Search

Spirotetramat, an insecticide derived from cycloketone and extensively utilized in agricultural production, has been reported to be toxic to an array of aquatic organisms. Previous studies have indicated that spirotetramat can cause toxicity such as impaired ovarian development and apoptosis in zebrafish, but its toxicological effects on lipid metabolism and liver health in zebrafish remain unclear. In this study, we explored the effects of spirotetramat exposure on zebrafish (Danio rerio) by examining key markers of lipid metabolism, alterations in gene expression related to this process, and histological characteristics of the liver. Spirotetramat significantly reduced the condition factor, triglycerides and low-density lipoprotein cholesterol levels at 2 mg/L. The expression of genes related to fatty acid synthesis (acacb), β-oxidation (acox1, pparda) and pro-inflammatory cytokines (tnf-α, il-1β) was downregulated. However, the expression of genes related to lipid transport and uptake (cd36, ppara) and output (apob) was upregulated. The activity of alanine aminotransferase was significantly inhibited. Histopathology results showed that spirotetramat exposure led to liver cell vacuolation and necrosis. In addition, molecular docking results of spirotetramat and lipid transport related protein (ACC, ApoB) in both zebrafish and human showed the binding energy of human proteins is lower than that for zebrafish, and that the number of hydrogen bonds formed was higher. It is speculated that spirotetramat may also pose a significant potential hazard to humans, potentially affecting human lipid metabolism and health. This study expunge shed light on the ecological toxicity of spirotetramat by showing how it disrupts lipid metabolism and causes tissue damage specifically in zebrafish liver, contributing to a deeper understanding of its harmful effects in aquatic environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The excessive and frequent use of insecticides has led to serious problems with insecticide residues, impacting nontarget organisms such as the parasitoid Encarsia formosa. This study examined the growth, development, and enzyme activity of E. formosa exposed to spirotetramat at LC 10 , LC 30 , and LC 50 . The regression equation for the toxicity of spirotetramat toward E. formosa was Y = 5.25X-11.07. After exposure to spirotetramat, the survival rates of E. formosa sharply decreased, which occurred earlier than those in the control batch. Although the maximum daily parasitism quantity of E. formosa increased and the average parasitism number, enumerated from the 1st to the 5th day, was 53.97 after being exposed to spirotetramat at LC 10 , the life span of its F 1 generation adults was only 8.47 days, which was significantly shorter than that in the control batch. After being exposed to spirotetramat at LC 50 , the average parasitism number of E. formosa was 63.30, and the developmental time of its F 1 generation, enumerated from the 1st to the 5th day after exposure to spirotetramat, was significantly longer than that of the control batch. The activities of mixed function oxidase, acetylcholinesterase, carboxylesterase, and catalase increased significantly, and the rate of increase in enzyme activity was directly proportional to the increase in the concentration of spirotetramat. These results revealed that the parasitic ability of E. formosa decreased after exposure to spirotetramat at LC 10 , LC 30 , and LC 50 . This leads to a change in parasitoid control of pests, revealing the potential environmental threat of insecticide residues to nontarget organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the sensitivity of the prawn Palaemon argentinus to the pyrethroid cypermethrin (CYP) and the tetramic acid spirotetramat (STM). These treatments were compared with prawns collected at a reference site to define their basal physiological state. Initially, physicochemical parameters and several pollutants at the selected site were analyzed. The LC 50 -96 h was determined in adult prawns. Then, prawns were exposed for 96 h to sublethal concentrations of CYP (0.0005 μg/l) and STM (0.44 mg/l) to evaluate the effects on some biochemical endpoints. A treatment combining both pesticides was also added at 5 % of these values. Controls with and without solvent (acetone) were included. The LC 50 -96 h values were 0.005 μg/l and 4.43 mg/l for CYP and STM, respectively. Moreover, some biomarkers linked to oxidative and energy metabolism were analyzed in the hepatopancreas and muscle of both essayed prawns and those at the basal state. The STM caused a significant decrease in total protein content (32 %) in contrast to the increase of protein carbonyl content (71 %) (p < 0.05). Also, glutathione S-transferase (52 %) and catalase (61 %) activities in the hepatopancreas of exposed prawns were higher compared to both the control and state basal groups (p < 0.05). In muscle, only a significant decrease in the lactate content (69 %) was caused by STM (p < 0.05). In addition, CYP caused a significant increase in the lactate dehydrogenase activity (110 %) in muscle and triacylglycerol content (73 %) in the hepatopancreas (p < 0.05). The integrated biomarker index (IBRv2) analysis showed that STM caused greater damage than CYP. Besides, the combined treatment showed an antagonistic interaction between both insecticides. The differential response of biomarkers to both CYP and STM exposure with respect to their basal levels shows a high sensitivity of P. argentinus demonstrating its potential role as a bioindicator organism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Azaspiracids (AZAs) are lipid biotoxins produced by the marine dinoflagellates Azadinium and Amphidoma spp. that can accumulate in shellfish and cause food poisoning in humans. However, the mechanisms underlying the tolerance of shellfish to high levels of such toxins remain poorly understood. This study investigated the combined effects of detoxification metabolism and stress-related responses in scallops Chlamys farreri exposed to AZA. Scallops accumulated a maximum of 361.81 μg AZA1 eq/kg and 41.6 % AZA residue remained after 21 days of exposure. A range of AZA2 metabolites, including AZA19, AZA11, and AZA23, and trace levels of AZA2-GST, were detected. Total hemocyte counts significantly increased and ROS levels remained consistently high until gradually decreasing. Immune system activation mediated mitochondrial dysfunction and severe energy deficiency. DEGs increased over time, with key genes CYP2J6 and GPX6 contributing to AZA metabolism. These transcriptome and metabolic results identify the regulation of energy metabolism pathways, including inhibition of the TCA cycle and activation of carbohydrates, amino acids, and lipids. AZA also induced autophagy through the MAPK-AMPK signaling pathways, and primary inhibited PI3K/AKT to decrease mTOR pathway expression. Our results provide additional insights into the resistance of C. farreri to AZA, characterized by re-establishing redox homeostasis toward a more oxidative state., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Compatibility studies of insecticides and natural enemies usually focus on short-term lethal effects, without considering the long-term sublethal effects (including progeny). Even less-explored are the effects of commercial insecticides formulated with more than one active product. Short- and long-term lethal and sublethal effects were studied for the first time on the progeny of commercial formulations of spirotetramat, imidacloprid and a commercial mixture of these active ingredients on pupae of Diaeretiella rapae (M'ntosh) (Hymenoptera: Braconidae), an endoparasitoid of aphids considered to be a potential biological control agent. Insecticides were exposed topically on aphid mummies in which the parasitoid was in the pupal stage., Results: Imidacloprid reduced adult emergence by more than 30% and prolonged intra-host development time with respect to control from half the maximum recommended field dose (MFRD). Spirotetramat and commercial mixture only showed significant effects on these endpoints at doses above the MFRD. The tested formulations did not affect adult longevity, sex ratio, and percentage of parasitism in the exposed generation. At low concentrations the active ingredients in the commercial mixture behave synergistically, whereas at medium and high concentrations they behave antagonistically. Considering the 10% lethal dose (LD 10 ), imidacloprid showed the highest hazard coefficient, whereas the commercial mixture was more hazardous when considering the LD 50 and LD 90 . The commercial mixture and imidacloprid induced higher adult emergence and altered the sex ratio in the progeny., Conclusions: The following order of toxicity on D. rapae can be established: imidacloprid > commercial mixture > spirotetramat. Joint use of this species with imidacloprid and commercial mixture should be avoided in integrated pest management programs. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

