Coordination regulation of enhanced performance reveals the tolerance mechanism of Chlamys farreri to azaspiracid toxicity.

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.

Basal levels of biochemical biomarkers in the freshwater prawn Palaemon argentinus and their alterations due to the exposure of both insecticides cypermethrin and spirotetramat.

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 LC50-96 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 LC50-96 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.

Short- and long-term effects of commercial formulations of imidacloprid, spirotetramat, and mixtures of these active ingredients on pupae of Diaeretiella rapae (Hymenoptera: Braconidae) and its progeny.

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 (LD10), imidacloprid showed the highest hazard coefficient, whereas the commercial mixture was more hazardous when considering the LD50 and LD90. 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.

Current Research Status of Azaspiracids.

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.

Azaspiracid accumulation in Japanese coastal bivalves and ascidians fed with Azadinium poporum producing azaspiracid-2 as the dominant toxin component.

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.

Gene regulation by morpholines and piperidines in the cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.

Ovicidal activity of spirotetramat and its effect on hatching, development and formation of Frankliniella occidentalis egg.

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 LC50 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.

In vitro anti-Leishmania activity and molecular docking of spiro-acridine compounds as potential multitarget agents against Leishmania infantum.

Leishmaniasis is an infectious disease with several limitations regarding treatment schemes. This work reports the anti-Leishmania activity of spiroacridine compounds against the promastigote (IC50 = 1.1 to 6.0 µg / mL) and amastigote forms of the best compounds (EC50 = 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.

Design, synthesis and biological evaluation of exiguamine A analogues as IDO1 inhibitors.

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 IC50 values at the level of 10-7-10-8 M. Compound 21f was the most potent IDO1 inhibitor with an IC50 value of 65.3 nM, which was comparable with the positive control drug epacadostat (IC50 = 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.

In vitro investigation of the genotoxicity of portimine, a cyclic imine toxin produced by the dinoflagellate Vulcanodinium rugosum, on human hepatic HepaRG cells.

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.

Pre-imaginal exposure to Oberon® disrupts fatty acid composition, cuticular hydrocarbon profile and sexual behavior in Drosophila melanogaster adults.

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.

Evaluation of potential miticide toxicity to Varroa destructor and honey bees, Apis mellifera, under laboratory conditions.

The honey bee, Apis mellifera L., is the world's most important managed pollinator of agricultural crops, however, Varroa mite, Varroa destructor Anderson and Trueman, infestation has threatened honey bee survivorship. Low efficacy and development of Varroa mite resistance to currently used Varroacides has increased the demand for innovative, effective treatment tool options that exhibit high efficacy, while minimizing adverse effects on honey bee fitness. In this investigation, the toxicity of 16 active ingredients and 9 formulated products of registered miticides for use on crops from 12 chemical families were evaluated in comparison to amitraz on Varroa mites and honey bees using contact surface and topical exposures. It was found that fenpyroximate (93% mortality), spirotetramat (84% mortality) and spirodiclofen (70% mortality) had greater toxicity to Varroa mites, but high dose rates caused high bee mortality (> 60%). With this in mind, further research is needed to investigate other options to minimize the adverse effect of these compounds on bees. The results also found high toxicity of fenazaquin and etoxazole against Varroa mites causing 92% and 69% mortality, respectively; and were found to be safe on honey bees. Collectively, it is recommended that fenazaquin and etoxazole are candidates for a potential Varroacide and recommended for further testing against Varroa mites at the colony level.

Detection of Cyclic Imine Toxins in Dietary Supplements of Green Lipped Mussels (Perna canaliculus) and in Shellfish Mytilus chilensis.

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.

Discovery of chemical markers for improving the quality and safety control of Sinomenium acutum stem by the simultaneous determination of multiple alkaloids using UHPLC-QQQ-MS/MS.

Sinomenium acutum stem is a popular traditional Chinese medicine used to treat bone and joint diseases. Sinomenine is considered the only chemical marker for the quality control of S. acutum stem in mainstream pharmacopeias. However, higenamine in S. acutum stem is a novel stimulant that was banned by the World Anti-Doping Agency in 2017. Therefore, enhancing the quality and safety control of S. acutum stem to avoid potential safety risks is of utmost importance. In this study, a fast, sensitive, precise, and accurate method for the simultaneous determination of 11 alkaloids in S. acutum stem by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) was established. This method successfully analyzed thirty-five batches of S. acutum stem samples. The average contents of sinomenine, magnoflorine, coclaurine, acutumine, higenamine, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine, and jatrorrhizine were 24.9 mg/g, 6.35 mg/g, 435 µg/g, 435 µg/g, 288 µg/g, 44.4 µg/g, 22.5 µg/g, 21.1 µg/g, 15.8 µg/g, 9.30 µg/g, and 8.75 µg/g, respectively. Multivariate analysis, including principal component analysis (PCA), orthogonal partial least square method-discriminant analysis (OPLS-DA), and hierarchical cluster analysis (HCA), were performed to characterize the importance and differences among these alkaloids in S. acutum stem samples. As a result, sinomenine, magnoflorine, coclaurine, acutumine, and higenamine are proposed as chemical markers for quality control. Higenamine and coclaurine are also recommended as chemical markers for safety control. This report provides five alkaloids that can be used as chemical markers for improving the quality and safety control of S. acutum stem. It also alerts athletes to avoid the risks associated with consuming S. acutum stem.

Structure-Based Drug Discovery of N-((R)-3-(7-Methyl-1H-indazol-5-yl)-1-oxo-1-(((S)-1-oxo-3-(piperidin-4-yl)-1-(4-(pyridin-4-yl)piperazin-1-yl)propan-2-yl)amino)propan-2-yl)-2'-oxo-1',2'-dihydrospiro[piperidine-4,4'-pyrido[2,3-d][1,3]oxazine]-1-carboxamide (HTL22562): A Calcitonin Gene-Related Peptide Receptor Antagonist for Acute Treatment of Migraine.

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.

Structure-Activity Relationship of Antischistosomal Ozonide Carboxylic Acids.

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.

Acute Oral Toxicity of Pinnatoxin G in Mice.

Pinnatoxin G (PnTx-G) is a marine cyclic imine toxin produced by the dinoflagellate Vulcanodiniumrugosum, 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 LD50 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.

Paraherquamide J, a new prenylated indole alkaloid from the marine-derived fungus Penicillium janthinellum HK1-6.

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.