Embryotoxic effects of Rovral® for early chicken (Gallus gallus) development.

Rovral® is a fungicide used to control pests that affect various crops and little is known regarding its effects on embryonic development of amniotes. Thus, this study aimed to determine the influence of Rovral® during chicken organogenesis using acute in ovo contamination. Fertilized eggs were inoculated with different concentrations of Rovral® (100, 300, 500 or 750 µl/ml), injected into the egg's air chamber. After 7 days, embryos were examined for possible malformations, staging, weight and mortality. Subsequently, head, trunk, limbs and eyes were measured for morphometry and asymmetry. For blood analysis, eggs were treated with 300 µl/ml Rovral® and glucose, presence of micronuclei and erythrocyte nuclei abnormalities determined. Treatments with Rovral® affected the mortality rate in a concentration-dependent manner. LC50 value was found to be 596 µl/ml which represents 397-fold higher than the recommended concentration for use. Rovral® produced several malformations including hemorrhagic, ocular and cephalic abnormalities. No significant changes were observed in body weight, staging, body measurements, symmetry and glucose levels of live embryos, which indicates this fungicide presents low toxicity under the analyzed conditions. Changes in erythrocyte nuclei were noted; however significant difference was observed only for presence of binucleated erythrocytes. It is important to point out that possibly more significant changes may have occurred at lower concentrations through chronic contamination. Therefore, caution is needed in the use of this fungicide, since it presents teratogenic and mutagenic potential.

Dose-independent genotoxic response in A549 cell line exposed to fungicide Iprodione.

The fungicide Iprodione is widely applied in vegetables and raises concern for human health. The A549 human lung carcinoma cell line is a suitable model for assessing the toxicological effects of drugs. The goal of this work was to evaluate the genotoxicity and oxidative stress in the A549 cell line exposed to sublethal concentrations from 3 to 100 µg/mL Iprodione considering LC50 = 243.4 µg/mL Iprodione, as determined by the MTT assay. Generalized Linear Mixed Models (GLMM) were performed to determine the association between the responses NDI, MNim and MNib and the explanatory variables. Iprodione and solvent were relativized to the control whereas the concentration was included as numeric variable. ANOVA was used for the comparison of treatments. The coefficients of linear association between the explanatory variables and NDI, and the coefficients of logistic association between explanatory variables and MNim were not significant. However, these coefficients showed significant association with MNib only for Iprodione treatment but not for Iprodione concentration, indicating lack of dose-response relationship. Genotoxicity risk assessment indicated that the increase in Iprodione concentrations increased slightly the probability of belonging to the genotoxic category. ANOVA showed significant differences in MNib, and non-significant differences in NDI and MNim among treatments. The oxidative stress analysis performed at 3, 12, and 25 µg/mL Iprodione showed a significant and linear increase in SOD, and a significant and linear decrease in GSH and GST. The Dunnett test was significant for GSH at 12 and SOD at 25 µg/mL.

N-acetylcysteine reduced the immunotoxicity effects induced in vitro by azoxystrobin and iprodione fungicides in mice.

Azoxystrobin (AZO) and Iprodione (IPR) fungicides are extensively used worldwide, and therefore, contaminate all environmental compartments. The toxicity and the mechanisms by which they affected immune cells are complex and remain unknown. This study investigated the impact of AZO and IPR on the in vitro function of mice peritoneal macrophages including lysosomal enzyme activity and tumor necrosis factor (TNF)α and nitric oxide (NO) production in response to lipopolysaccharide (LPS) stimulation, the proliferation of mice splenocytes stimulated by concanavalin (Con)A and LPS, and the production of the Th1cytokine interferon-gamma (IFNγ) and the Th2 cytokine interleukin (IL)-4 and IL-10 by ConA-activated splenocytes. This is the first report indicating that AZO and IPR fungicides dose-dependently inhibited mice macrophage lysosomal enzyme activity and LPS-stimulated production of TNFα and NO. Mitogen-induced proliferation of mice splenocytes was also suppressed by AZO and IPR in a dose-dependent manner. More pronounced impact was observed on ConA-induced response. The production of IFNγ by ConA-stimulated splenocytes was dose-dependently inhibited; however, the production of IL-4 and IL-10 increased in the same conditions. These results suggested that AZO and IPR polarized Th1/Th2 cytokine balance towards Th2 response. Overall, marked immunosuppressive effects were observed for AZO. The immunomodulatory effects caused by AZO and IPR were partially reversed by the pharmacological antioxidant N-acetylcysteine (NAC), suggesting that both fungicides exerted their actions through, at least in part, oxidative stress-dependent mechanism. Collectively, our data showed that AZO and IPR fungicides exerted potent immunomodulatory effects in vitro with eventually strong consequences on immune response and immunologically based diseases.

