Mechanisms Mediating the Combined Toxicity of Paraquat and Maneb in SH-SY5Y Neuroblastoma Cells.

Epidemiological and experimental studies have demonstrated that combined exposure to the pesticides paraquat (PQ) and maneb (MB) increases the risk of developing Parkinson's disease. However, the mechanisms mediating the toxicity induced by combined exposure to these pesticides are not well understood. The aim of this study was to investigate the mechanism(s) of neurotoxicity induced by exposure to the pesticides PQ and MB isolated or in association (PQ + MB) in SH-SY5Y neuroblastoma cells. PQ + MB exposure for 24 and 48 h decreased cell viability and disrupted cell membrane integrity. In addition, PQ + MB exposure for 12 h decreased the mitochondrial membrane potential. PQ alone increased reactive oxygen species (ROS) and superoxide anion generation and decreased the activity of mitochondrial complexes I and II at 12 h of exposure. MB alone increased ROS generation and depleted intracellular glutathione (GSH) within 6 h of exposure. In contrast, MB exposure for 12 h increased the GSH levels, the glutamate cysteine ligase (GCL, the rate-limiting enzyme in the GSH synthesis pathway) activity, and increased nuclear Nrf2 staining. Pretreatment with buthionine sulfoximine (BSO, a GCL inhibitor) abolished the MB-mediated GSH increase, indicating that MB increases GSH synthesis by upregulating GCL, probably by the activation of the Nrf2/ARE pathway. BSO pretreatment, which did not modify cell viability per se, rendered cells more sensitive to MB-induced toxicity. In contrast, treatment with the antioxidant N-acetylcysteine protected cells from MB-induced toxicity. These findings show that the combined exposure of SH-SY5Y cells to PQ and MB induced a cytotoxic effect higher than that observed when cells were subjected to individual exposures. Such a higher effect seems to be related to additive toxic events resulting from PQ and MB exposures. Thus, our study contributes to a better understanding of the toxicity of PQ and MB in combined exposures.

Investigation on the mancozeb toxicity in adult zebrafish (Danio rerio).

Agriculture has gained increasing importance in response to the continuous growth of the world population and constant need for food. To avoid production losses, farmers commonly use pesticides. Mancozeb is a fungicide used in agriculture as this compound is effective in combating fungi that harm crops. However, this fungicide may also produce damage to non-target organisms present in soil and water. Therefore, this study aimed to investigate the influence of exposure to mancozeb on survival rate, locomotor activity, behavior, and oxidative status utilizing adult zebrafish (Danio rerio) as a model following exposure to environmentally relevant concentrations of this pesticide. The experimental groups were negative control, positive control, and mancozeb (0.3; 1.02; 3.47; 11.8 or 40 µg/L). Zebrafish were exposed to the respective treatments for 96 hr. Exposure to mancozeb did not markedly alter survival rate and oxidative status of Danio rerio. At a concentration of 11.8 µg/L, the fungicide initiated changes in locomotor pattern of the animals. The results obtained suggest that the presence of mancozeb in the environment might produce locomotor alterations in adult zebrafish, which subsequently disrupt the animals' innate defense mechanisms. In nature, this effect attributed to mancozeb on non-target organisms might result in adverse population impacts and ecological imbalance.

Identification and Fungicide Screening of Fungal Species Associated with Walnut Anthracnose in Shaanxi and Liaoning Provinces, China.

