Fungicidal; Bax; Bcl-2; autophagy; AMPK/mTOR

Herein, the toxicological risks of Pyraclostrobin toward HepG2 cells and the mechanisms of intoxication in vitro were investigated. The liver toxicity of pyraclostrobin in zebrafish larvae was also evaluated. It was found that pyraclostrobin induced DNA damage and reactive oxygen species generation in HepG2 cells, indicating the potential genotoxicity of pyraclostrobin. The results of fluorescent staining experiments and the expression of cytochrome c, Bcl-2 and Bax demonstrated that pyraclostrobin induced mitochondrial dysfunction, resulting in cell apoptosis. Monodansylcadaverine staining and autophagy marker-related proteins LC3, p62, Beclin-1 protein expression showed that pyraclostrobin promoted cell autophagy. Furthermore, immunoblotting analysis suggested that pyraclostrobin induced autophagy accompanied with activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/mTOR signaling pathway.[1]

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Effects of Pyraclostrobin on the survival rate and proliferation of HepG2 cells [1]
MTT assay was used to determine the cytotoxicity of pyraclostrobin on HepG2 cells. HepG2 cells were treated with different concentrations of pyraclostrobin for 24 h, the cell survival rate was negatively correlated with the concentration of pyraclostrobin and presented a concentration-dependent manner (Fig. 1A). The IC50 value of pyraclostrobin on HepG2 cells was estimated to be 30.22 μmol/L (Table 1).

The effect of Pyraclostrobin on human HepG2 cells proliferation by cell cloning experiment (Fig. 1B). As shown in Fig. 1D, after 6 h of exposure to pyraclostrobin (10, 20, 40, and 80 μmol/L), the cell clonal formation was 79.31, 31.03, 6.89, and 1.72%, respectively. The cell clonal formation rate decreased sharply with the increase of pyraclostrobin concentration, which showed a concentration-dependent relationship. These above data suggested that pyraclostrobin fungicide dramatically inhibited the survival and proliferation of HepG2 cells.

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Pyraclostrobin induced DNA damage in HepG2 cells [1]
Single cell gel electrophoresis (SCGE) assay was the most sensitive and rapid way to detect DNA damage. The degree of DNA damage can be evaluated by detecting migration optical density, tail length and tail moment. The parameters of neutral comet assay were summarized in Table 2, indicating that the phenomenon of comet (tail DNA %) is quite noticeable. As illustrated in Fig. 1E, HepG2 cells treated with 10–80 μmol/L pyraclostrobin significantly increased the tail DNA content and the formed tail length of comets compared to untreated cells. Meanwhile, Fig. 1C displayed the ratio of comet-positive cells increasing with a dose-dependent manner. When the exposure concentrations of pyraclostrobin were 0, 10, 20, 40, and 80 μmol/L, the percentages of DNA damaged cells were 7.12, 33.69, 46.64, 67.31, and 77.74%, respectively. The results showed that pyraclostrobin caused DNA single-strand break in HepG2 cells.

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Pyraclostrobin induced mitochondrial dysfunction in HepG2 cells [1]
Mitochondrial membrane potential (MMP) maintains the normal structure and function of mitochondria by regulating the selectivity and permeability of mitochondrial membrane (Tait and Green, 2012). To investigate whether mitochondrial dysfunction occurred in HepG2 cells exposure to pyraclostrobin for 6 h, the quantitative analysis of mitochondrial membrane potential (ΔΨm) was examined by fluorescent microscopy. As can be evidence from Fig. 2B and D, the green fluorescence intensity in HepG2 cells stained with Rho-123 showed a declining trend in a concentration-dependent manner, suggesting that pyraclostrobin led to ΔΨm collapse in HepG2 cells.

Mitochondrial damage can bring about excessive generation of ROS. Accordingly, ROS-sensitive probe DCFH-DA was used for detecting the intracellular ROS production in HepG2 cells. The fluorescent intensity can reflect the intracellular ROS levels of cells. As depicted in Fig. 2A and C, the DCF fluorescence signal intensity was significantly increased in Pyraclostrobin-treated HepG2 cells compared with the control cells, demonstrating that pyraclostrobin induced intracellular ROS production in a dose-dependent manner. Taken together, these findings suggested that pyraclostrobin induced mitochondria dysfunction, resulting in overproduction of ROS.

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Effects of Pyraclostrobin on apoptosis-related protein levels in HepG2 cells [1]
In order to explore the underlying mechanism of pyraclostrobin-induced apoptosis, hepatocellular carcinoma cells were treated with different concentrations of pyraclostrobin and the expressions of apoptosis-associated proteins were analyzed by immunoblotting. As exhibited in Fig. 3C and D, the content of cytochrome c (Cyt c) in the cytoplasm was increased in a concentration-dependent way with increasing concentration of pyraclostrobin, which proved that pyraclostrobin accelerated the release of Cyt c. In addition, pro-apoptotic protein Bax expression was decreased and anti-apoptosis protein Bcl-2 expression was down-regulated simultaneously (Fig. 3A and B). The above results showed that pyraclostrobin impaired the mitochondrial membrane, leading to the change of Bax/Bcl-2 to activate an apoptotic pathway.

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Effects of Pyraclostrobin on autophagy vesicles and autophagy-related proteins in HepG2 cells [1]
The morphological feature of autophagy is the formation of autophagy vesicles. MDC, an autofluorescent dye, was used to label pyraclostrobin-treated HepG2 cells, further observe autophagolysosomes under a fluorescence microscope. The images displayed an increase of fluorescent intensity in HepG2 cells, indicating that the number of autophagic vacuoles increased in the treatment of pyraclostrobin (Fig. 4 A). The results showed that pyraclostrobin was able to induce the formation and accumulation of autophagosomes in human HepG2 cells, and the promoting effect of pyraclostrobin was concentration-dependent.

