WP6 is focused on cellular models to study the potential toxicity of nanomaterials. The main aims are to assess effects of nanomaterials on the immune system (immunotoxicity) and to study deleterious effects on the genetic content of cells (genotoxicity), a harbinger of cancer development. Another aim will be to develop so-called high-throughput assays for rapid screening of large numbers of nanomaterials.
The work is conducted by a highly international constellation of nine partner institutes from seven different countries on five continents: Finland, Sweden, Germany, China, South Africa, Brazil and the United States. WP6 is coordinated by Karolinska Institutet in Stockholm, Sweden. The work is closely aligned with activities in WP5 (on bio-nanointeractions) and WP10-11 (on systems biology).
Nanomaterials that are accidentally or intentionally introduced into the human body invariably come into contact with the immune system. The interaction of nanomaterials with immune cells will further determine the fate of the nanomaterials and whether they are eliminated from the body or will accumulate in tissues and organs potentially leading to toxicity.
State-of-the art approaches will be applied to monitor the cellular uptake of nanomaterials by immune competent cells, their subcellular localisation, and subsequent responses of cells.
Another key end-point that raises significant concern for human health is DNA damage and genotoxicity that may, in turn, lead to cancer. Traditional genotoxicity assays in combination with novel, automated techniques provided by one of our industrial partners belonging to WP6 will be used to estimate the impact of nanomaterials on DNA, including unwinding and breaks of DNA strands and damage at the chromosomal level. For all of these studies, we will utilise mainly human primary cells or cell lines.
Outcomes of the workpackage
The overall aim of WP6 has been to apply a range of in vitro (cell-based) models and methods for the assessment of ENM toxicity, focusing on immunotoxicity/inflammogenic potential and genotoxicity/carcinogenic potential, and, moreover, to adapt selected in vitro assays for high-throughput screening. Additionally, WP6 has delivered samples for further assessment using so-called omics technologies in WP10. WP6 is unique in the sense that the 10 partner institutes were from 5 different geographic regions (Europe, North America, South America, Africa, and Asia). Moreover, in addition to academic partner institutes, two small companies (in Finland and Germany) were actively involved, not least in the high-throughput screening task. Overall, WP6 successfully fulfilled all of the expected objectives and the partners have thus generated a comprehensive set of results on a panel of 31 ENMs using a range of cell-based models and methods. These results have been transferred to WP11 to support the development of the Nanosafety Classifier. The work performed in WP6 has been presented at several international conferences and the WP leader also organized a conference on systems biology approaches in nanosafety research at the Nobel Forum at Karolinska Institutet in Stockholm in November 2015. The meeting provided an opportunity for scientists from different EU-funded projects as well as other international colleagues to present and discuss the latest findings in this emerging field.
The immune system is the first line of defense against foreign materials that enter our body, and this is why the work in WP6 was focused on possible adverse effects of ENM on immune-competent cells. The partners in WP6 have used transformed cell lines mimicking normal monocytes as well as primary human macrophages isolated from healthy donors. Using these models, we studied not only cell death by using conventional assays as well as label-free assays (using the xCELLigence Real-time Cell Analyser), but also the secretion of multiple soluble inflammatory mediators (cytokines and chemokines). In addition, exposure via inhalation represents the most likely route of exposure to ENMs in the occupational setting and there is a concern that certain ENMs may cause lung disease. For this reason, the lung cell line, BEAS-2B was employed both for conventional in vitro testing and for the high-throughput screening approaches. Another central question is whether ENMs may have carcinogenic properties, especially upon inhalation. For this reason, the work in WP6 also included genotoxicity assessment (i.e., assessment of DNA damaging effects) as a precursor to possible cancer development. To this end, lung cells as well as immune cells were utilized and the well-known comet and micronucleus assays were applied. In addition, we used mesenchymal stem cells to assess the impact of ENMs on cell differentiation and genotoxic effects. These cells possess a high capacity for self-replication and have the potential to differentiate into various different cell types when placed in an appropriate environment. Cancer is increasingly viewed as a stem cell disease, due to the misappropriation of homeostatic mechanisms that govern tissue repair and stem cell self-renewal, and the study of ENM effects on stem cells is therefore of considerable relevance. The work in WP6 has taken as its starting point methods and protocols established in several other previous EU-funded projects including FP7-NANOMMUNE, FP7 NANODEVICE, and FP7-MARINA. Because WP6 was tasked with the delivery of samples to WP10 to enable global proteomic, transcriptomic, and epigenomic assessment of ENM effects, we opted for well-established cell models to ensure that the models would yield reliable and reproducible results. To this end, two cell lines were selected: the human monocyte-like cell line, THP.1 and the human lung epithelial cell line, BEAS-2B, and samples exposed to the full panel of 31 ENMs were delivered to WP10.
In addition to the use of conventional testing approaches, the partners in WP6 also developed novel high-throughput screening platforms for rapid screening of large numbers of ENMs. Indeed, the enormous diversity of ENMs in terms of their different sizes, shapes, compositions and coatings necessitates high-throughput screening protocols to test for potential hazards. HTS methods are automated methods based on robust in vitro assays capable of simultaneously assessing large numbers of chemicals or particles. In WP6, more than 100.000 data-points were generated using HTS assays for cell viability and other critical end-points. WP6 partners also adapted an HTS assay for genotoxicity assessment of ENMs. With such HTS platforms, large numbers of ENMs can be tested; this may not only speed up the hazard assessment of ENMs, but could also provide a valuable source of data for computer modelling to predict the hazard potential of ENMs. These assays are thus a tangible output of the work in WP6.
With respect to the results, one of the key findings in WP6 is that surface modification of ENMs plays a role for cytotoxicity, with amino-functionalized materials generally displaying more cytotoxicity. This was evident when the immune cell models were employed and a similar pattern, albeit not as straightforward, was seen for the lung cell models. In addition, the core chemistry (i.e., whether the particles tested were made of gold, or copper, or of other composition) also played a role. The work has also shown that the impact on stem cell differentiation of different ENMs is dependent on their surface chemistry; however, not all ENMs affect stem cell differentiation. Overall, the results generated in WP6 represent one of the largest data sets on cytotoxicity-immunotoxicity and genotoxicity of ENMs to date. These results were shared with WP11 in order to promote the development of the Nanosafety Classifier and the results are also important for the interpretation of the omics results produced in WP10.
Dr. Bengt Fadeel
Head of the Unit Molecular Toxicology at The Institute of Environmental Medicine of the Karolinska Institute, Stockholm, Sweden, EU
His research interests include the assessment of immunotoxicity of engineered nanomaterials; and cell death signaling, its role in human disease. He has published more than 180 peer reviewed papers and review articles.
His laboratory participates in several national and EU-funded research projects with a focus on the safety of engineered nanomaterials, and the Flagship Project GRAPHENE, in the workpackage on Health & Environment.