Quercetin alleviates the toxicity of difenoconazole to the respiratory system of carp by reducing ROS accumulation and maintaining mitochondrial dynamic balance.

Difenoconazole (DFZ) is a fungicidal pesticide extensively employed for the management of fungal diseases in fruits, vegetables, and cereal crops. However, its potential environmental impact cannot be ignored, as DFZ accumulation is able to lead to aquatic environment pollution and harm to non-target organisms. Quercetin (QUE), a flavonoid abundant in fruits and vegetables, possesses antioxidant and anti-inflammatory properties. In this article, carp were exposed to 400 mg/kg QUE and/or 0.3906 mg/L DFZ for 30 d to investigate the effect of QUE on DFZ-induced respiratory toxicity in carp. Research shows that DFZ exposure increases reactive oxygen species (ROS) production in the carp's respiratory system, leading to oxidative stress, inflammation, and damage to gill tissue and tight junction proteins. Further research demonstrates that DFZ induces mitochondrial dynamic imbalance and gill cell apoptosis. Notably, QUE treatment significantly reduces ROS levels, alleviates oxidative stress and inflammation, and mitigates mitochondrial dynamics imbalance and mitochondrial apoptosis. This study emphasizes the profound mechanism of DFZ toxicity to the respiratory system of common carp and the beneficial role of QUE in mitigating DFZ toxicity. These findings contribute to a better understanding of pesticide risk assessment in aquatic systems and provide new insights into strategies to reduce their toxicity.

The alleviation of difenoconazole-induced kidney injury in common carp (Cyprinus carpio) by silybin: Involvement of inflammation, oxidative stress, and apoptosis.

Triazole fungicides, such as difenoconazole (DFZ), are frequently used to control fungus in crops that pollute water. The common carp (Cyprinus carpio) (hereafter referred to as "carp") is an excellent bio-indicator of water quality. The seeds of the silymarin plant contain a flavonolignan called silybin (SYB), which is used to treat liver disease. To explore SYB's involvement in DFZ-triggered kidney damage in carps, an H&E assay was conducted, and ROS level was also examined. The results demonstrated that SYB alleviated DFZ-induced destruction of kidney tissue structure in carps, as well as alleviating the elevation of kidney ROS level in carps. RT-qPCR and Western blot were used to detect inflammation-, oxidative stress- and apoptosis-related factors at mRNA level and protein level. The experimental findings indicated that relative to the DFZ group, SYB + DFZ co-treatment reduced inflammation-related mRNA level of il-6, il-1ß and tnf-α, elevated mRNA level of il-10. It also reduced protein expression levels of NF-κB and iNOS. In addition, SYB + DFZ co-treatment reduced DFZ-induced increase in the oxidative stress-related mRNA indicators sod and cat, and decreased the protein expression levels of Nrf2 and NQO1. SYB reduced the DFZ-induced increase in pro-apoptotic gene Bax mRNA and protein expression levels and the DFZ-induced decrease in anti-apoptotic gene Bcl-2 mRNA and protein expression levels. In summary, SYB potentially mitigates DFZ-induced kidney damage in carp by addressing inflammation, oxidative stress, and apoptosis. Our results establish a theoretical foundation for the clinical advancement of freshwater carp feeds.

Protective effect of feed additive ferulic acid on respiratory depression and oxidation imbalance of carp induced by pesticide difenoconazole via ROS/NF-κB/NLRP3 axis.

