Catalytic role of histidine-114 in the hydrolytic dehalogenation of chlorothalonil by Pseudomonas sp. CTN-3.

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile; TPN) is an environmentally persistent fungicide that sees heavy use in the USA and is highly toxic to aquatic species and birds, as well as a probable human carcinogen. The chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, UniProtKB C9EBR5) degrades TPN to its less toxic 4-OH-TPN analog making it an exciting candidate for the development of a bioremediation process for TPN; however, little is currently known about its catalytic mechanism. Therefore, an active site residue histidine-114 (His114) which forms a hydrogen bond with the Zn(II)-bound water/hydroxide and has been suggested to be the active site acid/base, was substituted by an Ala residue. Surprisingly, ChdH114A exhibited catalytic activity with a kcat value of 1.07 s-1, ~ 5% of wild-type (WT) Chd, and a KM of 32 µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and ChdH114A active sites were examined using UV-Vis and EPR spectroscopy on the catalytically competent Co(II)-substituted enzyme as well as all-atomistic molecular dynamics (MD) simulations. Combination of these data suggest His114 can quickly and reversibly move nearly 2 Å between one conformation that facilitates catalysis and another that enables product egress and active site recharge. In light of experimental and computational data on ChdH114A, Asn216 appears to play a role in substrate binding and preorganization of the transition-state while Asp116 likely facilitates the deprotonation of the Zn(II)-bound water in the absence of His114. Based on these data, an updated proposed catalytic mechanism for Chd is presented.

Identification of potential chemical biomarkers of hexaconazole using in vitro metabolite profiling in rat and human liver microsomes and in vivo confirmation through urinary excretion study in rats.

Hexaconazole (HEX) is an azole fungicide widely used in agricultural practices across various countries and numerous studies have reported the toxic effects of HEX, such as endocrine disruption, immunotoxicity, neurotoxicity and carcinogenicity. Despite its widespread agricultural use and toxic effects, the metabolism of HEX is not completely understood, and information on urinary elimination of HEX or its metabolites is limited. Therefore, in the present study, we aimed to identify HEX metabolites in rat and human liver microsomes followed by their in vivo confirmation using a urinary excretion study in rats to identify potential candidate for exposure biomarkers for human biomonitoring studies. From the in vitro assay, a total of 12 metabolites were observed, where the single oxidation metabolites (M5 and M6) were the most abundant metabolites in both rat and human liver microsomes. The triple oxidation followed by dehydration metabolite, M8 (which could also be hexaconazole acid or hydroxy keto-hexaconazole), and the double oxidation metabolite (M9) were the major metabolites found in rat urine and were detectable in rat urine longer than the parent. These metabolites increased with decreasing concentrations of HEX in the rat urine samples. Therefore, metabolites M8, M9 and M5 could be pursued further as potential biomarkers for assessing and monitoring human exposure to HEX.

Stimulation of estrogen receptor-alpha by hydroxyanilide fungicide, fenhexamid promotes lipid accumulation in 3 T3-L1 adipocyte.

Fenhexamid are fungicides that act against plant pathogens by inhibiting sterol biosynthesis. Nonetheless, it can trigger endocrine disruption and promote breast cancer cell growth. In a recent study, we investigated the mechanism underlying the lipid accumulation induced by fenhexamid hydroxyanilide fungicides in 3 T3-L1 adipocytes. To examine the estrogen receptor alpha (ERα)-agonistic effect, ER transactivation assay using the ERα-HeLa-9903 cell line was applied, and fenhexamid-induced ERα agonist effect was confirmed. Further confirmation that ERα-dependent lipid accumulation occurred was provided by treating 3 T3-L1 adipocytes with Methyl-piperidino-pyrazole hydrate (MPP), an ERα-selective antagonist. Fenhexamid mimicked the actions of ERα agonists and impacted lipid metabolism, and its mechanism involves upregulation of the expression of transcription factors that facilitate adipogenesis and lipogenesis. Additionally, it stimulated the expression of peroxisome proliferator-activated receptor (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), and sterol regulatory element-binding protein 1 (SREBP1) and significantly elevated the expression of fatty acid-binding protein 4 (FABP4). In contrast, in combination with an ERα-selective antagonist, fenhexamid suppressed the expression of adipogenic/lipogenic transcription factors. These results suggest that fenhexamid affects the endocrine system and leads to lipid accumulation by interfering with processes influenced by ERα activation.

