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Translocation of TiO2 nanoparticles through different models of gastrointestinal epithelium
Abstracts / Toxicology Letters 205S (2011) S60–S179
P1310 Translocation of TiO2 nanoparticles through different models of gastrointestinal epithelium M. Carrière 1,∗ , E. Brun 2 , D. Jaillard 3 , S. Sorieul 4 , B. Fayard 5 , A. Flank 6 , A. Mabondzo 7 , N. Herlin-Boime 8 1
Laboratoire Lésion Des Acides Nucléiques (lan), CEA, Grenoble, France, 2 Laboratoire Structure Et Dynamique Par Résonance Magnétique, CEA, Gif sur Yvette, France, 3 Ccme, univ Paris Sud, Orsay, France, 4 Umr5797 Cnrs-univ Bordeaux I, CENBG, Gradignan, France, 5 Lps, univ Paris Sud, Orsay, France, 6 Lucia Beamline, Synchrotron SOLEIL, Saint Aubin, France, 7 Laboratoire D’etude Du Métabolisme Du Médicament, CEA, Gif sur Yvette, France, 8 Laboratoire Francis Perrin, CEA, Gif sur Yvette, France Nanoparticles (NPs) are presently introduced in a fast growing number of commercial products. Among them, titanium dioxide is found in daily use hygiene products or orally administered drugs. However its effects on gastrointestinal tract are poorly investigated. Here we focused on the translocation of TiO2 NPs through different models of gastrointestinal epithelia: Caco-2 enterocytes, HT29-MTX mucus-secreting cells and a co-culture of both types, more representative of the complexity of the digestive barrier. Once their integrity and functionality checked, they were exposed to anatase and rutile NPs, well characterized in terms of purity, size and crystallinity. After exposure, cyto- and genotoxicity were assessed together with the activity of enzymes responsible for cell redox balance preservation. Internalization was followed by TEM and -XRF elemental mapping, coupled to XAS analysis of Ti speciation, allowing to get insights on NPs fate after internalization. Ti contents in apical and basolateral compartments were determined by ICP-MS. TiO2 NPs surface modification after exposure to gastrointestinal fluids was investigated. A digestion model was thus developed, simulating the different compartments of a gastrointestinal tract. The originality of this project relies on the panel of techniques implemented to investigate digestive barrier translocation, bringing together biologists, chemists and physicists in a pluridisciplinary research program. doi:10.1016/j.toxlet.2011.05.544
P1311 A comparative evaluation of HCE-T cells cytotoxicity assay and HET-CAM assay using cosmetic surfactants for eye irritation potential S.A. Cho Safety Research Teatm, AmorePacific Corporation R&D Center, Yongin, Republic of Korea The Council of the European Union has banned marketing cosmetics and its ingredients tested on animals since March 2009. Following this regulation, various studies for alternatives to animal experimentation to test efficacy or toxicity of cosmetics and other products have been developed. Using human corneal cell line (HCE-T cells), we assessed the eye irritating potential of cosmetic ingredients by two evaluation criteria. We exposed HCE-T cells to 10 surfactants by serial dilution for 1 h as an endpoint of eye irritation potential and measured the cell viability (CV) at each concentration of the surfactant. Using the cell viability values, we calculated the CV50. We also exposed HCE-T cells to 10 surfactants (5%, 0.5% and 0.05%) for 1 h and measured the cell viability (CV) at each concentration of the surfactant. Using the cell viability
S155
values at 5%, 0.5% and 0.05%, we calculated the TEIS (total eye irritation score). With the results of CV 50 and TEIS, we assessed the rank correlation with the results of HET-CAM (Hens Egg Test on the Chorio-Allantoic Membrane) assay (INVITTOX Protocol Nr. 96, ECVAM) at 1% concentration. The results of both values (CV50 and TEIS) showed good positive correlations with the results of HETCAM assay (R2 = 0.8155 at CV50, R2 = 0.8598 at TEIS) and also could suggest the objective data compare to those of HET-CAM assay. We concluded that the new animal alternative test using HCE-T cells could be a good model for the prediction of eye irritation. doi:10.1016/j.toxlet.2011.05.545
