Toxicity Assessment of the Xanthid Crab Demania cultripes from Cebu Island, Philippines

Several cases of poisoning resulting in human fatalities and stemming from the ingestion of coral reef crabs have been reported from the Indo-Pacific region. We assessed the toxicity of the unidentified xanthid crab collected from the Camotes Sea off the eastern coast of Cebu Island, central Visayas region of Philippines from the food hygienic point of view. All seven specimens, which were identified with Demania cultripes, collected in 2006 were toxic to mice irrespective of the season of collection and induced paralytic symptoms typical of tetrodotoxin (TTX) and paralytic shellfish poison (PSP). The activity was expressed in mouse unit (MU) being defined as the amount of TTX to kill a 20 g ddY male mice in 30 min after i.p. injection. Toxicity scores for viscera and appendages of specimens were 18.2 ± 16.0 (mean ± S.D.) and 4.4 ± 2.6 MU/g, respectively. The highest individual toxicity scores observed for viscera and appendages were 52.1 and 7.7 MU/g, respectively. The frequency of toxic samples was 100%. Toxin profiles as determined by high-performance liquid chromatography-fluorescent detection analysis (HPLC-FLD) revealed that TTX was the main toxic principle accounting for about 90% of the total toxicity along with 4-epi TTX and 4,9-anhydroTTX. Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis revealed mass fragment ion peaks at m/z 376, 392 and 407, which were characteristic of the quinazoline skeleton (C9-base) specific to TTX. In addition, only a small amount of PSP containing gonyautoxins1–4 and hydroxysaxitoxin was detected. To our knowledge, this is the first report presenting evidence of occurrence of TTX and PSP in the xanthid crab D. cultripes inhabiting waters surrounding Cebu Island. From food hygienic point of view, people in coastal areas should be warned of the potential hazard of this crab in order to prevent its intentional or accidental consumption

イシダイのマイトジェン刺激リンパ球によるマクロファージ活性因子の産生と本因子の特性

Spleen lymphocytes were prepared from Japanese parrotfish (Oplegnathus fasciatus) and incubated with 100 mg/ml of Concanavalin A bound to Sepharose 4B beads for 48h, and then the stimulated cell supernatants were collected. Thyoglycolate induced, allogenic, peritoneal macrophages were incubated with or without stimulated cell supernatants. Then, the phagocytic activity against inactivated yeast and peroxidase production were measured. Stimulated cell supernatants increased the phagocytic activity and the peroxidase production of allogenic peritoneal macrophages. The activities of these stimulated cell supernatants were lost completely when they were heated at 56℃ for 30 min or dialyzed against a pH 2 buffer for 24h-similar to IFN-γ. Stimulated cell supernatants did not increase phagocytic activity and peroxidase production of heterogenic mouse peritoneal macrophages.These findings suggest that the spleen lymphocytes of Japanese parrotfish can produce a macrophage activating factor in vitro with Concanavalin A bound to Sepharose 4B beads, and that this factor may be IFN-γ.イシダイの脾臓細胞中のリンパ球をコンカナバリンA（Con A）セファロース4B とともに48時間培養し，その上清によるマクロファージ活性化を検討し，以下の結果を得た。Con A 培養上清で培養したイシダイ腹腔マクロファージの貪食能と細胞内のペルオキシダーゼ活性の両機能は共に亢進していた。イシダイCon A 培養上清をホ乳類でINF-γ 失活処理とされている熱（56℃）あるいは酸（pH2）処理を行ったところ，マクロファージの活性化が消失した。イシダイCon A 培養上清の腹腔マクロファージ活性化の種特異性について，マウス腹腔マクロファージを用いて検討したところ，後者は活性化能を持たず，イシダイCon A培養上清の種特異性が存在した。以上のことより，養殖イシダイ脾臓細胞中のリンパ球は，Con A で培養するとINF-γ を産生すると推察され，イシダイの魚体内でINF-γ，マクロファージの免疫防御機構の存在が示唆された

Dietary supplementation with alkylresorcinols prevents muscle atrophy through a shift of energy supply

It has been reported that phytoextracts that contain alkylresorcinols (ARs) protect against severe myofibrillar degeneration found in isoproterenol-induced myocardial infarction. In this study, we examined the effect of dietary ARs derived from wheat bran extracts on muscle atrophy in denervated mice. The mice were divided into the following four groups: (1) sham-operated (control) mice fed with normal diet (S-ND), (2) denervated mice fed with normal diet (D-ND), (3) control mice fed with ARs-supplemented diet (S-AR) and (4) denervated mice fed with ARs-supplemented diet (D-AR). The intake of ARs prevented the denervation-induced reduction of the weight of the hind limb muscles and the myofiber size. However, the expression of ubiquitin ligases and autophagy-related genes, which is associated with muscle proteolysis, was slightly higher in D-AR than in D-ND. Moreover, the abundance of the autophagy marker p62 was significantly higher in D-AR than in D-ND. Muscle atrophy has been known to be associated with a disturbed energy metabolism. The expression of pyruvate dehydrogenase kinase 4 (PDK4), which is related to fatty acid metabolism, was decreased in D-ND as compared with that in S-ND. In contrast, dietary supplementation with ARs inhibited the decrease of PDK4 expression caused by denervation. Furthermore, the abnormal expression pattern of genes related to the abundance of lipid droplets-coated proteins that was induced by denervation was improved by ARs. These results raise the possibility that dietary supplementation with ARs modifies the disruption of fatty acid metabolism induced by lipid autophagy, resulting in the prevention of muscle atrophy

