Ashutosh Kumar
Research interests
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InterestsBacteria, Oxidative Stress, Nanoparticles, Ecotoxicity
Other
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LanguagesEnglish, Hindi
Publications
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4.81Impact points
Mechanism of inhibition of the ATPase domain of human topoisomerase IIα by 1,4-benzoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, and 9,10-phenanthroquinone.
Toxicological sciences : an official journal of the Society of Toxicology. 01/2012;
The inhibition of human topoisomerase IIα (Hu-TopoIIα), a major enzyme involved in maintaining DNA topology, repair and chromosome condensation/decondensation results in loss of genomic integrity. In the present study, the inhibition of ATPase domain of Hu-TopoIIα as a possible mechanism of genotoxi... [more] The inhibition of human topoisomerase IIα (Hu-TopoIIα), a major enzyme involved in maintaining DNA topology, repair and chromosome condensation/decondensation results in loss of genomic integrity. In the present study, the inhibition of ATPase domain of Hu-TopoIIα as a possible mechanism of genotoxicity of 1,4-benzoquinone (BQ), hydroquinone (HQ), naphthoquinone (1,2-NQ and 1,4-NQ) and 9,10-phenanthroquinone (9,10-PQ) was investigated. In silico modeling predicted that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ could interact with Ser-148, Ser-149, Asn-150 and Asn-91 residues of the ATPase domain of Hu-TopoIIα. Biochemical inhibition assays with the purified ATPase domain of Hu-TopoIIα revealed that 1,4-BQ is the most potent inhibitor followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ. Ligand binding studies using isothermal titration calorimetry revealed that 1,4-BQ, HQ, 1,4-NQ, 1,2-NQ and 9,10-PQ enter into four sequentially binding site model inside the domain. 1,4-BQ exhibited strongest binding followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ as revealed by their average K(d) values. The cellular fate of such inhibition was further evidenced by an increase in the number of Hu-TopoIIα-DNA cleavage complexes in the human lung epithelial cells (BEAS-2B) using trapped in agarose DNA immunostaining (TARDIS) assay which utilizes antibody specific for Hu-TopoIIα. Further, the increase in γ-H2A.X levels quantitated by flow cytometry and visualized by immunofluorescence microscopy illustrated that accumulation of DNA double strand breaks (DSBs) inside the cells can be attributed to the inhibition of Hu-TopoIIα. These findings collectively suggest that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ, inhibit the ATPase domain and potentially result in Hu-Topo IIα mediated clastogenic and leukemogenic events.
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5.74Impact points
TiO(2) nanoparticles induce oxidative DNA damage and apoptosis in human liver cells.
Nanotoxicology. 11/2011;
Abstract Titanium dioxide nanoparticles (TiO(2) NPs), widely used in consumer products, paints, pharmaceutical preparations and so on, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses in vitro and in vivo. The present study revealed that TiO(2) NPs induce significant (... [more] Abstract Titanium dioxide nanoparticles (TiO(2) NPs), widely used in consumer products, paints, pharmaceutical preparations and so on, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses in vitro and in vivo. The present study revealed that TiO(2) NPs induce significant (p < 0.05) oxidative DNA damage by the Fpg-Comet assay even at 1 µg/ml concentration. A corresponding increase in the micronucleus frequency was also observed. This could be attributed to the reduced glutathione levels with concomitant increase in lipid peroxidation and reactive oxygen species generation. Furthermore, immunoblot analysis revealed an increased expression of p53, BAX, Cyto-c, Apaf-1, caspase-9 and caspase-3 and decreased the level of Bcl-2 thereby indicating that apoptosis induced by TiO(2) NPs occurs via the caspase-dependent pathway. This study systematically shows that TiO(2) NPs induce DNA damage and cause apoptosis in HepG2 cells even at very low concentrations. Hence the use of such nanoparticles should be carefully monitored.
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5.33Impact points
miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2.
The Journal of biological chemistry. 08/2011; 286(43):37347-57.
