Dithiocarbamate Fungicides Research Articles

RETRACTED: Mancozeb-induced genotoxicity and apoptosis in cultured human lymphocytes

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

Gold nanorods as surface enhanced Raman spectroscopy substrates for sensitive and selective detection of ultra-low levels of dithiocarbamate pesticides

We report the use of gold nanorods as solution-based SERS substrates for the detection of ultralow-levels of three different dithiocarbamate fungicides: thiram, ferbam and ziram. Gold nanorods are attractive to use as SERS substrates due to the ability to tune the surface plasmon resonance of the nanoparticles to the laser excitation wavelength of the Raman spectrometer equipped with a 785 nm diode laser. The gold nanorods are synthesized using a seed-mediated growth method and characterized using UV-Visible spectroscopy, zeta potential, and TEM. The gold nanorods have an aspect ratio of 2.19 ± 0.21 and have an average length of 37.81 ± 4.83 nm. SERS spectra are acquired at different concentrations of each fungicide and calibration curves are obtained by monitoring the intensity of the band arising from the ν(C-N) stretching mode coupled to the symmetric δ(CH(3)) motion. The limits of detection and limits of quantitation are obtained for each fungicide. The limits of detection are 11.00 ± 0.95 nM, 8.00 ± 1.01 nM, and 4.20 ± 1.22 nM for thiram, ferbam, and ziram respectively. The limits of quantitation are 34.43 ± 0.95 nM, 25.61 ± 1.01 nM, and 12.94 ± 1.22 nM for thiram, ferbam, and ziram respectively. It can be seen that the three different dithiocarbamates can be detected in the low nM range based on the limits of detection that are achieved.

Mechanism of ziram-induced apoptosis in human T lymphocytes

Ziram as a dithiocarbamate fungicide is widely used throughout the world in agriculture. We previously found that ziram significantly inhibited cytotoxic T lymphocyte activity in a dose-dependent manner. To explore the mechanism of this inhibition, we investigated ziram-induced apoptosis in human T lymphocytes. Jurkat T cells were treated with ziram at 0.031-1 μM for 2-24 h. Freshly isolated primary human T cells were treated with ziram at 0.0625-1 μM for 15 and 24 h. Apoptosis was determined by FITC-Annexin V/PI staining and the TUNEL assay. To explore the mechanism of apoptosis, intracellular levels of active caspases 3, 3/7, 8, and 9 and pan-caspase and mitochondrial cytochrome-c release were determined by flow cytometry. Disruption to mitochondrial transmembrane potential was determined with a MitoLight(™) Apoptosis Detection Kit. We found that ziram induced apoptosis in a time- and dose-dependent manner in both Jurkat cells and primary human T cells. The primary human T cells were more sensitive to ziram than the Jurkat cell line. Ziram induced increases in active caspases 3, 3/7, 8, and 9 and pan-caspase in a dose-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, partially but significantly inhibited the apoptosis. Moreover, a general caspase inhibitor, Z-VAD-FMK, significantly and almost completely blocked the apoptosis. Ziram also disrupted mitochondrial transmembrane potential and caused mitochondrial cytochrome-c release. These findings indicate that ziram can induce apoptosis in human T cells, and the apoptosis is mediated by both the caspase-cascade and the mitochondria/cytochrome-c pathways.

Dithiocarbamate fungicide zineb induces oxidative stress and apoptosis in Chinese hamster lung fibroblasts

Dithiocarbamate fungicide zineb leads to disturbances in many cellular processes in Chinese hamster fibroblasts (V79 cells). We observed high apoptotic activity and oxidative stress induced in vitro by exposure of V79 cells to zineb demonstrated by statistically significant increase in lipid peroxidation (measured as TBARS – thiobarbituric acid reactive substances) as well as disturbances in glutathione homeostasis. Zineb also inhibited activity of topoisomerase I. Zineb did not show any effect on protein oxidation (measured by protein carbonyls content). N-acetyl- l-cysteine (NAC) suppressed cellular changes induced by this fungicide in V79 cells. NAC pre-treatment prevented TBARS production and significantly decreased the number of apoptotic cells induced by zineb. These results show that zineb can disturb many cellular processes via oxidative damage.