Difenoconazole (DFZ), classified as a "low-toxicity pesticide," has seen widespread application in recent years. Nevertheless, the non-target toxicity of the substance, particularly towards aquatic creatures, has generated considerable apprehension. The anti-inflammatory and antioxidant effects of Ferulic Acid (FA) have attracted considerable study in this particular setting. This study established a chronic exposure model to DFZ and investigated the protective effects of FA on chronic respiratory inhibition leading to gill damage in freshwater carp. Histological analyses via HE staining indicated that FA effectively alleviated gill tissue damage induced by chronic DFZ exposure. The qRT-PCR results showed that the addition of FA reduced the expression of IL-1ß, IL-6 and TNF-α while boosting the expression of IL-10 and TGF-ß1. Biochemical analyses and DHE staining revealed that FA reduced MDA levels and increased CAT and GSH activities, along with T-AOC, decreased ROS accumulation in response to chronic DFZ exposure. The results obtained from Western blotting analysis demonstrated that the addition of FA effectively suppressed the activation of the NF-κB signalling pathway and the NLRP3 inflammasome pathway in the gills subjected to prolonged exposure to DFZ. In summary, FA ameliorated gill tissue inflammation and blocked ROS accumulation in carp exposed to chronic DFZ, mitigating tissue inflammation and restoring redox homeostasis through the NF-κB-NLRP3 signaling pathway. Hence, the application of FA has been found to be efficacious for improving respiratory inhibition and mitigating gill tissue inflammation and oxidative stress resulting from DFZ pollution in aquatic habitats.

The effect of formulation composition and adjuvant type on difenoconazole dislodgeable foliar residue.

Rigorous risk assessments for those exposed to pesticides are carried out to satisfy crop protection regulatory requirements. Non-dietary risk assessments involve estimating the amount of residue which can be transferred from plant foliage to the skin or clothes, known as dislodgeable foliar residues (DFRs). DFR data are less available than crop residue data as studies are costly and limited by seasonality. European regulatory authorities are reticent to allow extrapolation of study data to different scenarios as the contributory factors have hitherto been poorly identified. This study is the first to use a new laboratory DFR method to investigate how one such factor, pesticide formulation, may affect DFR on a variety of crops. The study used the active substance difenoconazole as both an emulsifiable concentrate (EC 10%) and a wettable powder (WP 10%) with and without adjuvants (Tween 20 and organophosphate tris(2-ethylhexyl)phosphate TEHP) on tomato, French bean and oilseed rape. A comparable DFR% was retained from the WP and EC formulation on most crops except for tomato, where lower DFR% was retained in the case of WP (39 ± 4.7%) compared to EC (60 ± 1.2%). No significant effect of adjuvant addition was observed for either formulation except when mixing TEHP (0.1% w/v) to the EC 10% on French bean, resulting in 8% DFR reduction compared to the EC formulation alone. This research demonstrates the value of a unique DFR laboratory technique in investigating the importance of the formulation and in-tank adjuvants as factors that affect DFR.

Crosstalk of oxidative stress, inflammation, apoptosis, and autophagy under reactive oxygen stress involved in difenoconazole-induced kidney damage in carp.

Difenoconazole is a commonly used triazole fungicide in agricultural production. Because of its slow degradation and easy accumulation in the environment, it seriously endangers both animal health and the ecological environment. Therefore, it is hoped that the effects on carp kidneys can be studied by simulating difenoconazole residues in the environment. The experiment was designed with two doses (0.488 mg/L, 1.953 mg/L) as exposure concentrations of difenoconazole for 4 d. Histopathological results showed that difenoconazole could cause severe damage to the kidney structure and extensive inflammatory cell infiltration in carp. Elevated levels of Creatinine, and BUN suggested the development of kidney damage. The DHE fluorescence probe's result suggested that difenoconazole might cause reactive oxygen species (ROS) to accumulate in the kidney of carp. Difenoconazole was found to increase MDA levels while decreasing the activities of CAT, SOD, and GSH-PX, according to biochemical indicators. In addition, difenoconazole could up-regulate the transcription levels of inflammatory factors tnf-α, il-6, il-1ß, and inos. At the same time, it inhibited the transcription level of il-10 and tgf-ß1. The TUNEL test clearly showed that difenoconazole induced apoptosis in the kidney and vastly raised the transcript levels of apoptosis-related genes p53, caspase9, caspase3, and bax while inhibiting the expression of Bcl-2, fas, capsase8. Additionally, TEM imaging showed that clearly autophagic lysosomes and autophagosomes were formed. Elevated levels of LC3II protein expression, increased transcript levels of the autophagy-related gene atg5 as well as decreased transcript levels of p62 represented the generation of autophagy. In conclusion, the study illustrated that oxidative stress, inflammation, apoptosis, and autophagy all played roles in difenoconazole-induced kidney injury in carp, which was closely linked to ROS production. This work provides a valuable reference for studying the toxicity of difenoconazole to aquatic organisms.