When humans consume seafood contaminated by lipophilic polyether phycotoxins, such as azaspiracids (AZAs), the toxins are mainly leached and absorbed in the small intestine, potentially causing intestinal damage. In this study, human intestinal epithelial Caco-2 cells were used to investigate the adverse effects of azaspiracid-2 (AZA-2) on human intestinal epithelial cells. Cell viability, apoptosis, oxidative damage and mitochondrial ultrastructure were investigated, and ribonucleic acid sequence (RNA-seq) analysis was applied to explore the potential mechanisms of AZA-2 toxicity to Caco-2 cells. Results showed that AZA-2 significantly reduced the proliferation of Caco-2 cells in a concentration-dependent response, and the 48 h EC 50 of AZA-2 was 12.65 nmol L -1 . AZA-2 can induce apoptosis in Caco-2 cells in a dose-dependent manner. Visible mitochondrial swelling, cristae disintegration, membrane rupture and autophagy were observed in Caco-2 cells exposed to AZA-2. Reactive oxygen species (ROS) and malondialdehyde (MDA) content were significantly increased in Caco-2 cells after 48 h of exposure to 1 and 10 nmol L -1 of AZA-2. Transcriptome analysis showed that KEGG pathways related to cellular oxidative damage and lipid metabolism were affected, mainly including mitophagy, oxidative phosphorylation, cholesterol metabolism, vitamin digestion and absorption, bile secretion and the peroxisome proliferator-activated receptor signaling pathway. The cytotoxic effects of AZA-2 on Caco-2 cells may be associated with ROS-mediated autophagy and apoptosis in mitochondrial cells. Results of this study improve understanding of the cytotoxicity and molecular mechanisms of AZA-2 on Caco-2 cells, which is significant for protecting human health.

The presence and impact of toxins have been detected in various regions worldwide ever since the discovery of azaspiracids (AZAs) in 1995. These toxins have had detrimental effects on marine resource utilization, marine environmental protection, and fishery production. Over the course of more than two decades of research and development, scientists from all over the world have conducted comprehensive studies on the in vivo metabolism, in vitro synthesis methods, pathogenic mechanisms, and toxicology of these toxins. This paper aims to provide a systematic introduction to the discovery, distribution, pathogenic mechanism, in vivo biosynthesis, and in vitro artificial synthesis of AZA toxins. Additionally, it will summarize various detection methods employed over the past 20 years, along with their advantages and disadvantages. This effort will contribute to the future development of rapid detection technologies and the invention of detection devices for AZAs in marine environmental samples.

The filter-feeding bivalves often accumulate marine toxins by feeding on toxic dinoflagellates that produce marine toxins. Azaspiracids (AZAs) are a group of lipophilic polyether toxins which have been detected in a variety of organisms in many countries. In our present study, accumulation kinetics and toxin distributions in the tissues of seven bivalve species and ascidians relevant to Japanese coastal waters were investigated by experimentally feeding a toxic dinoflagellate Azadinium poporum, which produces azaspiracid-2 (AZA2) as the dominant toxin component. All bivalve species and ascidians investigated in this study had the capability to accumulate AZA2 and no metabolites of AZA2 were detected in the bivalves and the ascidians. Japanese short-neck clams, Japanese oysters, Pacific oysters and ascidians accumulated AZA2 with the highest concentrations on the hepatopancreas, whereas the highest concentrations of AZA2 were found on the gills in surf clams and horse clams. Hard clams and cockles accumulated high levels of AZA2 in both the hepatopancreas and the gills. As far as we know, this is the first report describing detailed tissue distribution of AZAs in several bivalve species other than mussels (M. edulis) and scallops (P. maximus). Variation of accumulation rates of AZA2 in Japanese short-neck clams on different cell densities or temperatures were observed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A series of exiguamine A analogues were designed and synthesized via 15 steps. Their inhibitory activities against IDO1 were tested and the structure-activity relationships were studied. Most compounds exhibited potent IDO1 inhibitory activities with IC 50 values at the level of 10 -7 -10 -8  M. Compound 21f was the most potent IDO1 inhibitor with an IC 50 value of 65.3 nM, which was comparable with the positive control drug epacadostat (IC 50  = 46 nM). Moreover, compound 21f showed higher selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO) and no cytotoxicity at its effective concentration, rending it justifiable for further optimization and evaluation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Frankliniella occidentalis (Pergande) has become an important vegetable pest worldwide because of its economic damage to crop production. However, it is difficult to control due to its unique living habits. In this study, the eggs of F. occidentalis were used as the target to explore the ovicidal activity of spirotetramat on the thrips and its effect on hatching, development and formation. After the treatment of spirotetramat, the LC 50 value descreased with increased egg age using egg dipping method, and showed the same trend as the leaf dipping method verified on living plants. Through ultra-depth-of-field microscopy, scanning electron microscopy and transmission electron microscopy, the egg shell and internal structures of F. occidentalis eggs were studied. Spirotetramat can destroy the egg shells of F. occidentalis, resulting in shrinkage of the egg surface, sunken pores, egg deformities, egg shell rupture and other phenomena. This allows spirotetramat to enter the egg and destroy the egg structure, making the egg internal structure flocculent, fuzzy and unevenly distributed, which affects embryonic development and causes the nymphs to die before hatching. Therefore, the prevention and control of F. occidentalis using spirotetramat before damage is caused to crops should have a better effect., (© 2021. The Author(s).)