Synthesis, Antibacterial and Antifungal Activities of Some Carbazole Dithiocarbamate Derivatives

In this study, we aimed at the synthesis of new N,N-disubstituted dithiocarbamoic acid, 2-[(9-ethyl-9Hcarbazol- 3-yl)amino]-2-oxoethyl esters and their antimicrobial evaluations. The chemical structures of the compounds were elucidated by IR, 1H NMR, 13C NMR, MS spectral data and elemental analysis. The antibacterial and antifungal activities of the synthesised derivatives were tested against Gram (+), Gram (-) bacteria and yeasts such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Bacillus cereus, Methicillin-resistant Staphylococcus aureus (MRSA), Candida albicans (two strains), Candida glabrata, Candida tropicalis, Candida parapsilosis and Saccharomyces cerevisiae using a microbroth dilution technique. The results showed that the compounds displayed different degrees of antimicrobial activities; however, when compared almost all of the compounds displayed better antifungal activities (minimum inhibitory concentrations, 312.5- > 1250 μg mL-1). Keywords: Antibacterial activity, Antifungal activity, Carbazole, Dithiocarbamate, Dithiocarbamate fungicides, nonselective pestici, Cladosporium cucumerinum, phytoalexins, conjugated compounds, aromatic compounds, heterocyclic compounds, N,N-disubstituted dithiocarbamoic acid, Shimadzu 8400S spectrophotometer, tetramethylsilane (TMS)

Direct analysis of dithiocarbamate fungicides in fruit by ambient mass spectrometry

Dithiocarbamates (DTCs) are fungicides that require a specific single-residue method for detection and verification of compliance with maximum residue limits (MRLs) as established for fruit and vegetables in the EU. In this study, the use of ambient mass spectrometry was investigated for specific determination of individual DTCs (thiram, ziram) in fruit. Two complementary approaches have been investigated for their rapid analysis: (i) direct analysis in real time (DART) combined with medium-high resolution/accurate mass time-of-flight mass spectrometry (TOFMS) and high-resolution/accurate mass Orbitrap MS, and (ii) desorption electrospray ionization (DESI) combined with tandem-in-time mass spectrometry (MS2). With both techniques, thiram deposited on a glass surface (DART) or Teflon (DESI) could be directly detected. With DART, this was also possible for ziram. Before the instrumental analysis of fruit matrix, an extract had to be prepared following a straightforward procedure. The raw extracts were deposited on a slide (DESI), or rods were dipped into the extracts (DART), after which thiram and ziram could be rapidly detected (typically 10 samples in a few minutes). In the case of thiram, the lowest calibration levels were 1 mg kg−1 (DART–TOFMS, DESI–MS2) and 0.1 mg kg−1 (DART–Orbitrap MS). For ziram, the achieved lowest calibration levels were 0.5 mg kg−1 (DART–TOFMS) and 1 mg kg−1 (DART–Orbitrap MS). In all cases, this was sufficiently low to test samples against EU-MRLs for a number of fruit crops. Using an internal standard, (semi)quantitative results could be obtained.

Effect of Dithiocarbamate Fungicide Mancozeb on Development, Reproduction and Ultrastructure of Fat Body of Agrotis segetum Moths

Sublethal effects of ethylene bis dithiocarbamate fungicide mancozeb on development and reproduction of turnip moth ( Agrotis segetum ) was investigated. Larvae were exposed to the fungicide in a diet. Exposure did not cause massive mortality of larvae. However, we observed various sublethal effects. Larval development was longer than in control, also mortality of pupae and time of pupation was altered; exposure to mancozeb affected fecundity, too. Scanning electron microscopy revealed that females had laid fewer eggs, and they showed various malformations, which might affect hatching success. These changes were also in tune with observations of the larval fat body ultrastructure (transmission electron microscopy). Fat body cells showed a range of malformations: envelopes of nuclei were invaginated and swallen. In cytoplasm, glycogen content was decreased, ER showed swelling and cytoplasm became more lucent. All these changes had been observed before the mortality of larvae was noticed. Therefore, we think that ultrastructural changes may be an important marker of stress caused by mancozeb in the environment.