Meptyldinocap induces implantation failure by forcing cell cycle arrest, mitochondrial dysfunction, and endoplasmic reticulum stress in porcine trophectoderm and endometrial luminal epithelial cells.

Meptyldinocap is a dinitrophenol fungicide used to control powdery mildew. Although other dinitrophenol pesticides have been found to exhibit reproductive toxicity, studies of meptyldinocaps are scarce. This study investigated the adverse effects of meptyldinocap on porcine trophectoderm (pTr) and porcine endometrial luminal epithelial (pLE) cells, which play crucial roles in implantation. We confirmed that meptyldinocap decreased cell viability, induced apoptosis, and inhibited proliferation by decreasing proliferation-related gene expression and inducing changes in the cell cycle. Furthermore, meptyldinocap treatment caused mitochondrial dysfunction, endoplasmic reticulum stress, and disruption of calcium homeostasis. Moreover, it induces alterations in mitogen-activated protein kinase signaling cascades and reduces the migration ability, leading to implantation failure. Our findings suggest that meptyldinocap reduces the cellular functions of pTr and pLE cells, which are important for the implantation process, and interferes with interactions between the two cell lines, potentially leading to implantation failure. We also propose a mechanism by which the understudied fungicide meptyldinocap exerts its cytotoxicity.

Potential anti-Pythium insidiosum therapeutics identified through screening of agricultural fungicides.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Clinical manifestations of pythiosis include an eye, blood vessel, skin, or gastrointestinal tract infection. Pythiosis has been increasingly reported worldwide, with an overall mortality rate of 28%. Radical surgery is required to save patients' lives due to the limited efficacy of antimicrobial drugs. Effective medical treatments are urgently needed for pythiosis. This study aims to find anti-P. insidiosum agents by screening 17 agricultural fungicides that inhibit plant-pathogenic oomycetes and validating their efficacy and safety. Cyazofamid outperformed other fungicides as it can potently inhibit genetically diverse P. insidiosum isolates while exhibiting minimal cellular toxicities. The calculated therapeutic scores determined that the concentration of cyazofamid causing significant cellular toxicities was eight times greater than the concentration of the drug effectively inhibiting P. insidiosum. Furthermore, other studies showed that cyazofamid exhibits low-to-moderate toxicities in animals. The mechanism of cyazofamid action is likely the inhibition of cytochrome b, an essential component in ATP synthesis. Molecular docking and dynamic analyses depicted a stable binding of cyazofamid to the Qi site of the P. insidiosum's cytochrome b orthologous protein. In conclusion, our search for an effective anti-P. insidiosum drug indicated that cyazofamid is a promising candidate for treating pythiosis. With its high efficacy and low toxicity, cyazofamid is a potential chemical for treating pythiosis, reducing the need for radical surgeries, and improving recovery rates. Our findings could pave the way for the development of new and effective treatments for pythiosis.IMPORTANCEPythiosis is a severe infection caused by Pythium insidiosum. The disease is prevalent in tropical/subtropical regions. This infectious condition is challenging to treat with antifungal drugs and often requires surgical removal of the infected tissue. Pythiosis can be fatal if not treated promptly. There is a need for a new treatment that effectively inhibits P. insidiosum. This study screened 17 agricultural fungicides that target plant-pathogenic oomycetes and found that cyazofamid was the most potent in inhibiting P. insidiosum. Cyazofamid showed low toxicity to mammalian cells and high affinity to the P. insidiosum's cytochrome b, which is involved in energy production. Cyazofamid could be a promising candidate for the treatment of pythiosis, as it could reduce the need for surgery and improve the survival rate of patients. This study provides valuable insights into the biology and drug susceptibility of P. insidiosum and opens new avenues for developing effective therapies for pythiosis.

Complete biodegradation of fungicide carboxin and its metabolite aniline by Delftia sp. HFL-1.

