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 75% of compounds that were classified as photoclastogenic in mammalian assays were negative in the standard 3T3 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 3D tissue models have allowed for a much greater representation of living tissues. We propose to evaluate in vitro models consisting of hanging drop 3D liver cell culture and 3D reconstructed human epidermal (RHE) tissues. 3D Multi-cell human liver microtissues (InSphero) and 3D differentiated model of the human epidermis (EpiDerm" - MatTek Corp) will be evaluated for their ability to predict photogenotoxins. Evaluation of phosphorylation of histone H2AX will be quantitatively measured as an indicator of DNA damage. DNA damage induces phosphorylation of Ser139 of the carboxy terminus of histone H2AX (?-H2AX). Detection of ?-H2AX 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 3D liver microtissues; and 3D reconstructed human epidermal (RHE) tissue to represent more physiological relevant test conditions than 2D monolayer cultures. DNA damage induces phosphorylation of Ser139 of the carboxy terminus of histone H2AX (?-H2AX). We will monitor ?-H2AX as a mechanistic and quantitative endpoint of DNA damage caused by photogenotoxicity in the 3D tissues.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
Project #
1R43ES025098-01
Application #
8832781
Study Section
Special Emphasis Panel (ZRG1-OTC-R (11))
Program Officer
Shaughnessy, Daniel
Project Start
2014-09-25
Project End
2015-02-28
Budget Start
2014-09-25
Budget End
2015-02-28
Support Year
1
Fiscal Year
2014
Total Cost
$88,608
Indirect Cost
Name
MB Research Laboratories, Inc.
Department
Type
DUNS #
077070712
City
Spinnerstown
State
PA
Country
United States
Zip Code
18968