In vitro evaluation of pyrethroid-mediated changes on neuronal burst parameters using microelectrode arrays.

Effects of pyrethroids (beta-cyfluthrin, bifenthrin, cypermethrin, deltamethrin, lambda-cyhalothrin, and permethrin) on the burst parameters (mean burst rate [MBR], percent spikes in burst [PSB], mean burst duration [MBD], mean spikes in burst [MSB], mean interspike interval in burst [MISIB], and mean interburst interval [MIBI]) have been investigated using the microelectrode array technique. Rat cortical neuronal networks (between 24 and 35 DIV) were exposed to the five accumulative concentrations of pyrethroids (0.01µM, 0.1µM, 1µM, 10µM, and 100µM) after initially recording the baseline activity. When compared to the baseline, the burst parameter that had undergone the most change (either increase/decrease) at the initial concentrations was MBR, followed by MIBI and PSB. The other burst parameters (MSB, MBD, and MISIB) did not undergo much change (either increase/decrease) by the pyrethroids at the initial concentrations when compared to the baseline. The MBR of all pyrethroids rose at initial concentrations followed by decrease at higher concentrations. A drop in the MIBI accompanied the rise in the MBR. The rank orders of relative potency of pyrethroids on the IC50 of different burst parameters clearly distinguish type-1 pyrethroids (bifenthrin, permethrin) from the type-2 pyrethroids (beta-cyfluthrin, cypermethrin, deltamethrin, lambda-cyhalothrin), with type-2 being more potent. The rank order of relative potency of pyrethroids based on the IC50 of MBR was beta-cyfluthrin>lambda-cyhalothrin>deltamethrin>cypermethrin>bifenthrin>permethrin.

Exposure to cypermethrin and mancozeb alters the expression profile of THBS1, SPP1, FEZ1 and GPNMB in human peripheral blood mononuclear cells.

The complex immune system displays a coordinated transcriptional response to xenobiotic exposure by altering expression of designated transcription factors that, in turn, trigger immune responses. Despite the identification of several transcription factors that contribute to regulatory response, very little is known about the specific role of factors that are triggered due to exposure to obnoxious pesticides. Here, for the first time, alterations in human peripheral blood lymphocyte expression of transcriptional factors - thrombospondin-1 (THBS-1), secretory phospho-protein-1 (SPP-1), glycoprotein non-metastatic-ß (GPNMB) and fasciculation and elongation factor ζ-1 (FEZ-1), due to in vitro exposure to the crop protection chemicals cypermethrin and mancozeb are reported. Results revealed significant changes in expression profiles due to mancozeb exposure, supporting its immune dysfunction potential; in contrast, cypermethrin exposure did not cause significant changes. Based on these effects on gene expression across the doses tested, it was likely key components of immune mechanisms such as proliferation, cell adhesion, apoptosis and cell activation in human PBMC were affected. Although these data are from in vitro experiments, the results point out the potential role for changes in these factors in the etiology of defective T-cell immune function seen in humans occupationally exposed to crop protection chemicals like mancozeb. These studies suggest the involvement of transcription factors in regulation of pesticide-induced immune dysfunction; these studies also represent a novel approach for identifying potential immune-related dysfunctions due to exposure to pesticides. Further studies are needed to better understand the functional significance of these in vitro findings.

Human mesenchymal stem cells as a novel platform for simultaneous evaluation of cytotoxicity and genotoxicity of pharmaceuticals.

The in vitro micronucleus test is a well-known test for the screening of genotoxic compounds. However until now, most studies have been performed on either human peripheral lymphocytes or established cancer cell lines. This study provides human mesenchymal stem cells as an alternative to the conventional micronucleus test. We grew umbilical cord mesenchymal stem cells (UC-MSCs) on coverslips eliminating the cumbersome technique involving hypotonic treatment, fixation and preparing smears required for suspension culture (lymphocytes). The background frequency of nuclear blebs and micronuclei in UC-MSCs was found to be 7±5, in lymphocytes 16±3.5 and 9±3 and that for A549 cell line was 65±5 and 15±5 per 1000 cells, respectively, suggesting differences in the repair mechanism of normal and cancer cell lines. We inspected the cytotoxic and genotoxic effects of two known mutagens, mitomycin-C and hydrogen peroxide (H2O2), on UC-MSCs, lymphocytes and A549 cells. Treatment with mitomycin-C and H2O2 demonstrated drastic differences in the degree of cytotoxicity and genotoxicity suggesting a constitutional difference between normal and cancer cells. In addition we tested two solvents, dimethyl sulfoxide (DMSO) and ethanol, and two drugs, metformin and rapamycin. DMSO above 1% was found to be cytotoxic and genotoxic, whereas ethanol at same concentration was neither cytotoxic nor genotoxic indicating the minimal non-toxic level of the solvents. This study thus offers UC-MSCs as a better substitute to peripheral lymphocytes and cancer cell lines for high throughput screening of compounds and reducing the animal studies.

In vitro myelotoxic effects of cypermethrin and mancozeb on human hematopoietic progenitor cells.

