Development of human derived liver cell lines for genotoxicity tests

According to current international legislation, chemicals have to be tested in regard to their genotoxic properties. At present, chemicals are tested in a first stage under in vitro conditions. The animal experiments are conducted if positive results are obtained. Due to the high number of false-positive results obtained in the in vitro experiments, up to 80% of animal studies could be avoided if more reliable in vitro models would be available. The number of animals which are used in the EU per year due to false-positives is approximately 16,000. The main reason for the low reliability of the in vitro modelsis poor representation of drug-metabolizing enzymes converting chemicalsto genotoxic/carcinogenic metabolites or detoxify their reaction products, therefore activation mixtures are used which reflect the situation in the human body only partly. The currently used cells are lacking enzymes catalyzing detoxification processes. Preliminary investigations showed that the activities of drug-metabolizing enzymes are at least partly retained in human-derived cell lines. Therefore, we will select from ca. 20 different cell lines the most promising ones and investigate their suitability for the detection of genotoxic properties of selected model compounds (primary screening with time- and cost-efficient single cell gel electrophoresis assays). In parallel, we will try to develop hepatocyte-like cells from stem cells which possess drug-metabolizing enzymes and test them in the primary screening trial. In the next step, the most promising cell lines will be characterized with molecular-biological methods (determination of the activities of various enzymes, investigation of the expression of proteins proteomics and transcription of genes transcriptomics in comparison to primary human liver cell lines). Selected cell lines will be improved by transfection of genes encoding for transcription factors which control the activities of drug-metabolizing enzymes. Subsequently, a protocol will be developed for micronucleus assays. This procedure is used according to OECD guidelines for routine testing of chemicals. In the last phase of the project, a newly developed cell line will be validated, i.e. micronucleus assays will be conducted with 20 different chemicals including compounds which give false-negative results. These chemicals will be taken from a table which was developed by ECVAM experts. Therefore, our results can be compared with those obtained with other cell lines. In addition to the reduction of the use of laboratory animals, it will be also possible to use the new cell lines for variety of other toxicological purposes (to investigate combined effects, synergistic and antagonistic, of chemicals) since the drug metabolizing enzymes will be (in contrast to other lines) represented in inducible form. Further areas of use are investigations of acute toxic properties of chemicals and of the molecular mechanisms which cause these effects, and additionally also studies concerning the metabolism of new drugs.

One of the most relevant Problems in toxicological investigations is the inadequate representation of metabolic processes in cell lines which are currently used for experiments with toxins and pharmaceuticals. This is also true for investigations with new chemicals which are realized in order to find out if they damage the genetic material. Such effects can cause cancer, but also infertility and heritable diseases. Enzymes that activate or detoxify DNA-reactive chemicals are not active in currently used cell lines; therefore quite often false-positive results are obtained which lead to subsequent animal experiments. These in vivo studies could be avoided, if more reliable in vitro models are available. We investigated in the frame of a bilateral project (in cooperation with the National Institute of Biology in Ljubljana) 12 human liver-derived cell lines in regard to their capacity to detect DNA-reactive carcinogens. One of the lines (Huh6) was found to be highly suitable, i.e. it "recognized" carcinogenic compounds more efficiently than other cell lines, which are currently used and also the number of false-positive results was lower. Comparison with the results of animal studies in which DNA-damage is monitored in bone marrow cells (the most frequently used in vivo procedure with laboratory rodents) showed that investigations with the cell line Huh6 lead to more reliable results. In the frame of the project, it was possible to develop a model which can be used in the future for the investigation of chemicals and our findings indicate that it could contribute substantially to the reduction of animals in routine testing of compounds. Furthermore, it may be possible to use this cell line also for acute toxicity studies and investigations concerning the metabolism of xenobiotics. Results which were obtained in the frame of the study were published in a number of top journals.