Fungicide carboxin was commonly used in the form of seed coating for the prevention of smut, wheat rust and cotton damping-off, leading carboxin and its probable carcinogenic metabolite aniline to directly enter the soil with the seeds, causing residual pollution. In this study, a novel carboxin degrading strain, Delftia sp. HFL-1, was isolated. Strain HFL-1 could use carboxin as the carbon source for growth and completely degrade 50 mg/L carboxin and its metabolite aniline within 24 h. The optimal temperatures and pH for carboxin degrading by strain HFL-1 were 30 to 42 °C and 5 to 9, respectively. Furthermore, the complete mineralization pathway of carboxin by strain HFL-1 was revealed by High Resolution Mass Spectrometer (HRMS). Carboxin was firstly hydrolyzed into aniline and further metabolized into catechol through multiple oxidation processes, and finally converted into 4-hydroxy-2-oxopentanoate, a precursor of the tricarboxylic acid cycle. Genome sequencing revealed the corresponding degradation genes and cluster of carboxin. Among them, amidohydrolase and dioxygenase were key enzymes involved in the degradation of carboxin and aniline. The discovery of transposons indicated that the aniline degradation gene cluster in strain HFL-1 was obtained via horizontal transfer. Furthermore, the degradation genes were cloned and overexpressed. The in vitro test showed that the expressed degrading enzyme could efficiently degrade aniline. This study provides an efficient strain resource for the bioremediation of carboxin and aniline in contaminated soil, and further revealing the molecular mechanism of biodegradation of carboxin and aniline.

New insights into triazole fungicide-caused hematopoietic abnormality in zebrafish based on GRα screening developmental toxicity.

Triazole fungicides (TFs) are known to be common environmental contaminants that can be toxic to aquatic animals, but their developmental toxicity is not fully understood. To address this gap, we first used a glucocorticoid receptor α (GRα)-mediated dual luciferase reporter gene system to explore the possible development toxicity of ten TFs and found that flusilazole (FLU) exhibited stronger agonistic activity against GRα. Subsequent transcriptome sequencing showed that FLU exposure affected GRα activation and hematopoiesis associated with a variety of biological processes, including responses to corticosteroid release, embryonic hematopoiesis, erythroid differentiation, and the development of hematopoietic or lymphoid organs. Furthermore, based on in situ hybridization and staining techniques, we clarified that FLU decreased the expression of the primitive hematopoietic marker genes gata1 and pu.1. and caused the defects in the posterior blood island (PBI), thereby impacting intermediate hematopoietic processes. Also, FLU significantly reduced the expression of the crucial hematopoietic gene cmyb and disrupted the production of erythrocytes and bone marrow cells during definitive hematopoiesis. Consistently, we found that FLU induced lesions in the kidney, a hematopoietic organ, including the infiltration of inflammatory cells, tubular collapse, reduced tubular filtration area, and interstitial hydronephrosis. We also found that FLU increased aberrant red blood cells in the peripheral blood of zebrafish. These findings provide new insights into the developmental toxicity and ecotoxicological risk of TFs.

Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Introduction: Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Design: Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12µg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48µg/kg.d; KTZ 3-12-48mg/kg.d). Results: Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. Conclusion: nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

The interaction effects of pesticides with Saccharomyces cerevisiae and their fate during wine-making process.

Pesticide residues in grapes could be transferred to fermentation system during the wine-making process, which may interfere the normal proliferation of Saccharomyces cerevisiae and subsequently affect the safety and quality of wine products. However, the interaction between pesticides and Saccharomyces cerevisiae is still poorly understood. Herein, the fate, distribution and interaction effect with Saccharomyces cerevisiae of five commonly-used pesticides during the wine-making process were evaluated. The five pesticides exerted varied inhibition on the proliferation of Saccharomyces cerevisiae, and the order of inhibition intensity was difenoconazole > tebuconazole > pyraclostrobin > azoxystrobin > thiamethoxam. Compared with the other three pesticides, triazole fungicides difenoconazole and tebuconazole showed stronger inhibition and played a major role in binary exposure. The mode of action, lipophilicity and exposure concentration were important factors in the inhibition of pesticides. Saccharomyces cerevisiae had no obvious impacts on the degradation of target pesticides in the simulated fermentation experiment. However, the levels of target pesticides and their metabolite were significantly reduced during the wine-making process, with the processing factors ranged from 0.030 to 0.236 (or 0.032 to 0.257) during spontaneous (or inoculated) wine-making process. As a result, these pesticides were significantly enriched in the pomace and lees, and showed a positive correlation (R2 ≥ 0.536, n = 12, P < 0.05) between the hydrophobicity of pesticides and distribution coefficients in the solid-liquid distribution system. The findings provide important information for rational selection of pesticides on wine grapes and facilitate more accurate risk assessments of pesticides for grape processing products.

Do Fungicides Affect Alkaloid Production in Catharanthus roseus (L.) G. Don. Seedlings?