Neoplastic Alterations Induced in Mammalian Skin Following Mancozeb Exposure UsingIn VivoandIn VitroModels

Mancozeb, ethylene(bis)dithiocarbamate fungicides, has been well documented in the literature as a multipotent carcinogen, but the underlying mechanism remains unrevealed. Thus, mancozeb has been selected in this study with the objective to decipher the molecular mechanism that culminates in carcinogenesis. We employed two-dimensional gel electrophoresis and mass spectrometry to generate a comparative proteome profile of control and mancozeb (200 mg/kg body weight) exposed mouse skin. Although many differentially expressed proteins were found, among them, two significantly upregulated proteins, namely, S100A6 (Calcyclin) and S100A9 (Calgranulin-B), are known markers of keratinocyte differentiation and proliferation, which suggested their role in mancozeb-induced neoplastic alterations. Therefore, we verified these alterations in the human system by using HaCaT cells as an in vitro model for human skin keratinocyte carcinogenesis. Upregulation of these two proteins upon mancozeb (0.5 μg/mL) exposure in HaCaT cells indicated its neoplastic potential in human skin also. This potential was confirmed by increase in number of colonies in colony formation and anchorage-independent growth assays. Modulation of S100A6/S100A9 targets, elevated phosphorylation of extracellular signal regulated kinase (ERK1/2), Elk1, nuclear factor- kappa B and cell division cycle 25 C phosphatase, and cyclin D1 and cyclooxygenase-2 upregulation was seen. In addition, PD98059 (ERK1/2 inhibitor) reduced cell proliferation induced by mancozeb, confirming the involvement of ERK1/2 signaling. Conclusively, we herein present the first report asserting that the mechanism involving S100A6 and S100A9 regulated ERK1/2 signaling underlies the mancozeb-induced neoplastic potential in human skin.

Solution-based direct readout surface enhanced Raman spectroscopic (SERS) detection of ultra-low levels of thiram with dogbone shaped gold nanoparticles

We report the use of two different sizes of dogbone shaped gold nanoparticles as colloidal substrates for surface enhanced Raman spectroscopy (SERS) based detection of ultra-low levels of thiram, a dithiocarbamate fungicide. We demonstrate the ability to use a solution based, direct readout SERS method as a quantitative tool for the detection of ultra-low levels of thiram. The two different sizes of dogbone shaped gold nanoparticles are synthesized by using the seed-mediated growth method and characterized by using UV-visible spectroscopy and transmission electron microscopy (TEM). The smaller dogbone shaped nanoparticles have an average size of 43 ± 13 nm. The larger dogbone shaped gold nanoparticles have an average size of 65 ± 15 nm. The nanoparticle concentration is 1.25 × 10(11) nanoparticles per mL for the smaller dogbone shaped gold nanoparticles and is 1.13 × 10(11) nanoparticles per mL for the larger dogbone shaped gold nanoparticles. Different concentrations of thiram are allowed to bind to the two different sizes of dogbone shaped gold nanoparticles and the SERS spectra are obtained. From the calibration curve, the limit of detection for thiram is 43.9 ± 6.2 nM when the smaller dogbone shaped gold nanoparticles are used as colloidal SERS substrates In the case of the larger dogbone shaped gold nanoparticles, the limit of detection for thiram is 11.8 ± 3.2 nM. The lower limit of detection obtained by using the larger dogbone shaped gold nanoparticles as colloidal substrates is due to the lightning rod effect, higher contributions from the electromagnetic enhancement effect, and larger number of surface sites for thiram to bind.

