Caspase 3/8/9; antifungal

As the outermost layer of the body, the skin plays an important role in exposure to pesticides, which could have negative impacts on human health. Trifloxystrobin is a widely used fungicide of the strobilurin class, however, there is little information regarding the skin contact-associated toxic mechanism. Therefore, the present study was performed in order to identify the skin toxicity mechanism of trifloxystrobin using HaCaT (keratinocyte of human skin) cells. Following 24 or 48 h treatment, cell viability, and subsequent Annexin V-FITC/propidium iodide assay, TUNEL assay and Western blotting were performed to investigate the cell death mechanism of trifloxystrobin. Exposure to trifloxystrobin resulted in diminished viability of HaCaT cells in both a time- and concentration-dependent manner. The cell death was derived through apoptotic pathways in the HaCaT cells. Furthermore, we explored the effect of trifloxystrobin on TRAIL-mediated extrinsic apoptosis using siRNA transfection. Knockdown of death receptor 5 suppressed trifloxystrobin-provoked apoptosis. These results indicate that trifloxystrobin induces TRAIL-mediated apoptosis and has an inhibitory effect in keratinocytes that can interfere with the barrier function and integrity of the skin. This could be proposed as a mechanism of skin toxicity by trifloxystrobin and considered in the management of pesticide exposure.[1]

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Strobilurin fungicides are quinone outside inhibitors (QoI) used to treat fungal pathogens for agricultural and residential use. Here, we compared the potential for neurotoxicity of the widely used strobilurins, azoxystrobin (AZS) and trifloxystrobin (TFS), in differentiated human SH-SY5Y cells. Fungicides did not include cytotoxicity up to 200 µM but both induced loss of cell viability at 48 h, with TFS showing slightly higher toxicity that AZS. Caspase 3/7 activity was induced in SH-SY5Y cells by both fungicides at 48 h (50 µM for AZS and 25 µM for TFS). ATP levels were reduced following a 24-hour exposure to > 25 µM AZS and > 6.25 µM TFS and both fungicides rapidly impaired oxidative respiration (~12.5 µM for AZS and ~3.125 µM TFS) and decreased oligomycin-induced ATP production, maximal respiration, and mitochondrial spare capacity. AZS at 100 µM showed a continual impairment of mitochondrial membrane potential (MMP) between 4 and 48 h while TFS at > 50 µM decreased MMP at 24 h. Taken together, TFS exerted higher mitochondrial toxicity at lower concentrations compared to AZS in SH-SY5Y cells. To discern toxicity mechanisms of strobilurin fungicides, lipidomics was conducted in SH-SY5Y cells following exposure to 6.25 µM and 25 µM AZS, and a total of 1595 lipids were detected, representing 49 different lipid classes. Lipid classes with the largest proportion of lipids detected in SH-SY5Y cells included triglycerides (17%), phosphatidylethanolamines (8%), ether-linked triglycerides (8%), phosphatidylcholines (7%), ether-linked phosphatidylethanolamines (6%), and diacylglycerols (5%). Together, these 5 lipid classes accounted for over 50% of the total lipids measured in SH-SY5Y cells. Lipids that were increased by AZS included acyl carnitine, which plays a role in long chain fatty acid utilization for mitochondrial β-oxidation, as well as non-modified, ether linked, and oxidized triacylglycerols, suggesting compensatory upregulation of triglyceride biosynthesis. The ceramide HexCer-NS, linked to neurodegenerative diseases, was decreased in abundance following AZS exposure. In summary, strobilurin fungicides rapidly inhibit mitochondrial oxidative respiration and alter the abundance of several lipids in neuronal cells, relevant for understanding environmental exposure risks related to their neurotoxicity.[2]

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Theileria parasites are responsible for devastating cattle diseases, causing major economic losses across Africa and Asia. Theileria spp. stand apart from other apicomplexa parasites by their ability to transform host leukocytes into immortalized, hyperproliferating, invasive cells that rapidly kill infected animals. The emergence of resistance to the theilericidal drug Buparvaquone raises the need for new anti-Theileria drugs. We developed a microscopy-based screen to reposition drugs from the open-access Medicines for Malaria Venture (MMV) Pathogen Box. We show that Trifloxystrobin (MMV688754) selectively kills lymphocytes or macrophages infected with Theileria annulata or Theileria parva parasites. Trifloxystrobin treatment reduced parasite load in vitro as effectively as Buparvaquone, with similar effects on host gene expression, cell proliferation and cell cycle. Trifloxystrobin also inhibited parasite differentiation to merozoites (merogony). Trifloxystrobin inhibition of parasite survival is independent of the parasite TaPin1 prolyl isomerase pathway. Furthermore, modeling studies predicted that Trifloxystrobin and Buparvaquone could interact distinctly with parasite Cytochrome B and we show that Trifloxystrobin was still effective against Buparvaquone-resistant cells harboring TaCytB mutations. Our study suggests that Trifloxystrobin could provide an effective alternative to Buparvaquone treatment and represents a promising candidate for future drug development against Theileria spp[3].

