepared by dilution with solvent and non-toxic scallop extract, respectively, and were used to quantify the toxins by absolute calibration method. As a result of the validation, the results with ent-STX were found falling within all the guideline criteria (the trueness, repeatability, within laboratory re-producibility, and minimum applicable range). Moreover, ent-STX was used to confirm and quantify STX in mussels, scallops and noble scallops. The ent-STX was confirmed to be utilized as reference material. However, it can be sometimes detected an unidentified STX analogue in Japanese bivalve molluscs with retention time close to STX under the qualitative and quantitative selected reaction monitoring (SRM) conditions of STX which are often used in the previous reports. To avoid the assignment of the unidentified STX analogue to STX, appropriate SRM conditions of m/z 300.1>282.0 need to be selected.

Species diversity of amphidomatacean dinoflagellates belonging to Azadinium and Amphidoma was examined by microscopic observation and ITS-and LSU rDNA-based phylogeny, using 81 culture strains and two uncultured cells from Japanese waters during 2016-2024. In total, 11 species were found in Japanese waters. Of these, Azadinium caudatum, Az. cuneatum, Az. dexteroporum, and Az. spinosum were newly found in the Northwest Pacific, Az. dalianense was the first report in Japan, and Azadinium inconspicuum sp. nov. was a new species described in this study. Azadinium inconspicuum independently branched in the clade of Az. dexteroporum/Az. galwayense/Az. perfusorium but differed from Az. dexteroporum in shape of the Po plate (absent vs. present of the finger-like protrusion), from Az. galwayense in shape of the 2a plate (four-vs. five-sided), and from Az. perfusorium in position of pyrenoid (in the episome vs. at the antapex). It had a close resemblance to Az. luciferelloides in thecal morphology, but differed in detailed position of the ventral pore and shape of the posterior sulcal plate. Azaspiracids (AZAs) were detected in Azadinium poporum and Az. spinosum, but no trace was found in other amphidomatacean cultures, including Az. dexteroporum. Among 81 cultures examined, 47 cultures were assigned to Az. poporum, and belonged to four intraspecific ribotypes. Major AZA components in Japanese Az. poporum strains were AZA-59 (ribotype A1), AZA-2 (ribotype A2), AZA-2,-11,-36,-40 (ribotype B), and AZA-2 (ribotype C1). These results suggested that Az. poporum, particularly in ribotype C1, where high AZA amounts were detected, is the major AZA-producer along the Japanese coastal waters.

Diarrhetic shellfish toxins (DSTs) and azaspiracids (AZAs) are lipophilic shellfish toxins produced by toxic dinoflagellates and known to pose significant risks to shellfish consumers. This study investigates tissue-specific uptake, accumulation and retention of dissolved AZA2, okadaic acid (OA), and dinophysistoxin-1 (DTX1) in Manila clam (Ruditapes philippinarum). Manila clam feeding on toxic dinoflagellate showed significant accumulation of AZA2 in the digestive glands. On the other hand, dissolved AZA2 was accumulated significantly in the gills and showed strong tissue retention. Manila clam accumulated only trace levels of the dissolved OA and DTX1 in the digestive glands in all of its tissues. These results suggested that Manila clam had different uptake mechanisms for AZAs and DSTs. Temperature significantly influenced dissolved AZA2 uptake, with higher accumulation at warmer conditions conducive to Manila clam activity. These findings offer the first comparative analysis of dissolved AZAs and DSTs uptake in Manila clam, highlighting their potential role in environmental toxin monitoring and food safety risk assessment.

Three novel hypoxanthine derivatives (1–3) were obtained from the Okinawan cyanobacterium Okeania hirsuta. The structures of these compounds were elucidated mainly based on the spectroscopic data, including 1D and 2D NMR, as well as high-resolution mass spectrometry. In particular, the amounts of obtained compounds 2 and 3 were only 200 μg and much less than 50 μg, respectively. Therefore, some carbons signals could not be observed on 13C NMR spectra of these compounds. However, the detailed analysis of HSQC and HMBC spectra allowed us to elucidate their structures. For NMR measurements of compound 3, it was found that using an 800 MHz NMR machine equipped with a cryogenic probe and acetic acid-d4 as a solvent is essential. Compounds (1–3) were N-3′-carbonylbutyl group-connected hypoxanthines.