Leishmaniasis is an infectious disease with several limitations regarding treatment schemes. This work reports the anti-Leishmania activity of spiroacridine compounds against the promastigote (IC 50  = 1.1 to 6.0 µg / mL) and amastigote forms of the best compounds (EC 50  = 4.9 and 0.9 µg / mL) inLeishmania (L.) infantumand proposes an in-silico study with possible selective therapeutic targets for L. infantum. The substituted dimethyl-amine compound (AMTAC 11) showed the best leishmanicidal activity in vitro, and was found to interact with TryRandLdTopoI. comparisons with standard inhibitors were performed, and its main interactions were elucidated. Based on the biological assessment and the structure-activity relationship study, the spiroacridine compounds appear to be promisinganti-leishmaniachemotherapeutic agents to be explored., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Portimine, a recently identified cyclic imine produced by the dinoflagellate Vulcanodinium rugosum, has been described as a potent apoptotic agent in contrast to most of the cyclic imines that are well-known to be neurological toxins. As apoptosis can be a consequence of a high level of DNA lesions, we investigated the responses of portimine on several endpoints aimed at detecting DNA damage in the hepatic cell line HepaRG. Portimine induced phosphorylation of H2AX, which could possibly be consistent with the previously published induction of apoptosis with this toxin. In addition, detection of apoptosis through the activation of caspase-3, the induction of strand breaks detected by the comet assay as well as chromosome and genome mutations using the micronucleus assay were addressed. Surprisingly, portimine treatment resulted in increases in only γH2AX in differentiated HepaRG cells whereas no effects on the other endpoints were detected. These increases in γH2AX in the absence of genotoxic effects in the other tests could indicate that portimine could possibly induce a DNA replication stress and/or that the compound can be detoxified by the HepaRG cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Oberon® is a commercial formulation of spiromesifen, a pesticide inhibitor of lipid biosynthesis via acetyl CoA carboxylase, widely used in agricultural crop protection. However, its mode of action requires further analysis. We currently examined the effect of this product on Drosophila melanogaster as a non-target and model organism. Different concentrations of spiromesifen were administered by ingestion (and contact) during pre-imaginal development, and we evaluated its delayed action on adults. Our results suggest that spiromesifen induced insecticidal activity on D. melanogaster. Moreover, spiromesifen treatment significantly increased the duration of larval and pupal development at all tested concentrations while it shortened longevity in exposed males as compared to control males. Also, pre-imaginal exposure to spiromesifen quantitatively affected fatty acids supporting its primary mode of action on lipid synthesis. In addition, this product was found to modify cuticular hydrocarbon profiles in exposed female and male flies as well as their sexual behavior and reproductive capacity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Seafood represents a significant part of the human staple diet. In the recent years, the identification of emerging lipophilic marine toxins has increased, leading to the potential for consumers to be intoxicated by these toxins. In the present work, we investigate the presence of lipophilic marine toxins (both regulated and emerging) in commercial seafood products from non-European locations, including mussels Mytilus chilensis from Chile, clams Tawerea gayi and Metetrix lyrate from the Southeast Pacific and Vietnam, and food supplements based on mussels formulations of Perna canaliculus from New Zealand. All these products were purchased from European Union markets and they were analyzed by UPLC-MS/MS. Results showed the presence of the emerging pinnatoxin-G in mussels Mytilus chilensis at levels up to 5.2 µg/kg and azaspiracid-2 and pectenotoxin-2 in clams Tawera gayi up to 4.33 µg/kg and 10.88 µg/kg, respectively. This study confirms the presence of pinnatoxins in Chile, one of the major mussel producers worldwide. Chromatograms showed the presence of 13-desmethyl spirolide C in dietary supplements in the range of 33.2-97.9 µg/kg after an extraction with water and methanol from 0.39 g of the green lipped mussels powder. As far as we know, this constitutes the first time that an emerging cyclic imine toxin in dietary supplements is reported. Identifying new matrix, locations, and understanding emerging toxin distribution area are important for preventing the risks of spreading and contamination linked to these compounds.

Structure-based drug design enabled the discovery of 8 , HTL22562, a calcitonin gene-related peptide (CGRP) receptor antagonist. The structure of 8 complexed with the CGRP receptor was determined at a 1.6 Å resolution. Compound 8 is a highly potent, selective, metabolically stable, and soluble compound suitable for a range of administration routes that have the potential to provide rapid systemic exposures with resultant high levels of receptor coverage (e.g., subcutaneous). The low lipophilicity coupled with a low anticipated clinically efficacious plasma exposure for migraine also suggests a reduced potential for hepatotoxicity. These properties have led to 8 being selected as a clinical candidate for acute treatment of migraine.

Semisynthetic artemisinins and other bioactive peroxides are best known for their powerful antimalarial activities, and they also show substantial activity against schistosomes-another hemoglobin-degrading pathogen. Building on this discovery, we now describe the initial structure-activity relationship (SAR) of antischistosomal ozonide carboxylic acids OZ418 ( 2 ) and OZ165 ( 3 ). Irrespective of lipophilicity, these ozonide weak acids have relatively low aqueous solubilities and high protein binding values. Ozonides with para -substituted carboxymethoxy and N -benzylglycine substituents had high antischistosomal efficacies. It was possible to increase solubility, decrease protein binding, and maintain the high antischistosomal activity in mice infected with juvenile and adult Schistosoma mansoni by incorporating a weak base functional group in these compounds. In some cases, adding polar functional groups and heteroatoms to the spiroadamantane substructure increased the solubility and metabolic stability, but in all cases decreased the antischistosomal activity.

A new prenylated indole alkaloid, named paraherquamide J ( 1 ), together with four known compounds ( 2 - 5 ), were isolated from the mangrove rhizosphere soil-derived fungus Penicillium janthinellum HK1-6. The planar structure and relative configuration of 1 were determined by detailed analysis of the spectroscopic data especially the NOESY spectrum. The absolute configuration of 1 was determined by ECD spectra. Compound 2 was first isolated as a natural product and named as paraherquamide K. All isolated metabolites were evaluated for their antibacterial, topoisomerase I (topo I) inhibitory activities and lethality towards brine shrimp Artemia salina .

