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 overtime the UVA1- induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of the two endogenous biomarkers 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 We measure NAD(P)H and FAD fluorescence lifetime and fraction of bound coenzymes both in keratinocytes in the epidermis basal layer and in fibroblasts in the dermis superficial layer. 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 OXPHOS or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamics and sensitivities to skin cell types and UVA1 dose. While FAD biomarker is UVA1 dose-dependent in keratinocytes, NAD(P)H biomarker shows earlier time points modulation in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter MCE prevents UVA1 metabolic stress response from occurring.
Human skin is constantly exposed to environmental stresses such as UV light and pollution. These agents cause
oxidative stress associated with reactive oxygen species (ROS) generation, that will interfere with the normal cellular
redox equilibrium. As ROS are mainly produced within mitochondria, the cellular metabolic activity could be impacted
by UV light.
We dynamically assessed UVA light (representing the majority of solar UV rays reaching Earth surface) effects on
cellular metabolic activity of reconstructed human skin using multiphoton fluorescence lifetime imaging microscopy
(FLIM).
Multiphoton FLIM offers non-invasive, label-free quantitative functional information on cellular metabolic activity
based on the endogenous two-photon excited fluorescence (2PEF) of NADH (reduced form of nicotinamide adenine
dinucleotide) and FAD (flavine adenine dinucleotide) metabolic coenzymes.
The experiments were performed in both stratum granulosum and spinosum layers (T-Skin™ model, Episkin™), before
and after (30 min and 2 h) UVA exposure (20 J/cm²; 20 min exposure; 320 – 400 nm).
We observed quasi similar effects in both epidermal layers after UVA exposure:
• Decrease of RedOx ratio NADH / (NADH + FAD) at 30 min and 2 h;
• Increase in the proportion of protein-bound NADH at 2 h, and in the proportion of free FAD as early as 30 min
after UVA exposure;
This study shows that the effects of UVA light on epidermis, can be non-invasively evidenced and followed overtime
using NADH/FAD multiphoton FLIM imaging method. Altogether, these data suggest that epidermal cells respond to
UVA light by promoting oxidative phosphorylation, the most efficient metabolic pathway for ATP production.
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