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Dufulin is a new type of chiral antiplant virus agent independently developed in China. The present study was conducted to determine the effects of different concentrations of rac‐dufulin and dufulin enantiomers (1, 5, and 10 mg/L) on oxidative stress in Tubifex after exposure for 3, 7, and 14 d. Results showed that rac‐dufulin and individual enantiomers had no significant effects on total protein content and glutathione reductase activities. Increased superoxide dismutase demonstrated the generation of superoxide anion radical. The increase in glutathione S‐transferase may be due to detoxification mechanisms. The different changes in catalase activities could be due to oxidative stress. The increase in malondialdehyde may be due to the accumulation and toxicity of contaminations. The degradation behavior of dufulin enantiomers was studied through spiked‐water and spiked‐soil tests. The degradation rate of S‐(+)‐dufulin was faster than that of R‐(−)‐dufulin. The present study demonstrated the occurrence of enantioselectivity in the degradation and oxidative stress of dufulin to Tubifex. In spiked soil, the concentrations of dufulin enantiomers in underlying soil were significantly higher than those in overlying water; but after 5 d of degradation, the bioturbation of Tubifex could facilitate part of dufulin diffusing from the underlying soil into the overlying water and altered the partitioning of dufulin. The present study provided a basis for conducting environmental safety risk assessments and rationally using dufulin as a chiral pesticide. Environ Toxicol Chem 2020;39:2136–2146. © 2020 SETAC [ABSTRACT FROM AUTHOR]

A rapid method for the determination of dufulin residues in the carp and crayfish by QuEChERS-high performance liquid chromatography-tandem mass spectrometry was developed. The samples were extracted with acetonitrile, and cleaned up by 50 mg PSA, 50 mg C18 and 50 mg MgSO4. The samples were separated on a Eclipse XDB-C18 column, and detected under positive polarity multiple reactions monitoring (MRM) mode. The matrix matched external standard calibration curves were used for quantitative analysis. The result indicated that the good linearities of dufulin was in the concentration range of 1.0-50 μg/L with correlation coefficients higher than 0.999. At the fortified levels of 10, 50 and 100 μg/kg, the average recoveries of dufulin in the carp and crayfish, were in the range of 88%-103%, with intra- and inter-day relative standard deviations varies from 1.2% to 5.4%. The limit of detections(LOD) for carp and crayfish were 0.4 and 0.8 μg/kg, respectively. And the limit of quantifications(LOQ) of dufulin for them were 2 and 3 μg/kg, respectively. This method was simple, accurate, economic and friendly to the environment, which could meet the requirements for rapid determination of dufulin residues in the carp and crayfish [ABSTRACT FROM AUTHOR]

ern rice black streaked dwarf virus (SRBSDV) causes severe harm to rice production. Unfortunately, studies on effective antiviral drugs against SRBSDV and interaction mechanism of antiviral molecule targeting on SRBSDV have not been reported. This study found dufulin (DFL), an ideal anti-SRBSDV molecule, and investigated the interactions of DFL targeting on the nonstructural protein P9-1. The biological sequence information and bonding characterization of DFL to four kinds of P9-1 protein were described with fluorescence titration (FT) and microscale thermophoresis (MST) assays. The sequence analysis indicated that P9-1 had highly-conserved C- and N-terminal amino acid residues and a hypervariable region that differed from 131 aa to 160 aa. Consequently, wild-type (WT-His-P9-1), 23 C-terminal residues truncated (TR-ΔC23-His-P9-1), 6 N-terminal residues truncated (TR-ΔN6-His-P9-1), and Ser138 site-directed (MU-138-His-P9-1) mutant proteins were expressed. The FT and MST assay results indicated that DFL bounded to WT-His-P9-1 with micromole affinity and the 23 C-terminal amino acids were the potential targeting site. This system, which combines a complete sequence analysis, mutant protein expression, and binding action evaluating system, could further advance the understanding of the interaction abilities between antiviral drugs and their targets.

