Benomyl-induced development and cardiac toxicity in zebrafish embryos.

Benomyl is a highly effective broad-spectrum fungicide widely used worldwide to control vegetable, fruit, and oil crop diseases. However, the mechanism of its toxicity to aquatic organisms and humans remains unknown. In this study, zebrafish were used to determine the toxicity of benomyl. It was found to be highly toxic, with a 72-h post-fertilization (hpf) lethal concentration 50 (LC50) of 1.454 mg/L. Benomyl induced severe developmental toxicity, including shorter body length, slower heart rate, and a reduced yolk absorption rate. Benomyl also increased oxidative stress in zebrafish, especially in the heart and head, as well as increasing malondialdehyde (MDA) content and decreasing catalase (CAT) and superoxide dismutase (SOD) activities. This indicates that benomyl induced reactive oxygen species (ROS) production and cell membrane peroxidation in vivo. Acridine orange (AO) staining and apoptosis factor detection further indicated that benomyl induced apoptosis in zebrafish. Overall, these findings demonstrate that benomyl disrupts cellular homeostasis by activating oxidative stress in zebrafish, resulting in an imbalance of cardiac development-related gene expression and apoptosis, which causes severe developmental toxicity and cardiac dysfunction. This study evaluated the in vivo toxicity of benomyl, which is a potential threat to aquatic organisms and humans. Possible toxicity mechanisms are explored, providing a valuable reference for the safe use of benomyl.

The TOG protein Stu2 is regulated by acetylation.

Stu2 in S. cerevisiae is a member of the XMAP215/Dis1/CKAP5/ch-TOG family of MAPs and has multiple functions in controlling microtubules, including microtubule polymerization, microtubule depolymerization, linking chromosomes to the kinetochore, and assembly of γ-TuSCs at the SPB. Whereas phosphorylation has been shown to be critical for Stu2 localization at the kinetochore, other regulatory mechanisms that control Stu2 function are still poorly understood. Here, we show that a novel form of Stu2 regulation occurs through the acetylation of three lysine residues at K252, K469, and K870, which are located in three distinct domains of Stu2. Alteration of acetylation through acetyl-mimetic and acetyl-blocking mutations did not impact the essential function of Stu2. Instead, these mutations lead to a decrease in chromosome stability, as well as changes in resistance to the microtubule depolymerization drug, benomyl. In agreement with our in silico modeling, several acetylation-mimetic mutants displayed increased interactions with γ-tubulin. Taken together, these data suggest that Stu2 acetylation can govern multiple Stu2 functions, including chromosome stability and interactions at the SPB.

The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa.

γ-Tubulin ring complexes (γ-TuRC) mediate nucleation and anchorage of microtubules (MTs) to microtubule organizing centers (MTOCs). In fungi, the spindle pole body (SPB) is the functional equivalent of the centrosome, which is the main MTOC. In addition, non-centrosomal MTOCs (ncMTOCs) contribute to MT formation in some fungi like Schizosaccharomyces pombe and Aspergillus nidulans. In A. nidulans, MTOCs are anchored at septa (sMTOC) and share components of the outer plaque of the SPB. Here we show that the Neurospora crassa SPB is embedded in the nuclear envelope, with the γ-TuRC targeting proteins PCP-1Pcp1/PcpA located at the inner plaque and APS-2Mto1/ApsB located at the outer plaque of the SPB. PCP-1 was a specific component of nuclear MTOCs, while APS-2 was also present at the septal pore. Although γ-tubulin was only detected at the nucleus, spontaneous MT nucleation occurred in the apical and subapical cytoplasm during recovery from benomyl-induced MT depolymerization experiments. There was no evidence for MT nucleation at septa. However, without benomyl treatment MT plus-ends were organized in the septal pore through MTB-3EB1. Those septal MT plus ends polymerized MTs from septa in interphase cells Thus we conclude that the SPB is the only MT nucleation site in N. crassa, but the septal pore aids the MT network arrangement through the anchorage of the MT plus-ends through a pseudo-MTOC.

A distinct P-body-like granule is induced in response to the disruption of microtubule integrity in Saccharomyces cerevisiae.

