Low concentrations of ethylene bisdithiocarbamate pesticides maneb and mancozeb impair manganese and zinc homeostasis to induce oxidative stress and caspase-dependent apoptosis in human hepatocytes.

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

Pre-imaginal exposure to mancozeb induces morphological and behavioral deficits and oxidative damage in Drosophila melanogaster.

Mancozeb (MZ), a manganese/zinc containing ethylene-bis-dithiocarbamate, is a broad-spectrum fungicide. Chronic exposure to MZ has been related to several organisms' neurological, hormonal, and developmental disorders. However, little is known about the post-natal effects of developmental exposure to MZ. In this study, Drosophila melanogaster was subjected to a pre-imaginal (eggs-larvae-pupae stage) model of exposure to MZ at 0.1 and 0.5 mg/mL. The emergence rate, body size, locomotor performance, sleep patterns, and molecular and biochemical parameters were evaluated in post-emerged flies. Results demonstrate that pre-imaginal exposure to MZ significantly impacted early emerged flies. Additionally, reduced progeny viability, smaller body size and delaying in emergence period, locomotor impairment, and prolonged sleep time were observed. Content of glucose, proteins, and triglycerides were altered, and the bioenergetics efficiency and oxidative phosphorylation at complex I were inhibited. mRNA stade state levels of genes responsive to stress, metabolism, and regulation of circadian cycle (Nrf2, p38, Hsp83, Akt1, GPDH, tor, per, tim, dILP2, and dILP6) were augmented, pointing out to stimulation of antioxidant defenses, insulin-dependent signaling pathway activation, and disruption of sleep regulation. These data were followed by increased lipid peroxidation and lower glutathione levels. In addition, the activity of catalase and glutathione-S-transferase were induced, whereas superoxide dismutase was inhibited. Together, these results demonstrate that developmental exposure to MZ formulation led to phenotype and behavioral alterations in young flies, possibly related to disruption of energetic metabolism, oxidative stress, and deregulation of genes implied in growth, sleep, and metabolism.

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.

The ethylene bisdithiocarbamate fungicides mancozeb and nabam alter essential metal levels in liver and kidney and glutathione enzyme activity in liver of Sprague-Dawley rats.

Mancozeb is a fungicide of the ethylene bisdithiocarbamate (EBDC) class complexed to the metals manganese and zinc. Nabam is the sodium salt of the EBDC backbone. The purpose of this study was to determine if these EBDC compounds alter essential metal homeostasis and glutathione status in Sprague-Dawley rats. Our findings indicate EBDCs caused accumulation of copper in kidneys, but not liver. EBDC compounds also increased glutathione reductase activity in liver, but not kidneys, whereas only mancozeb increased glutathione peroxidase activity in the liver. Mancozeb and nabam increased total glutathione in liver, but only mancozeb increased total glutathione in the kidney. Neither mancozeb nor nabam altered glutathione ratio in either liver or kidney compared to control. Our data suggest that the EBDC backbone of mancozeb, and not the zinc or manganese moieties, is responsible for changes in glutathione status and alteration of essential metal homeostasis in rat liver and kidney.

Multiple toxicity of propineb in developing zebrafish embryos: Neurotoxicity, vascular toxicity, and notochord defects in normal vertebrate development.

A dithiocarbamate (DTC) fungicide, propineb, affects thyroid function and exerts immunotoxicity, cytotoxicity, and neurotoxicity in humans. Long-term exposure to propineb is associated with carcinogenicity, teratogenicity, malfunction of the reproductive system, and abnormalities in vital signs during organ development. However, there is no evidence of acute toxicity attributable to propineb in zebrafish. Therefore, in the present study, we assessed the toxicity of propineb in zebrafish by studying its adverse effects on embryo development, angiogenesis, and notochord development. Embryos with propineb exposure developed morphological and physiological defects and in larvae, apoptosis and notochord defects were induced in the early development stage. Transgenic fli1:eGFP zebrafish exposed to propineb showed abnormal larval development with defects in angiogenesis and deformed vasculature. Propineb induced irreversible damage to the neural development of embryos and neurogenic defects in developing zebrafish in transgenic olig2:dsRED zebrafish. These results show that exposure to propineb triggers abnormalities in different organ systems of zebrafish and suggests the physiological complexity of the response to propineb.