Abstract: A simple and rapid method for enantioselective determination of dufulin in cucumber and soil was developed by liquid chromatography with tandem mass spectrometry. The enantiomers were separated on a Superchiral S‐OD chiral cellulose tris(3,5‐dimethylphenylcarbamate) column at 20°C, with a mixture of acetonitrile and water (0.1% formic acid; 52:48, v/v) as mobile phase at a flow rate of 0.65 mL/min. The pretreatment conditions were optimized using an orthogonal test, and the optimized method showed good linearity and sensitivity. The limits of detection and limits of quantification of two dufulin enantiomers were 0.006 and 0.02 mg/kg, respectively. The average recoveries of S‐enantiomer and R‐enantiomer in cucumber and soil were 80.61–99.83% and 80.97–102.96%, respectively, with relative standard deviations of 1.30–9.72%. The method was successfully applied to determine dufulin in real cucumber and soil samples. The results demonstrate that the method could facilitate further research on the differences between individual dufulin enantiomers with respect to metabolites and environmental fate and finally help reveal the complex interactions that exist between dufulin, humans and the environment. [ABSTRACT FROM AUTHOR]

Carbon-14 labelled Dufulin, diethyl ((2-fluorophenyl)((4-methylbenzo[2-14C]thiazol-2-yl) amino)methyl)phosphonate was synthesized from barium [14C]carbonate via Grignard reaction, Curtius arrangement, nucleophilic addition, thionation reaction, cyclization reaction, etc. The labelled product (38.3 mCi) was obtained after RP-HPLC purification in the overall radiochemical/chemical yield of 10%. The product was characterized by ESI-MS, 1H NMR and HPLC-FSA. Its technical data (radiochemical purity > 98%, chemical purity > 98%, specific activity 58.0 mCi/mmol) was determined by TLC-IIA, HPLC-LSC, HPLC-FSA/PDA/MS and LSC. This carbon-14 labelled Dufulin can be used as radiotracer in the study on its metabolism and environmental behavior by employing radioisotope tracing techniques. [ABSTRACT FROM AUTHOR]

An effective and sensitive chiral analytical method was established to investigate the stereoselective dissipation of rac-dufulin in watermelon using ultra high performance liquid chromatography with a superchiral S-OD chiral column (4.6 × 150 mm i.d., 5 μm) coupled with high-resolution mass spectrometry. To optimize the pretreatment method for detecting rac-dufulin in the three matrixes, different extraction solvents, extractant volumes, extraction times, and absorbents were investigated to improve extraction efficiency. Moreover, analysis of variance was used to perform method validation for determination of the two dufulin enantiomers in the three matrixes. Using the optimized method, good linearity was obtained (determination coefficient > 0.999). The limits of detection and quantification of the two dufulin enantiomers in soil, watermelon, and pulp were 0.15 and 0.5 μg/kg, respectively. The average recoveries of the two enantiomers in the three matrixes at four spiked levels ranged from 75.0 to 107.8%, with intra- and inter-day relative standard deviations of 0.4-10.4%. In field trials, the R enantiomer was preferentially dissipated in watermelon. These method validation results confirmed that the developed method was convenient and reliable for the stereoselective determination of enantiomers of rac-dufulin in watermelon. [ABSTRACT FROM AUTHOR]

Dufulin (DFL) is a potent, new-generation agrochemical that protects crops, such as tobacco, from viruses. Herein, for the first time, we developed a sensitive and specific polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect DFL residues in samples. The assay showed high sensitivity and specificity to DFL, with a half maximal inhibitory concentration (IC50) of 48.5 ng ml−1 and limit of detection (IC20) of 2.5 ng ml−1. The average recovery of DFL in different spiked samples, such as tobacco, rice, tomato, cucumber, soil and water, was in the range of 70.7–138.8%. A statistically significant correlation was observed between our developed ELISA and previously established methodology of high-performance liquid chromatography with a diode array detector. These results indicate our assay is a potentially useful analytical tool for rapid detection of DFL residue in actual samples. [ABSTRACT FROM AUTHOR]

