Mechanism of Anti-Cancer Activity of Benomyl Loaded Nanoparticles in Multidrug Resistant Cancer Cells.

Polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles loaded with benomyl as anticancer drug formulation against multidrug-resistant EMT6/AR1 cells were synthesized by amine-carboxylate reaction. Using transmission electron microscopy, the average size of chitosan-poly(D,L-lactide-co-glycolide) nanoparticles and benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles was estimated to be 155 ± 20 nm and 160 ± 25 nm, respectively. Fourier transform infrared spectroscopy revealed that poly(D,L-lactide-co-glycolide) and chitosan are linked by covalent bonds. Zeta potentials of benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles at pH 4, 7.2, and 10 were 30 ± 1.8, 19 ± 0.65, and -22 ± 0.15 mV, respectively, indicating the formation of stable, hydrophilic nanoparticles. The release of benomyl from benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles followed pH-dependent kinetics. The uptake of fluorescein isothiocyanate-labeled chitosan-poly(D,L-lactide-co-glycolide) nanoparticles was concentration-dependent in both MCF-7 and multidrug-resistant EMT6/AR1 cells. EMT6/AR1 cells showed 10-fold higher resistance to benomyl compared to MCF-7 cells; in contrast, benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles effectively inhibited proliferation of MCF-7 and EMT6/AR1 cells with a half-maximal inhibitory concentration of 4 ± 0.5 and 9 ± 0.5 pM, respectively. In the presence of a P-glycoprotein inhibitor, the activity of benomyl was increased, suggesting that benomyl is a substrate for P-glycoprotein. Further, benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles depoly-merized microtubules both in interphase and mitosis. It blocked cell cycle progression at G2/M and induced apoptosis in EMT6/AR1 cells, suggesting that benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles have chemotherapeutic activity against multidrug-resistant cancer cells.

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.

Combined bromodeoxyuridine immunocapture and terminal-restriction fragment length polymorphism analysis highlights differences in the active soil bacterial metagenome due to Glomus mosseae inoculation or plant species.

High numbers of bacteria are associated with arbuscular mycorrhizal (AM) fungi, but their functions and in situ activities are largely unknown and most have never been characterized. The aim of the present study was to study the impact of Glomus mosseae inoculation and plant type on the active bacterial communities in soil by using a molecular approach, bromodeoxyuridine (BrdU) immunocapture in combination with terminal-restriction fragment length polymorphism (T-RFLP). This approach combined with sequence information from clone libraries, enabled the identification of actively growing populations, within the total bacterial community. Distinct differences in active bacterial community compositions were found according to G. mosseae inoculation, treatment with an antifungal compound (Benomyl) and plant type. The putative identities of the dominant bacterial species that were activated as a result of G. mosseae inoculation were found to be mostly uncultured bacteria and Paenibacillus species. These populations may represent novel bacterial groups that are able to influence the AM relationship and its subsequent effect on plant growth.

Genotoxicity of benomyl and its residues in somatic and germ cells of mice fed on treated stored wheat grains.

Swiss mice were fed for 2, 4 and 8 weeks wheat grains treated with 1, 2, and 4 g benomyl/kg and stored for 6 and 12 weeks. The maximum effect of benomyl on the induction of chromosomal aberrations was observed after feeding mice for 8 weeks with wheat grains treated with 4 g benomyl/kg and stored for 12 weeks. Its proportion differed significantly in bone marrow and spermatocyte cells, 15+/-0.51% vs. 13.4+/-0.66%, respectively, from that in nontreated mice (background level), 4.4+/-0.24% and 3.8+/-0.20%, respectively. Lengthening the storage period of treated wheat grains caused a dose-dependent increase in the frequency of sister chromatid exchanges: 8.61+/-0.34 vs. 4.16+/-0.06/cell. The proportion of sperm-head abnormalities increased by lengthening the period of storage and feeding: 7.7+/-0.41% vs. 3.25+/-0.12%. In another experiment mice were orally treated by gavage with benomyl at 50, 100, 150, 200 mg/kg; a significant and dose-dependent increase in sperm-head abnormalities was observed. These findings demonstrate that benomyl (a 50% wettable powder formulation) and its residues in wheat grains are genotoxic in mice.

Incidence and severity of frogeye leaf spot and associated yield losses in soybeans in agroecological zone II of Zambia.

