Responsible Toxin Research Articles

Development of a membrane-disruption assay using phospholipid vesicles as a proxy for the detection of cellular membrane degradation

Snakebite envenoming is a global health issue that affects millions of people worldwide, and that causes morbidity rates surpassing 450,000 individuals annually. Patients suffering from snakebite morbidities may experience permanent disabilities such as pain, blindness and amputations. The (local) tissue damage that causes these life-long morbidities is the result of cell- and tissue-damaging toxins present in the venoms. These compounds belong to a variety of toxin classes and may affect cells in various ways, for example, by affecting the cell membrane. In this study, we have developed a high-throughput in vitro assay that can be used to study membrane disruption caused by snake venoms using phospholipid vesicles from egg yolk as a substrate. Resuspended chicken egg yolk was used to form these vesicles, which were fluorescently stained to allow monitoring of the degradation of egg yolk vesicles on a plate reader. The assay proved to be suitable for studying phospholipid vesicle degradation of crude venoms and was also tested for its applicability for neutralisation studies of varespladib, which is a PLA2 inhibitor. We additionally made an effort to identify the responsible toxins using liquid chromatography, followed by post-column bioassaying and protein identification using high-throughput venomics. We successfully identified various toxins in the venoms of C. rhodostoma and N. mossambica, which are likely to be involved in the observed vesicle-degrading effect. This indicates that the assay can be used for screening the membrane degrading activity of both crude and fractionated venoms as well as for neutralisation studies.

Determination of ibotenic acid and muscimol in species of the genus Amanita section Amanita from China

The genus Amanita sect. Amanita harbors approximately 150 species in the world, and 27 species have been recognized in China. Some of the species in China have continuously caused poisoning. The responsible toxins should be ibotenic acid (IBO) and muscimol (MUS). However, species of the section Amanita containing IBO and MUS and their systematic positions are unclear. In this study, the contents of IBO and MUS in 84 samples of 24 species in section Amanita were detected using UPLC‒MS/MS, and the distribution of toxin-containing species in the molecular phylogeny was analyzed by the combined (ITS, nrLSU, RPB2, TUB2 and TEF1-α) dataset using maximum likelihood (ML) analysis and Bayesian inference (BI). Our results indicated that 10 of the 24 species contained IBO and MUS ranging from 0.6125 to 32.0932 and 0.0056–5.8685 g/kg dry weight, respectively. Among these 10 species, the toxins of eight species, including Amanita altipes, A. concentrica, A. flavopantherina, A. griseopantherina, A. pseudopantherina, A. rubrovolvata, A. subglobosa and A. sychnopyramis, were detected for the first time. In addition, the IBO and MUS contents of A. subglobosa in different growth stages showed that both toxins decreased in the mature stage. The phylogenetic analysis showed that all species of sect. Amanita from China were divided into 5 groups. And IBO- and MUS-containing species were gathered in clades Ⅰ and Ⅳ, but not all of the species in the two clades contain the toxins. No presence of IBO and MUS in the species of clades Ⅱ, Ⅲ and Ⅴ were confirmed.

Haff disease in the pandemic COVID-19 period in Brazil

Objective: This study describes and characterize the cases of Haff disease reported in Brazil during the COVID-19 pandemic period. Descriptive and cross-sectional study using public data from the National Center for Strategic Information and Response in Health Surveillance nacional. The Risk Communication number 17 of 09/15/2021 was accessed. The description of suspected cases was extended to all Brazilian states where notifications occurred, from January 1, 2020, to September 15, 2021. There was an increase in cases of Haff's disease in Brazil, from 2020 onwards, with 46 in the Northeast region of Brazil. Eighty-five cases and three deaths were reported between January and September 2021 in the North, Northeast, South and Midwest regions. Most cases occurred in regions of greater heat such as the coastal region of the Northeast and around regions of the Amazon River Basin. A critical period for coping with the SARS-CoV-2 virus pandemic in Brazil, cases of disease of Haff could be reported in various regions of country. Despite the etiological suspicions, studies, and follow-up of the cases, it is still not possible to affirm that the disease is caused by biomolecules of the Palytoxin-Like Toxins group. The possible identification of the responsible toxin(s) will be essential for a complete understanding of the etiopathogenic mechanism of the disease and adequate development of prevention, treatment, and cure measures.