Walnut is cultivated around the world for its precious woody nut and edible oil. Recently, walnut infected by Colletotrichum spp. resulted in a great yield and quality loss. In August and September 2014, walnut fruits with anthracnose were sampled from two commercial orchards in Shaanxi and Liaoning provinces, and five representative isolates were used in this study. To identify the pathogen properly, four genes per region (internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, actin, and chitin synthase) were sequenced and used in phylogenetic studies. Based on multilocus phylogenetic analysis, five isolates clustered with Colletotrichum fioriniae, including its ex-type, with 100% bootstrap support. The results of multilocus phylogenetic analyses, morphology, and pathogenicity confirmed that C. fioriniae was one of the walnut anthracnose pathogens in China. All 13 fungicides tested inhibited mycelial growth and spore germination. Flusilazole, fluazinam, prochloraz, and pyraclostrobin showed the strongest suppressive effects on the mycelial growth than the others, the average EC50 values ranged from 0.09 to 0.40 µg/ml, and there was not any significant difference (P < 0.05). Pyraclostrobin, thiram, and azoxystrobin were the most effective fungicides on spore germination (P < 0.05), and the EC50 values ranged from 0.01 to 0.44 µg/ml. Pyraclostrobin, azoxystrobin, fluazinam, flusilazole, mancozeb, thiram, and prochloraz exhibited a good control effect on walnut anthracnose caused by C. fioriniae, and preventive activities were greater than curative activities. Pyraclostrobin at 250 a.i. µg/ml and fluazinam at 500 a.i. µg/ml provided the highest preventive and curative efficacy, and the values ranged from 81.3 to 82.2% and from 72.9 to 73.6%, respectively. As a consequence, mancozeb and thiram could be used at the preinfection stage, and pyraclostrobin, azoxystrobin, flusilazole, fluazinam, and prochloraz could be used at the early stage for effective prevention and control of walnut anthracnose caused by C. fioriniae. The results will provide more significant instructions for controlling the disease effectively in northern China.

Integrin Mac1 mediates paraquat and maneb-induced learning and memory impairments in mice through NADPH oxidase-NLRP3 inflammasome axis-dependent microglial activation.

INTRODUCTION: The mechanisms of cognitive impairments in Parkinson's disease (PD) remain unknown. Accumulating evidence revealed that brain neuroinflammatory response mediated by microglial cells contributes to cognitive deficits in neuropathological conditions and macrophage antigen complex-1 (Mac1) is a key factor in controlling microglial activation. OBJECTIVES: To explore whether Mac1-mediated microglial activation participates in cognitive dysfunction in PD using paraquat and maneb-generated mouse PD model. METHODS: Cognitive performance was measured in wild type and Mac1-/- mice using Morris water maze test. The role and mechanisms of NADPH oxidase (NOX)-NLRP3 inflammasome axis in Mac1-mediated microglial dysfunction, neuronal damage, synaptic degeneration and phosphorylation (Ser129) of α-synuclein were explored by immunohistochemistry, Western blot and RT-PCR. RESULTS: Genetic deletion of Mac1 significantly ameliorated learning and memory impairments, neuronal damage, synaptic loss and α-synuclein phosphorylation (Ser129) caused by paraquat and maneb in mice. Subsequently, blocking Mac1 activation was found to mitigate paraquat and maneb-elicited microglial NLRP3 inflammasome activation in both in vivo and in vitro. Interestingly, stimulating activation of NOX by phorbol myristate acetate abolished the inhibitory effects of Mac1 blocking peptide RGD on paraquat and maneb-provoked NLRP3 inflammasome activation, indicating a key role of NOX in Mac1-mediated NLRP3 inflammasome activation. Furthermore, NOX1 and NOX2, two members of NOX family, and downstream PAK1 and MAPK pathways were recognized to be essential for NOX to regulate NLRP3 inflammasome activation. Finally, a NLRP3 inflammasome inhibitor glybenclamide abrogated microglial M1 activation, neurodegeneration and phosphorylation (Ser129) of α-synuclein elicited by paraquat and maneb, which were accompanied by improved cognitive capacity in mice. CONCLUSIONS: Mac1 was involved in cognitive dysfunction in a mouse PD model through NOX-NLRP3 inflammasome axis-dependent microglial activation, providing a novel mechanistic basis of cognitive decline in PD.

Mancozeb induces nephrotoxicity by impairing the oxidative phosphorylation pathway: A transcriptome study.