To further clarify the mechanism of Pyraclostrobin triggering autophagy, Western blotting was performed to determine the expression of major autophagy-related proteins in HepG2 cells treated with pyraclostrobin for 6 h. The LC3 protein possesses two forms: LC3-I and LC3-II, and the transformation of LC3-I to LC3-II is considered as a marker of autophagy. Beclin-1 is essential for autophagy membrane nucleation, and its binding with autophagy precursor makes it a key protein for autophagy initiation and progression (Hao et al., 2019). Compared to the control group, the expression ratio of LC3-II/I and Beclin-1 protein were both increased, while the expression of p62 was remarkably descended (Fig. 4B and C). These results powerfully confirmed that pyraclostrobin promoted autophagy in HepG2 cells. Furthermore, as shown in Fig. 4D and E, the phosphorylated levels of mTOR and p70s6k after exposure to pyraclostrobin were gradually inhibited, and the AMPK phosphorylation was significantly raised in a dose-dependent way. Based on the above data, the pyraclostrobin-mediated autophagy in HepG2 cells involved the AMPK/mTOR signaling pathway.

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Pyraclostrobin induced fluorescence colocalization of mitochondria and lysosomes [1]
To further determine if the damaged mitochondria were bound to lysosomes, fluorescent probes were used to detect the lysosome mass. As shown in Fig. 5, the fluorescence intensity from Lyso-tracker Red, which evaluated lysosome activity, was gradually increased in the pyraclostrobin-treated groups. The merging photos of mitochondria and lysosomal fluorescence displayed that mitochondria were gradually degraded by lysosomes. The co-localization results showed that the damaged mitochondria in pyraclostrobin-induced cells might be engulfed by lysosomes.

Pyraclostrobin is a highly effective and broad-spectrum strobilurin fungicide. With the widespread use of pyraclostrobin to prevent and control crop diseases, its environmental pressure and potential safety risks to humans have attracted much attention. Visualization of zebrafish liver and oil red staining indicated that pyraclostrobin could induce liver degeneration and liver steatosis in zebrafish. Collectively, these results help to better understand the hepatotoxicity of pyraclostrobin and provide a scientific basis for its safe applications and risk control.[1]

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The aim of the present study was to assess the toxic effects of Pyraclostrobin on DNA damage and antioxidant enzymatic activities in the zebrafish (Danio rerio) liver. Based on the 96-h median lethal concentration (96 h LC50, 0.056 mg/L) of this chemical, fish were exposed to three doses (0.001, 0.01, and 0.02 mg/L) and sampled on days 7, 14, 21 and 28 after the initiation of a subchronic toxicity test. The levels of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), glutathione S-transferase (GST), reactive oxygen species (ROS) and DNA damage were determined. The amount of pyraclostrobin residue in the water was also measured. The concentrations in the three treatment groups varied no more than 5% during the exposure periods, indicating that pyraclostrobin is relatively stable during this time in an aquatic environment. ROS and MDA levels significantly changed in a dose dependent manner during the experiment. Enzymatic activities were inhibited to a certain extent. DNA damage was significantly enhanced. These results collectively indicate that pyraclostrobin induces oxidative stress and DNA damage in zebrafish.[2]

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Pyraclostrobin is widely used to control crop diseases, and was reported to be highly toxic to aquatic organisms. The molecular target of pyraclostrobin to fungus is the mitochondrion, but its effect on mitochondria of aquatic organisms has rarely been investigated. In this study, zebrafish larvae at 4 days post fertilization (dpf) were exposed to a range of pyraclostrobin for 96 h to assess its acute toxicity and effects on mitochondria. Pyraclostrobin at 36 μg/L or higher concentrations caused significant influences on larval heart and brain including pericardial edema, brain damage malformations, histological and mitochondrial structural damage of the two organs. The results of RNA-Seq revealed that the transcripts of genes related to oxidative phosphorylation, cardiac muscle contraction, mitochondrion, nervous system development and glutamate receptor activity were significantly influenced by 36 μg/L pyraclostrobin. Further tests showed that pyraclostrobin at 18 and 36 μg/L reduced the concentrations of proteins related to cardiac muscle contraction, impaired cardiac function, inhibited glutamate receptors activities and suppressed locomotor behavior of zebrafish larvae. Negative changes in mitochondrial complex activities, as well as reduced ATP content were also observed in larvae treated with 18 and 36 μg/L pyraclostrobin. These results suggested that pyraclostrobin exposure caused cardiotoxicity and neurotoxicity in zebrafish larvae and mitochondrial dysfunction might be the underlying mechanism of pyraclostrobin toxicity [3].

Cell viability assay [1]
As described in the literature, MTT assay can detect HepG2 cell viability (Grela et al.). The HepG2 cells were harvested by trypsinization. The cell density was adjusted to 1 × 105 cells/mL with a cell counting apparatus. Pour 100 μL cell suspension onto a 96-well plate and incubated it at 37 °C in a 5% CO2 incubator of 24 h. Then Pyraclostrobin was added with a series concentration (10, 20, 40, and 80 μmol/L). After 24 h of treatment, 20 μL MTT reagent (5 mg/mL) was added to each well. Let stand for 4 h in the incubator, the upper MTT solution and the medium solution were absorbed, followed adding 150 μL DMSO to dissolve formazan. Then, the absorbance at 492 and 630 nm of each well was measured using a Synergy H1 microplate reader (Bio-Teck, Winooski, VT, USA).

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Cell proliferation assay [1]
Colony formation assay is an essential marker of cytotoxicity. HepG2 cells were seeded in a 6-cm cell culture dish at a density of 500 cells/ml for 24 h and then inoculated with 10, 20, 40, and 80 μmol/L Pyraclostrobin. The control group was fresh medium containing 0.1% DMSO. After 10 days, the medium is sucked out of the pore. Then 5% glutaraldehyde was used for fixation, 10% Giemsa staining, and the colony count was examined by an atomical microscope.