Pesticide-tolerant bacteria isolated from a biopurification system to remove commonly used pesticides to protect water resources.

In this study, we selected and characterized different pesticide-tolerant bacteria isolated from a biomixture of a biopurification system that had received continuous applications of a pesticides mixture. The amplicon analysis of biomixture reported that the phyla Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were predominant. Six strains grew in the presence of chlorpyrifos and iprodione. Biochemical characterization showed that all isolates were positive for esterase, acid phosphatase, among others, and they were identified as Pseudomonas, Rhodococcus and Achromobacter based on molecular and proteomic analysis. Bacterial growth decreased as both pesticide concentrations increased from 10 to 100 mg L-1 in liquid culture. The Achromobacter sp. strain C1 showed the best chlorpyrifos removal rate of 0.072-0.147 d-1 a half-life of 4.7-9.7 d and a maximum metabolite concentration of 2.10 mg L-1 at 120 h. On the other hand, Pseudomonas sp. strain C9 showed the highest iprodione removal rate of 0.100-0.193 d-1 a half-life of 4-7 d and maximum metabolite concentration of 0.95 mg L-1 at 48 h. The Achromobacter and Pseudomonas strains showed a good potential as chlorpyrifos and iprodione-degrading bacteria.

The fungicide iprodione affects midgut cells of non-target honey bee Apis mellifera workers.

The honey bee Apis mellifera is an important pollinator of agricultural crops and natural forests. Honey bee populations have declined over the years, as a result of diseases, pesticides, and management problems. Fungicides are the main pesticides found in pollen grains, which are the major source of protein for bees. The objective of this study was to evaluate the cytotoxic effects of the fungicide iprodione on midgut cells of adult A. mellifera workers. Bees were fed on iprodione (LD50, determined by the manufacturer) for 12 or 24 h, and the midgut was examined using light and transmission electron microscopies. The expression level of the autophagy gene atg1 was assessed in midgut digestive cells. Cells of treated bees had signs of apoptosis: cytoplasmic vacuolization, apical cell protrusions, nuclear fragmentation, and chromatin condensation. Ultrastructural analysis revealed some cells undergoing autophagy and necrosis. Expression of atg1 was similar between treated and control bees, which can be explained by the facts that digestive cells had autolysosomes, whereas ATG-1 is found in the initial phases of autophagy. Iprodione acts by inhibiting the synthesis of glutathione, leading to the generation of reactive oxygen species, which in turn can induce different types of cell death. The results indicate that iprodione must be used with caution because it has side effects on non-target organisms, such as pollinator bees.

Toxicological effects of comercial formulations of fungicides based on procymidone and iprodione in seedlings and root tip cells of Allium cepa.

In this study the phytotoxic, cytotoxic, genotoxic and mutagenic effects of two commercial fungicide-active compounds, procymidone (PR) and iprodione (IP), were determined. The parameters evaluated were germination and root growth, mitotic index, chromosomal and nuclear aberrations, and molecular analyses were also performed in the model plant Allium cepa L. The results demonstrated that the active compounds PR and IP were phytotoxic, delaying germination and slowing the development of A. cepa seedlings. Moreover, PR and IP showed cytogenotoxicity towards A. cepa meristematic cells, inducing chromosomal changes and cell death. The mutagenic activity of the active compounds was demonstrated by the detection of DNA changes in simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers in the treated cells compared to the negative control. Together, these results contribute to a better understanding of the damage caused by these substances in living organisms and reveal a promising strategy for prospective studies of the toxic effects of environmental pollutants.

AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.

