In vitro and in vivo impairment of embryo implantation by commonly used fungicide Mancozeb.

The fungicide Mancozeb is an endocrine-disrupting chemical and the mode of action of Mancozeb on embryo implantation is largely unknown. Mancozeb (1 and 3 µg/ml) significantly reduced Jeg-3 trophoblastic spheroids attachment to endometrial epithelial Ishikawa cells. Mancozeb treatment from gestation day (GD) 1 to GD8 or from GD4 to GD8 significantly lowered the number of implantation sites with higher incidence of morphological abnormalities in the reproductive tissues. However, these were not seen in the treatment from GD1 to GD4. Mancozeb at 30 mg/kg BW/d did not alter the expression of p53, COX-2, or PGFS transcripts in the uterus, but down-regulated the PGES transcript and protein. Mancozeb treatment in human endometrial stromal cells did not alter the decidualization response, but the morphological transformation was impaired. Taken together, exposure to Mancozeb affected embryo implantation probably through the modulation of decidualization and to delineate the exact mode of action needs further investigations.

Eco-friendly PEG-based controlled release nano-formulations of Mancozeb: Synthesis and bioefficacy evaluation against phytopathogenic fungi Alternaria solani and Sclerotium rolfsii.

Controlled release (CR) nano-formulations of Mancozeb (manganese-zinc double salt of N,N-bisdithiocarbamic acid), a protective fungicide, have been prepared using laboratory-synthesized poly(ethylene glycols) (PEGs)-based functionalized amphiphilic copolymers without using any surfactants or external additives. The release kinetics of the developed Mancozeb CR formulations were studied and compared with that of commercially available 42% suspension concentrate and 75% wettable powder. Maximum amount of Mancozeb was released on 42nd day for PEG-600 and octyl chain, PEG-1000 and octyl chain, and PEG-600 and hexadecyl chain, on 35th day for PEG-1000 and hexadecyl chain, on 28th day for PEG-1500 and octyl chain, PEG-2000 and octyl chain, PEG-1500 and hexadecyl chain, and PEG-2000 and hexadecyl chain in comparison to both commercial formulations (15th day). The diffusion exponent (n value) of Mancozeb in water ranged from 0.42 to 0.62 in tested formulations. The half-release (t1/2) values ranged from 17.35 to 35.14 days, and the period of optimum availability of Mancozeb ranged from 18.54 to 35.42 days. Further, the in vitro bioefficacy evaluation of developed formulations was done against plant pathogenic fungi Alternaria solani and Sclerotium rolfsii by poison food technique. Effective dose for 50% inhibition in mgL-1 (ED50) values of developed formulations varied from 1.31 to 2.79 mg L-1 for A. solani, and 1.60 to 3.14 mg L-1 for S. rolfsii. The present methodology is simple, economical, and eco-friendly for the development of environment-friendly CR formulations of Mancozeb. These formulations can be used to optimize the release of Mancozeb to achieve disease control for the desired period depending upon the matrix of the polymer used. Importantly, the maximum amount of active ingredient remains available for a reasonable period after application. In addition, the developed CR formulations were found to be suitable for fungicidal applications, allowing use of Mancozeb in lower doses.

The Effect of Ascorbic Acid on Mancozeb-Induced Toxicity in Rat Thymocytes.