Single clusters of self-assembled silver nanoparticles for surface-enhanced Raman scattering sensing of a dithiocarbamate fungicide

Hydrophobic silver (Ag) nanoparticles of ∼16 nm diameter were self-assembled as building blocks in an emulsion to form large spherical clusters upon the removal of organic solvents. The self-assembled clusters of Ag nanoparticles have diameters in the range 0.5–1.0 μm and are composed of thousands of densely packed Ag nanoparticles, leading to the generation of multiple active sites or hot spots for surface-enhanced Raman scattering (SERS) in a single cluster, as clearly observed using confocal Raman microscopy. Such single clusters of Ag nanoparticles show significant SERS activity for Rhodamine-6G and dithiocarbamates such as thiram. The enhancement factor for R6G was calculated to reach 1 × 109, which is possible for the observation of SERS signals of a single molecule of R6G according to literature reports. The as-prepared individual clusters of Ag nanoparticles have been demonstrated for the SERS detection of the agricultural chemical thiram. The results show that the detection limit for thiram is as low as 0.024 ppm, which is much lower than the maximal residue limit (MRL) of 7 ppm in fruit prescribed by U.S. Environmental Protection Agency (EPA). The system also possesses the ability to selectively detect dithiocarbamate compounds over other types of agricultural chemical. Furthermore, spiked and recovery tests show that the Ag nanoparticle clusters can be used to detect thiram in natural lake water and commercial apple juice without much interference.

Fourth Derivative Spectrophotometric Method for the Determination of Fungicide Maneb Using Sodium Molybdate

A highly sensitive fourth derivative spectrophotometric method is presented for the residue analysis of ethylenebis dithiocarbamate fungicide Maneb. The fungicide maneb forms a blue complex with sodium molybdate on heating which is studied spectrophotometrically. The method has been successfully applied to maneb determination in wheat grains and soft drinks with high recoveries. The analytical sensitivity of the present method has been found to be 0.0011 μg/mL.

Dietary risk assessment of organophosphorus and dithiocarbamate pesticides in a total diet study at a Brazilian university restaurant

In this study ready-to-eat food samples were collected in the production line of the university restaurant of the University of Brasilia, Brazil, which serves non-vegetarian and vegetarian meals daily. Samples were analysed for the presence of ten organophosphorus insecticides (OPs) by GC/FPD, after extraction with ethyl acetate and anhydrous sodium sulfate (LOQ = 0.002 mg kg–1), and for dithiocarbamate fungicides, as CS2, using the spectrophotometric method (LOQ = 0.05 mg kg−1). About 43% of the 175 samples analysed contained at least one OP compound at levels up to 1.83 mg kg−1. Methamidophos was the compound most detected (37.7%), present in most of the soup, soybean and salad samples. No OP residues were found in fruit juice, beans and bran rice samples. The cumulative acute intake of OPs was estimated using methamidophos and acephate as index compounds (IC). The total cumulative intake represented 9.1% and 47.7% of the methamidophos ARfD for the non-vegetarian and vegetarian diets, respectively. When acephate was used as IC, the total intakes represented 20.7% and 116% of the ARfD for the non-vegetarian and vegetarian diets, respectively. Dithiocarbamates were detected in 70% of the 177 samples analysed, at levels up to 0.51 mg kg−1 CS2; all salad samples were positive and no residues were found in fruit juice. The chronic intake of dithiocarbamates represented 8.6 and 8.9% of the ADI (mancozeb) for the vegetarian and non vegetarian diets, respectively.

Effects of mancozeb and other dithiocarbamate fungicides on Saccharomyces cerevisiae: the role of mitochondrial petite mutants in dithiocarbamate tolerance

Saccharomyces cerevisiae as model system was used to evaluate the occurrence of resistant mutants and adaptation mechanism to mancozeb (MZ), a widespread fungicide of the dithiocarbamate class with a broad spectrum of action and multiple cell targets. We were unable to isolate mutants resistant to inhibitory concentration of MZ but found an unusually large number of mitochondrial defective petite mutants among cells incubated in the presence of subinhibitory MZ concentration. Similar results were obtained with two other dithiocarbamate fungicides. Comparison of wild type and petite mutants showed that the latter were more resistant to toxic effects of MZ, highlighting the role of mitochondria in MZ-tolerance. The data suggest that petite cells, arising by exposure to sub-inhibitory MZ concentration, are not induced by fungicides but are spontaneous mutants already present in the population before the contact with the fungicide.