Dufulin is a newly developed plant antiviral agent, which is widely used in the control of many viral crop diseases. Existing research mainly focuses on its antiviral effect, but research in relation to resistance to abiotic stress is unclear. This study was based on the treatment of rice with salt (NaCl), and exogenous application of Dufulin as a stress-resistant agent. The effect of Dufulin on salt stress of rice was revealed. There were 1997 differential genes detected, including 1449 up-regulated and 548 down-regulated. After the application of Dufulin to rice, when salt stress was applied, peroxidase activity was increased and superoxide dismutase activity was reduced; GO and KEGG analyses indicated that the stimulated genes are related to the stress resistance pathway, thus improving the ability of rice to resist salt stress. Quantitative real-time PCR analysis was used to verify the dynamic changes of growth- and stress-resistance-related genes, among which integral membrane protein DUF6 containing protein, OsHKT1;4 (Na+ transporter) and zinc-finger protein were verified to increase by more than three times, and OsIAA1 and OsIAA9 were verified as down-regulated. Measuring the length of root, stem and leaf, and OsIAA1 and OsIAA9 expression showed that Dufulin promoted rice growth. After that, Dufulin could enhance the salt resistance of rice by regulating the expression of integral membrane protein DUF6 containing protein, OsHKT1;4, zinc-finger protein and other related genes under salt stress. The results elucidated the mechanism of Dufulin action during salt stress in rice at the transcriptional level. [Display omitted] • Dufulin plays an active role in rice under salt stress. • Dufulin can increase peroxidase activity and reduce superoxide dismutase activity in rice under salt stress. • Differential genes were obtained by transcriptome sequencing of samples treated with Dufulin. [ABSTRACT FROM AUTHOR]

Southern rice black-streaked dwarf virus (SRBSDV) is an important rice virus that can cause considerable crop losses. During infection, SRBSDV P9-1 accumulates viroplasms in SRBSDV-infected plant and insect cells, which may facilitate the formation of SRBSDV particles. Our field trial results showed that Dufulin inhibited infection activity in SRBSDV-infected rice. However it is not clear how Dufulin inhibits the infection activity. Therefore we studied the interactions between Dufulin and SRBSDV using P9-1 as a potential target, and found that Dufulin inhibited SRBSDV P9-1 gene expression and had a micromole affinity for the arginine 175 residue. Substituting arginine with glycine resulted in a mutant that was significantly less sensitive to Dufulin. Based on these results, we speculate that Dufulin inhibits the virulence of SRBSDV by binding with arginine 175 in the internal pore of P9-1 octameric protein. [ABSTRACT FROM AUTHOR]

Dufulin is a biologically derived antiviral agent chemically synthesized by α-phosphoramidate in sheep and is effective against viral diseases in plants such as tobacco, rice, cucumber and tomato. However, the environmental behaviors and fate of Dufulin under different cultivation systems remain unknown. This study investigates the absorption, translocation and accumulation of 14C-Dufulin stereoisomers introduced by pesticide leaf daubing and by mixing the pesticide with soil in different tissues of cherry radish. We particularly focused on whether the behaviors of Dufulin enantiomers in plants were stereoselective. In the leaf uptake experiments, S-Dufulin and R-Dufulin were transported both up and down, while more than 93% of the pesticide remained in the labeled leaves. During the radicular absorption experiments, both enantiomers of Dufulin were taken up by radish roots and moved to the upper part of the plant, while less than 0.2% Dufulin was absorbed from the soil. Hence, it was easier for Dufulin to enter plants through the leaf surface than through the roots. However, we found in this trial that the stereoisomers of Dufulin underwent nonstereoselective absorption and translocation, which implies that rac-Dufulin and its metabolites should be a major research priority. Overall, our results provide a relatively accurate prediction of the risk assessment of Dufulin, which will help guide its rational use in the environment as well as ensure eco-environmental safety and human health. [Display omitted] • Most of the Dufulin enantiomers absorbed by leaves remained in the labeled leaf. • Only a small amount of Dufulin enantiomers transported to other tissues. • Dufulin enantiomers absorbed into the plant through the roots were extremely few. • Non-stereoselectivity was found in distribution either absorbed by leaves or roots. [ABSTRACT FROM AUTHOR]

