Secretory glands of tetrodotoxin in the skin of the Japanese newt Cynops pyrrhogaster.

Intra-tissue distribution of tetrodotoxin (TTX) in the skin of larva, juvenile, and adult Japanese newt Cynops pyrrhogaster was investigated by means of a monoclonal antibody-based immunoenzymatic technique. In the investigation, TTX was localized at immature glands in juvenile, and at the granular cells composing of granular and mixed glands in adult specimens under a light microscope. No specific stain was recognized in larval section. A duct like structure extending from the granular gland towards super epithelial layer was visualized in the toluidine blue treated skin section of adult newt. When stimuli by wiping with gauze ('handling stimulus') were given, C. pyrrhogaster was found to secrete an applicable amount of TTX and 6-epiTTX from the skin, suggesting that the newt has a granular gland of TTX to secrete it towards the body surface possibly as a biological defensive agent.     

Toxicity and paralytic shellfish toxin profiles of the xanthid crabs, Lophozozymus pictor and Zosimus aeneus, collected from some Australian coral reefs

Purification of toxic aqueous extracts from the xanthid crabs Zosimus aeneus and Lophozozymus pictor, collected from Australian waters, yielded paralytic shelfish toxins, including saxitoxin (STX), neosaxitoxin (neoSTX) and gonyautoxins 1, 2 and 4 (GTX1,2,4). No more than two paralytic shellfish toxins were found in any of the purified extracts from any specimen. Four specimens of Z. aeneus and one specimen of L. pictor each contained more toxic material than the suggested human oral lethal dose. The moult of a specimen of L. pictor was toxic, which may indicate a route in crabs for toxin removal.     

Toxicity assessment of cyanobacterial toxin mixtures

A method is presented for the toxicity assessment of mixtures of cyanobacterial toxins with hepatotoxic, neurotoxic, and carcinogenic effects. The method is based on the derivation of toxicity equivalent factors obtained from toxicological data presented for cyanobacterial toxins in the literature and on the use of toxicity equivalents in analogy with the method used for polychlorinated dibenzo[p]dioxins and dibenzofurans. Because the method easily can include all toxicological data for cyanobacterial toxins at any time, the quality of the toxicity assessment of these toxins can be increased continuously depending on data availability. Generally, all toxicologically relevant cyanobacterial toxins for which data are available should be included in this assessment. The method is considered useful in the toxicity assessment of cyanobacterial toxin mixtures in surface water bodies and in drinking water. When using the total sum of the toxicity equivalents, the approach proposed here seems to lead to a more realistic toxicity assessment of cyanobacterial toxin mixtures than does the worst-case approach. However, the availability of data about acute and, especially, chronic toxicity must be increased significantly in the future in order to establish toxicologically validated exposure limit values for cyanobacterial toxins, especially those with hepatotoxic, neurotoxic, and carcinogenic effects.     

Paralytic shellfish toxin profiles and toxin variability of the genus Alexandrium (Dinophyceae) isolated from the Southeast China Sea.

Paralytic shellfish toxin (PST) profiles of 16 Alexandrium isolates from the Southeast China Sea were analyzed by high-pressure liquid chromatography. Toxin content and composition of three A. tamarense isolates, ATDH01, ATGX02 and ATMJ02, were also investigated at different growth phases and under various culture conditions. Our results showed that six strains of A. affine were non-toxic, while 10 strains of A. tamarense and A. catenella were toxic. These toxic isolates grown in the same culture conditions consistently produced an unusually high proportion of the N-sulfocarbamoyl toxin C1/2 (around 60-80% of total toxins) and medium amounts of gonyautoxin GTX5 (around 15-30% of total) with only trace quantities (<5% of total) of other saxitoxin derivatives (i.e. GTX1, GTX3, GTX4 and neoSTX). The toxin composition of three A. tamarense isolates did not vary with the growth phases, although higher toxin contents (Qt, fmolcell(-1)) were found in the exponential phase. Variations in temperature, salinity and nutrient levels affected toxin content of three A. tamarense isolates but they did not have pronounced effects on the toxin composition (mole %). These results indicate that toxin composition remained relatively constant under various culture conditions, suggesting that toxin composition could be used as a stable biomarker for the Alexandrium species in this region. However, comparison of toxin profiles between isolates from different localities require special caution since isolates even from the same region can have distinct toxin profiles.     

