Ecotoxicological Impact of the Marine Toxin Palytoxin on the Micro-Crustacean Artemia franciscana.

Palytoxin (PLTX) is a highly toxic polyether identified in various marine organisms, such as Palythoa soft corals, Ostreopsis dinoflagellates, and Trichodesmium cyanobacteria. In addition to adverse effects in humans, negative impacts on different marine organisms have been often described during Ostreopsis blooms and the concomitant presence of PLTX and its analogues. Considering the increasing frequency of Ostreopsis blooms due to global warming, PLTX was investigated for its effects on Artemia franciscana, a crustacean commonly used as a model organism for ecotoxicological studies. At concentrations comparable to those detected in culture media of O. cf. ovata (1.0-10.0 nM), PLTX significantly reduced cysts hatching and induced significant mortality of the organisms, both at larval and adult stages. Adults appeared to be the most sensitive developmental stage to PLTX: significant mortality was recorded after only 12 h of exposure to PLTX concentrations > 1.0 nM, with a 50% lethal concentration (LC50) of 2.3 nM (95% confidence interval = 1.2-4.7 nM). The toxic effects of PLTX toward A. franciscana adults seem to involve oxidative stress induction. Indeed, the toxin significantly increased ROS levels and altered the activity of the major antioxidant enzymes, in particular catalase and peroxidase, and marginally glutathione-S-transferase and superoxide dismutase. On the whole, these results indicate that environmentally relevant concentrations of PLTX could have a negative effect on Artemia franciscana population, suggesting its potential ecotoxicological impact at the marine level.

Peripheral Pain Modulation of Chrysaora pacifica Jellyfish Venom Requires Both Ca2+ Influx and TRPA1 Channel Activation in Rats.

The venom of jellyfish triggers severe dermal pain along with inflammation and tissue necrosis, and occasionally, induces internal organ dysfunction. However, the basic mechanisms underlying its cytotoxic effects are still unknown. Here, we report one of the mechanisms involved in peripheral pain modulation associated with inflammatory and neurotoxic oxidative signaling in rats using the venom of jellyfish, Chrysaora pacifica (CpV). This jellyfish is identified by brown tentacles carrying nematocysts filled with cytotoxic venom that induces severe pain, pruritus, tentacle marks, and blisters. The subcutaneous injection of CpV into rat forepaws in behavioral tests triggered nociceptive response with a decreased threshold for mechanical pain perception. These responses lasted up to 48 h and were completely blocked by verapamil and TTA-P2, T-type Ca2+ channel blockers, or HC030031, a transient receptor potential cation ankyrin 1 (TRPA1) channel blocker, while another Ca2+ channel blocker, nimodipine, was ineffective. Also, treatment with Ca2+ chelators (EGTA and BaptaAM) significantly alleviated the CpV-induced pain response. These results indicate that CpV-induced pain modulation may require both Ca2+ influx through the T-type Ca2+ channels and activation of TRPA1 channels. Furthermore, CpV induced Ca2+-mediated oxidative neurotoxicity in the dorsal root ganglion (DRG) and cortical neurons dissociated from rats, resulting in decreased neuronal viability and increased intracellular levels of ROS. Taken together, CpV may activate Ca2+-mediated oxidative signaling to produce excessive ROS acting as an endogenous agonist of TRPA1 channels in the peripheral terminals of the primary afferent neurons, resulting in persistent inflammatory pain. These findings provide strong evidence supporting the therapeutic effectiveness of blocking oxidative signaling against pain and cytotoxicity induced by jellyfish venom.

Peptide Blocker of Ion Channel TRPV1 Exhibits a Long Analgesic Effect in the Heat Stimulation Model.

