The photoprotective properties of α-tocopherol phosphate against long-wave UVA1 (385 nm) radiation in keratinocytes in vitro

UVA1 radiation (340-400 nm), especially longwave UVA1 (> 370 nm), is often ignored when assessing sun protection due to its low sunburning potential, but it generates Reactive Oxygen Species (ROS) and is poorly attenuated by sunscreens. This study aimed to investigate if α-tocopherol phosphate, (α-TP) a promising new antioxidant, could protect against long-wave UVA1 induced cell death and scavenge UVA1 induced ROS in a skin cell model. HaCaT keratinocyte cell viability (24 h) was assessed with Alamar Blue and Neutral Red assays. The metabolism of α-TP into α-T, assessed using mass spectrometry, and the compound's radical scavenging efficacy, assessed by the dichlorodihydrofluorescein (H2DCFDA) ROS detection assay, was monitored in HaCaTs. The mechanism of α-TP ROS scavenging was determined using non-cell based DPPH and ORAC assays. In HaCaT keratinocytes, irradiated with 226 J/cm² UVA1 in low-serum (2%, starved) Cell Culture medium, pretreatment with 80 µM α-TP significantly enhanced cell survival (88%, Alamar Blue) compared to control, whereas α-T pre-treatment had no effect survival (70%, Alamar Blue). Pre-treatment of cells with 100 μM α-TP or 100 μM α-T before 57 J/cm² UVA1 also significantly reduced ROS generation over 2 h (24.1% and 23.9% respectively) compared to the control and resulted in α-TP bioconversion into α-T. As α-TP displayed weak antioxidant activity in the cell-free assays thus its photoprotection was assigned to its bioconversion to α-T by cellular phosphatases. Through this mechanism α-TP prevented long-wave UVA1 induced cell death and scavenged UVA1 induced ROS in skin cells when added to the starved Cell Culture medium before UVA1 exposure by bioconversion into α-T.