Genetic dissection of complex traits using hierarchical biological knowledge.

Despite the growing constellation of genetic loci linked to common traits, these loci have yet to account for most heritable variation, and most act through poorly understood mechanisms. Recent machine learning (ML) systems have used hierarchical biological knowledge to associate genetic mutations with phenotypic outcomes, yielding substantial predictive power and mechanistic insight. Here, we use an ontology-guided ML system to map single nucleotide variants (SNVs) focusing on 6 classic phenotypic traits in natural yeast populations. The 29 identified loci are largely novel and account for ~17% of the phenotypic variance, versus <3% for standard genetic analysis. Representative results show that sensitivity to hydroxyurea is linked to SNVs in two alternative purine biosynthesis pathways, and that sensitivity to copper arises through failure to detoxify reactive oxygen species in fatty acid metabolism. This work demonstrates a knowledge-based approach to amplifying and interpreting signals in population genetic studies.

Transcriptome analysis of Apis mellifera under benomyl stress to discriminate the gene expression in response to development and immune systems.

The health and safety of the honeybees are seriously threatened due to the abuse of chemical pesticides in modern agriculture and apiculture. In this study, the RNA Seq approach was used to assess the effects of the honeybees treated with benomyl. The results showed that there were a total of 11,902 differentially expressed genes (DEGs). Among them, 5,759 DEGs were up-regulated and involved in the functions of immunity, detoxification, biological metabolism, and regulation. The DEGs were clustered in the GO terms of epidermal structure and response to external stimuli, and most of the DEGs were enriched in 15 pathways, such as light conduction, MAPK, calcium ion pathway, and so on. Moreover, the pathway of the toll signal transduction was activated. The data investigated that the expression of functional genes involved in the growth, development, foraging, and immunity of honeybees were significantly affected by benomyl stress, which would seriously threaten the health of the honeybees. This study provided a theoretical basis for revealing the response mechanism of honeybees to pesticides stress.

Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease.

Parkinson disease (PD) is a neurodegenerative disorder particularly characterized by the loss of dopaminergic neurons in the substantia nigra. Pesticide exposure has been associated with PD occurrence, and we previously reported that the fungicide benomyl interferes with several cellular processes potentially relevant to PD pathogenesis. Here we propose that benomyl, via its bioactivated thiocarbamate sulfoxide metabolite, inhibits aldehyde dehydrogenase (ALDH), leading to accumulation of the reactive dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), preferential degeneration of dopaminergic neurons, and development of PD. This hypothesis is supported by multiple lines of evidence. (i) We previously showed in mice the metabolism of benomyl to S-methyl N-butylthiocarbamate sulfoxide, which inhibits ALDH at nanomolar levels. We report here that benomyl exposure in primary mesencephalic neurons (ii) inhibits ALDH and (iii) alters dopamine homeostasis. It induces selective dopaminergic neuronal damage (iv) in vitro in primary mesencephalic cultures and (v) in vivo in a zebrafish system. (vi) In vitro cell loss was attenuated by reducing DOPAL formation. (vii) In our epidemiology study, higher exposure to benomyl was associated with increased PD risk. This ALDH model for PD etiology may help explain the selective vulnerability of dopaminergic neurons in PD and provide a potential mechanism through which environmental toxicants contribute to PD pathogenesis.

Benomyl treatment decreases fecundity of ant queens.

Methyl benzimidazole carbamate fungicides, including benomyl, are widely used in agriculture, and to eliminate entomopathogenic infections. We treated queens of Myrmica rubra (Hymenoptera:Formicidae) infected or not by Rickia wasmannii (Laboulbeniales:Laboulbeniaceae) with benomyl, 1mg/ml p.o. for six weeks. Benomyl did not treat the infection, and the treatment alone caused strong decrease in the fecundity of control healthy queens from 18.0±8.4 to 3.7±5.2eggs per healthy queen. This is the first evidence on severe adverse effects of methyl benzimidazole carbamate fungicide on the fecundity of insects, which might be responsible for altered species composition of ant assemblages in the cultural landscape.

Benomyl, aldehyde dehydrogenase, DOPAL, and the catecholaldehyde hypothesis for the pathogenesis of Parkinson's disease.

