RESUMO
DPM (diesel particulate matter) is ubiquitously present in the mining environment and is known for mutagenicity and carcinogenicity to humans. However, its health effects in surface coal mines are not well studied, particularly in India. In this study, DPM exposure and corresponding exposure biomarkers were investigated in four different surface coal mines in Central India. To document and evaluate the DPM exposure in surface coal miners, we characterized 1-NP (1-nitropyrene) in the mining environment as surrogate for DPM using Sioutas Cascade Impactor. Exposure biomarkers were analyzed by collecting post work shift (8-h work shift) urine samples and determining the concentrations of 1-aminopyrene (1-AP) as a metabolite of 1-NP and 8-hydroxydeoxyguanosine (8OHdG) as DNA damage marker. We observed high concentration of 1-NP (7.13-52.46 ng/m3) in all the mines compared with the earlier reported values. The average creatinine corrected 1-AP and 8OHdG levels ranged 0.07-0.43 [Formula: see text]g/g and 32.47-64.16 [Formula: see text]g/g, respectively, in different mines. We found 1-AP in majority of the mine workers' urine (55.53%) and its level was higher than that reported for general environmental exposure in earlier studies. Thus, the study finding indicates occupational exposure to DPM in all the four mines. However, the association between 1-NP level and exposure biomarkers (1-AP and 8OHdG) was inconsistent, which may be due to individual physiological variations. The data on exposure levels in this study will help to understand the epidemiological risk assessment of DPM in surface coal miners. Further biomonitoring and cohort study are needed to exactly quantify the occupational health impacts caused by DPM among coal miners.
Assuntos
Poluentes Ocupacionais do Ar , Minas de Carvão , Mineradores , Exposição Ocupacional , Poluentes Ocupacionais do Ar/análise , Carvão Mineral , Estudos de Coortes , Monitoramento Ambiental , Humanos , Índia , Exposição Ocupacional/análise , Material Particulado/análise , Pirenos , Emissões de Veículos/análiseRESUMO
Nitrobenzene, nitrotoluenes and nitrobenzoic acid are toxic and mutagenic. Their removal from the environment is necessary to avoid health and environmental damage. In this study, Cupriavidus strain a3 was found to utilize 2-nitrotoluene (2NT), 3-nitrotoluene (3NT), 4-nitrotoluene (4NT), nitrobenzene (NB) and 2-nitrobenzoic acid (2NBA) as carbon and nitrogen source, resulting in their detoxification. The metabolism involved reductive transformation of nitroaromatics to the corresponding amines followed by cleavage of amino group to release ammonia. Cell free extract showed nitroreductase activity in the range of 310-389 units/mg. NB was reduced to form benzamine and 4-aminophenol, 2NT was reduced to 2-aminotoluene, whereas 2NBA was reduced to form 2-aminobenzoic acid. Similarly, 3NT was metabolized to 3-aminotoluene and 2-amino-4-methylphenol, while 4NT was reduced to 4-nitrosotoluene and 4-aminotoluene. Cytotoxicity and apoptosis assays using Jurkat cell line, and Ames test were used to evaluate the detoxification of nitroaromatics during biodegradation. Biodegradation with Cupriavidus resulted in 2.6-11 fold increase in cell viability, 1.3-2.3 fold reduction in apoptosis, 1.6-55 fold reduction in caspase-3 activation, and complete disappearance of mutagenic activity. In soil microcosm, bioaugmentation with Cupriavidus resulted in 16-59% degradation of various nitroaromatics, as against <14% degradation without bioaugmentation. Thus, the present study reflects promising capability of Cupriavidus strain a3 in degradation and detoxification of multiple nitroaromatics.
