Dominique Lison1, Saloua Ibouraadaten2, Sybille van den Brule2, Milica Todea3,4, Adriana Vulpoi3, Flaviu Turcu3, Christina Ziemann5, Otto Creutzenberg5, James C Bonner6, Marcel Ameloot7, Hannelore Bové7. 1. Louvain centre for Toxicology and Applied Pharmacology (LTAP), Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Brussels, Belgium. dominique.lison@uclouvain.be. 2. Louvain centre for Toxicology and Applied Pharmacology (LTAP), Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Brussels, Belgium. 3. Interdisciplinary Research Institute in Bio- Nano- Sciences, Babes-Bolyai University (BBU), Cluj-Napoca, Romania. 4. Department of Molecular Sciences, Faculty of Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Germany. 5. Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany. 6. Toxicology Program, Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, USA. 7. Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium.
Abstract
BACKGROUND: In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs). METHODS AND RESULTS: We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1β by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1β responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples. CONCLUSION: The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.
BACKGROUND: In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs). METHODS AND RESULTS: We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1β by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1β responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples. CONCLUSION: The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.
Authors: Dominique Lison; Leen C J Thomassen; Virginie Rabolli; Laetitia Gonzalez; Dorota Napierska; Jin Won Seo; Micheline Kirsch-Volders; Peter Hoet; Christine E A Kirschhock; Johan A Martens Journal: Toxicol Sci Date: 2008-04-08 Impact factor: 4.849
Authors: Julie Muller; François Huaux; Antonio Fonseca; Janos B Nagy; Nicolas Moreau; Monique Delos; Encarnacion Raymundo-Piñero; François Béguin; Micheline Kirsch-Volders; Ivana Fenoglio; Bice Fubini; Dominique Lison Journal: Chem Res Toxicol Date: 2008-07-18 Impact factor: 3.739
Authors: Sumit Gangwal; James S Brown; Amy Wang; Keith A Houck; David J Dix; Robert J Kavlock; Elaine A Cohen Hubal Journal: Environ Health Perspect Date: 2011-07-25 Impact factor: 9.031