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In vitro cytotoxicity and genotoxicity investigation of differently sized TiO2 nanoparticles
Abstracts / Toxicology Letters 258S (2016) S62–S324
for public health safety agencies and consumers, investigation of the potential impact of these products on human health has been lacking. There is an urgent need to clarify the toxic effects of these materials and to identify the mechanisms involved in their toxicity. Considering the large number of MNMs currently being used in consumer products, high throughput screening approaches aimed at predicting and assessing toxicity are clearly needed. The objective of the present study was to apply a high content analysis based approach to evaluate the toxicity of a series of six MNMs (TiO2 : NM-103 and NM-104, SiO2 : NM-200 and NM203, Zinc oxide: NM-110, and nano-silver NM-300K) in Caco-2 and A549 cells. Following a 24 h treatment of cells with MNMs (1–100 g/mL), cells were processed for high content analysis. A total of 8 cellular markers were evaluated including cell count, nuclear size and intensity, cell cycle, apoptosis, ␥H2AX, p53 phospho S15, and ATM phospho S1981. No significant toxicity or changes in these markers were observed for either TiO2 or SiO2 nanomaterials. However, ZnO and nano silver particles induced significant toxicity and concentration dependent effects on the 8 cellular markers investigated. These results suggest that high content analysis based approaches could be useful for toxicity screening of nanomaterials. Research was funded by the European Union Seventh Framework Programme (FP7/2007–2013) under the project NANoREG, grant agreement 310584. http://dx.doi.org/10.1016/j.toxlet.2016.06.1955 P17-036 Epigenetic effects of SW and MW carbon nanotubes in vitro P. Hoet ∗ , D. Öner, M. Gosh, L. Godderis Department of Public Health and Primary Care, Laboratory Environment and Health, KU Leuven, Leuven, Belgium Production and use of carbon nanotubes (CNTs) augmented the last decade, increasing the risk of exposure. We investigated cyto- and genotoxicity and epigenetic effects induced by single-walled-CNTs (SWCNTs) and multi-walled-CNTs (MWCNTs) in vitro (human cell lines). Two doses (25–100 g/ml) were evaluated by means of the FpG Comet assay and the micronucleus assay with and without incubation of CytochalasinB in 16HBE (bronchial epithelial) and THP1 (monocytic) cell lines, exposed for 24 h. We further, analysed the DNA-methylation. Results: CNTs induced significant tail DNA and tail moment in both cells types after 24 h exposure compared to controls (up to 10-fold). Moreover, oxidative DNA damage was observed. Frequency of micronucleus was increased when 16HBE cells were exposed to SWCNTs and MWCNTs (1.5-fold). No significant increase in micronucleated cells in THP1 cells were observed after 24 h. CBPI (CytochalasinB block proliferation index) was not significantly altered with CytB incubation in tested conditions. However, cell cycle phases were significantly disturbed in 16HBE cells. Although, global DNA-methylation was not significantly altered after 24 h exposure, gene (and cell) specific alterations were observed. Conclusion: The current study revealed epigenetic machinery is susceptible to CNT administration. We observed CNTs induced DNA damage in cells in vitro. Changes in DNA-methylation are complex and show gene specific alterations. Sponsor: Stichting tegen Kanker (N◦ 2012-218 & 2014-147). http://dx.doi.org/10.1016/j.toxlet.2016.06.1956
S273
P17-037 In vitro cytotoxicity and genotoxicity investigation of differently sized TiO2 nanoparticles O.C. Ulker ∗ , A. Ustundag, Y. Duydu, C.O. Yalcin, A. Karakaya Department of Toxicology, Faculty of Pharmacy, Ankara University, Ankara, Turkey Nanoparticles have been used in a range of products (cosmetics, food, drug products, etc.) due to their unique properties derived from their small sizes. Titanium dioxide (TiO2 ) is one of the most frequently applied nanomaterials and primarily used as a pigment. The previous studies have investigated both the cytotoxic and genotoxic effects of TiO2 , but the results of these studies are not compatible with each other. We aimed to investigate the cytotoxic and genotoxic effects of three different nanosize of TiO2 nanoparticles (20 nm, <100 nm, <150 nm) on HaCaT cell line by performing cell viability assay (NRU) and comet assay and micronucleus