Toxicity of coarse and fine particulate matter from sites with contrasting traffic profiles

Residence in urban areas with much traffic has been associated with various negative health effects. However, the contribution of traffic emissions to these adverse health effects has not been fully determined. Therefore, the objective of this in vivo study is to compare the pulmonary and systemic responses of rats exposed to particulate matter (PM) obtained from various locations with contrasting traffic profiles. Samples of coarse (2.5 microm-10 microm) and fine (0.1 microm-2.5 microm) PM were simultaneously collected at nine sites across Europe with a high-volume cascade impactor. Six PM samples from various locations were selected on the basis of contrast in in vitro analysis, chemical composition, and traffic profiles. We exposed spontaneously hypertensive (SH) rats to a single dose (3 mg PM/kg body weight or 10 mg PM/kg body weight) of either coarse or fine PM by intratracheal instillation. We assessed changes in biochemical markers, cell differentials, and histopathological changes in the lungs and blood 24 h postexposure. The dose-related adverse effects that both coarse and fine PM induced in the lungs and vascular system were mainly related to cytotoxicity, inflammation, and blood viscosity. We observed clear differences in the extent of these responses to PM from the various locations at equivalent dose levels. There was a trend that suggests that samples from high-traffic sites were the most toxic. It is likely that the toxicological responses of SH rats were associated with specific PM components derived from brake wear (copper and barium), tire wear (zinc), and wood smoke (potassium).     

Toxicity and recovery in the pregnant mouse after gestational exposure to the cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN) is a tricyclic alkaloid toxin produced by fresh water cyanobacterial species worldwide. CYN has been responsible for both livestock and human poisoning after oral exposure. This study investigated the toxicity of CYN to pregnant mice exposed during different segments of gestation. The course of recovery and individual responses to the toxin were evaluated. Adverse effects of CYN were monitored up to 7 weeks post‐dosing by clinical examination, histopathology, biochemistry and gene expression. Exposure on gestational days (GD) 8–12 induced significantly more lethality than GD13–17 exposure. Periorbital, gastrointestinal and distal tail hemorrhages were seen in both groups. Serum markers indicative of hepatic injury (alanine amino transferase, aspartate amino transferase and sorbitol dehydrogenase) were increased in both groups; markers of renal dysfunction (blood urea nitrogen and creatinine) were elevated in the GD8–12 animals. Histopathology was observed in the liver (centrilobular necrosis) and kidney (interstitial inflammation) in groups exhibiting abnormal serum markers. The expression profiles of genes involved in ribosomal biogenesis, xenobiotic and lipid metabolism, inflammatory response and oxidative stress were altered 24 h after the final dose. One week after dosing, gross, histological and serum parameters had returned to normal, although increased liver/body weight ratio and one instance of gastrointestinal bleeding was found in the GD13–17 group. Gene expression changes persisted up to 2 weeks post‐dosing and returned to normal by 4 weeks. Responses of individual animals to CYN exposure indicated highly significant inter‐animal variability within the treated groups. Copyright © 2010 John Wiley & Sons, Ltd.     

Toxicity of ricin toxin A chain in rats

Ricin toxin A chain (RTA) is the cytotoxic component of the dimeric protein, ricin, one of the most potent and deadly plant toxins extracted from the seeds of Ricinus communis. RTA has been investigated as a potential candidate for cancer chemotherapy, in the form of immunotoxins, and as a method for depleting macrophages in vivo. The toxicity of RTA immunotoxins is mostly characterized by inflammation and necrosis and has been attributed to the RTA moiety of the conjugate. The present study was carried out to investigate the toxicity of intravenously (i.v.) administered RTA alone and to assess whether the observed tissue injuries are associated with increases in oxidative stress (OS) and inflammation. RTA (10 or 90 µg/kg body weight) was administered to animals i.v., and 5 or 24 hours later, liver, lungs, kidneys, and hearts were examined. RTA, at a dose of 90 µg/kg (i.v.), resulted in significant increases (P < 0.05) in an inflammatory response (i.e., increases in hepatic and lung myeloperoxidase activity) and increases in oxidant response (increases in lipid peroxidation and decreases in glutathione levels in hepatic and lung homogenates). These data suggest that i.v. administration of RTA resulted in organ injuries that were associated with inflammation and OS.     

