Abstract
Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.
Highlights
Solar ultraviolet (UV) rays constitute one of the most important environmental stresses to which human skin is constantly exposed
To approach the question whether UVA1 exposure has a specific effect on the metabolic state of basal epidermal keratinocytes and of fibroblasts in the superficial dermis, we studied reconstructed human skin samples exposed to different external stimuli: control, UVA1 25 J/cm[2], and UVA1 40 J/cm[2] and we measured their metabolic fingerprint in both skin layers with 2c-2PEF fluorescence lifetime microscopy (FLIM) of NAD(P)H and flavin adenine dinucleotide (FAD) at specific time points: before (T0) and following UVA exposure, at 30 min (Texpo + 30 min) and 2 h (Texpo + 2 h) (Figs. 3, 4)
We first optimized NAD(P)H and FAD excitation efficiencies by enhancing FAD signal level independently of NAD(P)H through two-color twophoton excitation (Fig. 1) by wavelength mixing[39] obtaining comparable fluorescence intensities with a good signal-to-noise ratio in the two channels, in a one-shot measurement and without motion artifacts (Fig. 2c). We applied this approach to the simultaneous measurement of NAD(P)H and FAD lifetimes in keratinocytes in the epidermis basal layer and in fibroblasts in the superficial dermis layer of reconstructed human skin exposed to different doses of UVA1 light or control-sham exposure (Fig. 1b)
Summary
Solar ultraviolet (UV) rays constitute one of the most important environmental stresses to which human skin is constantly exposed. Longwave UVA (UVA1, 340–400 nm) exposure has several important consequences both at cellular and molecular level as the photoexcited states of skin endogenous photosensitizers generate reactive oxygen species (ROS) (Figure S1)[5]. Our group recently implemented simultaneous two-photon excitation of NAD(P)H and FAD by wavelength mixing to acquire FLIM data of the two biomarkers at the same time and perform multiparametric metabolic imaging in dynamic biological s ystem[26]. The goal of this study is to assess at different time scales the effects of UVA1 light exposure on the cellular metabolic activity of different cell types in reconstructed human skin using simultaneous fluorescence lifetime imaging of NAD(P)H and FAD. We present measurements of NAD(P)H and FAD fluorescence lifetimes changes after short-term exposure to UVA1 rays in keratinocytes and fibroblasts. We show that the metabolic response is biomarker, cell type and dose-dependent
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