Flusilazole-induced damage to SerW3 cells via cytotoxicity, oxidative stress and lipid metabolism: An in vitro study.

Flusilazole (C16H15F2N3Si) is a triazole fungicide and it is being used widely in recent years to control fungal infections in various fruits and vegetables. This study aims to evaluate the impact of flusilazole on cytotoxicity, ATP-dependent cassette transporter proteins (ABC transporter proteins) in SerW3 cells. In this study, SerW3 cells have administrated with 25, 100, and 200 µM flusilazole, cell viability was performed. The quantity of the cellular lipids was evaluated spectrophotometrically. Moreover, the expression of the ABCA1 and ABCB1 proteins determined by immunofluorescence microscopy. Furtherly, evaluation of the cell death type and measurement of the activity of the antioxidant enzymes was performed. According to the results, flusilazole treatment gave rise to inhibition in cell viability, increase in apoptotic cell number, reduction in cellular lipids, and inhibition in the expression of ABCA1 and ABCB1 proteins. Furthermore, it caused decreases in antioxidant enzyme activities. It may be concluded that flusilazole administration may cause infertility/subfertility. The mechanism of action can be due to cytotoxicity, impairment of the detoxification mechanisms, lipid metabolism, and dysregulation of cell functions.

Baseline sensitivity and action mechanism of the sterol demethylation inhibitor flusilazole to Valsa mali.

The apple Valsa canker caused by Valsa mali is a devastating branch disease that has seriously threatened the development of the apple industry worldwide. In current study, a total of 115 V. mali strains collected from different apple orchards in Shaanxi Province of China during 2016 and 2017 were tested for their sensitivity to flusilazole. The average EC50 (effective concentrations causing 50% mycelial growth inhibition) value of all tested strains for flusilazole was 0.0892 (±0.0036) µg/mL and the frequency distribution of the EC50 values was unimodal. Flusilazole exhibited both excellent protective and curative activity on detached apple branches, which was significantly better than the commonly used fungicide thiophanate-methyl. After flusilazole treatment, mycelia twisted with offshoot of top increased, the V. mali strains lost the ability of fruiting body production, and cell membrane permeability of the mycelia increased while ergosterol content and pectinase activity decreased. The expression of pectinase genes involved in virulence down-regulated after flusilazole treatment. This study is the first report on the baseline sensitivity of V. mali to flusilazole. These results indicated that flusilazole has a great potential to play an important role in the management of Valsa canker.

Baseline Sensitivity and Toxic Action of the Sterol Demethylation Inhibitor Flusilazole Against Botrytis cinerea.

In the present study, a total of 95 Botrytis cinerea single-spore strains collected from different hosts in Shaanxi Province of China were characterized for their sensitivity to the sterol demethylation inhibitor fungicide flusilazole. The effective concentration for 50% inhibition of mycelial growth (EC50) of flusilazole ranged from 0.021 to 0.372 µg/ml, with an average value of 0.093 µg/ml. Cross-resistance between flusilazole and commonly used fungicides was not detected, and no flusilazole-resistant mutants were induced. Both on detached strawberry leaves and in greenhouse experiments, flusilazole was more effective than the commonly used fungicide carbendazim at reducing gray mold. After culture on PDA plates or detached strawberry leaves, no difference in sclerotia production or pathogenicity was detected between two strains, WG12 (most sensitive to flusilazole) and MX18 (least sensitive to flusilazole). After treatment with flusilazole, however, the two strains lost the ability to produce sclerotia, and oxalic acid and ergosterol contents in mycelium decreased. Interestingly, the inhibition rate of ergosterol content in MX18 was significantly lower than that in WG12. Expression of Cyp51, BcatrD, and Bcmfs1 genes all increased after treatment with flusilazole, especially the Cyp51 and BcatrD genes. However, the expression of Cyp51 gene or BcatrD gene in WG12 and MX18 were significantly different from each other after treatment with flusilazole. In addition, no point mutations in Cyp51 gene were found in MX18. These data suggest flusilazole is a promising fungicide for resistance management of gray mold and also provided novel insights into understanding the resistance mechanism of flusilazole against plant pathogens.

Simultaneous detection of fluquinconazole and flusilazole in lettuce using gas chromatography with a nitrogen phosphorus detector: decline patterns at two different locations.