This study analyzed the mechanism underlying mancozeb (MCZ)-induced kidney injury by detecting kidney function indicators, combined with transcriptome and metabolome sequencing. Twenty mice were randomly assigned into two groups (control and MCZ groups) to explore the MCZ-induced kidney toxicity. The control group was gavaged with 0.2 mL of deionized water, and the MCZ group with 0.2 mL of 100 mg/kg MCZ for 30 days. The kidney structure of the MCZ group was damaged, with slight hyaline degeneration in the kidney tubular epithelial envelope. The creatinine (CRE) and uric acid (UA) were significantly increased in the MCZ group than in the control group. Moreover, the reactive oxygen species (ROS) significantly accumulated in the MCZ group kidneys. Compared to the control group, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were significantly decreased in the MCZ group, while the MDA content was substantially increased. The differentially expressed genes (DEGs) in the MCZ group were mainly enriched in the oxidative phosphorylation pathway. Besides, in the MCZ group, ndufs1 and ndufab1 genes were significantly up-regulated, while cox5b, ndufa5, and ndufa6 genes were significantly down-regulated, consistent with the PCR verification results. The metabolomic analysis identified cGMP-PKG signaling pathway of MCZ-induced nephrotoxicity, with Guanosine monophosphate and Adenosine 5'-monophosphate as the main altered metabolites. These results indicated that MCZ impairs the mice kidneys by obstructing the oxidative phosphorylation pathway, which increases oxidative stress in the kidneys, resulting in kidney injury.

Effective Management of Citrus Melanose Based on Combination of Ecofriendly Chemicals.

Citrus melanose, caused by the ascomycete fungus Diaporthe citri, is one of the most important diseases in China that affects not only the production but also the quality of citrus. In China, mancozeb is recommended to control melanose disease at the dose of 1.34 g/liter. However, it is widely applied in practice at the dose of 2.66 g/liter or even 4 g/liter, because reduced efficacy of the recommended dose was observed in regions severely damaged by melanose. In this study, some ecofriendly chemicals for melanose management were evaluated. First, the sensitivity to fungicides was screened in the laboratory based on the inhibition of mycelial growth and conidial germination of D. citri. Results showed that both quinone outside inhibitor (QoI) fungicides kresoxim-methyl and trifloxystrobin inhibited conidial germination of D. citri up to 100% at 0.1 µg/ml. The in vivo control efficacy on detached fruit indicated that treatments with elastic nanocopolymer film at 2 g/liter, mancozeb at 1 g/liter, and kresoxim-methyl at 0.1 g/liter significantly inhibited the infection process compared with the control treatment of mineral oil alone. In field trials, the efficacy of kresoxim-methyl at 0.1 g/liter and elastic nanocopolymer film at 2 g/liter mixed with mancozeb at 1 g/liter was equal to that of mancozeb at 2.66 g/liter. The use of mancozeb could be reduced greatly, and the newly developed fungicide combinations are more environmentally friendly due to the low toxicity of both QoI fungicides and elastic nanocopolymer film. The newly developed method with ecofriendly chemicals should play an important role in the management of citrus melanose in the future.

L-carnitine attenuates acoustic startle reflex dysfunction in adult male rats exposed to mancozeb.

The fungicide mancozeb increases oxygen-free radicals in the central nervous system. As an antioxidant, L-carnitine protects DNA and cell membranes from damage caused by oxygen-free radicals. The present study investigated how L-carnitine affected the acoustic startle response (ASR) in rats exposed to mancozeb. In this experimental study, male Wistar rats were gavaged orally with mancozeb (500, 1000, and 2000 mg/kg), L-carnitine (100, 200, and 400 mg/kg), or L-carnitine (200 mg/kg) + mancozeb (500 mg/kg) three times in 1 week. In the sham group, saline (0.9%, 10 mL/kg) was gavaged at a volume equivalent to that of the drugs. The control group did not receive any treatment. The results showed that locomotor activity and the percentage of prepulse inhibition in the mancozeb groups decreased compared to the sham group while these parameters increased in the L-carnitine group (200 mg/kg) compared to sham rats. In conclusion, mancozeb may increase the risk factor for cognitive diseases such as schizophrenia in people exposed to it while pretreatment with L-carnitine can attenuate the toxic effect.

The protective function of taurine on pesticide-induced permanent neurodevelopmental toxicity in juvenile rats.