Ferulic acid attenuated difenoconazole-induced immunotoxicity in carp by inhibiting TRAF/TAK1/NF-κB, Nrf2 and p53 pathways.

Difenoconazole (DFZ) is a classical triazole fungicide that causes immunosuppression in non-target organisms. Ferulic acid (FA) is a polyphenolic molecule found in nature that has antioxidant and anti-inflammatory activities. The purpose of this investigation was to see if FA could prevent DFZ-induced immunosuppression and to identify the potential mechanisms. Carp were exposed to 1/10 LC50 of DFZ as well as fed normal feed or feed containing dietary additive FA for 30 d. It was found that DFZ-induced immunosuppression could be improved by FA, as evidenced by upregulation of Hb, C3 and IgM and downregulation of LDH. It was then investigated that FA could ameliorate DFZ-induced splenic injury through p53-mediated apoptosis. At the same time, enhancing the levels of CAT, GSH and T-AOC in spleen and transcription levels Nrf2 signaling pathway related genes indicated that FA reduced oxidative damage caused by DFZ by blocking the Nrf2 signaling pathway. In addition, FA inhibited the inflammatory response triggered by TRAF/TAK1/NF-κB signaling pathway, downregulated the transcript levels of pro-inflammatory factors (il-1ß, tnf-α, il-6) and the level of NLRP3 inflammasome (NRLP3, ASC, Caspase 1), and upregulated the transcript levels of anti-inflammatory factors (tgf-ß1, il-10). In conclusion, the above results suggested that FA mediated TRAF/TAK1/NF-κB, Nrf2, and p53 pathways to attenuate DFZ-induced inflammation, oxidative stress, and apoptosis thereby enhancing the immune capacity of carp.

Nano-TiO2 aggravates bioaccumulation and developmental neurotoxicity of difenoconazole in zebrafish larvae via oxidative stress and apoptosis: Protective role of vitamin C.

Titanium dioxide nanoparticles (n-TiO2) could enhance the bioavailability and toxicity of coexisting organic contaminants in the aquatic environment. This study attempted to investigate the combined effects of n-TiO2 and difenoconazole (DIF) on the neurodevelopment of zebrafish and the underlying mechanisms. In this study, zebrafish embryos were exposed to n-TiO2 (100 µg/L), DIF (0, 0.1 and 0.5 mg/L) and their mixtures from 4 to 96 h post fertilization (hpf) and neurotoxicity was evaluated. Our results indicated that n-TiO2 adsorbed DIF into the brain of zebrafish and significantly enhanced the bioaccumulation of DIF and n-TiO2 in the 0.5 mg/L co-exposure group. 100 µg/L n-TiO2 was not developmentally toxic to the zebrafish larvae, but it exacerbated DIF-induced neurobehavioral alterations in the zebrafish larvae. n-TiO2 also aggravated DIF-induced suppression of central nervous system (CNS) neurogenesis in Tg (HuC:egfp) zebrafish, motor neuron axon length in Tg (hb9:egfp) zebrafish, and downregulation of neurodevelopmental genes (elavl3, ngn1, gap43, gfap and mbp). In addition, DIF elevated oxidative stress by accumulation of reactive oxygen species (ROS) and inhibition of antioxidant enzymes, and triggered apoptosis by upregulation of p53, bax, bcl-2 and caspase-3, which were markedly intensified in the presence of n-TiO2. Moreover, vitamin C (VC) ameliorated n-TiO2/DIF-induced abnormal locomotor behaviors and neurotoxicity by inhibiting oxidative stress and apoptosis, indicating that oxidative stress and apoptosis are involved in n-TiO2/DIF-induced neurotoxicity. Taken together, our data indicated that n-TiO2 enhanced the accumulation of DIF and heightened oxidative stress and apoptosis, thereby inducing neurotoxicity. This study exemplifies the importance of the toxicity assessment of chemical mixtures and novel insights to mitigate their combined toxicity.

