Projects / Programmes
Development of new hepatic cell lines for genotoxicity and carcinogenicity studies: new hope for human health and laboratory animal welfare protection
| Code |
Science |
Field |
Subfield |
| 1.03.00 |
Natural sciences and mathematics |
Biology |
|
| Code |
Science |
Field |
| B200 |
Biomedical sciences |
Cytology, oncology, cancerology |
| Code |
Science |
Field |
| 1.06 |
Natural Sciences |
Biological sciences |
genotoxicity, metabolic transformation, in vitro testing, human liver cells, stem cells, toxicogenomic, laboratory animals
Researchers (14)
Organisations (2)
Abstract
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.
Significance for science
In the 21st Century the toxicity testing is rapidly moving from in vivo tests with animals to in vitro methodologies. However, the currently available cell based in vitro test models, which are used in toxicological 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 was aimed to achieve. Among numerous hepatic cell lines available, which are all of cancerous origin, we identified two with promising properties: HepG2, which are already used in genetic toxicology, and HuH6 that has not been used in this application so far. With the comparative toxicogenomic analysis of the expression of key enzymes, involved in metabolic transformation of xenobiotics (phase I and phase II metabolic enzymes, transporters and liver specific transcription factors), we found important differences between HepG2 and HuH6 cells and primary human hepatocytes. This new knowledge represents a valuable information for further studies of factors that regulate the activation of metabolic enzymes in the in vitro conditions. On one hand, it can be the basis for further development of improved test systems. On the other hand, based on the knowledge of mechanisms of action of genotoxic substances with concurrent knowledge of the weakness of test cells, it is possible to assess the relevance of the obtained results for the in vivo situation. An important scientific achievement of the project is the development of hepatocyte like cells by differentiation of mesenchymal stem cells, and establishment of a stable cell line with immortalization. We have shown that integration of differentiation and imortalization with transformation with hTERT is possible. This new metabolically active cell line has the potential to be used in many applications, not only in routine testing of toxicity and genotoxicity, but also in the studies of biochemical mechanisms of toxicity. An important advantage compared to other metabolically active cell lines, which are all cancerous origin, is that these cells do not have the properties of cancer cells. In cancer cells the cellular processes, including responses to chemicals, differ significantly from responses of normal cells, which may also be one of the reasons for uncertainties of currently used in vitro test systems. The research results of this project contribute to important new findings relevant for the development of 3D experimental models in toxicology, which are currently one of the most prominent fields of research and development in vitro testing systems, in which we are already actively involved.
Significance for the country
As described in the justification of the proposal, one of the main difficulty of the current regulatory strategy for genotoxicity testing is a high rate of false positive results that leads to a high number of follow-up in vivo tests to confirm these results. This leads not only to the use of a large number of experimental animals, but also to high costs of the follow-up testings and delay of the product marketing. This is particularly relevant for pharmaceutical industries and cosmetics production. In cosmetics production compounds that are positive in the in vitro genotoxicity tests are immediately banned from further development, as in vivo test are not allowed according to the 7th Amendment to the EU Cosmetics Directive (http://ec.europa.eu/consumers/sectors/cosmetics/files/doc/200315/200315_en.pdf), and thus eventual false positive results cannot be identified. In this project, we identified two hepatic cell lines and developed a hepatocyte like cell line, which possess very promising properties for the use in genotoxicity testing. The results demonstrated more accurate predictivity compared to currently available assays, which will significantly contribute to the reduced costs in development different product. From the public health point of view, it is notable that more reliable in vitro genotoxicity assays will lead to the production of safer and cheaper products, which will be available much faster. In terms of the society, the reduction of animal testing will assist in the reduction of problems created by the activities of the animal rights activists. The new cells have also the potential to be used for the development of reporter based genotoxicity assays in automated high throughput genotoxicity screening. This is currently highly desirable, particularly in pharmaceutical industry and the early phase of drug development. The demand for fast and reliable genotoxicity tests on the international market is higher from the current offer therefore the results of this project represent also direct business opportunity for the establishment of spinout company, what will significantly contribute to the development of NIB as well as Slovenian SMEs. Another business opportunity is the possibility to use the new cell line for commercial in vitro production of human metabolic enzyme mixtures, which is currently not available in routine genotoxicity testing. Taken together, the realisation of the project will contribute to the reduction of the use of experimental animals in genetic toxicology, and increase the efficiency of toxicological studies and accuracy of the results. This will significantly contribute to the reduction of the costs in new product development, protection of human health and bring the business opportunity that may attract foreign investors (particularly pharmaceutical and cosmetic industries).
Most important scientific results
Annual report
2014,
final report
Most important socioeconomically and culturally relevant results
Final report