P1312 NaCl-induced hyperosmotic stress as an in vitro dry eye model potentiated by synergic toxic effects of Benzalkonium chloride on conjunctival epithelial cells C. Clouzeau ∗ , D. Godefroy, L. Riancho, W. Rostene, C. Baudouin, F. Brignole-Baudouin Institut De La Vision Inserm UMR S598, Paris, France Purpose: Benzalkonium chloride (BAK), a commonly used preservative in eye drops, was shown to promote tear hyperosmolarity and induce dry eye syndrome. Our aim was to develop a toxicological in vitro model of dry eye using NaCl-induced hyperosmolar conditions (HO) and to investigate the respective and synergistic cytotoxic effects of these two deleterious stimulations on conjunctival epithelial cells. Methods: Conjunctival cells were cultured for 24 h in HO (400-425-500 mOsM) or in nonor cytotoxic concentrations of BAK (10–4%–3.10 to 4%–5.10–4%) or in combination of both. Cell viability (Neutral red), cell death (AnnexinV-FITC/7AAD, membrane permeability-YO-PRO1 and chromatin condensation-Hoechst33342/propidium iodide) and oxidative stress (H2DCFDA, Hydroethidine) were assessed using spectrofluorimetry and/or flow cytometry. Immunohistochemistry was performed for actin cytoskeleton, active-caspase3, PARP-1, cytochrome c and AIF. Calcium flux was assessed to investigate intracellular calcium movement. Results of the study: HO induced a decrease in cell proliferation and viability, illustrated by an increase in membrane permeability, cell shrinkage, cell blebbing and chromatin condensation. A caspase-dependent apoptosis was observed with cytochrome c release from mitochondria to cytoplasm, leakage of PARP-1 from nucleus and caspase-3 activation. AIF, a caspase-independent apoptosis factor, was translocated from mitochondria to nucleus. Intracellular calcium increased in a HO/BAK dependant manner. This in vitro study shows that NaClinduced HO and BAK, even at non cytotoxic concentration each, act synergistically to induce cell toxicity. This hyperosmolarity model highlights the risk of inducing a vicious circle in clinical practice and as consequence the importance of avoiding BAK in clinical dry eye conditions. doi:10.1016/j.toxlet.2011.05.546
P1313 In vitro blood–brain barrier model adapted to repeated dose toxicological screening A. da Costa ∗ , J. Hachani, C. Landry, R. Cecchelli, M. Culot Laboratoire de la barrière hémato-encéphalique (LBHE), Lens, France Toxicity to the CNS is required by many regulatory programs. Cell lines derived from brain cells have shown promise as alterna-
P1310 Translocation of TiO2 nanoparticles through different models of gastrointestinal epithelium M. Carrière 1,∗ , E. Brun 2 , D. Jaillard 3 , S. Sorieul 4 , B. Fayard 5 , A. Flank 6 , A. Mabondzo 7 , N. Herlin-Boime 8 1
Laboratoire Lésion Des Acides Nucléiques (lan), CEA, Grenoble, France, 2 Laboratoire Structure Et Dynamique Par Résonance Magnétique, CEA, Gif sur Yvette, France, 3 Ccme, univ Paris Sud, Orsay, France, 4 Umr5797 Cnrs-univ Bordeaux I, CENBG, Gradignan, France, 5 Lps, univ Paris Sud, Orsay, France, 6 Lucia Beamline, Synchrotron SOLEIL, Saint Aubin, France, 7 Laboratoire D’etude Du Métabolisme Du Médicament, CEA, Gif sur Yvette, France, 8 Laboratoire Francis Perrin, CEA, Gif sur Yvette, France Nanoparticles (NPs) are presently introduced in a fast growing number of commercial products. Among them, titanium dioxide is found in daily use hygiene products or orally administered drugs. However its effects on gastrointestinal tract are poorly investigated. Here we focused on the translocation of TiO2 NPs through different models of gastrointestinal epithelia: Caco-2 enterocytes, HT29-MTX mucus-secreting cells and a co-culture of both types, more representative of the complexity of the digestive barrier. Once their integrity and functionality checked, they were exposed to anatase and rutile NPs, well characterized in terms of purity, size and crystallinity. After exposure, cyto- and genotoxicity were assessed together with the activity of enzymes responsible for cell redox balance preservation. Internalization was followed by TEM and -XRF elemental mapping, coupled to XAS analysis of Ti speciation, allowing to get insights on NPs fate after internalization. Ti contents in apical and basolateral compartments were determined by ICP-MS. TiO2 NPs surface modification after exposure to gastrointestinal fluids was investigated. A digestion model was thus developed, simulating the different compartments of a gastrointestinal tract. The originality of this project relies on the panel of techniques implemented to investigate digestive barrier translocation, bringing together biologists, chemists and physicists in a pluridisciplinary research program. doi:10.1016/j.toxlet.2011.05.544
