Human and rat gonadal 3ß-hydroxysteroid dehydrogenases are suppressed by dithiocarbamate fungicides via interacting with cysteine residues.

Dithiocarbamates have been widely used in various industrial applications, such as insecticides (ferbam) or drug (disulfiram). This study explored the inhibitory effects of dithiocarbamates on human and rat gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD) and investigated the structure-activity relationship and mechanistic insights. The inhibitory activity of six dithiocarbamates and thiourea on the conversion of pregnenolone to progesterone was evaluated using human KGN cell and rat testicular microsomes, with subsequent progesterone measurement using HPLC-MS/MS. The study found that among the tested compounds disulfiram, ferbam, and thiram exhibited significant inhibitory activity against human 3ß-HSD2 and rat 3ß-HSD1, with ferbam demonstrating the highest potency. The mode of action for these compounds was characterized, showing mixed inhibition for human 3ß-HSD2 and mixed/noncompetitive inhibition for rat 3ß-HSD1. Additionally, it was observed that dithiothreitol dose-dependently reversed the inhibitory effects of dithiocarbamates on both human and rat gonadal 3ß-HSD enzymes. The study also delved into the penetration of these dithiocarbamates through the human KGN cell membrane and their impact on progesterone production, highlighting their potency in inhibiting human 3ß-HSD2. Furthermore, bivariate correlation analysis revealed a positive correlation of LogP (lipophilicity) with IC50 values for both enzymes. Docking analysis indicated that dithiocarbamates bind to NAD+ and steroid-binding sites, with some interactions with cysteine residues. In conclusion, this study provides valuable insights into the structure-activity relationship and mechanistic aspects of dithiocarbamates as inhibitors of human and rat gonadal 3ß-HSDs, suggesting that these compounds likely exert their inhibitory effects through binding to cysteine residues.

A Fungicide, Fludioxonil, Formed the Polyploid Giant Cancer Cells and Induced Metastasis and Stemness in MDA-MB-231 Triple-Negative Breast Cancer Cells.

Fludioxonil, an antifungal agent used as a pesticide, leaves a measurable residue in fruits and vegetables. It has been identified to cause endocrine disruption, interrupt normal development, and cause various diseases such as cancers. In this study, fludioxonil was examined for its effects on the development and metastasis of breast cancer cells. On fludioxonil exposure (10-5 M) for 72 h, mutant p53 (mutp53) MDA-MB-231 triple-negative breast cancer (TNBC) cells significantly inhibited cell viability and developed into polyploid giant cancer cells (PGCCs), with an increase in the number of nuclei and expansion in the cell body size. Fludioxonil exposure disrupted the normal cell cycle phase ratio, resulting in a new peak. In addition, PGCCs showed greater motility than the control and were resistant to anticancer drugs, i.e., doxorubicin, cisplatin, and 5-fluorouracil. Cyclin E1, nuclear factor kappa B (NF-κB), and p53 expressions were remarkably increased, and the expression of cell cycle-, epithelial-mesenchymal-transition (EMT)-, and cancer stemness-related proteins were increased in the PGCCs. The daughter cells obtained from PGCCs had the single nucleus but maintained their enlarged cell size and showed greater cell migration ability and resistance to the anticancer agents. Consequently, fludioxonil accumulated Cyclin E1 and promoted the inflammatory cytokine-enriched microenvironment through the up-regulation of TNF and NF-κB which led to the transformation to PGCCs via abnormal cell cycles such as mitotic delay and mitotic slippage in mutp53 TNBC MDA-MB-231 cells. PGCCs and their daughter cells exhibited significant migration ability, chemo-resistance, and cancer stemness. These results strongly suggest that fludioxonil, as an inducer of potential genotoxicity, may induce the formation of PGCCs, leading to the formation of metastatic and stem cell-like breast cancer cells.

Deciphering the role of nitric oxide in mitigation of systemic fungicide induced growth inhibition and oxidative damage in wheat.

