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c 3T3 A31-1-1 cell transformation assay
Abstracts / Toxicology Letters 229S (2014) S40–S252
Gold and silver nanoparticles (AuNPs and AgNPs) raised remarkable interest in several research areas, especially in medicine. They have unique optical and magnetic properties, which combined with their chemical stability and lack of evidence of significant toxicity make them a primary choice to various biomedical applications. Peptide functionalization has proven to increase biocompatibility, half-life and decrease NPs toxicity (Huang et al., 2010; Khlebtsov et al., 2011). This work reports the evaluation of cytotoxicity of AuNPs and AgNPs in a human intestinal epithelial cell line (Caco2). The nanoparticles (20 nm) were coated with either citrate, or pentapeptides CALNN and CALNS. The calcein–AM assay, the propidium iodide assay, the neutral red uptake assay, and the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay were used to evaluate the cytotoxicity after incubation with a wide range of concentrations [1–100 M] for 6–72 h. The results of all experiments demonstrated that all tested AuNPs did not cause toxicity regardless of the surface coating. For AgNPs, there was an evident toxic effect only at the highest concentration after 48 h of exposure. Overall, it can be concluded that under our experimental conditions the tested nanoparticles are not cytotoxic in this cell line, even at high concentrations. Given the known interference of nanomaterials in several classical in vitro cytotoxicity tests, the multiple and complementary assays chosen for the present work give further support to these conclusions. Acknowledgments: Pest-C/EQB/LA0006/2013 References Huang, X., et al., 2010. In: Grobmyer, S.R., Moudgil, B.M. (Eds.), Cancer Nanotechnology. Humana Press, pp. 343–357. Khlebtsov, N., et al., 2011. Chem. Soc. Rev. 40, 1647–1671.
http://dx.doi.org/10.1016/j.toxlet.2014.06.640 P-3.129 Carcinogenic potential of Ag-NP using Balb/c 3T3 A31-1-1 cell transformation assay Wun Hak Choo 1,∗ , Jeong Eun Lee 1 , Han Soo Cho 1 , Seung Min Oh 2 1
Sungkyunkwan University, Suwon, Republic of Korea, 2 Hoseo University, Asan, Republic of Korea The prediction of the carcinogenic potential for humans has been mostly based on animal experiments. For the last few decade years, the scientific community has paid great attention to alternative strategies in compliance with common moral and ethical values. To use cell transformation assay (CTA) as alternative methods for carcinogenesis, the European Centre for the Validation of Alternative Methods (ECVAM) carried out a validation study in CTA Balb/c 3T3 mouse fibroblast cell line. In this study, we carried out cell transformation assay in Balb/c 3T3 A31-1-1 cells to evaluate the carcinogenic potential of silver nanoparticle. We chose the test chemicals such as positive control (3-methylcholanthrene; 3-MC, benzo(a)pyrene; BaP), negative control (anthracene; An), test compounds (silver nanoparticle; Ag-NP). The colony forming efficiency (CFE) test, and the crystal violet (CV) test were performed in order to find the appropriate dose-range for the cell transformation assay. CTA assay was performed in Balb/c 3T3 A31-1-1 exposed to 3-MC, BaP, An, and Ag-NP for 72 h. And then the cells exposed to chemicals were incubated for 23–30 days with 10 mL of fresh 10% DMEM/FI2 complete medium and stained by 0.4% Giemsa staining for evaluating cell transformation. Our results showed that 3-MC and B(a)P induced cell transformation, whereas An did not. Ag-NP did not
S189