Strobilurins is the most widely used class of fungicides, but is reported highly toxic to some aquatic organisms. In this study, zebrafish embryos were exposed to a range concentrations of three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) for 96 h post-fertilization (hpf) to assess their aquatic toxicity. The 96-h LC50 values of pyraclostrobin, trifloxystrobin and picoxystrobin to embryos were 61, 55, 86 μg/L, respectively. A series of symptoms were observed in developmental embryos during acute exposure, including decreased heartbeat, hatching inhibition, growth regression, and morphological deformities. Moreover, the three fungicides induced oxidative stress in embryos through increasing reactive oxygen species (ROS) and malonaldehyde (MDA) contents, inhibiting superoxide dismutase (SOD) activity and glutathione (GSH) content as well as differently changing catalase (CAT) activity and mRNA levels of genes related to antioxidant system (Mn-sod, Cu/Zn-sod, Cat, Nrf2, Ucp2 and Bcl2). In addition, exposure to the three strobilurins resulted in significant upregulation of IFN and CC-chem as well as differently changed expressions of TNFa, IL-1b, C1C and IL-8, which related to the innate immune system, suggesting that these fungicides caused immunotoxicity during zebrafish embryo development. The different response of enzymes and genes in embryos exposed to the three fungicides might be the cause that leads to the difference of their toxicity. This work made a comparison of the toxicity of three strobilurins to zebrafish embryos on multi-levels and would provide a better understanding of the toxic effects of strobilurins on aquatic organisms.[3]

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Trifloxystrobin is a new type of fungicide, which is extensively used due to its excellent antifungal activity. In this study, oxidative stress and DNA damage induced by trifloxystrobin exposure was evaluated using Eisenia fetida at subchronic toxicity concentrations in artificial soil and brown soil (0.1-2.5 mg/kg). Throughout the exposure period (days 7, 28 and 56), six biochemical indicators including reactive oxygen species (ROS), antioxidant enzymes (SOD and CAT), glutathione S-transferase (GST), lipid peroxidation and DNA damage (8-hydroxydeoxyguanosine) were measured. In addition, the integrated biomarker response (IBR) index was calculated to make comparison of toxicological response between artificial and brown soils. Results indicated that trifloxystrobin can induce oxidative stress and DNA damage to earthworms with subchronic toxicity greater in brown soil compared to artificial soil as determined through integrated calculations for six biochemical indicators. Trifloxystrobin toxicological experiments in artificial soil may not accurately evaluate its toxicity in natural soil ecosystems, as the toxicity of trifloxystrobin to Eisenia fetida was underestimated[4].

Enzyme activity assays [3]
The zebrafish embryos/larvae samples (96 hpf) were homogenized (1:9,w/v) using an electric homogenizer in PBS buffer (7.4) and centrifuged at 12,000 × g for 15 min at 4 °C to obtain the supernatant for biochemical analysis. The activities of superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) content were measured using SOD Assay Kit, CAT Assay Kit and GSH Assay Kit, respectively, according to the manufacturer's instructions. SOD activity was determined based on WST-1 method using a microplate reader at 450 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit the oxidation reaction by 50% and was expressed as U/mg protein. The activity of CAT was monitored at 405 nm on a UV spectrophotometer. One unit of CAT activity was defined as the amount of enzyme required to consume 1 μmol H2O2 in 1 s and was expressed as U/mg protein. The GSH content was determined based on DTNB method at 405 nm using a microplate reader and was expressed as μmol/g protein. The total protein content was measured using a Bradford Protein Assay Kit following the manufacturer's instructions, with bovine serum albumin (BSA) as standard.