The ion channel TRPV1, which is one of the most important integrators of pain and inflammatory stimuli, is considered a promising therapeutic target in the treatment of pain conditions. In this work, we performed a comparative study of the analgesic effect in the "hot plate" test of recombinant analogues of Kunitz-type peptides from the sea anemone Heteractis crispa venom: APHC1-modulator of TRPV1 and HCRG21-a full blocker of TRPV1. As a result of biological tests, it was shown that the full blocker HCRG21, despite the higher value of 50% effective concentration of TRPV1 inhibition, had an equal analgesic ability with the APHC1 upon intramuscular administration and retained it for 13 h of observation. The analgesic effect of APHC1 at a dose of 0.1 mg/kg when administered intramuscularly developed very quickly in 5 min but lasted 3 h. The differences in the pharmacodynamic profile of the peptides are in good agreement with different mechanisms of binding to TRPV1.

Effects of palytoxins extracted from Ostreopsis ovata on the oxidative stress and immune responses in Pacific white shrimp Litopenaeus vannamei.

Palytoxins (PLTXs) are a group of complex and poisonous marine natural products that are toxic to marine life and even human beings. In the present study, the oxidative stress and immune response in the hepatopancreas and gills of Litopenaeus vannamei were assessed for 72 h after injection with PLTX extracts. Chemical and physiological parameters, e.g., the respiratory burst (O2-), activities of antioxidant enzymes, oxidative damage to lipids, carbonylation of proteins, and immune gene mRNA expression levels, were analysed. The results showed that the PLTX extract was not fatal to the shrimp but could reduce their mobility. The O2- levels in the gills gradually increased after exposure to PLTX extracts and were significantly higher than those in the control from 6 to 72 h. The malondialdehyde content, lipid peroxidation, protein carbonyl levels, and total antioxidant capacity in the gills all peaked at 12 h. At the same time, the gills were loosely connected, there was a clear disintegration of the epithelial tissue, and the stratum corneum disappeared after 12 h. In addition, compared to those in the control group, the PLTX extract treatment increased the O2- content, malondialdehyde content, lipid peroxidation, and protein carbonyl levels from 12 to 72 h, 24-48 h, 12-24 h, and 12-72 h after injection in the hepatopancreas of the shrimp, respectively. Both the Crustin and Toll gene expression levels significantly increased in the hepatopancreas compared to those in the control 6-72 h after injection of the toxin. In parallel, the expression levels of the manganese superoxide dismutase gene gradually decreased from 6 to 48 h and returned to normal levels after 72 h. Interestingly, the total antioxidant capacity also significantly increased compared to that in the control from 6 to 72 h. Our results indicate that although PLTX extracts cause lipid peroxidation and carbonylation of proteins in hepatopancreatic cells, leading to their damage, they did not cause a decrease in the total antioxidant capacity of the hepatopancreas.

Novel Adjuvant Based on the Pore-Forming Protein Sticholysin II Encapsulated into Liposomes Effectively Enhances the Antigen-Specific CTL-Mediated Immune Response.

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.

Effects of intranasal administration of the peptide antagonist of type I vaniloid receptor (TRPV1) in the rodent central nervous system.

Intranasal administration of the polypeptide APHC3, an antagonist of the TRPV1 receptor, had acute anxiolytic and antidepressant effects, as well as an ability to modify the microglial response to proinflammatory stress and cytokine profile of the hippocampus. However, the acute antidepressant effect of the polypeptide was not related to the attenuation of neuroiflammation and probably had a different mechanism. The use of intranasal administration of the APHC3 peptide as a therapeutic approach aimed at decreasing depression symptoms needs additional studies in order to find the mechanism of action of this polypeptide in the central nervous system (CNS).

Preliminary Study on Nematocyst Types and Venom Isolation of Cassiopea andromeda Forskål, 1775 (Scyphozoa, Cnidaria) from Turkey.