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.

Thyroid disrupting pesticides mancozeb and fipronil in mixture caused oxidative damage and genotoxicity in lymphoid organs of mice.

The interference in endocrine signaling in particular of hypothyroid-pituitary-thyroid axis during embryonic/neonatal development increases the risk of long-lasting immune dysfunctioning. Anticipating that, environmentally realistic exposure of established thyroid disrupting pesticides of dithiocarbamate group mancozeb and phenylpyrazole fipronil was given to mice as individual and as mixtures (MIX-I/MIX-II) during the critical initiation phase of the immune response from postnatal day (PND) 31 till PND 60 (maturation phase). The direct exposure effect was assessed at PND 61 and the persistent effect was assessed at PND 91. Pronounced oxidative stress/genotoxicity in lymphoid organs at even low dose mixture exposure of pesticides (MIX-I/ MIX-II) continued to suppress the immune system till adulthood; might be due to the synergistic/additive action. The oxidative stress/genotoxicity effect was prevented on T4 supplementation to inhibit immunotoxicity as T4 is an immune enhancer and antioxidants. Oxidative stress/genotoxicity is suggested as a mechanism of thyroid disruption mediated immune suppression.

Thyroxine modulation of immune toxicity induced by mixture pesticides mancozeb and fipronil in mice.

AIM: The cross regulation between neuroendocrine system, particularly Hypothalamus-Pituitary-Thyroid (HPT) axis and immune system during embryonic/early neonatal developmental stages shapes the functional attribute of immune response throughout the life. Thus, disruption of immune system was anticipated on exposure to thyroid disrupting pesticides (TDPs) mancozeb (MCZ) and fipronil (FPN) during critical windows of early postnatal days (PND) development. MAIN METHODS: Mice were exposed to MCZ and FPN as individual (0.5% LD 50 each) and as mixtures (0.25% and 0.5% LD 50 each) from PND 31 (initiation phase of immune response) till PND 60 (Maturation phase). Thyroxine (T4) supplementation was given from PND 51 to PND 60. Assessment was done at PND 61 as well as at PND 91 (adults). KEY FINDINGS: Plasma level of thyroid hormones (T3 and T4) was reduced but pituitary hormone (TSH) increased till adulthood on exposure to mixture pesticides but not on individual exposure. Mixture pesticides also increased body weight gain and reduced survival rate in adults. Exposure of individual pesticides exert immunotoxicity but more pronounced immune suppression was observed in mixture pesticides exposed group as reflected in reduced relative weight and cellularity in spleen and thymus, reduced in vitro mitogenic (Con A/LPS) response of splenocytes and thymocytes (reduced proliferative index and increased apoptotic/necrotic death). T4 supplementation ameliorated thyroid disruptive and immunotoxic effect of pesticides. SIGNIFICANCE: The additive/synergistic toxicity as well as hypothyroidism induced by mixture pesticides has produced pronounced immune suppression that reflected till adulthood. Supplementation of T4 prevented thyroid axis disruption mediated immunosuppression.

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.

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.

Mancozeb induces testicular dysfunction through oxidative stress and apoptosis: Protective role of N-acetylcysteine antioxidant.

