Study of in vitro Genotoxicity of Nanoparticles: Cellular uptake, DNA interaction, and chromosome breakage

Abstract

Nanotechnology has attracted considerable attention in the scientific community ever since its emergence as a powerful basic and applied science tool. Nanoparticles have many special physicochemical properties, and thereby may contribute to extraordinary hazards for human health and the environment. The current major applications of TiO2 nanoparticles includes; selfcleaningcements, glass and paints; water purification systems; antifogging coatings for glass; and as a UV attenuating ingredient in lotions, sunscreens and cosmetics. The unique properties of TiO2 nanoparticles may alter the way they interact with biological molecules and consequently, their toxicity. According to IARC guideline the TiO2 NPs are classified under group 2-B, which means possibly carcinogen. The genotoxic effects of any particle could be of two types: Direct and Indirect. Direct method includes the ROS generation and the indirect method includes DNA binding. These all applications lead to the concern about the genotoxic effects of the TiO2 NPs. Here we aimed to study the clastogenicity of nanoparticles on metaphase chromosomes of short term cultured blood cells using chromosome aberration and COMET, the in vitro DNA interaction with TiO2 nanoparticles, the human blood cells with TiO2 nanoparticles in vitro and assess the cellular uptake through fluorescence microscopy The present study focuses on In vitro assessment of genotoxicity of Titanium Dioxide Nanoparticle by human cultured peripheral blood cells in terms of Chromosomal Aberration assay. The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells. Structural aberrations may be of two types, chromosome or chromatid. We aimed to check the genotoxicity risk assessment for TiO2 NPs. For that we had done two different assays: chromosome aberration assay, in which we got the dose dependent increase in aberration, and second COMET assay which is for checking DNA damage, in that we got significant increase in DNA damage than the control group. In case of DNA and TiO2 NPs binding, the uv spectrophometer was done. In that we got the native DNA at 259nm. After addition of TiO2 NPs with 25 to 100 (micromolar concentration), it indicated hyperchromicity (increase in absorption) with increase in the concentration of TiO2 NPs. That indicates the change in the complex as the TiO2 binds with DNA. For checking of cellular uptake of TiO2 NPs, we had coat them with tween 80, which is a surfactant with auto fluorescent properties. The coated NPs were checked under UV spectrophotometer which also had given us different absorption and fluorescence 32 | P a g e microscopy had given us fluorescence under blue filter. But after culture set up of 24 hours, and harvesting, the cellular uptake was not observed under the microscope. It could be because of less time incubation of the culture.