It has been shown that 5-amino-4-imidazolecarboxamide riboside (AICAr) can inhibit cell proliferation and induce apoptosis in childhood acute lymphoblastic leukemia (ALL) cells. Although AICAr could regulate cellular energy metabolism by activating AMPK, the cytotoxic mechanisms of AICAr are still unclear. Here, we knocked out TP53 or PRKAA1 gene (encoding AMPKα1) in NALM-6 and Reh cells by using the clustered regularly interspaced short palindromic repeats/Cas9 system and found that AICAr-induced proliferation inhibition was independent of AMPK activation but dependent on p53. Liquid chromatography-mass spectrometry analysis of nucleotide metabolites indicated that AICAr caused an increase in adenosine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphosphate levels by up-regulating purine biosynthesis, while AICAr led to a decrease in cytidine triphosphate, uridine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate levels because of reduced phosphoribosyl pyrophosphate production, which consequently impaired the pyrimidine biosynthesis. Ribonucleoside triphosphate (NTP) pool imbalances suppressed the rRNA transcription efficiency. Furthermore, deoxy-ribonucleoside triphosphate (dNTP) pool imbalances induced DNA replication stress and DNA double-strand breaks, followed by cell cycle arrest and apoptosis in ALL cells. Exogenous uridine could rebalance the NTP and dNTP pools by supplementing pyrimidine and then attenuate AICAr-induced cytotoxicity. Our data indicate that RNA transcription inhibition and DNA replication stress induced by NTP and dNTP pool imbalances might play a key role in AICAr-mediated cytotoxic effects on ALL cells, suggesting a potential clinical application of AICAr in future ALL therapy.-Du, L., Yang, F., Fang, H., Sun, H., Chen, Y., Xu, Y., Li, H., Zheng, L., Zhou, B.-B. S. AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.

Disruption of oocyte maturation by selected environmental chemicals in zebrafish.

Oocyte maturation can be a target of endocrine disruption by environmental chemicals capable of acting as hormone mimics, receptor blockers, and/or enzyme inhibitors. Six environmental chemicals (genistein, endosulfan, malathion, iprodione, carbaryl, and glyphosate) were selected to determine their ability to interfere with oocyte maturation in zebrafish. The translucent oocytes undergoing germinal vesicle (nucleus) breakdown (GVBD) were counted and expressed as a ratio of oocytes undergoing GVBD and total oocytes exposed. The GVBD increased significantly in oocytes exposed to 10 IU/ml to 100 IU/ml human chorionic gonadotropin (hCG). The lowest effective concentration of genistein that inhibited hCG-induced GVBD was 30 µM, while endosulfan inhibited it at 0.03 µM concentration. In addition, malathion inhibited hCG-induced GVBD at the lowest concentration of 60 µM. These inhibitory effects were likely due to the chemicals acting as estrogen mimics, induction of estrogen receptors, or increase in aromatase activity resulting in enhanced estrogen action. Fungicide iprodione, possibly acting as a progestin mimic, promoted hCG-induced GVBD at the lowest concentration of 20 µM, while the weed killer glyphosate inhibited hCG-induced GVBD starting at the 50 µM concentration. These results demonstrate the feasibility of using fully grown zebrafish oocytes arrested at the prophase I stage in an in vitro incubation system to evaluate the effects of a variety of environmental chemicals on oocyte maturation.

The effects of iprodione fungicide on survival, behavior, and brood development of honeybees (Apis mellifera L.) after one foliar application during flowering on mustard.

A semifield study to assess the effects of iprodione on honeybees at label use rates was conducted on a bloom mustard crop. The present study followed the Organisation for Economic Co-operation and Development guideline 75 tunnel test and consisted of 3 groups: the iprodione-treated group, the untreated control group, and the toxic reference item group. In addition to the tunnels used for biological assessments, a tunnel was set up in the treatment and control groups to determine the level of residues in flowers, nectar, and pollen. The major endpoints to assess the effects of the application of iprodione were mortality, flight intensity, behavior, condition of the colonies, and development of the brood. Residue analysis showed that honeybees were exposed to significant residues of iprodione. However, no adverse effects were observed on overall mortality, flight intensity, behavior, or brood development of honeybees compared to control. It is concluded that iprodione does not adversely affect the health of honeybees when applied in agriculture at commercially relevant rates in a worst-case exposure scenario. Environ Toxicol Chem 2018;37:3086-3094. © 2018 SETAC.

Deciphering the toxic effects of iprodione, a fungicide and malathion, an insecticide on thiol protease inhibitor isolated from yellow Indian mustard seeds.