Astaxanthin Prevents Atrophy in Slow Muscle Fibers by Inhibiting Mitochondrial Reactive Oxygen Species via a Mitochondria-Mediated Apoptosis Pathway

Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in the lipid bilayer. This study aimed to investigate the effects of AX on muscle-atrophy-mediated disturbance of mitochondria, which have a lipid bilayer. Tail suspension was used to establish a muscleatrophied mouse model. AX diet fed to tail-suspension mice prevented loss of muscle weight, inhibited the decrease of myofiber size, and restrained the increase of hydrogen peroxide (H2O2) production in the soleus muscle. Additionally, AX improved downregulation of mitochondrial respiratory chain complexes I and III in the soleus muscle after tail suspension. Meanwhile, AX promoted mitochondrial biogenesis by upregulating the expressions of adenosine 5\u27-monophosphate-activated protein kinase (AMPK) α-1, peroxisome proliferator-activated receptor (PPAR)-γ, and creatine kinase in mitochondrial (Ckmt) 2 in the soleus muscle of tail-suspension mice. To confirm the AX phenotype in the soleus muscle, we examined its effects on mitochondria using Sol8 myotubes derived from the soleus muscle. We found that AX was preferentially detected in the mitochondrial fraction; it significantly suppressed mitochondrial reactive oxygen species (ROS) production in Sol8 myotubes. Moreover, AX inhibited the activation of caspase 3 via inhibiting the release of cytochrome c into the cytosol in antimycin A-treated Sol8 myotubes. These results suggested that AX protected the functional stability of mitochondria, alleviated mitochondrial oxidative stress and mitochondriamediated apoptosis, and thus, prevented muscle atrophy

Maturation-associated changes in toxicity of the pufferfish Takifugu poecilonotus.

From October 2006 to December 2007, wild specimens of the pufferfish Takifugu poecilonotus (93 females, 45 males) were collected from the Ariake Sea. Tissue toxicity was examined by mouse bioassay, and tetrodotoxin (TTX) content in the blood plasma by enzyme-linked immunosorbent assay. The relationship between toxicity and maturation was investigated based on changes in the gonadosomatic index: December-March in females and November-March in males, the \u27maturation period\u27; April, \u27just after spawning\u27; and the other months, the \u27ordinary period\u27. Toxicity of both sexes was high throughout the year, but sharply declined in April. In all tissues examined (skin, liver, and ovary) other than testis, toxicity exceeded 1000MU/g or 10,000MU/individual in many individuals. Seasonal profiles of tissue toxicity differed markedly between sexes. In females, liver toxicity was high during the ordinary period, and ovary toxicity was high during the maturation period. In males, little maturation-associated change in the toxin distribution was observed. Plasma TTX levels were similar between the sexes (1.59-15.1MU/ml), and fluctuated largely throughout the year without corresponding changes in tissue toxicity. The percentage of TTX binding to high molecular-weight substances in the plasma varied in association with maturation; the binding ratio fluctuated at relatively low levels during the ordinary period, and stabilized at a high level during the maturation period

Toxins of Pufferfish That Cause Human Intoxications

Many marine pufferfish possess a potent neurotoxin, tetrodotoxin (TTX). In general, they have strong toxicity in the liver and ovary, leading to a frequent occurrence of human poisonings. TTX is originally produced by marine bacteria and distributes over a wide variety of aquatic animals. In pufferfish, TTX is derived from the food chain that consists of these TTXbearing organisms (i.e., their prey). The transfer, accumulation, and elimination mechanisms of TTX taken up into the pufferfish body via prey remain unclear. Recent studies have revealed that the liver of pufferfish has a specific TTXuptake mechanism, and TTX introduced into the pufferfish body is first absorbed in the liver and then transferred to the skin through the circulatory system. This inter-tissue transfer and accumulation of TTX are greatly affected by the state of maturation. TTX-bearing organisms show extremely high resistance to TTX, and seem to possess TTX as a biological defense mechanism. Furthermore, TTX may involved in the control of information transmission in the central nervous system of pufferfish. TTX poisonings due to small scavenging gastropods have so far occurred in Taiwan and China. Recently, one such gastropod, Nassarius glans, caused food poisoning incidents in Kyushu, Japan. N. glans is highly toxic, and possesses a large amount of TTX not only in the viscera but also in the muscle. After 1990, a total of 9 poisoning incidents due to ingestion of boxfish (pufferfish of the family Ostraciidae) occurred in southwestern Japan, involving 13 patients and 1 death. The symptoms are very similar to those of parrotfish poisoning (a unique variety of food poisoning that has sporadically occurred in Japan), suggesting that the causative substance is a palytoxin (PTX)-like toxin as in the parrotfish poisoning. Freshwater pufferfish and some marine pufferfish possess paralytic shellfish poison (PSP) instead of or in addition to TTX, and may cause ‘paralytic shellfish poisoning by pufferfish’. The toxins of the above mentioned fish and shellfish are all exogenous, and their toxicity may be greatly affected by a change in the marine environment, such as elevations in water temperature due to global worming. We need to enhance the information/collaboration network among East Asian countries and vigilantly monitor how our changing climate is affecting the toxicity and distributions of these organisms.Nagasaki University Major Research Project: Restoration of Marine Environment and Resources in East Asi