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Studies on the mode of action of Multi-Walled Carbon Nanotubes onto human bronchial epithelial cells

: Walter, Dorothee; Niehof, Monika; Hackbarth, Anja; Hansen, Tanja

ALTEX Proceedings 2 (2013), Nr.2, S.129
ISSN: 2194-0479
European Congress on Alternatives to Animal Testing <18, 2013, Linz>
Fraunhofer ITEM ()

With a production rate of several hundred tons per year, Multi-Walled Carbon Nanotubes (MWCNTs) account for the largest part in the increasing production of nanomaterials. Serious concerns have been raised about the safety of MWCNTs due to their asbestos-like structure. As the most likely route of human exposure to MWCNTs is inhalation, the influence of MWCNTs on the alveolar epithelial cell line A549 has already been widely discussed within the scope of toxicity screenings and is associated with the induction of ROS and other cytotoxic events. In contrast, only few publications exist, which discuss the effects of MWCNTs on cells of the bronchial system. Because of MWCNT's mode of deposition, investigation of the impact of MWCNTs in this area is of great importance. Therefore this project presents the results of several in vitro cytotoxicity assays and gene expression analysis carried out with custom made MWCNTs (length 8.57.
For separation of agglomerated MWCNTs into single ones, MWCNTs were suspended in Dulbecco's cell culture medium using a sonotrode twice (90% duty cycle, 100% amplitude) for five minutes. Calu-3 cells were incubated for 24 or 48 h with MWCNTs at a concentration of 5, 10, or 20 decrease in ATP.
The induction of oxidative stress and the activation of the NF?B signaling pathway are generally considered to be important for the formation of fiber-mediated inflammation. Our studies on oxidative stress showed no MWCNT-dependent increase of ROS. Pathway-specific real time PCR arrays (Human NF?B Signaling Pathway, Human Oxidative Stress and Antioxidant Defense, Human Stress & Toxicity PathwayFinder, SABioscience/Qiagen) were carried out to determine MWCNT’s influence on gene expression levels. Our studies identified the regulation of a few genes which are thought to be involved in the regulation of oxidative stress. Additionally, MWCNTs changed the expression of several genes which are involved in DNA repair or regulation of immune response and down regulated the expression of the tumor suppressor gene EGR1. Due to their tight growth in monolayers, Calu-3 cells form an epithelial-like structure, which can be used for studies on transepithelial resistance. In the present study, no effect of MWCNTs was detected on the strength of the transepithelial resistance. It can be concluded that, unlike A549 cells, Calu-3 cells are relatively insensitive to exposure to MWCNTs. In order to establish a predictive toxicity screening for MWCNTs, the choice of a sensitive cell model is essential.