Method validations in addition to decline patterns of fluquinconazole and flusilazole in lettuce grown under greenhouse conditions at two different locations were investigated. Following the application of fluquinconazole and flusilazole at a dose rate of 20 mL/20 L water, lettuce samples were collected randomly for up to 7 days post-application, and simultaneously extracted with acetone, purified through solid-phase extraction, analyzed via gas chromatography with a nitrogen phosphorus detector, and confirmed through gas chromatography-mass spectrometry. The linearity was excellent, with determination coefficients (R(2) ) between 0.9999 and 1.0. The method was validated in triplicate at two different spiking levels (0.2 and 1.0 mg/kg) with satisfactory recoveries between 75.7 and 97.9% and relative standard deviations of <9. The limit of quantification was 0.01 mg/kg. Both analytes declined very quickly, as can be seen from the short half-life time of <4 days. Statistical analysis revealed significant differences between residues at different days of sampling, except at 7 days post-application (triple application). At that point, the decline patterns of fluquinconazole and flusilazole were independent of application rate, location, temperature and humidity. Copyright © 2015 John Wiley & Sons, Ltd.

Genotoxicity effects of Flusilazole on the somatic cells of Allium cepa.

The aim of this study was to evaluate the effects of the fungicide flusilazole on somatic cells of Allium cepa. For evaluation of cytogenetic effects, root meristem cells of A. cepa were treated with 10, 20, 30 and 45 ppm (EC50 concentration) for 24, 48 and 72 h. The mitotic index and different types of chromosomal abnormalities such as bridges, stickiness and laggards were determined in both control and test groups. Acridine orange/Ethidium bromide double staining and fluorescence microscope was used to determine the stability of chromosome structure. Data obtained from staining process indicated that ratio of necrotic cells significantly increased by the flusilazole presoaking. The RAPD-PCR method was used and the higher doses treated-group (45 ppm) was more distant to the control group compare with others.

Adsorption behavior of azole fungicides on polystyrene and polyethylene microplastics.

Agricultural plastic films and triazole fungicides are widely used in agricultural production process. Exposure to natural environment, agricultural plastic films will degrade into micron plastic particles, which will adsorb pesticide molecules and may affect their toxicity, biological activity and persistence. The long-term coexistence of microplastics (MPs) and triazole fungicides will bring potential harms to the agricultural ecological environment. Therefore, two kinds of triazole fungicides flusilazole (FLU) and epoxiconazole (EPO) were selected as cases and the adsorption behaviors of them on polystyrene and polyethylene were investigated. A series of factors which could affect the adsorption behavior were evaluated. Specifically, the particle size of MPs could affect its adsorption capacity, and the smaller the particle size, the stronger the adsorption capacity. Moreover, with the increase of pH value from 6.0 to 9.0, the adsorption capacity of MPs to target compounds gradually increased. The effect of ionic strength was evaluated by NaCl, and 0.05% of NaCl was beneficial to the adsorption process, while the continuous increase of NaCl concentration inhibited the adsorption. Oxalic acid and humic acid decreased the adsorption capacity of flusilazole on PE by 15.99-32.00% and PS by 35.02-48.67%, respectively. In addition, compared with the single pesticide system, the adsorption capacity of MPs for flusilazole and epoxiconazole in the binary pesticides system decreased by 36.13-37.93% and 44.36-51.35%, respectively, indicating that competitive adsorption occurred between the two pesticides. Meanwhile, the adsorption process was evaluated by adsorption kinetics and adsorption isotherms and were consistent with pseudo-second-order kinetic model and Freundlich isotherm model, respectively. Finally, several characterization analyses were conducted to investigated the adsorption mechanism, and hydrogen, halogen bonding and hydrophobic interaction proved to play an important role. The study on the adsorption behavior and mechanism of pesticide on MPs was the basis of assessing the risk of joint exposure.

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

Objectives: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

Cellulose nanofibers coated with silver nanoparticles as a flexible nanocomposite for measurement of flusilazole residues in Oolong tea by surface-enhanced Raman spectroscopy.

Cellulose nanofibers (CNF) coated with inorganic nanoparticles are novel hybrid nanocomposites that have great potential in various areas including agriculture and food science. The objectives of this study were to synthesize nanocomposites consisted of CNF coated with silver nanoparticles (AgNPs), which can be used as a surface-enhanced Raman spectroscopy (SERS) platform for measuring pesticides in Oolong tea. CNF were coated with AgNPs to form uniform CNF-AgNP nanocomposites that were investigated by transmission electron microscopy. Three-dimensional and porous CNF structures were loaded with AgNPs with an average size of 41 nm. CNF-AgNP substrates were applied in characterization and measurement of flusilazole in Oolong tea samples by SERS. A detection limit of 0.5 mg/kg for flusilazole was obtained based on partial least squares (PLS) regression analysis. These results indicate that CNF-AgNP nanocomposites combined with SERS is an accurate, sensitive, and efficient technique for identification and quantification of pesticide residues in Oolong tea.

Fungicide Treatments to Control Seed-borne Fungi of Sunflower Seeds.

