Program

Selenoneine, 2-selenyl-Nα,Nα,Nα-trimethyl-L-histidine, is an antioxidant found in the tissues of tuna and other fish that can mediate the accumulation, demethylation, and metabolism of methylmercury (MeHg). We measured concentrations of selenoneine, total selenium, MeHg, and inorganic mercury (Hg) in red blood cells (RBC), livers and kidneys of tuna and dolphins. Yellowfin tuna and other tuna species contained total mercury at 0.30-1.30 mg/kg in the muscles and total selenium at 6-26 mg/kg, and almost part of selenium was due to selenoneine. In dolphins, RBC contained the high levels of selenoneine at 0.63–6.8 mg Se/kg, whereas plasma concentrations contained less than 0.13 mg Se/kg, indicating that selenoneine is highly concentrated in RBC. Selenoneine was also detected in liver and kidney tissue. Selenoneine was the primary chemical form of selenium found in dolphin tissues. In addition, liver tissue contained high levels of total selenium, ranging from 6.0–96.8 mg/kg, and inorganic Hg, ranging from 6.8–96.4 mg/kg. Kidney tissue contained 3.45–8.72 mg/kg total selenium and 1.40–6.61 mg/kg inorganic Hg. These findings suggest that mercury selenide (HgSe) might be formed as a MeHg-detoxified product in these tissues. Selenoneine-MeHg complex was detected in the dolphin liver, and this complex might be an intermediate in the MeHg detoxification mechanism.

Exosomes are extracellular small granule vesicles produced by cells, and might mediate a signal transduction mechanisms to transfer the information between cells and organisms. They contain various molecular constituents, such as, proteins, DNA, mRNA and miRNA, and transfer molecules from cells through membrane vesicle trafficking. We found exosome-mediated methylmercury (MeHg) excretion pathway accelerated by 2-selenyl-trimethyl-histidine, selenoneine, in zebrafish embryos. Here, we report evidence that a selenoneine accelerates the excretion of MeHg though exosomal secretion, and dynamics of secreted exosomes following MeHg exposure in zebrafish models. The embryos (8 hpf) were microinjected with MeHg-Cys (10−200 µg Hg/ml) into yolk sac, and cultured for 16 h. Exosomes were purified by ultracentrifugation at 100,000g for 1-3 h. Exosomal markers were measured. To visualize the production of exosomes in vivo, cd63-GFP expressing zebrafish lines were generated. Exosomes were released by MeHg exposure in a dose dependent manner. Scanning probe and electron microscopic observation revealed that the exosomes were vesicles of 20-50 nm in diameter. Hg (0.05-0.1 ng Hg/embryo) was contained in the exosomes. Selenoneine (3 µM) treatment accelerated exosomal secretion and Hg excretion. Exosomal secretion pathway was accelerated by selenoneine incorporated into cells and played an essential role for methylmercury detoxification. Selenoneine is incorporated into cells by an organic cations/carnitine transporter-1 (OCTN1). In the OCTN1 knockout fish (octn1-/-), octn1-/- had hypersensitivity against MeHg treatment, the exosomal secretion and Hg excretion were suppressed, and exosomal secretion by selenoneine was disappeared. In cd63-GFP transgenic zebrafish lines, the fluorescent signals was observed in central nervous system, yolk sac and somite, and enhanced signals were detected by MeHg injection and selenoneine treatment. Therefore, The exosomal secretory pathway was accelerated by selenoneine incorporated into cells through OCTN1 and might mediate MeHg detoxification.

Concentrations of trace metals were determined in the muscle tissue, digestive gland and gills of Mediterranean mussels (Mytilus galloprovincialis) collected from four different locations (distant zone: upstream and downstream; cage zone: cage bottom panel and upper frame) around an offshore copper alloy fish farm. Levels of copper (Cu), zinc (Zn), manganese (Mn) and iron (Fe) as mg/kg wet weight in the edible part of the mussels collected from distant zone (upstream Zn7.33>Fe2.8>Cu0.13>Mn0.07, downstream Zn9.9>Fe5.67>Cu0.18>Mn0.17) were significantly lower (p<0.05) than those sampled from the cage zone (bottom panel Zn22.25>Fe13.75>Cu2.39>Mn0.85, cage upper frame Zn17.1>Fe8.74>Cu1.39>Mn0.26). Overall, metal contents in mussels differed with sampling locations, resulting in significantly higher (p<0.05) concentrations in the samples from cage frame and the bottom panel of the copper alloy mesh pen, compared to those from distant areas both upstream and downstream. Trace metal concentrations in mussels used as a bio-indicator were lowest for the unaffected upstream area, while highest in mussels scattered on the bottom panel of the copper alloy mesh pen. However, the rates of target hazard quotients (THQ) for all tested trace metals from all locations in the present study were smaller than “one” (THQ<1), indicating that the consumption of mussels grown around a copper alloy cage fish farm were within safe limits and did not exceed maximum levels suggested by the US Food and Drug Administration (USFDA) and European Union (EU) regulations for seafood consumption.

