Quantification of alpha-amanitin in biological samples by HPLC using simultaneous UV- diode array and electrochemical detection

α-Amanitin is a natural bicyclic octapeptide, from the family of amatoxins, present in the deadly mushroom species Amanita phalloides. The toxicological and clinical interests raised by this toxin, require highly sensitive, accurate and reproducible quantification methods for pharmacokinetic studies. In the present work, a high-performance liquid chromatographic (HPLC) method with in-line connected diode-array (DAD) and electrochemical (EC) detection was developed and validated to quantify α-amanitin in biological samples (namely liver and kidney). Sample pre-treatment consisted of a simple and unique deproteinization step with 5% perchloric acid followed by centrifuga tion at 16,000. ×g, 4. °C, for 20. min. The high recovery found for α-amanitin (≥96.8%) makes this procedure suitable for extracting α-amanitin from liver and kidney homogenates. The resulting supernatant was collected and injected into the HPLC. Mobile phase was composed by 20% methanol in 50. mM citric acid, and 0.46. mM octanessulfonic acid, adjusted to pH 5.5. The chromatographic runs took less than 22. min and no significant endogenous interferences were observed at the α-amanitin retention time. Calibration curves were linear with regression coefficients higher than 0.994. The overall inter- and intra-assay precision did not exceed 15.3%.The present method has low interferences with simple and fast processing steps, being a suitable procedure to support in vivo toxicokinetic studies involving α-amanitin. In fact, the validated method was successfully applied to quantify α-amanitin in biological samples following intraperitoneal α-amanitin administration to rats. Moreover, human plasma was also used as matrix and the purposed method was adequate for detection of α-amanitin in that matrix. The results clearly indicate that the proposed method is suitable to investigate the pharmacokinetic and tissue distribution of α-amanitin. Additionally, the method will be very useful in the development of novel and potent antidotes against amatoxins poisoning and to improve the knowledge of α-amanitin toxicity.This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project Pest- C/EQB/LA0006/2013. Juliana Garcia and Vera Marisa Costa thank FCT - Foundation for Science and Technology - for their PhD grant (SFRH/BD/74979/ 2010 ) and Post-doc grant (SFRH/BPD/63746/2009), respectively.info:eu-repo/semantics/publishedVersio

Amanita phalloides poisoning: Mechanisms of toxicity and treatment

Amanita phalloides, also known as 'death cap', is one of the most poisonous mushrooms, being involved in the majority of human fatal cases of mushroom poisoning worldwide. This species contains three main groups of toxins: amatoxins, phallotoxins, and virotoxins. From these, amatoxins, especially α-amanitin, are the main responsible for the toxic effects in humans. It is recognized that α-amanitin inhibits RNA polymerase II, causing protein deficit and ultimately cell death, although other mechanisms are thought to be involved. The liver is the main target organ of toxicity, but other organs are also affected, especially the kidneys. Intoxication symptoms usually appear after a latent period and may include gastrointestinal disorders followed by jaundice, seizures, and coma, culminating in death. Therapy consists in supportive measures, gastric decontamination, drug therapy and, ultimately, liver transplantation if clinical condition worsens. The discovery of an effective antidote is still a major unsolved issue. The present paper examines the clinical toxicology of A. phalloides, providing the currently available information on the mechanisms of toxicityinvolved and on the current knowledge on the treatment prescribed against this type of mushrooms. Antidotal perspectives will be raised as to set the pace to new and improved therapy against these mushrooms.This work was supported by the Fundação para a Ciência e Tecnologia (FCT ) – project PTDC/DTPFTO/4973/2014 – and the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project Pest-C/EQB/LA0006/2013. Juliana Garcia and Vera Marisa Costa thank FCT for their PhD grant (SFRH/BD/74979/2010) and Post-doc grants (SFRH/BPD/63746/2009 and SFRH/BPD/110001/2015), respectively.info:eu-repo/semantics/publishedVersio

Co-ingestion of amatoxins and isoxazoles-containing mushrooms and successful treatment: A case report