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DNA damage assay [1]
The alkaline comet assay is the most sensitive and rapid method to detect DNA damage (Cetinkaya et al., 2016; Zhang et al., 2019). Pyraclostrobin was diluted to 0, 10, 20, 40 and 80 μmol/L. Added 2 mL of the test solution to each well and put it back into the incubator for 12 h. Then the cells were put into a centrifuge at 4 °C to collect precipitation. PBS was washed the precipitation for 3 times to remove pyraclostrobin, and the preheated low melting point agarose gel was mixed with cells containing PBS in a ratio of 1 : 5. After blending, 100 μL gel was dripped onto the slide (Ghassemi-Barghi et al., 2016). Agarose was coagulated at 4 °C for 15 min, and the slides were immersed in the fresh lysate (10% DMSO, 10 mM Trise-HCl, 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, pH 10) at 4 °C for 30 min. After lysis, the slides were rinsed three times with deionized water, and soaked in fresh alkaline electrophoretic solution at 4 °C for 10 min. Electrophoresis was performed at 20 V for 20 min. After electrophoresis, the slides were rinsed with neutralization buffer for 3 times and then with deionized water for 3 times. Then PI reagent (20 mg/mL) was added to stain for 5 min. Finally, the slides were examined with a fluorescence microscope and the degree of DNA damage was analyzed by an image analysis system.

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Mitochondrial membrane potential analysis [1]
Rhodamine123 (Rho-123) was used to detect Δψm to analyze whether mitochondrial damage HepG2 cells (Ferlini and Scambia, 2007). Cells were treated with Pyraclostrobin at a specified concentration in a 6-well plate for 12 h. The cell surface was washed with PBS buffer for 3 times, then stained with Rho-123 for 15 min in darkness. Fluorescence intensity of pyraclostrobin treated HepG2 cells were detected by fluorescence microscopy.

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Intracellular ROS measurement [1]
DCFH-DA is a common way to detect changes in intracellular ROS levels. Pyraclostrobin was treated with a specified concentration of pyraclostrobin for 6 h, and HepG2 cells were washed twice by cold PBS buffer. Then 1 mL DCFH-DA (10 M) staining solution was added to each well and incubated in an incubator at 5% CO2 and 37 °C for 30 min. The fluorescence (excitation at 488 and 530 nm) intensity of ROS was observed and recorded by fluorescence microscope.

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Western blotting [1]
In order to investigate the potential mechanism of Pyraclostrobin ether-induced HepG2 cell death, Western blot was used to analyze the specific proteins. After treatment with pyraclostrobin at a specified concentration for 6 h, the cells were washed with cold PBS (pH 7.4) for 3 times and harvested. Then the cells were centrifuged at 4 °C, 12,000 rpm for 15 min. We collected the supernatant and determined the protein concentration by BCA. After 8–15% SDS-PAGE treatment, the same volume of protein was transferred to polyvinylidene fluoride (PVDF) membrane by electrophoresis. The membrane was sealed with 5% milk in Tris-buffered saline-Tween (TBST; 10 mM Tris-HCl, 150 mM NaCl, 0.1% of Tween-20, pH 7.5) for 2 h. Then, the membrane was incubated at 4 °C with primary antibodies overnight and incubated at room temperature with secondary antibody for 1 h. After treatment with enhanced chemiluminescence (ECL) reagent, visualized signals came out. Finally, all the protein bands were scanned by ImageJ software, and the IDVS were quantified and normalized to β-actin.

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Autophagy analysis [1]
Monodansylcadaverine (MDC) can specifically mark the formation of autophagy vesicles (Cárdenas et al., 2010). Pyraclostrobin was diluted to 0, 10, 20, 40, and 80 μmol/L. Added 2 mL of the test solution to each well and put it back into an incubator at 5% CO2 and 37 °C for 6 h. Then the drug-containing medium in the suction hole was used to wash the cell surface with PBS solution for 3 times, followed adding 1 mL of MDC dye to each well. After incubation in the incubator for 30 min, MDC staining solution was sucked out, and the cell surface was cleaned with PBS solution for 3 times. MDC fluorescence intensity was observed and recorded by fluorescence microscopy.

Zebrafish larvae toxicity testing [1]
The AB-wild type adult zebrafish and Tg (fabp10a:dsRed; ela3l:EGFP) transgenic line were purchased from China Zebrafish Resource center. Zebrafish were cultured in a recirculating culture system (the temperature was at 28 °C; the light-dark cycle of 14:10 h). Male and female zebrafish were chosen in equal proportions for spawning, following previously established procedures (Lu et al., 2022). The collected zebrafish larvae (72 h post fertilization, 72 hpf) were subjected to Pyraclostrobin (0, 0.01, 0.02, 0.04, and 0.08 μmol/L) exposure persisting until 72 hpf, and there were 20 zebrafish larvae in each group. Following exposure, the zebrafish were then observed and imaged by a fluorescence microscope.

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The fish were fed bait each day at regular intervals until 24 h before the acute and subchronic tests were performed. Half of the water was replaced at the time every 2 days, and feces, redundant bait and dead fish were extracted using the siphon method to avoid interference. The acute toxicity test is a static test that was performed to acquire the 96 h LC50 of Pyraclostrobin. The concentrations that led to acute toxicity were 0, 0.001, 0.01, 0.05, 0.06, 0.07, 0.08 and 0.1 mg/L. Each sample consisted of ten randomly selected fish and 1.5 L of exposed solution. Based on Passino and Smith (1987), the resulting 96 h LC50 was used to evaluate the acute toxicity (mg/L) of the pesticides in zebrafish as follows: less than 1, highly toxic; 1–10, moderately toxic; 10–100, slightly toxic; 100–1,000, practically harmless; and greater than 1,000, relatively harmless. The subchronic toxicity test for pyraclostrobin was performed a control group and three groups exposed to different levels of pyraclostrobin (i.e., 0.001, 0.01 and 0.02 mg/L). One hundred and twenty fish were randomly selected and assigned to a vessel containing 20 L of water at one of the three concentrations. The subchronic toxicity test is a semistatic test, and half of the exposed solution was replaced at the same time every 2 days to maintain the concentration of pyraclostrobin throughout the subchronic toxicity experiment. The fish were sampled in triplicate to analyze the levels of ROS, SOD, CAT, GST, MDA, and DNA damage on days 7, 14, 21 and 28. The control was set up using 1 mL of acetone dissolved in the same source of dechlorinated tap water to prevent interference from the solvent. Three replicates were performed for each trial in both the acute and the subchronic toxicity tests [2].