A sensitive and selective analytical method for dufulin detection was developed using ultra-performance liquid chromatography coupled with photo-diode array detection (UPLC-PDA). Dufulin analysis was carried out with 1 μl sample injection on BEH C18 column (50.0 mm × 2.1 mm i. d., 1.7 μm film thickness) thermostatted at 40°C with a mobile phase of methanol/water (58/42 by volume) at flow rate of 0.3 ml min−1. Dissipation rates of dufulin in paddy plant, soil, water, and brown rice in three districts in China were studied using the method developed. Dufulin (30% WP) was applied at two dosages, 337.50 g a. i. ha−1(recommended dosage) and 506.25 g a. i. ha−1(1.5 times the recommended dosage) in experimental fields in Guizhou, Anhui, and Tianjin provinces in China. The limit of detection (LOD) and the limit of quantification (LOQ) of dufulin were 2.5 and 7.5 μg kg−1, respectively. The average recoveries were 72.76–95.43, 97.84–101.7, 96.99–100.8, and 80.91–100.5% in paddy plant at four spiking levels (0.04, 0.20, 2.0, 50.4 mg kg−1), in water at three spiked levels (0.0025, 0.025, 0.25 mg l−1), in soil at three spiking levels (0.04, 0.13, 1.3 mg kg−1), and in brown rice at four spiking levels (0.05, 0.20, 2.0, 10.0 mg kg−1), respectively. Our results showed that half-lives of dufulin in paddy plant and water in the three districts were 7.7–11.3 and 2.1–4.8 days, respectively. At harvest, dufulin residues in brown rice samples collected 35 days after the last dufulin application at the recommended dosage were below 0.5 mg kg−1. Our results provided data to help establish appropriate application frequency and harvest intervals in the use of dufulin for rice pest control in China. [ABSTRACT FROM AUTHOR]

Dufulin is a newly developed antiviral agent (or pesticide) that activates systemic acquired resistance of plants. This pesticide is widely used in China to prevent abroad viral diseases in rice, tobacco and vegetables. In this study, the potential impacts such as soil type, moisture, temperature, and other factors on Dufulin degradation in soil were investigated. Degradation of Dufulin followed the first-order kinetics. The half-life values varied from 2.27 to 150.68 days. The dissipation of Dufulin was greatly affected by soil types, with DT (Degradation half time) varying between 17.59, 31.36, and 43.32 days for Eutric Gleysols, Cumulic Anthrosols, and Dystric Regosols, respectively. The elevated moisture accelerated the decay of Dufulin in soil. Degradation of Dufulin increased with temperature and its half-life values ranged from 16.66 to 42.79 days. Sterilization of soils and treatment with HO resulted in a 6- and 8-fold decrease in degradation rates compared to the control, suggesting that Dufulin degradation was largely governed by microbial processes. Under different light spectra, the most effective degradation occurred with 100-W UV light (DT = 2.27 days), followed by 15-W UV light (DT = 8.32 days) and xenon light (DT = 14.26 days). Analysis by liquid chromatography-mass spectroscopy (LC-MS) revealed that 2-amino-4-methylbenzothiazole was one of the major decayed products of Dufulin in soils, suggesting that elimination of diethyl phosphate and 2-fluorobenzaldehyde was most like the degradation pathway of Dufulin in Eutric Gleysols. [ABSTRACT FROM AUTHOR]

[Display omitted] • A functional MOF is designed for sensing the novel pesticide Dufulin. • A fluorescent Dufulin sensing strategy is realized sensitively and selectively. • The sensor shows the detection of Dufulin with rapidity and low detection limit. • The enzyme-based system provide a paradigm for detecting enzyme and its inhibitor. • The proposed strategy can be extended as a general platform against pesticide. Developing a rapid and accurate strategy of sensing Dufulin is a vital challenge for risk assessment and food crops along with its spreading usage. Herein a dye-encapsulated azoterephthalate metal–organic framework (MOF)-based fluorescent sensing system was designed for Dufulin analysis by acid phosphatase (ACP) enzyme-controlled collapse of MOF framework and subsequent release of the encapsulated dye. The fluorescence intensity of the DMOF/AAP/ACP system was negatively related to the dosage of Dufulin (0–5 μg mL−1) with detection limit of 2.96 ng mL−1. The sensing system able to rapidly and sensitively sense the activity of ACP and Dufulin, and was also applicable for assessment of the real samples including paddy water and soil, polished rice and cucumber. Accordingly, this study illustrated the feasibility and the potential of MOF-derived nanosensors for improving pesticide analysis and opening up the design of the enzyme-based probes for pesticide sensing in environmental assessment and food safety. [ABSTRACT FROM AUTHOR]