Occurrence of tetrodotoxin in the causative gastropod polinices didyma and another gastropod natica lineata collected from Western Taiwan

Attempts were made to elucidate the responsible toxins in the gastropod Polinices didyma which recently caused food poisoning incidents in western Taiwan. The remained (cooked) and captured (live) specimens of both gastropods were assayed for toxicity (as tetrodotoxin = TTX). Average toxicity of cooked and live specimens was 118 ± 105 and 47 ± 28 MU/specimen, respectively. In addition, another species Natica lineata collected from the same area was also found to be toxic. The toxin of each gastropod was partially purified from the methanolic extract of the gastropod by ultrafiltration and Bio-Gel P-2 column chromatography. HPLC and GC-MS analyses demonstrated that the toxin consisted of TTX. It was concluded that the causative agent of the above food poisoning was TTX.

Genetics, Toxicity, and Distribution of Enterohemorrhagic Escherichia coli Hemolysin.

The ability to produce enterohemolysin is regarded as a potential virulence factor for enterohemorrhagic Escherichia coli (EHEC) and is frequently associated with severe human diseases such as hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). The responsible toxin, which has also been termed EHEC-hemolysin (EHEC-Hly, syn. Ehx), belongs to the Repeats in Toxin (RTX)-family of pore-forming cytolysins and is characterized by the formation of incomplete turbid lysis zones on blood agar plates containing defibrinated sheep erythrocytes. Besides the expression of Shiga toxins (Stx) and the locus of enterocyte effacement (LEE), EHEC-Hly is a commonly used marker for the detection of potential pathogenic E. coli strains, although its exact role in pathogenesis is not completely understood. Based on the current knowledge of EHEC-Hly, this review describes the influence of various regulator proteins, explains the different mechanisms leading to damage of target cells, discusses the diagnostic role, and gives an insight of the prevalence and genetic evolution of the toxin.

Chemical Analysis of Plants that Poison Livestock: Successes, Challenges, and Opportunities.

Poisonous plants have a devastating impact on the livestock industry as well as human health. To fully understand the effects of poisonous plants, multiple scientific disciplines are required. Chemical analysis of plant secondary compounds is key to identifying the responsible toxins, characterizing their metabolism, and understanding their effects on animals and humans. In this review, we highlight some of the successes in studying poisonous plants and mitigating their toxic effects. We also highlight some of the remaining challenges and opportunities with regards to the chemical analysis of poisonous plants.

Accumulation and depletion of indospicine in calves (Bos taurus) fed creeping indigo (Indigofera spicata)

Prolonged consumption of Indigofera pasture plants can cause both hepatotoxicosis and reproductive losses in grazing animals with the responsible toxin indospicine forming persistent tissue residues. Separate accumulation and depletion feeding trials were undertaken in calves fed Indigofera spicata (3 mg indospicine/kg bodyweight) to ascertain the appearance and elimination of indospicine from various tissues. In the accumulation trial indospicine concentrations increased throughout the 42-day feeding period with maximum levels of 15 mg/L in plasma and 19 and 33 mg/kg in liver and muscle, respectively. In the depletion trial, calves were fed I. spicata for 35 days, after which the plant was withdrawn from the diet. The rate of elimination was relatively slow with estimates of half-life being 31, 25 and 20 days for muscle, liver and plasma, respectively. Indospicine levels measured in bovine tissues in this trial are comparable with levels in horsemeat and camel meat reported to cause fatal hepatoxicity in dogs, a species known to be susceptible to this toxin. The persistence of indospicine residues in bovine tissues and the widespread distribution of Indigofera species in tropical and sub-tropical grazing lands warrant further investigation, as indospicine has been established as causing reproductive losses and likely contributes to calf losses in these regions.