The Processing-body is a conserved membraneless organelle that has been implicated in the storage and/or decay of mRNAs. Although Processing-bodies have been shown to be induced by a variety of conditions, the mechanisms controlling their assembly and their precise physiological roles in eukaryotic cells are still being worked out. In this study, we find that a distinct subtype of Processing-body is induced in response to conditions that disrupt microtubule integrity in the budding yeast, Saccharomyces cerevisiae. For example, treatment with the microtubule-destabilizing agent, benomyl, led to the induction of these novel ribonucleoprotein granules. A link to microtubules had been noted previously and the observations here extend our understanding by demonstrating that the induced foci differ from traditional P-bodies in a number of significant ways. These include differences in overall granule morphology, protein composition, and the manner in which their induction is regulated. Of particular note, several key Processing-body constituents are absent from these benomyl-induced granules, including the Pat1 protein that is normally required for efficient Processing-body assembly. However, these novel ribonucleoprotein structures still contain many known Processing-body proteins and exhibit similar hallmarks of a liquid-like compartment. In all, the data suggest that the disruption of microtubule integrity leads to the formation of a novel type of Processing-body granule that may have distinct biological activities in the cell. Future work will aim to identify the biological activities of these benomyl-induced granules and to determine, in turn, whether these Processing-body-like granules have any role in the regulation of microtubule dynamics.

Suppression of arbuscular mycorrhizal fungi increased lead uptake in maize leaves and loss via surface runoff and interflow from polluted farmland.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous in farmland. But the knowledge on AMF impact on lead (Pb) migration in farmland is limited. A field experiment was conducted in the rainy season (May-October) for two years in a Pb-polluted farmland. Benomyl was used to specifically suppress the native AMF growth in the farmland. The effect of benomyl-induced AMF suppression on the Pb uptake in maize, and Pb loss via surface runoff and interflows (20 cm and 40 cm depth) from the farmland was investigated. The benomyl significantly inhibited the AMF growth, resulting in decreases in the colonization rate, spore number, and contents of total and easily extractable glomalin-related soil protein (GRSP); and promoted the Pb migration into maize shoots and mainly enriched in leaves. The particulate Pb accounted for 83.2%-90.6% of Pb loss via surface runoff, while the proportion of particulate Pb loss via interflow was decreased and the proportion of dissolved Pb loss increased with the increase of soil depth. The AMF suppression led to a decrease in dissolved Pb concentration and loss, but an increase in particulate Pb concentration and loss, and enhanced the total Pb loss via surface runoff and interflows. Moreover, significant or very significant negative correlations were observed between the AMF colonization rate in roots with the Pb uptake in leaves, and the content of easily extractable GRSP with the particulate Pb loss. These results indicated the native AMF contributed to immobilizing Pb in soil and inhibited its migration to crops and the surrounding environment.

In Silico Docking of Novel Phytoalkaloid Camalexin in the Management of Benomyl Induced Parkinson's Disease and its In Vivo Evaluation by Zebrafish Model.

BACKGROUND: Parkinson's Disease (PD) exhibits the extrapyramidal symptoms caused due to the dopaminergic neuronal degeneration in the substantia nigra of the brain and depletion of Aldehyde Dehydrogenase (ALDH) enzyme. OBJECTIVE: This study was designed to enlighten the importance of the Aldehyde dehydrogenase enzyme in protecting the dopamine levels in a living system. Camalexin, a potentially active compound, has been evaluated for its dopamine enhancing and aldehyde dehydrogenase protecting role in pesticide-induced Parkinson's disease. METHODS: AutoDock 4.2 software was employed to perform the docking simulations between the ligand camalexin and standard drugs Alda-1, Ropirinole with three proteins 4WJR, 3INL, 5AER. Consequently, the compound was evaluated for its in vivo neuroprotective role in the zebrafish model by attaining Institutional Animal Ethical Committee permission. The behavioral assessments and catecholamine analysis in zebrafish were performed. RESULTS: The Autodock result shows that the ligand camalexin has a lower binding energy (-3.84) that indicates a higher affinity with the proteins when compared to the standard drug of proteins (-3.42). In the zebrafish model, behavioral studies provided evidence that camalexin helps in the improvement of motor functions and cognition. The catecholamine assay has proved that there is an enhancement in dopamine levels, as well as an improvement in aldehyde dehydrogenase enzyme. CONCLUSION: The novel compound, camalexin, offers a protective role in Parkinson's disease model by its interaction with neurochemical proteins and also in alternative in vivo model.

Scedo-Select III: a new semi-selective culture medium for detection of the Scedosporium apiospermum species complex.