Assuntos
Biodegradação Ambiental , Cupriavidus/fisiologia , Poluentes Ambientais/metabolismo , Nitrobenzenos , Solo , Tolueno/análogos & derivados , ToluidinasRESUMO
Fluoride is an essential trace element required for proper bone and tooth development. Systemic high exposure to fluoride through environmental exposure (drinking water and food) may result in toxicity causing a disorder called fluorosis. In the present study, we investigated the alteration in DNA methylation profile with chronic exposure (30 days) to fluoride (8â¯mg/l) and its relevance in the development of fluorosis. Whole genome bisulfite sequencing (WGBS) was carried out in human osteosarcoma cells (HOS) exposed to fluoride. Whole genome bisulfite sequencing (WGBS) and functional annotation of differentially methylated genes indicate alterations in methylation status of genes involved in biological processes associated with bone development pathways. Combined analysis of promoter DNA hyper methylation, STRING: functional protein association networks and gene expression analysis revealed epigenetic alterations in BMP1, METAP2, MMP11 and BACH1 genes, which plays a role in the extracellular matrix disassembly, collagen catabolic/organization process, skeletal morphogenesis/development, ossification and osteoblast development. The present study shows that fluoride causes promoter DNA hypermethylation in BMP1, METAP2, MMP11 and BACH1 genes with subsequent down-regulation in their expression level (RNA level). The results implies that fluoride induced DNA hypermethylation of these genes may hamper extracellular matrix deposition, cartilage formation, angiogenesis, vascular system development and porosity of bone, thus promote skeletal fluorosis.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Doenças Ósseas/induzido quimicamente , Metilação de DNA/efeitos dos fármacos , Água Potável/química , Exposição Ambiental/efeitos adversos , Fluoretos/toxicidade , Desenvolvimento Ósseo/genética , Doenças Ósseas/genética , Doenças Ósseas/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , Oligoelementos , Transcriptoma/efeitos dos fármacosRESUMO
The current study addresses the removal of an emerging environmental contaminant (primidone) in batch adsorption experiments using commercial-grade powdered activated charcoal (PAC). The experiments for the removal of primidone were performed to identify the effect of various adsorption parameters. The second-order rate expression best represented the adsorption kinetics data. The Freundlich isotherm equation was best fitted to the experimental adsorption data at equilibrium for removal of primidone using PAC. The values for change in entropy (ΔSo) were positive, which indicates that the degree of freedom of the process increases. The negative values of change in enthalpy (ΔHo) and change in Gibb's free energy (ΔGo) indicate that the physical adsorption is a dominant phenomenon, and the process is feasible and spontaneous. The negative value of ΔHo also represented the exothermicity of the adsorption process. The Taguchi optimization technique calculated the influence of variation of different process parameters, viz., initial pH (pH0), PAC dosage (m), initial adsorbate concentration (C0), solution temperature (T), and process contact time (t), on the removal of primidone by adsorption from aqueous solution. Each of the above parameters was examined at three levels to study their effects on the adsorptive uptake of primidone using PAC (qe, mg g-1), and the optimum value necessary to maximize qe was determined. The findings from the ANOVA indicate that the PAC dose (m) is the most notable parameter contributing 62.16% to qe and a 71.96% to the signal to noise (S/N) ratio data, respectively. The confirmation experiments performed at the optimum parameter condition validated the applicability of the Taguchi design of experiments. The percent removal and adsorptive uptake at the optimal condition were 86.11% and 0.258 mg g-1, respectively.
