Automated high content imaging for in vitro assessment of nanomaterial toxicity
1
Abstracts / Toxicology Letters 211S (2012) S35–S42 S41 aspiration exposure. We used long tangled, long needle-like CNT and crocidolite asbestos and a dose of 10 or 40 g per mouse. The mice were sacrificed 4 or 16 h or 28 days after the exposure. To find out the importance of major inflammatory markers TNF- and IL-1, we used Etanercept and Anakinra as antagonists. The results showed that all the materials elicited neutrophilia at 16 h, but the needle-like CNT stood out with significantly higher levels already after 4 h. At 16 h the percentage of neutrophils in BAL was close to 40%. Marked neutrophilia was accompanied by the pro- duction of several chemokines specific to needle-like CNT, such as IL-1, CCL3 and CXCL9. Lung morphology supported these results and after 28 days we saw pulmonary granulomas, goblet cells and changes in intracellular structures. Both antagonists resulted in a significant decrease in neutrophilia and mRNA levels of IL-1 and TNF-. Our results suggest that long needle-like CNT are clearly more hazardous than tangled ones, or even asbestos. Even though many CNT in recent research have been proven to be relatively safe, making reliable risk assessment of CNT seems to be ever more important. doi:10.1016/j.toxlet.2012.03.170 OS3-5 Automated high content imaging for in vitro assessment of nanomaterial toxicity Georgina Harris, Taina Palosaari, Milena Mennecozzi, Jean-Michele Gineste, Roman Liska, Luis Saavedra, Anne Milcamps, Maurice Whelan JRC – European Commission, Italy Keywords: Nanotoxicology; In vitro; High content fluorescence- based imaging Purpose: Today the growing pressure to avoid animal use for toxicological assessment of substances and nanoparticles is evi- dent and there are an increasing number of studies using in vitro approaches to determine the possible cellular effects of nanoma- terials. We propose automated high content fluorescence-based imaging as a useful quantitative tool to screen nanomaterials for their adverse toxicological effects on cells in culture. Additionally, the technical challenges and pitfalls associated with automated high throughput/content in vitro nanotoxicity testing are consid- ered. Methods: Over 20 nanomaterials (including titanium oxides, zinc oxides, silicon dioxides, iron oxides and carbon nano-tubes) were tested in a series of assays, measuring three endpoints relevant for safety assessment, namely, reactive oxygen species production, DNA double-strand breaks and micronucleus forma- tion. Impact: The reliability and relevance of the results obtained will be discussed in the context of the potential of this approach to advance safety assessment science in the support of regulatory decision making specific to nanomaterials. doi:10.1016/j.toxlet.2012.03.171 OS3-6 A comprehensive evaluation platform to assess nanoparticle toxicity in vitro Cordula Hirsch 1 , Tina Buerki-Thurnherr 1 , Lisong Xiao 2 , Osman Arslan 2 , Bruno Wampfler 1 , Sanjay Mathur 2 , Matthias Roesslein 1 , Peter Wick 1 , Harald F. Krug 1 1 Empa, Switzerland, 2 University of Cologne, Germany Purpose: The unique properties of engineered nanomaterials (ENM) render them suitable for various applications. Even though studies on biological effects of ENM are available standardized and validated test systems are still missing. In contrast to sol- uble chemicals the toxicity testing of ENM requires additional controls. These assess unintended interference reactions and/or aggregation/solubilization behavior of the material in order to avoid false positive and false negative results. The goal here is to provide an in vitro platform of harmonized, robust and compre- hensively validated tools to reliably investigate toxicological effects of ENM. The platform comprises four key aspects of cytotoxicity: viability, inflammation, genotoxicity and oxidative stress. Meth- ods: Initial experiments focused on the viability of A549 (human lung epithelial) cells after treatment with commercially avail- able ZnO nanoparticles (IBUtec). Dose–response relationships of nanoparticulate ZnO in comparison to soluble ZnCl 2 were carried out using AnnexinV/Propidium Iodide doublelabeling to analyze apoptotic/necrotic cell death. Further studies addressing the mech- anism(s) of ZnO-induced toxicity were (for technical reasons) carried out in Jurkat (T) cells and include measurement of reac- tive oxygen species (ROS; DCF-assay), chelation experiments and the use of mutant cell lines as well as additional ZnO nanoparticles. Results and conclusion: We found similar dose–response curves for ZnO nanoparticles and soluble ZnCl 2 indicating that mainly Zn ions released from the ENM are responsible for ZnO toxicity in A549 as well as Jurkat