test. No cytotoxic effect was observed at the tested concentrations of TiO2 (5,10,50,100 g/mL). However at <100 nm and <20 nm size of TiO2 nanoparticles’ IC20 were calculated at the highest concentration. Comet scores obtained from TiO2 nanoparticles exposed cells showed that 20 nm particle sized TiO2 nanoparticles induced DNA damage in all tested concentrations. Size of <100 nm and <150 nm TiO2 nanoparticles induced DNA damage at 5 and 50 g/mL concentrations respectively. No significant induction of micronuclei formation was observed in the treated HaCaT cell lines using micronucleus test. These findings may suggest that the cytotoxicity and genotoxicity of TiO2 nanoparticles is size and concentration dependent, while smallest size and higher concentrations significantly induced DNA strand breaks in HaCaT cell lines. This project was supported by TUBITAK (Project number: 113S229). http://dx.doi.org/10.1016/j.toxlet.2016.06.1957 P17-038 Effects of CuO nanoparticles on an in vitro model of intestinal barrier F. Caloni 1,∗ , C. Cortinovis 2 , G. Colombo 3 , I. Dalle Donne 3 , P. Mantecca 4 , A. Gedanken 5 , I. Perelshtein 5 , N. Bellitto 1 , M. Perego 1 , M. Albonico 1 1 Department of Veterinary Medicine (DIMEVET), Università degli Studi di Milano, Via Celoria, 10, 20133 Milan, Italy 2 Department of Health, Animal Science and Food Safety (VESPA), Università degli Studi di Milano, Via Celoria, 10, 20133 Milan, Italy 3 Department of Biosciences, Università degli Studi di Milano, via Celoria 26, I-20133 Milan, Italy 4 Department of Earth and Environmental Science, POLARIS Research Center, University of Milano Bicocca, Milan 20126, Italy 5 Department of Chemistry and Nanomaterials, Bar-Ilan University Center for Advanced Materials and Nanotechnology, Ramat-Gan, Israel
The rapid growth in development and use of nanotechnological products increases the likelihood of human exposure to nanoparticles (NPs). Thus, this study aimed to investigate the effects of CuO NPs on the intestinal barrier using Caco-2 cells. CuO NPs were synthesized by sonochemistry and fully characterized by HRTEM, XRD, TCA and ICP-MS. Caco-2 cells were cultured on semi-permeable inserts and, after differentiation, treated for 24 h with CuO NPs (10 g/ml, 50 g/ml and 100 g/ml) from the apical (Ap) or baso-
for public health safety agencies and consumers, investigation of the potential impact of these products on human health has been lacking. There is an urgent need to clarify the toxic effects of these materials and to identify the mechanisms involved in their toxicity. Considering the large number of MNMs currently being used in consumer products, high throughput screening approaches aimed at predicting and assessing toxicity are clearly needed. The objective of the present study was to apply a high content analysis based approach to evaluate the toxicity of a series of six MNMs (TiO2 : NM-103 and NM-104, SiO2 : NM-200 and NM203, Zinc oxide: NM-110, and nano-silver NM-300K) in Caco-2 and A549 cells. Following a 24 h treatment of cells with MNMs (1–100 g/mL), cells were processed for high content analysis. A total of 8 cellular markers were evaluated including cell count, nuclear size and intensity, cell cycle, apoptosis, ␥H2AX, p53 phospho S15, and ATM phospho S1981. No significant toxicity or changes in these markers were observed for either TiO2 or SiO2 nanomaterials. However, ZnO and nano silver particles induced significant toxicity and concentration dependent effects on the 8 cellular markers investigated. These results suggest that high content analysis based approaches could be useful for toxicity screening of nanomaterials. Research was funded by the European Union Seventh Framework Programme (FP7/2007–2013) under the project NANoREG, grant agreement 310584. http://dx.doi.org/10.1016/j.toxlet.2016.06.1955 P17-036 Epigenetic effects of SW and MW carbon nanotubes in vitro P. Hoet ∗ , D. Öner, M. Gosh, L. Godderis Department of Public Health and Primary Care, Laboratory Environment and Health, KU Leuven, Leuven, Belgium Production and use of carbon nanotubes (CNTs) augmented the last decade, increasing the risk of exposure. We investigated cyto- and genotoxicity and epigenetic effects induced by single-walled-CNTs (SWCNTs) and multi-walled-CNTs (MWCNTs) in vitro (human cell lines). Two doses (25–100 g/ml) were evaluated by means of the FpG Comet assay and the micronucleus