In chronic alcoholism, brain shrinkage and cognitive defects because of neuronal death are well established, although the sequence of molecular events has not been fully explored yet. We explored the role of microRNAs (miRNAs) in ethanol-induced apoptosis of neuronal cells. Ethanol-sensitive miRNAs ... [more] In chronic alcoholism, brain shrinkage and cognitive defects because of neuronal death are well established, although the sequence of molecular events has not been fully explored yet. We explored the role of microRNAs (miRNAs) in ethanol-induced apoptosis of neuronal cells. Ethanol-sensitive miRNAs in SH-SY5Y, a human neuroblastoma cell line, were identified using real-time PCR-based TaqMan low-density arrays. Long-term exposure to ethanol (0.5% v/v for 72 h) produced a maximum increase in expression of miR-497 (474-fold) and miR-302b (322-fold). Similar to SH-SY5Y, long-term exposure to ethanol induced miR-497 and miR-302b in IMR-32, another human neuroblastoma cell line. Using in silico approaches, BCL2 and cyclin D2 (CCND2) were identified as probable target genes of these miRNAs. Cotransfection studies with 3'-UTR of these genes and miRNA mimics have demonstrated that BCL2 is a direct target of miR-497 and that CCND2 is regulated negatively by either miR-302b or miR-497. Overexpression of either miR-497 or miR-302b reduced expression of their identified target genes and increased caspase 3-mediated apoptosis of SH-SY5Y cells. However, overexpression of only miR-497 increased reactive oxygen species formation, disrupted mitochondrial membrane potential, and induced cytochrome c release (mitochondria-related events of apoptosis). Moreover, ethanol induced changes in miRNAs, and their target genes were substantially prevented by pre-exposure to GSK-3B inhibitors. In conclusion, our studies have shown that ethanol-induced neuronal apoptosis follows both the mitochondria-mediated (miR-497- and BCL2-mediated) and non-mitochondria-mediated (miR-302b- and CCND2-mediated) pathway.
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6.08Impact points
Engineered ZnO and TiO(2) nanoparticles induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli.
Free radical biology & medicine. 08/2011; 51(10):1872-81.
Extensive use of engineered nanoparticle (ENP)-based consumer products and their release into the environment have raised a global concern pertaining to their adverse effects on human and environmental health. The safe production and use of ENPs requires improvement in our understanding of environme... [more] Extensive use of engineered nanoparticle (ENP)-based consumer products and their release into the environment have raised a global concern pertaining to their adverse effects on human and environmental health. The safe production and use of ENPs requires improvement in our understanding of environmental impact and possible ecotoxicity. This study explores the toxicity mechanism of ZnO and TiO(2) ENPs in a gram-negative bacterium, Escherichia coli. Internalization and uniform distribution of characterized bare ENPs in the nano range without agglomeration was observed in E. coli by electron microscopy and flow cytometry. Our data showed a statistically significant concentration-dependent decrease in E. coli cell viability by both conventional plate count method and flow cytometric live-dead discrimination assay. Significant (p<0.05) DNA damage in E. coli cells was also observed after ENP treatment. Glutathione depletion with a concomitant increase in hydroperoxide ions, malondialdehyde levels, reactive oxygen species, and lactate dehydrogenase activity demonstrates that ZnO and TiO(2) ENPs induce oxidative stress leading to genotoxicity and cytotoxicity in E. coli. Our study substantiates the need for reassessment of the safety/toxicity of metal oxide ENPs.
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3.03Impact points
A flow cytometric method to assess nanoparticle uptake in bacteria.
Cytometry. Part A : the journal of the International Society for Analytical Cytology. 06/2011; 79(9):707-12.
Toxicity of engineered nanomaterials (ENMs), such as metal oxides, has been of concern among environmental and health scientists. For ecotoxicity studies of ENMs, it is important to assess nanoparticle uptake and correlate it with the cellular response. However, due to nonavailability of adequate me... [more] Toxicity of engineered nanomaterials (ENMs), such as metal oxides, has been of concern among environmental and health scientists. For ecotoxicity studies of ENMs, it is important to assess nanoparticle uptake and correlate it with the cellular response. However, due to nonavailability of adequate methods for assessing cellular uptake of ENMs, there is a lack of information in this important area. In the present study, a method has been developed using flow cytometry, which allows for rapid detection of ENM internalization in live bacteria under different experimental conditions for several generations. Our data demonstrate significant internalization of Zinc oxide (ZnO) and Titanium (IV) oxide (TiO(2) ) nanoparticles (NPs) in Escherichia coli in a dose-dependent manner. ZnO NPs treatment exhibited a significant increase in the intensity of side scatter (SSC) with liver-S9 fraction (76, 94, and 181% increase) rather than without S9 (10.5, 24.5, and 125.9% increase) at 10, 40, and 80 μg/ml concentrations, respectively. This was due to the protein coating of NPs by the S9 fraction. A similar response was also observed on exposure to TiO(2) NPs (139 and 203% with S9 and 128 and 198% without S9). In a multigeneration study, this new method was able to detect the presence of ENMs in E. coli up to four generations. Our data demonstrate that this method can be used for assessing the uptake of ENMs in bacteria and provides a handle to toxicologists for ecotoxicity studies of economically important ENMs to ensure safer products in the market.
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1.59Impact points
Titanium dioxide nanoparticles induce oxidative stress-mediated apoptosis in human keratinocyte cells.