Cell viability assay [1]
Cell viability assay was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. About 4000 cells per well were seeded on 96-well plates. Cells were treated with medium as a control and 0.1% DMSO as a vehicle control. Trifloxystrobin/TRF was serially diluted from 500 μM to 2 μM. After 24 or 48 h, 20 μl of MTT solution (5 mg/ml) was added to each well. Following additional incubation in the dark for 2 h at 37 °C, the supernatants were removed and DMSO was added to dissolve the formed formazan crystals. The absorbance of the samples was detected at 540 nm using a microplate spectrophotometer. The values were calculated as a percentage of the control.

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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay [1]
Apoptotic cells were detected by the TUNEL assay using a DeadEnd™ Colorimetric TUNEL System kit. After 48 h of treatment with 0.5 μM TRF/Trifloxystrobin, the TUNEL assay was performed according to the protocol of the manufacturer. The TUNEL assay can detect apoptotic cells in situ based on the measurement of nuclear DNA fragmentation and chromatin condensation using biotinylated nucleotide and terminal deoxynucleotidyl transferase (TdT) to label the 3′-OH DNA ends of double-stranded DNA breaks. The images were obtained using a microscope.

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siRNA transfection [1]
HaCaT cells were suspended and transfected with 500 nM siRNA per 2.5 × 105 cells using e Neon® transfection system based on electroporation technology. The following day, the cells were treated with 0.05% DMSO or 0.5 μM Trifloxystrobin/TRF. Scrambled siRNA (siScr) and DR5 siRNA were used. The DR5 siRNA sequence was sense 5′-CAGACUUGGUGCCCUUUGA-3′ and antisense 5′-UCAAAGGGCACCAAGUCUG-3′.

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Apotox Glo assay: cell viability, cytotoxicity, and apoptosis [2]
For the ApoTox-Glo™ Triplex Assay, cells were pelleted by centrifugation at 200 g for 5 min at room temperature (RT) and then counted on a hemocytometer. Cells were seeded in 100 µL of media containing ~10,000 cells into each well of three black 96 well plates. Four wells were seeded with 100 µL of media lacking cells and were used to correct background fluorescence and luminescence. Differentiated cells were treated with one concentration of either fungicide over a dose range of 25–200 µM for azoxystrobin and 25–200 µM for Trifloxystrobin (n = 4 wells/dose). At 4, 24, and 48 h, 100 µL of media was removed to bring the final volume down to 100 µL, then 20 µL of reconstituted Viability/Cytotoxicity Reagent was added to each well. After 30 s of orbital shaking at 400 rpm, the plate was incubated for 1.5 h in a 37 °C CO2 incubator.

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ATP and metabolically active cells [2]
The CellTiter-Glo® Luminescent Cell Viability Assay determines ATP levels in SH-SY5Y cells which is an indicator of metabolically active cells. SH-SY5Y cells were first collected and seeded in 100 µL containing ~10,000 cells into each well of three white 96 well culture plates. Differentiated cells were treated for 6, 24, and 48 h. Antimycin (AM) was used as a positive control for the assay because it binds to cytochrome c reductase and inhibits cellular respiration and ATP production. Fluazinam was used as a positive control because it is a potent mitochondrial uncoupler which lowers ATP levels. Cells were exposed to either media only, 0.1% DMSO solvent control, positive controls (10 μM antimycin or 25 and 50 μM fluazinam), or one dose of azoxystrobin of Trifloxystrobin at 0.1–200 μM. Each chemical was assessed under approximately 8 or more different concentrations at each time point. Cells were incubated with the chemicals for the appropriate time in an incubator at 5% CO2 at 37 °C. After azoxystrobin and trifloxystrobin treatments, an equal volume of CellTiter-Glo® reagent was added to the media in each well, followed by orbital shaking at 400 rpm for 30 s and incubation at room temperature for 10 min. The luminescence was then read using a Synergy™ 4 Hybrid Microplate Reader (BioTek), using an automatic gain adjustment.

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Mitochondrial membrane potential (MMP) [2]
The Mitochondrial Membrane Potential Kit (MAK160–1KT) was used to measure the effect of fungicides on MMP in SH-SY5Y cells at 4, 24, and 48 h (n = 4/experimental group) and followed our protocol (Sanchez et al., 2020). Cells were seeded in 100 µL with 10,000 cells in each well of three black 96 well plates, differentiated, and treated with one concentration of either azoxystrobin or Trifloxystrobin (6.25 up to 100 μM) or either solvent control (0.1% DMSO), media, or a positive control (4 and 8 μM FCCP or 10 μM antimycin, AM). Fluorescence excitation/emission intensities of 540/590 nm (red) and 490/525 nm (green) were measured using a Synergy™ 4 Hybrid Microplate Reader (BioTek) set to automatic gain adjustment. Graphs represent the mean proportion of red/green fluorescence intensities across wells per treatment group from three independent experiments.