The incidence and severity of frogeye leaf spot of soybean (Glycine max (L.) Merr.) was studied in agroecological region II of Zambia during the 1997/98 crop growing season. A survey was conducted on farmers' fields on SCSI Kaleya, Magoye and Hernon-147 cultivars. Disease incidence and severity was assessed by monitoring disease increments at two weeks interval (beginning of January to April) from nine fields, three from each province. Soybean cultivars were evaluated for yield losses resulting from frogeye leaf spot. Field plots of each cultivar were either sprayed twice with benomyl (benlate) or not sprayed at all. The results showed that the incidence of frogeye leaf spot was highest in Southern province (5.1), followed by Lusaka province (4.9) while Central province had the lowest disease incidence (1.8). Values for area under disease progress curve (AUDPC) were significantly greater (P < 0.05) for Lusaka and Southern provinces than for Central province. Yields in benomyl protected plots ranged from 1444 kg ha-1 to 2320 kg ha-1 and were significantly different among the cultivars. Average yields of non protected plants were reduced by 30.5% for Kaleya, 35.6% for Hernon-147 and 37.2% for SCS1. Incidence and severity increased with time and varied depending on weather parameters and susceptibility of cultivars to the disease. Yield losses due to frogeye leaf spot occurred through a reduction in seed size. Differences in weather conditions and amount of inocula are believed to contribute to the observed variation in incidence and severity of the disease at different locations.

An LC/MS/MS method for improved quantitation of the bound residues in the tissues of animals orally dosed with [(14)C]Benomyl.

Livers of goats orally dosed with [phenyl(U)-(14)C]benomyl contained radioactive residues which were not extractable using conventional, solvent-based extraction methods. We report a new residue method capable of enhanced extraction of benomyl-derived residues with selective and sensitive quantitation capability for methyl 4-hydroxybenzimidazol-2-ylcarbamate (4-HBC), methyl 5-hydroxybenzimidazol-2-ylcarbamate (5-HBC), and methyl benzimidazol-2-ylcarbamate (MBC). This method involves rigorous Raney-nickel reduction of hypothesized thioether bonds between benomyl residues and polar cellular components. Following acidic dehydration (desulfurization), the polar benomyl-derived residues are extracted into ethyl acetate and analyzed by LC/MS/MS. We have shown this method to be superior to alternative extraction approaches. When applied to goat liver tissue containing [phenyl(U)-(14)C]benomyl-bound residues, the extraction efficiency of total radioactive residues was approximately 30%, and the major benomyl-derived residue was 5-HBC (91-95% of extractable residue) with minor levels of carbendazim (MBC) (5-9%). HPLC/LSC data were consistent with the LC/MS/MS data. The overall method satisfies U.S. regulatory requirements in extraction efficiency, selectivity in detection, and limits of quantitation for benomyl-bound residues.

The role of the benomyl metabolite carbendazim in benomyl-induced testicular toxicity.

The present study has investigated the role of benomyl (BNL) vs carbendazim (CBZ) in BNL-induced testicular toxicity. Equivalent molar concentrations of BNL and CBZ were administered to rats intraperitoneally (859 mumol/kg) or by direct injection into the testis (1.37 mumol/testis). Whereas no significant testicular damage was observed both 1 and 2 hr after BNL administration by the ip route, CBZ administration resulted in sloughing of the seminiferous epithelium after 1 hr, which increased in severity at the 2-hr time point. Intratesticular treatment of BNL caused little testicular damage after 1 hr whereas an equimolar amount of CBZ elicited severe disruption of the seminiferous epithelium. Testicular levels of CBZ and BNL were measured at various times after both routes of administration. The AUC from the concentration of CBZ in the testis vs time plot showed an excellent relationship to the number of tubules which exhibited slouging. The BNL AUC also showed a straight-line relationship to severity of lesion. However, when the contribution of CBZ to the BNL response was subtracted, no effect of BNL was discernible. The effect of BNL and CBZ on testicular microtubule assembly was then investigated. IC50 for CBZ was 5 microM and that for BNL was 75 microM. Again, the effect of BNL on microtubule assembly could be largely accounted for by the presence of the CBZ breakdown product. These results strongly suggest that the BNL metabolite CBZ, and not BNL itself, is the mediator of BNL-induced testicular toxicity and inhibitor of testicular microtubule assembly.

Estudo da influência de agrotóxicos sobre o desenvolvimento do Rhizobium leguminosarum biovar phaseoli / Study of the agrotoxics influence over the development of the Rhizobium leguminosarum biovar phaseoli

As bactérias fixadoras de nitrogênio atmosférico, via processo simbiótico, repassam N2 às leguminosas sob forma de amônia, proporcionando aumento na produçäo de alimentos protéicos, reciclagem biológica de nitrogênio do ar, evitando o alto custo da adubaçäo nitrogenada e o efeito potencialmente poluidor do nitrato lixiviado. Testou-se quatro estirpes de Rhizobium phaseoli frente a quatro fungicidas indicados para o tratamento de sementes de Phaseolus vulgaris L. (feijoeiro), no Estado do Rio Grande o sul (Brasil). Considerando-se as dosagens recomendadas dos fungicidas, houve crescimento bacteriano das quatro estirpes frente a Benomil e inibiçäo total frente a Captan; porém, para PCNB e Thiram, a resistência dependeu do tipo de estirpe. Observou-se que näo há influência de fungicidas sobre o desenvolvimento do Rhizobium phaseoli quando seleciona-se a estirpe adequada ao fungicida corretamente dosado.