Nematocyst types of Cassiopea andromeda were investigated. Medusae samples were taken from Güllük Bay, Mugla, Turkey. Nematocyst samples from oral arms of C. andromeda were observed on light microscope and photographed. Birhopaloid and a-isorhiza nematocyst types were found in C. andromeda. Moreover, it was seen that nematocyst sizes increased with increasing the bell diameters of the individuals. Also, the venom of the species was isolated and injected intramuscularly to Cyprinus carpio juveniles. Signs of partial paralysis, raking, and immobilized fins were observed in the juveniles consequently. Death was observed for the fishes which were 3-4 g in the range of weight. This study is a preliminary work on nematocysts and venom of C. andromeda. Further studies on neurotoxic effects of nematocyst venoms of this species should follow.

Buccal mucosal delivery of a potent peptide leads to therapeutically-relevant plasma concentrations for the treatment of autoimmune diseases.

Stichodactyla helianthus neurotoxin (ShK) is an immunomodulatory peptide currently under development for the treatment of autoimmune diseases, including multiple sclerosis and rheumatoid arthritis by parenteral administration. To overcome the low patient compliance of conventional self-injections, we have investigated the potential of the buccal mucosa as an alternative delivery route for ShK both in vitro and in vivo. After application of fluorescent 5-Fam-ShK to untreated porcine buccal mucosa, there was no detectable peptide in the receptor chamber using an in vitro Ussing chamber model. However, the addition of the surfactants sodium taurodeoxycholate hydrate or cetrimide, and formulation of ShK in a chitosan mucoadhesive gel, led to 0.05-0.13% and 1.1% of the applied dose, respectively, appearing in the receptor chamber over 5h. Moreover, confocal microscopic studies demonstrated significantly enhanced buccal mucosal retention of the peptide (measured by mucosal fluorescence associated with 5-Fam-ShK) when enhancement strategies were employed. Administration of 5-Fam-ShK to mice (10mg/kg in a mucoadhesive chitosan-based gel (3%, w/v) with or without cetrimide (5%, w/w)) resulted in average plasma concentrations of 2.6-16.2nM between 2 and 6h, which were substantially higher than the pM concentrations required for therapeutic activity. This study demonstrated that the buccal mucosa is a promising administration route for the systemic delivery of ShK for the treatment of autoimmune diseases.

Role of TRPV1 and ASIC3 in formalin-induced secondary allodynia and hyperalgesia.

BACKGROUND: In the present study we determined the role of transient receptor potential V1 channel (TRPV1) and acid-sensing ion channel 3 (ASIC3) in chronic nociception. METHODS: 1% formalin was used to produce long-lasting secondary allodynia and hyperalgesia in rats. Western blot was used to determine TRPV1 and ASIC3 expression in dorsal root ganglia. RESULTS: Peripheral ipsilateral, but not contralateral, pre-treatment (-10min) with the TRPV1 receptor antagonists capsazepine (0.03-0.3µM/paw) and A-784168 (0.01-1µM/paw) prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in the ipsilateral and contralateral paws. Likewise, peripheral ipsilateral, but not contralateral, pre-treatment with the non-selective and selective ASIC3 blocker benzamil (0.1-10µM/paw) and APETx2 (0.02-2µM/paw), respectively, prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. Peripheral ipsilateral post-treatment (day 6 after formalin injection) with capsazepine (0.03-0.3µM/paw) and A-784168 (0.01-1µM/paw) reversed 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. In addition, peripheral ipsilateral post-treatment with benzamil (0.1-10µM/paw) and APETx2 (0.02-2µM/paw), respectively, reversed 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. TRPV1 and ASIC3 proteins were expressed in dorsal root ganglion in normal conditions, and 1% formalin injection increased expression of both proteins in this location at 1 and 6 days compared to naive rats. CONCLUSIONS: Data suggest that TRPV1 and ASIC3 participate in the development and maintenance of long-lasting secondary allodynia and hyperalgesia induced by formalin in rats. The use of TRPV1 and ASIC3 antagonists by peripheral administration could prove useful to treat chronic pain.

Acid mediates a prolonged antinociception via substance P signaling in acid-induced chronic widespread pain.