Mancozeb, as a dithiocarbamate fungicide, has been found to exhibit toxicological manifestations in different cells, mainly by generation of free radicals which may alter antioxidant defence systems in cells. The effect of mancozeb on the cells of a primary lymphoid organ has not been studied. In the present study, the effects of mancozeb (0.2, 2 and 5 µg/ml) or mancozeb+ascorbic acid (100 µg/ml), or ascorbic acid alone or control medium alone on the levels of cell viability, apoptosis, intracellular reactive oxygen species production (ROS), mitochondrial membrane potential (MMP) and ATP levels in rat thymocytes were examined in vitro. Cells treated with mancozeb displayed a concentration-dependent increase of hypodiploid cells and ROS production followed by markedly decreased viability of the cells, MMP and ATP levels. Application of ascorbic acid significantly reduced cytotoxicity in cell cultures treated with 0.2 and 2 µg/ml of mancozeb, together with significantly decreased ROS levels and increased MMP and ATP levels. In cells treated with 5 µg/ml of mancozeb, ascorbic acid failed to reduce toxicity while simultaneously increasing the apoptosis rate of thymocytes. These results suggest that ROS plays a significant role in mancozeb-induced toxicity, through alteration of mitochondrial function. Ascorbic acid administration reduced the toxicity rate in cells treated with lower mancozeb concentrations, while it may have the ability to shift cells from necrosis to apoptosis in the presence of highest mancozeb concentrations.

Mancozeb-induced genotoxicity and apoptosis in cultured human lymphocytes.

AIMS: Mancozeb is a dithiocarbamate fungicide known to be genotoxic and induces tumors in rodents at various sites. There is no report in the literature about its genotoxicity in humans. Here, we investigated the association between mancozeb exposure and induction of genotoxic and proapoptotic changes in cultured human lymphocytes (CHLs). MAIN METHODS: Lymphocytes were isolated from peripheral blood of healthy non-smoking donors. Induction of micronuclei and chromosomal aberrations was recorded both by conventional and flow cytometric methods. Annexin-V FITC was used for the differentiation of apoptotic and necrotic cells by flow cytometry. KEY FINDINGS: Mancozeb exposure (0.5, 2 and 5 µg/ml) to CHLs leads to significant induction in the frequency of chromosomal aberrations (CAs) and micronuclei (MN), in a dose-dependent manner. Concomitantly, pro-oxidant potential of mancozeb was also recorded, by increase in the levels of reactive oxygen species (ROS) generation. Our results demonstrated that ROS plays a critical role in the initiation of mancozeb induced apoptosis in CHLs through two ways, primarily through mitochondria-mediated pathway including induction of ROS, decrease in mitochondrial membrane potential (ΔΨm), along with cytochrome c release from mitochondria, and activation of the caspase cascade. The other pathway includes increase in ROS, which resulted in activation of NF-κB, expression of FasL and triggered FasL-dependent pathway, which also involves caspase-8. Therefore, exposure to mancozeb can lead to induction of apoptosis in CHLs through both mechanisms. SIGNIFICANCE: The results of study confirm that mancozeb exposure can induce genotoxicity and apoptosis in CHLs, thus pose a potential risk to exposed human population.

Combined exposure to endocrine disrupting pesticides impairs parturition, causes pup mortality and affects sexual differentiation in rats.

Risk assessment is currently based on the no observed adverse effect levels (NOAELs) for single compounds. Humans are exposed to a mixture of chemicals and recent studies in our laboratory have shown that combined exposure to endocrine disrupters can cause adverse effects on male sexual development, even though the doses of the single compounds are below their individual NOAELs for anti-androgenic effects. Consequently, we have initiated a large project where the purpose is to study mixture effects of endocrine disrupting pesticides at low doses. In the initial range-finding mixture studies, rats were gavaged during gestation and lactation with five doses of a mixture of the fungicides procymidone, mancozeb, epoxyconazole, tebuconazole and prochloraz. The mixture ratio was chosen according to the doses of each individual pesticide that produced no observable effects on pregnancy length and pup survival in our laboratory and the dose levels used ranged from 25 to 100% of this mixture. All dose levels caused increased gestation length and dose levels above 25% caused impaired parturition leading to markedly decreased number of live born offspring and high pup perinatal mortality. The sexual differentiation of the pups was affected at 25% and higher as anogenital distance was affected in both male and female offspring at birth and the male offspring exhibited malformations of the genital tubercle, increased nipple retention, and decreased prostate and epididymis weights at pup day 13. The results show that doses of endocrine disrupting pesticides, which appear to induce no effects on gestation length, parturition and pup mortality when judged on their own, induced marked adverse effects on these endpoints in concert with other pesticides. In addition, the sexual differentiation of the offspring was affected. This as well as the predictability of the combination effects based on dose-additivity modelling will be studied further in a large dose-response study.

