Flusilazole Research Articles

New insights into triazole fungicide-caused hematopoietic abnormality in zebrafish based on GRα screening developmental toxicity

Triazole fungicides (TFs) are known to be common environmental contaminants that can be toxic to aquatic animals, but their developmental toxicity is not fully understood. To address this gap, we first used a glucocorticoid receptor α (GRα)-mediated dual luciferase reporter gene system to explore the possible development toxicity of ten TFs and found that flusilazole (FLU) exhibited stronger agonistic activity against GRα. Subsequent transcriptome sequencing showed that FLU exposure affected GRα activation and hematopoiesis associated with a variety of biological processes, including responses to corticosteroid release, embryonic hematopoiesis, erythroid differentiation, and the development of hematopoietic or lymphoid organs. Furthermore, based on in situ hybridization and staining techniques, we clarified that FLU decreased the expression of the primitive hematopoietic marker genes gata1 and pu.1. and caused the defects in the posterior blood island (PBI), thereby impacting intermediate hematopoietic processes. Also, FLU significantly reduced the expression of the crucial hematopoietic gene cmyb and disrupted the production of erythrocytes and bone marrow cells during definitive hematopoiesis. Consistently, we found that FLU induced lesions in the kidney, a hematopoietic organ, including the infiltration of inflammatory cells, tubular collapse, reduced tubular filtration area, and interstitial hydronephrosis. We also found that FLU increased aberrant red blood cells in the peripheral blood of zebrafish. These findings provide new insights into the developmental toxicity and ecotoxicological risk of TFs.

Joint toxic effects of thiamethoxam and flusilazole on the adult worker honey bees (Apis mellifera L.)

Insect pollinators are routinely exposed to a complex mixture of many pesticides. However, traditional environmental risk assessment is only carried out based on ecotoxicological data of single substances. In this context, we aimed to explore the potential effects when worker honey bees (Apis mellifera L.) were simultaneously challenged by thiamethoxam (TMX) and flusilazole (FSZ). Results displayed that TMX possessed higher toxicity to A. mellifera (96-h LC50 value of 0.11 mg a. i. L−1) than FSZ (96-h LC50 value of 738 mg a. i. L−1). Furthermore, the mixture of TMX and FSZ exhibited an acute synergistic impact on the pollinators. Meanwhile, the activities of SOD, caspase 3, caspase 9, and PPO, as well as the expressions of six genes (abaecin, dorsal-2, defensin-2, vtg, caspase-1, and CYP6AS14) associated with oxidative stress, immune response, lifespan, cell apoptosis, and detoxification metabolism were noteworthily varied in the individual and mixture challenges than at the baseline level. These data revealed that it is imminently essential to investigate the combined toxicity of pesticides since the toxicity evaluation from individual compounds toward honey bees may underestimate the toxicity in realistic conditions. Overall, the present results could help understand the potential contribution of pesticide mixtures to the decline of bee populations.

Environmentally friendly etchant of in situ plasmon-activated water to improve SERS sensing of pesticides

Wet etching, the most commonly used technology in the etching industry, is simple and convenient to use, and is currently the most widely used technical method of etching. However, etching solvents used in etching technology are harmful to living organisms and the environment. Another frequently used etching technique is dry etching. Dry etching is often used to prepare array substrates for surface-enhanced Raman scattering (SERS). However, the disadvantage of dry etching technology is its complicated operation and expensive equipment required. In order to resolve the shortcomings of the above etching technologies, we propose a new technical concept, in situ vapor etching with room-temperature plasmon-activated water (PAW). The vapor of traditional deionized water (DIW) cannot perform etching of SERS-active substrates. In situ PAW vapor has characteristics of high energy and doping electrons which enable in situ PAW vapor to perform nanostructured treatment of SERS-active substrates. In addition, many properties of PAW vapor are also superior to those of DIW. Application of PAW vapor to the rhodamine 6 G (R6G) probe molecule and the imidacloprid (IM), acetamiprid (AC), and flusilazole (FL) pesticides resulted in higher signal acquisitions and lower relative standard deviations (RSDs). In our proposed innovative technique, the limit of detection (LOD) of IM was determined to be 10 ppb. Application of this technique yielded the same or better results than SERS-array substrates. This environmentally friendly etching method is an innovative technology that can provide a wider range of SERS applications.