Consento (CON) poses a significant environmental hazard as a systemic fungicide, adversely affecting the health of non-target organisms. Nitric oxide (NO), a signaling molecule, is known to play a crucial role in plant physiology and abiotic stress tolerance. However, whether NO plays any role to enhance fungicide CON tolerance in wheat seedlings is yet unclear. Therefore, we conducted a hydroponic experiment i) to investigate the morpho-physio-biochemical changes of wheat seedlings to fungicide CON stress, and ii) to examine the effects of NO and fungicide CON treatments on oxidative damage, antioxidant system, secondary metabolism and detoxification of systemic fungicide in wheat seedlings. The results showed that CON fungicide at the highest (4X) concentration significantly decreased wheat seedlings fresh weight (46.89%), shoot length (40.26%), root length (56.11%) and total chlorophyll contents (67.44%) in a dose response relationship. Moreover, CON significantly increased hydrogen peroxide, malondialdehyde, catalase, ascorbate peroxidase, glutathione-S-transferase, and peroxidase activities while decreased reduced glutathione (GSH) content. This ultimately impaired the redox homeostasis of cells, leading to oxidative damage in cell membrane. Under fungicide treatment, the addition of NO reduced the fungicide phytotoxicity, with an increase of over 60% in seedling growth. The NO application mitigated CON phytotoxicity as reflected by significantly increased chlorophyll pigments (69.88%) and decreased oxidative damage in wheat leaves. Indeed, the NO alleviatory effect was able to increase the tolerance of seedlings to fungicide, which resulted increments in antioxidant and detoxification enzymes activity, with the enhanced GSH level (78.54%). Interestingly, NO alleviated CON phytotoxicity through the phenylpropanoid pathway by enhancing the activity of secondary metabolism enzymes such as phenylalanine ammonia-lyase (47.28%), polyphenol oxidase (9%), and associated metabolites such as phenolic acids (77.62%), flavonoids (34.33%) in wheat leaves. Our study has provided evidence that NO plays a key role in the metabolism and detoxification of systemic fungicide in wheat through enhanced activity of antioxidants, detoxifications and secondary metabolic enzymes.

Network toxicological and molecular docking to investigate the mechanisms of toxicity of agricultural chemical Thiabendazole.

Food safety is closely linked to human health. Thiabendazole is widely used as a fungicide and deodorant on agricultural products like vegetables and fruits to prevent fungal infections during transport and storage. This study aims to investigate the toxicity and potential mechanisms of Thiabendazole using novel network toxicology and molecular docking techniques. First, the ADMETlab2.0 and ADMETsar databases, along with literature, predicted Thiabendazole's potential to induce cancer and liver damage. Disease target libraries were constructed using GeneCards and TCMIP databases, while Thiabendazole target libraries were constructed using Swiss Target Prediction and TCMIP databases. The Venn database identified potential targets associated with Thiabendazole-induced cancer and liver injury. Protein-protein interaction (PPI) networks were derived from the STRING database, and gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways were obtained from the DAVID database. Molecular docking assessed the binding affinity between Thiabendazole and core targets. The study revealed 29 potential targets for Thiabendazole-induced cancer and 30 potential targets for liver injury. PPI identified 5 core targets for Thiabendazole-induced cancers and 4 core targets for induced liver injury. KEGG analysis indicated that Thiabendazole might induce gastric and prostate cancer via cyclin-dependent kinase 2 (CDK2) and epidermal growth factor receptor (EGFR) targets, and liver injury through the same targets, with the p53 signaling pathway being central. GO analysis indicated that Thiabendazole-induced cancers and liver injuries were related to mitotic cell cycle G2/M transition and DNA replication. Molecular docking showed stable binding of Thiabendazole with core targets including CDK1, CDK2, EGFR, and checkpoint kinase 1 (CHEK1). These findings suggest Thiabendazole may affect the G2/M transition of the mitotic cell cycle through the p53 signaling pathway, potentially inducing cancer and liver injury. This study provides a theoretical basis for understanding the potential molecular mechanisms underlying Thiabendazole toxicity, aiding in the prevention and treatment of related diseases. Additionally, the network toxicology approach accelerates the elucidation of toxic pathways for uncharacterized agricultural chemicals.

Neurotoxicity of hexaconazole on rat brain: The aspect of biological rhythm.

Hexaconazole is a widely used and frequently detected fungicide which is also reported to be persistent in environment. The toxicity of Hex to non-organisms such as reproductive toxicity, endocrine disrupting toxicity, and carcinogenic toxicity had been reported. However, study on the Hex-induced neurotoxicity is rare and the mechanism is still unclear. Therefore, in this study, environmental related concentrations of Hex were chosen to investigate the effects of Hex on nervous system from the aspect of biological rhythm under 90 d sub-chronic exposure. The results showed that Hex significantly affected the cognitive function of rats resulting in the deterioration of learning and memory ability and induced oxidative stress in rat brain. Moreover, the notable changes of neurotransmitters in rat brain suggested the disorder of nerve signaling conduction induced by Hex. The influence of Hex on biological rhythm was further detected which showed that levels of rhythm regulatory genes and proteins significantly disturbed at four monitored time periods. Based on these results, it was supposed that the underlying mechanism of Hex-induced cognitive dysfunction might through oxidative stress pathway. Our findings could systematically and comprehensively clarify the effects of Hex on nervous system and were helpful for prevention neurological diseases induced by triazole pesticides.