Pesticides are being used globally to improve agricultural production. They are applied specifically to combat with pathogens that are a major threat for reduced optimum yield of crops. This study was carried out to see the effect of commercially used pesticides on a specific plant protein viz. phytocystatin isolated from yellow mustard seeds (YMP). Phytocystatin is a thiol proteinase inhibitor, which regulates endogenous and exogenous cysteine proteinases and plays a vital physiological role in plants. Different classes of pesticides like fungicide (iprodione) of dicarboximide class and an insecticide (malathion) of class organophosphate are retorted for our study. In the presence of these pesticides, biophysical and biochemical changes were observed in phytocystatin. These changes were evaluated making use of caseinolytic activity assay, UV-vis spectroscopy, fluorescence spectroscopy, FTIR, and circular dichroism. Isothermal titration calorimetry was employed to see interaction pattern of these pesticides with phytocystatin. The results obtained clearly depict that the pesticides bind with the phytocystatin thereby changing its native conformation and reducing its intrinsic property of inhibition on cysteine proteinase as evident by reduced anti-papain inhibition in the presence of pesticides. Furthermore, CD and FTIR spectroscopy results clearly show a decrease in α-helical content upon interaction with malathion and iprodione. Among the two pesticides, iprodione has far more pronounced effect on YMP evident from striking changes in UV, Fluorescence, CD and FTIR spectroscopy. 2,4-dinitrophenylhydrazine spectrophotometric assay was also carried out to check production of ROS, generation of ROS was observed in the presence of these pesticides thus implying that ROS might be responsible for changes in native structure of phytocystatin induced by pesticides.

Atrazine, chlorpyrifos, and iprodione effect on the biodiversity of bacteria, actinomycetes, and fungi in a pilot biopurification system with a green cover.

The use of biopurification systems can mitigate the effects of pesticide contamination on farms. The primary aim of this study was to evaluate the effect of pesticide dissipation on microbial communities in a pilot biopurification system. The pesticide dissipation of atrazine, chlorpyrifos and iprodione (35 mg kg-1 active ingredient [a.i.]) and biological activity were determined for 40 days. The microbial communities (bacteria, actinomycetes and fungi) were analyzed using denaturing gradient gel electrophoresis (DGGE). In general, pesticide dissipation was the highest by day 5 and reached 95%. The pesticides did not affect biological activity during the experiment. The structure of the actinomycete and bacterial communities in the rhizosphere was more stable during the evaluation than that in the communities in the control without pesticides. The rhizosphere fungal communities, detected using DGGE, showed small and transitory shifts with time. To conclude, rhizosphere microbial communities were not affected during pesticide dissipation in a pilot biopurification system.

Effects of low doses of carbendazim or iprodione either separately or in mixture on the pubertal rat seminiferous epithelium: An ex vivo study.

It has been shown that non-cytotoxic doses of Carbendazim (CBZ), a broad-spectrum benzimidazole fungicide, possess endocrine-disrupting (androgen-like) actions, ex vivo, on the pubertal rat seminiferous epithelium. Iprodione (IPR), a dicarboximide fungicide, is also known to be an endocrine-disrupter (anti-androgen). The effect of a mixture of these two pesticides was investigated in the validated rat seminiferous tubule culture model. Cultures were performed in the absence or presence of CBZ 50nM or IPR 50nM either alone or in mixture (Mix), over a 3-week period. Mix exerted a dramatic effect on two proteins (Connexin 43 and Claudin-11) of the blood-testis barrier and possessed similar effects to IPR on some germ cell populations. The presence of IPR together with CBZ (Mix) cancelled the effect of CBZ on the increase of the androgen-dependent TP1 and TP2 mRNAs and on the decrease of ERα, ERß mRNAs. Nevertheless, CBZ alone or IPR alone or Mix induced toxicity on spermatogenesis resulting in a decrease of round spermatids (the precursors of spermatozoa). These results strongly suggest that, even at these low concentrations, the effects of IPR and of CBZ are not solely dependent on their respective anti-androgenic and androgen-like effects and should involve several mechanisms of action.

Tradescantia as a biomonitor for pesticide genotoxicity evaluation of iprodione, carbaryl, dimethoate and 4,4'-DDE.