Numerous studies have confirmed that prenatal or early postnatal exposure to pesticides can lead to functional deficits in the developing brain. This study aimed to investigate whether combined exposure to paraquat (PQ) and maneb (MB) during puberty could cause permanent toxic effects in the neural system of rats. In addition, the neuroprotective function of taurine (T) and its possible mechanism were investigated. Rats were administered PQ + MB intragastrically for 12 continuous weeks, while taurine dissolved in water was fed to the rats for 24 continuous weeks. In the behavioral tests, the rats' trajectories became complex, and the reaction latencies and mistake frequencies increased. Significant changes were found in the hippocampal neurons of the PQ + MB groups but not in the taurine treatment groups. PQ + MB stimulated cAMP to reduce the production of protein kinase A (PKA) and inhibited the activation of other elements, such as brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), phospho-CREB (p-CREB), immediate-early genes (IEGs)Arc, and c-Fos. Importantly, taurine regulated the level of cAMP and the expression of the abovementioned proteins. Together, our findings implied that adolescent exposure to PQ + MB may impact the behavior and cognitive function of rats via the cAMP-PKA-CREB signaling pathway, while taurine may in turn exert neuroprotection by diminishing these impacts.

Low level of mancozeb exposure affects ovary in mice.

Mancozeb (MCZ) is widely used as a protective fungicide. This study aimed to explore the effects of low level MCZ exposure on ovary in mice. Twenty Kunming mice were randomly divided into control and MCZ groups (10 mice each). The mice in the MCZ group were given 100 mg/kg MCZ daily via gavage. The mice were sacrificed to collect serum and ovaries on day 31. The experimental indicators were then assessed. The weight of MCZ-exposed mice significantly reduced while ovarian index significantly increased compared with the control group. The FSH, LH, E2, P, CAT, SOD and MDA contents in the serum were significantly decreased and the content of estradiol significantly increased after MCZ exposure. Histological observation showed that the ovarian structure of mice exposed to MCZ was damaged, and the apoptosis was increased. Immunohistochemistry and RT-qPCR showed that the expression of Bax, caspase-3 and caspase-9 significantly increased in the MCZ- group. Conversely, Bcl-2 expression significantly decreased. Transcriptome sequencing showed that the expression of NADH dehydrogenase ND3, ND4L, ND6 subunits, Cyt b, and SDHC genes in mitochondria were down-regulated after MCZ exposure, similar to real-time PCR analysis. These results collectively indicate that the MCZ can affect the abnormal function of mitochondrial respiratory chain, lead to oxidative phosphorylation decoupling, produce oxidative stress, and finally cause ovarian injury and apoptosis in mice.

Neurotoxicity of pesticides - A link to neurodegeneration.

Parkinson's disease (PD) is a neurodegenerative disorder which mainly targets motor symptoms such as tremor, rigidity, bradykinesia and postural instability. The physiological changes occur due to dopamine depletion in basal ganglia region of the brain. PD aetiology is not yet elucidated clearly but genetic and environmental factors play a prominent role in disease occurrence. Despite of various environmental factors, pesticides exposure has been convicted as major candidate in PD pathogenesis. Among various pesticides 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been widely investigated in PD following with paraquat (PQ), maneb (MB), organochlorines (OC) and rotenone. Effect of these pesticides has been suggested to be involved in oxidative stress, alterations in dopamine transporters, mitochondrial dysfunction, α-synuclein (αSyn) fibrillation, and neuroinflammation in PD. The present review discusses the influence of pesticides in neurodegeneration and its related epidemiological studies conducted in PD. Furthermore, we have deliberated the common pesticides involved in PD and its associated genetic alterations and the probable mechanism of them behind PD pathogenesis. Hence, we conclude that pesticides play a prominent role in PD pathogenesis and advance research is needed to investigate the alterations in genetic and mechanistic aspects of PD.

Transcriptomics and metabolomics revealed the molecular mechanism of the toxic effect of mancozeb on liver of mice.