Deposition and dissipation of difenoconazole in pepper and soil and its reduced application to control pepper anthracnose.

The initial deposition amount, dissipation dynamics, retention rate, and field control efficacy of difenoconazole in pepper-soil system were studied with different application dosages, planting regions and patterns. The initial deposition amount of difenoconazole under the same application dosage showed the following order: fruits < cultivated soils < lower stems < upper stems < lower leaves < upper leaves, open field < greenhouse, and Changjiang < Cixi < Hefei < Langfang, respectively, which increased with increasing application dosage. The dissipation rates in leaves, stems, fruits and cultivated soils exhibited an initially fast and then slow trend, while the retention rates displayed a tendency of first increasing and then stabilizing with increasing application dosages. After 7 d of difenoconazole application, the retention rates at five concentrations were 10.3%- 39.1%, and the field efficacy mostly reached the minimum effective dose. These results suggested that difenoconazole could be reduced by 25% based on the minimum recommended dose meeting the requirements of field control efficacy for controlling pepper anthracnose.

Mechanisms regarding respiratory toxicity triggered by accumulation of ROS in carp exposed to difenoconazole.

Difenoconazole is a widely used but difficult-to-degrade fungicide that can directly affect aquatic ecosystems. Here, two doses (0.488 mg/L, 1.953 mg/L) of difenoconazole were used to study the toxicity to the respiratory system of carp at an exposure time of 96 h. The results showed that difenoconazole exposure resulted in severe structural damage to carp gill tissue with extensive inflammatory cell infiltration. Mechanistically, difenoconazole exposure led to excessive accumulation of ROS in carp gill tissue, which induced an inflammatory response in the gill tissue. Meanwhile, the activities of SOD and CAT were reduced and the NRF2 signaling pathway was activated to regulate the imbalance between oxidation and antioxidation. In addition, difenoconazole exposure further activated the mitochondrial pathway of apoptosis by upregulating cytochrome C, BAX, cleaved-caspase 9, and downregulating Bcl-2. More interestingly, exposure to difenoconazole increased autophagosomes, but lysosomal dysfunction prevented the late stages of autophagy from proceeding smoothly, resulting in a protective autophagic response that is not properly initiated. In summary, difenoconazole exposure caused respiratory toxicity including inflammation response, oxidative stress, apoptosis, and autophagy in carp through the accumulation of ROS. The present study expanded our understanding of the toxic effects of difenoconazole on organisms and its possible threat to the aquatic environment.

Integrative physiological, critical plant endogenous hormones, and transcriptomic analyses reveal the difenoconazole stress response mechanism in wheat (Triticum aestivum L.).

Difenoconazole (DFN) is widely utilized as a fungicide in wheat production. However, its accumulation in plant tissues has a profound impact on the physiological functions of wheat plants, thus severely threatening wheat growth and even jeopardizing human health. This study aims to comprehensively analyze the dynamic dissipation patterns of DFN, along with an investigation into the physiological, hormonal, and transcriptomic responses of wheat seedlings exposed to DFN. The results demonstrated that exposure of wheat roots to DFN (10 mg/kg in soil) led to a significant accumulation of DFN in wheat plants, with the DFN content in roots being notably higher than that in leaves. Accumulating DFN triggered an increase in reactive oxygen species content, malonaldehyde content, and antioxidant enzyme activities, while concurrently inhibiting photosynthesis. Transcriptome analysis further revealed that the number of differentially expressed genes was greater in roots compared with leaves under DFN stress. Key genes in roots and leaves that exhibited a positive response to DFN-induced stress were identified through weighted gene co-expression network analysis. Metabolic pathway analysis indicated that these key genes mainly encode proteins involved in glutathione metabolism, plant hormone signaling, amino acid metabolism, and detoxification/defense pathways. Further results indicated that abscisic acid and salicylic acid play vital roles in the detoxification of leaf and root DFN, respectively. In brief, the abovementioned findings contribute to a deeper understanding of the detrimental effects of DFN on wheat seedlings, while shedding light on the molecular mechanisms underlying the responses of wheat root and leaves to DFN exposure.