There is a current tendency to develop and apply environmentally friendly techniques that meet the requirements of green analytical chemistry as an alternative to conventional analytical methods. For toxicity evaluation, these alternatives may be found in bioassays such as Tradescantia. This technique, developed in the 1980s, is highly sensitive to evaluate environmental mutagens, simple and cheap. In this paper, the sensibility of both the Tradescantia micronucleus bioassay (Trad-MCN) and the Tradescantia stamen hair bioassay (Trad-SH) were studied for carbaryl, dimethoate and iprodione, common agricultural and domestic pesticides that are currently used in Chile, which have never been tested with such bioassays. Biomonitor exposures were performed by capillary absorption for each individual pesticide over a wide range of concentrations, from maximum residue limits (trace levels) up to the application dose in agricultural fields. In addition, the organochloride 4,4'-DDE was included but only in the concentration range from 0.01mgL-1 to 1mgL-1, mimicking residue concentrations since it is not a commercial product but, rather, the main breakdown product of the persistent organochloride pesticide 4,4-DDT, whose use was discontinued in Chile in the 1980s. The Trad-MCN bioassay revealed a significant increase in micronucleus frequency at the early tetrads of meiotic pollen mother cells of the biomonitor Tradescantia pallida var. purpurea, induced by 4,4'-DDE (for 1mgL-1), dimethoate (for 40mgL-1, 200mgL-1, 400mg/L-1) and carbaryl (for 889mgL-1). Iprodione did not generate any significant change at the tested concentration. Meanwhile, the Trad-SH bioassay was carried out by analysis of the phenotype variations of the stamen hair cells of the Tradescantia clone KU-20 for the same pesticides and doses. This bioassay was not sufficiently sensitive for toxicity evaluation of most of the pesticides tested, with exception of dimethoate in low doses (2 and 5mg/L-1).

Ex vivo assessment of testicular toxicity induced by carbendazim and iprodione, alone or in a mixture.

To measure the testicular toxicity of two fungicides (carbendazim and iprodione), alone or in a mixture, we used a rat ex vivo model of seminiferous tubules, greatly reducing the number of rodents used, in accordance with the 3R rule (Replacement, Reduction, and Refinement). This model allows the representation of puberty, a critical life period with regard to endocrine disruptors. The cellular modifications were followed for three weeks through transcriptomic and proteomic profiling analysis. A quantitative and comparative method was developed to estimate how known pathways were disturbed by each substance. This pathway-driven analysis revealed a strong alteration of steroidogenesis and an impairment of meiosis in all cases, albeit the initial molecular events were different for both substances. The ex vivo cytogenetic analysis confirmed that both fungicides alter the course of the first meiotic prophase. In addition, the mixture of both substances triggered effects greater than the sum of their cumulative effects and compromised future sperm motility after a shorter time of exposure compared with the fungicides tested separately. The alliance of an ex vivo culture with "omics" strategies complemented with a physiological examination is a powerful combination of tools for testing substances, separately or in a mixture, for their testicular toxicity. In particular, proteomics allowed the identification of systematically differentially expressed proteins in the secretomes of exposed cultures, such as FUCO and PEBP1, two proteins linked with the motility and fertilizing ability of spermatozoa, respectively. These proteins may be potential biomarkers of testicular dysfunction and infertility.

Effects of Fungicide and Adjuvant Sprays on Nesting Behavior in Two Managed Solitary Bees, Osmia lignaria and Megachile rotundata.

There is a growing body of empirical evidence showing that wild and managed bees are negatively impacted by various pesticides that are applied in agroecosystems around the world. The lethal and sublethal effects of two widely used fungicides and one adjuvant were assessed in cage studies in California on blue orchard bees, Osmia lignaria, and in cage studies in Utah on alfalfa leafcutting bees, Megachile rotundata. The fungicides tested were Rovral 4F (iprodione) and Pristine (mixture of pyraclostrobin + boscalid), and the adjuvant tested was N-90, a non-ionic wetting agent (90% polyethoxylated nonylphenol) added to certain tank mixtures of fungicides to improve the distribution and contact of sprays to plants. In separate trials, we erected screened cages and released 20 paint-marked females plus 30-50 males per cage to document the behavior of nesting bees under treated and control conditions. For all females in each cage, we recorded pollen-collecting trip times, nest substrate-collecting trip times (i.e., mud for O. lignaria and cut leaf pieces for M. rotundata), cell production rate, and the number of attempts each female made to enter her own or to enter other nest entrances upon returning from a foraging trip. No lethal effects of treatments were observed on adults, nor were there effects on time spent foraging for pollen and nest substrates and on cell production rate. However, Rovral 4F, Pristine, and N-90 disrupted the nest recognition abilities of O. lignaria females. Pristine, N-90, and Pristine + N-90 disrupted nest recognition ability of M. rotundata females. Electroantennogram responses of antennae of O. lignaria females maintained in the laboratory did not differ significantly between the fungicide-exposed and control bees. Our results provide the first empirical evidence that two commonly used fungicides and a non-ionic adjuvant can disrupt nest recognition in two managed solitary bee species.