Assuntos
Carvão Vegetal/química , Modelos Teóricos , Primidona/análise , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Adsorção , Concentração de Íons de Hidrogênio , Cinética , Temperatura , TermodinâmicaRESUMO
Anthracosilicosis (AS), a prevalent form of pneumoconiosis among coal miners, results from the accumulation of carbon and silica in the lungs from inhaled coal dust. This study investigated genotoxic effects and certain cytokine genes polymorphic variants in Russian coal miners with ÐS. Peripheral leukocytes were sampled from 129 patients with AS confirmed by X-ray and tissue biopsy and from 164 asymptomatic coal miners. Four single-nucleotide polymorphisms were genotyped in the extracted DNA samples: IL1ß T-511C (rs16944), IL6 C-174G (rs1800795), IL12b A1188C (rs3212227) and VEGFA C634G (rs2010963). Genotoxic effects were assessed by the analysis of chromosome aberrations in cultured peripheral lymphocytes. The mean frequency of chromatid-type aberrations and chromosome-type aberrations, namely, chromatid-type breaks and dicentric chromosomes, was found to be higher in AS patients [3.70 (95% confidence interval {CI}, 3.29-4.10) and 0.28 (95% CI, 0.17-0.38)] compared to the control group [2.41 (95% CI, 2.00-2.82) and 0.09 (95% CI, 0.03-0.15)], respectively. IL1ß gene T/T genotype (rs16944) was associated with AS [17.83% in AS patients against 4.35% in healthy donors, odds ratio = 4.77 (1.88-12.15), P < 0.01]. A significant increase in the level of certain chromosome interchanges among AS donors is of interest because such effects are typical for radiation damage and caused by acute oxidative stress. IL1ß T allele probably may be considered as an AS susceptibility factor among coal miners.
Assuntos
Antracossilicose/genética , Estudos de Associação Genética , Interleucina-1beta/genética , Exposição Ocupacional , Adulto , Antracossilicose/etiologia , Antracossilicose/patologia , Aberrações Cromossômicas/efeitos dos fármacos , Carvão Mineral/efeitos adversos , Minas de Carvão , Dano ao DNA/efeitos dos fármacos , Predisposição Genética para Doença , Genótipo , Humanos , Subunidade p40 da Interleucina-12/genética , Interleucina-6/genética , Masculino , Pessoa de Meia-Idade , Mineradores , Polimorfismo de Nucleotídeo Único/genética , Dióxido de Silício/isolamento & purificação , Dióxido de Silício/toxicidade , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Environmental occurrence of CECs poses a great threat to both aquatic life and human health. The aim of this study was to optimize and validate SPE/LC-(ESI)MS-MS method for simultaneous quantitative monitoring of two sub-classes of CECs (pharmaceuticals and hormones) and to estimate the concentrations of select CECs in environmental water samples. For all the tested analytes, recoveries in laboratory reagent water were greater than 81%. Average percent (relative standard deviation) RSD of the analytes in recovery, repeatability, and reproducibility experiments were ≤ 10%. Determination coefficients (r2) of primidone, diclofenac, testosterone, and progesterone were estimated to be 0.9979, 0.9972, 0.9968, and 0.9962, respectively. Limits of detection (LOD) for primidone, diclofenac, testosterone, and progesterone were 4.63 ng/L, 5.36 ng/L, 0.55 ng/L, and 0.88 ng/L, respectively. Limits of quantification (LOQ) for primidone, diclofenac, testosterone, and progesterone were 14.72 ng/L, 17.06 ng/L, 1.766 ng/L, and 2.813 ng/L, respectively. Average recoveries in environmental water and wastewater samples were greater than 74% and RSD were ≤ 7%. Trace levels (68.33-125.70 ng/L) of primidone were detected in four environmental water samples, whereas diclofenac was not detected in any of the tested sample. Trace levels of progesterone were observed in two environmental samples (16.64 -203.73 ng/L), whereas testosterone was detected in STP inlet sample (178.16 ng/L).