Flusilazole Induced Cytotoxicity and Inhibition of Neuronal Growth in Differentiated SH-SY5Y Neuroblastoma Cells by All-Trans-Retinoic Acid (Atra)

Flusilazole (FLUS) is a broad-spectrum organosilicon triazole fungicide used for protecting economically important cereals and orchard fruits. Considering the exposure route of pesticides, pesticide contamination of food is inevitable. Furthermore, excessive exposure to pesticides causes health problems in both target and non-target organisms. It was aimed to evaluate the effects of the triazole fungicide FLUS on cytotoxicity and neurite extension in differentiated SH-SY5Y neuroblastoma cells. The SH-SY5Y cells were differentiated into mature neurons using 10-µM all-trans-retinoic acid (RA) treatment for 7 days. Then the differentiated SH-SY5Y cells were treated with 50, 100 and 200 μM FLUS for 24 h. Afterwards, cell viability assays were performed including crystal violet, neutral red cell viability, and lactate dehydrogenase leakage assays. The morphological examinations were performed and neurite lenghts of the cells were measured in all experimental groups. FLUS treatment induced cytotoxicity in SH-SY5Y cells differentiated with RA. Significant decreases in cell viability percentages were observed. Furthermore, neurite lengths were negatively affected by the treatment of FLUS at the highest concentration. FLUS is a fungicide widely used in agriculture to protect crops from fungal diseases. However, the intensive use of these compounds causes a potential risk to human and environmental health. According to the results of the study, it can be concluded that high concentrations of FLUS cause neurotoxicity by causing neural cell death and adverse effects on neurite outgrowth in differentiated SH-SY5Y cells. FLUS exposure can cause neuronal degeneration in mammals.

Cell differentiation in the cardiac embryonic stem cell test (ESTc) is influenced by the oxygen tension in its underlying embryonic stem cell culture

Oxygen (O2) levels in the mammalian embryo range between 2.4% and 8%. The cardiac embryonic stem cell test (ESTc) is a model for developmental toxicity predictions, which is usually performed under atmospheric O2 levels of 20%. We investigated the chemical sensitivity of the ESTc carried out under 20% O2, using embryonic stem cells (ESC) cultured under either 20% O2 or 5% O2. ESC viability was more sensitive to valproic acid (VPA) but less sensitive to flusilazole (FLU) when cultured under 5% versus 20% O2. For beating cardiomyocyte differentiation, lower ID50 values were found for FLU and VPA when the ESCs had been cultured under 5% versus 20% O2. At differentiation day 4, gene expression values were primarily driven by the level of O2 during ESC culture instead of exposure to FLU. In addition, using ESCs cultured under 5% O2 tension, VPA enhanced Nes (ectoderm) expression. Bmp4 (mesoderm) was enhanced by VPA when using ESCs cultured under 20% O2. At differentiation day 10, using ESCs cultured under 5% instead of 20% O2, Nkx2.5 and Myh6 (cardiomyocytes) were less affected after exposure to FLU or VPA. These results show that O2 tension in ESC culture influences chemical sensitivity in the ESTc. This enhances awareness of the standard culture conditions, which may impact the application of the ESTc in quantitative hazard assessment of chemicals.

Enhancing Stability and Formulation Capability of Fungicides by Cocrystallization through a Novel Multistep Slurry Conversion Process

Solid agrochemicals with poor thermal stability pose a huge challenge to its production and application. Herein, two low melting point fungicides, pyraclostrobin (ZJ) and flusilazole (FZ), were des...

Flusilazole induces spatio-temporal expression patterns of retinoic acid-, differentiation- and sterol biosynthesis-related genes in the rat Whole Embryo Culture.

Embryotoxic responses are critically dependent on the timing of exposure during embryo development. Here, we examined the time- dependent developmental effects in rat embryos exposed to flusilazole (FLU), and their link to retinoic acid (RA) mediated pathways. To this end, we assessed the effects of 4h exposure of rat embryos in vitro to 300μM FLU during four developmental time windows (0-4, 4-8, 24-28 and 44-48h), evaluating morphological parameters, expression and localization of five genes directly or indirectly linked with the RA pathway. These were RA- (Cyp26a1 and Dhrs3), differentiation- (Gbx2 and Cdx1) and sterol biosynthesis- (Cyp51) related genes. Extended exposure for 48h to 300μM FLU resulted in morphological changes, typical for triazoles and RA, while the 4h exposure times did not. Time dependent significant upregulation of the five selected genes was observed. These results corroborate that the embryotoxic responses to FLU are correlated with the regulation of the RA pathway. Thus, these gene expression markers can be considered early biomarkers of FLU-induced potential developmental toxicity later in the development.

Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro

Conazole fungicides are widely used in agriculture despite their suspected endocrine disrupting properties. In this study, the potential (anti-)androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO), fluconazole (FLUC), flusilazole (FLUS), hexaconazole (HEXA), myconazole (MYC), penconazole (PEN), prochloraz (PRO), tebuconazole (TEBU), triadimefon (TRIA), and triticonazole (TRIT) were examined using murine Leydig (MA-10) cells and human T47D-ARE cells stably transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T) secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration-dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC50 = 12.4 μM) or TEBU (IC50 = 2.4 μM) in combination with LH. The expression of steroidogenic and cholesterol biosynthesis genes was not changed by conazole exposure. Also, there were no changes in reactive oxygen species (ROS) formation that could explain the altered T secretion after exposure to conazoles. Nine conazoles decreased T-induced AR activation (IC50s ranging from 10.7 to 71.5 μM) and effect potencies (REPs) were calculated relative to the known AR antagonist flutamide (FLUT). FLUC had no effect on AR activation by T. FLUS was the most potent (REP = 3.61) and MYC the least potent (REP = 0.03) AR antagonist. All other conazoles had a comparable REP from 0.12 to 0.38. Our results show distinct in vitro anti-androgenic effects of several conazole fungicides arising from two mechanisms: inhibition of T secretion and AR antagonism, suggesting potential testicular toxic effects. These effects warrant further mechanistic investigation and clearly show the need for accurate exposure data in order to perform proper (human) risk assessment of this class of compounds.