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Acute toxicity test [3]
Zebrafish larvae at 4 dpf were randomly transferred into 24-well plates and subjected to doses of 33, 36, 40, 44 and 48 μg/L Pyraclostrobin until 8 dpf, respectively. Both blank control and solvent control were set. Each plate contained twenty larvae with one larva in 2 mL solution and each concentration replicated three times (per plate as one replicate). All tested larvae were cultured in an incubator (27 ± 1 °C; 14:10 h light/dark photoperiod). Test solutions were renewed every 24 h. Mortality and abnormalities of larvae were examined daily under a light microscope (Olympus BH-2) and recorded by an inverted microscope. Percentage of deformed larvae was calculated by dividing malformed individuals by all surviving individuals in one replicate.

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Histological and subcellular structural analysis [3]
Zebrafish larvae were exposed to 0 and 36 μg/L Pyraclostrobin from 4 to 8 dpf under the same culture condition as that mentioned above. Each replicate contained 100 larvae and each concentration replicated three times. The dose of 36 μg/L pyraclostrobin was chosen mainly because death of zebrafish larvae was firstly observed at this concentration. At the end of the exposure, larvae were collected for histological and subcellular structural analysis, with both 15 larvae from each replicate (n = 3).
Larvae for histological analysis were fixed overnight with 4% paraformaldehyde (PFA) at 4 °C, then dehydrated using graded ethanol before paraffin embedding. Embedded larvae were sectioned (2–3 μm sections) and stained with hematoxylin and eosin (HE). Images were obtained with a NanoZoomer S210 and captured by an NDP. view 2.
Larvae for subcellular structural analysis were fixed in 2.5% glutaraldehyde for at least 2 h and washed with 0.1 M phosphate buffer (pH = 7.2) 3 times. Then, samples were fixed in 1% osmic acid for 2 h and washed 3 times with 0.1 M phosphate buffer (pH = 7.2). After dehydration in graded acetone, all the specimens were embedded in epoxy resin. Ultrathin sections taken from selected areas were prepared using an ultramicrotome and stained with uranyl acetate and lead citrate. Subcellular structure of the larvae was observed under Transmission Electron Microscopy.

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RNA-Seq analysis and RT-qPCR validation [3]
40 larvae that exposed to 0 and 36 μg/L Pyraclostrobin from 4 dpf to 8 dpf were collected and total RNA was extracted using a spin column method. RNA concentration and quality were determined using a NanoPhotometer spectrophotometer (Implen, Germany) and an Agilent Bioanalyzer 2100 (Agilent Technologies, USA). RNA-Seq of different samples was performed by Novogene company. Genes with adjusted p-value (padj) < 0.05 were defined as differentially expressed genes (DEGs). KEGG pathways and GO analysis were conducted using KOBAS (2.0) and GOseq (Release 2.12) based on the lists of DEGs (padj < 0.05) for each treatment, respectively. The detailed procedure of RNA-Seq analysis was presented in supplemental materials.

Fifteen candidate genes were chosen for validation by RT-qPCR using independent RNA samples from zebrafish larvae exposed to 0 and 36 μg/L Pyraclostrobin from 4 dpf to 8 dpf. Total RNA was extracted and 1 μg of RNA was used for first-stand cDNA synthesis using the FastQuant RT Kit (Tiangen Biotech, Beijing, China). Zebrafish-specific primers were designed for the genes of interest using Primer Premier 6.0 software (Table S2). The procedure of RT-qPCR was performed according to previous published protocols (Li et al., 2018b). mRNA levels of target genes were calculated and normalized against housekeeping gene β-actin by the 2−ΔΔCT method (Livak and Schmittgen, 2001). Three biological replicates and three technical replicates were performed for each sample. Negative controls (water blanks and total RNA without reverse transcription) were performed and thermal denaturation (melt curve analysis) were used to confirm product specificity (Fig. S15).

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Western blotting [3]
Zebrafish larvae were treated with 0, 9, 18 and 36 μg/L Pyraclostrobin for 96 h (n = 3 replicates, 40 larvae per replicate). At 8 dpf, larvae were homogenized in liquid nitrogen, and total protein was extracted for Western blot. Protein samples (about 50 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked and blots were incubated with mouse anti-DHPR (1:500) and rabbit anti- β-actin IgG (1:4000) followed by horseradish peroxidase (HRP) conjugated secondary antibodies (goat anti-mouse (1: 3000) and goat anti-rabbit (1: 3000). ECL reagent was applied to the membrane for 4 min. Chemiluminescence imaging system was used to evaluate the protein signal. The results of Western blot were quantified with Quantity One software.

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Larval locomotor behavior analysis [3]
Zebrafish larvae were treated with 0, 9, 18 and 36 μg/L Pyraclostrobin from 4 to 8 dpf (n = 3 replicates, 20 larvae per replicate) in 24-well plates under the same conditions as that in acute toxicity test. At the end of exposure, free swimming activities of larvae within 10 min were monitored using a USB 3.0 color video camera with an e2v CMOS sensor. The data of average velocity and moved distance were obtained from LoliTrack Version 4.2.0 software.