Assuntos
Monitoramento Ambiental/métodos , Poluentes Químicos da Água/análise , Cromatografia Líquida/métodos , Diclofenaco , Humanos , Índia , Limite de Detecção , Reprodutibilidade dos Testes , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Águas ResiduáriasRESUMO
Manganese (Mn) is an essential trace element required for optimal functioning of cellular biochemical pathways in the central nervous system. Elevated exposure to Mn through environmental and occupational exposure can cause neurotoxic effects resulting in manganism, a condition with clinical symptoms identical to idiopathic Parkinson's disease. Epigenetics is now recognized as a biological mechanism involved in the etiology of various diseases. Here, we investigated the role of DNA methylation alterations induced by chronic Mn (100 µM) exposure in human neuroblastoma (SH-SY5Y) cells in relevance to Parkinson's disease. A combined analysis of DNA methylation and gene expression data for Parkinson's disease-associated genes was carried out. Whole-genome bisulfite conversion and sequencing indicate epigenetic perturbation of key genes involved in biological processes associated with neuronal cell health. Integration of DNA methylation data with gene expression reveals epigenetic alterations to PINK1, PARK2 and TH genes that play critical roles in the onset of Parkinsonism. The present study suggests that Mn-induced alteration of DNA methylation of PINK1-PARK2 may influence mitochondrial function and promote Parkinsonism. Our findings provide a basis to further explore and validate the epigenetic basis of Mn-induced neurotoxicity .
Assuntos
Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Manganês/toxicidade , Doença de Parkinson/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neuroblastoma/genética , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Exposure to pre-concentrated inlet or outlet STP wastewater extracts at different concentrations (0.001% to 1%) induced dose-dependent toxicity in MCF-7 cells, whereas drinking water extracts did not induce cytotoxicity in cells treated. GC-MS analysis revealed the occurrence of xenobiotic compounds (Benzene, Phthalate, etc.) in inlet/outlet wastewater extracts. Cells exposed to inlet/outlet extract showed elevated levels of reactive oxygen species (ROS: inlet: 186.58%, p<0.05, outlet, 147.8%, p<0.01) and loss of mitochondrial membrane potential (Δψm: inlet, 74.91%, p<0.01; outlet, 86.70%, p<0.05) compared to the control. These concentrations induced DNA damage (Tail length: inlet: 34.4%, p<0.05, outlet, 26.7%, p<0.05) in treated cells compared to the control (Tail length: 7.5%). Cell cycle analysis displayed drastic reduction in the G1 phase in treated cells (inlet, G1:45.0%; outlet, G1:58.3%) compared to the control (G1:67.3%). Treated cells showed 45.18% and 28.0% apoptosis compared to the control (1.2%). Drinking water extracts did not show any significant alterations with respect to ROS, Δψm, DNA damage, cell cycle and apoptosis compared to the control. Genes involved in cell cycle and apoptosis were found to be differentially expressed in cells exposed to inlet/outlet extracts. Herein, we propose cell-based toxicity assays to evaluate the efficacies of wastewater treatment and recycling processes.
Assuntos
Água Potável/análise , Reciclagem , Águas Residuárias/toxicidade , Poluentes Químicos da Água/toxicidade , Purificação da Água/métodos , Apoptose/efeitos dos fármacos , Análise da Demanda Biológica de Oxigênio , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Citometria de Fluxo , Humanos , Índia , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Águas Residuárias/análise , Poluentes Químicos da Água/análiseRESUMO
Recent developments in nanotechnology have facilitated the synthesis of novel engineered nanoparticles (ENPs) that possess new and different physicochemical properties. These ENPs have been ex tensive ly used in various commercial sectors to achieve both social and economic benefits. However. the increasing production and consumption of ENPs by many different industries has raised concerns about their possible release and accumulation in the environment. Released EN Ps may either remain suspended in the atmosphere for several years or may accumulate and eventually be modified int o other substances. Settled nanoparticles can he easily washed away during ra in s. and therefore may easily enter the food chain via water and so il. Thus. EN Ps can contaminate air. water and soil and can subsequently pose adverse risks to the health of different organisms. Studies to date indicate that ENP transport to and within the ecosystem depend on their chemical and physical properties (viz .. size. shape and solubility) . Therefore. the EN Ps display variable behavior in the environment because of their individual properties th at affect their tendency for adsorption, absorption, diffusional and colloidal interaction. The transport of EN Ps also influences their fate and chemical transformation in ecosystems. The adsorption, absorption and colloidal interaction of ENPs affect their capacity to be degraded or transformed, whereas the tendency of ENPs to agglomerate fosters their sedimentation. How widely ENPs are transported and their environmental fate influence how tox ic they may become to environmental organisms. One barrier to fully understanding how EN Ps are transformed in the environment and how best to characterize their toxicity, is related to the nature of their ultrafine structure. Experiments with different animals, pl ants, and cell lines have revealed that ENPs induce toxicity via several cellular pathways that is linked to the size. shape. surface area, agglomeration state. and sur face charge of the ENP involved. Future research is needed to elucidate the mechanisms by which nanoparticles act to induce their tox ic effects aft er they reach various ecosystems. Moreover. work is needed to develop a holistic approach for better understanding the effects that ENPs produce at the cellular and genetic level.
