PCLS (Precision Cut Lung Slices) as a putative model for genotoxicity testing by application of the comet assay
Actually, precision cut lung slices are under extensive investigation to serve as a model for acute inhalation toxicology. Slices can be employed as an ex-vivo/in-vitro model as alternative method in the context of REACH and the 3Rs. Moreover, this in vitro model represents in vivo tissue including all naturally occurring cell types of the respiratory tract situated in their physiological environment. For the reason of these beneficial characteristics, PCLS may be an in vitro model of first choice in the future, especially with respect to studies on inflammatory changes in the lung. However, since safety control and toxicology demand studies on genotoxicity, we also started to establish a Comet assay with live lung tissue in vitro from mouse lungs to expand the experimental possibilities from investigations with PCLS on toxicity and inflammatory responses to genotoxicity. The work focused on optimization of a simple and fast cell separation method by enzymatic digestion of the lung tissue, application of the alkaline Comet assay and exposure of lung slices to culture media (negative control) and test compounds in a first series of experiments. Ethyl methanesulfonate (EMS) and formalin (FA) were tested as model substances known to induce DNA damage. These alterations can be observed in the Comet assay as, respectively, an increased rate of DNA breaks (EMS) or a reduced rate of DNA breaks by induction of cross-links (FA). Factors of concern for the reproducibility and meaningfulness of results obtained with the method established here to investigate genotoxicity induced in this in-vivo-like live complex cell system by application of the Comet assay to PCLS are discussed. These include the slice-to-slice- and individual-to-individual-reproducibility as well as the background signal and the dose-dependency of the effects induced by the model substances. In summary, the experiments showed that dose-dependent effects induced by EMS and FA could be detected in mouse PCLS, and by application of the Comet assay, effects from strand-breaking and cross-linking substances could be reproducibly discriminate.