Absorption, Distribution and Excretion
Oral administration. The absorption, distribution, and elimination of pyraclostrobin were studied in male and female Wistar rats (aged at least 7 weeks) after oral administration of pyraclostrobin (purity, >98%) radiolabelled with carbon-14 at either the tolyl or chlorophenyl rings. ... In a series of four experiments, the excretion of pyraclostrobin was studied in excreta collected at 6, 12 and 24 hr after dosing, and at 24 hr intervals thereafter for 168 hr, or until 90% of the applied radioactivity had been excreted. In the first three experiments, groups of four male and four female rats were given a single oral dose of 14C-tolyl- or 14C-chlorophenyl-labelled pyraclostrobin or unlabelled pyraclostrobin at 50 mg/kg bw. In the fourth experiment, four rats of each sex were given a single oral dose of 14C-tolyl-labelled pyraclostrobin at 5 mg/kg bw. At the end of each of these experiments, the animals were sacrificed and the heart, liver, spleen, bone, skin, lung, ovaries, bone marrow, carcass, muscle, kidney, testes, brain, pancreas, uterus, adipose tissue, stomach and contents, thyroid glands, adrenal glands, blood/plasma and intestinal tract and contents were assessed for radioactivity. Exhaled air was also collected from two males in each of the two experiments using radiolabelled pyraclostrobin in order to determine exhalation of 14C-labelled gases. Two additional experiments were conducted to examine blood concentrations of radioactivity after administration of 14C-tolyl-labelled pyraclostrobin at 5 or 50 mg/kg bw. Blood samples (100-200 uL) were taken from animals at 0.5, 1, 2, 4, 8, 24, 48, 72, 96 and 120 hr after dosing, and the amount of radioactivity in whole blood and plasma was assessed. Tissue distribution was examined in animals sacrificed at 0.5, 8, 20 and 42 hr after dosing at 5 mg/kg bw, and at 0.5, 24, 36 and 72 hr after dosing at 50 mg/kg bw. The heart, liver, spleen, bone, skin, lung, ovaries, bone marrow, carcass, muscle, kidney, testes, brain, pancreas, uterus, adipose tissue, stomach and contents, thyroid glands, adrenal glands, blood/plasma and intestinal tract and contents were assessed for radioactivity. To examine biliary excretion of pyraclostrobin, bile ducts of the animals were cannulated and bile was collected at 3 hr intervals until 48 hr after administration of 14C-tolyl-labelled pyraclostrobin at 5 or 50 mg/kg bw in four animals of each sex at each dose (the duration depended on the health of the animals and the excretion rate at later time-points). In rats given a single dose of 14C-tolyl-labelled pyraclostrobin at either 5 or 50 mg/kg bw, plasma concentrations of radioactivity initially peaked after 0.5 to 1 hr; there was a secondary peak after 8 hr in males at 5 or 50 mg/kg bw and females given 5 mg/kg bw, and after 24 hR in females given 50 mg/kg bw. The magnitude of the difference in the time to peak for females, given the high dose, is likely to be at least partially artifactual owing to the absence of a sampling point between 8 and 24 hr. After the second peak, plasma concentrations declined to <0.1 ug equivalent/g after 120 hr. The terminal half-lives were similar in males and females, but were 50% longer at 5 mg/kg bw than at 50 mg/kg bw. The area under the curve of plasma concentration-time was approximately proportional to dose for each sex, indicating that absorption was not saturated at the higher dose.
After a single oral dose of 14C-tolyl-labelled pyraclostrobin at 50 mg/kg bw, the highest concentrations of radioactivity /in rats/ were found in the gastrointestinal tract (gut, 28 to 39 ug equivalent/g; gut contents, 63 to 92 ug equivalent/g; stomach, 325 to 613 ug equivalent/g; stomach contents, 1273 to 1696 ug equivalent/g) after 0.5 hr. The liver (13 to 25 ug equivalent/g) had higher concentrations of radioactivity than the kidneys (4 to 7 ug equivalent/g) and plasma (2 to 6 ug equivalent/g), with lowest values being recorded in the bone (0.1 to 0.3 ug equivalent/g) and brain (1 to 2 ug equivalent/g). After 72 hr, tissues and organs contained <2.6 ug equivalent/g. After a dose of 5 mg/kg bw, the highest concentrations of radioactivity were also found in the gastrointestinal tract (gut, 5 ug equivalent/g; gut contents, 7 to 9 ug equivalent/g; stomach, 49 to 89 ug equivalent/g; stomach contents, 160 to 205 ug equivalent/g) after 0.5 hr. After 42 hr, tissues and organs contained <0.7 ug equivalent/g. In rats that were pretreated with unlabelled pyraclostrobin for 14 days and given a single oral dose of 14C-tolyl-labelled pyraclostrobin at 5 mg/kg bw, the highest concentrations of radioactivity after 120 hr were found in the thyroid gland (0.18 to 0.35 ug equivalent/g) and the liver (0.1 ug equivalent/g). In all other tissues, the concentration of radioactivity recorded was <0.1 ug equivalent/g. The rapid and essentially complete excretion of pyraclostrobin and the decline of tissue concentrations to low levels over the observation period, suggests a low potential for accumulation.
The overall recovery of radioactivity was 91 to 105% in all /four oral experiments in rats/. In the first 48 hr after a single oral dose of 14C-tolyl-labelled pyraclostrobin at 5 or 50 mg/kg bw, 10 to 13% of the administered radioactivity was excreted in the urine and 74 to 91% was excreted in the feces. The total amount of radioactivity excreted in the urine and feces after 120 hr was 11 to 15% and 81 to 92%, respectively. A similar pattern of excretion was observed in rats that were pre-treated with unlabelled pyraclostrobin for 14 days and given a single oral dose of 14C-tolyl-labelled pyraclostrobin at 5 mg/kg bw of (12 to 13% in the urine and 76 to 77% in the feces after 48 hr; 12 to 14% in the urine and 79 to 81% in the feces after 120 hr) and in rats given a single oral dose of chlorophenyl-labelled pyraclostrobin at 50 mg/kg bw (11 to 15% in the urine and 68 to 85% in the feces after 48 hr; 12 to 16% in the urine and 74 to 89% in the feces after 120 hr). There was no detectable radioactivity in the expired air from rats treated with 14C-tolyl- or 14C-chlorophenyl-labelled pyraclostrobin at 50 mg/kg bw. In tissues and organs, the radioactivity that remained after 120 hr was <1 mg equivalent/g at 50 mg/kg bw and <0.1 mg equivalent/g at 5 mg/kg bw. Within 48 hr after administration of 14C-tolyl-labelled pyraclostrobin at 5 or 50 mg/kg bw of, 35 to 38% of the administered radioactivity was excreted via the bile, indicating, in conjunction with observations on urinary excretion, that approximately 50% of the administered dose had been absorbed.
Dermal application. The absorption and, to a limited extent, the distribution and excretion of 14C-labelled pyraclostrobin (in Solvesso) in groups of 16 male Wistar rats was assessed after a single dermal application at a nominal dose of 0.015, 0.075 or 0.375 mg/cm2, corresponding to 0.15, 0.75 and 3.75 mg/animal or approximately 0.8, 4 and 18 mg/kg bw. Animals were exposed to the test material for 4 (four rats per group) or 8 (12 rats per group) hr and four rats per group were sacrificed at 4, 8, 24 or 72 hr after the start of the exposure. An area of approximately 10 cm2 on the shoulders was clipped free of hair and was washed with acetone 24 hr before dosing. A silicone ring was glued to the skin and the test substance preparation (10 uL/cm2) was administered with a syringe, which was weighed before and after application. A nylon mesh was then glued to the surface of the silicone ring and covered with a porous bandage. After the exposure period, the protective covers were removed and the exposed skin was washed with a soap solution. After sacrifice, the concentration of radioactivity in the excreta, blood cells, plasma, liver, kidneys, carcass, treated and untreated skin was assessed. Radioactivity in the cage and skin wash and the protective covering, including the silicone ring, was also assessed. In all groups, 99 to 110% of the radioactivity was recovered. At sacrifice at 72 hr, after an 8 hr exposure, 1.6 to 2.6% of the administered dose was absorbed, 22 to 26% was on the skin or in the skin wash, and 72 to 80% was recovered on the protective cover. Only 0.2 to 0.4% and 0.9 to1.8% was excreted in the urine and faeces, respectively.
For more Absorption, Distribution and Excretion (Complete) data for PYRACLOSTROBIN (6 total), please visit the HSDB record page.