Systematic health risks assessment of chiral fungicide famoxadone: Stereoselectivities in ferroptosis-mediated cytotoxicity and metabolic behavior.

Famoxadone is a chiral fungicide frequently found in the environment and agricultural products. However, the health risks of famoxadone enantiomers are not well understood. This study investigated the stereoselective cytotoxicity and metabolic behavior of famoxadone enantiomers in mammals. Results showed that R-famoxadone was 1.5 times more toxic to HepG2 cells than S-famoxadone. R-famoxadone induced more pronounced ferroptosis compared to S-famoxadone. It caused greater upregulation of genes related to iron transport and lipid peroxidation, and greater downregulation of genes related to peroxide clearance. Furthermore, R-famoxadone induced more severe lipid peroxidation and reactive oxygen species (ROS) accumulation through ACSL4 activation and GPX4 inhibition. Additionally, the bioavailability of R-famoxadone in mice was six times higher than that of S-famoxadone. Liver microsome assays, cytochrome P450 (CYP450) inhibition assays, human recombinant CYP450 assays, and molecular docking suggested that the lower binding affinities of CYP2C8, CYP2C19, and CYP2E1 for R-famoxadone caused its preferential accumulation. Overall, R-famoxadone poses a higher risk than S-famoxadone due to its greater cytotoxicity and persistence. This study provides the first evidence of ferroptosis-induced stereoselective toxicity, offering insights for the comprehensive health risk assessment of chiral famoxadone and valuable references for the application of high-efficiency, low-risk pesticide enantiomers.

Adverse outcome pathway-based approach to reveal the mechanisms of skin sensitization and long-term aging effects of chlorothalonil.

Chlorothalonil (CHT) is a widely used antifungal agent and is reported to be a sensitizer that can cause allergic contact dermatitis (ACD). ACD initiation is associated with various innate immune cell contributions and is usually accompanied by persistent inflammation, which is a potential contributing factor to skin damage. However, detailed information on the mechanisms by which CHT induces skin sensitization and damage is still insufficient. This study focused on investigating the possible sensitization process and mechanism of CHT and the adverse effects of repeated CHT exposure. CHT activates dendritic cells and promotes the proliferation of lymph cells in the skin sensitization phase, causing severe inflammation. Keratinocytes activate the NLRP3 inflammasome pathway to cause inflammation during CHT treatment, and macrophages also secrete inflammatory cytokines. In addition, CHT-induced inflammation triggered skin wrinkles, decreased epidermal thickness and decreased collagen. Cell experiments also showed that repeated exposure to CHT led to cell proliferation inhibition and senescence, and CHT-induced autophagy dysfunction was not only the reason for inflammation but also for senescence. This study defined the possible process through which CHT is involved in the skin sensitization phase and elucidated the mechanism of CHT-induced inflammation in innate immune responses. We also determined that repeated CHT exposure caused persistent inflammation, ultimately leading to skin aging.

Novel Insights into Stereoselective Reproductive Toxicity Induced by Mefentrifluconazole in Earthworms (Eisenia fetida): First Report of Estrogenic Effects.

Widespread use of the new chiral triazole fungicide mefentrifluconazole (MFZ) poses a threat to soil organisms. Although triazole fungicides have been reported to induce reproductive disorders in vertebrates, significant research gaps remain regarding their impact on the reproductive health of soil invertebrates. Here, reproduction-related toxicity end points were explored in earthworms (Eisenia fetida) after exposure for 28 d to soil containing 4 mg/kg racemic MFZ, R-(-)-MFZ, and S-(+)-MFZ. The S-(+)-MFZ treatment resulted in a more pronounced reduction in the number of cocoons and juveniles compared to R-(-)-MFZ treatment, and the expression of annetocin gene was significantly downregulated following exposure to both enantiomers. This reproductive toxicity has been attributed to the disruption of ovarian steroidogenesis at the transcriptional level. Further studies revealed that MFZ enantiomers were able to activate the estrogen receptor (ER). Indirect evidence for this estrogenic effect is provided by the introduction of 17ß-estradiol, which also induces reproductive disorders through ER activation.

Unveiling the ecotoxicological effects of azoxystrobin-based fungicides at realistic concentrations on the land snail, Theba pisana.