show the cell transformation at all treated doses (6.25, 25, 100, 200, 400 mg/mL). Therefore, to certainly evaluate carcinogenic potential of Ag-NP, further studies were needed to cell transformation in human cell lines chronically exposed to Ag-NP. http://dx.doi.org/10.1016/j.toxlet.2014.06.642 P-3.130 Assessment of the neurotoxic effects of aluminum hydroxide vaccine adjuvant injections in mice Guillemette Crépeaux 1,∗ , Housam Heidi 1,2 , Marie-Odile David 2 , Patrick Curmi 2 , Eleni Tzavara 3 , Bruno Giros 3 , Christopher A. Shaw 4 , Romain K. Gherardi 1 , Josette Cadusseau 1 Inserm U955, Créteil, France, 2 Inserm U829, Evry, France, 3 Inserm U1130, Cnrs UMR 8246, UPMC UM CR18, Paris, France, 4 University of British Columbia, Vancouver, Canada 1
Aluminum (Al) is a potent stimulator of the immune system able to increase the immune response to an antigen. Thus the nanocrystalline compound aluminum hydroxide has been used as vaccinal adjuvant for a century. Until recently, no rigorous animal studies of potential aluminum adjuvant neurotoxicity have been performed, whereas Al is a known neurotoxin, and long-term persistence of Al vaccine adjuvants can lead to cognitive dysfunction and autoimmunity in presumably susceptible adult humans. For instance, a small proportion of people vaccinated with aluminum hydroxide-containing vaccines present with delayed onset of diffuse myalgia, chronic fatigue and cognitive dysfunctions, and exhibit very long-term persistence of aluminum hydroxide nanoparticles loaded macrophages at the site of previous intramuscular immunization, forming a granulomatous lesion called macrophagic myofasciitis (MMF). Recent animal experiments indicate that biopersistent nanomaterials taken up by monocyte-lineage cells in tissues can first translocate to draining lymph nodes, then circulate in blood within phagocytes, reach the spleen, and finally accumulate in the brain. In this context, we addressed the question of Aluminum hydroxide neurotoxicity with behavioural studies in 8-week female CD-1 mice injected intra-muscularly in three equivalent-to-human doses. A battery of eight complementary tests was performed at 45 days, 90 days, 135 days and 180 days after injections. Tests were conducted in order to assess the levels of activity, anxiety and depression, the short-term memory performances, the muscular strength, pain sensitivity and locomotor coordination. No significant differences between groups were observed at D45. Experiments for later times are in progress. http://dx.doi.org/10.1016/j.toxlet.2014.06.643
Gold and silver nanoparticles (AuNPs and AgNPs) raised remarkable interest in several research areas, especially in medicine. They have unique optical and magnetic properties, which combined with their chemical stability and lack of evidence of significant toxicity make them a primary choice to various biomedical applications. Peptide functionalization has proven to increase biocompatibility, half-life and decrease NPs toxicity (Huang et al., 2010; Khlebtsov et al., 2011). This work reports the evaluation of cytotoxicity of AuNPs and AgNPs in a human intestinal epithelial cell line (Caco2). The nanoparticles (20 nm) were coated with either citrate, or pentapeptides CALNN and CALNS. The calcein–AM assay, the propidium iodide assay, the neutral red uptake assay, and the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay were used to evaluate the cytotoxicity after incubation with a wide range of concentrations [1–100 M] for 6–72 h. The results of all experiments demonstrated that all tested AuNPs did not cause toxicity regardless of the surface coating. For AgNPs, there was an evident toxic effect only at the highest concentration after 48 h of exposure. Overall, it can be concluded that under our experimental conditions the tested nanoparticles are not cytotoxic in this cell line, even at high concentrations. Given the known interference of nanomaterials in several classical in vitro cytotoxicity tests, the multiple and complementary assays chosen for the present work give further support to these conclusions. Acknowledgments: Pest-C/EQB/LA0006/2013 References Huang, X., et al., 2010. In: Grobmyer, S.R., Moudgil, B.M. (Eds.), Cancer Nanotechnology. Humana Press, pp. 343–357. Khlebtsov, N., et al., 2011. Chem. Soc. Rev. 40, 1647–1671.