Assuntos
Nanopartículas/metabolismo , Nanopartículas/toxicidade , Animais , Transporte Biológico , Linhagem Celular , Dano ao DNA , Espécies Reativas de Oxigênio/metabolismoRESUMO
Present study reports the identification of genomic and proteomic signatures of endosulfan exposure in hepatocellular carcinoma cells (HepG2). HepG2 cells were exposed to sublethal concentration (15µM) of endosulfan for 24h. DNA microarray and MALDI-TOF-MS analyses revealed that endosulfan induced significant alterations in the expression level of genes and proteins involved in multiple cellular pathways (apoptosis, transcription, immune/inflammatory response, carbohydrate metabolism, etc.). Furthermore, downregulation of PHLDA gene, upregulation of ACIN1 protein and caspase-3 activation in exposed cells indicated that endosulfan can trigger apoptotic cascade in hepatocellular carcinoma cells. In total 135 transcripts and 19 proteins were differentially expressed. This study presents an integrated approach to identify the alteration of biological/cellular pathways in HepG2 cells upon endosulfan exposure.
Assuntos
Carcinoma Hepatocelular/genética , Endossulfano/toxicidade , Genômica/métodos , Inseticidas/toxicidade , Neoplasias Hepáticas/genética , Proteínas/química , Proteômica/métodos , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/metabolismo , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/química , Neoplasias Hepáticas/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMO
Anthropogenic activities have substantially increased the level of greenhouse gases (GHGs) in the atmosphere and are contributing significantly to the global warming. Carbon dioxide (CO2 ) is one of the major GHGs which plays a key role in the climate change. Various approaches and methodologies are under investigation to address CO2 capture and sequestration worldwide. Carbonic anhydrase (CA) mediated CO2 sequestration is one of the promising options. Therefore, the present review elaborates recent developments in CA, its immobilization and bioreactor methodologies towards CO2 sequestration using the CA enzyme. The promises and challenges associated with the efficient utilization of CA for CO2 sequestration and scale up from flask to lab-scale bioreactor are critically discussed. Finally, the current review also recommends the possible future needs and directions to utilize CA for CO2 sequestration.
Assuntos
Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Biodegradação Ambiental , Reatores Biológicos , Biotecnologia/tendências , Enzimas Imobilizadas/metabolismo , Aquecimento Global/prevenção & controleRESUMO
Cattle grazing nearby coal-fired power stations are exposed to fly ash. The present investigation aims to assess the environmental and health impacts of fly ash containing mercury emitted from thermal power plant. The health effect of fly ash were studied using 20 lactating cattle reared within a 5-km radius of s thermal power plant for the possible effect of fly ash such as the alterations in hematological and biochemical parameters of blood, milk, and urine. Results indicated that the hemoglobin levels (6.65 ± 0.40 g/dl) were significantly reduced in all the exposed animals. Biochemical parameters viz., blood urea nitrogen (27.35 ± 1.19 mg/dl), serum glutamate oxaloacetate transaminase (43.39 ± 3.08 IU/l), albumin, and creatinine were found to be increased, whereas serum glutamate pyruvic transaminase (29.26 ± 2.02) and Ca(2+) were observed to be statistically insignificant in exposed animals. Mercury concentrations estimated in the blood, milk, and urine of exposed (n = 20) and control (n = 20) animals were 7.41 ± 0.86, 4.75 ± 0.57, 2.08 ± 0.18, and 1.05 ± 0.07, 0.54 ± 0.03, 0.20 ± 0.02 µg/kg, respectively. The significant increase (P < 0.01) in the levels of mercury in blood, milk, and urine of exposed animals in comparison to control indicated that the alterations of biochemical parameters in exposed cattle could be due to their long term exposure to fly ash mercury which may have direct or indirect impact on human populations via food chain.