Mancozeb (MZB) is one of the fungicides used in pest control programs that might affect human health including reproductive system. The aim of this study was to demonstrate the mechanisms through which MZB induces testicular tissue damage and the probable protective effect of N-acetylcysteine (NAC), a modified amino acid, with antioxidant property, against MZB toxicity in an animal model. Male albino mice ( n = 8) were exposed to different doses of MZB (250 and 500 mg/kg/day) by oral gavage without or with NAC (200 mg/kg, twice/week) for 40 days. Sub-chronic MZB dose-dependently decreased sperm motility and count. Exposure to MZB increased lipid peroxidation and protein carbonyl, while it reduced antioxidant enzymes activities, total antioxidant capacity, and glutathione content. The histopathological examination clearly showed deleterious changes in the testicular structure. At the molecular levels, the results of quantitative real time-poly chain reaction (qRT-PCR) showed that MZB upregulated oxidative stress markers inducible nitric oxide synthase (iNOS) and NADPH oxidase 4 (NOX4) and downregulated expression of the glutathione peroxidase 1 (Gpx1) gene as one of the most important antioxidant enzymes. MZB also induced apoptosis dose-dependently in the testes as determined by the terminal dUTP nick-end labeling assay and immunoblotting. NAC administration decreased the mRNA levels of both iNOS and NOX4 with a concomitant increase in Gpx1 expression. It also significantly decreased MZB-induced oxidative stress and apoptosis. Collectively, the present study showed MZB-induced oxidative damage in testes leading to apoptosis. It revealed that antioxidants such as NAC can mitigate oxidant injury induced by the dithiocarbamate pesticides in the reproductive system.

Transport of a manganese/zinc ethylene-bis-dithiocarbamate fungicide may involve pre-synaptic dopaminergic transporters.

Mancozeb (MZ), an organic-metal fungicide used predominantly on vegetables and fruits, has been linked to neurodegeneration and behavioral disruptions in a variety of organisms, including humans. Both γ-aminobutyric acid and dopamine neurons appear to be more vulnerable to MZ exposure than other neuronal populations. Based on these observations, we hypothesized that MZ may be differentially transported into these cells through their presynaptic neurotransmitter transporters. To test this, we pretreated Caenorhabditis elegans with transporter antagonists followed by exposure to various concentrations of MZ. Potential neuroprotection was monitored via green fluorescence associated with various neuron populations in transgenic worm strains. Neurodegeneration associated with subacute MZ treatment (30 min) was not altered by transporter antagonist pretreatment. On the other hand, pretreatment with a dopamine transporter antagonist (GBR12909) appeared to protect dopaminergic neurons from chronic (24 h) MZ treatment. These results are consistent with other reports that dopamine transporter levels or activity may modulate toxicity for neurotoxicants.

N-acetylcysteine inhibits Mancozeb-induced impairments to the normal development of zebrafish embryos.

Mancozeb (MZ), a manganese/zinc-containing ethylene-bis-dithiocarbamate (EBCD) fungicide has been claimed to present low acute toxicity and short environmental persistence, however, its effects on embryogenesis in non-target organisms is unclear. Here, we used zebrafish embryos (5 hpf) to assess the potential embryotoxic effects induced by MZ (up to 72 hpf) as well as the role of reactive oxygen species (ROS) in this process by pre-treatment with a classical antioxidant (N-acetylcysteine, NAC). Markers of reactive oxygen species production (ROS), glutathione (GSH) levels and glutathione S-transferase (GST) activity were measured along with genotoxicity (comet assay), cell death (Acridine Orange) and behavioral parameters (spontaneous movement, touch stimulation and swimming response), in order to determine potential mechanisms of embryotoxicity. According to results, MZ was able to induce morphological abnormalities such as body axis distortion, DNA damage, cell death, increased ROS generation and changes in behavioral endpoints during zebrafish development. All these toxic effects were inhibited by the pre-treatment with NAC indicating a key role of redox unbalance during MZ-induced embryotoxicity. At least in our knowledge, this is the first report on the deleterious effect of MZ to the normal embryogenesis of zebrafish. In addition, the importance of ROS generation during this pathophysiological condition was highlighted.

Effects of mancozeb, metalaxyl and tebuconazole on steroid production by bovine luteal cells in vitro.