The Scedosporium apiospermum complex is responsible for a large variety of infections in human. Members of this complex have become emerging fungal pathogens with an increasing occurrence in patients with underlying conditions such as immunosuppression or cystic fibrosis. A better knowledge of these fungi and of the sources of contamination of the patients is required and more accurate detection methods from the environment are needed. In this context, a highly selective culture medium was developed in the present study. Thus, various aliphatic, cyclic, or aromatic compounds were tested as the sole carbon source, in combination with some inorganic nitrogen sources and fungicides. The best results were obtained with 4-hydroxy-benzoate combined with ammonium sulfate and the fungicides dichloran and benomyl. This new culture medium called Scedo-Select III was shown to support growth of all species of the S. apiospermum complex. Subsequently, this new culture medium was evaluated successfully on water and soil samples, exhibiting higher sensitivity and selectivity than the previously described SceSel+ culture medium. Therefore, this easy-to-prepare and synthetic semi-selective culture medium may be useful to clarify the ecology of these fungi and to identify their reservoirs in patients' environment.

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.

Development and validation of benomyl birdseed agar for the isolation of Cryptococcus neoformans and Cryptococcus gattii from environmental samples.

One of the difficulties of isolating Cryptococcus neoformans and Cryptococcus gattii from environmental samples is the abundant overgrowth of other yeast and mold species that occurs on the plates. Here we report the application of benomyl to Guizotia abyssinica seed extract growth medium to improve the isolation of C. neoformans and C. gattii from environmental samples. We validated this medium by recovering C. neoformans and C. gattii from convenience soils and swabs from a region of the United States where these yeasts are endemic.

Functional genomics may allow accurate categorization of the benzimidazole fungicide benomyl: lack of ability to act via steroid-receptor-mediated mechanisms.

Although benomyl and its metabolite carbendazim have been shown to adversely affect male reproduction, the mechanisms of action do not appear to involve the endocrine system. However, few studies have been conducted using currently proposed tests specifically focused on endocrine disruption. Here, potential estrogen- and androgen-mediated activity of benomyl was therefore investigated in vitro and in vivo. Benomyl and carbendazim proved negative for agonistic and antagonistic activity in reporter gene assays for the human estrogen receptor alpha and androgen receptor. In uterotrophic and Hershberger assays using Crj:CD(SD)IGS rats, benomyl (100, 300 or 1000 mg/kg/day, p.o., N = 6) did not exert agonistic effects. However, the highest dose decreased uterine weights in the uterotrophic assay, and decreased weights of some androgen-related tissues of castrated rats receiving a testosterone propionate (TP, 0.2 mg/kg) injection in the Hershberger assay; the effects were less severe than those with p,p'-DDE (100 mg/kg/day). When 4 mg/kg/day of TP was injected, decrease of organ weights due to benomyl was attenuated but still observed. Thus, its influence in some tissues was more potent than that of p,p'-DDE. Benomyl had no apparent effects on serum androgen levels. Microarray analysis of the gene expression profile in the ventral prostate of TP-injected castrated rats treated with benomyl indicated clear differences from the patterns observed with p,p'-DDE and flutamide. Taken together, these findings suggest the decreased organ weights observed in vivo to be caused by mechanisms that are not steroid-receptor-mediated, such as interfering with assembly of microtubules by benomyl. The study furthermore suggests that functional genomics may provide a reliable evidence for accurate categorization of test chemicals.

Accelerated degradation of N, N'-dibutylurea (DBU) upon repeated application.

In a recent study on the degradation of N,N'-dibutylurea (DBU), a breakdown product of benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate], the active ingredient in Benlate fungicides, degradation half-lives of 1.4-46.5 days were observed across several soils incubated at various combinations of soil moisture potential (-0.03 and -0.1 MPa) and temperature (23, 33, and 44 degrees C) for a single DBU application of 0.08 and 0.8 microg g(-1) (Lee et al. 2004). However, Benlate can be applied as often as every 7 days resulting in the repeated application of DBU likely to be present in the Benlate over a growing season. In this study, the effect of seven repeated DBU applications on mineralization rate was investigated in two soils, which encompass the range in rates previously observed. For the slower degrading soil, repeated DBU application increased mineralization from 0.029 to 0.99 day(-1) at the 0.08 microg g(-1) rate, and 0.037 to 0.89 day(-1) at the 0.8 microg g(-1) rate. For the faster degrading soil, effects on mineralization of repeated DBU applications were small to negligible. For the latter soil, the effect on mineralization of applied DBU concentrations from 0.0008 to 80 microg g(-1) was also investigated. Mineralization rates decreased from 0.43 to 0.019 day(-1) with increasing DBU concentrations. However, the amount of DBU mineralized by day 70 was similar across concentrations and averaged 83% of applied. Microbial respiration was not affected by increasing DBU concentrations. These findings support the supposition that DBU is readily degraded by soil microorganisms, thus unlikely to accumulate in agricultural soils.