Mancozeb (MCZ), a broad-spectrum fungicide, has been widely used in crops (tomatoes and potatoes) in the past few decades, resulting in its bioaccumulation in the food web. However, the mechanism of MCZ on liver injury has not been reported yet. This study combined transcriptomics and metabolomics to explore the potential mechanism of MCZ on liver injury. MCZ group was given 100 mg/kg MCZ every day, and the C group was given 0.2 mL of deionized water every day. One hundred mg/kg MCZ led to unclear hepatocyte structure and hemorrhagic inflammatory cell infiltration. Transcriptomics and metabolomics analyses showed that the MCZ group resulted in 326 differentially expressed genes (DEGs) and 179 differential metabolites. Joint analysis showed that DEGs and differential metabolites were mainly enriched in the adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway. We found that MCZ could increase the content of reactive oxygen species (ROS) and reduce the activities of superoxide dismutase (SOD) and catalase (CAT). The contents of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in the liver decreased significantly, and the state of DNA methylation was significantly higher than the control (C) group (p < 0.05). Our results suggest that AMPK and mitogen­activated protein kinase (MAPK) signaling pathways play an important role in MCZ-induced liver injury and are the key mechanisms for understanding the hepatotoxicity of MCZ.

Cyclooxygenase-2 activates the free radical-mediated apoptosis of polymorphonuclear leukocytes in the maneb- and paraquat-intoxicated rats.

Overproduction of free radicals and inflammation could lead to maneb (MB)- and paraquat (PQ)-induced toxicity in the polymorphonuclear leukocytes (PMNs). Cyclooxygenase-2 (COX-2), an inducible COX, is imperative in the pesticides-induced pathological alterations. However, its role in MB- and PQ-induced toxicity in the PMNs is not yet clearly deciphered. The current study explored the contribution of COX-2 in MB- and PQ-induced toxicity in the PMNs and the mechanism involved therein. Combined MB and PQ augmented the production of free radicals, lipid peroxides and activity of superoxide dismutase (SOD) in the rat PMNs. While combined MB and PQ elevated the expression of COX-2 protein, activation of nuclear factor-kappa B (NF-κB) and phosphorylation of c-Jun N-terminal kinase (JNK), release of mitochondrial cytochrome c and levels of procaspase-3/9 were attenuated in the PMNs. Celecoxib (CXB), a COX-2 inhibitor, ameliorated the combined MB and PQ-induced modulations in the PMNs. MB and PQ augmented the free radical generation, COX-2 protein expression, NF-κB activation and JNK phosphorylation and reduced the cell viability of cultured rat PMNs and human leukemic HL60. MB and PQ elevated mitochondrial cytochrome c release and poly (ADP-ribose) polymerase cleavage whilst procaspase-3/9 levels were attenuated in the cultured PMNs. MB and PQ also increased the levels of phosphorylated c-jun and caspase-3 activity in the HL60 cells. CXB; SP600125, a JNK-inhibitor and pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, rescued from MB and PQ-induced changes in the PMNs and HL60 cells. However, CXB offered the maximum protection among the three. The results show that COX-2 activates apoptosis in the PMNs following MB and PQ intoxication, which could be linked to NF-κB and JNK signaling.

Phenolic-rich extract of avocado Persea americana (var. Colinred) peel blunts paraquat/maneb-induced apoptosis through blocking phosphorylation of LRRK2 kinase in human nerve-like cells.

It is increasingly evident that LRRK2 kinase activity is involved in oxidative stress (OS)-induced apoptosis-a type of regulated cell death and neurodegeneration, suggesting LRRK2 inhibition as a potential therapeutic target. We report that a phenolic-rich extract of avocado Persea americana var. Colinred peel (CRE, 0.01 mg/ml) restricts environmental neurotoxins paraquat (1 mM)/maneb (0.05 mM)-induced apoptosis process through blocking reactive oxygen species (ROS) signaling and concomitant inhibition of phosphorylation of LRRK2 in nerve-like cells (NLCs). Indeed, PQ + MB at 6 h exposure significantly increased ROS (57 ± 5%), oxidation of protein DJ-1cys106SOH into DJ-1Cys106SO3 ([~3.7 f(old)-(i)ncrease]), augmented p-(S935)-LRRK2 kinase (~20-f(old) (i)ncrease), induced nuclei condensation/fragmentation (28 ± 6%), increased the expression of PUMA (~6.2-fi), and activated CASPASE-3 (CASP-3, ~4-fi) proteins; but significantly decreased mitochondrial membrane potential (ΔΨm, ~48 ± 4%), all markers indicative of apoptosis compared to untreated cells. Remarkably, CRE significantly diminished both OS-signals (i.e., DCF+ cells, DJ-1Cys106SO3) as well as apoptosis markers (e.g., PUMA, CASP-3, loss of ΔΨm, p-LRRK2 kinase) in NLCs exposed to PQ + MB. Furthermore, CRE dramatically reestablishes the transient intracellular Ca2+ flow (~300%) triggered by dopamine (DA) in neuronal cells exposed to PQ + MB. We conclude that PQ + MB-induced apoptosis in NLCs through OS-mechanism, involving DJ-1, PUMA, CASP-3, LRRK2 kinase, mitochondria damage, DNA fragmentation, and alteration of DA-receptors. Our findings imply that CRE protects NLCs directly via antioxidant mechanism and indirectly by blocking LRRK2 kinase against PQ + MB stress stimuli. These data suggest that CRE might be a potential natural antioxidant.

