The objective of this Phase 1 STTR research is to quantify genotoxicity in basal cell keratinocytes from organotypic cultures (Epiderm) in response to commonly used chemical agents. The proposed product is a Comet Chip assay that measures DNA damage in basal cells derived from a reconstructed human epidermis. Technical questions that will be addressed are 1) Can we modify our currently used Comet Chip assay to incorporate extracellular matrix proteins or antibodies? 2) Can we isolate individual basal keratinocytes from a 3D organotypic skin culture on the basis of their preferential adhesion to these matrix proteins, including collagen I and IV, or by using immobilized antibodies to integrin ?1? 3) Can we confirm their identity by quantum dot-coupled antibodies specific for ?2 or ?1 (collagen ligand), integrins? 4) Can we use the isolated, antibody-labeled epidermal basal cells to detect and quantify levels of DNA damage in response to known environmental genotoxic agents? 5) Can we use our Immuno-CometChip assay to screen large numbers of agents currently or proposed to be marketed? The impact of the proposed research will be to reduce animal model use for toxic agent screening, since human organotypic culture has been shown to be almost identical to human skin with respect to its cytokine profile in response to corrosive or irritating agents. The market for screening skin genotoxic agents is immense, since the current screening procedures cannot keep pace with the number of new agents currently being introduced. Aim 1 will be to develop a new method for the isolation of basal keratinocytes, and an immunostaining method for simultaneous visualization of specific antigens, including ?1 integrin and DNA damage. Aim 2 will be to validate the Immuno-CometChip assay using known DNA damaging agents, including H2O2. This adds three new parameters to the Comet assay. The first is obtaining 96 treatment groups of 240 single basal epidermal keratinocytes from an organotypic culture on a single chip, the second is verifying their identity with surface markers, and the third is the simultaneous reproducible assay for DNA damage.
Public Health Relevance Statement: Public Health Relevance: The Comet assay has appeal for many reasons. The assay is rapid, simple, sensitive, reliable, and fairly inexpensive. Its use for testing for skin toxicity hs been hampered by a number of factors, including background DNA damage incurred during the normal process of differentiation in organotypic skin models, which mimic human skin. We therefore propose to develop a Comet Chip assay, a high throughput, reproducible, and quantitative method to isolate basal keratinocytes and measure genotoxicity in response to commonly used chemical agents.
NIH Spending Category: Bioengineering; Genetics; Nanotechnology; Prevention
Project Terms: Address; Adhesions; Alkalies; Allergic; Animal Model; Antibodies; Antigens; Basal Cell; base; Binding; Biological Assay; Caliber; Cell Separation; Cells; Chemical Agents; Chemicals; chromatin immunoprecipitation; cold temperature; Collagen; Comet Assay; Corrosives; Coupled; Cryopreserved Cell; cytokine; Deposition; DNA Damage; Epidermis; Extracellular Matrix Proteins; fluorescence microscope; Gel; genotoxicity; Goals; Grant; Household; Human; Hydrogen Peroxide; Hypersensitivity skin testing; Imagery; Individual; Integrins; interest; keratinocyte; Label; Ligands; Light; Market Research; Marketing; Measures; Methods; Modeling; Monitor; Mutagenicity Tests; Mutagens; nanoparticle; New Agents; Peptide Hydrolases; Phase; Process; Proteins; Protocols documentation; public health relevance; Quantum Dots; rapid technique; Research; research and development; response; screening; Screening procedure; Sepharose; Skin; Small Business Technology Transfer Research; Staging; Stem cells; success; Surface; Techniques; Testing; Time; Toxic effect; Toxicity Tests; treatment group; Universities; Validation
