Stage-specific phenotypic and transcriptional alterations in HaCaT keratinocytes exposed to acute and chronic blue light

Despite evidence that visible light (VL) has similar effects on human skin as those of UVA, VL is often viewed as harmless. High SPF sunscreen prevents erythema but can lead to overexposure to UVA and VL, with unknown consequences. To explore the impact of chronic blue light exposure, we irradiated (50 J/cm², λ= 408 nm, three times a week) human immortalized keratinocytes under acute (3 irradiations), intermediate (14 irradiations), and chronic (42 irradiations) blue-light exposure, monitoring phenotypic and gene expression changes. Chronically exposed keratinocytes exhibit increased nuclei area, chromatin alterations, higher proliferation, and apoptosis resistance, mirroring the consequences of chronic UVA exposure. While acute exposure upregulated keratinization and downregulated tissue repair and apoptosis genes, chronically exposed cells had upregulated genes involved with energy metabolism and oxidative phosphorylation and downregulated genes were enriched for immune and inflammatory responses. Specific transcription factors were identified in both the acute and chronic stages, some of which have been associated with UVB exposure. In the acute stage, IRF1, EGR1, ELF3, and FOSL1 were noted, while in the chronic stage, CENPX, SRF, CEBPB, and KLF4 were identified. We identified some changes in chronically irradiated keratinocytes similar to the malignant transformation, emphasizing the need for further research on the long-term impacts of blue light exposure on human skin. HaCaT cells (5 x 10⁵ cells per 100 mm plate) were exposed to a blue light LED source (λ = 408 nm, irradiance = 4.6 mW.cm-2) developed by the Optics and Photonics Research Center at the Institute of Physics, University of São Paulo. The cells were irradiated in phosphate-buffered saline at an irradiation of 50 J/cm² three times a week for up to 14 weeks. Light dose was constant through the experiment and chosen to provide 50% cell death in HaCaT cells. After each irradiation, the culture medium was reintroduced until the next session. Cells were passaged every 3-5 days.