In the present study, the dose-effect and time-effect relationships of Dufulin racemate (rac - DFL) and its R(S) -enantiomers in rats were investigated after oral administration to evaluate their safety. A total of six doses (2.5, 5.0, 10.0, 20.0, 50.0, and 100.0 mg/kg) were administered and seven time-intervals (1 h, 3 h, 1 d, 3 d, 5 d, 7 d, and 14 d) were considered to observe the effects of rac - DFL , (R) - DFL, and (S) - DFL on general behavioral characteristics, liver and kidney functions, pathological changes, and lipid metabolism in rats. The results showed that the rats in each group exhibited a good mental state, agile activity, smooth and shiny fur, and normal diet. Viscera indices of heart, liver, spleen, lung, and kidney were 5.10–5.56, 4.15–4.59, 0.24–0.28, 6.08–6.48, and 11.02–11.98 mg/g for dose-effect relationships, and 5.01–5.94, 4.11–4.79, 0.24–0.30, 6.00–6.87, and 11.02–11.99 mg/g for time-effect relationships, respectively. Values of ALT, AST, TBil, DBil, IBil, BUN, Scr, β2-MG, and UA were 33.02–38.93 U/L, 108.17–126.53 U/L, 16.22–17.94 μmol/L, 5.75–8.12 μmol/L, 9.50–10.94 μmol/L, 4.03–5.85 mmol/L, 19.42–21.61 μmol/L, 48.16–52.73 mg/L, and 68.51–78.65 μmol/L, respectively. The statistical results showed that there were no significant differences in organ indices as well as liver and kidney function indices among different groups. In terms of pathological morphology, liver and kidney tissue sections of different groups of rats demonstrated normalcy. Rac - DFL , (R) - DFL, and (S) - DFL in the range of 2.5–100.0 mg/kg exerted no significant effect on lipid metabolism. Compared with the blank group, 35, 55, and 14 differential lipids were screened from rac - DFL, (S) - DFL, and (R)- DFL groups, respectively. These lipid changes completely returned to normalcy within 3 h. There were no significant differences at 1, 3, 5, 7, and 14 d after gavage. These results will aid further evaluation of the safety of dufulin and for provision of scientific evidence for its application as a pesticide. [ABSTRACT FROM AUTHOR]

Microplastics and pesticides are identified as two environmental pollutants that have an adverse impact on the environment. The knowledge about the combined exposure of pesticides and microplastics may facilitate further assessment of their ecotoxicity. In this study, we investigated joint effects of microplastic and dufulin on bioaccumulation, oxidative stress and metabolic profile of the earthworm. Bioaccumulation analysis showed that the bio-soil accumulation factor of dufulin in earthworms reached its maximum value on the 14th day, and microplastics could significantly increase the bioaccumulation of dufulin in earthworms. Biochemical analysis showed that the oxidative damage of earthworms could be observed on the 14th day of the exposure to dufulin, while the oxidative damage of earthworms could be observed on the 7th day of the combined exposure to microplastics and dufulin, and it could still be observed on the 14th day. 1H-NMR-based metabolomics revealed that the exposure of dufulin significantly altered the relative abundances of 14 metabolites and two metabolic pathways, but the combined exposure of dufulin and microplastics significantly changed the relative abundances of 21 metabolites and three metabolic pathways. It could be seen that microplastics could aggravate the oxidative damage and the interference with the metabolic profile caused by dufulin to earthworms. The results of this study could provide effective information for the risk assessment of dufulin and microplastic in environmental safety. Image 1 • Microplastics could increase the bioaccumulation of dufulin in earthworms. • Microplastics could aggravate the oxidative damage caused by dufulin to earthworms. • Microplastics could aggravate the metabolic disorders caused by dufulin to earthworms. [ABSTRACT FROM AUTHOR]