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Intracellular reactive oxygen species in differentiated SH-SY5Y cells [2]
Reactive oxygen species (ROS) production was measured using the ROS-Glo™ H2O2 Assay following the manufacturer’s protocol. Our methods followed that outlined in (Sanchez et al., 2020) (n = 4/experimental group). Cells were seeded at 10,000 cells and exposed to one dose of either 6.25, 12.5, 25, 50, or 100 μM azoxystrobin or 6.25, 12.5, 25, 50, or 100 μM Trifloxystrobin for 4-hour by adding 10 µL of 10X stocks together with 20 µL H2O2 substrate solution. The plate was then returned to a 37 °C incubator for 4 h, after which 100 µL of reconstituted ROS-Glo™ Detection Solution was added to each well. The plate was then incubated at room temperature for 20 min before luminescence was measured using a Synergy™ 4 Hybrid Microplate Reader set to automatic gain adjustment. The experiment was repeated independently three times under the same conditions. Menadione at two different concentrations was used as a positive control for ROS production.

Exposure for embryos acute toxicity [3]
Acute-toxicity test of zebrafish embryo was conducted according to the OECD Draft Proposal-Fish Embryo Toxicity (FET) Test (OECD, 2013) and a previously proposed method (Fraysse et al., 2006). Embryos at 2 hpf were randomly distributed in 24-well culture plates (2 mL solution and 1 embryo per well) for exposure to the test solutions (pyraclostrobin: 30.0, 37.5, 47.0, 58.6, 73.0 μg/L; Trifloxystrobin: 30.0, 37.5, 47.0, 58.6, 73.0 μg/L; picoxystrobin: 60, 69, 79, 91, 105 μg/L) for 96 h. Test concentrations were designed based on pre-experiment data (data not shown). Reconstituted water was used to prepare all test solutions, which was also served as blank control. Solvent control was arranged containing the same acetone and Tween-80 contents as that in the test solutions with the highest concentrations of each fungicide. In each 24-well plate, 20 wells contained test solution, and 4 wells contained reconstituted water as the internal control. Each concentration and control replicated three times (per plate as one replicate) and contained 60 embryos. All tested 24-well plates were placed in an incubator (27 ± 1 °C; 14:10 h light/dark photoperiod). The plates were covered with transparent lids to prevent evaporation. The exposure solution was renewed every 24 h to keep the appropriate concentration of fungicides and water quality. Dead individuals were immediately removed during exposure. Morphological development and abnormalities were checked daily and recorded using an inverted microscope. The heartbeat rates were measured by counting the number of heartbeat of surviving zebrafish embryos/larvae at 72 hpf in a 20 s period using a microscope. Hatching rate of embryos was calculated as a percentage of the hatched eggs at 72 hpf. The body length of 96 hpf larvae was measured by using Aigo GE-5.

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Exposure for enzyme activity and gene expression tests [3]
Embryos at 2 hpf were randomly transferred into test solutions (pyraclostrobin: 0, 10, 20, 40 μg/L; Trifloxystrobin: 0, 10, 20, 40 μg/L; picoxystrobin: 0, 15, 30, 60 μg/L) in 1 L beakers. The concentrations were selected based on the results of acute toxicity and some reported environmental concentrations. The lowest concentration was about 1/6 of the 96 h-LC50 value and lower than that detected in paddy water in China (Cao et al., 2015; Guo et al., 2016); the highest concentration was about 2/3 of the 96 h-LC50 value and had adverse effects on embryos. Each beaker contained 500 mL of exposure solution and 200 embryos, and there were 3 beakers in each concentration treatment. The external conditions during exposure, including the temperature, humidity and light cycle, were the same as that in the acute toxicity test. The exposure solution was renewed every 24 h to keep the appropriate concentration of fungicides and water quality. At 96 hpf, embryos (120 for antioxidant index measurement; 30 for RNA extraction) from each replicate were collected and washed twice with reconstituted water. The embryo samples were stored at −80 °C for further study.