The presence of endophytes in plants is undeniable, but how significant their involvement is in the host plant biosynthetic pathways is still unclear. The results reported from fungicide treatments in plants varied. Fungicide treatment in Taxus was found to decrease the taxol content. In Ipomoea asarifolia, Pronto Plus and Folicur treatments coincided with the disappearance of ergot alkaloids from the plant. In Narcissus pseudonarcissus cv. Carlton, a mixture of fungicide applications decreased the alkaloids concentration and altered the carbohydrate metabolism. Jacobaea plants treated with Folicur reduced the pyrrolizidine alkaloids content. There have not been any studies into the involvement of endophytic fungi on alkaloids production of Catharanthus roseus until now. Though there is a report on the isolation of the endophytic fungi, Fusarium oxysporum from C. roseus, which was reported to produce vinblastine and vincristine in vitro. To detect possible collaborations between these two different organisms, fungicides were applied to suppress the endophytic fungi in seedlings and then measure the metabolomes by 1HNMR and HPLC analysis. The results indicate that endophytic fungi were not directly involved in alkaloids biosynthesis. Treatment with fungicides influenced both the primary and secondary metabolism of C. roseus. The systemic fungicides Pronto Plus and Folicur caused an increase in loganin and secologanin levels. In contrast, control samples had higher level of catharanthine and vindoline. This means that fungicide treatments cause changes in plant secondary metabolism.

Mining an O-methyltransferase for de novo biosynthesis of physcion in Aspergillus nidulans.

Physcion is one of natural anthraquinones, registered as a novel plant-derived fungicide due to its excellent prevention of plant disease. However, the current production of physcion via plant extraction limits its yield promotion and application. Here, a pair of polyketide synthases (PKS) in emodin biosynthesis were used as probes to mining the potential O-methyltransferase (OMT) responsible for physcion biosynthesis. Further refinement using the phylogenetic analysis of the mined OMTs revealed a distinct OMT (AcOMT) with the ability of transferring a methyl group to C-6 hydroxyl of emodin to form physcion. Through introducing AcOMT, we successfully obtained the de novo production of physcion in Aspergillus nidulans. The physcion biosynthetic pathway was further rationally engineered by expressing the decarboxylase genes from different fungi. Finally, the titer of physcion reached to 64.6 mg/L in shake-flask fermentation through enhancing S-adenosylmethionine supply. Our work provides a native O-methyltransferase for physcion biosynthesis and lays the foundation for further improving the production of physcion via a sustainable route. KEY POINTS: • Genome mining of the native O-methyltransferase responsible for physcion biosynthesis • De novo biosynthesis of physcion in the engineered Aspergillus nidulans • Providing an alternative way to produce plant-derived fungicide physcion.

Transcriptomic Analysis of Resistant and Wild-Type Botrytis cinerea Isolates Revealed Fludioxonil-Resistance Mechanisms.

Botrytis cinerea, the causal agent of gray mold, is one of the most destructive pathogens of cherry tomatoes, causing fruit decay and economic loss. Fludioxonil is an effective fungicide widely used for crop protection and is effective against tomato gray mold. The emergence of fungicide-resistant strains has made the control of B. cinerea more difficult. While the genome of B. cinerea is available, there are few reports regarding the large-scale functional annotation of the genome using expressed genes derived from transcriptomes, and the mechanism(s) underlying such fludioxonil resistance remain unclear. The present study prepared RNA-sequencing (RNA-seq) libraries for three B. cinerea strains (two highly resistant (LR and FR) versus one highly sensitive (S) to fludioxonil), with and without fludioxonil treatment, to identify fludioxonil responsive genes that associated to fungicide resistance. Functional enrichment analysis identified nine resistance related DEGs in the fludioxonil-induced LR and FR transcriptome that were simultaneously up-regulated, and seven resistance related DEGs down-regulated. These included adenosine triphosphate (ATP)-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, and the high-osmolarity glycerol (HOG) pathway homologues or related genes. The expression patterns of twelve out of the sixteen fludioxonil-responsive genes, obtained from the RNA-sequence data sets, were validated using quantitative real-time PCR (qRT-PCR). Based on RNA-sequence analysis, it was found that hybrid histidine kinase, fungal HHKs, such as BOS1, BcHHK2, and BcHHK17, probably involved in the fludioxonil resistance of B. cinerea, in addition, a number of ABC and MFS transporter genes that were not reported before, such as BcATRO, BMR1, BMR3, BcNMT1, BcAMF1, BcTOP1, BcVBA2, and BcYHK8, were differentially expressed in the fludioxonil-resistant strains, indicating that overexpression of these efflux transporters located in the plasma membranes may associate with the fludioxonil resistance mechanism of B. cinerea. All together, these lines of evidence allowed us to draw a general portrait of the anti-fludioxonil mechanisms for B. cinerea, and the assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of B. cinerea resistance to fludioxonil.

