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Genotoxicity induced by nanoparticles
- 1. Anju Surendranath Division of Toxicology GENOTOXICITY INDUCED BY NANOPARTICLES GENOTOXICITY INDUCED BY NANOPARTICLES
- 2. Nanoparticles Nanoparticle research is currently an area of intense scientific research, due to a wide variety of potential applications in biomedical, optical, and electronic fields. A nanoparticle is a microscopic particle with at least one dimension less than 100 nm which attributes to its typical properties.
- 5. SURFACE FUNCTIONALISATION Donaldson et al., Env Health Persp, 2008
- 7. NANOMATERIAL TOXICOLOGY Oberdoster et al, Env health persp, 2008
- 8. MECHANISM OF NP ENTRY Asharani et al., ACS Nano, 2008
- 10. NP Exposure Asharani et al., ACS Nano, 2008
- 11. NP s Tan et al., ACS Nano, 2018
- 17. DNA LADDERING ASSAY Donaldson et al., Nanotox, 2004
- 18. COMET ASSAY & MICRONUCLEUS
- 23. Micronucleus Study Donaldson et al., Nanotox, 2004This assay is now recognized as one of the most successful and reliable assays for genotoxic carcinogens
- 26. Research Paper Title: High Throughput Screening Platform for Engineered Nanoparticle- Mediated Genotoxicity Using Comet Chip Technology Aim: Designing a CometChip platform to enable efficient and reliable measurement of NP-mediated DNA damage for nano-genotoxicity studies
- 28. METHODOLOGY ENPs synthesis and characterization Microfabrication of a microwell array ENP suspension preparation and cell exposure Agarose encapsulation of exposed cells and electrophoresis Comet imaging Assessing NP mediated DNA damage using comet chip platform Comparison of DNA damage using traditional Comet assay
- 30. SSA (specific surface area by BET method), XRD for particle diameter. DLS for hydrodynamic size determination, Polydispersity index and zeta potential for both MEM and RPMI dispersed ENPs.
- 31. TEM image of ENP. (A) Silver, (B) Iron oxide, (C) Silica, (D) Cerium Oxide and (E) Zinc Oxide nanoparticles
- 33. Qualitative images of ENP mediated damage in TK1 cells using both comet chip and standard comet assays. (A) media treated control cells, (B) TK6 cells were exposed to industrially relevant ENPs at 5, 10 and 20ug/ml concentrations for 4h and evaluated using Comet Chip technology. Expanded view shows ZnO NPs treated TK6 cells revealing significant DNA damage. (C) traditional comet assay of TK6 cells treated with ZnO NPs at 20ug/ml for 4hr for comparison. . Scale bar 100um.
- 34. Evaluation of TK6 cells and H973 cells exposed to 5, 10 and 20ug/ml concentrations of ENPs where (A,D) DNA damage, (B,E) cellular viability and (C,F) CyQuant NF assessments were performed at 4h and 24h post exposure.
- 35. CONCLUSION •Demonstrated the efficacy of high throughput platform for assessing DNA damage in mammalian cells induced by ENP exposure. •In addition to its high throughput nature, the proposed comet chip approach has advantages over traditional comet assay. •The microfabricated cell array, 96 well plate format and automated image processing reduces noise, bias and labor but more importantly increases efficiency by two folds compared to traditional comet assay. •Observed significant levels of single stranded DNA damage in both TK6 and H9T3 cells demonstrating the efficacy of this platform to produce robust sets of data to capture initial molecular events. •Altogether the CometChip method could prove to be a powerful tool for assessing DNA damage and possible gene mutations of ENPs enhancing its ability to predict in vivo genotoxicity.
- 36. REFERENCES • Tan, K.X., Barhoum, A., Pan, S. and Danquah, M.K., 2018. Risks and toxicity of nanoparticles and nanostructured materials. In Emerging ACS Nano, 121-139. • Donaldson, K., Stone, V., Tran, C.L., Kreyling, W. and Borm, P.J., 2004. Nanotoxicology. • Oberdörster, G., Oberdörster, E. and Oberdörster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environmental health perspectives, 113(7), pp.823- 839. • AshaRani, P.V., Low Kah Mun, G., Hande, M.P. and Valiyaveettil, S., 2008. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS nano, 3(2), pp.279-290.
Editor's Notes
- The in vivo test normally uses mouse bone marrow or mouse peripheral blood. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. Simple Giemsa staining was originally used for MN scoring. Later, cytokinesis-block micronucleus (CBMN) method was reported, where Cyt-B, an inhibitor of the spindle assembly, was used to prevent cytokinesis occurred after nuclear division. CBMN method is used for the assessment of chromosomal loss, breakage, and associated apoptosis and necrosis induced by different mutagens. A micronucleus is the erratic (third) nucleus that is formed during the anaphase of mitosis or meiosis. cytoplasmic bodies having a portion of acentric chromosome or whole chromosome which was not carried to the opposite poles during the anaphase. Their formation results in the daughter cell lacking a part or all of a chromosome. These chromosome fragments or whole chromosomes normally develop nuclear membranes and form as micronuclei as a third nucleus. There is a chance of more than one micronucleus forming when more genetic damage has happened. The micronucleus test is used as a tool for genotoxicity assessment of various chemicals
- When DNA damage, whether it is endogenous or exogenous, forms double stranded breaks (DSBs), it is always followed by the phosphorylation of the histone, H2AX. H2AX is a variant of the H2A protein family, which is a component of the histone octomer in nucleosomes. It is phosphorylated by kinases such as ataxia telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) in the PI3K pathway. This newly phosphorylated protein, gamma-H2AX, is the first step in recruiting and localizing DNA repair proteins. DSBs can be induced by mechanisms such as ionizing radiation or cytotoxic agents and subsequently, gamma-H2AX foci quickly form. These foci represent the DSBs in a 1:1 manner and can be used as a biomarker for damage. An antibody can be raised against gamma-H2AX which can therefore be visualized by immunofluorescence through secondary antibodies. The detection and visualization of gamma-H2AX by flow cytometry allow the assessment of DNA damage, related DNA damage proteins and DNA repair. Gamma-H2AX also has other applications in the detection of genomic damage caused by cytotoxic chemical agents and environmental and physical damage, especially in the context of cancer treatment and therapy.