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Soil and earthworms [4]
As recommended by Organization of Economic and Co-operation Development (OECD, 2016) guidelines, artificial soil prepared for this study was composed of silica sand (70%), kaolin (20%) and sphagnum peat (10%), with a pH of 5.5–6.5, adjusted using calcium carbonate. By addition of deionized water, the moisture content was set as 35% by weight of dry soil. The natural soil selected for this research was a brown soil, which was collected from farm-oriented fields in Taian City (Shandong Province, China, 36°09′N, 117°09′E). The soil is classified as brown soil according to the genetic soil classification of China (GSCC). The district where the brown soil was collected had no history of Trifloxystrobin application. The brown soil was sieved less to than 2.0 mm for use in this study. Essential data about artificial and brown soils is provided in the “Supplementary Material” Table S1. Earthworms (Eisenia fetida), procured from Guyongjin Earthworm farm, were raised for two weeks to acclimated to laboratory conditions prior to toxicity testing. Healthy individuals (weight between 0.3 and 0.6 g) with fully-developed clitella were chosen at random. Ahead of exposure to Trifloxystrobin, earthworms were placed in non-toxic target soils for 24 h.

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Toxicity experiment design [4]
Subchronic toxicity tests were performed simultaneously in artificial soil and brown soil. Soils were prepared by spiking 50 g of soil with 1 mL of Trifloxystrobin-acetonitrile solution. Following mixing and acetonitrile volatilization, spiked soil was mixed with additional soil to prepare 500 g aliquots containing 0.1, 1.0 and 2.5 mg/kg Trifloxystrobin. Control soil were prepared with uniform manner without the addition of Trifloxystrobin. Soil moisture was made adjustment to 60% field capacity through the addition of deionized water. For each exposed dose, three replicates were prepared with 10 earthworms added and incubated at 20 ± 1 °C for up to 56 days. Beakers were sealed with holed plastic film to ensure ventilation with moisture content monitored throughout the exposure period (gravimetrically) and adjusted if necessary. On days 7, 28 and 56, nine earthworms were sampled randomly from each concentration (3 per replicate) and depurated overnight prior to the measurement of exposure indices (ROS, SOD, CAT, GST, MDA and 8-OHdG). Three Eisenia fetida were used for ROS content determination, three Eisenia fetida for 8-OHdG content determination, and three Eisenia fetida for the other enzyme indices. During the entire exposure period, the mortality of earthworms in all controls was 0 and that in all exposed groups was ≤90%, which was consistent with the OECD (2016) guidelines.

Absorption, Distribution and Excretion
Male and female rats were dosed by gavage with either [Glyoxyl-Phenyl-(U)-(14)C] (spec. act. range: 54.3 to 63.5 uCi/mg, radiochemical purity range: >97 to >99%) or [Trifluormethyl-Phenyl-(U)-14C] /trifloxystrobin/ (CGA- 279202) (spec. act.: 59.2 uCi/mg, radiochemical purity: >99%). For all of the groups except D2, the animals were dosed with [Glyoxyl-Phenyl-(U)-(14)C]-/trifloxystrobin/ (CGA 279202). In Groups B1 and D1, urine and feces samples were collected up to 7 days from 5 animals/sex dosed with 0.5 or 100 mg/kg of the test material, respectively. For Group C1, 5 animals/sex were pretreated for 14 days with 0.5 mg/kg of unlabelled /trifloxystrobin/ (CGA 279202) (purity: 99.7%), followed by 0.5 mg/kg of the labelled test material. Urine and feces samples were likewise collected from these animals for up to 7 days. In Group D2, 5 animals/sex were dosed with 100 mg/kg of [Trifluormethyl-Phenyl-(U)-(14)C] /trifloxystrobin/ (CGA-279202) and urine and feces samples were collected up to 7 days. Twelve animals/sex were dosed with either 0.5 or 100 mg/kg of the test material in Groups F1 and 5 and Groups F2 and 6, respectively. Tissue residues were determined at 4 time points based upon pharmacokinetic determinations derived from the previous groups. The bile ducts of animals in Group G were cannulated. In Groups G1 and 3, 6 males and 5 females were treated with 0.5 mg/kg of the test material. Six males and 4 females were dosed with 100 mg/kg of the test material in Groups G2 and 4, respectively. At the low dose level, 56 to 65% of the dose was absorbed with 41 to 47% of the dose recovered from the bile. In the high dose group, 25 to 45% of the dose was absorbed with 19 to 35% of the dose recovered from the bile. In the low dose treatment, 18 to 19% and 79% of the dose was excreted in the urine and feces, respectively, of the males. For the females, 35 to 42% was excreted in the urine and 56 to 63% in the feces. Pretreatment with unlabelled test material did not alter the pattern of excretion. In the high dose groups, the males excreted 10 to 12% and 82 to 84% in the urine and feces, respectively. The females excreted 27% in the urine and 64 to 66% in the feces. Very minimal levels of radiolabel were recovered from the expired air of the animals in Group D2. The half lives for the depletion of radiolabel from the tissues ranged from 13 to 42 hours except for the spleen and blood of the high dose females (68 and 82 hours, respectively). The times to maximal concentration of the test material in the blood were either 12 to 24 hours after dosing. The times to maximal concentration ranged from 23 to 67 hours after dosing. Residual retention of the radiolabel in the carcass after 7 days was very minimal with 0.3 to 0.5% of the dose administered recovered.
Trifloxystrobin was moderately absorbed from the gastrointestinal tract and rapidly distributed. In the low-dose group, approximately 56% and 65% administered dose (AD) was absorbed in males and females respectively (based on the total recovery from urine, feces, bile and tissues), with 41 and 47% being in bile of males and females, respectively. In the high-dose, group, the degree of absorption was 41 and 27%, while the bile content was 35% and 19%, respectively for males and females. The blood kinetics revealed a moderate absorption rate in both sexes with two peaks (after 0.5 and 12 hours at the low dose and 12 and 24 hours at the high dose). The highest residues were found in blood, kidneys, spleen and liver and were comparable between sexes. Excretion of the radioactivity was rapid. Approximately 85-96% of the dose was excreted within 48 hours. The route of elimination was influenced by the sex of the animals, females eliminated twice the amount with the urine than males, accounting for 27-42% and 12-19% of the dose, respectively. The amounts excreted via feces were 79-82% and 56-64% of the dose in males and females, respectively. In both sexes biliary excretion was the major route of elimination. The involvement of an enterohepatic shunt mechanism in the elimination process is indicated.

