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.

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.

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.

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.

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.

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.

Integrin CD11b mediates locus coeruleus noradrenergic neurodegeneration in a mouse Parkinson's disease model.

BACKGROUND: The loss of locus coeruleus noradrenergic (LC/NE) neurons in the brainstem is reported in multiple neurodegenerative disorders, including Parkinson's disease (PD). However, the mechanisms remain unclear. Strong evidence suggested that microglia-mediated neuroinflammation contributes to neurodegeneration in PD. We recently recognized integrin CD11b, the α-chain of macrophage antigen complex-1 (Mac-1, also called CR3), as a key regulator for microglial activation. However, whether CD11b is involved in LC/NE neurodegeneration in PD remains to be investigated. METHODS: LC/NE neurodegeneration and microglial activation were compared between wild type (WT) and CD11b KO mice after treated with paraquat and maneb, two pesticides that widely used to create PD model. The role of NLRP3 inflammasome in CD11b-mediated microglial dysfunction and LC/NE neurodegeneration was further explored. LC/NE neurodegeneration, microglial phenotype, and NLRP3 inflammasome activation were determined by using Western blot, immunohistochemistry, and RT-PCR technologies. RESULTS: Paraquat and maneb co-exposure elevated the expressions of CD11b in the brainstem of mice, and CD11b knockout significantly reduced LC/NE neurodegeneration induced by paraquat and maneb. Mitigated microglial activation and gene expressions of proinflammatory cytokines were also observed in paraquat and maneb-treated CD11b-/- mice. Mechanistically, CD11b-mediated NLRP3 inflammasome activation contributes to paraquat and maneb-induced LC/NE neurodegeneration. Compared with WT controls, CD11b deficiency reduced paraquat and maneb-induced NLRP3 expression, caspase-1 activation, and interleukin-1ß production in mice. Furthermore, inhibition of NLRP3 inflammasome by glybenclamide, a sulfonylurea inhibitor of NLRP3 inflammasome, was found to be able to suppress microglial proinflammatory activation and nuclear factor-κB activation induced by paraquat and maneb. Moreover, reduced reactive oxygen species production, NADPH oxidase, and inducible nitric oxide synthase expressions as well as 4-hydroxynonenal and malondialdehyde levels were detected in combined glybenclamide and paraquat and maneb-treated mice compared with paraquat and maneb alone group. Finally, we found that glybenclamide treatment ameliorated LC/NE neurodegeneration and α-synuclein aggregation in paraquat and maneb-treated mice. CONCLUSION: Our findings suggested that CD11b mediates LC/NE neurodegeneration through NLRP3 inflammation-dependent microglial proinflammatory activation in a two pesticide-induced mouse PD model, providing a novel insight into the immune pathogenesis of LC/NE neuronal damage in related disorders.

Mancozeb impaired male fertility in rabbits with trials of glutathione detoxification.

The study aims to evaluate the potential reproductive toxicity induced by mancozeb fungicide in male rabbits and to examine the ameliorative effect of glutathione (GSH), a non-enzymatic antioxidant, against mancozeb reproductive toxicity. Mancozeb is a member of the dithiocarbamates group currently in use in the management of fungal diseases of plants. To achieve these aims, mature male White New-Zealand rabbits of 4-5 months old were randomly assigned to four groups of 9 animals each: control, mancozeb only, mancozeb and GSH, and GSH only. This study discovered a significant reduction in serum FSH, LH, testosterone and testicular LDH, ACP, and ALP levels in the groups of mancozeb-treated rabbits compared with control. The mancozeb-treated groups also showed significant losses in sperm viability, along with a significant increase in the number of abnormal sperms. Finally, an upregulation in steroidogenic 3ß-HSD enzyme activity was noted in mancozeb-treated rabbits. Histopathological inspection of the testicles established disruption of the germinal epithelium with vacuolization of Leydig cells and reduced spermatogenic cells. GSH co-administration increased serum concentrations of FSH, LH, testosterone, and levels of the testicular enzymes: LDH, ACP, and ALP. Improved steroidogenesis was indicated in this group by a significant improvement in the testicular 3ß-HSD enzyme, by a significant increase in sperm viability, and by a significant decrease in the number of abnormal sperms. The findings of this study suggest that mancozeb exposure has anti-spermatogenic and anti-steroidogenic adverse effects in rabbits and administration of GSH may alleviate the reproductive toxicity.

