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TiO2 nanoparticles induced micronucleus formation in human liver (HepG2) cells: comparison of conventional and flow cytometry based methods
© Vallabani et al; licensee BioMed Central Ltd. 2014
Published: 21 January 2014
TiO2 nanoparticles (TiO2 NPs) are extensively used metal oxide NPs in cosmetics, as an additive in pharmaceuticals, food colorant etc. Being widely used NPs, their toxicity assessment studies help in understanding the adverse effects to the humans. It is likely that NPs exposure to the humans can be through different routes but will finally reach the liver. Therefore, an attempt was made to explore the genotoxicity of TiO2 NPs on human liver cells (HepG2).
Materials and methods
TiO2 NPs were characterized by transmission electron microscopy (TEM) for their primary size, shape and dynamic light scattering for their size, size distribution and zeta potential in culture medium. Cellular uptake of TiO2 NPs in HepG2 cells was detected using flow cytometry method. Moreover, ultrathin sections of cells were analysed using TEM to visualise the internalisation of TiO2 NPs. The genotoxic potential of TiO2 NPs was assessed by micronucleus assay using the conventional and flow cytometry methods.
TEM analysis of TiO2 NPs revealed a mean diameter size of 30-70 nm and DLS measurements showed a mean hydrodynamic diameter and zeta potential of 192.5 ± 10 nm and -11.4 ± 1.2 mV, respectively. The electron microscopy and flow cytometry studies for particle internalisation showed a significant cellular uptake of TiO2 NPs in the human liver cells (HepG2). A significant (p < 0.05) induction in micronucleus formation was observed at 20 µg/ml of TiO2 NPs exposure when compared to control cells. However further treatment to HepG2 with higher concentrations (40 and 80 µg/ml) showed a decrease in micronucleus formation than 20 µg/ml.
In contrary, the flow cytometric results exhibited a significant concentration dependent induction of micronucleus in HepG2 cells exposed to all concentrations (20, 40 and 80 µg/ml) of TiO2 NPs than control.
The difference in the micronucleus frequency obtained from conventional and flow cytometry methods in HepG2 may be due to the accumulation of TiO2 NPs on prepared slides, which hinders the counting of micronucleus (in conventional method). However, in the flow cytometry analysis, these nanoparticles do not interfere with the optics. Hence, it is proposed that in the case of NPs treatment, flow cytometry based micronucleus assay should be used instead of the conventional method.
The financial assistance for the Centre for Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute for Chemical Technology (Grant no. ILS/GICT/2013/003) is acknowledged.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.