Applicant:

Dr. Suzanne Kadereit

Group Leader, Stem Cell Group

Doerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine

University of Konstanz, Germany

Co-Applicant:

Prof. Dr. Marcel Leist

Doerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine

University of Konstanz, Germany 

Background:

Engineered nanoparticles (NP) are entering consumer products and our environment at a fast pace. Nanoparticles can be found in food items, cosmetics, electronics and are likely to be used increasingly in medicine, where they will be actively introduced into the body. In addition, it has been shown that NP can cross the blood brain barrier and the placenta, and reach the adult as well as the fetal brain. Currently, there is no unified regulation governing nanoparticle-based products (not even separate CAS-numbers). In Europe, NP are included as chemicals under REACH, however, no separate testing is mandated yet. This is currently under discussion, as toxicity of NP is not comparable to that of their bulk counterparts. NP toxicity is significantly higher than that of the bulk product. Moreover, not much is known about the effects of long-term exposure to NP. This cannot be tested in current cell models, and is tested in animal models. It is to be expected that once a consensus has been reached on how to test for NP toxicity, animal usage will increase dramatically, including likely life-time exposure protocols, and suffering of countless animals. There is however still the possibility to press forward to establish in vitro testing as the standard testing method, as in the field of nanotoxicity animal testing has not yet been established as validated method. It is therefore urgent to define human biomarkers for nanotoxicity that could allow to define novel in vitro assay systems free of animal testing. Animal testing could then be reduced to assess metabolic turn-over and bioaccumulation, only for NP that passed prior in vitro testing.

Method/approach:

We have developed a human developmental toxicity (DNT) model, based on differentiation of human embryonic stem cells. In this system we can show toxicity of NP and propose now to investigate this toxicity in more detail. We will investigate whether we can define a set of marker genes affected by exposure to methylmercury and different NP, in order to define a biomarker profile which is specific for nano-DNT.

Relevance to 3R:

Testing for nanotoxicity is as of now still unregulated and not mandatory. As increasing evidence for toxicity of nanomaterials emerges, it is to be expected that testing, at least for certain types of NP, will become mandatory. As each NP will likely have to be tested separately, as with each modification toxic properties are likely to change, a gigantic increase in animal usage can be expected. As in the field of nanotoxicity testing no ‘gold standards’ (such as animal experiments) have yet been established, there is the unprecedented opportunity to establish mandatory in vitro testing as the first line of experiments to be used. That way, only nanomaterials which did not elicit noticeable toxicity in vitro will be tested in animals. As a consequence, large numbers of animals can be spared of painful exposure to nanomaterials. This will be particularly of interest for NP which have been surface/shape modified for additional functionality. Also, with the herein proposed system and approach (identification of biomarkers for nano-DNT), long-term exposure experiments can also be shifted from animals to in vitro systems. Currently, long-term exposure scenarios cannot be tested in conventional cell culture systems and have to be tested in animals. Moreover, novel test strategies as proposed herein will not only eliminate a large number of animal experiments, but also reduce suffering in the animals, as testing will then only take place in settings where non-toxic to low-toxic NP will be tested for bioaccumulation and turnover, at low doses to evaluate long-term burden and consequences in the body.

Project report published in Archives of Toxicology 2013 Apr; 87(4):721-33

Summary:

Nanoparticles (NPs) have been shown to accumulate in organs, cross the blood-brain barrier and placenta, and have the potential to elicit developmental neurotoxicity (DNT). Here, we developed a human embryonic stem cell (hESC)-derived 3-dimensional (3-D) in vitro model that allows for testing of potential developmental neurotoxicants. Early central nervous system PAX6(+) precursor cells were generated from hESCs and differentiated further within 3-D structures. The 3-D model was characterized for neural marker expression revealing robust differentiation toward neuronal precursor cells, and gene expression profiling suggested a predominantly forebrain-like development. Altered neural gene expression due to exposure to non-cytotoxic concentrations of the known developmental neurotoxicant, methylmercury, indicated that the 3-D model could detect DNT. To test for specific toxicity of NPs, chemically inert polyethylene NPs (PE-NPs) were chosen. They penetrated deep into the 3-D structures and impacted gene expression at non-cytotoxic concentrations. NOTCH pathway genes such as HES5 and NOTCH1 were reduced in expression, as well as downstream neuronal precursor genes such as NEUROD1 and ASCL1. FOXG1, a patterning marker, was also reduced. As loss of function of these genes results in severe nervous system impairments in mice, our data suggest that the 3-D hESC-derived model could be used to test for Nano-DNT.