The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is detoxified mainly by aldehyde dehydrogenase (ALDH). We find that the fungicide benomyl potently and rapidly inhibits ALDH and builds up DOPAL in vivo in mouse striatum and in vitro in PC12 cells and human cultured fibroblasts and glial cells. The in vivo results resemble those noted previously with knockouts of the genes encoding ALDH1A1 and 2, a mouse model of aging-related Parkinson's disease (PD). Exposure to pesticides that inhibit ALDH may therefore increase PD risk via DOPAL buildup. This study lends support to the "catecholaldehyde hypothesis" that the autotoxic dopamine metabolite DOPAL plays a pathogenic role in PD.

Benomyl induced oxidative stress related DNA damage and apoptosis in H9c2 cardiomyoblast cells.

Benomyl, benzimidazole group pesticide, has been prohibited in Europe and USA since 2003 due to its toxic effects and it has been still determined as food and environmental contaminant. In the present study, the toxic effect mechanisms of benomyl were evaluated in rat cardiomyoblast (H9c2) cells. Cytotoxicity was determined by MTT and NRU assay and, oxidative stress potential was evaluated by reactive oxygen species (ROS) production and glutathione levels. DNA damage was assessed by alkaline comet assay. Relative expressions of apoptosis related genes were evaluated; furthermore, NF-κB and JNK protein levels were determined. At 4 µM concentration (at which cell viability was >70%), benomyl increased 2-fold of ROS production level and 2-fold of apoptosis as well as DNA damage. Benomyl down-regulated miR21, TNF-α and Akt1 ≥ 48.75 and ≥ 97.90; respectively. PTEN, JNK and NF-κB expressions were upregulated. The dramatic changes in JNK and NF-κB expression levels were not observed in protein levels. These findings showed the oxidative stress related DNA damage and apoptosis in cardiomyoblast cells exposed to benomyl. However, further mechanistic and in vivo studies are needed to understand the cardiotoxic effects of benomyl and benzimidazol fungucides.

The budding yeast protein Sum1 functions independently of its binding partners Hst1 and Sir2 histone deacetylases to regulate microtubule assembly.

The budding yeast protein Sum1 is a transcription factor that associates with the histone deacetylase Hst1p or, in its absence, with Sir2p to form repressed chromatin. In this study, SUM1 has been identified as an allele-specific dosage suppressor of mutations in the major alpha-tubulin-coding gene TUB1. When cloned in a 2mu vector, SUM1 suppressed the cold-sensitive and benomyl-hypersensitive phenotypes associated with the tub1-1 mutation. The suppression was Hst1p- and Sir2p-independent, suggesting that it was not mediated by deacetylation events associated with Sum1p when it functions along with its known partner histone deacetylases. This protein was confined to the nucleus, but did not colocalize with the microtubules nor did it bind to alpha- or beta-tubulin. Cells deleted of SUM1 showed hypersensitivity to benomyl and cold-sensitive growth, phenotypes exhibited by mutants defective in microtubule function and cytoskeletal defects. These observations suggest that Sum1p is a novel regulator of microtubule function. We propose that as a dosage suppressor, Sum1p promotes the formation of microtubules by increasing the availability of the alphabeta-heterodimer containing the mutant alpha-tubulin subunit.

Rhinitis associated with pesticide exposure among commercial pesticide applicators in the Agricultural Health Study.

OBJECTIVES: Rhinitis is common, but the risk factors are not well described. To investigate the association between current rhinitis and pesticide use, we used data from 2245 Iowa commercial pesticide applicators in the Agricultural Health Study. METHODS: Using logistic regression models adjusted for age, education and growing up on a farm, we evaluated the association between current rhinitis and 34 pesticides used in the past year. RESULTS: 74% of commercial pesticide applicators reported at least one episode of rhinitis in the past year (current rhinitis). Five pesticides used in the past year were significantly positively associated with current rhinitis: the herbicides 2,4-D, glyphosate and petroleum oil, the insecticide diazinon and the fungicide benomyl. The association for 2,4-D and glyphosate was limited to individuals who used both in the past year (OR 1.42, 95% CI 1.14 to 1.77). Both petroleum oil and diazinon showed consistent evidence of an association with rhinitis, based on both current use and exposure-response models. We saw no evidence of confounding by common agricultural rhinitis triggers such as handling grain or hay. CONCLUSIONS: Exposure to pesticides may increase the risk of rhinitis.

Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo.

Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up-regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 x 10(-6), 40 x 10(-6) and 80 x 10(-6) M of benomyl-treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase-based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 x 10(-6) M concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds.

A novel yeast-based tool to detect mutagenic and recombinogenic effects simultaneously.

In this work, we describe a new yeast-based assay to allow efficient detection of a comprehensive spectrum of genotoxicity events. The constructed diploid Saccharomyces cerevisiae strain allows the simultaneous monitoring of forward mutations, mitotic recombination events and chromosome loss or non-disjunction by direct selection in an easy and highly reproducible approach. The strain contains a DNA module consisting of a single functional copy of the URA3 gene and the kanMX4 gene inserted at the ADE2 locus on the right arm of chromosome XV. The changes of the genotype within the marker region were primarily selected on 5-fluoroorotic acid (5-FOA) agar plates. Further simple phenotypic tests of the 5-FOA-resistant ura3 clones make it possible to analyze the genetic configuration in detail (e.g. point mutations in URA3, gene conversion, crossing-over and chromosome loss). We demonstrate the successful application of our test system by studying the effects of well-known genotoxic agents (UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, aniline and benomyl). We found that the various agents induced mutations and recombination events with different relative frequencies. The integration of the module has generated a hot spot region of mutation and recombination at the borders of the artificially integrated URA3 kanMX4 cassette, which makes the system more sensitive towards DNA-damaging agents. Unlike other test systems, our S. cerevisiae strain is capable to detect a mutagenic effect caused by aniline.

Effects of three pesticides on the avoidance behavior of earthworms in laboratory tests performed under temperate and tropical conditions.

Little research has been performed on the impact of pesticides on earthworms under tropical conditions. Taking into consideration the often-limited resources in tropical countries, simple screening tests are needed. Therefore, it was investigated whether three pesticides relevant for the Brazilian Amazon (benomyl, carbendazim, lambda-cyhalothrin) affect the avoidance behavior of the earthworm Eisenia fetida. The tests were performed for two days according to ISO guideline 17512 but were adapted to tropical conditions (i.e. test substrate, test organism and temperature). The results indicate that this test gives reproducible and reliable results. Toxicity values (NOEC, EC50) are lower than those determined in 14 day-acute mortality tests and are approximately in the same range such as those found in 56 day-chronic reproduction tests with the same earthworm species, which were performed in parallel. Therefore, the use of the earthworm avoidance tests is recommended as a screening tool for the risk assessment of pesticides.

Avoidance tests with earthworms and springtails: defining the minimum exposure time to observe a significant response.

Based on the ability of organisms to avoid contaminated soils, avoidance tests have a great potential as early screening tools in lower tier levels of ERA schemes. Aiming at their standardization, the definition of the minimum exposure time necessary to observe an avoidance response to a contaminant is needed. To fill this gap, avoidance tests with earthworms (Eisenia andrei) and springtails (Folsomia candida), comparing distinct time periods (from 1-7 to 1-14 days, respectively), were performed using the artificial OECD soil and reference chemicals for each test organism. Results showed that for both organisms a clear response within 24 h of exposure can be obtained. This rapid response enhances the utility of the test for "on site" analysis to evaluate contaminated sites.

Formulation factors that can reduce the formation of the phytotoxic impurity, N,N'-dibutylurea, from benomyl.