EVALUATION OF THE NANOSILVER COLLOIDAL SOLUTION IN COMPARISON WITH THE REGISTERED FUNGICIDE TO CONTROL GREENHOUSE CUCUMBER DOWNY MILDEW DISEASE IN THE NORTH OF IRAN

ISHS VI International Postharvest Symposium EVALUATION OF THE NANOSILVER COLLOIDAL SOLUTION IN COMPARISON WITH THE REGISTERED FUNGICIDE TO CONTROL GREENHOUSE CUCUMBER DOWNY MILDEW DISEASE IN THE NORTH OF IRAN

Ziram induces apoptosis and necrosis in human immune cells

Ziram as a dithiocarbamate fungicide is widely used throughout the world in agriculture and as an accelerating agent is used in latex production. In order to investigate ziram-induced apoptosis/necrosis and its underlying mechanism in human immune cells, a human monocyte-like cell line (U937) was treated with ziram at 0.0312-2 μM for 2-24 h at 37 °C in a 5% CO₂ incubator. Apoptosis/necrosis induced by ziram was determined by analysis of FITC-Annexin-V/PI staining and the intracellular level of active caspase-3 by flow cytometry and DNA fragmentation analysis. We found that ziram induced apoptosis/necrosis in U937 in a time- and dose-dependent manner, as shown by FITC-Annexin-V/PI staining. DNA fragmentation was detected when cells were treated with 0.5, 1, or 2 μM ziram for 24 h. Ziram also induced an increase in intracellular active caspase-3 in U937 cells in a dose-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, significantly inhibited the ziram-induced apoptosis. Moreover, it was found that ziram induced mitochondrial cytochrome c release in U937 cells. These findings indicate that ziram can induce apoptosis/necrosis in U937 cells, and this effect is partially mediated by activation of intracellular caspase-3 and mitochondrial cytochrome c release.

Development and validation of a cellular biosensor detecting pesticide residues in tomatoes

Two of the most important categories of pesticides used in agricultural practice are organophosphates and dithiocarbamates. Their extensive and inappropriate use has rendered their reliable monitoring at trace levels more and more necessary. This study presents the construction of a rapid and sensitive cellular biosensor test based on the measurement of changes of the cell membrane potential of immobilized cells, according to the working principle of the Bioelectric Recognition Assay (BERA). The cells were immobilized by entrapment in a sodium alginate bead and directly applied in different pesticide dilutions and agricultural samples. The pesticides used were the organophosphate insecticide diazinon and the dithiocarbamate fungicide propineb. Two different cell types, N2a (neuroblastoma) and Vero (fibroblast) were used as the biosensory elements in order to investigate their differential response against the pesticides. In this way, we hoped to increase the selectivity of the assay. Based on the observed patterns of response, we demonstrate that the sensor can be used for the qualitative and, in some concentrations, quantitative detection of the pesticides with a high degree of reproducibility. The lowest detected concentration was 3nM. Finally, for the investigation of the effects of different pesticides on the accumulation of cytosolic Ca(2+), we conducted a fluorescent assay on N2a cells treated with tomato sample extracts, which were replicates of the E.U. proficiency test sample. The tomato samples were either organically grown or contained 14 different pesticides. The experimental results showed a higher increase of the intracellular Ca(2+) concentration in cells treated with non-organic samples compared to the cells treated with organic samples.