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Metabolism / Metabolites
In rats, the absorbed compound was rapidly cleared with extensive metabolism, particularly through hydrolysis of the ester group. Other significant metabolic routes were O-demethylation of the methoxyimino group and oxidation of the methyl side chain to the corresponding alcohol and carboxylic acid.
Male and female rats were dosed by gavage with either [Glyoxyl-Phenyl-(U)-14C] (radiochemical purity range: >97 to >99%) or [Trifluormethyl-Phenyl-(U)-14C]-/trifloxystrobin/ (CGA- 279202) (radiochemical purity: >99%). For all of the groups except D2, the animals were dosed with [Glyoxyl-Phenyl-(U)-14C]-/trifloxystrobin/ (CGA 279202). ...Thirty five metabolites were isolated and identified from the urine, feces and bile samples. Major metabolic pathways included 1) hydrolysis of the methyl ester to the corresponding acid, 2) O-demethylation of the methoxyimino group, and 3) oxidation of the methyl side chain to a primary alcohol, followed by further oxidation to the carboxylic acid. This last reaction was a more prominent metabolic pathway in the female rats with the resultant isolation of major sex-specific urinary metabolites. Cleavage of the glyoxyl-phenyl and trifluoromethyl-phenyl moieties accounted for 10% of the dose. For the trifluoromethyl phenyl fragment, oxidation of the hydroxyimino group led to the formation of a nitro compound and oxidation of the methyl group resulted in the formation of the carboxylic acid. In addition, hydrolysis of the imino group formed an intermediate ketone with succeeding reactions ultimately leading to trifluoromethyl benzoic acid. For the glyoxyl-phenyl moiety, oxidation resulted in the formation of a benzoic acid. O-demethylation of the methoxyimino group resulted in the hydroxyimino compound. Hydrolysis of the imino group yielded the a-keto acid followed by decarboxylation to the phthalic acid. Conjugates with glucuronide or sulfate were isolated from the bile. Four to 7% and 31 to 47% of the low and high doses, respectively, were eliminated in feces as the unmetabolized test material. The absorbed dose was predominantly isolated in the bile. Further processing returned the test material and/or metabolites to the intestinal tract and elimination in the feces or reuptake via the enterohepatic pathway.

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Biological Half-Life
The tissue half-lives /in rats/ ranged from 13 to 42 hours.