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Metabolism / Metabolites
Tissues, excreta and bile from animals used in the toxicokinetics studies and from additional groups given a single dose at 50 mg/kg bw per day (to provide more material for analysis) were analysed for metabolites of pyraclostrobin. In order to determine the metabolites in the plasma, liver and kidneys, additional groups were treated with a single dose of 14C-tolyl- or 14C-chlorophenol ring-labelled pyraclostrobin at 5 and 50 mg/kg bw and sacrificed 8 hr later. Metabolites were identified using high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The metabolism of pyraclostrobin proceeded through three main pathways primarily involving alterations to the three major portions of the pyraclostrobin molecule. The methoxy group on the tolyl-methoxycarbamate moiety was readily lost, with few major metabolites retaining this group. Hydroxylation of the aromatic and/or pyrazole rings was followed by glucuronide and occasionally sulfate conjugation, and many metabolites were derived from the chlorophenol-pyrazole or tolyl-methoxycarbamate moieties of pyraclostrobin, following cleavage of the ether linkage, with subsequent ring hydroxylation and glucuronide or sulfate conjugation. Metabolites were similar in both sexes and at all doses. No unchanged parent compound was found in the bile or urine and only small amounts in the faeces. Compounds dominating the identified metabolites recovered from the urine were: ring-hydroxylated pyraclostrobin; the chlorophenol pyrazole moiety hydroxylated on the pyrazole ring with or without a sulfate conjugate; a glucuronide of the tolyl-methoxycarbamate moiety; and a benzoic acid derivative of the tolyl-methoxycarbamate moiety. In the feces, the dominant metabolite was a demethoxylated and pyrazole ring hydroxylated pyraclostrobin. In the bile, the primary metabolite was a glucuronide of pyraclostrobin hydroxylated on the pyrazole ring at the 4' position and this compound, together with the demethoxylated derivative found in the faeces, was also the dominant metabolite isolated from the plasma and the liver. Demethoxylation of the methoxycarbamate moiety appeared to occur primarily in the gut, as the major metabolite in the bile retains this group intact whereas in the feces the major metabolite is the demethoxylated derivative. Most of the radiolabel isolated from the kidneys was in the form of the unchanged parent compound and a demethoxylated derivative.
Wistar rats were dosed ... with chlorophenyl-labeled pyraclostrobin (>98% chemical purity, >98% radiochemical purity) or tolyl-labeled pyraclostrobin (>98% chemical purity, >98% radiochemical purity), adjusted with unlabeled pyraclostrobin (BAS 500 F), 99.8 % purity to desired dose. ... Tissue samples were collected 8 hr after dosing, to achieve maximal tissue levels for analysis. Data did not demonstrate sex differences. Dose levels (5 or 50 mg/kg) and treatment history (2 week pre-treatment with 50 mg/kg/day pyraclostrobin) had no apparent effect on metabolic disposition. The most abundant fecal metabolite was 500M08 (de-methoxylated ai, which is hydroxylated in the 4-position of the pyrazole ring), accounting for about 38% of total administered dose. Other significant fecal metabolites were further hydroxylated: usually on the chlorophenyl ring and sometimes also on the tolyl ring. The major biliary metabolite was 500M46 (formed by hydroxylation followed by glucuronidation of carbon 4 of the pyrazole group of the ai). The majority of lesser biliary metabolites were also glucuronides. No single urinary metabolite comprised more than about 3% of administered dose. Predominant urinary metabolites were various products of cleavage of the ether oxygen (often to form a glucuronide or benzoic acid derivative), or 500M06 (de-methoxylated 500M46). Detectable plasma residues were limited to 500M06 and 500M46 (representing about 0.02% of administered dose). These metabolites plus parent pyraclostrobin were found in liver in higher amounts (these 3 residues combined representing about 0.5% of dose). Only pyraclostrobin could be detected in kidneys, to the extent of about 0.03% of dose. Thus absorbed pyraclostrobin is efficiently metabolized to polar products and is cleared effectively from the body.
Metabolite /is/ methyl-N-(((1- (4-chlorophenyl) pyrazol-3-yl)oxy]otolyl) carbamate (BF 500-3)
Major routes of metabolism involved demethoxylation and hydroxylation of the pyrazole and other ring systems followed by glucuronidation.