Assuntos
Poluentes Atmosféricos/toxicidade , Cinza de Carvão/toxicidade , Mercúrio/toxicidade , Centrais Elétricas , Poluentes Atmosféricos/análise , Poluentes Atmosféricos/metabolismo , Animais , Aspartato Aminotransferases/sangue , Bovinos , Cinza de Carvão/análise , Creatinina/sangue , Monitoramento Ambiental , Mercúrio/análise , Mercúrio/metabolismo , Material Particulado/análise , Material Particulado/metabolismo , Material Particulado/toxicidade , Albumina Sérica/metabolismoRESUMO
Multi-target-multi-drug approaches are needed to accelerate the process of drug discovery screening and to design efficient therapeutic strategies against diseases that involve alterations in multiple cellular targets. Herein we report single-cell cotransfection imaging cytometry to quantitatively screen drug-induced off-target effects. Vascular endothelial growth factor (VEGF) and histone deacetylase (HDAC) genes amplified from the genomic DNA were cloned in fluorescently tagged gene constructs (RFP-HDAC/YFP-VEGF). These gene constructs were cotransfected in HEK-293 cells to explore the possibility of off-target effects of 4-phenylbutyrate and Iressa on the expression of VEGF and HDAC through single-cell imaging cytometry. Iressa (10 µM) treatment at the time of cotransfection or 48 h after cotransfection of RFP-HDAC/YFP-VEGF plasmids in HEK-293 cells resulted in off-target effects on HDAC expression. These results suggest possible applications of Iressa in the treatment of diseases in which expression of both HDAC and VEGF should be inhibited. 4-Phenylbutyrate (2.0 mM) did not show any off-target effects on VEGF expression. The developed quantitative multicolor live single-cell cotransfection imaging can be employed to select better drug combinations for faster screening and greater accuracy in multi-target-multi-drug analysis by increasing the on-target/desired off-target effects and eliminating the undesirable off-target effects.
Assuntos
Descoberta de Drogas/métodos , Inibidores de Histona Desacetilases/farmacologia , Citometria por Imagem/métodos , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Gefitinibe , Células HEK293 , Histona Desacetilases/genética , Humanos , Fenilbutiratos/farmacologia , Transfecção/métodos , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Microplastics are considered to be ubiquitous and widespread emerging contaminants. They are persistent in the nature and pose considerable harm to the environment. Their omnipresence is documented in almost all aquatic habitats, several atmospheric and terrestrial environments, and also in human consumables. The objective of this review is to provide an overview of the environmental prevalence of the microplastics in all environmental compartments, and their possible adverse impacts. It also presents review of the studies conducted in India and the epitome of potential mitigation measures. The need and direction of future research are highlighted. The review will help in determining the exposure levels, environmental consequences, and risk estimations, and will guide the researchers and policymakers.