Toxicity Summary
IDENTIFICATION AND USE: Trifloxystrobin is a white powder. It is used as agricultural fungicide. HUMAN EXPOSURE AND TOXICITY: Trifloxystrobin is harmful if absorbed through skin. Prolonged or frequently repeated skin contact may cause allergic reactions in some individuals. ANIMAL STUDIES: Trifloxystrobin was considered to be of low acute toxicity by the oral route in mice and rats, by the dermal route in rabbits, and by the inhalation route in rats. Trifloxystrobin was not selectively neurotoxic following acute gavage or subchronic dietary administration in rats. There were no treatment-related effects upon the mating, fertility, or litter size in rats. It was not mutagenic in S. typhimurium strains TA98, TA100, TA102, TA1535, or TA1357, or in E. coli strain WP2 uvrA with and without metabolic activation. ECOTOXICITY STUDIES: Trifloxystrobin is toxic to nontarget aquatic organisms. Trifloxystrobin could affect the activities of antioxidant enzymes, disrupt photosynthesis in Chlorella vulgaris, and damage cellular structure. Trifloxystrobin is highly toxic to fish embryos. It is very toxic to Daphnia magna and can cause harm to D. magna at environmentally relevant concentrations. Trifloxystrobin was very highly toxic to sheepshead minnows.

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Toxicity Data
LC50 (rat) > 4,646 mg/m3

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Non-Human Toxicity Values
LD50 Rabbit dermal >2000 mg/kg
LD50 Rat oral >4000 mg/kg
LD50 Rat oral >5000 mg/kg
LD50 Rat dermal >2000 mg/kg

[1]. Trifloxystrobin induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HaCaT, human keratinocyte cells . Drug and Chemical Toxicology, 2017, 40(1): 67-73.

[2]. Neurotoxicity assessment of QoI strobilurin fungicides azoxystrobin and trifloxystrobin in human SH-SY5Y neuroblastoma cells: Insights from lipidomics and mitochondrial bioenergetics. Neurotoxicology, 2022, 91: 290-304.

[3]. Developmental toxicity, oxidative stress and immunotoxicity induced by three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) in zebrafish embryos . Chemosphere, 2018, 207: 781-790.

[4]. Oxidative stress and DNA damage induced by trifloxystrobin on earthworms (Eisenia fetida) in two soils . Science of The Total Environment, 2021, 797: 149004.

[5]. Trifloxystrobin blocks the growth of Theileria parasites and is a promising drug to treat Buparvaquone resistance . Communications Biology, 2022, 5(1): 1253.

Mechanism of Action
... The present study was performed in order to identify the skin toxicity mechanism of trifloxystrobin using HaCaT (keratinocyte of human skin) cells. Following 24 or 48 hr treatment, cell viability, and subsequent Annexin V-FITC/propidium iodide assay, TUNEL assay and Western blotting were performed to investigate the cell death mechanism of trifloxystrobin. Exposure to trifloxystrobin resulted in diminished viability of HaCaT cells in both a time- and concentration-dependent manner. The cell death was derived through apoptotic pathways in the HaCaT cells. Furthermore, we explored the effect of trifloxystrobin on TRAIL-mediated extrinsic apoptosis using siRNA transfection. Knockdown of death receptor 5 suppressed trifloxystrobin-provoked apoptosis. These results indicate that trifloxystrobin induces TRAIL-mediated apoptosis and has an inhibitory effect in keratinocytes that can interfere with the barrier function and integrity of the skin ...
Strobilurin fungicidal activity inhibits mitochondrial respiration by disrupting the cytochrome complex, thus blocking electron transfer. /Strobilurin fungicides/

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Trifloxystrobin is the methyl ester of (2E)-(methoxyimino)[2-({[(E)-{1-[3-(trifluoromethyl)phenyl]ethylidene}amino]oxy}methyl)phenyl]acetic acid. A foliar applied fungicide for cereals which is particularly active against Ascomycetes, Deuteromycetes and Oomycetes It has a role as a mitochondrial cytochrome-bc1 complex inhibitor and an antifungal agrochemical. It is an oxime O-ether, an organofluorine compound, a methyl ester and a methoxyiminoacetate strobilurin antifungal agent.
Benzeneacetic acid, alpha-(methoxyimino)-2-[[[(E)-[1-[3-(trifluoromethyl)phenyl]ethylidene]amino]oxy]methyl]-, methyl ester, (alphaE)- has been reported in Apis cerana with data available.
Trifloxystrobin is a foliar applied fungicide for cereals which is particularly active against Ascomycetes, Deuteromycetes and Oomycetes. It has a broad spectrum of action with preventative and curative action. It is a respiration inhibitor (QoL fungicide).