Investigating the Toxicity of the Aeruginosin Chlorosulfopeptides by Chemical Synthesis.

Harmful algal blooms are becoming more prevalent all over the world, and identification and mechanism-of-action studies of the responsible toxins serve to protect ecosystems, livestock, and humans alike. In this study, the chlorosulfopeptide aeruginosin 828A, which rivals the well-known toxin microcystin LR in terms of crustacean toxicity, has been synthesized for the first time. Furthermore, three congeners with different permutations of the chloride and sulfate groups were prepared, thereby enabling toxicity studies without the risk of contamination by other natural toxins. Toxicity assays with the sensitive crustacean Thamnocephalus platyurus demonstrated that the introduction of a sulfate group leads to pronounced toxicity, and NMR spectroscopic evidence suggests that the chloride substituent modulates the conformation, which in turn might influence protease inhibition.

Identification of Cyclopropylacetyl-(R)-carnitine, a Unique Chemical Marker of the Fatally Toxic Mushroom Russula subnigricans.

A toxic mushroom, Russula subnigricans, causes fatal poisoning by mistaken ingestion. In spite of the potent bioactivity, the responsible toxin had not been identified for about 50 years since its first documentation. Recently, we isolated an unstable toxin and determined the structure. The slow elucidation was partly due to the instability of the toxin and also due to misidentification of R. subnigricans for similar mushrooms. To discriminate genuine Russula subnigricans from similar unidentified Russula species, we searched for a unique chemical marker contained in the mushroom. Cyclopropylacetyl-(R)-carnitine specific to R. subnigricans was identified as a novel compound whose (1)H-NMR signals appearing in the upfield region were easily recognizable among the complicated signals of the crude extract.

Food Poisonings by Ingestion of Cyprinid Fish

Raw or dried gallbladders of cyprinid fish have long been ingested as a traditional medicine in the Asian countries, particularly in China, for ameliorating visual acuity, rheumatism, and general health; however, sporadic poisoning incidences have occurred after their ingestion. The poisoning causes complex symptoms in patients, including acute renal failure, liver dysfunction, paralysis, and convulsions of limbs. The causative substance for the poisoning was isolated, and its basic properties were examined. The purified toxin revealed a minimum lethal dose of 2.6 mg/20 g in mouse, when injected intraperitoneally. The main symptoms were paralysis and convulsions of the hind legs, along with other neurological signs. Liver biopsy of the euthanized mice clearly exhibited hepatocytes necrosis and infiltration of neutrophils and lymphocytes, suggesting the acute dysfunction of the liver. Blood tests disclosed the characteristics of acute renal failure and liver injury. Infrared (IR) spectrometry, fast atom bombardment (FAB) mass spectrometry, and 1H- and 13C-nuclear magnetic resonance (NMR) analysis indicated, a molecular formula of C27H48O8S, containing a sulfate ester group for the toxin. Thus, we concluded that the structure of carp toxin to be 5α-cyprinol sulfate (5α-cholestane-3α, 7α, 12α, 26, 27-pentol 26-sulfate). This indicated that carp toxin is a nephro- and hepato- toxin, which could be the responsible toxin for carp bile poisoning in humans.

Abstract PL02-02: Aristolochic acid-induced nephropathy and urothelial carcinoma: A preventable global disease