Yeast adaptation to mancozeb involves the up-regulation of FLR1 under the coordinate control of Yap1, Rpn4, Pdr3, and Yrr1.

FLR1 gene, encoding a multidrug resistance (MDR) transporter of the major facilitator superfamily (MFS) was found to confer resistance to the fungicide mancozeb in Saccharomyces cerevisiae. This agrochemical has been linked to the development of Parkinson disease and cancer. Yeast response to mancozeb was proved to involve the strong activation of FLR1 transcription (20-fold) during the fungicide-induced growth latency. This activation of FLR1 transcription is fully dependent on Yap1p and is reduced (by 50%) in the absence of Rpn4p, Yrr1p or Pdr3p. A model for the coordinate action over FLR1 transcription activation, in response to mancozeb, of these transcription factors that mediate oxidative stress response (Yap1p), proteasome gene expression (Rpn4p), and pleiotropic drug resistance (Pdr3p and Yrr1p), is proposed.

Morphometric analysis of follicular growth and biochemical constituents in albino rats exposed to mancozeb.

Mancozeb, an ethylenebisdithiocarbamate (EBDC), was administered orally at a dose of 700 mg/kg body weight/day to female virgin rats for 5, 10, 20, and 30 days to examine the effect on ovarian follicular development. No significant change occurred in the number of estrous cycles and the duration of proestrus, estrus, and metestrus, but mancozeb treatment for 5 days significantly increased the duration of diestrus. Mancozeb treatment for 10 days significantly increased the number of estrous cycle and the duration of estrus, with a concomitant significant increase in diestrus, but no change in proestrus and metestrus. With mancozeb treatment for 20 and 30 days, the number of estrous cycles and duration of proestrus, estrus, and metestrus were significantly decreased, with a concomitant significant increase in the duration of diestrus. Exposure of rats to mancozeb for 5 days resulted in a significant decrease in stage II and the total number of healthy follicles but no change in atretic follicles. Mancozeb treatment for 10 days resulted in a significant decrease in stages I, II, and IV and in the total number of healthy follicles. A significant increase in atretic follicles was found in rats treated with mancozeb for 20 and 30 days. No significant change occurred in body and organ weights in any group, but the thyroid weight of 20 and 30 days mancozeb-treated rats was significantly increased. The level of protein in the ovary was significantly decreased, but no change was found in the uterus and liver of mancozeb-treated animals. The level of glycogen was significantly decreased in the ovary and the uterus with mancozeb treatment, but not in the liver. With mancozeb treatment, the levels of phospholipids and neutral lipids were significantly increased in the liver but significantly decreased in the uterus. The change in the biochemical constituents of ovary, uterus, and liver was duration dependent. The results of the study thus indicate a marked disruption of the estrous cycle, pathological changes in the gonads, and organ-specific biochemical changes in rats after exposure to mancozeb.

Inhalation toxicity of propineb. Part I: Results of subacute inhalation exposure studies in rats.