BACKGROUND: Substance P is an important neuropeptide released from nociceptors to mediate pain signals. We recently revealed antinociceptive signaling by substance P in acid-sensing ion channel 3 (ASIC3)-expressing muscle nociceptors in a mouse model of acid-induced chronic widespread pain. However, methods to specifically trigger the substance P antinociception were still lacking. RESULTS: Here we show that acid could induce antinociceptive signaling via substance P release in muscle. We prevented the intramuscular acid-induced hyperalgesia by pharmacological inhibition of ASIC3 and transient receptor potential V1 (TRPV1). The antinociceptive effect of non-ASIC3, non-TRPV1 acid signaling lasted for 2 days. The non-ASIC3, non-TRPV1 acid antinociception was largely abolished in mice lacking substance P. Moreover, pretreatment with substance P in muscle mimicked the acid antinociceptive effect and prevented the hyperalgesia induced by next-day acid injection. CONCLUSIONS: Acid could mediate a prolonged antinociceptive signaling via the release of substance P from muscle afferent neurons in a non-ASIC3, non-TRPV1 manner.

Pelagia noctiluca (Scyphozoa) crude venom injection elicits oxidative stress and inflammatory response in rats.

Cnidarian toxins represent a rich source of biologically active compounds. Since they may act via oxidative stress events, the aim of the present study was to verify whether crude venom, extracted from the jellyfish Pelagia noctiluca, elicits inflammation and oxidative stress processes, known to be mediated by Reactive Oxygen Species (ROS) production, in rats. In a first set of experiments, the animals were injected with crude venom (at three different doses 6, 30 and 60 µg/kg, suspended in saline solution, i.v.) to test the mortality and possible blood pressure changes. In a second set of experiments, to confirm that Pelagia noctiluca crude venom enhances ROS formation and may contribute to the pathophysiology of inflammation, crude venom-injected animals (30 µg/kg) were also treated with tempol, a powerful antioxidant (100 mg/kg i.p., 30 and 60 min after crude venom). Administration of tempol after crude venom challenge, caused a significant reduction of each parameter related to inflammation. The potential effect of Pelagia noctiluca crude venom in the systemic inflammation process has been here demonstrated, adding novel information about its biological activity.

Case series: inhaled coral vapor--toxicity in a tank.

INTRODUCTION: Palytoxin (PTX) is considered a severe marine toxin. Although rare, reports of human exposure from consumption of PTX have described significant morbidity and mortality. PTX is the suspected agent in Haff disease, in which rhabdomyolysis occurs within 24 h of eating contaminated fish such as buffalo fish. PTX is primarily present in soft corals or in dinoflagellates, and it can contaminate crustaceans and other fish as it bioaccumulates up the food chain. Only 23 cases have been reported in the USA, including two recent cases in New York City. Reports of inhalational exposure to PTX are uncommon. CASE REPORTS: We describe a case series of six patients, including four adults and two children, with inhalational exposure to PTX aerosolized from Palythoa corals. Their symptoms included some degree of respiratory involvement, myalgias, paresthesias, low-grade fevers, and gastrointestinal symptoms. Fortunately, there were no serious outcomes and all patients survived without sequelae. DISCUSSION: Although rare, exposure to palytoxin is not restricted to people visiting marine environments because of Palythoa coral in some home aquariums. Routes of exposure go beyond consumption of fish that feed on the coral and include dermal as well as inhalational exposure. Palytoxin exposure should be considered in the differential diagnosis of patients who own or work with fish tanks and present with symptoms that include respiratory complaints, myalgias, neuromuscular dysfunction, hemolysis, and cardiac toxicity. There is no known antidotal therapy and treatment should focus on meticulous supportive care.

protective effect of tetracycline against dermal toxicity induced by Jellyfish venom.