Hepatoprotective Effect of Moringa Oil on Rats under Fungicide Toxicity.

The present study is designed to evaluate whether pretreatment with moringa would have a protective effect on thioacetamide (TAA)-induced liver fibrosis, assessing biochemical and histopathological changes in Wistar male rats. Exposure to TAA induced notable biochemical and histopathological alterations. Liver fibrosis induced by TAA, along with associated biochemical and histological damage, has not been previously investigated in male rats supplemented with moringa oil. The experiment involved forty male rats distributed across four groups, each comprising ten rats. Group 1 served as controls and received intraperitoneal injections of saline solution twice weekly for six weeks. Group 2 rats were injected with 300 mg/kg body weight of TAA (Sigma-Aldrich Corp.) twice weekly for the same duration. Group 3 rats were orally supplemented with moringa oil at 800 mg/kg body weight/day and received intraperitoneal injections of TAA at the same dosage as Group 2 for six weeks. Finally, Group 4 rats were injected with saline solution twice weekly and orally supplemented with moringa oil at 800 mg/kg body weight/day for the same period. At the end of the experiment, we determined body weight and performed liver function analysis. Additionally, we examined the liver histology of the different groups. Results showed that moringa oil treatment protected rat livers from TAA toxicity by improving liver function analysis and preventing liver fibrosis. Moringa oil can be considered a promising agent for protection against TAA toxicity.

Fludioxonil, a phenylpyrrol pesticide, induces Cytoskeleton disruption, DNA damage and apoptosis via oxidative stress on rat glioma cells.

Pesticides products are widely used to increase food productivity and to decrease food-borne diseases. Fludioxonil is a worldwide used phenylpyrrol fungicide. This pesticide can induce serious effects on human health especially on nervous system. We assessed the role of oxidative stress in the toxicity of Fludioxonil and examined its apoptotic mechanism of action on rat neural cells (F98). We have shown that the increasing concentration of Fludioxonil reduces the percentage of living F98 cells viability and increases the levels of reactive oxygen species and malondialdheydes. The reduction of cells proliferation was demonstrated with an accumulation in G2/M phase. The immunocytochemical analysis has shown that Fludioxonil induced the disruption of the cytoskeleton. DNA damage was also provoked in a concentration dependent manner as illustrated by the comet assay. The depolarization of the mitochondria and the positive Annexin V FITC-PI confirmed the apoptosis induced by this fungicide. Interestingly, the F98 cells viability and ROS levels were restored with N-acetylcysteine pre-treatment. These results highlight the involvement of oxidative stress in the toxicity induced by this fungicide, and that free radicals generation plays a key role in the induction of apoptosis probably induced via the mitochondrial pathway.

Whole-genome analysis and secondary metabolites production of a new strain Brevibacillus halotolerans 7WMA2: A potential biocontrol agent against fungal pathogens.

The extensive usage of synthetic fungicides against fungal diseases has caused adverse impacts on both human and agricultural crops. Therefore, the current study aims to establish a new bacterium 7WMA2, as a biocontrol agent to achieve better antifungal results. The strain 7WMA2 was isolated from marine sediment, displayed a broad spectrum of several fungi that includes Alternaria alternata, Cladosporium sp., Candida albicans, Fusarium oxysporum, Trichosporon pullulans, and Trichophyton rubrum. The 16S rRNA phylogeny inferred that strain 7WMA2 was a member of Brevibacillus. The phylogenetic and biochemical analyses revealed that the strain 7WMA2 belongs to the species of Brevibacillus halotolerans. The complete genome sequence of Brevibacillus halotolerans 7WMA2 consists of a circular chromosome of 5,351,077 bp length with a GC content of 41.39 mol %, including 4433 CDS, 111 tRNA genes, and 36 rRNA genes. The genomic analysis showed 23 putative biosynthetic secondary metabolite gene clusters responsible for non-ribosomal peptides, polyketides and siderophores. The antifungal compounds concentrated from cell-free fermentation broth demonstrated strong inhibition of fungi, and the compounds are considerably thermal stable and adaptable to pH range 2-12. This complete genome sequence has provided insight for further exploration of antagonistic ability and its secondary metabolite compounds indicated feasibility as biological control agents against fungal infections.