http://dx.doi.org/10.1016/j.toxlet.2014.06.640 P-3.129 Carcinogenic potential of Ag-NP using Balb/c 3T3 A31-1-1 cell transformation assay Wun Hak Choo 1,∗ , Jeong Eun Lee 1 , Han Soo Cho 1 , Seung Min Oh 2 1
Sungkyunkwan University, Suwon, Republic of Korea, 2 Hoseo University, Asan, Republic of Korea The prediction of the carcinogenic potential for humans has been mostly based on animal experiments. For the last few decade years, the scientific community has paid great attention to alternative strategies in compliance with common moral and ethical values. To use cell transformation assay (CTA) as alternative methods for carcinogenesis, the European Centre for the Validation of Alternative Methods (ECVAM) carried out a validation study in CTA Balb/c 3T3 mouse fibroblast cell line. In this study, we carried out cell transformation assay in Balb/c 3T3 A31-1-1 cells to evaluate the carcinogenic potential of silver nanoparticle. We chose the test chemicals such as positive control (3-methylcholanthrene; 3-MC, benzo(a)pyrene; BaP), negative control (anthracene; An), test compounds (silver nanoparticle; Ag-NP). The colony forming efficiency (CFE) test, and the crystal violet (CV) test were performed in order to find the appropriate dose-range for the cell transformation assay. CTA assay was performed in Balb/c 3T3 A31-1-1 exposed to 3-MC, BaP, An, and Ag-NP for 72 h. And then the cells exposed to chemicals were incubated for 23–30 days with 10 mL of fresh 10% DMEM/FI2 complete medium and stained by 0.4% Giemsa staining for evaluating cell transformation. Our results showed that 3-MC and B(a)P induced cell transformation, whereas An did not. Ag-NP did not
S189
show the cell transformation at all treated doses (6.25, 25, 100, 200, 400 mg/mL). Therefore, to certainly evaluate carcinogenic potential of Ag-NP, further studies were needed to cell transformation in human cell lines chronically exposed to Ag-NP. http://dx.doi.org/10.1016/j.toxlet.2014.06.642 P-3.130 Assessment of the neurotoxic effects of aluminum hydroxide vaccine adjuvant injections in mice Guillemette Crépeaux 1,∗ , Housam Heidi 1,2 , Marie-Odile David 2 , Patrick Curmi 2 , Eleni Tzavara 3 , Bruno Giros 3 , Christopher A. Shaw 4 , Romain K. Gherardi 1 , Josette Cadusseau 1 Inserm U955, Créteil, France, 2 Inserm U829, Evry, France, 3 Inserm U1130, Cnrs UMR 8246, UPMC UM CR18, Paris, France, 4 University of British Columbia, Vancouver, Canada 1
Aluminum (Al) is a potent stimulator of the immune system able to increase the immune response to an antigen. Thus the nanocrystalline compound aluminum hydroxide has been used as vaccinal adjuvant for a century. Until recently, no rigorous animal studies of potential aluminum adjuvant neurotoxicity have been performed, whereas Al is a known neurotoxin, and long-term persistence of Al vaccine adjuvants can lead to cognitive dysfunction and autoimmunity in presumably susceptible adult humans. For instance, a small proportion of people vaccinated with aluminum hydroxide-containing vaccines present with delayed onset of diffuse myalgia, chronic fatigue and cognitive dysfunctions, and exhibit very long-term persistence of aluminum hydroxide nanoparticles loaded macrophages at the site of previous intramuscular immunization, forming a granulomatous lesion called macrophagic myofasciitis (MMF). Recent animal experiments indicate that biopersistent nanomaterials taken up by monocyte-lineage cells in tissues can first translocate to draining lymph nodes, then circulate in blood within phagocytes, reach the spleen, and finally accumulate in the brain. In this context, we addressed the question of Aluminum hydroxide neurotoxicity with behavioural studies in 8-week female CD-1 mice injected intra-muscularly in three equivalent-to-human doses. A battery of eight complementary tests was performed at 45 days, 90 days, 135 days and 180 days after injections. Tests were conducted in order to assess the levels of activity, anxiety and depression, the short-term memory performances, the muscular strength, pain sensitivity and locomotor coordination. No significant differences between groups were observed at D45. Experiments for later times are in progress. http://dx.doi.org/10.1016/j.toxlet.2014.06.643