Spirotetramat (SPT) is a new tetronic acid derivative insecticide used to control scales and aphids; the potential for endocrine disruptor effects in fish could not be finalized with the available data. In this study, zebrafish were selected to assess the endocrine-disrupting effects. Significant decrease of plasma estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT) were observed in both male and female following the spirotetramat exposure; the vitellogenin (VTG) level in females significantly decreased. The expression of the hypothalamic-pituitary-gonad (HPG) axis genes fshr, lhr and esr1 showed significant increase in the gonads, which expression in males is higher than in females. In addition, the activities of capspase-3 and caspase-9 significantly decreased in both males and females liver, while the capspase-3 and caspase-9 were increased in male testis, the mRNA expression levels of genes expression related to the apoptosis pathway were also significantly altered after the spirotetramat exposure. Additionally, we found the parental zebrafish exposed to spirotetramat induced the development delay of its offspring. Above all, the adverse effects induced by spirotetramat suggesting that spirotetramat is a potential exogenous hazardous agent., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Spiromesifen (Oberon® 240 SC), a pesticide widely used to control pests like mites and whiteflies, was investigated using Drosophila melanogaster Meigen, 1830 (Diptera, Drosophilidae) as a model organism. The compound was applied topically at two concentrations (LC 10 : 21.45 and LC 25 : 39.53 μg active ingredient/pupa), on newly molted pupae and assessed on morphometric measurements of ovaries and the progeny of surviving adults. Results showed that spiromesifen inhibited the growth and development of ovaries, reducing at the highest dose (LC 25 ) the number of oocytes, the volume of basal oocytes and ovarian weight. Biochemical analysis revealed that the tested compound reduced the ovarian levels of carbohydrates and glycogen during the sexual maturation. Moreover, fecundity, fertility and number of descendants from parents that survived to the treatment of pupae were significantly reduced. The sex ratio determined indicated a significant decrease in treated series and males seemed more sensitive to spiromesifen than females. Lastly, the compound was found to affect the sexual behavior., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pinnatoxin G (PnTx-G) is a marine cyclic imine toxin produced by the dinoflagellate Vulcanodinium rugosum , frequently detected in edible shellfish from Ingril Lagoon (France). As other pinnatoxins, to date, no human poisonings ascribed to consumption of PnTx-G contaminated seafood have been reported, despite its potent antagonism at nicotinic acetylcholine receptors and its high and fast-acting toxicity after intraperitoneal or oral administration in mice. The hazard characterization of PnTx-G by oral exposure is limited to a single acute toxicity study recording lethality and clinical signs in non-fasted mice treated by gavage or through voluntary food ingestion, which showed differences in PnTx-G toxic potency. Thus, an acute toxicity study was carried out using 3 h-fasted CD-1 female mice, administered by gavage with PnTx-G (8-450 µg kg - 1 ). At the dose of 220 µg kg - 1 and above, the toxin induced a rapid onset of clinical signs (piloerection, prostration, hypothermia, abdominal breathing, paralysis of the hind limbs, and cyanosis), leading to the death of mice within 30 min. Except for moderate mucosal degeneration in the small intestine recorded at doses of 300 µg kg - 1 , the toxin did not induce significant morphological changes in the other main organs and tissues, or alterations in blood chemistry parameters. This acute oral toxicity study allowed to calculate an oral LD 50 for PnTx-G equal to 208 g kg - 1 (95% confidence limits: 155-281 µg kg - 1 ) and to estimate a provisional NOEL of 120 µg kg - 1 ., Competing Interests: The authors declare no conflict of interest.

This work demonstrates a novel chemosensor, SPTPA, that exhibits fluorescence and colorimetric dual sensing of hypochlorite with an ICT ON strategy. Additionally, the chemosensor successfully detected and imaged endogenous hypochlorite in living cells, various types of bacteria, and zebrafish.

The antinociceptive effects of spirocyclopiperazinium salt compound DXL-A-24 on neuropathic pain and chemical-stimulated pain were investigated in this study. After the administration of DXL-A-24, the paw withdrawal latency (PWL) and mechanical withdrawal threshold (MWT) were increased in rats suffering from neuropathic pain (chronic constriction injury, CCI) on days 1, 3, 5, 7 and 14 after surgery, and pain responses were inhibited in mice stimulated with chemicals (formalin or acetic acid). In the analysis of antinociceptive targets, the effect of DXL-A-24 was blocked by a peripheral nicotinic acetylcholine receptor (nAChR) antagonist (hexamethonium, Hex) or α7 nAChR antagonist (methyllycaconitine, MLA) in the formalin test. Meanwhile, the effect of DXL-A-24 was also blocked by a peripheral muscarinic acetylcholine receptor (mAChR) antagonist (atropine methylnitrate, Amn) or M4 mAChR antagonist (tropicamide, TRO). The antinociceptive signalling pathway was explored using molecular biology methods in ipsilateral dorsal root ganglions (DRGs) of CCI rats after the administration of DXL-A-24 for 7 days. Western blot analyses showed that the increased levels of phosphorylation of calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) and cAMP response element-binding protein (CREB) were eliminated, and the qRT-PCR assay showed that the increase in the expression of Tumor necrosis factor alpha (TNF-α) mRNA was reduced. Meanwhile, immunofluorescence staining revealed that the increase in calcitonin gene related peptide (CGRP) expression was inhibited by the administration of DXL-A-24, and the effect was blocked by MLA or TRO. In conclusion, DXL-A-24 exerts significant antinociceptive effects on neuropathic pain and chemical-stimulated pain. The antinociceptive effect of DXL-A-24 is probably attributed to the activation of peripheral α7 nAChR and M4 mAChR, the subsequent inhibition of the CaMKIIα/CREB signalling pathway, and finally the inhibition of TNF-α and CGRP expression.

Spirotetramat is a toxic commercially known as Movento used to control pistachio psylla pests. In the present study, the effects of Movento on passive avoidance learning of rats and their ability to explore the novel object in the novel object recognition test were investigated. The changes in the concentration of hippocampal brain-derived neurotrophic factor (BDNF) proteins were evaluated, too. Male Wistar rats were gavaged at different dosages of the Movento (50, 100, 250, 500, 1000, 1250, and 1500 mg/kg) or saline for 7 days (administered every 2 days). We showed that Movento caused 50 and 100% mortality at the dose of 1250 and 1500 mg/kg, respectively. At the dose of 1000 mg/kg, Movento significantly decreased locomotor activity (P < 0.05). These rats also displayed a significant decrease in the number of training trials in the shuttle box and the ability to recognize a novel object compared with the control group (P < 0.01). The BDNF protein level of hippocampus also showed a significant decrease in the Movento (1000 mg/kg) compared with the control group (P < 0.01) while the number of pancellular necrosis pyramidal CA1 cells increased significantly in the Movento group (P < 0.001). We concluded that exposure to Movento can decline sensory, motor, and learning in rats.

Azaspiracids (AZAs) are marine biotoxins including a variety of analogues. Recently, novel AZAs produced by the Mediterranean dinoflagellate Azadinium dexteroporum were discovered (AZA-54, AZA-55, 3-epi-AZA-7, AZA-56, AZA-57 and AZA-58) and their biological effects have not been investigated yet. This study aimed to identify the biological responses (biomarkers) induced in mussels Mytilus galloprovincialis after the bioaccumulation of AZAs from A. dexteroporum . Organisms were fed with A. dexteroporum for 21 days and subsequently subjected to a recovery period (normal diet) of 21 days. Exposed organisms accumulated AZA-54, 3-epi-AZA-7 and AZA-55, predominantly in the digestive gland. Mussels' haemocytes showed inhibition of phagocytosis activity, modulation of the composition of haemocytic subpopulation and damage to lysosomal membranes; the digestive tissue displayed thinned tubule walls, consumption of storage lipids and accumulation of lipofuscin. Slight genotoxic damage was also observed. No clear occurrence of oxidative stress and alteration of nervous activity was detected in AZA-accumulating mussels. Most of the altered parameters returned to control levels after the recovery phase. The toxic effects detected in M. galloprovincialis demonstrate a clear biological impact of the AZAs produced by A. dexteroporum , and could be used as early indicators of contamination associated with the ingestion of seafood.

