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 alpha-tocopherol phosphate, (alpha-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 alpha-TP into alpha-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 alpha-TP ROS scavenging was determined using non-cell based DPPH and ORAC assays. In HaCaT keratinocytes, irradiated with 226 J/cm(2) UVA1 in low-serum (2%, starved) cell culture medium, pretreatment with 80 mu M alpha-TP significantly enhanced cell survival (88%, Alamar Blue) compared to control, whereas alpha-T pre-treatment had no effect survival (70%, Alamar Blue). Pre-treatment of cells with 100 mu M alpha-TP or 100 mu M alpha-T before 57 J/cm(2) UVA1 also significantly reduced ROS generation over 2 h (24.1% and 23.9% respectively) compared to the control and resulted in alpha-TP bioconversion into alpha-T. As alpha-TP displayed weak antioxidant activity in the cell-free assays thus its photoprotection was assigned to its bioconversion to alpha-T by cellular phosphatases. Through this mechanism alpha-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 alpha-T.