Toxicity and oxidative stress induced by T-2 toxin and HT-2 toxin in broilers and broiler hepatocytes

T-2 and HT-2 toxins belong to mycotoxins which are found in human foods and animal chow. We investigated the toxicity and oxidative stress induced by T-2/HT-2 in broilers and chicken hepatocytes. Maize cultures of Fusarium poae was fed to broilers for 42 d, and the physiological index, biochemical index and expression of mRNAs related to oxidative stress were analyzed. Chicken hepatocytes were treated with different levels of T-2/HT-2, and the following parameters were detected: cell viability, GSH and MDA concentration, LDH leakage, activities of ALT/AST, ROS, GSH-PX, SOD and CAT, and expression of mRNA related to oxidative stress. In vivo, high levels of mycotoxins (4 mg/kg T-2 and 0.667 mg/kg HT-2) in feed caused significant reductions in body weight, weight gain, and serum total protein, and significant increases in feed conversion ratio, ALP, ALT/AST activities, and expression of mRNA related to oxidative stress. In vitro, cells treated with T-2/HT-2 showed reductions of GSH concentration and significant increases in LDH leakage, ALT/AST ROS, GSH-PX, SOD and CAT activities, MDA concentration, and expression of mRNA related to oxidative stress. Consequently, F. poae culture material and T-2/HT-2 induced toxicity and oxidative stress in vivo and in vitro, respectively.     

Toxicity of microcystin-LR, a cyanobacterial toxin, to multiple life stages of the burrowing mayfly, Hexagenia, and possible implications for recruitment

Burrowing mayflies, genus Hexagenia, were extirpated from the major water bodies of North America in the early 1950s, paralleling an increase in eutrophication and organic pollution, and a decrease in dissolved oxygen concentrations. Burrowing mayflies recolonized the western basin of Lake Erie, but remain absent in other former habitats such as Oneida Lake, New York. Eutrophication is commonly associated with a shift in the phytoplankton community toward dominance by cyanobacteria, and therefore, one class of cyanobacterial toxins, microcystins, were investigated as a contributing factor to Hexagenia's eradication or as an impediment to recolonization. Laboratory experiments were conducted to determine if microcystin-LR (MC-LR) produced negative effects on Hexagenia at three points within its life cycle: egg, hatchling nymph (<24-h old, <1 mm total length), and pre-emergence nymph (>17 mm). Treatment concentrations ranged from the guideline set by the World Health Organization for drinking water (0.001 microg mL(-1)) to 0.1 microg mL(-1) for the egg experiment and 10 microg mL(-1) for the nymph trials. Eggs showed a delay in hatching and an altered distribution of hatching over the study period when submerged in 0.1 microg mL(-1) MC-LR (an elevated concentration representative of bloom scum). The 72-h (1.1 microg mL(-1)) and 96-h (0.049 microg mL(-1)) LC(50) values for hatchling nymphs exceeded typical bloom concentrations of North American lakes, (0.01 microg mL(-1)). Large nymphs were more tolerant of the toxin, as indicated by 100% survival over seven days exposure to 10 microg mL(-1), suggesting older larvae can withstand brief encounters with high microcystin levels for at least short periods of time. The sensitivity of young nymphs and eggs to MC-LR may have implications for the recruitment of the genus in water bodies with persistent summer cyanobacterial blooms.     

Inhibition of Na currents by the toxin extracts from puffer fishes captured in the sea coast of Japan

The inhibitory effects of toxin extracted from muscle or liver of five different puffer fishes (hereafter referred as puffer(s)) captured on the Japanese sea coast were examined on voltage-dependent sodium current (I_Na) recorded from dissociated single rat hippocampal CA1 neurons. The inhibitory effects estimated from IC₅₀ values of toxin extracts on I_Na were in the order of Takifugu vermicularis > Lagocephalus wheeleri > Canthigaster rivulata > Takifugu rubripes > Arothron reticularis from muscle and T. vermicularis > T. rubripes > L. wheeleri > A. reticularis > C. rivulata from liver, thereby indicating that the amount of toxin in the liver or muscle differs between puffers. In addition, the present results indicate that the muscle of T. vermicularis, which is eaten in Japan, contains relatively higher amounts of toxin compared to those of T. rubripes, also eaten. This observation suggests that caution should be taken concerning the maximal edible amount of muscle prepared from T. rubripes.     