Mushroom poisonings occur when ingestion of wild mushrooms containing toxins takes place, placing the consumers at life-threatening risk. In the present case report, an unusual multiple poisoning with isoxazoles- and amatoxins-containing mushrooms in a context of altered mental state and poorly controlled hypertension is presented. A 68-year-old female was presented to São João hospital (Portugal) with complaints of extreme dizziness, hallucinations, vertigo and imbalance, 3 h after consuming a stew of wild mushrooms. The first observations revealed altered mental state and elevated blood pressure. The examination of cooked mushroom fragments allowed a preliminary identification of Amanita pantherina. Gas chromatography-mass spectrometry (GC-MS) showed the presence of muscimol in urine. Moreover, through high-performance liquid chromatography-ultraviolet detection (HPLC-UV) analysis of the gastric juice, the presence of α-amanitin was found, showing that amatoxins-containing mushrooms were also included in the stew. After 4 days of supportive treatment, activated charcoal, silybin and N-acetylcysteine, the patient recovered being discharged 10 days post-ingestion with no organ complications. The prompt and appropriate therapy protocol for life-threatening amatoxins toxicity probably saved the patient's life as oral absorption was decreased and also supportive care was immediately started.This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project Pest-C/EQB/LA0006/2013. Juliana Garcia and Vera Marisa Costa thank FCT e Foundation for Science and Technology e for their PhD grant (SFRH/BD/74979/2010) and Post-doc grant (SFRH/BPD/63746/2009), respectively.info:eu-repo/semantics/publishedVersio

Reactivity of paraquat with sodium salicylate: formation of stable complexes

Sodium salicylate (NaSAL) has been shown to be a promising antidote for the treatment of paraquat (PQ) poisonings. The modulation of the pro-oxidant and pro-inflammatory pathways, as well as the antithrombogenic properties of NaSAL are probably essential features for the healing effects provided by this drug. Nevertheless, a possible direct chemical reactivity between PQ and NaSAL is also a putative pathway to be considered, this hypothesis being the ground of the present study. In accordance, it is shown, for the first time that PQ and NaSAL react immediately in aqueous medium and within 2–3 min in the solid state. Photographs and scanning electron photomicrographs indicated that a new chemical entity is formed when both compounds are mixed. This assumption was corroborated by the evaluation of the melting point, and through several analytical techniques, namely ultraviolet/visible spectroscopy, nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry/mass spectrometry (GC/MS/MS), liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) and infrared spectroscopy, which revealed that stable charge-transfer complexes are formed when PQ is mixed with NaSAL. LC/ESI/MS/MS allowed obtaining the stoichiometry of the charge-transfer complexes. In order to increase resolution, single value decomposition, acting as a filter, showed that the chargetransfer complexes withm/z 483, 643 and 803 correspond to the pseudo-molecular ions, respectively 1:2, 1:3 and 1:4 (PQ:NaSAL). In conclusion, these results provided a new and important mechanism of action of NaSAL against the toxicity mediated by PQ

[1-(3-Chloro­phen­yl)-1H-1,2,3-triazol-4-yl]methanol hemihydrate

The asymmetric unit of the title hydrate, C9H8ClN3O·0.5H2O, comprises two independent 1,2,3-triazole mol­ecules and a water mol­ecule of crystallization. The dihedral angles between the six- and five-membered rings in the 1,2,3-triazole mol­ecules are 12.71 (19) and 17.3 (2)°. The most significant different between them is found in the relative orientations of the terminal CH2OH groups with one being close to perpendicular to the five-membered ring [N—C—C—O torsion angle = 82.2 (5)°], while in the other mol­ecule, a notable deviation from a perpendicular disposition is found [torsion angle = −60.3 (5)°]. Supra­molecular chains feature in the crystal packing sustained by O—H⋯(O,N) inter­actions along the a-axis direction. The chains are connected via C—H⋯N inter­actions and the resultant layers stack along the b axis

Mitochondrial Cumulative Damage Induced by Mitoxantrone: Late Onset Cardiac Energetic Impairment

Mitoxantrone (MTX) is a chemotherapeutic agent, which presents late irreversible cardiotoxicity. This work aims to highlight the mechanisms involved in the MTX-induced cardiotoxicity, namely the effects toward mitochondria using in vivo and in vitro studies. Male Wistar rats were treated with 3 cycles of 2.5 mg/kg MTX at day 0, 10, and 20. One treated group was euthanized on day 22 (MTX22) to evaluate the early MTX cardiac toxic effects, while the other was euthanized on day 48 (MTX48), to allow the evaluation of MTX late cardiac effects. Cardiac mitochondria isolated from 4 adult untreated rats were also used to evaluate in vitro the MTX (10 nM, 100 nM, and 1 lM) direct effects upon mitochondria functionality. Two rats of MTX48 died on day 35, and MTX treatment caused a reduction in relative body weight gain in both treated groups with no significant changes in water and food intake. Decreased levels of plasma total creatine kinase and CK-MB were detected in the MTX22 group, and increased plasma levels of lactate were seen in MTX48. Increased cardiac relative mass and microscopic changes were evident in both treated groups. Considering mitochondrial effects, for the first time, it was evidenced that MTX induced an increase in the complex IV and complex V activities in MTX22 group, while a decrease in the complex V activity was accompanied by the reduction in ATP content in the MTX48 rats. No alterations in mitochondria transmembrane potential were found in isolated mitochondria from MTX48 rats or in isolated mitochondria directly incubated with MTX. This study highlights the relevance of the cumulative MTX-induced in vivo mitochondriopathy to the MTX cardiotoxicity.This work was supported by the Fundação para a Ciência e Tecnologia (FCT)—project (EXPL/DTP-FTO/0290/ 2012)—QREN initiative with EU/FEDER financing through COMPETE— Operational Programme for Competitiveness Factors. LGR, VMC, and RJD-O thank FCT for their PhD Grant (SFRH/BD/63473/ 2009) and Post-doc Grants (SFRH/BPD/63746/2009) and (SFRH/ BPD/36865/2007), respectively. The authors are grateful to Fundação para a Ciência e Tecnologia for grant no. Pest C/EQB/LA0006/2011