In the past two decades, hematologic and immunologic disorders in humans have been increasingly reported as a result of pesticide exposures. Therefore, safety assessment is required to assess the effects on hematopoiesis and thus on the immune system in addition to routine toxicity evaluation. Currently, the data available on effects of pesticides on hematopoiesis in humans is limited. In the study here, cypermethrin and mancozeb were evaluated for their possible effects on hematopoiesis in vitro. Hematopoietic stem or progenitor cells from human cord blood were isolated and then exposed for 14 days to cypermethrin or mancozeb at non-cytotoxic doses (0.9-16 µM), and the effect on hematopoiesis screened via a methylcellulose-based clonogenic assay. Results indicated there were significant concentration-related decreases in clonogenic potentials of erythroid and granulocyte-macrophage colony formation. The inhibitory concentration (IC50) value with erythroid progenitors for cypermethrin was 8.7 [± 0.2 µM; mean [± SE]) and for mancozeb 6.2 [± 0.2] µM. Similarly, IC50 values with granulocyte-macrophage progenitors for cypermethrin and mancozeb were 19.2 [± 1.0] and 8.1 [± 0.2] µM, respectively. These data suggest that erythroid progenitors are perhaps more sensitive to these pesticides. Still, further studies are needed to understand the functional significance of these in vitro findings. For now, these data, albeit preliminary, emphasize the need to include an expanded battery of tests to understand effects on immune parameters in pre-clinical safety studies with pesticides. This study also emphasizes the utility of human cord blood in assessing potential effects on hematopoiesis in vitro.

Proliferation and TH1/TH2 cytokine production in human peripheral blood mononuclear cells after treatment with cypermethrin and mancozeb in vitro.

In recent times, human cell-based assays are gaining attention in assessments of immunomodulatory effects of chemicals. In the study here, the possible effects of cypermethrin and mancozeb on lymphocyte proliferation and proinflammatory (tumor necrosis factor (TNF-) α) and immunoregulatory cytokine (interferon- (IFN-) γ, interleukins (IL) 2, 4, 6, and 10) formation in vitro were investigated. Human peripheral blood mononuclear cells (PBMC) were isolated and exposed for 6 hr to noncytotoxic doses (0.45-30 µM) of cypermethrin or mancozeb in the presence of activating rat S9 fraction. Cultures were then further incubated for 48 or 72 hr in fresh medium containing phytohemagglutinin (10 µg/mL) to assess, respectively, effects on cell proliferation (BrdU-ELISA method) and cytokine formation (flow cytometric bead immunoassays). Mancozeb induced dose-dependent increases in lymphocyte proliferation, inhibition of production of TNFα and the TH2 cytokines IL-6 and IL-10, and an increase in IFNγ (TH1 cytokine) production (at least 2-fold compared to control); mancozeb also induced inhibition of IL-4 (TH2) and stimulated IL-2 (TH1) production, albeit only in dose-related manners for each. In contrast, cypermethrin exposure did not cause significant effects on proliferation or cytokine profiles. Further studies are needed to better understand the functional significance of our in vitro findings.

Toxicity of zinc oxide nanoparticles through oral route.

This experiment was aimed to determine the significance of dose by comparing acute oral toxicological potential of nano-sized zinc oxide (20 nm) with its micro-sized zinc oxide. Sprague Dawley rats, 8 to 9 weeks old, were administered with 5, 50, 300, 1000 and 2000 mg/kg body weight (b.w.) of nano- and micro-sized zinc oxide suspended in distilled water once through oral gavage. The effects of the micro- and nano-sized zinc oxide on biochemical and hematological parameters were analyzed on day 14 of administration. The organs were collected for histopathology. Interestingly, inverse dose-dependent increase was noted in aspartate aminotransferase, alanine aminotransferase serum levels of nano-size zinc oxide groups when compared with their micro-sized zinc oxide. Clotting time was effected in all the male groups of nano-size zinc oxide, except in 1000 mg/kg b.w. The incidences of microscopic lesions in liver, pancreas, heart and stomach were higher in lower doses of nano-size zinc oxide compared to higher dose. However, the incidences of above lesions were higher in rats treated with a high dose of micro-sized zinc oxide. We conclude that nano-size zinc oxide exhibited toxicity at lower doses, thus alarming future nanotoxicology research needs to be focused on importance of dose metrics rather following the conventional methods while conducting in vivo experiments.

Cytogenetic analysis of 52 Indian patients with de novo myelodysplastic syndromes-a comparative analysis of results with reports from Asia.

Most published data on myelodysplastic syndromes (MDS) are derived from Western countries, which report MDS as a disease of the elderly. However, it was observed that Asian MDS patients were younger than subjects in Western reports. With this in mind, the study was conducted prospectively on 52 Indian patients to define chromosomal abnormalities and to understand ethno-geographical differences, if any, underlying the pathogenesis of MDS among this Asian population. Cytogenetic analysis was performed using GTG banding and karyotyped according to the International System for Human Cytogenetic Nomenclature (ISCN). The incidence of MDS was predominant in the age group of 41-60 years (44.23%), with a median age at diagnosis of 55 years. The disease was more frequent in males (33 patients, 63.46%) than females (19 patients, 36.53%). Of 48 patients successfully karyotyped, 17 had normal karyotype (35.4%) and 31 patients (64.5%) had a chromosomal abnormality. The most frequent chromosome abnormalities were del 5q/-5 in 13 patients (42%), -7/7q- in 10 patients (32.2%), +8 and del 20q- in 6 cases each (19.3%) and i(17)(q10) in 1 patient (3.2%). In addition to these non-random chromosomal abnormalities, some rare abnormalities were also encountered. A higher rate of transformation to acute myeloid leukaemia (AML) was observed in the Chinese population compared to other Asian countries. The incidence of chromosomal abnormalities varied considerably across the different Asian populations. The overall frequency of chromosomal abnormalities in our study was comparable to most Western reports. Further prospective studies are warranted to elucidate precisely the ethnic differences in the pathogenesis of MDS in the Indian population.