assay with and without incubation of CytochalasinB in 16HBE (bronchial epithelial) and THP1 (monocytic) cell lines, exposed for 24 h. We further, analysed the DNA-methylation. Results: CNTs induced significant tail DNA and tail moment in both cells types after 24 h exposure compared to controls (up to 10-fold). Moreover, oxidative DNA damage was observed. Frequency of micronucleus was increased when 16HBE cells were exposed to SWCNTs and MWCNTs (1.5-fold). No significant increase in micronucleated cells in THP1 cells were observed after 24 h. CBPI (CytochalasinB block proliferation index) was not significantly altered with CytB incubation in tested conditions. However, cell cycle phases were significantly disturbed in 16HBE cells. Although, global DNA-methylation was not significantly altered after 24 h exposure, gene (and cell) specific alterations were observed. Conclusion: The current study revealed epigenetic machinery is susceptible to CNT administration. We observed CNTs induced DNA damage in cells in vitro. Changes in DNA-methylation are complex and show gene specific alterations. Sponsor: Stichting tegen Kanker (N◦ 2012-218 & 2014-147). http://dx.doi.org/10.1016/j.toxlet.2016.06.1956
S273
P17-037 In vitro cytotoxicity and genotoxicity investigation of differently sized TiO2 nanoparticles O.C. Ulker ∗ , A. Ustundag, Y. Duydu, C.O. Yalcin, A. Karakaya Department of Toxicology, Faculty of Pharmacy, Ankara University, Ankara, Turkey Nanoparticles have been used in a range of products (cosmetics, food, drug products, etc.) due to their unique properties derived from their small sizes. Titanium dioxide (TiO2 ) is one of the most frequently applied nanomaterials and primarily used as a pigment. The previous studies have investigated both the cytotoxic and genotoxic effects of TiO2 , but the results of these studies are not compatible with each other. We aimed to investigate the cytotoxic and genotoxic effects of three different nanosize of TiO2 nanoparticles (20 nm, <100 nm, <150 nm) on HaCaT cell line by performing cell viability assay (NRU) and comet assay and micronucleus test. No cytotoxic effect was observed at the tested concentrations of TiO2 (5,10,50,100 g/mL). However at <100 nm and <20 nm size of TiO2 nanoparticles’ IC20 were calculated at the highest concentration. Comet scores obtained from TiO2 nanoparticles exposed cells showed that 20 nm particle sized TiO2 nanoparticles induced DNA damage in all tested concentrations. Size of <100 nm and <150 nm TiO2 nanoparticles induced DNA damage at 5 and 50 g/mL concentrations respectively. No significant induction of micronuclei formation was observed in the treated HaCaT cell lines using micronucleus test. These findings may suggest that the cytotoxicity and genotoxicity of TiO2 nanoparticles is size and concentration dependent, while smallest size and higher concentrations significantly induced DNA strand breaks in HaCaT cell lines. This project was supported by TUBITAK (Project number: 113S229). http://dx.doi.org/10.1016/j.toxlet.2016.06.1957 P17-038 Effects of CuO nanoparticles on an in vitro model of intestinal barrier F. Caloni 1,∗ , C. Cortinovis 2 , G. Colombo 3 , I. Dalle Donne 3 , P. Mantecca 4 , A. Gedanken 5 , I. Perelshtein 5 , N. Bellitto 1 , M. Perego 1 , M. Albonico 1 1 Department of Veterinary Medicine (DIMEVET), Università degli Studi di Milano, Via Celoria, 10, 20133 Milan, Italy 2 Department of Health, Animal Science and Food Safety (VESPA), Università degli Studi di Milano, Via Celoria, 10, 20133 Milan, Italy 3 Department of Biosciences, Università degli Studi di Milano, via Celoria 26, I-20133 Milan, Italy 4 Department of Earth and Environmental Science, POLARIS Research Center, University of Milano Bicocca, Milan 20126, Italy 5 Department of Chemistry and Nanomaterials, Bar-Ilan University Center for Advanced Materials and Nanotechnology, Ramat-Gan, Israel
The rapid growth in development and use of nanotechnological products increases the likelihood of human exposure to nanoparticles (NPs). Thus, this study aimed to investigate the effects of CuO NPs on the intestinal barrier using Caco-2 cells. CuO NPs were synthesized by sonochemistry and fully characterized by HRTEM, XRD, TCA and ICP-MS. Caco-2 cells were cultured on semi-permeable inserts and, after differentiation, treated for 24 h with CuO NPs (10 g/ml, 50 g/ml and 100 g/ml) from the apical (Ap) or baso-