Mancozeb, metalaxyl and tebucanazole are widely used pesticides in agriculture and industry to treat plant pathogenic fungi. Livestock may be exposed to such substances by consuming contaminated plants. The present study was designed to evaluate the effects of these three fungicides on bovine luteal cell steroidogenesis. Luteal slices from mid-cycle corpus luteum were dissociated into single cell suspension in aerated (O2) culture media (DMEM/F12) by enzymatic digestion. The cells were incubated in newborn calf serum (10%) for 18 h and then with serum-free media containing mancozeb (0.01 µM, 0.1 µM, 1 µM), tebuconazole (1 µM, 10 µM, 100 µM) or metalaxyl (100 µM, 500 µM, 2500 µM) for additional 96 h. The medium was replaced on day 1 and 3; and the retrieved medium was stored at -20 °C until progesterone assay. Treatment of cells with three different fungicides induced dose dependent variable decrease in steroid synthesis during incubation periods. Incubation of cells with 1 µM mancozeb exhibited a 33% decline in steroid synthesis on day 3 and 48% decline on day 5 compared with controls. Treatment of cells with 100 µM tebuconazole and 500 µM metalaxyl resulted in a 65% and 31% decrease, respectively, in progesterone accumulation on day 5 of incubation. Fungicide induced suppressive effects on luteal steroidogenesis were as metalaxyl < tebuconazole < mancozeb. Results of the present study suggest that designated concentrations of all three fungicides studied might have varying degrees of adverse effects on luteal steroidogenesis.

Assessment of protective potential of Nigella sativa oil against carbendazim- and/or mancozeb-induced hematotoxicity, hepatotoxicity, and genotoxicity.

Nigella sativa oil (NSO) possesses antioxidant activity. However, its protective role against the hazards of fungicides has been poorly studied. Therefore, the present work aimed at determining the ameliorative potential of NSO against hepatotoxicity induced by carbendazim (CBZ) and/or mancozeb (MNZ) in female rats. In the present study, about 120 adult female Sprague-Dawley rats were randomly divided into eight equal groups. One group of animals was kept as a negative control (Gp. 1); groups 2, 3 and 4 orally received CBZ (200 mg/kg body wt) and/or MNZ (300 mg/kg body wt) daily for 2 weeks (positive groups). In order to assess the hepatoprotective potential of NSO, in comparison with NSO-treated rats (Gp. 5), groups 6, 7 and 8 were CBZ- and/or MNZ-exposed groups pre-treated orally with NSO (2 ml/kg body wt) daily for 2 weeks (prophylactic groups). All groups were kept further for 15 days without medications to observe the withdrawal effect. At the end of exposure and withdrawal periods, the body weight of all experimental rats was recorded and blood samples were collected for hematological, clinico-biochemical, and micronucleus assays. The animals were then sacrificed, and the liver and bone marrow were harvested for oxidative stress bioassay, chromosomal aberrations, DNA fragmentation, and histopathological examinations. The results suggested that pre-treatment with NSO remarkably diminished CBZ- and MNZ-induced macrocytic hypochromic anemia, leukocytosis, lymphocytosis, eosinophilia, and neutropenia. Besides, it also minimized the elevated liver enzymes, lipid peroxidation, micronucleus incidence, DNA damage, and chromosomal aberration frequency. Conversely, NSO significantly stimulated the CBZ- and/or MNZ-induced antioxidant system suppression. The NSO also normalized the hepatic structural architecture. As far as withdrawal effect is concerned, there was almost disappearance of the bad effects of these fungicides and the values were close to the normal range especially with the use of NSO. Ultimately, the results revealed that N. sativa oil is an effective hepatoprotective agent due to its genoprotective and free radical scavenging activities.

Forskolin ameliorates mancozeb-induced testicular and epididymal toxicity in Wistar rats by reducing oxidative toxicity and by stimulating steroidogenesis.

In the present study, we have tested the beneficial effects of forskolin in protecting the mancozeb-induced reproductive toxicity in rats. Adult male Wistar rats were exposed to either mancozeb (500 mg/kg body weight/day) or forskolin (5 mg/kg body weight/day) or both for 65 days and analyzed for spermatogenesis and steroidogenesis and testicular and epididymal oxidative toxicity. A significant decrease in daily sperm production, epididymal sperm count, motile, viable, and hypo-osmotic swelling-tail swelled sperm was observed in mancozeb-treated rats. The activity levels of testicular 3ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase and circulatory testosterone levels were significantly decreased in mancozeb-treated rats. Exposure to mancozeb resulted in a significant decrease in glutathione levels and superoxide dismutase and catalase activity levels with an increase in lipid peroxidation levels in the testes and epididymis. Coadministration of forskolin mitigated the mancozeb-induced oxidative toxicity and suppressed steroidogenesis and spermatogenesis.