cells. The more detailed mechanistic study in Jurkat cells revealed a caspase-independent alternative apoptosis path- way induced by Zn ions that is independent of the formation of ROS. doi:10.1016/j.toxlet.2012.03.172 OS3-7 Biocompatible micro cavity chip for noninvasive toxicity studies on the cellular level Yvonne Kohl 1 , Yvonne Kohl 1 , Oostingh Gertie 2 , Albert Duschl 2 , von Briesen Hagen 1 1 Fraunhofer IBMT, Germany, 2 University of Salzburg, Austria One focus of the European Regulation of chemical substances, REACH, is the support of alternative test methods for the risk assess- ment of chemicals. Also, nanomaterials have to be regulated by REACH. Hence, the goal of this study was to establish and evalu- ate a miniaturised in vitro system to investigate nanoparticulate and viral effects on the cellular level as new screening method for assessing the toxic potential of nanoscaled materials. By semiconductor process technology, an array system with miniaturised cell culture chambers has been established. Each chamber has an 800 nm thick silicon nitride membrane (0.27 m 2 ) on which a small, but statistically significant, cell number was cul- tured. The biocompatibility of the micro cavity chip (MCC) was determined by analysing the viability and morphology of differ- ent cell lines (human stem cells, neuronal cells, human lung cells, stably transfected reporter gene cells) after culturing and differen- tiating in the MCC. A transfected reporter gene cell line was used to investigate nanoparticulate and viral effects on an inflammatory response. All tested cell lines proliferate and differentiate successfully in the MCCs. No negative effect of miniaturisation on the cell behaviour was indicated. Therefore, the new in vitro system was evaluated as a system for noninvasive nanotoxicological real time analysis of individual adherent cells in a defined population. The application of the MCC-based system for investigating time- and
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Transcript of Automated high content imaging for in vitro assessment of nanomaterial toxicity
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Abstracts / Toxicology L
spiration exposure. We used long tangled, long needle-like CNTnd crocidolite asbestos and a dose of 10 or 40 �g per mouse. Theice were sacrificed 4 or 16 h or 28 days after the exposure. To
nd out the importance of major inflammatory markers TNF-� andL-1�, we used Etanercept and Anakinra as antagonists.
The results showed that all the materials elicited neutrophiliat 16 h, but the needle-like CNT stood out with significantly higherevels already after 4 h. At 16 h the percentage of neutrophils in BAL
as close to 40%. Marked neutrophilia was accompanied by the pro-uction of several chemokines specific to needle-like CNT, such as
L-1�, CCL3 and CXCL9. Lung morphology supported these resultsnd after 28 days we saw pulmonary granulomas, goblet cells andhanges in intracellular structures. Both antagonists resulted in aignificant decrease in neutrophilia and mRNA levels of IL-1� andNF-�.
Our results suggest that long needle-like CNT are clearly moreazardous than tangled ones, or even asbestos. Even though manyNT in recent research have been proven to be relatively safe,aking reliable risk assessment of CNT seems to be ever more
mportant.
oi:10.1016/j.toxlet.2012.03.170
S3-5utomated high content imaging for in vitro assessment ofanomaterial toxicity
eorgina Harris, Taina Palosaari, Milena Mennecozzi,ean-Michele Gineste, Roman Liska, Luis Saavedra, Anne
ilcamps, Maurice Whelan
JRC – European Commission, Italy
Keywords: Nanotoxicology; In vitro; High content fluorescence-ased imaging
Purpose: Today the growing pressure to avoid animal use foroxicological assessment of substances and nanoparticles is evi-ent and there are an increasing number of studies using in vitropproaches to determine the possible cellular effects of nanoma-erials. We propose automated high content fluorescence-basedmaging as a useful quantitative tool to screen nanomaterials forheir adverse toxicological effects on cells in culture. Additionally,he technical challenges and pitfalls associated with automatedigh throughput/content in vitro nanotoxicity testing are consid-red. Methods: Over 20 nanomaterials (including titanium oxides,inc oxides, silicon dioxides, iron oxides and carbon nano-tubes)ere tested in a series of assays, measuring three endpoints
elevant for safety assessment, namely, reactive oxygen speciesroduction, DNA double-strand breaks and micronucleus forma-ion. Impact: The reliability and relevance of the results obtainedill be discussed in the context of the potential of this approach
o advance safety assessment science in the support of regulatoryecision making specific to nanomaterials.