In our present study, we developed quantitative 1H-nuclear magnetic resonance (1H-qNMR) spectroscopy to quantify several hydrophilic components related to fish quality indices and applied it to evaluate the characteristics of Japanese horse mackerel. The K-values from several fish, as determined by 1H-qNMR, closely matched those obtained by conventional high-performance liquid chromatography (HPLC) method. Following that, we applied 1H-qNMR to Japanese horse mackerel stored for up to 24 h. Although the K-values remained nearly constant within 24 h, the lactate content changed drastically, which was also reflected in the sensory evaluation of sourness. This study demonstrated that 1H-qNMR is applicable for a comprehensive assessment of fish quality and is highly useful in evaluating freshness on the basis of K-values, as well as other quality indices such as taste and fish odor.

A new lipopeptide, N-desmethylmajusculamide B (1), was isolated from the Okinawan cyanobacterium Okeania hirsuta along with 2 known compounds majusculamide A (2) and majusculamide B (3). The planar structure of (1) was elucidated by a detailed analysis of mass spectrometry and nuclear magnetic resonance spectra. The absolute configurations of the amino acid residues were determined using Marfey's analysis. The configuration of C-16 in the α-methyl-β-keto-decanoyl moiety was determined unambiguously to be S by conducting a semisynthesis of N-desmethylmajusculamide B from 3. The cytotoxicity against mouse L1210 leukemia cells was evaluated for majusculamides (1-3).

Lipophilic marine biotoxin azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters, and detailed toxin profiles of two strains (mdd421 and HM536) among them were clarified by several detection techniques on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). In our present study, AZA analogues in seven strains of A. poporum from Japanese coastal waters (including two previously reported strains) were determined by these detection techniques. The dominant AZA in the seven strains was AZA2 accompanied by small amounts of several known AZAs and twelve new AZA analogues. Eight of the twelve new AZA analogues discovered in our present study were detected as bi-charged ions on the positive mode LC/MS/MS. This is the first report describing AZA analogues detected as bi-charged ions with hexose and sulfate groups in their structures.

Absolute configuration at 12 stereocenters in the 36-membered macrocyclic ring portion of poecillastrin C (1) was disclosed by chemical degradation and NMR analyses of 1, chemical synthesis, and molecular modeling techniques.

Amphidoma languida, a marine thecate dinoflagellate that produces the lipophilic toxin azaspiracids (AZAs), is primarily found in the Atlantic. Although this species has not been recorded in the Asian Pacific, environmental DNAs related to Am. languida have been widely detected in the region by metabarcoding analysis. Their morphology and AZA production remain unclear. In this study, the morphology, ultrastructure, phylogeny, and AZA production of nine Amphidoma strains isolated from Japan, Malaysia, and Philippines were investigated. Phylogenetic trees inferred from rDNAs (SSU, ITS, and LSU rDNA) showed monophyly of the nine Pacific strains and were sister to the Am. languida clade, including the toxigenic strains from the Atlantic. Cells were ellipsoid, 8.7-16.7 µm in length and 7.4-14.0 µm in width, with a conspicuous apical pore complex. A large nucleus in the hyposome, parietal chloroplast with a spherical pyrenoid in the episome, and refractile bodies were observed. Thecal tabulation was typical of Amphidoma, Po, cp, X, 6', 6'', 6C, 5S, 6''', 2''''. A ventral pore was located on the anterior of 1' plate, beside the suture to 6' plate. The presence of a ventral depression, on the anterior of anterior sulcal plate, was different from Am. languida. A large antapical pore, containing approximately 10 small pores, was observed. Cells were apparently smaller than Am. trioculata, a species possessing three pores (ventral pore, ventral depression, and antapical pore). TEM showed the presence of crystalline structures, resembling guanine crystals, and cytoplasmic invaginations into the pyrenoid matrix. Flagellar apparatus lacking the striated root connective is similar to peridinioids and related dinoflagellates. AZAs were not detected from the Pacific strains by LC-MS/MS. This non-toxigenic Amphidoma species, here we propose as Amphidoma fulgens sp. nov., is widely distributed in the Asian Pacific. Moreover, molecular comparison also suggested that most of the environmental DNA sequences previously reported as Am. languida or related sequences from the Asian Pacific were attributable to Am. fulgens.