This article addresses results from repeated 1- and 4-wk inhalation exposure studies in Wistar rats with solid aerosol (dust) atmospheres of propineb, a zinc bisdithiocarbamate homopolymer that is used as an agrochemical fungicide. Groups of 10 rats/sex were exposed nose-only to mean concentrations of 3.97, 11.24, and 21.95 mg propineb/m(3) using an exposure regimen of 6 h/day, 5 days/wk for 4 wk. Concentrations were selected based on results from a pilot study in which rats were exposed under identical conditions on 5 consecutive days for 6 h/day to mean concentrations of 10.1, 19.9, 38.1, and 78.7 mg/m(3). Both studies demonstrated that with respect to muscular effects female rats were remarkably more susceptible as compared to males. Female rats exposed to 11.24 mg/m(3) and above displayed characteristic signs of toxicity that included weakness and flaccid paralysis of hindlegs and ensuing immobilization that was considered to be the cause of emaciation and ensuing mortality in some rats. There was an apparent reciprocal relationship of concentration and the manifestation of clinical evidence of muscular dysfunction; that is, the onset in female rats exposed to 11.24, 21.95, 38.1, and 78.7 mg/m(3) was on days 15, 8, 4, and 3, respectively. In contrast, none of the male rats elaborated comparable effects up to 38.1 mg/m(3). Neuromuscular measures included leg grip strength and supplemented the clinical findings, whereas the landing foot splay was only minimally affected. Hematology and clinical pathology endpoints, including those addressing thyroidal function, were unobtrusive up to and including 78.7 mg/m(3). Lung weights were significantly increased in groups exposed to 21.95 mg/m(3) and above, especially in male rats. The microscopic examinations made in the 4-wk study demonstrated an increased incidence of intraalveolar material and enlarged, foamy alveolar macrophages at 3.97 mg/m(3) and above. Especially in female rats an atrophy of thigh muscle fibers, including increased nuclei and focal degeneration, occurred at 11.24 mg/m(3) and above. TTCA (2-thiazolidinethione-4-carboxylic acid) in urine, a metabolite and biomarker of exposure to CS(2), which is a putative breakdown product of propineb, was proportionally higher in the female rats exposed to 11.24 mg/m(3) and above. This biomarker appears to accumulate with time. This finding provides indirect evidence that the etiopathologic cause of neuromuscular changes is related to intermediary levels of CS(2). The data of this investigation suggest that the toxicity of inhaled propineb is characterized by two independent effects, namely, responses occurring at the alveolar level and muscular weakness, especially in female rats. With respect to the latter finding, the no-observed-adverse-effect level (NOAEL) of the 4-wk study is 3.97 mg/m(3). Further study is needed to clarify whether the pulmonary response observed at this exposure level is consistent with an adaptive or an early adverse effect.

Inhalation toxicity of propineb. Part II: Results of mechanistic studies in rats.

Previous repeated inhalation exposure studies revealed two independent organotropic effects of inhaled propineb dust: One was restricted to the lung, the other to muscle weakness of hindlimbs. These effects were believed to be causally related to the principle decomposition products of this type of dithiocarbamate in the biological milieu and related to zinc and carbon disulfide. Two mechanistic 1-wk inhalation studies were performed, each focusing on one of these findings. The 7 x 6-h/day repeated-exposure inhalation study analyzed whether the nature of the response occurring at the alveolar level is "adaptive" or "early adverse" and whether soluble zinc is the causative agent. Groups of 18 female rats were exposed nose-only to mean concentrations of 0, 1.1, 5.5, and 25.8 mg propineb/m(3) and 6.9 mg ZnO/m(3). On postexposure days 1, 3, and 15 the time course of responses was analyzed by bronchoalveolar lavage (BAL), including quantification of Zn and metallothionein (MT) in BAL cells. Clinical evidence of muscular weakness was investigated separately in 20 female Wistar rats exposed to 70 mg propineb/m(3) on 5 consecutive days (6 h/day), followed by a 2-wk postexposure period. Clinical signs, body weights, and feed and water consumption were recorded as frequently as technically feasible. Fifty percent of rats received an oral cysteine supplementation to verify/refute the hypothesis that the incapacitation observed in previous studies is the cause of emaciation and associated impairment of CS(2) detoxification. The findings of the first study are consistent with this hypothesis, namely, that soluble Zn triggers a series of pulmonary events that is consistent with the homeostasis of this essential metal. It is concluded, accordingly, that the adjusted maximal workplace level for ZnO is also valid for propineb to preclude Zn-mediated responses to occur in the lung. With respect to muscular effects, this mechanistic study demonstrates further that the increased detoxification capacity afforded by oral supplementation of cysteine mitigates markedly the toxic potency of propineb. Procedural variables specific to the inhalation bioassay appear to be decisive for the elicitation of muscular effects. The major variable is considered to be the large drop in body weights associated with each exposure session and the concomitantly decreased uptake of essential nutritional factors (e.g., cysteine) involved in the detoxification of this compound. Accordingly, the muscular deficits elicited by high concentrations of propineb are viewed to be secondary effects in an animal species likely to be more susceptible to this type of change than humans (Pauluhn & Rosenbruch, 2003).