Fungicide benomyl is easily decomposed to carbendazim (MBC) and butyl isocyanate (BIC) in formulation, BIC is further hydrolyzed to butylamine. The BIC also reacts with butylamine to form N,N'-dibutylurea (DBU), a phytotoxic compound. The purpose of this study was to investigate the effects of selected additives and the manufacturing method of benomyl water dispersible granules (WG) on reducing DBU content in benomyl formulations. The manufacturing methods studied were granulation by extrusion, fluid bed spray, and spray dry. For the extrusion method, each benomyl powder formulation was homogenized by kneading with 20% v/w of 95% ethanol instead of water. After granulation, the percentages of the active ingredient benomyl and its degradation product carbendazim in each formulation were determined. For the fluid bed spray method, two formulations of wettable powders were formed. The first sample was granulated using 5% Na(2)SO(4) as the binder solution; the second sample used 2% urea. Changes in the active ingredient content after granulation were determined for each sample. For the spray dry method, four basic formulations of 70% benomyl, 5% sodium dodecyl sulfate (SDS) and 10% or 20% sodium sulfate were prepared, to study the effects of HMTA, urea and dispersant on reducing DBU formation in formulation. The DBU content of each formulation was measured for the fresh samples and after 1 year of storage. The results showed that urea had a stabilizing effect on benomyl, and reduced DBU formation. BIC increased benomyl yield during manufacturing, which reduced DBU content in fresh samples but allowed a greater potential for future DBU formation since it did not stabilize the extra benomyl. HMTA was found to reduce DBU in both aqueous BIC and prepared formulations. The study discusses how each of the selected constituents affected DBU formation and how commercial formulations can be improved to reduce DBU formation. From this study, it is clear that a safer benomyl formulation can be developed.

Potential teratogenic effects of benomyl in rat embryos cultured in vitro.

A possible association between environmental exposure to benomyl and anophthalmia has been suggested. The aim of the present study was to investigate potential teratogenic effects of benomyl using the 9.5 day rat embryo culture method using rat and human serum. Explanted rat embryos were cultured in rat serum (n=121) or human serum (n=90) with differing concentrations of benomyl [170 nM to 13.6 microM], dissolved in ethanol (0.136%), at least five embryos per concentration being cultured. In addition, 18 embryos were cultured in both human and rat serum with the equivalent concentration of ethanol to act as a vehicle control. The cultured embryos were then measured and scored for growth and differentiation by two blinded observers. Embryotoxic effects were considered to be demonstrated by a decrease in parameters of growth such as crown rump length, yolk sac diameter and protein content, whereas embryopathic effects were considered to be those causing a decease in parameters of differentiation such as morphological score, somite number and optic development. Benomyl [> or =5 microM] produced a significant concentration dependent deterioration in morphological score, somite number and optic development. Gross toxic effects were noticed at concentrations of >12 microM in rat serum and >10microM in human serum as indicated by a significant effect on parameters measuring size (crown rump length; yolk sac diameter and protein content). This study provides evidence that benomyl is a potential developmental toxicant, affecting many parameters of differentiation, including optic development at levels below those that could be considered embryotoxic.

Quantitative kinetics of damage and recovery of cytoskeletal structure by means of image analysis.

The cytoskeleton is a network of proteins which structurally and dynamically organise the cytoplasm of living cells. Microtubules are among its constituents. Morphological alterations of microtubules are related to functional impairment. Therefore cytoskeletal morphology is a valuable indicator of cell injury and functionality. This paper focuses on the comparison between normal and altered cytoskeletal microtubules by means of image analysis and classification with the aim of replacing visual assessment. Morphology has been quantified by the extraction of some descriptors yielded by spatial differentiation, fractal analysis and Fourier analysis followed by non-linear filtering. The principal component analysis of these descriptors has led to image recognition and has been applied to hepatocytes and fibroblasts exposed to some xenobiotics. In the case of hepatocytes, images have been ranked according to the severity of cytoskeletal damage, a dose-response relation has been derived from the regression of the first principal component and the percentage of structural recovery after exposure has been estimated.

Avoidance behaviour of Enchytraeus albidus: effects of benomyl, carbendazim, phenmedipham and different soil types.

Enchytraeids are typical inhabitants of many soils, contributing to vital processes of this environmental compartment. Indirectly they are involved in regulating the degradation of organic matter, as well as improving the pore structure of the soil. Due to their behaviour, they are able to avoid unfavourable environmental conditions. Avoidance tests with enchytraeids, initially developed with earthworms by several authors, are quick and easy to perform. With these tests a first assessment of the toxicity of a (contaminated or spiked) soil is possible in just 48 h by using the reaction of the enchytraeids as measurement endpoint. In this period of time the organisms can choose between the control soil and the other soil (a contaminated or spiked or another soil with different physico-chemical properties). In the tests reported here, the enchytraeids were exposed to control soils spiked with the fungicides Benomyl and Carbendazim and the herbicide Phenmedipham. Several chemical concentrations were tested in order to evaluate the avoidance behaviour to toxic substances. In fact, often these short-term screening tests gave results showing avoidance at concentrations in a range similar to the acute test results but, higher than in chronic tests. Further tests are needed to decide whether the results gained in this study can be extrapolated to other chemicals. It is proposed to standardize the Enchytraeid Avoidance Test as it is currently done for the Earthworm Avoidance Test by the International Standard Organization (ISO).