Abstract Balkan endemic nephropathy (BEN) is a chronic renal disease affecting residents of rural farming villages located near tributaries of the Danube river. Otherwise rare urothelial carcinomas of the upper urinary tract (UTUC) develop in ~ 50% of patients with BEN. Our studies of the etiology of this disease were informed by reports of a similar syndrome affecting ~ 100 Belgian women who ingested Aristolochia fangchi as part of a weight loss routine (1). In this case, the responsible toxin proved to be aristolochic acid (AA), a component of all Aristolochia plants. In the Balkans, a pilot epidemiologic study confirmed earlier reports that Aristolochia clematitis (birthwort) often is found as a weed growing in cultivated fields. Traditional methods used for harvesting and milling of wheat allowed seeds of this plant to contaminate grain used to prepare bread, a dietary staple among residents of the endemic region. Aristolactam (Al)-DNA adducts were detected in the renal cortex and urothelial tumor tissue of patients with BEN (2). Moreover, a pattern of A:T to T:A mutations unique to AA was demonstrated first in TP53 (3, 4) and, subsequently throughout the genome (5). Based on these observations, it was proposed that the name, BEN, be changed to aristolochic acid nephropathy (AAN). With respect to prevention of BEN, scientific designation of this environmental toxin is important to public health authorities as it legitimizes efforts to change traditional agricultural practices. The experimental methods developed in our studies proved indispensable in our subsequent efforts to demonstrate that AAN and UTUC are prevalent in Asian countries, where Aristolochia herbs have been used for medicinal purposes for centuries. For example, in Taiwan, where the incidence of UTUC and of chronic kidney disease are the highest in the world, at least one-third of the population has been prescribed herbs containing AA (6). Remarkably, the unique TP53 mutational signature for AA established in our studies of BEN was recapitulated in cases of UTUC from Taiwan (7). Moreover, aristolactam-DNA adducts, which persist for decades in the renal cortex, were detected in patients with the signature mutation spectrum in TP53, thereby documenting the relationship between exposure to AA and its mutagenic effects. Millions of people in China are estimated to have ingested Aristolochia herbal remedies, highlighting the potential morbiditity and mortality associated with the medicinal use of this family of herbs (8). Studies designed to document AA exposure in countries where Aristolochia herbs are used in the practice of traditional Chinese medicine are currently underway. Importantly, the nephrotoxic effects of AA are irreversible and its carcinogenic effects may not become manifest until 30 or more years after exposure. Thus, the development of non-invasive, biomarker-based tests that can be used to detect AA-induced UTUC are highly desirable, as this cancer is often curable by surgery if detected early in the course of the disease. (Research supported by grants from the NIEHS (ES04068) Fogarty International Center, Croatian Ministry of Science, Taiwan Dept of Health, Zickler Family Foundation and Henry and Marsha Laufer).

Pregabalin beneficial effects on sleep quality or health-related quality of life are poorly correlated with reduction on pain intensity

Pregabalin beneficial effects on sleep quality or health-related quality of life are poorly correlated with reduction on pain intensity

Interannual variability in the timing of New England shellfish toxicity and relationships to environmental forcing

Routine monitoring along the coast of the Gulf of Maine (GoM) reveals shellfish toxicity nearly every summer, but at varying times, locations, and magnitudes. The responsible toxin is known to be produced by the dinoflagellate Alexandrium fundyense, yet there is little apparent association between Alexandrium abundance and shellfish toxicity. One possibility is that toxic cells are persistent in offshore areas and variability in shellfish toxicity is caused not by changes in overall abundance, but rather by variability in transport processes. Measurements of offshore Alexandrium biomass are scarce, so we bypass cell abundance as an explanatory variable and focus instead on the relations between shellfish toxicity and concurrent metrics of GoM meteorology, hydrology, and oceanography. While this yields over two decades (1985–2005) of data representing a variety of interannual conditions, the toxicity data are gappy in spatial and temporal coverage. We address this through a combination of parametric curve fitting and hierarchical cluster analysis to reveal eight archetypical modes of seasonal toxicity timing. Groups of locations are then formed that have similar interannual patterns in these archetypes. Finally, the interannual patterns within each group are related to available environmental metrics using classification trees. Results indicate that a weak cross-shore sea surface temperature (SST) gradient in the summer is the strongest correlate of shellfish toxicity, likely by signifying a hydrological connection between offshore Alexandrium populations and near-shore shellfish beds. High cumulative downwelling wind strength early in the season is revealed as a precursor consistent with this mechanism. Although previous studies suggest that alongshore transport is important in moving Alexandrium from the eastern to western GoM, alongshore SST gradient is not an important correlate of toxicity in our study. We conclude by discussing the implications of our results for designing efficient and effective shellfish monitoring programs along the GoM coast.