Thyroid disrupting pesticides impair the hypothalamic-pituitary-testicular axis of a wildlife bird, Amandava amandava.

The effect of two thyroid disrupting pesticides (TDPs) mancozeb (MCZ) and imidacloprid (IMI) on the hypothalamic-pituitary-gonadal/testicular (HPG) axis of a seasonally breeding bird, Amandava amandava has been evaluated. Male birds (n=8/group) were exposed to each of the pesticide (0.25% LD50 of respective pesticide) as well as to their two equimixture doses (0.25% of LD50 of each and 0.5% LD50 of each) through food for 30d during pre-breeding stage of the reproductive cycle. Reduction in weight, volume and other histopathological features revealed testicular regression. Suppression of gonadotropin releasing hormone, increased expression of gonadotropin inhibitory hormone in the hypothalamus of exposed groups as well as impairment of plasma levels of the reproduction related hormones indicated the disruption of the HPG axis. The pesticides interference of the thyroid function during the critical phase of reproductive development impaired the HPG axis; more significantly in co-exposed groups suggesting the cumulative toxicity.

Disruption of the hypothalamic-pituitary-thyroid axis on co-exposures to dithiocarbamate and neonicotinoid pesticides: Study in a wildlife bird, Amandava amandava.

Non-target organisms, including human and wildlife, are susceptible to deleterious effects of pesticide mixtures in their environment. Present study demonstrated the disruption of the hypothalamic-pituitary-thyroid (HPT) axis in a seasonally breeding wildlife bird Amandava amandava on co-exposure to dithiocarbamate mancozeb/MCZ and neonicotinoid imidacloprid/IMI, at concentrations even lower than respective environmentally realistic exposure level of each of the pesticide. Adult male birds (n=8/group) were exposed individually to 0.25% LD50 of each of MCZ (0.14mg) and IMI (2.75µl) followed by co-exposure to their equimixture as MIX-I (0.25% LD50 of each) and MIX-II (0.5% LD50 of each) through food for 30d in preparatory phase of reproductive cycle. Disruptions of thyroid gland and pituitary-thyroid axis were evident. Altered thyroid weight and volume, follicles with inactive colloids and lesions, decrease of height and nucleus-to-cytoplasm ratio of follicular epithelial cells were noted. Plasma levels of T4 and T3 were decreased, more significant in mixture groups than in individually exposed groups. Within co-exposed groups, comparatively high plasma T4 and T3 levels in MIX-II than MIX-I indicated dose non-responsiveness of the pesticides in mixtures; a characteristic displayed by endocrine disrupters. Plasma TSH level was increased in MCZ- and IMI- but decreased in MIX-I and MIX-II suggesting the disruption of the negative feedback and impairment of the HPT axis in co-exposed groups. Effects were more prominent in co-exposed groups due to combinatorial action and cumulative toxicity of pesticides. Considering the role of thyroid hormones in reproductive development, pesticides even in low dose could affect the thyroid homeostasis and reproductive axis.

Determinación de etilentiourea en acelgas tratadas con Zineb: Efecto del calor húmedo por ebullición / Determination of ethylenethiourea in chard treated with Zineb: Effect of wet heat by boiling