Assuntos
Microplásticos , Poluentes Químicos da Água , Monitoramento Ambiental , Humanos , Índia , Plásticos , Prevalência , Poluentes Químicos da Água/análiseRESUMO
In recent years, quantum dots (Qdot), with their unique physical, chemical, and optical properties, have been used extensively as probes to visualize several cell membrane receptors and extracellular biomolecules. However, Qdot-based intracellular imaging has always been associated with vital lacunas. High affinity between quantum dots may induce serious aggregation in the cytoplasm; as a result, quantum dot aggregates are usually misinterpreted as quantum dot-probed intracellular molecules. Moreover, due to the more viscous nature of the cytoplasm versus the extracellular aqueous media, aggregation issues become more severe during intracellular studies. In this work, we suggest direct nondestructive serotonin imaging in an intact cell using the quantum dot-based immunoassay with a rapid tunable multicolor imaging system based on the acousto-optic tunable filter. Any false-positive intracellular serotonin molecules that appeared due to the aggregation of quantum dots could be completely discriminated from the real intracellular serotonin granules through multicolor cellular imaging. The developed method is quick and has wide applicability in targeting various intracellular proteins, coenzymes, and micronutrients.
Assuntos
Microscopia de Fluorescência/métodos , Pontos Quânticos , Serotonina/química , Linhagem Celular Tumoral , Fixadores/química , Corantes Fluorescentes/química , Humanos , ImunoensaioRESUMO
Implementation of quantitative analytical approaches to image-based cellular assays remains a major challenge. We disclose a tool to achieve automatic rapid quantitative cellular imaging analysis based on uniform threshold intensity distribution. An acousto-optic tunable filter-based, quantitative multivariate imaging cytometer was set up to elucidate drug-induced cell death dynamics via cell viability and apoptosis/necrosis measurements in the human myeloid leukemia cell line, HL-60. Cells were treated with various drugs (camptothecin, naringenin, sodium salicylate) at different concentrations and time intervals. The developed protocol can directly depict and quantitate targeted cellular moieties, subsequently enabling a method that is applicable to various cellular assays with special reference to next generation drug discovery screening. This may also complement certain flow-cytometric measurements in studying quantitative physiology of cellular systems.
Assuntos
Citometria por Imagem/instrumentação , Citometria por Imagem/métodos , Sobrevivência Celular , Células HL-60 , HumanosRESUMO
Oligonucleotide chip-based assays can be a sample-thrifty, time-saving, routine tool for evaluation of chemical-induced DNA strand breaks. This article describes a novel approach using an oligonucleotide chip to determine photosensitizer-induced DNA single-strand breaks. Surface coverage of fluorophore-labeled oligonucleotides on silicon dioxide chip surfaces was determined on alkaline phosphatase digestion. Fluorescence maxima (at 520 nm) of the solutions were converted to molar concentrations of the fluorescein-modified oligonucleotide by interpolation from a predetermined standard linear calibration curve. The photosensitizing activity of chlorpromazine and triflupromazine toward DNA single-strand breaks was then studied at different drug doses and also as a function of photoirradiation time. Photoinduced single-strand breaks calculated using the method described here agreed with values predicted by theoretical extrapolation of the single-strand breaks obtained for plasmid DNAs from agarose gel electrophoresis, and thereby indirectly validated the chip-based assays. Under UV irradiation (>or=93.6 kJ/m2) chlorpromazine (>or=0.08 mM) was found to have significant photogenotoxicity. However, triflupromazine did not exhibit any (photo)genotoxicity over the concentration range studied (0.04-0.20mM). The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of breaks in DNA.