Effects of mancozeb on citrus rhizosphere bacterial community.

Multiple and consecutive application of fungicide might damage the rhizosphere bacterial community of citrus. In order to evaluated effect of mancozeb on the chemical properties of citrus-cultivated soil and the richness and diversity of rhizosphere bacterial community. The abundance response of rhizosphere bacterial groups without application or with application of 1.333 g mg-1 mancozeb for 2, 4, 6 and 8 times were investigated, and further studied the relationship between the rhizosphere bacterial community and chemical properties of citrus-cultivated soil. We found the rhizosphere bacterial composition and diversity were distinct between soil planted with citrus and without citrus, in addition, the abundance of rhizosphere-associated bacterial species in the soil planted with citrus increased significantly. Meanwhile, the chemical properties and the richness and diversity of rhizosphere bacterial community of the soil planted with citrus did not significantly change among different application frequence of mancozeb. Moreover, with the increased applying times of mancozeb, the relative abundance of Candidatus, Saccharibacteria, Parcubacteria, and Proteobacteria increased but the abundance of Nitrospirae decreased. In our one-year trial, there were less adverse effects of mancozeb on the citrus-cultivated rhizosphere by the repeated application of mancozeb. Therefore, mancozeb, as a fungicide, could be used multiple times to control citrus disease.

Cardiac developmental toxicity and transcriptome analyses of zebrafish (Danio rerio) embryos exposed to Mancozeb.

Mancozeb (MZ), an antibacterial pesticide, has been linked to reproductive toxicity, neurotoxicity, and endocrine disruption. However, whether MZ has cardiactoxicity is unclear. In this study, the cardiotoxic effects of exposure to environment-related MZ concentrations ranging from 1.88 µM to 7.52 µM were evaluated at the larval stage of zebrafish. Transcriptome sequencing predicted the mechanism of MZ-induced cardiac developmental toxicity in zebrafish by enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Consistent with morphological changes, the osm, pfkfb3, foxh1, stc1, and nrarpb genes may effect normal development of zebrafish heart by activating NOTCH signaling pathways, resulting in pericardial edema, myocardial fibrosis, and congestion in the heart area. Moreover, differential gene expression analysis indicated that cyp-related genes (cyp1c2 and cyp3c3) were significantly upregulated after MZ treatment, which may be related to apoptosis of myocardial cells. These results were verified by real-time quantitative RT-qPCR and acridine orange staining. Our findings suggest that MZ-mediated cardiotoxic development of zebrafish larvae may be related to the activation of Notch and apoptosis-related signaling pathways.

The fungicide Mancozeb reduces spheroid attachment onto endometrial epithelial cells through downregulation of estrogen receptor ß and integrin ß3 in Ishikawa cells.