Immunomodulatory effect of mancozeb, chlorothalonil, and thiophanate methyl pesticides on macrophage cells.

Mancozeb (MZ), chlorothalonil (CT), and thiophanate methyl (TM) are pesticides commonly used in agriculture due to their efficacy, low acute toxicity to mammals, and short environmental persistence. Although the toxic effects of these pesticides have been previously reported, studies regarding their influence on the immune system are limited. As such, this study focused on the immunomodulatory effect of MZ, CT, and TM pesticides on macrophage cells. RAW 264.7 cells were exposed to a range of concentrations (0.1-100 µg/mL) of these pesticides. CT exposure promoted an increase in reactive oxygen species (ROS) and nitric oxide (NO) levels. The MTT and ds-DNA assay results demonstrated that MZ, CT, and TM exposure induced macrophage proliferation. Moreover, MZ, CT, and TM promoted cell cycle arrest at S phase, strongly suggesting macrophage proliferation. The levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IFN-γ) and caspases (caspase 1, 3, and 8) in macrophages exposed to MZ, CT, and TM pesticides increased, whereas the anti-inflammatory cytokine levels decreased. These results suggest that MZ, CT, and TM exert an immunomodulatory effect on the immune system, inducing macrophage activation and enhancing the inflammatory response.

Toxicity of lead and mancozeb differs in two monophyletic Daphnia species.

Lead and mancozeb are two important chemicals used for different human purposes and activities worldwide. Hazard assessment in different areas of the world is carried out with different but phylogenetically similar species, adapted to different climatic conditions, in order to increase relevance. This study evaluated the sensitivity of two monophyletic species, the tropical species Daphnia similis and the temperate species Daphnia magna, to the two chemicals lead and mancozeb. Standard acute and chronic ecotoxicological tests (reproduction and growth), as well as other sublethal measurements such as the intrinsic rate of population increase (r), feeding rate (FR) and O2 consumption, were recorded along with the analysis of the AChE activity to determine the neurotoxicity of both contaminants. Albeit their similar evolutionary status, D. magna generally presented a lower sensitivity to Pb in comparison to D. similis. Despite the differences in sensitivity, both species presented similar patterns of response under Pb exposure, with diminished reproductive outputs, feeding impairment, reduced O2 consumption and no effect on AChE activity. Mancozeb decreased the reproduction, rate of population increase and feeding rate, increased the AChE activity in both species and increased O2 consumption only in D. magna. While D. magna increased O2 consumption under mancozeb exposure, no effects were observed for D. similis. Thus, species may present different responses and sensitivities to different pollutants, regardless of their phylogeny. Therefore, the use of ecotoxicological assays with native species is crucial for a better ecological risk assessment in contaminated areas.

Evaluation of cytotoxicity, genotoxicity and ecotoxicity of nanoemulsions containing Mancozeb and Eugenol.