Toxicity Data
LC50 (rat) > 310 mg/m3/4h < 1,070 mg/m3/4h

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Non-Human Toxicity Values
LC50 Rat (Wistar male & female) dermal >2000 mg/kg bw (no deaths)
LC50 Rat (Wistar male & female) inhalation (head and nose only), 4 hr >0.310 mg/L, <1.070 mg/L
LD50 Rat (Wistar male & female) oral >5000 mg/kg bw (no deaths)

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Non-Human Toxicity Values
LC50 Rat (Wistar male & female) dermal >2000 mg/kg bw (no deaths)
LC50 Rat (Wistar male & female) inhalation (head and nose only), 4 hr >0.310 mg/L, <1.070 mg/L
LD50 Rat (Wistar male & female) oral >5000 mg/kg bw (no deaths)

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Human Toxicity Excerpts
/SIGNS AND SYMPTOMS/ May be fatal if swallowed. Causes substantial but temporaly eye injury. Causes skin irritation. Harmful if absorbed through skin. /Headline/

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Non-Human Toxicity Excerpts /LABORATORY ANIMALS: Acute Exposure/ Skin sensitization in a Magnusson-Kligman maximization test, intradermal injections (2 x 0.1 mL) of Freund adjuvant in a 0.9% aqueous solution of sodium chloride (1 : 1), 5% pyraclostrobin in Freund adjuvant and 5% pyraclostrobin in 1% Tylose CB 30 000 in Aqua bidest (Tylose) were given to the left and right shoulders of each of 20 guinea-pigs. Sites were evaluated 24 hr after injections were given. One week later, 5% pyraclostrobin in Tylose (1 mL) was applied to a gauze patch of surface area 2 x 4 cm and administered topically to the same sites, then covered with an occlusive dressing for 48 hr, after which time the sites were assessed. On day 22, all animals were challenged with 0.5 mL of 1% pyraclostrobin in Tylose (right flank) and Tylose alone (left flank). A second challenge was performed on day 29, when the test substance was applied to the left flank and the vehicle applied to the right flank. All challenge sites were evaluated 24 and 48 hr after removal of the occlusive dressings. There were no deaths and all animals gained body weight normally over the study. Although intradermal injections of Freund adjuvant, 5% pyraclostrobin in Freund adjuvant and 5% pyraclostrobin in Tylose caused moderate and confluent erythema (Draize score = 2) and swelling in all animals, as did an occluded topical application of 5% pyraclostrobin in Tylose, the first and second challenges with 1% pyraclostrobin in Tylose and Tylose alone caused no effect in any animal at 24 or 48 h. The sensitivity of the procedure was confirmed in an assay with the positive controls technical-grade alpha-hexyl cinnamaldehyde technical (85%) and Lutrol E 400 DAB (Lutrol). Pyraclostrobin was not a skin sensitizer in guinea-pigs in this study.

/LABORATORY ANIMALS: Acute Exposure/ Ocular irritation. Pyraclostrobin (0.1 mL; purity, 98.2%) was instilled into the conjunctival sac of the right eye of one male and five female New Zealand white rabbits. After 24 hr, the test material was washed out with tap water. The left eye was not treated and served as a control. There were no deaths during the study. Conjunctival redness (score = 1-3) was observed in all animals up to 3 days after treatment, with swelling observed in five out of six rabbits at 1 hr (score = 1), six out of six rabbits on day 1 (average score = 1.2), three out of six rabbits on day 2 (score = 1), and two out of six rabbits on day 3 (score = 1). Discharge (score = 1) occurred in one out of six rabbits at 1 hr. There were no corneal or iridal effects and all conjunctival effects had resolved by day 8. Loss of hair at the margins of the eyelids occurred in six out of six rabbits from 1 day after treatment. Under the conditions of the study, pyraclostrobin was a slight ocular irritant in rabbits.[JMPR Toxicological Monograph. Pesticide residues in food - 2003 - Joint FAO/WHO Meeting on Pesticide Residues PYRACLOSTROBIN.

/LABORATORY ANIMALS: Acute Exposure/ Oral administration. Clinical signs after oral administration of pyraclostrobin consisted of dyspnea, staggering, piloerection, and diarrhea in all animals, resolving by day 6. There were no pathology findings. In a study of acute inhalation using acetone as the solvent, all animals at 1.070 and 5.300 mg/L died on the day of exposure. At 0.310 mg/L, bloody discharge from the nose (two males), piloerection and smeared fur (10 out of 10 animals) were observed. All effects had resolved in surviving animals by day 7. Where Solvesso was used as the solvent, all males and four out of five females at 7.3 mg/L died, and one out of 10 animals died at each of the two lower doses. There were no deaths at 0.89 mg/L.