Mancozeb is a metal-containing ethylene bis-dithiocarbamate fungicide widely used in agriculture. Ethylene thiourea (ETU) is the primary metabolite of Mancozeb. Mancozeb has been associated with spontaneous abortions and abnormal menstruation in women. However, the effects of Mancozeb and ETU on embryo attachment remain unknown. The human blastocyst surrogate trophoblastic spheroids (JEG-3), endometrial epithelial surrogate adenocarcinoma cells (Ishikawa), or human primary endometrial epithelial cells (EECs) monolayer were used in the spheroid attachment models. Ishikawa and EECs were pretreated with different concentrations of Mancozeb or ETU for 48 h before the attachment assay. Gene expression profiles of Ishikawa cells were examined to understand how Mancozeb modulates endometrial receptivity with Microarray. The genes altered by Mancozeb were confirmed by qPCR and compared with the ETU treated groups. Mancozeb and ETU treatment inhibited cell viability at 10 µg/mL and 5000 µg/mL, respectively. At non-cytotoxic concentrations, Mancozeb at 3 µg/mL and ETU at 300 µg/mL reduced JEG-3 spheroid attachment onto Ishikawa cells. A similar result was observed with human primary endometrial epithelial cells. Mancozeb at 3 µg/mL modified the transcription of 158 genes by at least 1.5-fold in Microarray analysis. The expression of 10 differentially expressed genes were confirmed by qPCR. Furthermore, Mancozeb decreased spheroid attachment possibly through downregulating the expression of endometrial estrogen receptor ß and integrin ß3, but not mucin 1. These results were confirmed in both overexpression and knockdown experiments and co-culture assay. Mancozeb but not its metabolite ETU reduced spheroid attachment through modulating gene expression profile and decreasing estrogen receptor ß and integrin ß3 expression of endometrial epithelial cells.

Autonomous regulation of inducible nitric oxide synthase and cytochrome P450 2E1-mediated oxidative stress in maneb- and paraquat-treated rat polymorphs.

Maneb (MB)- and paraquat (PQ)-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) is regulated in parallel by cytochrome P450 2E1 (CYP2E1) and inducible nitric oxide synthase (iNOS). However, mechanism underlying their regulation is not yet understood. The study investigated the role of nuclear factor- kappa B (NF-κB) and mitogen-activated protein kinase/extracellular signal regulated kinase/protein kinase C (MEK/ERK/PKC) pathway in the regulation of iNOS- and CYP2E1-induced oxidative stress in PMNs. MB + PQ-induced changes in nitrite content, lipid peroxidation (LPO), iNOS expression/activity and inflammatory mediators were alleviated by aminoguanidine (AG), an iNOS inhibitor, without any change in CYP2E1. Alternatively, diallyl sulphide (DAS), a CYP2E1 inhibitor, rescued from MB + PQ-induced changes in CYP2E1 activity/expression, free radical generation, superoxide dismutase (SOD) activity, LPO and pro-inflammatory cytokines without any alterations in nitrite content and iNOS activity/expression. Pyrrolidine dithiocarbamate (PDTC), NF-κB inhibitor, did not alter CYP2E1 but mitigated free radical generation, SOD activity, LPO, nitrite content, iNOS activity/expression and levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukine-1ß and interleukine-4). Ex-vivo treatment with MEK inhibitor (PD98059), ERK1/2 inhibitor (AG126) or PKC inhibitor (rottlerin) ameliorated MB + PQ-induced increase in free radical generation and CYP2E1 activity/expression in PMNs. While PD98059 and AG126 abated MB + PQ-induced increase in ERK1/2, PKC-α/δ and CYP2E1 levels, rottlerin restored PKC-α/δ and CYP2E1 towards normalcy without affecting ERK1/2 level in MB + PQ-treated group. The results suggest that iNOS and CYP2E1 contributing to MB + PQ-induced oxidative stress in rat PMNs exhibit differential regulatory mechanisms. The inflammatory mediators regulate iNOS expression while CYP2E1 expression is triggered via MEK-ERK1/2-PKC pathway.

Optimizing the Integration of a Biopesticide (Bacillus subtilis QST 713) with a Single-Site Fungicide (Benzovindiflupyr) to Reduce Reliance on Synthetic Multisite Fungicides (Captan and Mancozeb) for Management of Apple Scab.

