The human body is permanently exposed to substances that can damage the DNA and lead to tumours, whether it be through the influence of chemicals, drugs or UV irradiation. Cellular repair processes have a key role in maintaining cell function in the human body. In the molecular biology laboratory at the Albstadt-Sigmaringen University of Applied Sciences, a team led by Prof. Dr. Jörg Bergemann is developing and optimising assays that enable the quantitative determination of DNA damage and the investigation of repair mechanisms. The researchers exclusively use in vitro test systems. Using tissue engineering methods, the researchers are developing a skin model for pharmacological efficacy and biocompatibility studies. Industry is also extremely interested in such tests, which include a modified host cell reactivation assay.
The number of skin tumour diseases has continued to increase over the last few years. The most frequent skin tumours are basaliomas, squamous cell carcinomas and melanomas. UV irradiation can result in a broad range of DNA alterations, for example the development of characteristic dimers (cyclobutane pyrimidine dimers/CPDs). Healthy people possess several highly efficient systems in their cells that contribute to the complete elimination of UV-related DNA damage. However, repair of the DNA is impossible when the diseases are caused by genetic defects. Affected people develop different types of skin cancers very early on in life. Such cases include Xeroderma pigmentosum sufferers (also called midnight children) who must avoid sun light in order to prevent mutations due to the body's inability to repair DNA damage. Research and industry are increasingly focussed on developing pharmaceutical substances, for both cosmetics and drugs, that are able to strengthen the body's repair mechanisms.
Prof. Jörg Bergemann, head of the Biomedical Engineering study programme at the Albstadt-Sigmaringen University of Applied Sciences, and his team have developed different test systems to determine the ability of DNA to repair itself. One of these test systems is a modified host cell reactivation assay that is based on fluorescence activated cell sorting (FACS) and completely does away with animal experiments. "In contrast to currently used methods, our method is far more sensitive, more reproducible and we are also able to analyse individual cells as they undergo repair processes," said Prof. Jörg Bergemann summarising the advantages of their new method. The method enables the scientists to detect substances that stimulate the DNA's repair capacity as well as substances that damage the DNA. To do this, the researchers transfect, amongst other things, fibroblasts that are involved in the skin ageing process with reporter constructs that code for the proteins DsRed (irradiated reporter plasmids) and green fluorescent proteins pEGFP (non-irradiated plasmids). "The difference between irradiated and non-irradiated plasmids is that the protein on the irradiated plasmid can only be expressed following the successful repair of the DNA," said Prof. Jörg Bergemann.
The fibroblasts are transfected with a mixture of irradiated (DsRed) and non-irradiated pEGFP plasmids. "In principle, these are two alternative reporter constructs. It is also possible to use non-irradiated DsRed and irradiated pEGFP or some other plasmids that code for fluorescent proteins.
The repair capacity in the cells, assessed on the basis of the successful repair of UV-damaged reporters, can then be compared in a FACS analysis using cells that were either treated or not treated with an active substance under investigation. FACS (fluorescence activated cell sorting) relates to the analysis of cell properties through the emission of scattered light or fluorescence impulses as a reaction to laser irradiation. The cell suspension containing cells labelled with a fluorescent dye is directed into a thin stream, enabling the cells to pass in single file. Cells that are able to repair their DNA can express DsRed and pEGFP, while repair-deficient cells can only express pEGFP. "When combined with single cell analyses, the use of this internal standard leads to very reproducible experimental conditions," said Bergemann.
Using this method, the researchers were able to show, amongst other things, that cell-active folic acid promotes the repair capacity of dermal cells, which contributed to providing further evidence for claims in the field of anti-ageing. These investigations showed that active substances increased the repair capacity of skin cells in all of the 20 donors examined; due to the great variability of the cell donors, this is further evidence for the reproducibility of our assay," said Prof. Jörg Bergemann. The improvement varied between two and around 20 per cent. "Another advantage of this method is that we are able to produce large quantities of irradiated reporter genes and are thus able to use very similar damage profiles in our assays," said Prof. Jörg Bergemann.
The group of researchers uses a skin model as well as up to three-day-old human skin cells from people who have undergone surgery. These cells are then cultivated. "We preferentially work with keratinocytes, fibroblasts and melanocytes. We isolate these cells and also use them for our skin models," said Bergemann. Another major methodological development is the parallel isolation from sometimes very small samples of the three most important cell types occurring in human skin. "This allows us to tackle problems arising from a huge variety of donors when dealing with skin cells," said Bergemann. Artificial skin consists mainly of epidermal cells.
"Our experiments are mainly focused on UV-induced damage, but we can also expand our method to other damage profiles," highlighted Prof. Jörg Bergemann. Our methods are an excellent alternative to animal experiments and an expansion of alternative testing. This is not only good for ethical reasons, but also because it generates important and reliable measurement data, which animal experiments are unable to provide. "The substances that turn out to have no effect, do not need to be tested in animals in order to confirm their reliability," said the scientist. Bergemann believes that "it is not possible to completely do without animal experiments", but regards it as an important goal to reduce the number of animal experiments whenever possible, for example by further developing alternative methods.
"Due to the legal safety guidelines that are increasingly required to guarantee people's health and safety, I believe that an even larger number of animals will be used for tests. And this makes it even more important to work on reducing the number of animal tests," said the researcher. Prof. Jörg Bergemann and his team have the goal to simplify their modified host cell reactivation assay for use in drug screening, something that is currently only possible to a limited degree.