Ethylenethiourea in urine as an indicator of exposure to mancozeb in vineyard workers.

In the present study, the personal exposure to mancozeb and/or ethilenethiourea (ETU) in 13 Italian vineyard workers and in 13 subjects without occupational exposure to pesticides was investigated. With this aim, the level of ETU in urine and the dermal exposure to mancozeb were determined. Baseline urinary ETU results were lower than the analytical limit of detection for all controls (<0.5 microg/g creatinine) and for ten workers (median <0.5, range <0.5-3.4 microg/g creatinine). In workers, urinary ETU was significantly increased at the end of shift (2.5, <0.5-95.2 microg/g creatinine) compared with baseline levels. End-shift urinary ETU was higher in operators using open tractors (n=7) than in those using closed tractors (n=5) (16.2 vs. 2.4 microg/g creatinine), but the difference was not significant (P=0.073). End-shift urinary ETU was positively correlated with dermal exposure to mancozeb determined both over the clothes and on the skin (Spearman's rho=0.770 and 0.702, P=0.009 and 0.024, respectively). Wine consumption positively influenced the excretion of ETU.

Toxicity of a mancozeb containing fungicide formulation and CU-sulphate to chicken embryos after administration as single compounds or in combination.

Environmental pollution of metal modelled by copper-sulphate and a 80% mancozeb containing fungicide formulation (Dithane M-45) were studied on chicken embryos after administration as a single compounds or in combination. The test materials were injected into the air-chamber in a volume of 0.1 ml/egg on day 12 of incubation. The concentration of copper-sulphate was 0.01%. The applied concentration of Dithane M-45 fungicide formulation was 0.2%. Evaluation was done on day 19 of the hatching period. The combined administration of copper-sulphate and the fungicide formulation did not cause a significant reduction in body weight as compared to the control data and the results from individual toxicity study of the test materials. After the combined administration of copper-sulphate and the fungicide formulation the rate of embryomortality was 40%. The incidence of developmental anomalies were sporadic. Light microscopic findings exhibited a degenerative change in the liver tissue of combined administration group. Activities of GPT and GOT enzymes increased markedly in the combined administration group. In summary, it can be established that the interaction of copper-sulphate and an 80% mancozeb containing fungicide formulation (Dithane M-45) caused higher embryomortality with respect to the test of individual toxicity of copper-sulphate and fungicide in our study.

Synergism in mixtures of cymoxanil and mancozeb on Phytophthora infestans in vivo.

The preventive activity of 1:8 mixture of cymoxanil and mancozeb against Phytophthora infestans was higher than that of either the two single ingredients or the other nine mixtures. The synergistic interaction existed (synergy ratio 2.01) between the two at the mixing ratio of 1:8, whereas additive interaction (synergy ratios ranged from 0.73 to 1.34) existed at the mixing ratios ranging from 1:1 to 1:7, from 1:9 to 1:10, 1:8 was the optimal ratio. The preventive activity of 1:8 mixture was higher than the curative and the eradicative. In addition, the eradicative synergism of inhibiting sporangia production on lesions was stronger than the eradicative synergism of inhibiting lesion extension and suppressing infection of sporangia, and than the curative synergism of inhibiting lesion sporulation on detached potato leaflets.

Assessment of toxicological effects of mancozeb in male rats after chronic exposure.