Apple scab is one of the most economically important diseases of apple in temperate production regions. In the absence of durable host resistance in commercially preferred cultivars, considerable applications of fungicides are needed to manage this disease. With the sequential development of resistance to nearly all classes of single-site fungicides in the apple scab pathogen Venturia inaequalis, synthetic multisite fungicides, such as mancozeb and captan, often comprise the core of chemical management programs for apple scab. Although these fungicides have demonstrable benefits for both disease and fungicide resistance management, the sustainability movement within agriculture aims to reduce reliance on such fungicides because of their broader environmental impacts. In this study, we establish a framework to enhance the feasibility of chemical management programs that do not rely on use of synthetic multisite protectant fungicides to manage apple scab. Specifically, we wish to evaluate chemical programs that integrate the biopesticide Bacillus subtilis QST 713 (Serenade Opti) in rotation with benzovindiflupyr (Aprovia), a single-site fungicide belonging to the class of succinate dehydrogenase inhibitors (SDHI), to circumvent the need for applications of synthetic multisite fungicides. During implementation of these programs, disease incidence data were taken at biweekly intervals. Regardless of the seasonal challenges presented in the 2 years of this study, when Bacillus subtilis QST 713 was used in place of captan and mancozeb mixtures, we did not observe any significant differences (P > 0.05) in development of apple scab symptoms between any of the management programs for the vertical axis or super spindle orchards in either year. This potential for substituting synthetic multisite fungicides with biopesticides is best realized when the programs are used with a decision support system in a super spindle planting system, where trees have reduced canopy densities. This 2-year study shows the potential to achieve adequate disease control using the integration of SDHI fungicides and biological controls without the use of synthetic multisite fungicides.

Cytotoxicity of mancozeb on Sertoli-germ cell co-culture system: Role of MAPK signaling pathway.

Mancozeb (MZB) is a worldwide fungicide for the management of fungal diseases in agriculture and industrial contexts. Human exposure occurs by consuming contaminated plants, drinking water, and occupational exposure. There are reports on MZB's reprotoxicity such as testicular structure damage, sperm abnormalities, and decrease in sperm parameters (number, viability, and motility), but its molecular mechanism on apoptosis in testis remains limited. To investigate the molecular mechanisms involved in male reprotoxicity induced by MZB, we used primary cultures of mouse Sertoli-germ cells. Cells were exposed to MZB (1.5, 2.5, and 3.5 µM) for 3 h to evaluate viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, reactive oxygen species (ROS) generation, and oxidative stress parameters (lipid peroxidation). Cell death and mitogen-activated protein kinase (MAPK) signaling were measured in these cells using flow cytometry and western blotting. In addition, some groups were exposed to N-acetylcysteine (NAC, 5 mM) in the form of co-treatment with MZB. Mancozeb reduced viability and increased the level of intracellular ROS, p38 and c-Jun N-terminal kinases (JNK) MAPK proteins phosphorylation, and apoptotic cell death, which could be blocked by NAC as an inhibitor of oxidative stress. The present study indicated for the first time the toxic manifestations of MZB on the Sertoli-germ cell co-culture. Redox imbalance and p38 and JNK signaling pathway activation might play critical roles in MZB-induced apoptosis in the male reproductive system.

Effects of mancozeb on heat Shock protein 70 (HSP70) and its relationship with the thermal physiology of Physalaemus henselii (Peters, 1872) tadpoles (Anura: Leptodactylidae).

Negative impacts on amphibians have been reported due to contamination by agrochemicals. However, until now, no study has tested the effect of the fungicide mancozeb (MZ) on thermal tolerance and its relationship with the expression of heat shock proteins (HSPs). MZ is the best-selling broad-spectrum fungicide in the world, which negatively affects non-target organisms. Here, we tested for the first time the effects of MZ on critical thermal maximum (CTmax) and its relationship to the expression of heat shock protein 70 (HSP70) in tadpoles of Physalameus henselii, a colder-adapted species in southernmost of the Neotropical region. A sublethal concentration of 2 mg/L was used. We found that the CTmax of the MZ-treated group was lower than that of the control group. In addition, there was an increase in HSP70 expression in tadpoles exposed to MZ and in tadpoles that underwent heat treatment. However, tadpoles subjected to MZ and heat treatment showed no induced HSP70 protein expression. Our results demonstrated that sublethal doses of the fungicide MZ negatively affected the thermal physiology and heat shock protein expression in tadpoles of P. henselii by inducing an increase in HSP70 concentration and by reducing the critical CTmax supported by tadpoles. It is important to understand the relationship between environmental contamination and physiological thermal limits in our current scenario of high rates of habitat conversion associated with unrestricted use of agrochemicals, as well as the challenging environmental changes induced by global warming.