Fish roe, one of the popular seafood in Japan, is a major allergenic seafood. The existence of cross-reactivity between fish roe and muscle was suspected because some of salmon-roe-allergic patients showed hypersencitivity to fish meat. The objective of this work is to clarify distribution of salmon roe allergen, β’-component, in various organs and to investigate allergenic protein responsible for cross-reactivity between fish roe and muscle. Muscle, liver, kidney, testis (only male), and blood were sampled from matured chum salmon (male: 25, female: 25) and their β’-component concentration was determined by ELISA using anti-cum salmon β’-component IgG. As a result, protein reacted with IgG (β’-component like protein) was found in the organs of all females and in muscle of 17 female salmon. On the other hand, in male salmon, 17 samples did not contain β’-component like protein and a trace amount of that was found in muscle and internal organs of 8 males. Additionally, 170-kDa β’-component like protein was found in female muscle using immunoblotting, and β’-component like protein was identified as vitellogenin; which is a precursor of yolk protein containing β’-component and is carried in the bloodstream to oocytes in maturated female fish. These results suggest that small amount of vitellogenin contained in female muscle through blood or other organs results in spurious cross-reactivity between salmon roe and muscle. Therefore, female salmon meat has a food allergy risk for salmon-roe-allergic patients. Selective ingestion of male muscle would contribute to the reduction of food risk in the salmon roe allergic patients.

In order to investigate the antimicrobial mechanism of Ginkgo biloba leaves extracts (GBLE) against specific spoilage bacteria in aquatic products, minimal inhibitory concentration (MIC) of GBLE against Shewanella putrefaciens was measured by method of broth dilution combined with plate counting in this paper. The impact of GBLE on the growth of bacteria, the permeability of cell membrane and cell wall were also investigated by the methods, such as growth curve, content of alkaline phosphatase (AKP), electrical conductivity and content of protein. The structure of Shewanella putrefaciens cells was observed by scanning electron microscope (SEM). The results showed that the MIC of GBLE against Shewanella putrefaciens was 100mg/mL. The inhibitory rates of MIC and 2MIC concentration of GBLE against Shewanella putrefaciens were 36.11% and 100%. The growth curve, results of alkaline phosphatase (AKP) and electrical conductivity content showed that growth of bacteria was inhibited and the permeability of cell membrane increased significantly when compared with untreated group. The results of SEM revealed that the structure of Shewanella putrefaciens cells were destroyed and the limit of cells became blur, which suggested that the inhibition effect of GBLE against Shewanella putrefaciens was related with the damage of cell membrane and cell wall.

The circulation of perch was simulated and the effect of precooling treatment with slurry ice on the quality change of fresh perch during circulation was researched. Three circulation methods, such as Crush Ice (CK), Slurry Ice (SI) and Slurry Ice-No Ice-Crush Ice (SNI), were used for perch, which were processed by different precooling treatments. Proximate content, core temperature, sensory evaluation, physic-chemical (color, water-holding capacity (WHC) and total volatile Base Nitrogen (TVB-N)), microbial (Total viable counts (TVC)) parameters, which combine with scanning electron microscope (SEM) and Low field Nuclear Magnetic Resonance (LF-NMR), were measured for 4, 8, 12, 15, 18, 21d respectively so as to evaluate the influence of different precooling condition for the quality change of perch comprehensively. It showed that the chilling time of perch to 0℃°C in slurry ice was one half of that in crushed ice, the final temperature was -1.1℃°C. As for its quality, the effects of SI storage were no better than that of CK storage in initial time. However, the advantage of continuously precooling with slurry ice can be observed at the end of storage. When compared with the other two groups, WHC values, TVB-N and TVC of perch of SI storage can slow down significantly. Moreover, the water loss and muscle tissue was also inhibited, the shelf-life of SI group can be extented for 6 days. The muscle tissue, WHC value, TVB-N value of perch of SNI group were better than that of CK one, which had the similar storage time. Therefore, slurry ice was a rapid and efficient method for chilling and temperature control of aquatic products, the precooling with slurry ice can be applied in short-distance transportation of fresh perch.