: Seed-borne fungi in 69 sunflower cultivars were evaluated which comprised 52 confectionery and 17 oilseed types. Seed coats were placed on both NP-10 (Nonylphenol Ethoxylate based surfacant -10) and potato dextrose agar (PDA) media to culture fungi. The rate of contamination among the different varieties was calculated by counting seed coats with fungal colonies. The rate of contamination in the confectionary group (88%) was significantly (p ≤ 0.05) higher than in the oilseed group (71%). Of the 52 confectionery varieties, the dominant fungi recovered were Verticillium dahliae along with Alternaria spp., Fusarium spp., and Rhizopus spp., whereas the oilseed type varieties were contaminated with only V. dahliae. Molecular identification of fungal species via BLAST (Basic Alignment Search Tool) was performed on fungal sequences obtained from PCR (Polymerase Chain Reaction) analysis. The results included five Alternaria spp. that included Alternaria tenuissima, Alternaria alternata, Alternaria helianthiinficiens, Alternaria longipes, and Alternaria tamaricis, three Fusarium spp. such as Fusarium oxysporum, Fusarium incarnatum, and Fusarium proliferatum, and V. dahliae and Cladosporium cladosporioides. These were identified from pure fungal cultures recovered from seed coats. To efficiently control seed-borne fungi, four broad spectrum fungicides (carbendazim, triadimefon, caprio F-500, and flusilazole) were screened against V. dahliae isolate Gn3, which was isolated from a diseased LD 5009 sunflower plant. Flusilazole was selected based on its low half-maximal effective concentration value (EC50), 78.7 µg/mL. Seeds of diseased LD 5009 plants obtained from two different locations treated with formulated flusilazole fungicide at optimum parameters showed a significant (p ≤ 0.05) increase in seed germination and a decrease in contamination rate from 98% to less than 10%. The results affirmed that confectionery cultivars are much more susceptible to fungal contamination than oilseeds, and also that seed pretreatment is a suitable way to prevent the spread of soil- and seed-borne fungi in sunflower production.

Supramolecular Fluorescence Probe Based on Twisted Cucurbit[14]uril for Sensing Fungicide Flusilazole.

The host-guest complex of the common dye, thioflavin T (ThT), and twisted cucurbit[14]uril (tQ[14]) was selected as a fluorescent probe to determine non-fluorescent triazole fungicides, including flusilazole, azaconazole, triadimefon, tebuconazole, tricyclazole, flutriafol, penconazole, and triadimenol isomer A, in an aqueous solution. The experimental results reveal that the ThT@tQ[14] probe selectively responded to flusilazole with significant fluorescence quenching and a detection limit of 1.27 × 10-8 mol/L. In addition, the response mechanism involves not only a cooperation interaction-ThT occupies a side-cavity of the tQ[14] host and the triazole fungicide occupies another side-cavity of the tQ[14] host-but also a competition interaction in which both ThT and the triazole fungicide occupy the side-cavities of the tQ[14] host.

Rapid Detection of Flusilazole in Pears with Au@Ag Nanoparticles for Surface-Enhanced Raman Scattering.

Residual pesticides in vegetables or fruits have been become one of the world's most concerned food safety issues. Au-Ag core-shell nanoparticles (Au@Ag NPs) coupled with surface-enhanced Raman spectroscopy (SERS) was used for analysis of flusilazole which was widely applied in pears. Three different diameters of Au@Ag NPs were prepared to select the best SERS substrate for analyzing flusilazole. The Au@Ag NPs sizes of 90 ± 7 nm showed the highest enhancement effect and could be detected flusilazole standard solution and the minimum detectable concentration was 0.1 mg/L. Flusilazole in pear could also identified at as low as 0.1 µg/g. The amount of adsorbent is critical in the sample preparation process and the best amount of each absorber dosage was 0.6 g MgSO4, 0.2 g C18 and 0.2 g primary secondary amine (PSA). The experimental results indicated a good linear relationship between the Raman intensities of chief peaks and the concentrations of flusilazole solutions (R² = 0.924-0.962). This study shows that Au@Ag as SERS substrate has great potential to analyze of flusilazole in food matrices.

Reaction kinetics and mechanisms of organosilicon fungicide flusilazole with sulfate and hydroxyl radicals.