The ecotoxicological consequences of azoxystrobin on land snails have not yet been addressed. Therefore, the present study aims to provide novel data on the threat of a commercial grade azoxystrobin (AMISTAR) at two environmentally relevant concentrations (0.3 µg/ml) and tenfold (3 µg/ml) on the model species, Theba pisana by physiological, biochemical, and histopathological markers for 28 days. Our results showed a reduction in animal food consumption and growth due to exposure to both azoxystrobin concentrations. It also induced oxidative stress and led to a significant decrease in lipid peroxidation (LPO) levels after 7 days of exposure, while the opposite effect occurred after 28 days. Except for the 7-day exposure, all treated snails had significantly reduced glutathione (GSH) content and increased catalase (CAT) activity at all-time intervals. Glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities, and protein content (PC) were elevated in treated snails at all-time intervals. Moreover, alterations in acetylcholinesterase (AChE) activity between a decrease and an increase were noticed. Additionally, azoxystrobin exerted changes in T. pisana hepatopancreas architecture. Our study suggests that azoxystrobin may have negative ecological consequences for T. pisana and highlights its potential risks to the natural environment.

Exposure to pesticides used in rice farming (bentazone, chlorantraniliprole and tebuconazole) affects biochemical biomarkers and hepatic histopathological parameters of hammertoad tadpoles (Boana faber).

Pesticides used in rice cultivation can cause negative health effects to non-target organisms representative of natural biodiversity. In this context, the present study aimed to investigate the occurrence of pesticides in surface waters from a river that flows in the middle of a rice farming-dominated area. We were also interested in evaluate biochemical and histological effects caused by exposure (16 d) to the lower and higher concentrations of the main found herbicide (bentazone, BTZ), insecticide (chlorantraniliprole, CTP) and fungicide (tebuconazole, TBZ), isolated or mixed, in Boana faber tadpoles. No significant differences were observed in the development of the animals. Tadpoles exposed to the herbicide BTZ showed higher hepatic levels of malondialdehyde (MDA). In animals exposed to CTP, MDA levels were lower than controls. Animals exposed to the fungicide TBZ showed higher hepatic activity of glutathione S-transferase and carboxylesterase (CbE), as well as higher levels of carbonyl proteins and MDA. Animals exposed to Mix showed higher activity in CbE and glucose-6-phosphate dehydrogenase activity in the liver, as well as higher levels of MDA. In the brain and muscle of tadpoles exposed to Mix, acetylcholinesterase activity was higher. Histological changes were also observed in pesticide-exposed animals, such as increased occurrence of melanomacrophages, inflammatory infiltrates and congestion. Our data evidences the contamination of natural aquatic environments by rice pesticides, and the adverse effects of main ones in B. faber tadpoles, which suggests the contribution of pesticides derived from rice cultivation to the degradation of local biodiversity health.

Inhibition mechanism of Rhizoctonia solani by pectin-coated iron metal-organic framework nanoparticles and evidence of an induced defense response in rice.

Nanocrop protectants have attracted much attention as sustainable platforms for controlling pests and diseases and improving crop nutrition. Here, we reported the fungicidal activity and disease inhibition potential of pectin-coated metal-iron organic framework nanoparticles (Fe-MOF-PT NPs) against rice stripe blight (RSB). An in vitro bacterial inhibition assay showed that Fe-MOF-PT NPs (80 mg/L) significantly inhibited mycelial growth and nucleus formation. The Fe-MOF-PT NPs adsorbed to the surface of mycelia and induced toxicity by disrupting cell membranes, mitochondria, and DNA. The results of a nontargeted metabolomics analysis showed that the metabolites of amino acids and their metabolites, heterocyclic compounds, fatty acids, and nucleotides and their metabolites were significantly downregulated after treatment with 80 mg/L NPs. The difference in metabolite abundance between the CK and Fe-MOF-PT NPs (80 mg/L) treatment groups was mainly related to nucleotide metabolism, pyrimidine metabolism, purine metabolism, fatty acid metabolism, and amino acid metabolism. The results of the greenhouse experiment showed that Fe-MOF-PT NPs improved rice resistance to R. solani by inhibiting mycelial invasion, enhancing antioxidant enzyme activities, activating the jasmonic acid signaling pathway, and enhancing photosynthesis. These findings indicate the great potential of Fe-MOF-PT NPs as a new RSB disease management strategy and provide new insights into plant fungal disease management.