In the present study, we investigated toxin uptake/transfer profiles, and maturation-associated changes in the internal tetrodotoxin (TTX) distribution in the pufferfish Takifugu pardalis. Cultured specimens of the pufferfish (12 months old) were administered TTX or decarbamoylsaxitoxin (dcSTX) by oral gavage at a dose of 55 nmol/individual, and after 24 and/or 72 h, the toxins in each tissue were determined by LC-MS/MS and HPLC-FLD, respectively. The results revealed that more than 80% of the total TTX remaining in the body was retained in the skin in the TTX administration group, while 99% of the total dcSTX was in the digestive tract in the dcSTX administration group. From November to December 2014, female specimens of T. pardalis were collected from Omura Bay, Nagasaki Prefecture, Japan, and divided into four maturation stages (yolk vesicle stage, and yolk globule stages I, II and III) based on the formation of ovarian sections. LC-MS analyses revealed that from the yolk vesicle stage to yolk globule stage I, TTX concentration (MU/g) of the ovary, and from the yolk globule stage I to III, TTX amount (MU/individual) of the ovary was greatly increased. In the yolk vesicle stage, TTX of the skin accounted for 86% of the total TTX in the body, whereas in the yolk globule stage I-III, the ovarian TTX accounted for 53-76%. In female specimens collected from the same bay from February to May 2015, the TTX amount of the ovary greatly decreased after spawning, and instead the TTX amount of the liver increased obviously. The results suggested that T. pardalis specifically absorb TTX from food, and accumulate it mainly in the skin in the ordinary period, but in maturation period, TTX is actively transported and accumulated in the ovary, and after spawning, a part of remaining TTX is recovered to the liver.

Yotsu-Yamashita et al. (2005) reported that 4,9-anhydro-tetrodotoxin (4,9anh-TTX), a derivative of TTX found in puffer fish, reacts with biological thiols such as cystein (Cys) to form 4-S-Cys adduct of TTX. Not only Cys, various thiol compounds couple to 4,9anh-TTX. This finding enable us to design a new haptenic antigen essential to develop immuno-detection of TTX. Sulfhydryl moiety was introduced to tetrodotoxin (TTX) by a reaction between 4,9anh-TTX and (+) dithiothreitol (DTT). A new haptenic antigen was obtained from the mixture of a bifunctional coupling reagent (GMBS)-treated bovine serum albumin (BSA) and the DTT –TTX adduct. The ratio of TTX coupled to the carrier protein was estimated to be 14 % (w/w) by fluorescent intensity after sodium hydroxide treatment. In addition, 4,9anh-TTX was treated with 1,2-ethanedithiol (EDT) and resulting EDT-TTX adduct was introduced to GMBS-treated keyhole limpet hemocyanin (KLH). These two antigens (BSA-DTT-TTX and KLH-EDT-TTX) were inoculated biweekly to rabbits. High titer of anti-serum was observed within 4 months from the rabbits inoculated with KLH-EDT-TTX. The antiserum reacts well, not only with TTX, but also with 4epi-TTX, 11oxo-TTX as well as deoxy-TTX analogues which often found in TTX-bearing aquatic organisms such as puffer fish as well as toxic crab and gastropods. Separately, biotin-labeled TTX was prepared by a combination of maleimide-PEG2-biotin and DTT-TTX. By using the antiserum and biotin-labeled TTX, a new ELISA kit for TTX and its derivatives was designed.

【Purpose】 Some species of carnivorous or scavenger marine gastropods accumulate tetrodotoxin (TTX) and often cause TTX-poisoning in Japan and other countries. However, their toxin accumulation mechanism remains unclear. The purpose of this study is to assess TTX accumulation ability of gastropods by tissue culture method that we have newly established.
【Methods】 Japanese ivory shell Babylonia japonica was purchased at a fish market and used as a specimen. Digestive gland was removed and cut into a piece with approximately 1 g. The tissue was added into a culture medium containing 50 µM or 100 µM TTX and cultured at 18°C for 0-8 hours. Furthermore, concentration-dependence of TTX uptake into the digestive gland was examined by culture with 0-2,000 µM TTX for 1 hour. After culture, TTX was extracted and measured by LC-MS/MS method.
【Results】The commercial specimens did not contain TTX (< 25 ng TTX/g). TTX was detected from the digestive gland cultured with TTX, and the amount of toxin accumulated in the tissues increased with time. After 8 hours, the amount of toxin in the tissue was 5.14 ± 1.24 µg TTX/g and 50.2 ± 1.64 µg TTX/g when the tissues were cultured with 50 µM and 100 µM TTX, respectively. When the tissues were cultured with 0-2,000 µM TTX for 1 hour, the toxin uptake was linearly increased with the concentration in the medium up to 1,000 µM (129 ± 29.3 µg TTX/g at 1,000 µM TTX). The toxin amount at 2,000 µM TTX was almost the same as that at 1,000 µM TTX, indicating that TTX uptake of the digestive gland was saturable over 1,000 µM TTX. This method would be useful to assess TTX accumulation ability of gastropods. We are in progress to examine toxification in other species of gastropods.