Exposure to iprodione induces ROS production and mitochondrial dysfunction in porcine trophectoderm and uterine luminal epithelial cells, leading to implantation defects during early pregnancy.

Iprodione is a well-known fungicide used in the cultivation of strawberries, tomatoes, grapes, and green beans. In recent studies, neurotoxicity, cardiotoxicity, and endocrine toxicity of iprodione have been reported. Although reproductive toxicity of iprodione has been identified in animal studies, its effects are limited to male fertility. Also, the toxic effects of iprodione on pregnancy, especially the implantation process, have not been elucidated. This study demonstrated a series of cytotoxic responses of iprodione along with the alteration of implantation-related gene expression in porcine trophectoderm (pTr) and luminal epithelium (pLE) cells. In this study, iprodione suppressed cell viability, proliferation, and migration of these cells. Iprodione induced G1 phase arrest and attenuated spheroid formation by pTr and pLE cells. Furthermore, iprodione caused mitochondrial dysfunction and excessive reactive oxygen species generation, which resulted in an increase in mitochondrial calcium levels. Consequently, DNA damage and apoptotic cell death were induced by iprodione treatment in pTr and pLE cells. This stress-induced cell death was mediated by alterations in intracellular signal transduction, including the PI3K/AKT and MAPK signaling pathways. This finding suggests the potential of iprodione to impair the implantation capacity by exerting cytotoxic effects on fetal and maternal cells.

Sequential phase I metabolism of pyrethroids by duplicated CYP6P9 variants results in the loss of the terminal benzene moiety and determines resistance in the malaria mosquito Anopheles funestus.

Pyrethroid resistance in Anopheles funestus is threatening the eradication of malaria. One of the major drivers of pyrethroid resistance in An. funestus are cytochrome P450 monooxygenases CYP6P9a and CYP6P9b, which are found upregulated in resistant An. funestus populations from Sub-Saharan Africa and are known to metabolise pyrethroids. Here, we have functionally expressed CYP6P9a and CYP6P9b variants and investigated their interactions with azole-fungicides and pyrethroids. Some azole fungicides such as prochloraz inhibited CYP6P9a and CYP6P9b at nanomolar concentrations, whereas pyrethroids were weak inhibitors (>100 µM). Amino acid sequence comparisons suggested that a valine to isoleucine substitution at position 310 in the active site cavity of CYP6P9a and CYP6P9b, respectively, might affect substrate binding and metabolism. We therefore swapped the residues by site directed mutagenesis to produce CYP6P9aI310V and CYP6P9bV310I. CYP6P9bV310I produced stronger metabolic activity towards coumarin substrates and pyrethroids, particularly permethrin. The V310I mutation was previously also detected in a pyrethroid resistant field population of An. funestus in Benin. Additionally, we found the first metabolite of permethrin and deltamethrin after hydroxylation, 4'OH permethrin and 4'OH deltamethrin, were also suitable substrates for CYP6P9-variants, and were depleted by both enzymes to a higher extent than as their respective parent compounds (approximately 20% more active). Further, we found that both metabolites were toxic against An. funestus FANG (pyrethroid susceptible) but not towards FUMOZ-R (pyrethroid resistant) mosquitoes, the latter suggesting detoxification by overexpressed CYP6P9a and CYP6P9b. We confirmed by mass-spectrometric analysis that CYP6P9a and CYP6P9b are capable of cleaving phenoxybenzyl-ethers in type I pyrethroid permethrin and type II pyrethroid deltamethrin and that both enzymes preferentially metabolise trans-permethrin. This provides new insight into the metabolism of pyrethroids and a greater understanding of the molecular mechanisms of pyrethroid resistance in An. funestus.

Toxicological differences of trifloxystrobin and kresoxim-methyl on zebrafish in various levels of exposure routes, organs, cells and biochemical indicators.