New Syndromes in Mushroom Poisoning

Several new mushroom poisoning syndromes have been described since the early 1990s. In these syndromes, the onset of symptoms generally occurs >6 hours after ingestion. Treatment is mainly supportive. The syndrome induced by Amanita smithiana/proxima consists of acute tubulopathy, which appears earlier and does not have the same poor prognosis as the orellanine-induced syndrome. It has been described since 1992 in the US and Canada with A. smithiana; in France, Spain and Italy with A. proxima; and in Japan with A. pseudoporphyria. The responsible toxin is probably 2-amino-4,5-hexadienoic acid. The erythromelalgia syndrome has been described as early as the late 19th century in Japan and South Korea with Clitocybe acromelalga, and since 1996 in France and then Italy with C. amoenolens. Responsible toxins are probably acromelic acids identified in both species. Several cases of massive rhabdomyolysis have been reported since 1993 in France and 2001 in Poland after ingestion of large amounts of an edible and, until then, valuable species called Tricholoma equestre. These cases of rhabdomyolysis are associated with respiratory and cardiac (myocarditis) complications leading to death. Rhabdomyolysis with an apparently different mechanism was described in Taiwan in 2001 with Russula subnigricans. Finally, cases of encephalopathy were observed twice after ingestion of Hapalopilus rutilans in Germany in 1992 and Pleurocybella porrigens in Japan in 2004, where a convulsive encephalopathy outbreak was reported in patients with history of chronic renal failure.

Shiitake (Lentinula edodes)-Dermatitis

Shiitake an edible asian mushroom nowadays becomes popular in european cooking, too. Allergic diseases to shiitake occur as contact dermatitis, alveolitis or asthma. Consuming of raw shiitake causes toxicodermia. Along with this disease papules develop. The polysaccharide lentina probably is the responsible toxin. Within seven months, we found three patients developing skin lesions after eating this mushroom. A 45 years old patient who often prepared and consumed meals containing shiitake, showed recurrent erythematous papules on his face. A prick to prick test with a shiitake mushroom showed an immediate converting into a delayed reaction. Two other patients developed skin eruptions after eating raw shiitake. One of them had eaten fried shiitake before without any problems. Prick to prick tests with Shiitake turned out to be negative in both cases. At the side of testing, 24 hrs later a delayed reaction with severely itching papules developed. The first case is supposed to be an allergic reaction whether the two other cases may rather be a toxicodermia, because of their history and clinical symptoms. However, the positive skin tests in these two cases would suggest other possible mechanisms. Further studies are necessary to clarify theses different phenomenon.

Confirmation of domoic acid production of Pseudo-nitzschia multiseries isolated from Ofunato Bay, Japan

Production of domoic acid (DA), the responsible toxin for amnesic shellfish poisoning, was examined for 44 strains of Pseudo-nitzschia spp. isolated from Ofunato Bay, Japan. Only one strain which was identified as Pseudo-nitzschia multiseries produced DA in a level comparable to Canadian strains. No significant DA was detected in the rest of the strains, indicating that toxic P. multiseries does not bloom in a high density in the bay.

End-stage renal failure from mushroom poisoning with Cortinarius orellanus: Report of four cases and review of the literature

Mushrooms of the ubiquitous Cortinarius species (Cs) contain nephrotoxins that can cause acute and chronic renal failure by an unknown pathomechanism. Typical is a long symptom-free interval before the onset of clinical disease. A causal form of therapy is not known. Early hemodialysis can improve the prognosis of this potentially life-threatening condition. Diagnosis of Cs poisoning can be made by detecting the responsible toxin—orellanine—in plasma or renal tissue by fluorimetry after thin-layer chromatography or by identifying the spores of left-over mushrooms as Cs. Renal histology shows nonspecific changes such as tubular dilatation and flattening of the epithelium and signs of interstitial edema followed by interstitial fibrosis. We present four cases of Cs poisoning with different outcomes and a review of the literature.