Mancozeb is a fungicide widely used in agriculture, mostly against the pathogen Glomerella cingulata responsible for the rot of ripe grape, but presents high toxicity. Strategies are sought to reduce the toxicity of this fungicide and alternative treatments are welcome. An alternative could be the use of clove oil, which has Eugenol as its major compound, and has antifungal potential against G. cingulata, however, Eugenol is susceptible to degradation processes which may compromise its efficacy. The nanoencapsulation of Mancozeb and Eugenol is a possible strategy to overcome the limitations of toxicity, solubility and instability of these compounds. Therefore, the objective of this study is to develop nanoemulsions containing Mancozeb (0.1 mg/mL) and Eugenol (33 mg/mL), isolated or associated, and evaluate the safety of these formulations through cytotoxicity, genotoxicity and ecotoxicity tests. Nanoemulsions were developed by the spontaneous emulsification method, cytotoxicity and genotoxicity were evaluated in healthy human cells through MTT, Dichlorofluorescein diacetate and Picogreen tests, and ecotoxicity assessment was carried out using the chronic toxicity test in springtails. After preparation, the physicochemical characterization of the nanoemulsions were performed which presented mean particle size between 200 and 300 nm, polydispersity index less than 0.3, negative zeta potential and acid pH. The nanoencapsulation was able to avoid the reduction of the cell viability caused by Mancozeb, while Eugenol was shown to be safe for cell use in both free and nanostructured forms, however the association of the two active compounds showed toxicity in the higher doses of Mancozeb. In the ecotoxicity tests, both free Mancozeb and Eugenol forms presented high toxic potential for soil, whereas the nanoencapsulation of these compounds did not cause a reduction in number of springtails. Therefore, from the tests performed, it was possible to observe that nanoencapsulation of Mancozeb and Eugenol is a safe alternative for the application of these compounds mainly in agriculture.

Curcumin mitigates mancozeb-induced hepatotoxicity and genotoxicity in rats.

Mancozeb (MZ) is a widely used ethylene-bis-dithiocarbamate fungicide in agriculture causing hepatoxic and genotoxic effects in rats. Curcumin (CUR) has various pharmacological effects including antioxidant and anti-inflammatory properties. This study investigated the efficacy of CUR in mitigating MZ-induced hepatotoxicity and genotoxicity in rats. Twenty-four male rats were divided into four equal groups; group I (control) was given carboxymethyl cellulose, group II was orally administered CUR (100 mg/kg b.wt), group III was gavaged with MZ (750 mg/kg b.wt), and group IV was co-treated with MZ and CUR at the same doses daily for 10 weeks. As a result, the concurrent treatment with CUR and MZ minimized the increased levels of liver function markers in serum, lipid peroxidation, pro-inflammatory mediators and DNA damage parameters in liver. In addition, CUR administration improved the depleted markers of hepatic antioxidant status in MZ-treated rats. Moreover, CUR protected the liver against the histological alterations elicited by MZ exposure and also, reduced the immunopositive reactivity of pro-apoptotic p53 in cytoplasm of hepatocytes. The present findings suggest that CUR exerts a significant protective effect against MZ-induced hepatotoxicity and genotoxicity.

Developmental neurotoxicity of maneb: Notochord defects, mitochondrial dysfunction and hypoactivity in zebrafish (Danio rerio) embryos and larvae.

Broad applications and exposure to the fungicide maneb can lead to toxicity in non-target organisms. Maneb is also associated with neurogenerative diseases such as Parkinson's disease (PD). The objectives of this study were to determine the acute toxicity of maneb to zebrafish by measuring mitochondrial bioenergetics, locomotor activity, and the expression of genes related to the oxidative damage response, as well as those related to dopamine signaling due to its association with PD. Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to either solvent control (0.1% DMSO, v/v), or one dose of 0.1, 0.5, 1.0 and 10.0 µM maneb for 96 h. Maneb was moderately toxic to zebrafish embryos, and had a 96-h LC50 value of 4.29 µM (~ 1.14 mg/L). Maneb induced a dose-dependent increase in mortality, decreased hatching rate, and increased notochord deformity rate at both 1.0 and 10.0 µM after 72 and 96 h. Total body length was also significantly reduced with 1.0 µM maneb. A 50-60% decrease in mean basal oxygen consumption rate was also observed in embryos following a 24 hpf exposure to 10.0 µM maneb but oligomycin-induced ATP production and FCCP-induced maximum respiration remained unaffected. No change was detected in the expression levels of genes associated with oxidative stress (sod1 and sod2), nor those related to dopamine synthesis (th1), dopamine transporter (dat), dopamine receptors (drd1, drd2a, drd3, and drd4b). Thus, modifying the expression of these transcripts may not be a mechanism for maneb-induced developmental toxicity in zebrafish. To assess the potential for neurotoxicity, a dark photokinesis assay was conducted in larvae following 7 d exposure to 0.1, 0.5 and 1.0 µM maneb. Larvae exposed to 0.5 and 1.0 µM maneb showed signs related to hypoactivity, and this reduced activity is hypothesized to be associated with notochord defects as this deformity was prevalent at higher concentrations of maneb. Overall, these data demonstrate that maneb negatively affects embryonic development (i.e. notochord development), affects basal oxygen consumption rates of embryos, and induces hypoactivity in larval fish. This study improves understanding regarding the developmental neurotoxicity of the fungicide maneb to zebrafish.