Ultraviolet-vis degradation of iprodione and estimation of the acute toxicity of its photodegradation products.

The UV-vis photodegradation of iprodione in water was investigated with a high pressure mercury lamp photoreactor. Five photoproducts of iprodione were characterized by LC-HR-MS/MS and isotopic labeling; none of them has been reported in previous studies. Three of them result from the elimination of one or two chlorine atoms followed by hydroxy or hydrogen addition while the two others are cyclic isomers of iprodione. An ICR mass spectrometer was used for by-products identification; concentrations of photoproducts were estimated with a triple quadrupole instrument, using iprodione-D5 as an internal standard. Phototransformation mechanisms were postulated to rationalize photoproducts formation. In silico QSAR toxicity predictions were conducted with the Toxicity Estimation Software Tool (T.E.S.T.) considering oral rat LD50, mutagenicity and developmental toxicity. Low oral rat LD50 values of 350 mg/kg and 759 mg/kg were predicted for cyclic isomers of iprodione, compared to that of the parent molecule (2776 mg/kg). Toxicity estimations exhibited that all the iprodione photoproducts could be mutagenic while the parent compound is not. In vitro assays on Vibrio fischeri were achieved on both irradiated and non-irradiated aqueous solutions of iprodione and on HPLC fractions containing isolated photoproducts. Phenolic photoproducts were shown to be mainly responsible for toxicity enhancement with EC50 values of 0.3 and 0.5 ppm, for the bi- and mono-phenolic compounds issued from chlorine elimination.

Short-term effects of combined iprodione and vermicompost applications on soil microbial community structure.

The use of compost amendments to bioremediate potential organic pollutants in agricultural soils has recently become an increasingly important field of research. Although several fungicides have been extensively used to control a wide range of soil-borne fungal diseases, little is known about the impact of applying these pesticides on the structure and function of microbial communities in soils amended with vermicompost. The aim of this study was to evaluate the effect of a combined treatment of iprodione and vermicompost on soil microbiological parameters under laboratory conditions. The study was carried out on agricultural and grassland soils to identify the effect of iprodione application at field rate (FR) and 10-times FR (10 FR) with and without vermicompost (VCH) on iprodione breakdown, fluorescein diacetate activity (FDA), total fatty acid methyl ester (FAME) profiles, total protein content, and protein profiles by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Our results indicate that the addition of vermicompost decreased the iprodione breakdown at days 30 and 60 in non-sterilised agricultural soil and at 60 days in sterilised and non-sterilised grassland soil. Independent of vermicompost amended treatments, iprodione was found to mainly alter microbial communities after 30 days of incubation. On day 30, separation between communities treated with iprodione 10 FR and iprodione 10FR + VCH treatments were well defined in both agricultural and grassland soils. Within each soil type, our results showed no difference in the total protein content. However, the protein content in the grassland soil was clearly higher than in the agricultural soil. SDS-PAGE gels revealed that the treatments applied to the agricultural soil using iprodione at the highest dosages (iprodione 10FR and iprodione 10FR+VCH) resulted in an alteration of the band pattern. In conclusion, the experiments revealed that the addition of vermicompost may decrease the breakdown of iprodione in soils. Furthermore, elevated dosages of iprodione may potentially affect the microbial community structure and diversity of the soil, which may lead to the deterioration of soil quality and fertility.

AMP-activated protein kinase mediates effects of oxidative stress on embryo gene expression in a mouse model of diabetic embryopathy.