The toxin of diarrheic shellfish poisoning, okadaic acid, increases intestinal epithelial paracellular permeability

BACKGROUND & AIMS: Diarrhea associated with shellfish poisoning is poorly understood. The responsible toxin, dinophysistoxin 1, has been identified as okadaic acid, a potent phosphatase inhibitor, but its effects on intestinal epithelia have not been examined. The aim of this study was to investigate the effect of okadaic acid on intestinal epithelial function, both Cl- secretion and barrier function. METHODS: Cultured human intestinal epithelial T84 cell monolayers were used. The effect of okadaic acid on these monolayers was assessed by measuring electrophysiological parameters, lactate dehydrogenase release, and 22Na+ and [3H]mannitol flux rates. Protein phosphorylation studies were performed to identify potentially involved proteins. RESULTS: Okadaic acid does not directly stimulate Cl- secretion from intestinal epithelial cells. On the contrary, the response to well-characterized secretagogues is attenuated by okadaic acid. However, it does decrease transepithelial electrical resistance in a polarized fashion without inducing cytotoxicity. Sodium-mannitol flux studies suggest that the observed decrease in resistance is attributable to an increase in paracellular permeability. CONCLUSIONS: Okadaic acid, the toxin responsible for diarrheic shellfish poisoning, does not stimulate Cl- secretion but increases the paracellular permeability of intestinal epithelia. This alteration in intestinal epithelial physiology may contribute to the diarrhea of shellfish poisoning. (Gastroenterology 1997 Jan;112(1):100-8)

腸炎ビブリオVp-TRH毒素に対するモノクローナル抗体を用いたELISA法の開発と応用

Although it has been well established that Kanagawa phenomenon-positive Vibrio parahaemolyticus is a human enteropathogen, the Kanagawa phenomenon-negative one has been considered to be probably not pathogenic. We have found, however, an outbreak of gastroenteritis due to Kanagawa phenomenon-negative V. parahaemolyticus which produces a new toxin (Vp-TRH) resembling to Vp-TDH, a responsible toxin of Kanagawa phenomenon. In this study, we developed monoclonal antibodies against Vp-TRH which were used for development of an enzyme-linked immunosorbent assay (ELISA) for specifically detecting Vp-TRH. The ELISA was applied for analysis of production of Vp-TRH by various isolates of V. parahaemolyticus and we found that Vp-TRH-producing strains were derived mostly from human diarrheal stool, and not from the environment or sea foods. The results of the rabbit ileal loop test showed that Vp-TRH-producing (Kanagawa phenomenon-negative) strains, as well as Vp-TDH-producing (Kanagawa phenomenon-positive) strains could induce fluid accumulation. These results indicate the possibility that Vp-TRH-producing Kanagawa phenomenon-negative V. parahaemolyticus is a human enteropathogen.

Toxicity of the Trumpet Shell Charonia sauliae (“Boshubora”) Inhabiting the Coasts of Oita Prefecture, and Identification of the Responsible Toxin

A total of 105 specimens of the trumpet shell Charonia sauliae (“boshubora”) were collected from June 1984 through July 1985, at Kamae and Tsukumi in Oita Prefecture. Digestive glands were excised from these specimens and examined for toxicity (as tetrodotoxin; TTX).1) The frequencies of occurrence of toxic specimens at Kamae and Tsukumi were 43 and 55%, respectively.2) The average toxicity scores at Kamae and Tsukumi were 15 and 3.4MU/g digestive gland, the highest toxicity scores being 115 and 8.3MU/g digestive gland, respectively.3) The responsible toxin was partially purified by active charcoal treatment, followed by chromatography on Bio-Gel P-2. Analyses by TLC, cellulose acetate membrane electrophoresis and high performance liquid chromatography showed that the toxin was composed of TTX and related substances.