Comparison of in vitro toxicity in HepG2 cells: Toxicological role of Tebuconazole-tert-butyl-hydroxy in exposure to the fungicide Tebuconazole.

Fungicides are often used prophylactically, to control fungal diseases. Although fungicides have been designed to control pests/fungi, they frequently share molecular targets with non-target species, including humans. Tebuconazole, a fungicide belonging to the class of triazoles, is widely employed, has moderate to high persistence in soil, and can be found in different environmental levels. This fungicide is metabolized to the main hydroxy-derived metabolite, Tebuconazole-tert-butyl-hydroxy (or hydroxytebuconazole). This study aims to unveil the action mechanism of Tebuconazole and the role played by its metabolite, Tebuconazole-tert-butyl-hydroxy (5-(4-Chlorophenyl)-2,2-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)-1,3-pentanediol), within the expected spectrum of toxicity. In silico and in vitro analyses (MTT assay, cell cycle evaluation, annexin/PI assay, ROS accumulation assay, and mitochondrial membrane potential determination) were performed in HepG2 cells for 24 h and 48 h. Although in silico analysis suggested that both Tebuconazole and Tebuconazole-tert-butyl-hydroxy are potentially hepatotoxic, only Tebuconazole affected the tested cell line. Reduced MTT metabolism, and decreased mitochondrial membrane potential were the main findings. In conclusion, the action mechanism of Tebuconazole may be related to mitochondrial dysfunction. However, the findings of this study pointed out that Tebuconazole-tert-butyl-hydroxy does not play an important role in Tebuconazol toxicity. The study has generated new data that will help to understand how fungicides behave in the environment.

Myclobutanil induces cardiotoxicity in developing zebrafish larvae by initiating oxidative stress and apoptosis: The protective role of curcumin.

Myclobutanil (MYC) is a common triazole fungicide widely applied in agriculture. MYC extensively exists in the natural environment and can be detected in organisms. However, little is known about MYC-induced embryonic developmental damage. This study aimed to unravel the cardiotoxicity of MYC and the underlying mechanisms, as well as the cardioprotective effect of curcumin (CUR, an antioxidant polyphenol) using the zebrafish model. Here, zebrafish embryos were exposed to MYC at concentrations of 0, 0.5, 1 and 2 mg/L from 4 to 96 h post fertilization (hpf) and cardiac development was assessed. As results, MYC reduced the survival and hatching rate, body length and heart rate, but increased the malformation rate and spontaneous movement. MYC caused abnormal cardiac morphology and function in myl7:egfp transgenic zebrafish, and downregulated cardiac developmental genes. MYC promoted oxidative stress through excessive reactive oxygen species (ROS) accumulation and suppressed the activities of antioxidant enzymes, triggering cardiomyocytic apoptosis via upregulated expression of apoptosis-related genes. These adverse toxicities could be significantly ameliorated by the antioxidant properties of CUR, indicating that CUR rescued MYC-induced cardiotoxicity by inhibiting oxidative stress and apoptosis. Overall, our study revealed the potential mechanisms of oxidative stress and apoptosis in MYC-induced cardiotoxicity in zebrafish and identified the cardioprotection of CUR in this pathological process.

Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance.

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50-3000 ppm fluxapyroxad for 7 days and 250-3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250-3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1-100 µM fluxapyroxad or 500 µM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid ß-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1-100 µM fluxapyroxad or 500 µM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1-16 µM fluxapyroxad or 500 µM NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.

Dynamics of the content of reactive oxygen species and the state of the glutathione system in the oral cavity during subchronic intoxication wuth the fungicide thiram and its antioxidant correction.

Thiram is a dithiocarbamate derivative, which is used as a fungicide for seed dressing and spraying during the vegetation period of plants, and also as an active vulcanization accelerator in the production of rubber-based rubber products. In this study the content of reactive oxygen species (ROS) and the state of the glutathione system have been investigated in the oral fluid and gum tissues of adult male Wistar rats treated with thiram for 28 days during its administration with food at a dose of 1/50 LD50. Thiram induced formation of ROS in the oral cavity; this was accompanied by an imbalance in the ratio of reduced and oxidized forms of glutathione due to a decrease in glutathione and an increase in its oxidized form as compared to the control. Thiram administration caused an increase in the activity of glutathione-dependent enzymes (glutathione peroxidase, glutathione transferase, and glutathione reductase). However, the time-course of enzyme activation in the gum tissues and oral fluid varied in dependence on the time of exposure to thiram. In the oral fluid of thiram-treated rats changes in the antioxidant glutathione system appeared earlier. The standard diet did not allow the glutathione pool to be fully restored to physiological levels after cessation of thiram intake. The use of exogenous antioxidants resviratrol and an Echinacea purpurea extract led to the restoration of redox homeostasis in the oral cavity.