Triazole-induced gene expression changes in the zebrafish embryo

The zebrafish embryo is considered to provide a promising alternative test model for developmental toxicity testing. Most systems use morphological assessment of the embryos, however, microarray analyses may increase sensitivity and predictability of the test by detecting more subtle and detailed responses. In this study, we investigated the possibility of relating gene expression profiles of structurally similar chemicals tested in a single concentration, to a complete transcriptomic concentration–response of flusilazole (FLU). We tested five other triazoles, hexaconazole (HEX), cyproconazole (CYP), triadimefon (TDF), myclobutanil (MYC), and triticonazole (TTC) at equipotent concentrations based on morphological evaluation. Results showed that every compound had a different degree of regulation within their anti-fungal and developmental toxicity pathways, steroid biosynthesis and retinol metabolism, respectively. Assuming that the ratio between these pathways is relevant for efficacy compared to developmental toxicity, we found TTC was more efficient and CYP was more toxic compared to the other triazoles. With the approach used in this study we demonstrated that gene expression data allow more comprehensive assessment of compound effects by discriminating relative potencies using these specific gene sets. The zebrafish embryo model can therefore be considered a useful vertebrate model providing information of relevant pathways related to anti-fungal mechanism of action and toxicological activity.

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO–BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥687 genes) as well as a variety of GO–BP, as compared to MEHP, PHE and PENG (≤55 genes with no significant changes in GO–BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO–BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

Effect of fungicides on yielding and grain quality of malting barley cultivarsWpływ fungicydów na plonowanie i jakość ziarna browarnych odmian jęczmienia

Summary In field experiments with spring barley (Conchita, Mauritia and Sebastian cultivars) carried out in 2010–2011, the effect of fungicides Alert 375 SC (flusilazole, carbendazym), Artea 330 EC (propiconazole, cyproconazole) and Capalo 337,5 SE (fenpropimorph, metrafenone, epoxyconazole) on grain yield, yield components, diseases occurrence, grain filling and protein content in grain of spring barley cultivars were studied. All fungicides affected the increase in grain yield of spring barley cultivars, and reduced the intensity of fungal diseases caused by Pyrenophora tere, Rhynchosporium secalis and P. graminea. Yield increase was related to the increase of grain mass per ear and 1000 seed weight. The effect of applied fungicides on protein content in grain of spring barley cultivars was small. The positive effect of the applied fungicides on grain filling of spring barley was also stated.

Potential Use of Chitosan in the Control of Grapevine Trunk Diseases.

Summary. Due to its fungistatic or fungicidal potential, chitosan, a high molecular-weight polymer that is non-toxic and biodegradable, has become an alternative to conventional fungicides. In addition, chitosan is reported to elicit defense mechanisms in plant tissues. In this study, we explored the in vitro fungicidal effect of chitosan on some of the most important grapevine wood fungi, such as Botryosphaeria sp. (dieback and cane blight), Phomopsis sp. (Phomopsis cane and leaf spot), Eutypa lata (eutypa dieback), Neonectria liriodendri (black foot disease), Phaeomoniella chlamydospora (Petri disease and esca) and Fomitiporia sp. (esca). Inhibition of mycelial growth was evaluated at five concentrations 50, 25, 5, 2.5 and 0.5 mg a.i. l -1 of chitosan. Chitosan was effective in reducing mycelial growth of all the fungi. The lowest EC50 values were obtained with Pa. chlamydospora, Fomitiporia sp. and Botryosphaeria sp., and the highest with Neon. liriodendri. All these were inferior to the maximum recommended field rate (8.33 mg a.i. l -1 ) with exception of the value obtained with Neon. liriodendri. Greenhouse experiments were carried out to evaluate the efficacy of foliar sprays of chitosan on potted grapevine plants (cultivar Castelao) growing in a substrate artificially infested with Pa. chlamydospora or Neon. liriodendri. The effect of chitosan against Neon. liriodendri was similar to that achieved with some selected fungicides (carbendazim+flusilazole, cyprodinil+fludioxonil and tebuconazole). Chitosan significantly improved plant growth (plant height and number of roots) and decreased disease incidence compared with untreated plants. As regards Pa. chlamydospora, chitosan only reduced the disease incidence caused by this fungus.