Structure and other chemical characterizations of gila toxin,a lethal toxin from lizard venom

The complete primary structure of a lethal toxin, horridum toxin, from the venom of the lizard, Heloderma horridum horridum, was determined by Edman degradation. The amino acid sequence was deduced by overlapping peptide fragments generated by chemical and enzymatic digestions. Horridum toxin causes hemorrhage in internal organs and particularly in the eye, leading to exophthalmia, an effect that has not been observed for other toxins. It is a glycoprotein with a total of 210 residues. Examination of the primary sequence revealed that horridum toxin has considerable homology to tissue-type kallikrein and trypsin. Furthermore, synthetic substrates for trypsin, such as tosyl-l -arginine methyl ester, benzoyl-l -arginine ethyl ester and other p-nitroanilide substrates, were hydrolyzed. The toxin released bradykinin upon hydrolysis of kininogen. This enzymatic behavior is similar to that of plasma kallikrein; however, the presence of a characteristic “kallikrein-like” loop at 91-100 (GTIYNCNYVN) in the primary structure and other features similar to tissue kallikrein suggest that horridum toxin is more like tissue kallikrein. This toxin degraded all three chains of fibrinogen but did not form a clot, which suggests that it is different from thrombin. Moreover, it differs from another lethal factor from H. horridum horridum, gila toxin, which has 245 amino acid residues and does not cause exophthalmia.     

Nephrotoxicity of orellanine,a toxin from the mushroomCortinarius orellanus

Pure orellanine extracted from the mushroomCortinarius orellanus is highly toxic in mice both when given intraperitoneally (LD₅₀=12.5 mg/kg) or per os (LD₅₀ =90 mg/kg). The kidneys of mice injected i.p. with orellanine show similar tubular necrosis to that obtained with whole mushroom given per os. This demonstrates that orellanine is the true, principal toxin ofC. orellanus. The similarity of the structures of the toxin and of bipyridinium herbicides such as methylviologen (paraquat) or the nephrotoxic diquat led other authors to presume a similar mechanism for orellanine toxicity. Our study of the electrochemical behaviour of orellanine shows that its mechanism of action has to be different from that of these poisons.     

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish.

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques--mouse bioassay, ELISA, HPLC and mass spectrometry-we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6+/-12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6+/-0 5MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry.     

Toxicity Reduction Evaluation, Toxicity Identification Evaluation and Toxicity Tracking in Direct Toxicity Assessment

Toxicity reduction evaluations (TREs) in the River Esk and Lower Tees Estuary were based on the approach described by USEPA, but adapted to tackle the specific problems of the two sites. A combination of toxicity tracking and toxicity identification evaluation (TIE) was used at both locations to enhance the understanding of source and type of toxicants present. The assessment of toxicity at Langholm focussed on pesticides present in the sewerage network. The TIE programme indicated that the most likely toxic agents within the effluent were the organophosphate pesticides diazinon and to lesser extent propetamphos, although these did not account for all of the observed toxicity. The exact source of these toxicants was not clear although toxicity tracking identified two potential candidates. The TRE undertaken on the discharge to the lower Tees utilised high-throughput methods with standard test organisms to generate toxicity information throughout a complex sewerage network. The toxicity tracking information was used in conjunction with TIEs to identify a number of key sources of toxicity. Substantial toxicity was associated with a currently untreated industrial effluent. Chemical analysis and TIE highlighted cyanide as the likely toxicant in this effluent and its possible significance in the final discharge.     