Investigation of the effects of benomyl on rat nasal mucosa.

Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate, CAS Registry No. 17804-35-2] is a widely used agricultural fungicide. Previously, olfactory epithelial lesions were produced following a 45-day inhalation exposure to 50 and 200 mg/m3 benomyl. The present study, part of a range-finding study for a two-generation reproduction study, was conducted to determine if the previously reported effects on the nasal mucosa are the result of systemic toxicity or attributable to the inhalation route of exposure. Groups of 10 7-week-old male Crl:CD BR rats were fed diets containing 0, 5000, 10000, or 15000 ppm benomyl for 32 days. Individual body weights and food consumption were determined weekly and on the last day of the study. After 32 days on test, rats were euthanatized by pentobarbital anesthesia and exsanguination and were examined for gross alterations. The nasal cavity was processed for pathological examination. Mean body weight gain was statistically significantly decreased during the first week of treatment and the overall test period (Days 0-32) at the two highest dose levels. A significant decrease in food consumption also was seen during test interval Days 0-7 for the two highest dose groups. In addition, statistically significant decreases in food consumption were observed at the Day 7-14 interval for the 15,000 ppm dose group and at the 21-28 and 28-32 intervals for the two highest dose groups compared with controls. No histopathological lesions were noted in the nasal epithelium of any of the control or benomyl-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the fungicide benomyl on xenobiotic metabolism in rats.

The effect of benomyl administered orally (p.o.) and intraperitoneally (i.p.) on the activity of hepatic microsomal mixed-function oxidases (MFOs) was studied in rats. A dose of 100 mg/kg given i.p. reduced the activities of several hepatic drug-metabolizing enzymes 24 h following the treatment. A similar reduction in the activities of the MFOs was also noted 24 h following oral benomyl administration at a dose of 500 mg/kg. Furthermore, in vivo inhibition of drug metabolism by benomyl was demonstrated by increased pentobarbital sleeping-time 24 h after p.o. as well as i.p. dosing. No alterations were found in the serum sorbitol dehydrogenase (SDH) at 24 h after i.p. or oral benomyl indicating a lack of hepatotoxic effect. These results indicate that benomyl shows a route-independent effect on MFOs and is not toxic to the liver.

Rhizoxin resistant mutants with an altered beta-tubulin gene in Aspergillus nidulans.

Rhizoxin and ansamitocin P-3 (a maytansinoid compound), potent inhibitors of mammalian brain tubulin assembly, inhibit growth of a variety of fungi including Aspergillus nidulans. Mutants of A. nidulans, benA10 which is a benomyl resistant beta-tubulin gene mutant and tubA1 which is a benomyl supersensitive alpha-tubulin gene mutant, were both sensitive to rhizoxin and ansamitocin P-3 to the same extent as wild-type strains. We isolated 18 rhizoxin resistant mutants of A. nidulans. All of these mutants were cross-resistant to ansamitocin P-3, but not to benzimidazole antimitotic drugs. These mutants mapped to two loci, rhiA and rhiB, and all of those with high resistance mapped to rhiA. The fact that the protein extracts of rhiA mutants lost rhizoxin binding affinity and that rhiA was closely linked to benA, the major beta-tubulin gene in A. nidulans, indicated that rhiA must be a structural gene for beta-tubulin and that rhiA mutants are a new class of beta-tubulin gene mutants. All of this suggested that, in A. nidulans, these antimitotic drugs bind to beta-tubulin, and that rhizoxin and ansamitocin P-3 share the same binding site but the site does not overlap with the benzimidazole binding site. Protein extracts from a rhiB mutant retained rhizoxin binding affinity, therefore this rhizoxin resistance mechanism should not be a tubulin mediated process.

Evaluation of reproductive parameters in adult male Wistar rats after subchronic exposure (gavage) to benomyl.

Proven-breeder 102-d-old male Wistar rats were gavaged daily with 0, 1, 5, 15, or 45 mg/kg.d benomyl. The animals were bred to untreated females after 62 d and killed after 76-79 d for evaluation of selected male reproductive end points. Minimal to moderate changes were observed in rats dosed with 45 mg/kg.d; these included decreased testis and epididymis weight, reduced cauda sperm reserves, decreased sperm production, increased numbers of decapitated spermatozoa, and increased numbers of seminiferous tubules containing multinucleated giant cells. Reproductive performance, seminal vesicle and prostate weight, sperm motility, serum luteinizing hormone, follicle-stimulating hormone, prolactin, and androgen binding protein were not affected by any of the dosages tested. Based on these end points, the no-effect level was 15 mg/kg.d.