In the 21st Century toxicity testing is rapidly moving from in vivo test with animals to in vitro methodologies. However, currently available cell-based in vitro test models, which are used in toxiological investigations in general and in genotoxicity assays suffer from a series of limitations with reduced representation of xenobiotic drug metabolising enzymes being one of the most important. As stressed out by many scientists as well as regulators this is a problem that needs to be resolved, which is exactly what our project is aimed to achieve. In the proposed project we will not only develop metabolically active liver cells but additionally elucidate pathways that are under-represented in vitro test systems. The new knowledge will be of significant relevance for future development of in vitro test models as well as for the better understanding and interpretation of toxicological results, which important for the regulatory toxicology. Of particular scientific importance is our attempt to develop a novel approach that will enable the differentiation of stem cells into stable metabolically active liver cells, which (if successful) will be a major breakthrough not only in the field of genotoxicity testing but also in general toxicology as well as in the field of pharmacology. Least but not least the availability of metabolically active cells will be applicable not only for routine testing but also for mechanistic studies of the toxicity particularly for the investigations of hazards posed by exposure to mixtures of contaminants which is currently one of the hottest scientific topics in toxicology and risk assessment for human and environmental health
Significance for science
Current legislation requires genotoxicity testing of newly developed chemicals and of compounds such as drugs, cosmetics, food and feed additives, pesticides etc. The guidelines for genotoxicity testing require in the first stage a battery of in vitro tests with bacteria and mammalian cells, and when positive results are obtained follow-up experiments with rodents are conducted. According to the proposed strategy of the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) to “Avoid and Reduce Animal Use in Genotoxicity” a reduction of animals in genotoxicity/carcinogenicity testing can be achieved by the development of more reliable in vitro tests so that fewer in vivo follow-up tests are necessary, which is the aim of the proposed project. It has been estimated that at present, approximately 80 % of in vivo genotoxicity tests in the EU, which are performed due to false in vitro positives, could be avoided with more reliable tests. An important reason for the false results obtained with currently in vitro tests is the lack of mammalian cells lines, which possess phase I and phase II metabolic enzymes that catalyse in humans the activation and detoxification of genotoxins. The aim of the project is to develop human derived liver cells with phase I and phase II enzymes activities that will reflect biotransformation and genotoxic properties of chemicals in humans better than the cell lines which are currently used. Previous studies showed that many human derived hepatic cell lines retained the activities of certain drug metabolizing enzymes but are not suitable for routine testing as they are unstable and important enzymes are lacking. In the frame of the proposed project we will conduct a screening trial with a panel of human hepatoma cell lines, immortalized primary hepatocytes and stem cell derived hepatocyte like cells to identify promising candidate lines, which express the important phase I and phase II enzymes and detect genotoxic effects of representatives of different groups of carcinogenic genotoxins. The transcription of liver enriched transcription factors (LETFs), which control the transcription of xenobiotic drug metabolising enzymes, their activities and protein expression patterns will be monitored in the most suitable cell lines and compared with those of primary human hepatocytes. Subsequently, the sensitivity and specificity of the selected hepatic cell line to detect genotoxins will be improved by the use of a novel approach that is based on the re-activation of the expression of the key xenobiotic-metabolizing enzymes by transfecting hepatic cells with expression vectors encoding for the key LETFs involved in the regulation of xenobiotic-metabolizing enzymes. In the last stage of the project, a protocol for micronucleus assays will be established with the most suitable cell line. Cell line will be validated by testing chemicals from the list, which has been developed by an ECVAM expert group working on the validation of new in vitro genotoxicity tests. We expect that more reliable results (i.e. less false positives) will be obtained with the newly developed cell line. As a consequence, its use in genotoxicity testing will contribute substantially to a reduction of the use of laboratory rodents and to a more reliable safety evaluation of new chemicals and products that is needed for efficient human health protection. Furthermore the new cell line will be useful for acute toxicity studies and for pharmacological investigations concerning the metabolism of drugs. The innovative an ambitious project will be realized in the frame of a collaboration of Slovenian scientists from the National. Institute of Biology and from the Oncologic Institute Ljubljana and Austrian scientists from the Medical University Vienna through their complementary expertise in the fields of genetic toxicology, molecular biology, stem cell biology and the advanced “omics” technologies.