Heminecrolysin, the first hemolytic dermonecrotic toxin purified from scorpion venom

Envenomation caused by Hemiscorpius (H.) lepturus from Liochlidae family presents clinical features that have not been previously described for the Buthidae family scorpions. The most significant manifestations of H. lepturus envenomation are hemolysis and dermonecrosis which could lead in severe cases to renal, cardio-respiratory failure, and death. In this study, we aimed to identify and characterize the protein(s) causing these effects. We have purified a 33 kDa protein from the venom of H. lepturus and named it Heminecrolysin. Tryptic digestion and MS/MS analysis of obtained peptides showed homology with previously described brown spider sphingomyelinases D. Functional characterization of Heminecrolysin indicated a sphingomyelinase D, a complement-dependent hemolysis properties and a dermonecrosis activity. Heminecrolysin displayed higher hemolytic activity to human erythrocytes (ED50 of 0.025 μg/ml), a stronger inflammatory and dermonecrotic effects when injected intra-dermally to rabbit skins, while its efficiency to hydrolyze sphingomyelin seems weaker than other known spider dermonecrotic SMasesD (149 ± 32.5 nmol/mg). Step of sensitization of human erythrocytes by Heminecrolysin was shown to be Mg²⁺ and Ca²⁺-independent while hemolysis step in the presence of complement required both bivalent ions. Heminecrolysin is the first hemolytic dermonecrotic toxin identified in venom other than spiders. Except in spider Loxosceles genus and some pathogenic strains of Corynebacteria, sphingomyelinase D activity is unknown in the animal kingdom.     

Degradation of the cyanobacterial toxin cylindrospermopsin, from Cylindrospermopsis raciborskii, by chlorination.

Cylindrospermopsin, a potent cyanobacterial toxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies of Queensland, Australia. This study focussed on the effectiveness of chlorination as a water treatment procedure for cylindrospermopsin degradation. The results demonstrate that relatively low chlorine doses (<1 mg l(-1)) are sufficient for degradation of cylindrospermopsin, when the dissolved organic carbon content is low. However, if organic matter other than cylindrospermopsin is present in the solution, the effectiveness of chlorine for cylindrospermopsin degradation is reduced as other organic matter present consumes chlorine. Under the experimental conditions using samples with a solution pH of 6-9, a residual chlorine concentration of 0.5 mg l(-1)99% of cylindrospermopsin. Toxin degradation via chlorination occurs within the first minute and no difference was observable between degradation in an open system and in a closed system. With a decrease of the pH from 6 to 4 a reduction in the efficiency of chlorine for degradation of cylindrospermopsin was observable, a possible indication that cylindrospermopsin is more stable to chlorine degradation at lower pH. However, in normal water treatment this is not relevant since the pH is consistently higher than 6.     

Toxicity and oxidative stress induced by T-2 toxin in cultured mouse Leydig cells

To explore the toxic mechanism of T-2 toxin on Leydig cells of mice, we would investigate the toxicity and oxidative stress induced by T-2 toxin in the cells. Leydig cells were isolated and cultured with control or T-2 toxin (10^?7 M, 10^?8 M, or 10^?9 M) for 24?h, then cells and supernatants were harvested to examine cell viability, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activities, expression of messenger RNA (mRNA) related to oxidative stress, malondialdehyde (MDA) content and DNA damage. The cell viability was evaluated in mouse Leydig cells by MTT assay, MDA content and SOD, GSH-Px and CAT activities were measured by routine kits, expression of mRNA related to oxidative stress were examined by quantitative real-time polymerase chain reaction (PCR), and DNA damage was investigated by comet assay. Leydig cells treated with T-2 toxin showed significant reductions in cell viability, SOD, GSH-Px and CAT activities, and expression of mRNA related to oxidative stress, and remarkable increases in MDA content and levels of DNA damage. This study proves that T-2 toxin is toxic to Leydig cells of mice. Furthermore, oxidative stress plays an important role in the above-mentioned negative effects of T-2 toxin.     

Properties of a cytolytic toxin from the sea anemone, Stoichactis kenti

A cytolytic toxin (kentin) from the Indo-Pacific sea anemone, Stoichactis kenti, was purified to near homogeneity. The toxin is a basic polypeptide of molecular weight approximately 18,000. It broadly resembles cytotoxins from Stoichactis helianthus (helianthin), as well as similar toxins from a number of other anemones, namely Condylactis, Epiactis, Actinia, Pseudactinia, Tealia, Anthopleura, Radianthus and Gyrostoma. The amino acid composition of kentin shows considerable resemblance to that of helianthin, but there are also several significant differences. Neutralization tests indicate that kentin and helianthin are immunologically related but distinguishable. In contrast, no immunological relatedness was found between helianthin and cytolytic toxins from Condylactis gigantea and Epiactis prolifera.