Trifloxystrobin (TRI) and kresoxim-methyl (KRE), as quinone outside inhibitor fungicides (QoIs), have broad applications due to their effective activity against fungi. Excessive usages of agrochemicals trigger environmental risks, such as aquatic organisms (fish). Research performed in recent years has focused on the ecotoxicology of TRI and KRE in fish containing histologic morphology, enzyme activity, protein and gene expression under chronic toxicity conditions, whereas less is known about the underlying mechanisms of toxicity and differences between TRI and KRE in fish under acute toxicity conditions. In the present study, in comparison to different exposure routes [whole-body exposure (WBE), head exposure (HE), trunk exposure (TE), and Oral administration (OA)], the external substances TRI and KRE entered the fish body mainly via gill organs and led to fish toxicity. Furthermore, gill organs and gill cells were vulnerable to TRI and KRE exposure, which indicated that the gill is a vital impaired organ. The 96 h-LC50 (sublethal concentration) value of KRE was 289.8 µg L-1 (R2 = 0.9855) with an approximate 10-fold difference in TRI toxicity. The cytotoxicity exposed to TRI was higher than that in KRE at the same concentration. The potential mechanisms of toxic differences could be various toxic effects in terms of MCIII (mitochondrial complex III) activity, ATP (Adenosine triphosphate) content, MA (mitochondrial activity), ROS (reactive oxygen species) levels, and cellular respiration. Furthermore, the disorder in MCIII activity was probably the main potential mechanisms of toxic differences. To some extent, this research provides not only new insight into the underlying toxic mechanism of TRI and KRE in fish but also a basis for the guidance of agrochemicals considering aquatic risks.

Histopathological, immunohistochemical and biochemical alterations in liver tissue after fungicide-mancozeb exposures in Wistar albino rats.

PURPOSE: To evaluate the histopathological, immunohistochemical, and biochemical effects of liver changes after mancozeb administration. METHODS: Rats were divided into groups-the control group (n=7) and the mancozeb group (n=7)-, given 500 mg/kg mancozeb dissolved in corn oil daily for four weeks by an orogastric tube. Caspase-3 and tumor necrosis factor-alpha (TNF-α) primary antibodies were used for immunohistochemical analysis. RESULTS: Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) values of the mancozeb group increased significantly than ones of the control group. Venous dilatation, inflammation, hepatocyte degeneration, TNF-α, and caspase-3 expression scores increased significantly in the mancozeb group. In the mancozeb group, intensive caspase-3 expression was observed in hepatocyte cells around the central vein in the center of the liver lobule, and there was an increase in TNF-α expression in the inflammatory cells around the enlarged central vein and Kupffer cells and apoptotic hepatocyte cells. CONCLUSIONS: Subacute mancozeb exposure in rats leads to elevated toxicity with impaired liver function, increased inflammation in tissue and increased apoptosis due to cellular damage in the liver, and decreased liver regeneration ability due to congestion and degeneration of blood vessels.

Enantioselective effect of chiral fungicide prothioconazole on Fusarium graminearum: Fungicidal activity and DON biosynthesis.

Prothioconazole, a chiral triazole fungicide, is widely used to control Fusarium head blight (FHB) of wheat. Fusarium graminearum (F. graminearum), as the main pathogen of FHB, can produce many secondary metabolites including deoxynivalenol (DON), which threatens the health of humans and animals. However, some fungicides may stimulate F. graminearum to synthesize more DON under certain conditions. Until now, the fungicidal activity and enantioselective effect of prothioconazole enantiomers on DON production, transcriptome and metabolome of F. graminearum were unclear. The fungicidal activity of R-(-)-prothioconazole against F. graminearum was 9.12-17.73 times higher than that of S-(+)-prothioconazole under all conditions. Prothioconazole enantiomers can induce F. graminearum to synthesize more DON under 0.99 water activity (aw) and 30 °C, especially R-(-)-prothioconazole. The expression levels of TRI6, TRI10 and TRI101 under R-(-)-prothioconazole treatment were significantly higher than those under S-(+)-prothioconazole treatment. Most genes in glycolysis, pyruvate metabolism, the target of rapamycin (TOR) signaling transduction pathway and the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling transduction pathway showed higher expression levels under R-(-)-prothioconazole treatment than uner S-(+)-prothioconazole treatment and the control. The peroxisome pathway displayed higher transcriptional activity under S-(+)-prothioconazole treatment compared with R-(-)-prothioconazole and the control. Based on metabolomic data, R-(-)-prothioconazole can significantly influence phenylalanine metabolism, and no significantly enriched pathway was found under S-(+)-prothioconazole treatment. These results are helpful to understand the risk of prothioconazole enantiomers on DON production of F. graminearum and uncover the relevant underlying mechanisms of prothioconazole enantiomers.