BACKGROUND: Previously, we have reported that most, if not all, of the Scyphozoan jellyfish venoms contain multiple components of metalloproteinases, which apparently linked to the venom toxicity. Further, it is also well known that there is a positive correlation between the inflammatory reaction of dermal tissues and their tissue metalloproteinase activity. Based on these, the use of metalloproteinase inhibitors appears to be a promising therapeutic alternative for the treatment of jellyfish envenomation. METHODOLOGY AND PRINCIPAL FINDINGS: Tetracycline (a metalloproteinase inhibitor) has been examined for its activity to reduce or prevent the dermal toxicity induced by Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom (NnV) using in vitro and in vivo models. HaCaT (human keratinocyte) and NIH3T3 (mouse fibroblast) incubated with NnV showed decreases in cell viability, which is associated with the inductions of metalloproteinase-2 and -9. This result suggests that the use of metalloproteinase inhibitors, such as tetracycline, may prevent the jellyfish venom-mediated local tissue damage. In vivo experiments showed that comparing with NnV-alone treatment, tetracycline pre-mixed NnV demonstrated a significantly reduced progression of dermal toxicity upon the inoculation onto rabbit skin. CONCLUSIONS/SIGNIFICANCE: It is believed that there has been no previous report on the therapeutic agent of synthetic chemical origin for the treatment of jellyfish venom-induced dermonecrosis based on understanding its mechanism of action except the use of antivenom treatment. Furthermore, the current study, for the first time, has proposed a novel mechanism-based therapeutic intervention for skin damages caused by jellyfish stings.

Sea-anemone toxin ATX-II elicits A-fiber-dependent pain and enhances resurgent and persistent sodium currents in large sensory neurons.

BACKGROUND: Gain-of-function mutations of the nociceptive voltage-gated sodium channel Nav1.7 lead to inherited pain syndromes, such as paroxysmal extreme pain disorder (PEPD). One characteristic of these mutations is slowed fast-inactivation kinetics, which may give rise to resurgent sodium currents. It is long known that toxins from Anemonia sulcata, such as ATX-II, slow fast inactivation and skin contact for example during diving leads to various symptoms such as pain and itch. Here, we investigated if ATX-II induces resurgent currents in sensory neurons of the dorsal root ganglion (DRGs) and how this may translate into human sensations. RESULTS: In large A-fiber related DRGs ATX-II (5 nM) enhances persistent and resurgent sodium currents, but failed to do so in small C-fiber linked DRGs when investigated using the whole-cell patch-clamp technique. Resurgent currents are thought to depend on the presence of the sodium channel ß4-subunit. Using RT-qPCR experiments, we show that small DRGs express significantly less ß4 mRNA than large sensory neurons. With the ß4-C-terminus peptide in the pipette solution, it was possible to evoke resurgent currents in small DRGs and in Nav1.7 or Nav1.6 expressing HEK293/N1E115 cells, which were enhanced by the presence of extracellular ATX-II. When injected into the skin of healthy volunteers, ATX-II induces painful and itch-like sensations which were abolished by mechanical nerve block. Increase in superficial blood flow of the skin, measured by Laser doppler imaging is limited to the injection site, so no axon reflex erythema as a correlate for C-fiber activation was detected. CONCLUSION: ATX-II enhances persistent and resurgent sodium currents in large diameter DRGs, whereas small DRGs depend on the addition of ß4-peptide to the pipette recording solution for ATX-II to affect resurgent currents. Mechanical A-fiber blockade abolishes all ATX-II effects in human skin (e.g. painful and itch-like paraesthesias), suggesting that it mediates its effects mainly via activation of A-fibers.

Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis.