Influence of the carbamate fungicide benomyl on the gene expression and activity of aromatase in the human breast carcinoma cell line MCF-7.

The carbamate fungicide benomyl reportedly inhibited the growth of the human breast cancer cell line MCF-7 by inducing apoptosis. However, influence of benomyl on the expression and activity of aromatase of MCF-7 cells remains to be examined, since benomyl was identified as an endocrine disruptor. We here confirmed through cell cycle analysis and immunofluorescence staining that benomyl damaged microtubules and caused apoptosis. We also found that benomyl inhibited histone deacetylase (HDAC) 1 and accumulated acetylated histone H3 in MCF-7 cells. Additionally, benomyl enhanced the levels of aromatase protein and mRNA, albeit at high concentrations. It is thus likely that benomyl enhanced the promoter activity of the aromatase gene via acetylation of histone H3 as does the HDAC inhibitor Vorinostat. In conclusion, benomyl remains to be a risk factor as an endocrine disruptor for breast cancer.

Deleterious effects of benomyl and carbendazim on human placental trophoblast cells.

Benomyl and carbendazim are benzimidazole fungicides that are used throughout the world against a wide range of fungal diseases of agricultural products. There is as yet little information regarding the toxicity of benzimidazole fungicides to human placenta. In this study, we utilized human placental trophoblast cell line HTR-8/SVneo (HTR-8) to access the toxic effects of benomyl and carbendazim. Our data showed that these two fungicides decreased cell viability and the percentages of cells in G0/G1 phase, as well as induced apoptosis of HTR-8 cells. The invasion and migration of HTR-8 cells were significantly inhibited by benomyl and carbendazim. We further found that benomyl and carbendazim altered the expression of protease systems (MMPs/TIPMs and uPA/PAI-1) and adhesion molecules (integrin α5 and ß1) in HTR-8 cells. Our present study firstly shows the deleterious effects of benomyl and carbendazim on placental cells and suggests a potential risk of benzimidazole fungicides to human reproduction.

Histopathology of the male reproductive system induced by the fungicide benomyl.

Benomyl is an effective fungicide that has been in use for many years. This chemical and its primary metabolite, carbendazim, are microtubule poisons that are relatively nontoxic to all mammalian organs, except for the male reproductive system. Its primary effects, at moderate to low dosages, are on the testis, where it causes sloughing of germ cells in a stage-dependent manner. Sloughing is caused by the effects of the chemical on microtubules and intermediate filaments of the Sertoli cell. These effects spread to dividing germ cells and also lead to abnormal development of the head of elongating spermatids. At higher dosages, it causes occlusion of the efferent ducts, blocking passage of sperm from the rete testis to epididymis. The mechanism of occlusion appears to be related to fluid reabsorption, sperm stasis, followed by leukocyte chemotaxis, sperm granulomas, fibrosis and often the formation of abnormal microcanals. The occlusion results in a rapid swelling of the testis and ultimately seminiferous tubular atrophy and infertility. In conclusion, studies that reveal long term testicular atrophy following chronic or subchronic exposure to a toxicant should be re-examined for histopathological lesions in the efferent ductules and head of the epididymis. Lesions in the male track that cause blockage may induce permanent testicular damage and a decrease in sperm production.

Genotoxic effects of some systemic pesticides: in vivo chromosomal aberrations in bone marrow cells in rats.

The genotoxic effects of five pesticides (benomyl, 2,4-D, dimecron, monocrotophos, and vitavax) were evaluated in the rat bone marrow cytogenetic assay. The spectrum of aberrations observed included chromatid breaks, chromatid fragments, ring chromosomes, dicentric chromosomes, and chromosome fragments. It was observed that 2,4-D, dimecron, and vitavax were clastogenic, but the results obtained with benomyl and monocrotophos were equivocal.