Invasive fungal infections are one of the leading causes of nosocomial bloodstream infections with a limited treatment option. A series of derivatized spirooxindolo-pyrrolidine tethered indole and imidazole heterocyclic hybrids have been synthesized, and their antifungal activity against fungal strains were determined. Here we characterize the antifungal activity of a specific spirooxindolo-pyrrolidine hybrid, dubbed compound 9c, a spirooxindolo-pyrrolidine tethered imidazole synthesized with a 2-chloro and trifluoromethoxy substituent. The compound 9c exhibited no cytotoxicity against mammalian cell line at concentrations that inhibited fungal strains. Compound 9c also significantly inhibited the fungal hyphae and biofilm formation. Our results indicate that spirooxindolo-pyrrolidine heterocyclic hybrids potentially represent a broad class of chemical agents with promising antifungal potential., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Azaspiracids (AZAs) are marine toxins that are produced by Azadinium and Amphidoma dinoflagellates that can contaminate edible shellfish inducing a foodborne poisoning in humans, which is characterized by gastrointestinal symptoms. Among these, AZA1, -2, and -3 are regulated in the European Union, being the most important in terms of occurrence and toxicity. In vivo studies in mice showed that, in addition to gastrointestinal effects, AZA1 induces liver alterations that are visible as a swollen organ, with the presence of hepatocellular fat droplets and vacuoles. Hence, an in vitro study was carried out to investigate the effects of AZA1, -2, and -3 on liver cells, using human non-tumor IHH hepatocytes., Results: The exposure of IHH cells to AZA1, -2, or -3 (5 × 10 -12 -1 × 10 -7 M) for 24 h did not affect the cell viability and proliferation (Sulforhodamine B assay and 3 H-Thymidine incorporation assay), but they induced a significant concentration-dependent increase of mitochondrial dehydrogenases activity (MTT reduction assay). This effect depends on the activity of mitochondrial electron transport chain complex I and II, being counteracted by rotenone and tenoyl trifluoroacetone, respectively. Furthermore, AZAs-increased mitochondrial dehydrogenase activity was almost totally suppressed in the K + -, Cl - -, and Na + -free media and sensitive to the specific inhibitors of K ATP and hERG potassium channels, Na + /K + , ATPase, and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels., Conclusions: These results suggest that AZA mitochondrial effects in hepatocytes derive from an imbalance of intracellular levels of K + and, in particular, Cl - ions, as demonstrated by the selective reduction of toxin effects by CFTR chloride channel inhibition.

Peniciketal A (Pe-A), a spiroketal compound, is isolated from the saline soil-derived fungus Penicillium raistrickii. However, the underlying molecular mechanistic basis for the effects of Pe-A on leukemia is poorly understood. Here, we investigated that Pe-A reduced cell proliferation in three leukemia cell lines (THP-1, K562 and HL60). Importantly, Pe-A showed little cytotoxicity in primary mouse embryonic fibroblast (MEF) cells in a long-duration treatment. For the mechanistic research, we identified 3449 differentially expressed Pe-A-induced proteins through liquid chromatography-tandem mass spectrometry (LC-MS/MS) with TMT label in THP-1 cells. Results showed that many identified proteins were involved in apoptosis and/or autophagy. Then, we confirmed that Pe-A induced not only apoptosis via the mitochondrial pathway but also cytoprotective autophagy by activating the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway indeed. In addition, Pe-A also arrested the cell cycle at the G0-G1 phase by regulating the expressions of checkpoint protein. Collectively, these results provide new insights into the mechanisms that Pe-A may target autophagy-related or apoptosis-related pathways to suppress the development of human leukemia., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Anticancer therapeutics with profiles of high potency, low toxicity, and low resistance is of considerable interest. A new series of functionalized spirooxindole linked with 3-acylindole scaffold is reported, starting from chalcones derived from 3-acetyl indole with isatin, and l-4-thiazolidinecarboxylic acid. The reactions proceeded regioselectivity, stereoselectivity, without side products in high yield (71-89%). The new spirooxindole hybrids have been evaluated in vitro for their antiproliferative effects against colon cancer (HCT-116), hepatocellular carcinoma (HepG2) and prostate cancer (PC-3). The selectivity of their activity was evaluated. Some of the synthesized compounds showed considerable anticancer activities. Compound 4k proved to retain a high cytotoxic activity and selectivity against colon cancer cells HCT-116 (IC 50  = 7 ± 0.27 µM, SI: 3.7), and HepG2 (IC 50  = 5.5 ± 0.2 µM, SI: 4.7) in comparison to (IC 50  = 12.6 ± 0.5, SI: 0.4 and 5.5 ± 0.3 µM, SI: 0.9, respectively). Compound 4k was less active (IC 50  = 6 ± 0.3 µM, SI: 4.3) than cisplatin (IC 50  = 5 ± 0.56 µM, SI: 1.0) but showed greater selectivity towards prostate cancer cells PC-3 in comparison to cisplatin. The details of the binding mode of the active compounds were clarified by molecular docking. Ligand Efficiency (LE) and Ligand Lipophilic Efficiency (LLE) were evaluated and revealed that compound 4k had acceptable value., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The purpose of this work is to study fenspiride for the purposes and tasks of chemical and toxicological research. The technique of chemical-toxicological study of fenspiride in urine by preliminary and confirming methods is proposed. The authors proposed a method of thin - layer chromatography in the solvent system of acetone - 25% ammonia solution (9:1) in a preliminary study of fenspiride. The Rf value of fenspiride is 0.80±0.02. The degree of extraction when used as an extractant mixture of chloroform-propanol (9:1) is 28.1±1.4% The conditions for identification of fenspiride by gas chromatography-mass spectrometry (fenspiride retention time 11.1±0.1 minutes), followed by quantitative determination of the analyte, are proposed as a confirming method. The conditions for qualitative (retention time of fenspiride 8.1±0.1 minutes) and quantitative determination of fenspiride by high-performance liquid chromatography are proposed.