Oxidative stress and DNA alteration on the earthworm Eisenia fetida exposed to four commercial pesticides.

Modern agriculture is mainly based on the use of pesticides to protect crops but their efficiency is very low, in fact, most of them reach water or soil ecosystems causing pollution and health hazards to non-target organisms. Fungicide triazoles and strobilurins based are the most widely used and require a specific effort to investigate toxicological effects on non-target species. This study evaluates the toxic effects of four commercial fungicides Prosaro® (tebuconazole and prothioconazole), Amistar®Xtra (azoxystrobin and cyproconazole), Mirador® (azoxystrobin) and Icarus® (Tebuconazole) on Eisenia fetida using several biomarkers: lipid peroxidation (LPO), catalase activity (CAT), glutathione S-transferase (GST), total glutathione (GSHt), DNA fragmentation (comet assay) and lysozyme activity tested for the first time in E. fetida. The exposure to Mirador® and AmistarXtra® caused an imbalance of ROS species, leading to the inhibition of the immune system. AmistarXtra® and Prosaro®, composed of two active ingredients, induced significant DNA alteration, indicating genotoxic effects. This study broadened our knowledge of the effects of pesticide product formulations on earthworms and showed the need for improvement in the evaluation of toxicological risk deriving from the changing of physicochemical and toxicological properties that occur when a commercial formulation contains more than one active ingredient and several unknown co-formulants.

Myclobutanil induces neurotoxicity by activating autophagy and apoptosis in zebrafish larvae (Danio rerio).

Myclobutanil (MYC), a typical broad-spectrum triazole fungicide, is often detected in surface water. This study aimed to explore the neurotoxicity of MYC and the underlying mechanisms in zebrafish and in PC12 cells. In this study, zebrafish embryos were exposed to 0, 0.5 and 1 mg/L of MYC from 4 to 96 h post fertilization (hpf) and neurobehavior was evaluated. Our data showed that MYC decreased the survival rate, hatching rate and heart rate, but increased the malformation rate and spontaneous movement. MYC caused abnormal neurobehaviors characterized by decreased swimming distance and movement time. MYC impaired cerebral histopathological morphology and inhibited neurogenesis in HuC:egfp transgenic zebrafish. MYC also reduced the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and downregulated neurodevelopment related genes (gfap, syn2a, gap43 and mbp) in zebrafish and PC12 cells. Besides, MYC activated autophagy through enhanced expression of the LC3-II protein and suppressed expression of the p62 protein and autophagosome formation, subsequently triggering apoptosis by upregulating apoptotic genes (p53, bax, bcl-2 and caspase 3) and the cleaved caspase-3 protein in zebrafish and PC12 cells. These processes were restored by the autophagy inhibitor 3-methyladenine (3-MA) both in vivo and in vitro, indicating that MYC induces neurotoxicity by activating autophagy and apoptosis. Overall, this study revealed the potential autophagy and apoptosis mechanisms of MYC-induced neurotoxicity and provided novel strategies to counteract its toxicity.

Inhibition of human drug transporter activities by succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0-3.9 µM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 µM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 µM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.

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.

Impact of pesticides on marine coral reef foraminifera.

Our laboratory study looked into how pesticides affect the foraminifera species Heterostegina depressa and their obligatory algal endosymbionts. We incubated the foraminifera separately with different types of pesticides at varying concentrations (1 %, 0.01 % and 0.0001 %); we included the insecticide Confidor© (active substance: imidacloprid), the fungicide Pronto©Plus (tebuconazole), and the herbicide Roundup© (glyphosate). Our evaluation focused on the symbiont's photosynthetically active area (PA), and the uptake of dissolved inorganic carbon (DIC) and nitrogen (nitrate) to determine the vitality of the foraminifera. Our findings showed that even the lowest doses of the fungicide and herbicide caused irreparable damage to the foraminifera and their symbionts. While the insecticide only deactivated the symbionts (PA = 0) at the highest concentration (1 %), the fungicide, and herbicide caused complete deactivation even at the lowest levels provided (0.0001 %). The fungicide had the strongest toxic effect on the foraminiferal host regarding reduced isotope uptake. In conclusion, all pesticides had a negative impact on the holosymbiont, with the host showing varying degrees of sensitivity towards different types of pesticides.