Difenoconazole disrupts carp intestinal physical barrier and causes inflammatory response via triggering oxidative stress and apoptosis.

As a common fungicide, difenoconazole (DFZ) is widespread in the natural environment and poses many potential threats. Carp makes up a significant proportion of China's freshwater aquaculture population and are vulnerable to the DFZ. Therefore, this study investigated the effects of DFZ (0.488 mg/L and 1.953 mg/L) exposure for 4 d on the intestinal tissues of carp and explored the mechanisms. Specifically, DFZ exposure caused pathological damage to the intestinal tissues of carp, reducing the expression levels of intestinal tight junction proteins, and leading to damage to the intestinal barrier. In addition, DFZ exposure activated the NF-κB signaling pathway, increasing the levels of pro-inflammatory factors (TNF-α, IL-1ß, IL-6) and decreasing the levels of anti-inflammatory factors (IL-10, TGF-ß1). As disruption of the intestinal barrier is closely linked to oxidative stress and apoptosis, we have conducted research in both areas for this reason. The results showed that DFZ exposure elevated reactive oxygen species in carp intestines, decreased antioxidant enzyme activity, and suppressed the expression of oxidative stress-related genes. TUNEL results showed that DFZ induced the onset of apoptosis. In addition, the expression levels of apoptosis-related genes and proteins were examined. Western blotting results showed that DFZ could upregulate the protein expression levels of Bax, Cytochrome C and downregulate the protein levels of Bcl-2. qPCR results showed that DFZ could upregulate the transcript levels of Bax, Caspase-3, Caspase-8 and Caspase-9 and downregulate the transcript levels of Bcl-2 transcript levels. This suggests that DFZ can induce apoptosis of mitochondrial pathway in carp intestine. In conclusion, DFZ can induce oxidative stress and apoptosis in carp intestine, leading to the destruction of intestinal physical barrier and the occurrence of inflammation. Our data support the idea that oxidative stress and apoptosis are important triggers of pesticide-induced inflammatory bowel illness.

Analysis of the Difenoconazole-Resistance Risk and Its Molecular Basis in Colletotrichum truncatum from Soybean.

Anthracnose disease, caused by Colletotrichum truncatum, is a destructive fungal disease in soybean worldwide, and some demethylation inhibitor fungicides are used to manage it. In this study, the sensitivity of C. truncatum to difenoconazole was determined, and the risk for resistance development of C. truncatum to difenoconazole was also assessed. The results showed that the mean EC50 value was 0.9313 µg/ml, and the frequency of sensitivity formed a unimodal distribution. Six stable mutants with a mutation frequency of 8.33 × 10-5 were generated, and resistance factors ranged from 3.00 to 5.81 after 10 successive culture transfers. All mutants exhibited fitness penalties in reduced mycelial growth rate, sporulation, and pathogenicity, except for the Ct2-3-5 mutant. Positive cross-resistance was observed between difenoconazole and propiconazole but not between difenoconazole and prochloraz, pyraclostrobin, or fluazinam. One point mutation I463V in CYP51A was found in five resistant mutants. Surprisingly, the homologous I463V mutation has not been observed in other plant pathogens. CYP51A and CYP51B expression increased slightly in the resistant mutants as compared to wild-types when exposed to difenoconazole but not in the CtR61-2-3f and CtR61-2-4a mutants. In general, a new point mutation, I463V in CYP51A, could be associated with low resistance to difenoconazole in C. truncatum. In the greenhouse assay, control efficacy of difenoconazole on both parental isolates and the mutants increased in a dose-dependent manner. Collectively, the resistance risk of C. truncatum to difenoconazole is regarded to be low to moderate, suggesting that difenoconazole can still be reasonably used to control soybean anthracnose.