Sequence divergence of Entamoeba histolytica tubulin is responsible for its altered tertiary structure.

Atypical microtubular structures of the protozoan parasite Entamoeba histolytica (Eh) have been attributed to amino acid sequence divergence of Eh tubulin. To investigate if this sequence divergence leads to significant differences in the tertiary structure of the Eh alphabeta-tubulin heterodimer, we have modeled alphabeta-tubulin heterodimer of Eh based on the crystal structure of mammalian tubulin. The predicted 3D homology model exhibits an overall resemblance with the known crystal structure of mammalian tubulin except for the 16 residue long carboxy terminal region of Eh beta-tubulin. We propose that this C-terminal region may provide steric hindrance in the polymerization of Eh alphabeta-tubulin for microtubule formation. Using docking studies, we have identified the binding sites for different microtubule specific drugs on Eh beta-tubulin. Our model provides a rational framework, both for understanding the contribution of Eh beta-tubulin C-terminal region to alphabeta-tubulin polymerization and design of new anti-protozoan drugs in order to control amoebiasis.

A benzimidazole fungicide, benomyl, and its metabolite, carbendazim, induce aromatase activity in a human ovarian granulose-like tumor cell line (KGN).

Endocrine disruptor chemicals are known to cause a range of abnormalities in sexual differentiation and reproduction. One mechanism underlying such effects may be via alteration of aromatase activity, which is responsible for estrogen production. A good screening system for identifying endocrine disruptors has long been desired. We have recently established a human ovarian granulosa-like tumor cell line, KGN, which possesses a relatively high level of aromatase expression and is considered a useful mammalian model for investigating the in vitro effects of various chemicals on aromatase activity. In this study we screened 55 different candidate chemicals for endocrine disruptors by assaying aromatase activity. Only benomyl, known as both a benzimidazole fungicide and a microtubule-interfering agent, was found to induce aromatase activity in association with increased levels of aromatase mRNA in KGN cells. The effect of benomyl was presumed to be mediated by its metabolite carbendazim, because it produced an effect equivalent to that of benomyl. The mechanism underlying the benomyl-induced increase in aromatase activity appears independent of the cAMP-protein kinase A pathway. Treatment with taxol, another class of microtubule-interfering agents, also caused induction of aromatase in KGN cells. Both benomyl and taxol changed KGN cell morphology, including the development of cell roundness and a disorganized network of microtubules. These results indicate that benomyl is a potential endocrine disruptor that provides a novel estrogenicity and operates through a microtubule-interfering mechanism.

The yeast ubiquitin protease, Ubp3p, promotes protein stability.

Stu1p is a microtubule-associated protein required for spindle assembly. In this article we show that the temperature-sensitive stu1-5 allele is synthetically lethal in combination with ubp3, gim1-gim5, and kem1 mutations. The primary focus of this article is on the stu1-5 ubp3 interaction. Ubp3 is a deubiquitination enzyme and a member of a large family of cysteine proteases that cleave ubiquitin moieties from protein substrates. UBP3 is the only one of 16 UBP genes in yeast whose loss is synthetically lethal with stu1-5. Stu1p levels in stu1-5 cells are several-fold lower than the levels in wild-type cells and the stu1-5 temperature sensitivity can be rescued by additional copies of stu1-5. These results indicate that the primary effect of the stu1-5 mutation is to make the protein less stable. The levels of Stu1p are even lower in ubp3Delta stu1-5 cells, suggesting that Ubp3p plays a role in promoting protein stability. We also found that ubp3Delta produces growth defects in combination with mutations in other genes that decrease protein stability. Overall, these data support the idea that Ubp3p has a general role in the reversal of protein ubiquitination.

Structure-function relationships in yeast tubulins.

A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major alpha-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitive tub1 alleles were synthetically lethal in combination with tub3Delta, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of alpha-tubulin. The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast alpha-beta-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of beta-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of alpha-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the alpha-beta interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201 allele.

Monitoring of domestic and imported apples and rice by the U.S. Food and Drug Administration pesticide program.