Determination of thiram in natural waters using flow‐injection with cerium(IV)–quinine chemiluminescence system

A simple and rapid flow-injection chemiluminescence method has been developed for the determination of dithiocarbamate fungicide thiram based on the chemiluminescence reaction of thiram with ceric sulfate and quinine in aqueous sulfuric acid. The present method allowed the determination of thiram in the concentration range of 7.5-2500 ng/mL and the detection limit (signal-to-noise ratio = 3) was 7.5 ng/mL with sample throughput of 120/h. The relative standard deviation was 2.5% for 10 replicate analyses of 500 ng/mL thiram. The effects of foreign species including various anions and cations present in water at environmentally relevant concentrations and some pesticides were also investigated. The proposed method was applied to determine thiram in spiked natural waters using octadecyl bonded phase silica (C(18)) cartridges for solid-phase extraction. The recoveries were in the range 99 +/- 1 to 104 +/- 1%.

Surveillance of pesticide residues in fruits from Valencia during twenty months (2004/05)

The aim of this study was to investigate the pesticide residues in market fruits (oranges, tangerines, nectarines, peaches and khakis) from one Valencian Cooperative (Spain) and to conduct a monitoring of 32 organophosphorous, organonitrogen and organohalogenated pesticides and nine dithiocarbamate fungicides (DTCFs) usually applied on cultures of this area. Extracts were obtained by an official procedure for routine analysis based on ethyl acetate extraction. Residues of pesticides were determined by gas chromatography with nitrogen phosphorous detector (NPD), electron-capture detector (ECD) and mass spectrometry (MS) detectors. Mean recoveries obtained at fortification levels between 0.05 and 5 mg kg −1 were in range of 56–97% with relative standard deviations (RSDs) from 5% to 18%. The limits of quantification (LOQs) were in range of 0.1–140 μg kg −1 and lower than maximum residue limits (MRL) established by the Spanish legislation. 73% of the samples had no detectable residues. Of the contaminated samples, 13.8% exceeded the maximum residue limits (MRLs). Peaches and oranges showed the lowest contamination rates (13.9% and 21.3%, respectively). The contamination and violation rates were similar than the percentages recorded in previous monitoring studies in the same Community. Estimated daily intakes (EDIs) were calculated for these compounds in basis of European maximum residue limits (MRL) and residues found in the analysed samples and were compared with the acceptable daily intakes (ADIs).

Controlled release of the fungicide thiram from starch–alginate–clay based formulation

In order to modify the release of thiram, a dithiocarbamate fungicide, from starch–alginate beads and to minimize the agro-environmental pollution, kaolin and bentonite have been used as adsorbent in the bead formulations. Beads of different compositions were prepared by varying the amount of kaolin and bentonite and characterizing it by Fourier transform infrared spectroscopy (FTIR), scanning electron micrograph (SEM), energy dispersion analysis (EDAX), thermogravimetry and swelling studies. Formulation characteristics like entrapment efficiency, bead size, percentage equilibrium swelling of the beads and diffusion mechanism for thiram release have been evaluated. The incorporation of kaolin and bentonite in starch–alginate beads has controlled the release profile of thiram. The release from the bentonite-based formulations was slower than from the kaolin based formulations. The release proceeded as non-Fickian diffusion.

Photodegradation and Flow-Injection Determination of Dithiocarbamate Fungicides in Natural Water with Chemiluminescence Detection

A simple and rapid flow-injection method is reported for the determination of dithiocarbamate fungicides (maneb, nabam and thiram) based on chemiluminescence detection. The method involves the photodegradation of dithiocarbamate fungicides via UV light in an alkaline medium. Photoproducts are then reacted with luminol in the absence of an oxidant. Linear calibration graphs were obtained in the range 0.01 - 4.0 mg L(-1) for maneb and nabam and 0.05 - 1.0 mg L(-1) for thiram with relative standard deviations (n = 4) in the range 1.0 - 2.6%. The detection limits (S/N = 3) of maneb, nabam and thiram were 10, 8.0 and 5.0 ng mL(-1), respectively, with a sample throughput of 100 h(-1). The method was successfully applied to determine these dithiocarbamate fungicides in spiked natural water samples.