AIMS/HYPOTHESIS: Neural tube defects (NTDs) are a common malformation associated with diabetic embryopathy. Maternal hyperglycaemia-induced oxidative stress inhibits the expression of Pax3, a gene that is essential for neural tube closure, and increases the incidence of NTDs. Because oxidative stress can stimulate AMP-activated kinase (AMPK) activity, and AMPK can regulate gene transcription, we hypothesised that increased AMPK activity would mediate the adverse effects of maternal hyperglycaemia-induced oxidative stress on Pax3 expression and NTDs. METHODS: Pregnant mice were made transiently hyperglycaemic by glucose injection, or hypoxic by housing in a hypoxic chamber, or were treated with antimycin A to induce oxidative stress, and AMPK activity in the embryos was assayed. The effects of stimulating AMPK activity with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) on Pax3 expression and NTDs were determined. Vitamin E or glutathione ethyl ester was used to reduce oxidative stress, and compound C was used to inhibit AMPK activation. Murine embryonic stem cells were employed as an in vitro model to study the effects of oxidative stress on AMPK activity and the effects of AMPK stimulation on Pax3 expression. RESULTS: Maternal hyperglycaemia stimulated AMPK activity, and stimulation of AMPK with AICAR inhibited Pax3 expression (in vivo and in vitro) and increased NTDs (in vivo). Stimulation of AMPK by hyperglycaemia, hypoxia or antimycin A was inhibited by antioxidants. The AMPK inhibitor compound C blocked the effects of hyperglycaemia or AA on Pax3 expression and NTDs. CONCLUSIONS/INTERPRETATION: Stimulation of AMPK in embryos during a diabetic pregnancy mediates the effects of hyperglycaemia-induced oxidative stress to disturb the expression of the critical Pax3 gene, thereby causing NTDs.

Cumulative and antagonistic effects of a mixture of the antiandrogens vinclozolin and iprodione in the pubertal male rat.

Vinclozolin and iprodione are dicarboximide fungicides that display antiandrogenic effects in the male rat, which suggests that a mixture would lead to cumulative effects on androgen-sensitive end points. Iprodione is a steroid synthesis inhibitor, but androgen receptor antagonist activity, which is displayed by vinclozolin, has not been fully evaluated. Here, we demonstrate that iprodione binds to the human androgen receptor (IC(50) = 86.0 microM), reduces androgen-dependent gene expression, and reduces androgen-sensitive tissue weights in castrated male rats (Hershberger assay). Since vinclozolin and iprodione affect common targets in the pubertal male rat, we tested the hypothesis that a mixture would have cumulative antiandrogenic effects. An iprodione dose, that does not significantly affect androgen-dependent morphological end points, was combined with vinclozolin doses (2 x 5 factorial design). Sprague-Dawley rats were dosed by gavage with vinclozolin at 0, 10, 30, 60, and 100 mg/kg/day with and without 50 mg iprodione/kg/day from postnatal day (PND) 23 to 55-57 (n = 8 per group). The age at puberty (preputial separation [PPS]), organ weights, serum hormones, and ex vivo testis steroid hormone production were measured. Vinclozolin delayed PPS, reduced androgen-sensitive organ weights, and increased serum testosterone. The addition of iprodione enhanced the vinclozolin inhibition of PPS (PND 47.5 vs.49.1; two-way ANOVA: iprodione main effect p = 0.0002). The dose response for several reproductive and nonreproductive organ weights was affected in a cumulative manner. In contrast, iprodione antagonized the vinclozolin-induced increase in serum testosterone. These results demonstrate that these fungicides interact on common targets in a tissue-specific manner when coadministered to the pubertal male rat.

Toxicity of fungicides with different modes of action to Cladobotryum dendroides and Agaricus bisporus.

Isolates of Cladobotryum dendroides from Serbian mushroom farms and Agaricus bisporus F56 were tested for sensitivity to selected fungicides in vitro. Chlorothalonil was the most toxic fungicide to C. dendroides isolates (EC(50) values were below 1.68 mg L(-1)). Trifloxystrobin and kresoxim-methyl were not effective in growth inhibition of C. dendroides isolates (EC(50) values exceeded 300 mg L(-1)). Metalaxyl-M+mancozeb was the most toxic fungicide to strain F56 of A. bisporus, and iprodione the least toxic. The fungicide selectivity indexes for both C. dendroides and A. bisporus indicated that iprodione, chlorothalonil, captan and metalaxyl-M+mancozeb had satisfactory selective fungitoxicity. Iprodione had the best selectivity to both the pathogen and the host, although inferior than prochloraz manganese and carbendazim, fungicides officially recommended for mushroom cultivation in European Union (EU) countries.