Mancozeb, an ethylenebisdithiocarbamate fungicide was administered orally to male rats at doses 0, 500, 1000 and 1500 mg/kg/day for 90, 180 and 360 days produced dose dependent signs of poisoning, loss in body weight gain and mortality. However the signs of toxicity and mortality were more pronounced initially at 0-90 days as compared to 90-360 days of treatment period. A significant increase in the relative weight of liver and slight decrease in the kidney weight were observed in animals exposed to mancozeb (1000 and 1500 mg/kg/day) for 180 and 360 days associated with pathomorphological changes in liver, brain and kidney. Mancozeb has produced significant enzymatic changes in the activities of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE) throughout the period of study in a dose dependent manner. The alterations in the activity of enzymes associated with pathomorphological changes suggest that the chronic exposure of mancozeb produced significant toxicological effects in rats.

Effect of Ziram, Thiram, and Dithane M-45 on bone marrow cells of mice-assessed by micronucleus test.

Micronucleus test is an extensively used protocol to assess the mutagenicity of environmental chemicals. This was developed by Schmid and his co-workers (Matter and Schmid, 1971; Ledebur and Schmid, 1973). The micronucleus test is simple, quick and as sensitive as the chromosome aberration analysis. It is based on the principle that during anaphase, acentric chromatid and chromosome fragments lag behind, where as centric elements move towards the spindle pole. After telophase both the undamaged chromosomes and the centric fragments give rise to the daughter nuclei. The lagging elements are transferred into one or several secondary nuclei, which are as a rule much smaller than the main nucleus, and therefore called micronucleus (Schmid, 1973). The clastogenic effect of various chemicals is measured by micronucleus test. Erythrocytes are two types, the younger ones are polychromatic erythrocytes (PCE), which stain bluish and the older, the normo chromatic erythrocytes (NCE) which stain reddish. A few hours after the completion of last mitosis the erythroblasts expel their nucleus for unknown reasons and the micronucleus alone remains in the cytoplasm of the Polychromatic erythrocytes, and they are easily recognisable. Erythrocyte micronucleus represents the consequence of chromosomal aberrations induced during preceding mitotic division of erythrocytes (Matter and Grauwiler, 1974).

Effects of the subchronic administration of zinc ethylene-bis-dithiocarbamate (zineb) to rabbits.

The effects of subchronic administration (90 d) of zineb were studied in male New Zealand White rabbits. Rabbits were allotted to 3 groups of 8 animals each and offered diets containing 0, 0.3 or 0.6% zineb. A marked decline in weight gain, hemoglobin concentration, hematocrit, and erythrocyte and leucocyte counts occurred at the highest zineb dosage. There was a dose-related depression in circulating thyroid hormones, whereas serum lipid concentration, particularly that of cholesterol and triglycerides, increased. Hepatic lipid concentration was considerably reduced in rabbits exposed to 0.6% zineb. Neither serum testosterone nor the activities of selected testicular enzymes showed changes suggestive of testicular involvement. Pathological changes were in agreement with biochemical findings; there was a marked dose-related enlargement of the thyroid showing histological colloid struma. An increase in relative weight and moderate glycogenosis were detected in liver, whereas no lesions occurred in testes. It was concluded that thyroid and liver are the main targets for zineb toxicity in the rabbit. Unlike the results from previous studies conducted on other food-producing species, repeated exposure of rabbits to zineb failed to cause testicular damage. This might be related to the inability of zineb to significantly accumulate in the testes.

Carcinogenic activity of a carbamate fungicide, mancozeb on mouse skin.

Mancozeb, a polymeric complex of ethylene bis (dithiocarbamate) manganese with zinc salt is a protective fungicide. In the present study complete carcinogenic activity of mancozeb, has been observed following topical application on dorsal mouse skin. Female Swiss albino mice were exposed to mancozeb at a dose of 100 mg/kg body weight dissolved in 100 microliters dimethyl sulfoxide 3 times per week. Development of tumours was observed after 31 weeks (217 days) of mancozeb application. A high rate of mortality was observed after 54 weeks (378 days) of mancozeb application due to its toxicity and the study was terminated after 60 weeks. On histological examination, these tumours were found mostly to be benign in nature, e.g., squamous cell papillomas and keratoacanthomas.