oi:10.1016/j.toxlet.2012.03.171
S3-6comprehensive evaluation platform to assess nanoparticle
oxicity in vitro
ordula Hirsch 1, Tina Buerki-Thurnherr 1, Lisong Xiao 2, Osman
rslan 2, Bruno Wampfler 1, Sanjay Mathur 2, Matthiasoesslein 1, Peter Wick 1, Harald F. Krug 1Empa, Switzerland, 2 University of Cologne, Germany
211S (2012) S35–S42 S41
Purpose: The unique properties of engineered nanomaterials(ENM) render them suitable for various applications. Even thoughstudies on biological effects of ENM are available standardizedand validated test systems are still missing. In contrast to sol-uble chemicals the toxicity testing of ENM requires additionalcontrols. These assess unintended interference reactions and/oraggregation/solubilization behavior of the material in order toavoid false positive and false negative results. The goal here is toprovide an in vitro platform of harmonized, robust and compre-hensively validated tools to reliably investigate toxicological effectsof ENM. The platform comprises four key aspects of cytotoxicity:viability, inflammation, genotoxicity and oxidative stress. Meth-ods: Initial experiments focused on the viability of A549 (humanlung epithelial) cells after treatment with commercially avail-able ZnO nanoparticles (IBUtec). Dose–response relationships ofnanoparticulate ZnO in comparison to soluble ZnCl2 were carriedout using AnnexinV/Propidium Iodide doublelabeling to analyzeapoptotic/necrotic cell death. Further studies addressing the mech-anism(s) of ZnO-induced toxicity were (for technical reasons)carried out in Jurkat (T) cells and include measurement of reac-tive oxygen species (ROS; DCF-assay), chelation experiments andthe use of mutant cell lines as well as additional ZnO nanoparticles.Results and conclusion: We found similar dose–response curves forZnO nanoparticles and soluble ZnCl2 indicating that mainly Zn ionsreleased from the ENM are responsible for ZnO toxicity in A549 aswell as Jurkat cells. The more detailed mechanistic study in Jurkatcells revealed a caspase-independent alternative apoptosis path-way induced by Zn ions that is independent of the formation ofROS.
doi:10.1016/j.toxlet.2012.03.172
OS3-7Biocompatible micro cavity chip for noninvasive toxicitystudies on the cellular level
Yvonne Kohl 1, Yvonne Kohl 1, Oostingh Gertie 2, Albert Duschl 2,von Briesen Hagen 1
1 Fraunhofer IBMT, Germany, 2 University of Salzburg, Austria
One focus of the European Regulation of chemical substances,REACH, is the support of alternative test methods for the risk assess-ment of chemicals. Also, nanomaterials have to be regulated byREACH. Hence, the goal of this study was to establish and evalu-ate a miniaturised in vitro system to investigate nanoparticulateand viral effects on the cellular level as new screening method forassessing the toxic potential of nanoscaled materials.
By semiconductor process technology, an array system withminiaturised cell culture chambers has been established. Eachchamber has an 800 nm thick silicon nitride membrane (0.27 �m2)on which a small, but statistically significant, cell number was cul-tured. The biocompatibility of the micro cavity chip (MCC) wasdetermined by analysing the viability and morphology of differ-ent cell lines (human stem cells, neuronal cells, human lung cells,stably transfected reporter gene cells) after culturing and differen-tiating in the MCC. A transfected reporter gene cell line was usedto investigate nanoparticulate and viral effects on an inflammatoryresponse.
All tested cell lines proliferate and differentiate successfullyin the MCCs. No negative effect of miniaturisation on the cell
behaviour was indicated. Therefore, the new in vitro system wasevaluated as a system for noninvasive nanotoxicological real timeanalysis of individual adherent cells in a defined population. Theapplication of the MCC-based system for investigating time- and