Chitosan Enhances Low-Dosage Difenoconazole to Efficiently Control Leaf Spot Disease in Pseudostellaria heterophylla (Miq.) Pax.

Pseudostellaria heterophylla (Miq.) Pax is a popular clinical herb and nutritious health food. However, leaf spot disease caused by fungal pathogens frequently occurs and seriously influences the growth, quality, and yield of P. heterophylla. In this work, the field control roles of difenoconazole, chitosan, and their combination in the leaf spot disease in P. heterophylla and their effects on the disease resistance, photosynthetic capacity, medicinal quality, and root yield of P. heterophylla are investigated. The results manifest that 37% difenoconazole water-dispersible granule (WDG) with 5000-time + chitosan 500-time dilution liquid had a superior control capacity on leaf spot disease with the control effects of 91.17%~88.19% at 15~30 days after the last spraying, which significantly (p < 0.05) exceeded that of 37% difenoconazole WDG 3000-time dilution liquid and was significantly (p < 0.01) higher than that of 37% difenoconazole WDG 5000-time dilution liquid, chitosan 500-time dilution liquid, or chitosan 1000-time dilution liquid. Simultaneously, this combination could more effectively enhance the disease resistance, photosynthetic capacity, medicinal quality, and tuberous root yield of P. heterophylla compared to when these elements were applied alone, as well as effectively reduce difenoconazole application. This study emphasizes that chitosan combined with a low dosage of difenoconazole can be proposed as a green, efficient, and alternative formula for controlling leaf spot disease in P. heterophylla and enhancing its resistance, photosynthesis, quality, and yield.

Dissipation, residue and dietary risk assessment of difenoconazole in Rosa roxburghii.

Rosa roxburghii is a medicinal and edible plant, which is favored by consumers due to its rich vitamin C content. Residues and potential health risks of difenoconazole in the R. roxburghii ecosystem has aroused a concern considering its extensive use for controlling the powdery mildew of R. roxburghii. In this study, the residue of difenoconazole in R. roxburghii and soil was extracted by acetonitrile, purified by primary secondary amine and detected by liquid chromatography-tandem triple quadrupole mass spectrometry. The average recoveries in R. roxburghii and soil matrix varied from 82.59% to 99.63%, with relative standard deviations (RSD) of 1.14%-8.23%. The limit of quantification (LOQ) and detection (LOD) of difenoconazole in R. roxburghii and soil samples were 0.01 mg/kg. The dissipation of difenoconazole followed well the first-order kinetic, with a half-life of 3.99-5.57 d in R. roxburghii and 4.94-6.23 d in soil, respectively. And the terminal residues were <0.01-2.181 mg/kg and 0.014-2.406 mg/kg, respectively. The chronic and acute risk quotient values of difenoconazole were respectively 0.42% and 4.1%, which suggests that the risk was acceptable and safe to consumers. This study provides a reference for the safe and reasonable use of difenoconazole in R. roxburghii production.

A new laboratory method to study the impact of leaf texture on pesticide dislodgeable foliar residues (DFR).

Pesticides are vital in meeting the challenge of feeding the rapidly increasing world population. However, it is crucial that they are used in a way that does not compromise the safety of humans or the environment. Non-dietary worker risk assessments consider the amount of residue which can be transferred from plant foliage to the skin or clothes, known as dislodgeable foliar residues (DFRs). DFR data scarcity due to the costly and seasonal characteristics of DFR studies is an obstacle to the extrapolation of DFR data to different crops/leaves. This paper validates a new proof-of-concept technique to investigate factors that may affect DFR (leaf texture) using the fungicide difenoconazole EC 10% as an example on various leaves (i.e., French bean, soybean, tomato, oilseed rape, and wheat). DFR was the lowest in the case of oilseed rape (31.0 ± 3.4%) and the highest in French beans (82.0 ± 2.9%). This significant difference in DFR in the findings of this study sheds light on the importance of the leaf surface as a major factor affecting DFR and supports the application of the laboratory method for more extensive data generation. More data generation would enable the extrapolation saving money and resources.

