Dr. Kadereit, why is it so difficult to predict the toxicity of nanomaterials?

The toxicity of nanoparticles depends very much on the nanoparticles’ chemical properties, their surface properties, shape and size. Nanoparticles that have the same chemical composition but different shapes or sizes might have different effects on cells. The reactions of cells might differ and different cell types might also react differently. Different complex, highly regulated processes (e.g. development of the nervous system where cell division, cell differentiation and cell migration follow a highly orchestrated scheme) go on in a developing organism: DNA needs to be replicated, proteins need to be distributed to cells in a controlled fasion, cells need to divide at the right point in time, and so on. All these processes are extremely prone to the slightest changes occurring in the cell’s environment or inside it. The accumulation of even low quantities of toxic nanoparticles inside cells might lead to steric hindrance of processes related to the development of the nervous system, e.g. by way of sequestration or changes of neighbouring proteins.

Can you give me some information on how you develop a model for assessing nanotoxicity?

It is possible to model such complex processes in cell culture systems involving embryonic stem cells. We are trying to model the early development stage of the nervous system in order to test the toxicity of nanoparticles in detail. In this system, early brain precursor cells mature into neural precursor cells. The system allows the three-dimensional interaction of the cells, which is similar to the situation in tissue. As the cells mature, they go through important stages of early nervous system development.

What kind of damage can nanoparticles cause and what kind of animal experiments have been carried out to investigate the damage they cause?

In general, nanoparticles lead to inflammatory reactions. Nanoparticles that enter the human body along with particulate matter have also been linked with cardiovascular problems. They are also able to enter the brain or the placenta. Let me give you an example. The progeny of mice that were exposed to TiO₂ nanoparticles during pregnancy developed alterations in the gene expression pattern in the brain. The same progeny still had a lower than normal weight six weeks after birth and TiO₂ nanoparticles were detected in the testicles and the brain. Fullerenes have also been found to enter foetuses and to be passed on to the offspring by way of mother’s milk. The effects of nanoparticle exposure during human development are not yet well understood. In addition, there are currently no legal regulations requiring companies to assess the toxicity of the nanomaterials they produce.

Why did you choose to work with pluripotent stem cells of all other cells?

There is increasing evidence that animal experiments do not always provide information on whether certain nanomaterials that are toxic (or non-toxic) for animals are also toxic (or non-toxic) for humans. Animal experiments used for testing developmental neurotoxic effects are extremely time-consuming and costly. In addition, the results obtained in animal experiments cannot always be transferred to the situation in humans. Subtle forms of human developmental neurotoxicity, e.g. difficulties in drawing along shapes, cannot be modelled in mice or rats. Cell culture systems also have the advantage that they allow a closer look at cellular processes and the underlying mechanisms be investigated in greater detail. Biomarkers can be identified which would make it possible to identify initial signs of toxicity at an early stage. The use of pluripotent stem cells for cell culture experiments have three enormous advantages: first, they allow the generation of theoretically unlimited quantities of cells and second, even of different types of cells. And third, they also enable us to generate development models in a way that cannot be achieved with other cell types.

Why are embryonic stem cells suitable for modelling human development?

As far as embryonic stem cells are concerned, it has been shown that they perfectly recapitulate certain stages in the development of the nervous system in vitro. They are therefore excellently suited for modelling the development of the early nervous system and are used to an increasing extent for doing so. We have already shown that mouse embryonic stem cells are suitable for detecting extremely low quantities of toxicants that have a toxic effect on the development of the nervous system, mercury for example. We are now trying to do the same with human embryonic stem cells. Human cell culture systems offer a three-dimensional environment in which neural precursor cells can mature into precursors of nerve cells. As the cells mature, they receive signals and feedback from neighbouring cells. Three-dimensional cell cultures are a lot better than two-dimensional cell cultures when it comes to modelling the real situation of normal tissue.

Which methods and techniques are you using?

We mainly use conventional methods to find out whether the cells die as a result of their long-term exposure to nanoparticles and for determining the nanoparticle concentration that leads to the death of the cells. We then have a detailed look at the concentrations that have not led to the death of the cells and are investigating the processes that are happening in these cells. We also use fluorescent nanoparticles whose path and interactions we can observe inside the cells. We can also observe the effects of nanoparticles on neighbouring cells as these cells frequently respond to stress. 

Can you give me some more details about your cell-based in-vitro system?

The type of cell-based in-vitro systems used depends very much on the scientific question one hopes to solve. In general, I would say that the system used must not be too complex and also allow the effective resolution of questions using conventional molecular biology and biochemical methods. Unfortunately, this cannot always be achieved in developmental neurotoxicity models that are derived from pluripotent stem cells. On the other hand, these systems allow the investigation of interactions of individual and different cell types. Ideally, a system that the industry uses for screening purposes, must be designed such that it allows for automation and delivers results within a relatively short time and with little effort.

Why are your methods and findings alternatives to animal experiments?

Our department is specifically focused on developing alternatives to animal experiments. The development of our three-dimensional culture systems, which we use for modelling the early embryonic development of the human nervous system, was funded by the Doerenkamp-Zbinden Foundation for Realistic Animal Protection which supports research to reduce, replace and refine animal experimentation. As hardly any regulations or standards relating to the testing of the toxicity of nanoparticles are available and as it can be expected that nanotoxicity testing will become mandatory sometime in the future, I think we should start thinking about establishing culture systems for this purpose already now, rather than sacrificing any more animals. I would also like to point out that the number of animals that are available for experimental research in Europe is already insufficient for the large number of experiments and tests that need to be carried out. REACH (EU law on the “Registration, Evaluation, Authorisation and Restriction of Chemicals”), which has the aim to improve the protection of human health and the environment through the earlier identification of the intrinsic properties of chemical substances (REACH also makes clear that animal tests must only be used as a last resort), will require the toxicity of already marketed high volume chemicals to be tested within in the next few years. This means that effective testing systems need to become available.

How many animals are used for testing whether chemicals have hazardous effects on human health?

Studies have predicted that around 50 million animals, which is several millions more animals than the EU Commission had estimated, would be needed to test chemicals under REACH. It is worth noting that around 90,000 animals per year are sacrificed in Europe for the testing of chemicals. I also believe that the time frame the chemicals producers were given is too short in order to be able to test hundreds of thousands of chemicals. There is no way that the large number of chemicals can be tested using animals, which is both time-consuming and extremely costly. Alternatives to animal testing need to be urgently found. It does therefore not come as a surprise that the EU funds numerous projects and research consortia (including ESNATS of which we are also part) that have the goal of developing alternative methods to using animals for the testing of chemicals.

Contact:
Dr. Suzanne Kadereit
University of Konstanz
Tel: +49 (0)7531 / 88 - 5053
Fax: +49 (0)7531 / 88 - 5039
E-mail: suzanne.kadereit[at]uni-konstanz.de