Assessment of the application for renewal of the authorisation of PHYZYME® XP 10000 TPT/L (6-phytase) as a feed additive for all avian species and all swine species

PHYZYME\uae XP 10000 TPT/L is a feed additive that contains 6-phytase produced by a genetically modified strain of Schizosaccharomyces pombe. The applicant requested for the renewal of the authorisation for PHYZYME\uae XP 10000 TPT and L to be used as a feed additive for avian species for fattening/laying, weaned piglets, pigs for fattening and sows and for an extension of use to avian species reared for laying/breeding, suckling piglets and minor porcine species. To support the request or the renewal of the authorisation, the applicant provided evidence that the additive in the market complies with the conditions of the authorisation. According to the information provided by the applicant, no new evidence has been identified that would make the FEEDAP Panel reconsider the previous conclusions regarding the safety for the target species, consumer, user and environment. The application for renewal of the authorisation did not include a proposal for amending the conditions of use in those species for which an authorisation exists that would have an impact on the efficacy of the additive. Therefore, there was no need for assessing the efficacy of the additive in the context of the renewal of the authorisation. Regarding the new species/categories, the Panel concluded that the additive is safe and has a potential to be efficacious in avian species reared for laying/breeding at 250 FTU/kg feed and for suckling piglets and minor porcine species at 500 FTU/kg feed

Safety and efficacy of APSA PHYTAFEED® 20,000 GR/L (6-phytase) as a feed additive for chickens for fattening, chickens reared for laying and minor growing poultry species

APSA PHYTAFEED\uae 20,000 GR/L is a preparation of 6-phytase which is presented in solid and liquid forms. This additive is intended to be used as a zootechnical additive in chickens for fattening or reared for laying/breeding and minor poultry species for fattening or reared for laying/breeding. The 6-phytase present in the additive is produced by a genetically modified strain of Komagataella phaffii. The production strain and its recombinant DNA were not detected in intermediate products used to produce the additive. The final products do not trigger a safety concern with regard to the genetic modification. Based on the results obtained in a tolerance study in chickens for fattening and the data from a subchronic oral toxicity study the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive is safe for chickens for fattening. This conclusion was extended to chickens reared for laying/breeding and extrapolated to all minor poultry species for fattening or reared for laying/breeding. The FEEDAP Panel concluded that the use of APSA PHYTAFEED\uae 20,000 GR/L as a feed additive gives rise to no concern for consumers. The additive, in either form, is not toxic by inhalation or irritant for skin or eyes and it is not a dermal sensitizer, but it is considered a potential respiratory sensitizer. The use of the product as a feed additive is of no concern for the environment. The FEEDAP Panel evaluated three efficacy trials in which the retention of the phosphorus was studied. The data showed that the additive has the potential to improve the retention of phosphorus in the diets in chickens for fattening at 250 U/kg feed. This conclusion was extended to chickens reared for laying/breeding and extrapolated to all minor poultry species for fattening or reared for laying/breeding

Safety and efficacy of a natural mixture of dolomite plus magnesite and magnesium-phyllosilicates (Fluidol) as feed additive for all animal species

The additive, a natural mixture of dolomite plus magnesite and magnesium-phyllosilicates, is intended to be used as a technological additive (functional groups: anticaking agents) in feedingstuffs for all animal species. The additive is safe in complete feed for dairy cows, piglets and pigs for fattening at a maximum concentration of 20,000 mg/kg. No conclusions can be drawn for all the other animal species/categories. The additive is not genotoxic. As the additive is essentially not absorbed from the gut lumen, the Panel on Additives and Products or Substances used in Animal Feed considers that use of the additive in animal nutrition is safe for consumers of food products from animals fed diets containing the additive. The additive is not an irritant to the eyes and the skin, it is not a skin sensitiser and it is of low toxicity by the inhalation route. The components of the additive (dolomite, magnesite, talc and chlorite) are natural constituents of soil. Consequently, the use of the additive in animal nutrition will not pose a risk to the environment. The additive is effective as an anticaking agent at a minimum inclusion level of 5,000 mg/kg feed