As a new tetronic acid derivative insecticide, spirotetramat has been reported to be toxic to an array of aquatic organisms. However, the toxic effects of spirotetramat on zebrafish especially at ovary are still obscure. Hereby, the acute toxicity of spirotetramat towards zebrafish（Danio rerio），as well as the changes on biochemical and histological traits of ovary were investigated. The acute toxicity test results showed that the median lethal concentration (LC 50 ) value of spirotetramat were 9.61 mg/L and 7.21 mg/L at 72 h and 96 h, respectively, suggesting spirotetramat has moderate toxicity to zebrafish. In the following sub-lethal toxicity test, the gene expression of superoxide dismutase (SOD), catalase (CAT), and gonadotropic hormone receptor (FSHR and LHR) together with the content of malondialdehyde (MDA) in ovary were measured at 14, 21, and 28 days after exposure to 36, 360 and 720 μg/L. Under high concentration treatment (360 and 720 μg/L), MDA content, the relative transcription CAT and SOD gene level increased significantly in ovary (p < 0.05). That indicated sub-lethal doses spirotetramat caused oxidative stress and lipid peroxidation in zebrafish ovary during the entire experimental period. Under the exposure to spirotetramat at 720 μg/L after 14 days, the relative transcript FSHR gene level was down regulated, and the relative transcript LHR gene level was up regulated. Moreover, spirotetramat affected the oocyte development especially on the diameter size and maturation during the ovary tissue biopsies at 28 days. Taken together, these findings revealed the adverse effects of spirotetramat on fish from the biochemical and histological aspects., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Palmarumycin B 6 and its regioisomer were synthesized via 7- and 13-step routes using 2-chlorophenol and 4-chlorophenyl methyl ether as the starting materials in overall yields of 2.7% and 12%, respectively. Their structures were characterized by 1 H and 13 C NMR, HRESIMS, and X-ray diffraction data. The structure of palmarumycin B 6 was revised as 6-chloropalmarumycin CP 17 . The bioassay results showed that the larvicidal activity of palmarumycin B 6 with an LC 50 value of 32.7 μM was significantly higher than that of its 8-chloro isomer, with an LC 50 value of 227.3 μM.

The cytochrome P450 monooxygenases play a key role in detoxification mechanism for spirotetramat resistance in Aphis gossypii Glover. However, only one P450 genes (CYP6DA2), among thirty-five P450 genes identified from Aphis gossypii transcriptome database, has been reported to play important role in spirotetramat resistance in previous resistance level until now. In this study, after the confirmation of the rise of resistance level and important roles of P450s in spirotetramat resistance by the synergism analysis, the gene expression changes were determined for P450 genes in spirotetramat susceptible and resistant strains. Compared with the susceptible strain, CYP6CY4, CYP6CY14, CYP6CY18 and CYP6DC1 in CYP3 Clade were up-regulated in resistant nymphs, with the CYP6CY14, CYP6CY4, CYP6DC1, and CYP6CY18 increased to 2.54-, 1.51-, 1.31- and 1.29-fold, respectively. Eight genes in CYP3 Clade, three genes in CYP4 Clade and one gene in Mito Clade were down-regulated. In resistant adult aphids, CYP380C6 in CYP4 Clade, CYP353B1 in CYP2 Clade, and CYP307A1 in Mito Clade were up-regulated under spirotetramat stress, with the CYP380C6, CYP353B1 and CYP307A1 increased to 2.89-, 1.91-, and 1.38-fold, respectively. In contrast, the other P450 genes were almost down-regulated, especially these P450 genes in CYP3 Clade, CYP4 Clade and Mito Clade. RNA interference of CYP380C6 significantly increased the sensitivity of the resistant adults and nymphs to spirotetramat, while suppression of CYP6CY14 could not increase the toxicity of spirotetramat. These results indicate the possible involvement of the CYP380C6 genes in spirotetramat resistance at present very high resistance levels. Screening the expression changes of P450 genes under different spirotetramat resistance levels in the genome-scale will provide an overall view on the possible metabolic factors in the resistance development. The results may facilitate further work to validate the roles of P450 in spirotetramat resistance with heterologous expression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

With the purpose of guaranteeing the safe use of spirotetramat and preventing its potential health threats to consumers, a QuEChERS extraction method coupled with LC triple-quadrupole tandem MS was applied in this study to determine residual spirotetramat metabolites in different tissues of amaranth (Amaranthus tricolor) and in soil. The results indicate that the spirotetramat degraded into different types of metabolites that were located in different tissues of amaranth and in soil. B-keto, B-glu, and B-enol were the three most representative degradation products in the leaf of amaranth, and B-glu and B-enol were the two major degradation products found in the stem of amaranth; however, only B-enol was detected in the root of amaranth. B-keto and B-mono were the two products detected in the soil in which the amaranth grew. The cytotoxicity results demonstrate that spirotetramat and its metabolite B-enol inhibited cellular growth, and the toxicity of spirotetramat and its metabolite B-enol exceeded than that of the metabolites B-keto, B-mono, and B-glu. This investigation is of great significance to the safe use of spirotetramat in agriculture.

Azaspiracids (AZAs) are marine biotoxins produced by the genera Azadinium and Amphidoma, pelagic marine dinoflagellates that may accumulate in shellfish resulting in human illness following consumption. The complexity of these toxins has been well documented, with more than 40 structural variants reported that are produced by dinoflagellates, result from metabolism in shellfish, or are extraction artifacts. Approximately 34 μg of a new AZA with MW 823 Da (AZA26 (3)) was isolated from blue mussels ( Mytilus edulis), and its structure determined by MS and NMR spectroscopy. AZA26, possibly a bioconversion product of AZA5, lacked the C-20-C-21 diol present in all AZAs reported thus far and had a 21,22-olefin and a keto group at C-23. Toxicological assessment of 3 using an in vitro model system based on Jurkat T lymphocyte cells showed the potency to be ∼30-fold lower than that of AZA1. The corresponding 21,22-dehydro-23-oxo-analogue of AZA10 (AZA28) and 21,22-dehydro analogues of AZA3, -4, -5, -6, -9, and -10 (AZA25, -48 (4), -60, -27, -49, and -61, respectively) were also identified by HRMS/MS, periodate cleavage reactivity, conversion from known analogues, and NMR (for 4 that was present in a partially purified sample of AZA7).

A crude methanolic extract of the Indonesian sponge Clathria bulbotoxa showed a potent cytotoxic activity against the human epidermoid carcinoma A431 cells. An investigation of the active components led to the isolation of three new compounds named crambescidins 345 ( 1 ), 361 ( 2 ), and 373 ( 3 ), together with the known related metabolites crambescidins 359 ( 4 ), 657 ( 5 ), and 800 ( 6 ). The structures of the compounds were determined by spectroscopic analysis. These compounds 1 - 4 that possess a simple pentacyclic guanidine core exhibited moderate cytotoxicity against the A431 cells with the IC 50 values of 7.0, 2.5, 0.94, and 3.1 μM, respectively, while the known compounds 5 and 6 that possess a long aliphatic side chain were found to be significantly cytotoxic. On the other hand, in an anti-oomycete activity test against the fungus-like plant pathogen Phytophthora capsici , 1 - 4 showed a higher activity than that of 5 and 6 , suggesting that the long aliphatic side chain plays a significant role for cytotoxicity, but is not effective or suppressive for anti-oomycete activity., Competing Interests: The authors declare no conflict of interest.