/LABORATORY ANIMALS: Acute Exposure/ Dermal irritation: Undiluted pyraclostrobin (500 mg, purity 98.2%) was applied to the shaved, intact skin on the back/flanks of six New Zealand White rabbits under a semi occlusive bandage for 4 hr. At the end of the exposure period, the test substance was removed and the treated area was rinsed with polyethylene glycol and water. There were no mortalities. Erythema was observed in all animals from 1 hr after removal of the bandage and persisting in most animals until day 8, and in three animals until day 15. The maximum Draize score for erythema was 3 and the average scores at day 1 and 8 were 2 and 1.5 respectively. Oedema with a Draize score of 1 was observed in four out of six rabbits on day 1, resolving in all except two rabbits by day 8, but persisting in one rabbit until day 15. It was concluded that pyraclostrobin is a slight but prolonged skin irritant

[1]. Characterization of hepatotoxic effects induced by pyraclostrobin in human HepG2 cells and zebrafish larvae. Chemosphere, 2023, 340: 139732.
[2]. Acute and subchronic toxicity of pyraclostrobin in zebrafish (Danio rerio). Chemosphere, 2017, 188: 510-516.
[3]. Mitochondrial dysfunction-based cardiotoxicity and neurotoxicity induced by pyraclostrobin in zebrafish larvae. Environmental Pollution, 2019, 251: 203-211.

Pyraclostrobin is a carbamate ester that is the methyl ester of [2-({[1-(4-chlorophenyl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]methoxycarbamic acid. A fungicide used to control major plant pathogens including Septoria tritici, Puccinia spp. and Pyrenophora teres. It has a role as a mitochondrial cytochrome-bc1 complex inhibitor, a xenobiotic, an environmental contaminant and an antifungal agrochemical. It is a member of pyrazoles, a carbamate ester, an aromatic ether, a member of monochlorobenzenes, a methoxycarbanilate strobilurin antifungal agent and a carbanilate fungicide.
Pyraclostrobin has been reported in Ganoderma lucidum with data available.
Pyraclostrobin is a broad spectrum foliar fungicide belonging to the strobilurin chemical class. It acts by inhibition of mitochondrial respiration. This leads to a reduction of the available ATP quantity in the fungal cell. It is used for control or suppression of fungal diseases on many common crops including: Berries, Bulb, Cucurbit, Fruiting, and Root vegetables, and Cherries

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Mechanism of Action
Pyraclostrobin is a member of the strobilurin group of fungicides. The strobilurin fungicides act through inhibition of mitochondrial respiration by blocking electron transfer within the respiratory chain, which in turn causes important cellular biochemical processes to be severely disrupted, and results in cessation of fungal growth.

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In the current study, it was found that pyraclostrobin induced DNA damage and mitochondrial dysfunction leads to excessive generation of intracellular ROS, ultimately resulting mitochondrial-mediated cell apoptosis and producing toxic effects on HepG2 cells. A decrease in p62 protein levels and the accumulation of LC3-II and Beclin-1 proteins suggested that pyraclostrobin might induce autophagy. It was also revealed that the cytotoxicity of pyraclostrobin was associated with the AMPK/mTOR mediated autophagy and oxidative DNA damage. Moreover, pyraclostrobin induced liver injury and liver steatosis in zebrafish. This study has indicated that pyraclostrobin leads to genotoxicity of human liver cells and hepatotoxicity of zebrafish larvae, which provides a better understanding of the potential risk of pyraclostrobin to human safety and a theoretical basis for the mechanisms of hepatotoxicity induced by pyraclostrobin. [1]

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The results of the present study demonstrate the biochemical responses of and DNA damage caused in zebrafish (Danio rerio) exposed to pyraclostrobin. The primary conclusions are as follows:.
(1) Pyraclostrobin is highly toxic to zebrafish.
(2) Pyraclostrobin can induce oxidative stress and oxidative damage in the livers of zebrafish..
(3) The most sensitive biomarker in the present study was the OTMs obtained using comet assays..
(4) Pyraclostrobin was relatively stable in an aquatic environment throughout the experimental period.

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This study overall confirmed the mitochondrial-based toxic effects of pyraclostrobin on zebrafish larvae. It is a novel finding that pyraclostrobin damaged histological and subcellular structure of larval heart and brain, changed the expression level of cardiac muscle contraction pathway- and neural-related genes and proteins, impaired larval cardiac function and locomotor behavior. These events might result from mitochondrial dysfunction induced by pyraclostrobin. The results of present study facilitate a better understanding of pyraclostrobin toxicity to aquatic organisms.[3]

C19H18CLN3O4

387.82

387.098

C, 58.84; H, 4.68; Cl, 9.14; N, 10.84; O, 16.50

175013-18-0

6422843

Off-white to light yellow solid powder

1.3±0.1 g/cm3

501.1±60.0 °C at 760 mmHg

63.7-65.2°

256.8±32.9 °C

0.0±1.3 mmHg at 25°C

1.592

4.25

0

5

7

27

476

0

COC(=O)N(C1=CC=CC=C1COC2=NN(C=C2)C3=CC=C(C=C3)Cl)OC

HZRSNVGNWUDEFX-UHFFFAOYSA-N

InChI=1S/C19H18ClN3O4/c1-25-19(24)23(26-2)17-6-4-3-5-14(17)13-27-18-11-12-22(21-18)16-9-7-15(20)8-10-16/h3-12H,13H2,1-2H3

methyl N-[2-[[1-(4-chlorophenyl)pyrazol-3-yl]oxymethyl]phenyl]-N-methoxycarbamate

Pyraclostrobin; 175013-18-0; Pyraclostrobine; Headline; Cabrio; Pyrachlostrobin; BAS-500F; UNII-DJW8M9OX1H;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : 100 mg/mL (257.85 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.36 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 20.8 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: ≥ 2.08 mg/mL (5.36 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 20.8 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.)