Flusilazole is an organosilane fungicide used for treatments in agriculture and horticulture for control of diseases. The reaction kinetics and mechanism of flusilazole with sulfate and hydroxyl radicals were studied. The rate constant of the radicals with the fungicide were determined by laser flash photolysis of peroxodisulfate and hydrogen peroxide. The results were 2.0 × 109 s-1M-1 for the reaction of the fungicide with HO and 4.6 × 108 s-1 M-1 for the same reaction with SO4- radicals. The absorption spectra of organic intermediates detected by laser flash photolysis of S2O82- with flusilazole, were identified as α-aminoalkyl and siloxyl radicals and agree very well with those estimated employing the time-dependent density functional theory with explicit account for bulk solvent effects. In the continuous photolysis experiments, performed by photo-Fenton reaction of the fungicide, the main degradation products were: (bis(4-fluorophenyl)-hydroxy-methylsilane) and the non-toxic silicic acid, diethyl bis(trimethylsilyl) ester, in ten and twenty minutes of reaction, respectively.

Determination of eight pesticides in Lycium barbarum by LC-MS/MS and dietary risk assessment.

A LC-MS/MS method for determination of eight pesticides (triadimefon, sulfoxaflor, flusilazole, tebuconazole, difenoconazole, amitraz, azoxystrobin, and thiophanate-methyl) in Lycium barbarum was established. The samples were extracted with acetonitrile, and then cleaned up by primary secondary amine. The extracts were diluted with 0.1% formic acid in water. The results showed that at the fortified levels of 0.01-10mg/kg, the average recoveries of these pesticides ranged from 82.1% to 96.2% with the relative standard deviations lower than 7%. The half-lives of eight pesticides were 1.3-5.0days in Lycium barbarum fruits. The pre-harvest interval of all pesticides mentioned above were investigated. Tebuconazole (14days), sulfoxaflor (14days) and flusilazole (28days) have longer pre-harvest interval than the others which have 7days. The dietary risks, assessed as hazard quotients, were far below 100%. The results showed that the eight pesticides applied to Lycium barbarum were comparably safe for the consumer.

Infrared spectroscopy detects changes in an amphibian cell line induced by fungicides: Comparison of single and mixture effects.

Amphibians are regarded as sensitive sentinels of environmental pollution due to their permeable skin and complex life cycle, which usually involves reproduction and development in the aquatic environment. Fungicides are widely applied agrochemicals and have been associated with developmental defects in amphibians; thus, it is important to determine chronic effects of environmentally-relevant concentrations of such contaminants in target cells. Infrared (IR) spectroscopy has been employed to signature the biological effects of environmental contaminants through extracting key features in IR spectra with chemometric methods. Herein, the Xenopus laevis (A6) cell line was exposed to low concentrations of carbendazim (a benzimidazole fungicide) or flusilazole (a triazole fungicide) either singly or as a binary mixture. Cells were then examined using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy coupled with multivariate analysis. Results indicate significant changes in the IR spectra of cells induced by both agents at all concentrations following single exposures, primarily in regions associated with protein and phospholipids. Distinct differences were apparent in the IR spectra of cells exposed to carbendazim and those exposed to flusilazole, suggesting different mechanisms of action. Exposure to binary mixtures of carbendazim and flusilazole also induced significant spectral alterations, again in regions associated with phospholipids and proteins, but also in regions associated with DNA and carbohydrates. Overall these findings demonstrate that IR spectroscopy is a sensitive technique for examining the effects of environmentally-relevant levels of fungicides at the cellular level. The combination of IR spectroscopy with the A6 cell line could serve as a useful model to identify agents that might threaten amphibian health in a rapid and high throughput manner.

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.

Enhanced Dissipation of Triazole and Multiclass Pesticide Residues on Grapes after Foliar Application of Grapevine-Associated Bacillus Species.

Disease management in vineyards with fungicides sometimes results in undesirable residue accumulations in grapes at harvest. Bioaugmentation of the grape fructosphere can be a useful approach for enhancing the degradation rate and reducing the residues to safe levels. This paper reports the in vitro and in vivo biodegradation of three triazole fungicides commonly used in Indian vineyards, by Bacillus strains, namely, DR-39, CS-126, TL-171, and TS-204, which were earlier found to enhance the dissipation rate of profenophos and carbendazim. The strains utilized the triazoles as carbon source and enhanced their in vitro rate of degradation. Myclobutanil, tetraconazole, and flusilazole were applied in separate vineyard plots at field doses of 0.40 g L(-1), 0.75 mL L(-1), and 0.125 mL L(-1), respectively. Residue analysis of field samples from the treated fields reflected 87.38 and >99% degradations of myclobutanil and tetraconazole, respectively, by the strain DR-39, and 90.82% degradation of flusilazole by the strain CS-126 after 15-20 days of treatment. In the respective controls, the corresponding percent degradations were 72.07, 58.88, and 54.28, respectively. These Bacillus strains could also simultaneously degrade the residues of profenofos, carbendazim, and tetraconazole on the grape berries and can be useful in multiclass pesticide residue biodegradation.

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.