Assuntos
Quebras de DNA de Cadeia Simples/efeitos dos fármacos , DNA/metabolismo , Mutagênicos/toxicidade , Análise de Sequência com Séries de Oligonucleotídeos , Fármacos Fotossensibilizantes/toxicidade , Sequência de Bases , Clorpromazina/toxicidade , DNA/genética , Quebras de DNA de Cadeia Simples/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Propriedades de Superfície , Fatores de Tempo , Triflupromazina/toxicidade , Raios UltravioletaRESUMO
A new method was developed to determine the mutagenic efficacy of a suspected mutagen by employing green fluorescent protein (GFP) as a direct biosensor for mutation detection. Alterations in target gene (AcGFP1) expression after mutagen [(+/-)-7p,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)] treatment were measured to detect the mutagenic efficacy of the carcinogen. In contrast to mutagen treatment of the entire plasmid or cell culture, the target AcGFP1gene devoid of the plasmid backbone was exposed to BPDE (10-500 microM) to eliminate the need for an additional fusion gene. Shuttle vectors (pAcGFP-N1) were religated to the AcGFP1 gene with BPDE adducts (0-8.59 microM) and replicated in the eukaryotic host. This approach eliminated false-negative errors in target gene expression that arose from BPDE adduct formation in the residual plasmid backbone rather than in the AcGFP1 gene. Determination of the BPDE-AcGFP1 adducts allowed the quantitative mutagenic effect of the BPDE adducts on AcGFP1 gene expression to be monitored. The results obtained with flow cytometry and confocal microscopy validate our method and demonstrate efficient and direct use of GFP as a biosensor for mutation detection.
Assuntos
Técnicas Biossensoriais/métodos , Análise Mutacional de DNA/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/toxicidade , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Reações Falso-Negativas , Citometria de Fluxo , Humanos , Espaço Intracelular/metabolismo , Dados de Sequência Molecular , Mutagênicos/metabolismo , Mutagênicos/toxicidade , Plasmídeos/genética , Mutação PuntualRESUMO
The present study is aimed at evaluating the genotype frequency of detoxifying genes such as GSTM1, GSTT1 and NQO1 in Maharastrian population of central India. The study revealed about 64.6% of GSTM1-positive and 35.4% GSTM1-null population. GSTT1-positive genotype was found to be 87.5% and GSTT1-null showed 12.5%. The NQO1 genotype of Maharastrian population showed 52.3% of C/C, 42.48% C/T and 5.18% T/T. The NQO1 of this population does not deviate from the expected Hardy-Weinberg equilibrium. The genotype frequencies GSTM1 and GSTT1 of the population when compared with other ethnic groups of Asia and Caucasians show distinct nature of Maharastrian population from other Asian and Caucasian population.
Assuntos
Glutationa Transferase/genética , NAD(P)H Desidrogenase (Quinona)/genética , Polimorfismo Genético , População Branca/genética , Adulto , Genótipo , Humanos , Índia , Pessoa de Meia-IdadeRESUMO
The present study demonstrates apoptosis-inducing potential and mechanism of action of Tribulus terristris alkaloid extract in Jurkat E6-1 cancer cell line. Liquid Chromatography-Mass Spectrometry and High Resolution-Mass Spectrometry analysis identified the presence of four N-feruloyltyramine derivatives, namely trans-N-feruloyl-3-hydroxytyramine (1), trans-N-coumaroyltyramine (2), trans-N-feruloyltyramine (3) and trans-N-feruloyl-3-ethoxytyramine (4) in the alkaloid extract. Compounds 2 and 3 have not been yet reported in the alkaloid extract of T. terristris. In silico analysis revealed therapeutic potential of N-feruloyltyramine derivatives and strong binding efficiency to both chains of Tumor Necrosis Factor Receptor 1. Treatment of alkaloids extract to Jurkat E6-1 clone induced dose-dependent cytotoxicity (LC50 140.4 µg mL-1). Jurkat cells treated with alkaloids extract at sub-lethal concentration showed DNA fragmentation, enhancement in caspase-3 activity and phosphatidylserine translocation (apoptosis indicator) compared to control cells. Gene expression analysis using Human Apoptosis RT2 Profiler PCR Array analysis upon alkaloid treatment was found to significantly alter expression of critical genes such as TNFR1, FADD, AIFM, CASP8, TP53, DFFA and NFKB1. These genes are predicted to mediate apoptotic cell death via both intrinsic and extrinsic apoptosis pathway. In summary, we report the identification of new N-feruloyltyramine derivatives from alkaloid extract of T. terristris fruit with probable anti-leukemic and pharmacological potential.