In the end of March 2018, an unprecedented food poisoning incident due to ingestion of the visceral balls of geoduck Panopea japonica occurred in Japan. The patient, presented with symptoms of numbness on the lips and general weakness, was diagnosed as paralytic shellfish poisoning (PSP). The patient immediately treated with the mechanical ventilation recovered and left the hospital after 3 days treatment. Saxitoxins (STXs) in the plasma and urinary samples collected from the patient on the first and second day after hospitalization were analyzed by ultra high-performance liquid chromatograph coupled with tandem mass spectrometer (UHPLC/MS/MS) and liquid chromatograph with post-column fluorescent detector (LC/FLD). The STXs levels of 499.1 and 6.0 μg/L of STX dihydrochloride equivalent (STX·2HCl eq.) were quantitated by LC/FLD in the urinary samples on the first and second day, respectively. In addition, geoducks harvested from the same areas of the PSP causative specimens after the incident were analyzed by LC/FLD, and the results showed the level of STXs in their whole bodies of the geoducks exceeding 0.8 mg STX·2HCl eq./kg which is the maximum levels of STX in CODEX STAN 292-2008. Prominent toxins in STXs that detected in urinary and geoduck samples and identified by UHPLC/MS/MS and LC/FLD were gonyautoxin-1+4 (GTX1+4). These results concluded that the incident was the food poisoning due to STXs accumulated in the geoducks. This is the first PSP case caused by consumption of geoducks in Japan. This is also the first PSP case that causative toxins are detected in urinary samples of patients involved in PSP in Japan.

Recently, an instrumental analysis using LC-MS/MS has been developed and validated for paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) in bivalve molluscs in Japanese domestic and overseas. The method for 11 PSTs and TTX in scallops was validated in accordance with a previous report and CODEX-STAN. The samples were prepared by adding the standard mixture of PSTs and TTX to scallop (Patinopecten yessoensis) homogenates, extracted with 1% acetic acid and then cleaned up using an ENVI-Carb (250 mg/3 mL) cartridge. A single analyst extracted and analyzed in two samples per day on five successive days. As result of the validation, 11 PSTs were found falling within all the guideline criteria (the trueness, repeatability, within laboratory reproducibility, LOD, LOQ) in the tests using matrix-matched calibration or solvent calibration.However, the trueness of TTX was low value in the test using solvent standard. It would be due to the matrix effects at the LC-MS/MS analysis. The slope of the calibration curve at the matrix-matched standards of mussel was about the same as the slope at the solvent standards. Therefore, it is necessary to choose the most appropriate matrix depending on shellfish.

Food made by pickling pufferfish ovaries in salt and rice-bran is a tradition in Ishikawa Prefecture in Japan. The food is manufactured in two stages: curing in salt for a year and pickling in rice-bran for one or two years. Since pufferfish ovaries contain the potent neurotoxin tetrodotoxin (TTX), a toxicity test is imple-mented by mouse bioassay and only food that contains less than the quarantine limit (10 MU/g) are market-ed. However, testing by instrument analysis is internationally accepted for inspections for marine biotoxins including TTX. In the study, we developed a pretreatment method for TTX suitable for instrument analysis in food containing a large amount of salt, and then used the method to assess the level of TTX in food. Our experiments revealed that an activated charcoal cartridge is useful as the pretreatment method for TTXs in pufferfish ovaries: the recovery test of TTX using the food was 90%. We used the developed method to assess the level of TTX in commercially available foods made using pufferfish ovaries.