El Zineb es un plaguicida perteneciente a la familia de los etilenbisditiocarbamatos (EBDC) ampliamente utilizado en la provincia de Jujuy (Argentina). La toxicidad de este fungicida está dada por la etilentiourea (ETU) que es el principal producto de su degradación. La ETU tiene efectos mutagénicos, teratogénicos y cancerígenos en animales. La EPA la clasifica como probable cancerígeno humano. El objetivo del trabajo fue determinar la transformación del Zineb a ETU en acelgas cultivadas en Jujuy, por efecto del calor húmedo por tratamiento térmico. El método se basó en la extracción de la ETU con agua y su posterior reextracción con diclorometano a pH alcalino. El extracto se purificó por cromatografía en columna de alúmina y la ETU se cuantificó por HPLC con un detector UV-Visible a 232 nm. Para cumplir con el objetivo planteado se realizó un cultivo experimental de acelgas a las que se aplicó el fungicida Zineb, el cual luego se cuantificó a diferentes periodos de tiempo. La ETU se cuantificó con y sin aplicación de calor húmedo. La concentración de ETU en las acelgas sometidas a tratamiento térmico fue de 8,07 mg/kg y transcurridos 30 días disminuyó a 0,05 mg/kg, lo que representa una disminución de más del 99 % respecto a la concentración inicial. Simultáneamente, se determinó la concentración de Zineb en las acelgas y se obtuvo, inicialmente, 44 mg/kg y luego de 36 días la concentración de Zineb disminuyó a 3,83 mg/kg, lo que representa una disminución del 91 % respecto a la concentración del plaguicida al inicio. La detección de ETU en acelgas sometidas a tratamientos térmicos confirma la transformación del fungicida Zineb a un producto de degradación clasificado según el IARC en el grupo 3. Esto pone en discusión la reglamentación Argentina vigente, por cuanto al finalizar los tiempos de carencia no se evalúan la presencia de metabolitos o productos de degradación potencialmente tóxicos.

Zineb is a pesticide belonging to the family of ethylenebisdithiocarbamates (EBDC) widely used in the province of Jujuy, Argentina. The toxicity of this fungicide is given by the ethylenethiourea (ETU) which is the main product of degradation. The ETU has mutagenic, teratogenic and carcinogenic effects in animals. The EPA classifies it as a probable human carcinogen. The aim of the study was to determine the transformation of Zineb to ETU in cultivated chards in Jujuy, by treatment with humid heat. The method is based on extraction of ETU with water and subsequent extraction of the aqueous phase with dichloromethane. The extract was cleaned by alumina column chromatography and the ETU was quantified by HPLC with a UV-Visible detector at 232 nm. An experimental cultivation of chards was carried out and the Zineb fungicide was applied and then quantified at different time periods. The ETU was quantified with and without application of moist heat. The concentration of ETU in heat treated chards was 8.07 mg/kg and after 30 days decreased to 0.05 mg/kg, representing a decrease of more than 99 % over the initial concentration. Simultaneously, Zineb concentration in chard was initially determined as 44 mg/kg and, 36 days later, the Zineb concentration decreased to 3.83 mg/kg, representing a 91% decrease with respect to the concentration of the pesticide at the beginning. Detection of ETU in chards subjected to heat treatment confirms the transformation of the fungicide Zineb to a degradation product classified by the IARC as group 3. This puts into discussion the current Argentine regulations since, at the end of the deficiency times the presence of potentially toxic metabolites or degradation products is not evaluated.

Spectroscopic evaluation of the interaction between pesticides and chickpea cystatin: comparative binding and toxicity analyses.

The binding study of pesticides with proteins is of great importance in ecotoxicology. In this study, a comparative interaction mechanism of phytocystatin with three pesticides has been presented, each from a different class-glyphosate herbicide (GPS), chlorpyrifos insecticide (CPF), and mancozeb fungicide (MCZ). The interaction of purified chickpea cystatin (CPC) has been characterized by fluorescence, UV, and circular dichroism (CD) spectroscopic methods. The study revealed association constants (Ka) of 52 M(-1), 1.145 × 10(3) M(-1), and 36.12 M(-1) for the interaction of CPF, MCZ, and GPS with CPC, respectively, signifying the high affinity interaction for MCZ. Structural changes (at tertiary and secondary levels) were confirmed by UV-visible, intrinsic fluorescence and CD spectroscopy. The results showed that the effect on the CPC structure was more pronounced in the case of MCZ, which was followed by CPF and then GPS. The functional analysis of the pesticide treated inhibitor showed a decline in antipapain activity which varied with the time and dose as well as the class of pesticide. MCZ was relatively much more toxic as compared to CPF and GPS. Reactive oxygen species responsible for inhibitor damage were also analyzed. The results obtained implicate that the exposure of plants to pesticides may lead to physicochemical changes in proteins such as phytocystatins leading to physiological damage to the plant system.