Southern rice black-streaked dwarf virus (SRBSDV) causes disease in crops, which reduces the quality and yield. Several commercial antiviral agents are available to control the SRBSDV induced disease. However, the mechanism of antiviral agents controlling SRBSDV is largely unknown. Identifying targets in SRBSDV is a key step of antiviral agent discovery. Here, we investigated the potential protein target of the antiviral agent dufulin. We cloned and expressed a soluble viroplasmic P6 protein in the prokaryote Escherichia coli and the eukaryote Spodoptera frugiperda 9. The dissociation constants of dufulin with the purified P6 protein from E. coli and S. frugiperda 9 expression systems were 4.49 and 4.95 μM, respectively, indicating a strong binding affinity between dufulin and P6 protein. In vivo, dufulin significantly inhibited the expression of both P6 protein and P6 gene in the SRBSDV-infected rice leaves. This inhibition on P6 protein expression was also observed in transformed Nicotiana benthamiana where the P6 was overexpressed. Our data also showed that dufulin inhibited the duplication of SRBSDV in a dose-dependent manner in infected rice leaves with a half maximum effective concentration of 3.32 mM. It is therefore concluded that dufulin targets the viroplasmic protein P6 to inhibit the virulence of SRBSDV.

Dufulin is a plant antiviral agent with a novel molecular structure and has been used widely to prevent and control tobacco and rice viral diseases. In this study, an UHPLC-MS/MS method was developed for rapid determination of dufulin racemate ( rac-DFL) and its R ( S)-enantiomers in rat plasma, tissues, urine, and feces. A MALDI-MSI method was further used for visual research on tissue distribution after intragastric administration of the three analytes. Toxicokinetic study showed that both ( R)-enantiomer of dufulin (( R)-DFL) and ( S)-enantiomer of dufulin (( S)-DFL) had a faster ability to reach C max than that of rac-DFL. ( R)-DFL and ( S)-DFL had a similar T 1/2 , though both were significantly lower than rac-DFL. C max of rac-DFL was obviously higher than ( R)-DFL or ( S)-DFL. Meanwhile, C max of ( S)-DFL was only about 60% of ( R)-DFL. Rac-DFL and its R ( S)-enantiomers had a dose-dependent toxicokinetic profile. Tissue distribution results revealed rac-DFL, ( R)-DFL, and ( S)-DFL mainly distributed in the liver and kidney, but the maximum concentration was only ng/g grade and could significantly degrade within 3 h. This indicates that dufulin does not cause liver and kidney toxicity in animals. In addition, rac-DFL and its R ( S)-enantiomers have not been detected in brain tissue. Cumulative excretion of rac-DFL and its R ( S)-enantiomers within 24 h in urine and feces were less than 22.85% indicating that they mainly excreted as metabolites. These results could provide evidence for the in-depth toxicity evaluation of dufulin pesticide. In addition, its metabolic selectivity information in vivo has also been obtained.

In this study, the stereoselective bioaccumulation of rac-dufulin, pure S-(+)-dufulin, and pure R-(-)-dufulin in Tubifex (Oligochaeta, Tubificida) were analyzed in spiked-water and spiked-soil systems at low and high dose levels. In the bioaccumulation experiments treated with rac-dufulin, the enantioselective behaviors of the enantiomers show that the concentrations of R-(-)-dufulin are higher than those of S-(+)-dufulin at two dose levels. However, when treated with solely pure S-(+)-dufulin and R-(-)-dufulin, no significant difference of concentrations was detected in Tubifex. Furthermore, the calculated accumulation factors in Tubifex indicated that dufulin (racemic or the pure enantiomers) in the spiked-soil treatments had higher bioaccumulation potential than in the spiked-water treatments. The spiked-soil experiments revealed that the dissipation of dufulin in soil was not enantioselective at the enantiomer levels and Tubifex could reduce the concentrations of dufulin in the underlying solid matrix and accelerate its repair and detoxification process.