The objective of this STTR Phase II proposal is to utilize our proof-of concept results to develop and market an easy-to-use standardized platform to quantify genotoxicity specifically in basal cell skin keratinocytes from organotypic cultures (EpidermTM; MatTek) in response to commonly used chemical agents. The product is an automated high-throughput Immuno-CometChip assay that simultaneously isolates and verifies the basal layer origin of cells derived from an organotypic reconstructed human epidermis, and quantifies their level of DNA damage. The impact of the 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 response to corrosive, irritating, and allergic agents. The market for screening skin genotoxic agents is large, and current screening procedures cannot keep pace with new agents currently being introduced. Many of these agents are screened by companies, who will have an interest in a rapid method to determine at an early stage whether or not to proceed with research and development of lead compounds. We therefore propose the following Specific Aims:
Specific Aim 1 : To develop control cells to be included with each run of the Immuno-Comet Chip assay to ensure that our new method for the isolation of basal keratinocytes using a collagen I/agarose composite gel for our new CometChip platform (see below), will be successful for each run following an immunostaining method for simultaneous visualization of ?1 integrin along with DNA damage.
Specific Aim 2 : To develop computer software that only measures comets in integrin ?1-postive basal cells.
Specific Aim 3 : To determine activity of human epidermal cytochrome p450s (CYP) in the EpiDermTM organotypic model, in order to test if preincubation of procarcinogens and human S9 fractions is necessary.
Specific Aim 4 : To test 10 compounds each of carcinogens, procarcinogens and non-carcinogens from ?Toxicology in the 21st Century? (Tox21) in double-blinded fashion to determine if we can distinguish statistically significant difference between carcinogenic and non-carcinogenic compounds, and to determine if CYP enzymes are converting procarcinogens to carcinogens.

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 has 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.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Small Business Technology Transfer (STTR) Grants - Phase II (R42)
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Special Emphasis Panel (ZRG1)
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Shaughnessy, Daniel
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Amelia Technologies, LLC
United States
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