Effect of Bacillus cereus on the ecotoxicity of metal-based fungicide spiked soils: Earthworm bioassay.

Soil microorganisms exhibit varying levels of metal tolerance across a diverse range of environmental conditions. The use of metal-based fungicides such as mancozeb and copper oxychloride could potentially result in increased levels of manganese, zinc and copper which may adversely affect soil mesofauna. Under standardized earthworm bioassay conditions (temperature, pH, soil type and water content), we investigated the effect of Bacillus cereus on mancozeb and copper oxychloride ecotoxicity towards Eisenia andrei. A metal-tolerant Bacillus cereus strain previously isolated from a gold mining site was introduced into fungicide spiked soils. Earthworms were exposed to bacterial inoculated and non-inoculated substrates of mancozeb (8, 44, 800 and 1250 mg kg-1) and copper oxychloride (200, 450, 675 and 1000 mg kg-1). Experimental trials assessed avoidance-behavior, growth and reproduction utilizing standardized protocols (ISO and OECD). In the avoidance-behavior, E. andrei showed significant (p< 0.05) preference for inoculated substrates. Further, significant (p< 0.05) increases in biomass, survival, cocoons, juveniles and lower soil and tissue Mn, Cu and Zn contents were recorded at 8 and 44 mg kg-1 mancozeb and copper oxychloride 200 and 450 mg kg-1 inoculated soils compared to non-inoculated. However, at 800 and 1250 mg kg-1 mancozeb and 675 and 1000 mg kg-1 copper oxychloride concentrations, reproductive success in both inoculated and non-inoculated treatments was negatively (p< 0.05) affected. In conclusion, Bacillus cereus decreased the ecotoxicity of metal-based fungicides towards Eisenia andrei at 8 and 44 mg kg-1 mancozeb and 200 and 450 mg kg-1 copper oxychloride concentrations. The outcome observed with the inoculated substrates at elevated fungicides concentrations maybe as a result of the environmental conditions (pH and temperature).

Mancozeb exposure during development and lactation periods results in decreased oocyte maturation, fertilization rates, and implantation in the first-generation mice pups: Protective effect of vitamins E and C.

