Evaluating Photogenotoxicity using 3D Tissue Models

DESCRIPTION provided by applicant The objective of this proposal is to focus on evaluating materials for their ability to cause photogenotoxicity Photosensitivity is a harmful reaction that occurs when drugs or chemicals in the skin or eyes cause undesirable cellular damage when exposed to UV or visible light Photogenotoxicity is used as a means to screen a material for its photocarcinogenic potential Genotoxicity is one of the possible outcomes caused by phototoxins Although non photo genotoxicity has standardized testing methods currently there are no regulatory approved photogenotoxicity assays Applying the testing procedures used in genotoxicity testing towards photogenotoxicity is not a straightforward approach In a review of photoclastogenic UV induced chromosome disruption compounds more than of compounds that were classified as photoclastogenic in mammalian assays were negative in the standard T Neutral Red Update NRU phototoxicity assay demonstrating the high occurrence of pseudophotoclastogenicity A limitation of many of these assays is the extensive processing of cells to analyze DNA damage which can require hypotonic cell treatment or cell lysis DNA fixation or unwinding staining or electrophoresis then visual counting and scoring Cell lines have historically been used in most laboratory studies for genotoxicity assays Nonetheless the development of D tissue models have allowed for a much greater representation of living tissues We propose to evaluate in vitro models consisting of hanging drop D liver cell culture and D reconstructed human epidermal RHE tissues D Multi cell human liver microtissues InSphero and D differentiated model of the human epidermis EpiDermandquot MatTek Corp will be evaluated for their ability to predict photogenotoxins Evaluation of phosphorylation of histone H AX will be quantitatively measured as an indicator of DNA damage DNA damage induces phosphorylation of Ser of the carboxy terminus of histone H AX H AX Detection of H AX can be detected by both flow cytometry and immunofluorescence Monitoring a mechanistic endpoint of DNA repair will allow for a greater accuracy of evaluating Photogenotoxicity by allowing for a more quantitative endpoint PUBLIC HEALTH RELEVANCE The objective of this proposal is to focus on evaluating materials for their ability to cause photogenotoxicity Genotoxicity is one of the possible outcomes caused by phototoxins We propose to use in vitro methods consisting of hanging drop cell culture using D liver microtissues and D reconstructed human epidermal RHE tissue to represent more physiological relevant test conditions than D monolayer cultures DNA damage induces phosphorylation of Ser of the carboxy terminus of histone H AX H AX We will monitor H AX as a mechanistic and quantitative endpoint of DNA damage caused by photogenotoxicity in the D tissues