Abstract B035: Developing the UValidate platform to measure DNA damage and repair capacity in isogenic donor-derived skin keratinocytes, fibroblasts and melanocyte cell-lines with different Fitzpatrick phototypes

Abstract

Abstract DNA damage associated with ultraviolet (UV) radiation exposure causes accelerated skin aging and cancer-initiating mutations. Over-the-counter UVA and UVB filters are required in all sunscreens, but transdermal absorption of these compounds have been reported to impair development and reproduction and metabolize into carcinogenic/mutagenic chemical intermediates. To assess the effects of sunscreen active ingredients on UVR-induced DNA damage and repair, we developed a novel high throughput (HTP) screening platform, UValidate, employing mixed populations of isogenic skin cells, exposed to single or combinations of UV filters in the presence of UVB and UVA. UValidate contains a patented LED UVR DNA damage induction system (LUDIS:V3), facilitating programmable precise induction of DNA damage and repair at multiple time points and replicates, using a 96-well configuration capable of simultaneous delivering UVA and UVB. LUDIS:V3 optimization experiments surprisingly revealed that, unlike keratinocytes, skin fibroblasts only repaired UVA DNA damage in conditioned media. We then tested sunscreen active ingredient combinations for their capacity to alter DNA repair and found that: 1) Some sunscreen ingredients, including dioxybenzone, induce a significant increase in DNA damage above endogenous levels. 2) Fibroblasts and keratinocytes respond differently to the treatment protocols. 3) Some sunscreen active compounds contribute to reduction of UVA-induced DNA damage. 4) No sunscreen completely inhibits all UVA DNA damage. We are currently employing the LUDIS:V3 system with HTP comet assays to compare DNA damage and DNA repair capacities, +/- sunscreen active ingredients, of six isogenic sets of primary epidermal keratinocytes (NHEK), melanocytes (NHEM), and fibroblasts (NHDF)` with different Fitzpatrick phototypes I-VI, representative of the diverse American population. These cells were isolated from donor skin samples, and further purified to establish banks of isogenic NHEK, NHEM, and NHDF cell lines. UVA exposure with the LUDIS:V3 and comet assays revealed higher levels of DNA damage in NHEK and NHDF, compared to NHEM, likely due to the UVR-protective effects of melanin in NHEM. Consistently, NHEK with phototype V (from darkly pigmented skin with higher melanin levels) exhibit significantly less UVA-induced DNA damage compared with phototype II NHEK. To create cellular models of dermal disease for DNA repair assays, we have successfully knocked out XPA in multiple NHDF cell lines with different phototypes using a CRISPR-Cas9 KO strategy, as confirmed by immunoblot and NGS sequence analyses. These and other engineered DNA repair-deficient skin cells will be assayed as single or mixed cell-type 2D cultures, with fluorescent labeling allowing for identification of subpopulations in comet and alkaline diffusion assays. This technology addresses the need for better platforms for rapid genotoxicity testing of safer and more effective chemical UV filters to prevent cutaneous carcinogenesis in diverse populations. Citation Format: Dean S. Rosenthal, Elijah Finn, Devin Teehan, Nusrat Islam, Veerupaxagouda Patil, Bonnie Carney, Scott S. Rosenthal, Lucia Dussan, Cynthia M. Simbulan-Rosenthal, Peter Sykora. Developing the UValidate platform to measure DNA damage and repair capacity in isogenic donor-derived skin keratinocytes, fibroblasts and melanocyte cell-lines with different Fitzpatrick phototypes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B035.