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In summary, the sustained use of strobilurins in the agricultural sector has led to studies examining the potential adverse effects of these broad-spectrum fungicides on non-target humans and animals. These fungicides can potentially impact mitochondrial bioenergetics in neurons, and investigating their neurotoxicity is warranted, especially due to a direct causal link between mitochondrial dysfunction and neurodegenerative diseases. In fact, epidemiological studies have attributed transcriptional markers associated with autism, brain aging and neurodegeneration to the effects of environmental neurotoxicants, including strobilurins such as pyraclostrobin, trifloxystrobin, and fenamidone (Pearson et al., 2016). Mechanistic studies are therefore warranted that quantify the risk and investigate neurotoxic properties of strobilurin fungicides in neuronal cells following environmental and occupational exposures. [2]

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In summary, our results demonstrated that pyraclostrobin, trifloxystrobin and picoxystrobin exhibited high level of acute toxicity to zebrafish embryos. Embryos exposed to the three fungicides showed decreased heart rate, hatching inhibition, growth regression, and morphological deformities in a concentration-dependent manner. The underlying mechanisms of this developmental toxicity might be partly related to the abnormal generation of ROS, increase of MDA content, change of antioxidant enzymes activities and mRNA levels of genes related to oxidative stress and immune system. The different changes of these parameters might be responsible for the toxicity difference between the three fungicides. As the molecule target of strobilurins on fungus is mitochondria complex Ⅲ, the influence of these fungicides on fish mitochondria is needed in future study to fully understand the toxic mechanisms of strobilurin fungicides on aquatic organisms.[3]

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This study expanded the comprehension about toxicity mechanism of trifloxystrobin on earthworms, on the basis of which, differences in toxicity between artificial soil and brown soil were evaluated. After exposure to trifloxystrobin, ROS contents in earthworm accumulated which increased with increasing of exposure concentrations. Antioxidant enzymes (SOD, CAT) and detoxifying enzyme (GST) in Eisenia fetida showed different changes caused by the production of ROS. These results show that trifloxystrobin can induce oxidative stress on Eisenia fetida in artificial and brown soils. Moreover, trifloxystrobin can also cause lipid peroxidation and DNA damage (trifloxystrobin above 2.5 mg/kg), which were showed the increase both MDA content and 8-OHdG content in earthworms. The IBR values following 56 days exposure to 2.5 mg/kg trifloxystrobin showed that the trifloxystrobin toxicity in brown soil was greater than that in artificial soil. Ecotoxicological experiments in artificial soil may have underestimated the toxicity of trifloxystrobin on earthworm. Above results provided a theoretical foundation for further understanding the toxicological effects of strobilurins in real situation, and may be beneficial for assessing exposure of strobilurin fungicides in soil ecosystem.[4]

C20H19F3N2O4

408.3772

408.129

C, 58.82; H, 4.69; F, 13.96; N, 6.86; O, 15.67

141517-21-7

Trifloxystrobin-d6;2470226-50-5;Trifloxystrobin-d3

9578570

White to off-white solid powder

1.2±0.1 g/cm3

470.3±55.0 °C at 760 mmHg

72.9°

238.3±31.5 °C

0.0±1.2 mmHg at 25°C

1.511

5.11

0

9

8

29

607

0

C/C(=N\OCC1=CC=CC=C1/C(=N/OC)/C(=O)OC)/C2=CC(=CC=C2)C(F)(F)F

ONCZDRURRATYFI-QTCHDTBASA-N

InChI=1S/C20H19F3N2O4/c1-13(14-8-6-9-16(11-14)20(21,22)23)24-29-12-15-7-4-5-10-17(15)18(25-28-3)19(26)27-2/h4-11H,12H2,1-3H3/b24-13+,25-18-

methyl (2Z)-2-methoxyimino-2-[2-[[(E)-1-[3-(trifluoromethyl)phenyl]ethylideneamino]oxymethyl]phenyl]acetate

CGA 279202; 141517-21-7; Benzeneacetic acid, alpha-(methoxyimino)-2-[[[(E)-[1-[3-(trifluoromethyl)phenyl]ethylidene]amino]oxy]methyl]-, methyl ester, (alphaE)-; CHEBI:81833; CGA 279202; Consist; Trifloxystrobine; Zato; Trifloxystrobin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~306.09 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (5.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.17 mg/mL (5.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)