P1311 A comparative evaluation of HCE-T cells cytotoxicity assay and HET-CAM assay using cosmetic surfactants for eye irritation potential S.A. Cho Safety Research Teatm, AmorePacific Corporation R&D Center, Yongin, Republic of Korea The Council of the European Union has banned marketing cosmetics and its ingredients tested on animals since March 2009. Following this regulation, various studies for alternatives to animal experimentation to test efficacy or toxicity of cosmetics and other products have been developed. Using human corneal cell line (HCE-T cells), we assessed the eye irritating potential of cosmetic ingredients by two evaluation criteria. We exposed HCE-T cells to 10 surfactants by serial dilution for 1 h as an endpoint of eye irritation potential and measured the cell viability (CV) at each concentration of the surfactant. Using the cell viability values, we calculated the CV50. We also exposed HCE-T cells to 10 surfactants (5%, 0.5% and 0.05%) for 1 h and measured the cell viability (CV) at each concentration of the surfactant. Using the cell viability
S155
values at 5%, 0.5% and 0.05%, we calculated the TEIS (total eye irritation score). With the results of CV 50 and TEIS, we assessed the rank correlation with the results of HET-CAM (Hens Egg Test on the Chorio-Allantoic Membrane) assay (INVITTOX Protocol Nr. 96, ECVAM) at 1% concentration. The results of both values (CV50 and TEIS) showed good positive correlations with the results of HETCAM assay (R2 = 0.8155 at CV50, R2 = 0.8598 at TEIS) and also could suggest the objective data compare to those of HET-CAM assay. We concluded that the new animal alternative test using HCE-T cells could be a good model for the prediction of eye irritation. doi:10.1016/j.toxlet.2011.05.545
P1312 NaCl-induced hyperosmotic stress as an in vitro dry eye model potentiated by synergic toxic effects of Benzalkonium chloride on conjunctival epithelial cells C. Clouzeau ∗ , D. Godefroy, L. Riancho, W. Rostene, C. Baudouin, F. Brignole-Baudouin Institut De La Vision Inserm UMR S598, Paris, France Purpose: Benzalkonium chloride (BAK), a commonly used preservative in eye drops, was shown to promote tear hyperosmolarity and induce dry eye syndrome. Our aim was to develop a toxicological in vitro model of dry eye using NaCl-induced hyperosmolar conditions (HO) and to investigate the respective and synergistic cytotoxic effects of these two deleterious stimulations on conjunctival epithelial cells. Methods: Conjunctival cells were cultured for 24 h in HO (400-425-500 mOsM) or in nonor cytotoxic concentrations of BAK (10–4%–3.10 to 4%–5.10–4%) or in combination of both. Cell viability (Neutral red), cell death (AnnexinV-FITC/7AAD, membrane permeability-YO-PRO1 and chromatin condensation-Hoechst33342/propidium iodide) and oxidative stress (H2DCFDA, Hydroethidine) were assessed using spectrofluorimetry and/or flow cytometry. Immunohistochemistry was performed for actin cytoskeleton, active-caspase3, PARP-1, cytochrome c and AIF. Calcium flux was assessed to investigate intracellular calcium movement. Results of the study: HO induced a decrease in cell proliferation and viability, illustrated by an increase in membrane permeability, cell shrinkage, cell blebbing and chromatin condensation. A caspase-dependent apoptosis was observed with cytochrome c release from mitochondria to cytoplasm, leakage of PARP-1 from nucleus and caspase-3 activation. AIF, a caspase-independent apoptosis factor, was translocated from mitochondria to nucleus. Intracellular calcium increased in a HO/BAK dependant manner. This in vitro study shows that NaClinduced HO and BAK, even at non cytotoxic concentration each, act synergistically to induce cell toxicity. This hyperosmolarity model highlights the risk of inducing a vicious circle in clinical practice and as consequence the importance of avoiding BAK in clinical dry eye conditions. doi:10.1016/j.toxlet.2011.05.546
P1313 In vitro blood–brain barrier model adapted to repeated dose toxicological screening A. da Costa ∗ , J. Hachani, C. Landry, R. Cecchelli, M. Culot Laboratoire de la barrière hémato-encéphalique (LBHE), Lens, France Toxicity to the CNS is required by many regulatory programs. Cell lines derived from brain cells have shown promise as alterna-