BACKGROUND: Recent data have suggested a relationship between acute arthritic pain and acid sensing ion channel 3 (ASIC3) on primary afferent fibers innervating joints. The purpose of this study was to clarify the role of ASIC3 in a rat model of osteoarthritis (OA) which is considered a degenerative rather than an inflammatory disease. METHODS: We induced OA via intra-articular mono-iodoacetate (MIA) injection, and evaluated pain-related behaviors including weight bearing measured with an incapacitance tester and paw withdrawal threshold in a von Frey hair test, histology of affected knee joint, and immunohistochemistry of knee joint afferents. We also assessed the effect of ASIC3 selective peptide blocker (APETx2) on pain behavior, disease progression, and ASIC3 expression in knee joint afferents. RESULTS: OA rats showed not only weight-bearing pain but also mechanical hyperalgesia outside the knee joint (secondary hyperalgesia). ASIC3 expression in knee joint afferents was significantly upregulated approximately twofold at Day 14. Continuous intra-articular injections of APETx2 inhibited weight distribution asymmetry and secondary hyperalgesia by attenuating ASIC3 upregulation in knee joint afferents. Histology of ipsilateral knee joint showed APETx2 worked chondroprotectively if administered in the early, but not late phase. CONCLUSIONS: Local ASIC3 immunoreactive nerve is strongly associated with weight-bearing pain and secondary hyperalgesia in MIA-induced OA model. APETx2 inhibited ASIC3 upregulation in knee joint afferents regardless of the time-point of administration. Furthermore, early administration of APETx2 prevented cartilage damage. APETx2 is a novel, promising drug for OA by relieving pain and inhibiting disease progression.

Analgesic and antibutyrylcholinestrasic activities of the venom prepared from the Mediterranean jellyfish Pelagia noctiluca (Forsskal, 1775).

BACKGROUND: Toxins derived from jellyfishes have been exploited as a model for the development of new drug promising applications to treat neurodegenerative diseases. The present work is aimed to evaluate the acute toxicity of crude venom of Pelagia noctiluca and then to screen the analgesic and antibutyrylcholinestrasic (anti-BuChE) activities of the crude venom and its fractions. METHODS: Sephadex G75 gel was used to separate crude venom of Pelagia noctiluca, which led to some fractions. In addition, in vivo analgesic and in vitro plasma antibutyrylcholinestrasic activities were carried out with Pelagia crude venom and its fractions respectively. RESULTS: The crude venom and its fractions displayed analgesic and anti-BuChE activities at different doses without inducing acute toxicity. Fraction 2 possesses the highest analgesic and antibutyrylcholinestrasic properties. The crude venom and fraction 1 had shown to possess less significant inhibitory activity against analgesic and antibutyrylcholinestrasic models. CONCLUSIONS: Based on this study, the crude venom of Pelagia noctiluca is found to be a useful tool for probing pharmacological activity. The purification and the determination of chemical structures of compounds of active fractions of the venom are under investigation.

Target organ identification of jellyfish envenomation using systemic and integrative analyses in anesthetized dogs.

Proper treatment of jellyfish envenomed patients can be successfully achieved only from an understanding of the overall functional changes and alterations in physiological parameters under its envenomation. The majority of previous investigations on jellyfish venoms have covered only a couple of parameters at a time. Unlike most other fragmentary jellyfish studies, we employed an integrative toxicological approach, including hemodynamics, clinical chemistry and hematology analyses, using N. nomurai jellyfish venom (NnV) in dogs. After the baseline measurements for mean arterial pressure (MAP), cardiac output (CO) and heart rate (HR), NnV was intravenously administered to the dogs at doses of 0.1 or 0.3mg/kg body weight. The dogs showed significant decreases in MAP (-27.4±3.7 and -48.1±9.9 mmHg), CO (-1.1±0.1 L/min and -1.0±0.2 L/min), and HR (-4.5±0.3 and -9.9±3.1 beats/min) comparing with the respective baseline controls. The onset of systemic hypotension and bradycardia occurred within 1 min of NnV injection and they lasted for 1-35 min, depending on the NnV doses. Interestingly, serum biochemical analyses of envenomed dogs exhibited dramatic increases of alkaline phosphatase (ALP), creatine phosphokinase (CPK), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating its possible target organs. In conclusion, we have demonstrated simultaneously, for the first time, the multiple organ toxicities (cardiotoxic, myotoxic and hepatotoxic) of a scyphozoan jellyfish venom. Based on these results, an integrative toxinological approach using dogs appears to be effective in predicting jellyfish venom toxicities and designing their therapeutic strategies. We expect this method can be applied to other jellyfish venom research as well.