In 1993-94, the U.S. Food and Drug Administration (FDA) conducted a statistically based study of pesticide residues in domestic and imported fresh apples and processed rice. For apples, 769 domestic and 1062 imported samples were collected and analyzed; 85% of the domestic and 86% of the imported samples had detectable residues. Benomyl, a widely used fungicide, was found with greatest frequency in domestic apples, while diphenylamine was found most often in imported apples. One domestic and 4 imported samples contained violative residues of pesticides for which there are no U.S. tolerances on apples. The statistically weighted (by domestic packer throughput or import shipment size) violation rates for domestic and imported apples were 0.30% (0.13 unweighted) and 0.41% (0.38 unweighted), respectively. For rice, 598 domestic and 612 imported samples were collected and analyzed; 56% of the domestic and 12% of the imported samples had detectable residues. Malathion had the greatest frequency of occurrence in both groups of rice. Eight domestic and 9 imported samples were violative, all as a result of use of pesticides for which there are no U.S. tolerances on rice. The statistically weighted violation rates for domestic and imported rice were 0.43% (1.3 unweighted) and 1.1% (1.5 unweighted), respectively. Results of the statistically based study show that, as in FDA's regulatory monitoring, the levels of most pesticide residues found in these 2 commodities are generally well below U.S. tolerances, and few violative residues are found.

Amino acid alterations in the benA (beta-tubulin) gene of Aspergillus nidulans that confer benomyl resistance.

We report the cloning and sequencing of 18 mutant alleles of the benA, beta-tubulin gene of Aspergillus nidulans that confer resistance to the benzimidazole antifungal, antimicrotubule compounds benomyl, carbendazim, nocodazole, and thiabendazole. In 12 cases, amino acid 6 was changed from histidine to tyrosine or leucine. In four cases, amino acid 198 was changed from glutamic acid to aspartic acid, glutamine, or lysine. In two cases, amino acid 200 was altered from phenylalanine to tyrosine. These data, along with previous data indicating that amino acid 165 is involved in the binding of the R2 group of these compounds [Jung and Oakley, 1990: Cell Motil. Cytoskeleton 17:87-94], suggest that regions of beta-tubulin containing amino acids 6, 165, and 198-200 interact to form the binding site of benzimidazole antimicrotubule agents. These results also suggest that the presence of phenylalanine at amino acid 200 contributes to the great sensitivity of many fungi to benzimidazole antimicrotubule agents.

[Cytogenetic activity of metabolites of pesticides representing different classes of chemical compounds]. / Issledovanie tsitogeneticheskoi aktivnosti nekotorykh metabolitov riada pestitsidov--predstavitelei neskol'kikh klassov khimicheskikh soedinenii.

The cytogenetic activity of some substances formed in agricultural plants during metabolism of pesticides of four classes of chemical compounds was studied in the culture of human peripheric blood lymphocytes. Metabolites were shown either to have mutagenic properties similar to those of the initial compounds (ziramtetramethylthiourea, both being mutagens; captan-phthalimide, both possessing no cytogenetic activity) or to be considerably transformed in comparison with them as a result of deactivation (benomile-MBC) or activation (betanal-MHPC) processes. The latter variant if being determined for the genetic hazard of the pesticide necessitates to take into account data on the mutagenic character of those metabolites which really might enter the human organism.

Effect of benomyl on the reproductive development of male rats.

Benomyl, a systemic fungicide, was administered to male Sprague-Dawley rats during the prepuberal, pubertal, or postpubertal stage of reproductive development. Animals received 5 or 10 daily treatments of 0, 125, 200, 250, 500, or 1000 mg benomyl/kg . d by gavage. Observations were made at selected intervals after exposure and included hematological parameters, body weight, tissue weights, total epididymal sperm counts, vas deferens sperm concentration, serum follicle-stimulating hormone ( sFSH ) levels, and testicular histology. Data presented here suggest that there is an age-related difference in sensitivity to benomyl. Animals that received benomyl treatments during prepuberty showed no significant treatment effects in tissue weights, total epididymal sperm counts, vas deferens sperm concentration, or sFSH . Animals that received at least 250 mg/kg . d during puberty or postpuberty showed one or more of the following effects: decreased testicular or epididymal weights, decreased epididymal sperm count, decreased vas deferens sperm concentrations, and/or testicular lesions. Histological examination of testicular tissue indicated a higher incidence of diffuse hypospermatocytogenesis in pubertal (20% of the treated animals) and postpubertal (40% of the treated animals) animals that were exposed to benomyl. These values were compared with those of the treated prepubertal animals, which had a 10% incidence of diffuse hypospermatocytogenesis , and with all of the control animals, which had no occurrences of this testicular lesion.