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Azaspiracids (AZAs) are marine algal toxins that can be accumulated by edible shellfish to cause a foodborne gastrointestinal poisoning in humans. In the European Union, only AZA1, -2 and -3 are currently regulated and their concentration in shellfish is determined through their toxic equivalency factors (TEFs) derived from the intraperitoneal lethal potency in mice. Nevertheless, considering the potential human exposure by oral route, AZAs TEFs should be calculated by comparative oral toxicity data. Thus, the acute oral toxicity of AZA1, -2 and -3 was investigated in female CD-1 mice treated with different doses (AZA1: 135-1100μg/kg; AZA2 and AZA3: 300-1100μg/kg) and sacrificed after 24h or 14days. TEFs derived from the median lethal doses (LD 50 ) were 1.0, 0.7 and 0.5, respectively for AZA1, -2 and -3. In fact, after 24h from gavage administration, LD 50s were 443μg/kg (AZA1; 95% CL: 350-561μg/kg), 626μg/kg (AZA2; 95% CL: 430-911μg/kg) and 875μg/kg (AZA3; 95% CL: 757-1010μg/kg). Mice dead more than 5h after the treatment or those sacrificed after 24h (doses: ≥175μg AZA1/kg, ≥500μg AZA2/kg and ≥600μg AZA3/kg) showed enlarged pale liver, while increased serum markers of liver alteration were recorded even at the lowest doses. Blood chemistry revealed significantly increased serum levels of K + ions (≥500mg/kg), whereas light microscopy showed tissue changes in the gastrointestinal tract, liver and spleen. No lethality, macroscopic, tissue or haematological changes were recorded two weeks post exposure, indicating reversible toxic effects. LC-MS/MS analysis of the main organs showed a dose-dependency in gastrointestinal absorption of these toxins: at 24h, the highest levels were detected in the stomach and, in descending order, in the intestinal content, liver, small intestine, kidneys, lungs, large intestine, heart as well as detectable traces in the brain. After 14days, AZA1 and AZA2 were still detectable in almost all the organs and intestinal content., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The identification of a new suite of toxins, called azaspiracids (AZA), as the cause of human illnesses after the consumption of shellfish from the Irish west coast in 1995, resulted in interest in understanding the global distribution of these toxins and of species of the small dinoflagellate genus Azadinium, known to produce them. Clonal isolates of four species of Azadinium, A. poporum, A. cuneatum, A. obesum and A. dalianense were obtained from incubated sediment samples collected from Puget Sound, Washington State in 2016. These Azadinium species were identified using morphological characteristics confirmed by molecular phylogeny. Whereas AZA could not be detected in any strains of A. obesum, A. cuneatum and A. dalianense, all four strains of A. poporum produced a new azaspiracid toxin, based on LC-MS analysis, named AZA-59. The presence of AZA-59 was confirmed at low levels in situ using a solid phase resin deployed at several stations along the coastlines of Puget Sound. Using a combination of molecular methods for species detection and solid phase resin deployment to target shellfish monitoring of toxin at high-risk sites, the risk of azaspiracid shellfish poisoning can be minimized., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Crambescidin 816 is a guanidine alkaloid produced by the sponge Crambe crambe with known antitumoral activity. While the information describing the effects of this alkaloid in central neurons is scarce, Cramb816 is known to block voltage dependent calcium channels being selective for L-type channels. Moreover, Cramb816 reduced neuronal viability through an unknown mechanism. Here, we aimed to describe the toxic activity of Cramb816 in cortical neurons. Since calcium influx is considered the main mechanism responsible for neuronal cell death, the effects of Cramb816 in the cytosolic calcium concentration of cortical neurons were studied. The alkaloid decreased neuronal viability and induced a dose-dependent increase in cytosolic calcium that was also related to the presence of calcium in the extracellular media. The increase in calcium influx was age dependent, being higher in younger neurons. Moreover, this effect was prevented by glutamate receptor antagonists, which did not fully block the cytotoxic effect of Cramb816 after 24 h of treatment but completely prevented Cramb816 cytotoxicity after 10 min exposure. Therefore, the findings presented herein provide new insights into the cytotoxic effect of Cramb816 in cortical neurons.

The negative impact of conventional pesticides on the environment is already extensively discussed worldwide. Although the use of chemical agents for controlling agricultural pests remains as first-line strategy for pest control, novel biorational active insecticides, such as spirotetramat, have appeared in the pesticide market during recent years in Argentina. The aim of this study was to assess the toxicity of spirotetramat on two developmental stages of a Neotropical strain of Eretmocerus mundus, with the conventional insecticide cypermethrin as a positive control, and to determine spirotetramat's side effects on parasitoid demographic parameters. Lethal effects of both insecticides on pupae and adults were evaluated by adult emergency and survival, respectively; whereas sublethal effects on both development stages were assessed by adult longevity, reproduction capacity, sex ratio, and longevity of the first progeny. Spirotetramat proved less harmful than cypermethrin at both developmental stages studied, corroborating once more the high toxicity of this pyrethroid to natural enemies. Although spirotetramat did not affect the emergence and reproductive capacity of adults surviving pupal exposure, the longevity of the first progeny was reduced as was adult survival and longevity after exposure to residues. Spirotetramat also reduced all demographic parameters in the population evaluation. This work is the first report of spirotetramat toxicity at the population level and demonstrates the need to assess the total effect of pesticides on natural enemies.

Chemical study of the CH 2 Cl 2 -MeOH (1:1) extract from the sponge Monanchora unguiculata collected in Madagascar highlighted five new compounds, one acyclic guanidine alkaloid, unguiculin A (1) and four pentacyclic alkaloids, ptilomycalins E-H (2-5), along with four known compounds: crambescidin 800 (6) and crambescidin 359 (7), crambescidic acid (8), and fromiamycalin (9). Their structures were elucidated by 1D and 2D NMR spectra and HRESIMS data. All compounds were evaluated for their cytotoxicity against KB cells and their antiplasmodial activity. The new ptilomycalin E (2) and the mixture of the new ptilomycalins G (4) and H (5) showed promising cytotoxicity against KB cells with IC 50 values of 0.85 and 0.92 μM, respectively. Ptilomycalin F (3) and fromiamycalin (9) exhibited promising activity against Plasmodium falciparum with IC 50 values of 0.23 and 0.24 μM, respectively.