Difenoconazole causes spleen tissue damage and immune dysfunction of carp through oxidative stress and apoptosis.

As the use of pesticides increases year after year, so does the level of residual pesticides in the aquatic environment, posing a serious threat to non-target organisms. Difenoconazole (DFZ), a class of long-lasting fungicides and residues in the marine environment, has been shown to cause damaging effects on different organs of aquatic organisms. However, there is no research on the damage of DFZ to carp spleen tissue. This study aimed to investigate the acute toxic effects of DFZ on the spleen tissue of carp (Cyprinus carpio) by exposing juvenile carp to environmentally relevant concentrations of DFZ. We randomly selected 30 carp, divided them into the Control, Low, and High groups, and then exposed the three groups to 0, 0.488 mg/L DFZ, and 1.953 mg/L DFZ for 96 h respectively. We then investigated the toxic effects caused by DFZ on carp and spleen tissues by detecting changes in spleen histopathologic damage, apoptosis, oxidative stress, inflammation, and blood biochemical parameters. We found that DFZ causes severe histopathology in spleen tissue, including ballooning, structural relaxation, and giant mitochondria. In addition, we found that DFZ caused excessive apoptosis in spleen tissue by TUNEL staining and expression levels of apoptosis-related genes (caspase3, caspase8, caspase9, fas, bax, bcl-2, and p53). The activities and transcript levels of the antioxidant enzymes SOD, CAT, and GSH-Px were significantly down-regulated. In addition, DFZ led to a significant increase in activation of the NF-κB signaling pathway and mRNA levels of pro-inflammatory cytokines il-6, il-1ß, and tnf-α, and a substantial decrease in mRNA levels of anti-inflammatory cytokines il-10 and tgf-ß1 in spleen tissue. Blood biochemical parameters showed that DFZ exposure significantly reduced erythrocyte, leukocyte, hemoglobin, C3, and IgM levels. Collectively, DFZ exposure induced apoptosis, immunosuppression, oxidative stress, and inflammatory responses in the spleen tissue of carp, resulting in spleen tissue damage.

Difenoconazole disrupts the blood-brain barrier and results in neurotoxicity in carp by inhibiting the Nrf2 pathway mediated ROS accumulation.

Excessive use of hard-to-degrade pesticides threatens the ecological health of aquatic systems. This study aimed to investigate difenoconazole (DFZ) residues in the environment induced neurotoxicity in carp and the underlying mechanisms. A total of thirty-six carps were divided into three groups and exposed to 0, 0.5, and 2.0 mg/L DFZ for 96 h, respectively. The alterations in behavior and blood-brain barrier (BBB) were examined, and potential mechanisms were explored using immunological assays and biochemical methods. The results showed that DFZ exposure caused behavioral freezing, reduced feeding, and neuronal necrosis in carp. Mechanistically, DFZ triggered ROS accumulation and destroyed the balance between oxidation and antioxidation with increased lipid peroxidation product MDA contents and reduced antioxidant enzymes SOD and CAT activities in the carp brain by inhibiting the NF-E2-related factor 2 (Nrf2) pathway. The activation of oxidative stress further reduced tight junction proteins and MMP levels, thereby destroying BBB and leading to DFZ leakage into the brain. Increased BBB permeability additionally led to DFZ activation of nuclear factor kappa-B signaling-mediated inflammatory cytokine storm, exacerbating neuroinflammation. Meanwhile, DFZ exposure activated mitochondria-associated apoptosis in the carp's brain by up-regulating Bcl-2 associated X protein, cleaved-caspase3, and cytochrome C and decreasing B-cell lymphoma-2 levels. Interestingly, the carp's brain initiated a protective autophagic response via the PI3K/AKT/TOR pathway intending to counteract the neurotoxicity of DFZ. Overall, we concluded that accumulation of DFZ at high concentrations in the aquatic systems disrupted the BBB and resulted in neurotoxicity in carp through inhibition of Nrf2 pathway-mediated ROS accumulation. This study provides a reference for monitoring DFZ residues in the environment and a new target for the treatment of DFZ-induced neurotoxicity in carp.