Journal of biomedical nanotechnology. 02/2011; 7(1):100-1.
Titanium dioxide nanoparticles (TiO2 NPs) are the most commonly used metal oxide NPs in various industrial and commercial products. The present study has demonstrated a significant cellular uptake of TiO2 NPs in the human keratinocyte cells (HaCaT) using transmission electron microscopy and flow cyt... [more] Titanium dioxide nanoparticles (TiO2 NPs) are the most commonly used metal oxide NPs in various industrial and commercial products. The present study has demonstrated a significant cellular uptake of TiO2 NPs in the human keratinocyte cells (HaCaT) using transmission electron microscopy and flow cytometry. The data exhibited a significant (p < 0.05) concentration dependent decrease in cell viability and glutathione with concomitant increase in lipid peroxidation and reactive oxygen species. The increased oxidative stress further leads to apoptosis after 48 h of exposure. Our study demonstrates oxidative stress mediated apoptosis in human keratinocyte cells exposed to TiO2 NPs.
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1.59Impact points
Cellular response to metal oxide nanoparticles in bacteria.
Journal of biomedical nanotechnology. 02/2011; 7(1):102-3.
The exponential growth in the production and consumption of engineered nanoparticles (ENPs) has raised concern about their environmental fate. ENPs accumulation in ecosystems is likely to pose threat to specific and non-specific targets. In this study a novel approach using flow cytometry was valida... [more] The exponential growth in the production and consumption of engineered nanoparticles (ENPs) has raised concern about their environmental fate. ENPs accumulation in ecosystems is likely to pose threat to specific and non-specific targets. In this study a novel approach using flow cytometry was validated for detection of ENPs (ZnO and TiO2) uptake in live bacteria for several generations. These ENPs also induced frameshift mutation in S. typhimurium strains of Ames test, thus underscoring their possible carcinogenic potential.
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1.59Impact points
The need for novel approaches in ecotoxicity of engineered nanomaterials.
Journal of biomedical nanotechnology. 02/2011; 7(1):79-80.
The discovery of new diverse and novel applications for engineered nanomaterials (ENMs) creates possibilities of inadvertent release in the environment. The interactions of ENMs with living systems and possible impact/adverse effects cannot be predicted due to novel size and physiochemical propertie... [more] The discovery of new diverse and novel applications for engineered nanomaterials (ENMs) creates possibilities of inadvertent release in the environment. The interactions of ENMs with living systems and possible impact/adverse effects cannot be predicted due to novel size and physiochemical properties. The biological variability, dose dilemma, exposure methods, lack of suitable models and reference materials are certain important constraints in risk assessment of ENPs. Hence, we have explored the efficacy of microorganisms as models to understand uptake, accumulation and mechanism of adverse effects induced by selected nanoparticles.
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3.25Impact points
Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells.
Chemosphere. 02/2011; 83(8):1124-32.
Extensive production and consumption of nanomaterials such as ZnO and TiO(2) has increased their release and disposal into the environment. The accumulation of nanoparticles (NPs) in ecosystem is likely to pose threat to non-specific targets such as bacteria. The present study explored the effect of... [more] Extensive production and consumption of nanomaterials such as ZnO and TiO(2) has increased their release and disposal into the environment. The accumulation of nanoparticles (NPs) in ecosystem is likely to pose threat to non-specific targets such as bacteria. The present study explored the effect of ZnO and TiO(2) NPs in a model bacterium, Salmonella typhimurium. The uptake of ZnO and TiO(2) bare NPs in nano range without agglomeration was observed in S. typhimurium. TEM analysis demonstrated the internalization and uniform distribution of NPs inside the cells. Flow cytometry data also demonstrates that both ZnO and TiO(2) NPs were significantly internalized in the S. typhimurium cells in a concentration dependent manner. A significant increase in uptake was observed in the S. typhimurium treated even with 8 and 80 ng mL(-1) of ZnO and TiO(2) NPs with S9 after 60 min, possibly the formation of micelles or protein coat facilitated entry of NPs. These NPs exhibited weak mutagenic potential in S. typhimurium strains TA98, TA1537 and Escherichia coli (WP2uvrA) of Ames test underscoring the possible carcinogenic potential similar to certain mutagenic chemicals. Our study reiterates the need for re-evaluating environmental toxicity of ZnO and TiO(2) NPs presumably considered safe in environment.
Following (18)
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Gaurav Kumar
VIT University -
Anand Kumar Singh
CSIR-Indian Institute of Toxicology Research -
Premendra Dwivedi
Indian Institute of Toxicology Research -
Venu Gopal
National Institute of Pharmaceutical Education and Research -
Sandeep Kumar
Council of Scientific and Industrial Research (CSIR)