Yessotoxins (YTX) and azaspiracids (AZAs) are marine toxins produced by phytoplanktonic dinoflagellates that get accumulated in filter feeding shellfish and finally reach human consumers through the food web. Both toxin classes are worldwide distributed, and food safety authorities have regulated their content in shellfish in many countries. Recently, YTXs and AZAs have been described as compounds with subacute cardiotoxic potential in rats owed to alterations of the cardiovascular function and ultrastructural heart damage. These molecules are also well known in vitro inducers of cell death. The aim of this study was to explore the presence of cardiomyocyte death after repeated subacute exposure of rats to AZA-1 and YTX for 15 days. Because autophagy and apoptosis are often found in dying cardiomyocytes, several autophagic and apoptotic markers were determined by western blot in heart tissues of these rats. The results showed that hearts from YTX-treated rats presented increased levels of the autophagic markers microtubule-associated protein light chain 3-II (LC3-II) and beclin-1, nevertheless AZA-1-treated hearts evidenced increased levels of the apoptosis markers cleaved caspase-3 and -8, cleaved PARP and Fas ligand. Therefore, while YTX-induced damage to the heart triggers autophagic processes, apoptosis activation occurs in the case of AZA-1. For the first time, activation of cell death signals in cardiomyocytes is demonstrated for these toxins with in vivo experiments, which may be related to alterations of the cardiovascular function.

Azaspiracids (AZA) are a group of food poisoning phycotoxins that are known to accumulate in shellfish. They are produced by some species of the planktonic dinophycean taxon Amphidomataceae. Azaspiracids have been first discovered in Ireland but are now reported in shellfish from numerous global sites thus showing a wide distribution. In shellfish samples collected in 2009 near Huelva (Spain), AZA was also found along the Andalusian Atlantic coast for the first time. Analysis using LC-MS/MS revealed the presence of two different AZA analogues in different bivalve shellfish species (Chamelea gallina, Cerastoderma edule, Donax trunculus, and Solen vagina). In a number of samples, AZA levels exceeded the EU regulatory level of 160μg AZA-1 eq. kg -1 (reaching maximum levels of >500μg AZA-1 eq. kg -1 in Chamelea gallina and >250μg AZA-1 eq. kg -1 in Donax trunculus) causing closures of some local shellfish production areas. One dinophyte strain established from the local plankton during the AZA contamination period and determined as Amphidoma languida was in fact toxigenic, and its AZA profile disclosed it as the causative species: it contained AZA-2 as the main compound and the new compound AZA-43 initially detected in the shellfish. AZA-43 had the same mass as AZA-3, but produced different collision induced dissociation (CID) spectra. High resolution mass spectrometric measurements indicated that there is an unsaturation in the H, I ring system of AZA-43 distinguishing it from the classical AZA such as AZA-1, -2, and -3. Furthermore, the Spanish strain was different from the previously reported AZA profile of the species that consist of AZA-38 and AZ-39. In molecular phylogenetics, the Andalusian strain formed a monophyletic group together with other strains of Am. languida, but ITS sequences data revealed surprisingly high intragenomic variability. The first Andalusian case of AZA contamination of shellfish above the EU regulatory limit reported here clearly revealed the risk of azaspiracid poisoning (AZP) for this area and also for the Atlantic coast of Iberia and North Africa. The present study underlines the need for continuous monitoring of AZA and the organisms producing such toxins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Azaspiracids (AZAs) are the most recently discovered group of biotoxins and are the cause of azaspiracid shellfish poisoning (AZP) in humans. To date over thirty analogues have been identified. However, toxicological studies of AZAs are limited due to the lack of availability of toxins and toxin standards. Most data available are on acute toxicity and there are no data available on genotoxicity of AZAs. This study presents an integrated approach investigating the genotoxic potential of AZA1-3 in cell culture systems using the Comet assay combined with assays to provide information on possible apoptotic processes, cytotoxicity and changes in cell number. Results demonstrate a time and dose dependent increase in DNA fragmentation in most cell lines, indicating a genotoxic effect of AZA1-3. However, a significant reduction in cell number and a clear shift from early to late apoptosis was observed for all analogues in Jurkat T cells and HepG-2 cells; CaCo-2 cells did not show a clear apoptotic profile. Late apoptotic/necrotic cells correlate well with the percentage of tail DNA for all analogues in all three cell lines. All data taken together indicate that AZA1-3 is not genotoxic per se and demonstrate apoptotic/necrotic processes to be involved to some extent in AZAs toxicity. The sensitivities of cell lines and the different potencies of AZA1-3 are in agreement with the literature available. The order of sensitivity for all three AZAs tested in the present study is, in increasing order, CaCo-2 cells < HepG-2 cells < Jurkat T cells. The order of potency of AZA1-3 varies among the cell lines., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The cyclic imine toxin 20-methyl spirolide G (20-meSPX-G), produced by the toxigenic dinoflagellate Alexandrium ostenfeldii/Alexandrium peruvianum, has been previously reported to contaminate shellfish in various European coastal locations, as revealed by mouse toxicity bioassay. The aim of the present study was to determine its toxicological profile and its molecular target selectivity. 20-meSPX-G blocked nerve-evoked isometric contractions in isolated mouse neuromuscular preparations, while it had no action on contractions elicited by direct electrical stimulation, and reduced reversibly nerve-evoked compound muscle action potential amplitudes in anesthetized mice. Voltage-clamp recordings in Xenopus oocytes revealed that 20-meSPX-G potently inhibited currents evoked by ACh on Torpedo muscle-type and human α7 nicotinic acetylcholine receptors (nAChR), whereas lower potency was observed in human α4β2 nAChR. Competition-binding assays showed that 20-meSPX-G fully displaced [³H]epibatidine binding to HEK-293 cells expressing the human α3β2 (Ki = 0.040 nM), whereas a 90-fold lower affinity was detected in human α4β2 nAChR. The spirolide displaced [(125)I]α-bungarotoxin binding to Torpedo membranes (Ki = 0.028 nM) and in HEK-293 cells expressing chick chimeric α7-5HT₃ nAChR (Ki = 0.11 nM). In conclusion, this is the first study to demonstrate that 20-meSPX-G is a potent antagonist of nAChRs, and its subtype selectivity is discussed on the basis of molecular docking models., Competing Interests: The authors declare no conflict of interest.

TP53 also known as p53 is a tumor suppressor gene mutated in a variety of cancers. P53 is involved in cell cycle, apoptosis and DNA repair mechanisms and is thus tightly controlled by many regulators. Recently, strategies to treat cancer have focused on the development of MDM2 antagonists to induce p53 stabilization and restore cell death in p53 non-mutated cancers. However, some of these molecules display adverse effects in patients including induction of thrombocytopenia. In the present study, we have explored the effect of SAR405838 not only on human megakaryopoiesis but also more generally on hematopoiesis. We compared its effect to MI-219 and Nutlin, which are less potent MDM2 antagonists than SAR405838. We found that all these compounds induce a deleterious effect on all types of hematopoietic progenitors, as well as on erythroid and megakaryocytic differentiation. Moreover, they inhibit both early and late stages of megakaryopoiesis including ploidization and proplatelet formation. In conclusion, MDM2 antagonists induced a major hematopoietic defect in vitro as well as an inhibition of all stages of megakaryopoiesis that may account for in vivo thrombocytopenia observed in treated patients., Competing Interests: This work was supported in part by funds provided by Sanofi (Sanofi R&D Vitry-sur-Seine France).