Development of in vitro embrionic stem cell-based cell assays to screen for toxicity of emerging nanomaterials

2009 - 2011

Applicant:
Dr. Suzanne Kadereit
Group leader, Stem cell group
University of Konstanz
Box M657
78457 Konstanz
Germany

Background:
Nanomaterials and nanoparticles (NPs) are revolutionizing our environment and are starting to be incorporated massively into consumer goods, will likely be used on a large scale for soil and water remediation, and are very promising for future applications in medicine. Nevertheless, not much is known about their toxicity and potential for bioaccumulation in the food chain. It is likely that expected future regulation of nanomaterials will entail a large deployment of animal testing, with long to life-time, likely painful, exposure, and sacrificing of the animals to assess long-term effects and tissue distribution/accumulation. As it is already clear that many of the very small nanomaterials will end up within the cells, it is possible in this new emerging field of toxicity to establish in vitro, cell-based systems that have a high likelihood to become rapidly accepted standards as pre-screening of newly emerging materials. This will enable a largely reduced animal use and suffering, as only few and most promising, cell-non- toxic and non-inflammatory substances would then be tested in animals to assess metabolic turn-over and accumulation.

Methods/approach:
We propose to develop in vitro assay systems based on astrocytes derived from embryonic stem cells. Astrocytes are relatively easy to obtain from mouse embryonic stem cells (mESC) and from a neural progenitor line derived from mESC. As key inflammatory players in the brain, astrocytes are particularly well suited as read-out of potential toxicity in general and neurotoxicity in particular. Astrocytes will be derived from mESC and hESC, will be exposed to NP and cell stress and inflammatory read-outs will be measured with standard techniques such as fluorescent imaging, flow cytometry, ELISA. An inflammatory read-out profile relevant to the in vivo situation will be established that will allow for later scaling up and semi-high through-put screening of newly emerging NP.

Study plan:
We propose to develop in vitro systems with astrocytes as sensible environment-
sensing cells that will allow to gain novel insights into how NP influence inflammatory processes in general, and in the brain in particular. In a first time we will study the effects of certain already characterized NPs such as carbon black (CB), TiO2 fulluerol (a hydroxylated C60), polystyrene particles (PS) and quantum dots (QD). These NPs can be obtained in different sizes and surface modifications, and impact of size and surface will be monitored in more detail. We will first determine sub-lethal concentrations of the NPs and then assay even lower concentrations in order to gain insight on potential sub-acute toxicity that could occur after long-term exposure and accumulation of the NPs in certain sub-cellular compartments. Read-outs will include generation of reactive oxygen species as sign of oxidative stress in the cells, measurement of changes in intracellular Ca2+
, measurement of activation markers, histone acetylation to obtain insight into extend of modification of chromatin structure and potential effect on global gene expression (in case of major effects we will also include gene expression array studies), activation of the stress-sensing transcription factors NF-kB and AP-1, measurement of inflammatory cytokines, and testing of inflammation-stimulatory capacity of supernatants of NP-treated astrocytes on immune cells such as T cells. We will also test such supernatants on neurons in order to assess whether the tested Ns are capable of eliciting indirect toxicity (acute/sub-acute) on neurons. In these studies we will not monitor whether the particles enter the cells or not, as with their nanoscale size they can very well interact with and have an impact on extracellular matrix proteins and molecules involved in signaling and cellular communications, which can then result in aberrant signals, stress, toxicity, cell death and inflammation.

Relevance to 3R:
Nanotoxicity is a new area of toxicity testing which is as of yet still largely unregulated. With an estimated $1 trillion market share by 2012 of potentially unlimited numbers of different nanomaterials coming into consumer goods, medicine and the environment, a high use of experimental animal is to be anticipated. As in this new area not much is regulated yet, and not much experience is gained with animal experimentation, and thus no major prejudice against in vitro assays seems to be established yet (as is in other fields of research, where the animal experiment is firmly established as the ‘gold standard’), this is an unprecedented time and opportunity to establish in vitro assay systems as the first line of experiments without major opposition, and to minimize animal use drastically. For example, if it is clear that a nanomaterial elicits inflammation when entering a cell, there is no need to proceed with testing and to expose animals to painful instillation of particles into the lung (a major potential route of exposure to environmental particles) and later killing, to measure whether the particles elicited inflammation and cellular infiltration into the tissues. The test systems we propose to develop here are developed within a new interdisciplinary program in close collaboration with the chemists of the Chemical Biology Graduate Program of the University of Konstanz, and are anticipated to yield a robust first line of testing strategy for nanotoxicity, with the goal to establish new testing standards for this emerging field of toxicology. Such test systems will not only eliminate a large number of animal experiments, but also reduce suffering in the animals that are then used for testing only promising, non-toxic and non-inflammatory nanomaterials.