Ginger reserves testicular spermatogenesis and steroidogenesis in difenoconazole-intoxicated rats by conducting oxidative stress, apoptosis and proliferation.

Difenoconazole, a triazole fungicide, can induce reproductive toxicity in aquatic species, but the probable mechanisms of this hazard in mammals are not formally reported. Here, we have examined the possible ameliorative efficiency of the ginger aqueous extract against the reproductive toxicity of difenoconazole in male rats. Thirty-six animals were equally divided into six groups: control, ginger aqueous extract (50 mg/kg), difenoconazole (15 mg/kg), difenoconazole (30 mg/kg) and ginger co-treated with two doses of difenoconazole. Difenoconazole markedly decreased sperm count, motility and normality percentage, together with the Johnson score. Difenoconazole also significantly reduced serum testosterone, luteinizing hormone and follicle-stimulating hormone levels, as well as the activities of testicular steroidogenic acute regulatory protein and 17 ß-hydroxysteroid dehydrogenases. Furthermore, difenoconazole brought a significant decrease in the testicular activity of catalase, but it increased the activity of glutathione peroxidase. Moreover, difenoconazole upregulated the testicular transcripts of Bax and caspase-3, increased Ki-67 immunoreactivity and induced histoarchitecture alterations plus DNA damage. Remarkably, ginger co-treatment preserved sperm toxicity, restored hormone profiles, increased steroidogenic activity and prevented oxidative injury-promoted testicular apoptosis. In conclusion, phenolic acids and flavonoids of ginger can reserve spermatogenesis and steroidogenesis in difenoconazole-intoxicated rats by improving testicular redox status, inhibiting apoptosis and refining proliferation capacity.

DNA damage and cell apoptosis induced by fungicide difenoconazole in mouse mononuclear macrophage RAW264.7.

Difenoconazole (DFC) is a typical triazole fungicide. Because of its effective bactericidal activity, it has been widely used in agricultural products such as fruits and vegetables. This study revealed the cytotoxic effect of fungicide DFC on mouse monocyte macrophage RAW264.7. The results showed that the IC50 value of DFC on RAW264.7 cells was 37.08 µM (24 h). DFC can significantly inhibit the viability of RAW264.7 cells, induce DNA damage and enhance apoptosis. The established cytotoxicity test showed that DFC-induced DNA double strand breaks in RAW264.7 cells. DFC-treated cells showed typical morphological changes of apoptosis, including chromatin condensation and nuclear lysis. In addition, DFC can induce the release of Cyt c, promote the collapse of mitochondrial membrane potential and increase the Bax/Bcl-2 ratio in RAW264.7 cells. Through this research, people further understand the toxicity of DFC and provide a more scientific basis for its safety application and risk management.

Synergistic Effects of Bacillus amyloliquefaciens SDTB009 and Difenoconazole on Fusarium Wilt of Tomato.

Fusarium wilt is a destructive and widespread disease of tomatoes in China, and currently, there are no effective and environmentally friendly control measures. Combining biological control agents with fungicides has become an executable method for disease control. Here, Bacillus amyloliquefaciens SDTB009 showed excellent in vitro antagonistic activity against Fusarium oxysporum and tolerance to high concentrations of difenoconazole (200 mg/liter) in vitro. The combination of SDTB009 and difenoconazole exhibited more effectiveness in mycelial growth inhibition than either treatment alone. Compared with that in the SDTB009 bulk solution in vitro (5.22 g/liter), surfactin titer reached 7.15 g/liter in the 100 mg/liter of difenoconazole-containing medium. Interestingly, the upregulation of 20 genes in the surfactin biosynthesis pathway from 2-fold to 4-fold was observed, explaining the synergistic effect. The SDTB009 combined with varying concentrations of difenoconazole (60, 120, and 150 g a.i./ha) showed a synergistic effect in two consecutive years of field trials. These results show that the integration of difenoconazole with the biocontrol agent B. amyloliquefaciens SDTB009 synergistically increases the control efficacy of the fungicide against tomato Fusarium wilt.