Acute oral toxicity in mice of a new palytoxin analog: 42-hydroxy-palytoxin.

The acute oral toxicity of a new palytoxin congener, 42-hydroxy-palytoxin (42-OH-PLTX), was investigated in female CD-1 mice. The toxin (300-1697 µg/kg), administered by gavage, induced scratching, jumping, respiratory distress, cyanosis, paralysis and death of mice, with an LD50 of 651 µg/kg (95% confidence limits: 384-1018 µg/kg) within 24 h. Hematoclinical analyses showed increased plasma levels of lactate dehydrogenase and aspartate-aminotransferase at doses of 600 µg/kg and above, as well as of alanine-aminotransferase, creatine phosphokinase and potassium ions at ≥ 848 µg/kg. Histology revealed inflammatory lesions in the non-glandular area of the stomach of mice that survived up to 24 h after gavage (424-1200 µg/kg). Although no histological alterations were seen in skeletal and cardiac muscles, changes in some plasma biomarkers (creatine phosphokinase, lactate dehydrogenase) suggested involvement of these tissues in 42-OH-PLTX oral toxicity, in agreement with epidemiological data on seafood poisonings ascribed to palytoxins. Complete recovery of the tissue and hematological changes was observed two weeks post-exposure. Furthermore, 42-OH-PLTX induced in vitro delayed erythrocyte hemolysis at concentrations similar to those of PLTX (EC50 = 7.6 and 13.2 x 10⁻¹² M, respectively). This hemolysis could be completely neutralized by a monoclonal anti-PLTX antibody. The in vivo data, together with the in vitro data recorded for 42-OH-PLTX, seem to indicate Na+/K+-ATPase as one of the key cellular targets of this toxin.

Reversal of acid-induced and inflammatory pain by the selective ASIC3 inhibitor, APETx2.

BACKGROUND AND PURPOSE: Inflammatory pain is triggered by activation of pathways leading to the release of mediators such as bradykinin, prostaglandins, interleukins, ATP, growth factors and protons that sensitize peripheral nociceptors. The activation of acid-sensitive ion channels (ASICs) may have particular relevance in the development and maintenance of inflammatory pain. ASIC3 is of particular interest due to its restricted tissue distribution in the nociceptive primary afferent fibres and its high sensitivity to protons. EXPERIMENTAL APPROACH: To examine the contribution of ASIC3 to the development and maintenance of muscle pain and inflammatory pain, we studied the in vivo efficacy of a selective ASIC3 inhibitor, APETx2, in rats. KEY RESULTS: Administration of APETx2 into the gastrocnemius muscle prior to the administration of low pH saline prevented the development of mechanical hypersensitivity, whereas APETx2 administration following low-pH saline was ineffective in reversing hypersensitivity. The prevention of mechanical hypersensitivity produced by acid administration was observed whether APETx2 was applied via i.m. or i.t. routes. In the complete Freund's adjuvant (CFA) inflammatory pain model, local administration of APETx2 resulted in a potent and complete reversal of established mechanical hypersensitivity, whereas i.t. application of APETx2 was ineffective. CONCLUSIONS AND IMPLICATIONS: ASIC3 contributed to the development of mechanical hypersensitivity in the acid-induced muscle pain model, whereas ASIC3 contributed to the maintenance of mechanical hypersensitivity in the CFA inflammatory pain model. The contribution of ASIC3 to established hypersensitivity associated with inflammation suggests that this channel may be an effective analgesic target for inflammatory pain states.