This study aimed to evaluate the mancozeb (MNZ) impact on oocyte maturation of first-generation mice pups as well as their fertilization rate, embryo development, and implantation along with the preventative effect of vitamins E and C. Pregnant mice were randomly divided into six groups: control, vehicle, and MNZ (500 mg/kg body weight (BW)), vitamin E (200 mg/kg BW), MNZ plus vitamin E, MNZ plus vitamin C (100 mg/kg BW), and MNZ plus two vitamins. All treatments were conducted by oral gavage every 2 days from the second day of gestation until the end of lactation. Vitamin treatment was initiated 30 min before receiving MNZ. After birth, first-generation mice pups were kept until adulthood (8-10 W). Adult female mice pups superovulated and then the collected oocytes were examined for nuclear maturity status. After in vitro fertilization of metaphase II oocytes with sperm of the first-generation male mice pups, fertilization rate and embryo development were evaluated over 24 h. Also, the fecundity rate and the number of implanted embryos in vivo were studied on the eighth day of pregnancy. MNZ exposure during embryo development and lactation significantly decreased the total number of collected oocytes, oocyte maturation, fertilization rate, implantation rate, fecundity rate, and embryo development compared with the control group in the first-generation pups. In contrast, vitamin treatments significantly increased these parameters compared to the MNZ group. Reduction in the quality of oocyte, the rate of fertilization, embryo implantation, and development following MNZ exposure could decrease female reproductive success, while coadministration of vitamins E and C could prevent these complications.

Mancozeb fungicide-induced genotoxic effects, metabolic alterations, and histological changes in the colon and liver of Sprague Dawley rats.

The present study was designed to evaluate genotoxic markers of mancozeb exposure and withdrawal in colon and liver tissues together with histological changes in the gastrointestinal tract of Sprague Dawley rats. Thirty rats were divided into three equal groups; group I: treatment, 250 mg/kg mancozeb dissolved in corn oil administered twice weekly for 7 weeks; group II: withdrawal, the same treatment as group I after which animals were untreated for 5 weeks; group III: control, administered corn oil on the same schedule as group I for 7 weeks. All administrations were by oral gavage. Serum samples were analyzed for biochemical parameters. The comet assay and histopathological examinations were done on liver and colon specimens. The results demonstrated that mancozeb exposure caused significant increases in triglycerides and total cholesterol accompanied by decreases in glucose levels, with extensive DNA damage in liver and colon together with pathological changes in stomach, colon, and liver. Mancozeb withdrawal for 5 weeks improved the lipid and glucose profiles and decreased the degree of DNA damage and changes in the architecture of the stomach, colon, and liver. We concluded that discontinuing exposure to mancozeb fungicide for 5 weeks could ameliorate the adverse effects induced by 7 weeks of exposure to mancozeb. A longer withdrawal time may further reduce the observed genotoxicity.

Taurine protects noradrenergic locus coeruleus neurons in a mouse Parkinson's disease model by inhibiting microglial M1 polarization.

Beyond nigrostriatal dopaminergic system, the noradrenergic locus coeruleus (LC/NE) neurons are also degenerated in patients with Parkinson's disease (PD), the second most common neurodegenerative disorder. We previously reported that microglia-mediated neuroinflammation contributes to LC/NE neurodegeneration. The purpose of this study is aimed to test whether taurine, an endogenous amino acid, could be able to protect LC/NE neurons through inhibition of microglial activation using paraquat and maneb-induced mouse PD model. Taurine (150 mg/kg) was administrated (i.p) to mice 30 min prior to paraquat (10 mg/kg) and maneb (30 mg/kg) intoxication for consecutive 6 weeks (twice per week). The results clearly demonstrated that paraquat and maneb co-exposure resulted in loss of tyrosine hydroxylase-positive neurons in the LC in mice, which was significantly ameliorated by taurine. Mechanistically, inhibition of microglia-mediated neuroinflammation contributed to taurine-afforded neuroprotection. Taurine attenuated paraquat and maneb-induced microglial activation and M1 polarization as well as release of proinflammatory cytokines in brainstem of mice. Taurine also abrogated microglial NADPH oxidase activation and oxidative damage in paraquat and maneb-treated mice. Furthermore, inhibition of nuclear factor-κB (NF-κB) but not signal transducers and activators of transcription 1/3 (STAT1/3) signaling pathway participated in taurine-inhibited microglial activation. Collectively, taurine exerted LC/NE neuroprotection against microglia-mediated neurotoxicity. The robust neuroprotective effects of taurine suggest that taurine may be a promising candidate for potential therapy for patients suffering from PD.