Molecular toxicology of genotoxins and nanomaterials

Toxicology of nanoparticles (NPs) or research at the NanoBioInterface

As the understanding of materials at the nanoscale and the ability to control their structure improves, there appears an enormous potential to create a wide range of materials with novel characteristics and applications. Although the technological and economic benefits of nanomaterials are obvious, concern has also been raised that the very same properties that open a variety of applications might have adverse effects if such material is inhaled, ingested, applied to the skin or even released into the environment. These concerns have led to an increasing discussion in both the US and Europe about possible regulatory policies for NPs. These discussions show clearly the need to establish convincing scientific knowledge to assess the influence of NPs on human health and the ecosystem. Therefore the interaction of nanoparticles or nanostructured surfaces with living systems has become one of the most intriguing areas of basic and applied research at the interface of physics, chemistry and biology.

Previous and on-going work

So far different in vitro systems were used to assess the toxicity of NPs. As inhalation is one of the most relevant uptake routes for NPs we focussed on potential adverse effects of NPs in mammalian lung cells. Exposure systems included classical incubation of cells under submerged conditions which, however, are prone to produce artefacts as NPs e.g. start to agglomerate in cell culture media. Therefore a physiological more relevant exposure at the air liquid interface was developed by the ITC-TAB at KIT (Paur group). By this system nanoparticles are directly deposited onto human lung cells, thereby avoiding artefacts due to interaction of nanoparticles with biological media and allowing for exact dose determination at the same time. To monitor the amount of NPs deposited on-line a quartz crystal microbalance was developed and transferred to industry. The exposure system was equipped with an electrostatic deposition enhancement increasing deposition efficiencies by an order of magnitude.
A screening of defined metal-oxide nanoparticles, which were synthesized at KIT, has been performed by in vitro experiments. SiO₂- NPs were the most effective with regard to cytotoxicity, induction of anti-oxidative and inflammatory genes and surprisingly the presence of serum proteins significantly reduced the adverse effects. For identification of relevant serum proteins, which bind to NPs and thus suppress toxicity, NPs were mixed with serum and bound proteins were analysed by MALDI-TOF (in co-operation with IFG, Brenner-Weiss group). Several proteins could be identified which when bound to NPs mask their surface and alter their biological behaviour. Future studies need to characterize the physicochemical properties of the NP surface critical for the interactions with proteins and possibly also other components of biological membranes such as phospholipids. In summary, interactions of NPs with biological systems can only be understood if we explore what is happening at the nanobiointerface.
In vitro assays were further developed to monitor cellular toxicity by high-content and high-throughput microscopy. Those assays have been compared to conventional toxicity assays. Our novel assays are superior as they monitor more accurately adverse effects even at the single cell level and will allow in the future to screen different libraries (siRNA, cDNA, chemicals) for their effects on NP- mediated toxicity. The functional genomics approach should identify critical toxicity pathways that is the mechanism of action of NPs. A focus will be on cytotoxicity and inflammation induced by NPs as these endpoints are most relevant for particle-induced diseases. The effects of NPs will be compared to genotoxins which are known inducers of cell death and inflammation and thus are used as a reference for standardization of assays and interpretation of the results. Results on NP-induced toxicity in lung cell culture will be followed up also in vivo via instillation of NP suspensions into the lungs of mice.

Future plans to establish zebrafish embryos as a complementary model in addition to cells and rodents to study toxicity of nanoparticles

NPs might be released into the environment, in particular into the aquatic system. The effects of NPs in aquatic organisms, however, are still inadequately understood. We propose to study the effects of NPs in the zebrafish embryo. Zebrafish embryos are perfectly suited for the systematic study of teratogenic and embryo-toxic effects of chemicals. They allow efficient screening of many different substances and are already used e.g. in a standardized test for the analysis of wastewater in Germany to replace the traditional toxicity tests with adult fish. Considering the experimental advantages of zebrafish as small size of the embryo, availability of a sequenced genome and many mutants, the zebrafish is one of the most promising models for mechanistic teratology, toxicology and toxicogenomics in vertebrates. As they develop outside the mother, adverse effects of chemicals can be easily observed already at the first stages of development. In contrast to studies in cell culture the zebrafish embryo offers the analysis of toxic effects in a complex vertebrate organism. The zebrafish facility at KIT in Karlsruhe is one of the largest in the world and allows together with the existing high-throughput automated microscopy systems efficient toxicity screens in zebrafish embryos (in collaboration with the Strähle lab).

    
Model organims