Impact of cigarette smoke on physical-chemical and molecular proprieties of human skin in an ex vivo model

This study deals with the skin aging exposome focusing on the effect of cigarette smoke, one of the main harmful components. To that purpose, human skin living explants, an ex vivo model, were exposed to cigarette smoke of two cigarettes for 2hours using a patented lab-made exposure chamber, the Pollubox®. The effect on the surface physico-chemistry and molecular properties of the skin were thus deepened and for the first time reported. Basic Procedures: To evidence this effect, gene expression profile, MDA dosage and surface physiochemistry data (Surface free energy determination, Trans-Epidermal Water loss, skin pH and FTIR spectroscopy of the surface of the explants) were collected on non-treated and treated human skin explants Main Findings: Results exhibit a decrease of the total surface free energy of the treated skin explants. This decrease reflects higher interactions with polar compounds from the environment and as a consequence a decrease of the hydrophobia of the surface. Additionally, an increase of the trans epidermal water loss and skin pH was measured after treatment. The molecular analysis shows downregulation of specific genes combined with the increase of MDA in the cigarette smoke-treated skin explants. Principal Conclusions: Cigarette smoke induced an oxidation of the lipids at the surface of the skin explants. This implies an alteration of the skin barrier function: interactions with polar products are enhanced and lipid chain packing at the surface is modified. As a consequence, skin permeation could increase. While as expectedly genes involved in the AHR pathway were induced in CS-exposed human skin explants, our molecular analysis suggested that mitochondrial functions were strongly impacted and oxidized lipids failed to be eliminated promoting skin barrier alteration. Neonatal human epidermal keratinocytes (HEKn) are primary cells derived from neonatal foreskins. They are supplied by the American Type Culture Collection (ATCC) in the form of 5 × 10 5 viable cells vials from different donors in order to have a constant homogeneity of cell populations. These cells were cultured in a specific medium (Epilife, Life Technologies) supplemented with a plant-derived supplement (HKSdaFREE and HKGE, AvantBio) that replaces the sera of animal origin more commonly used in cell culture. The cells were cultured at 37 ° C in an incubator (5% CO2 / 95% air). Total RNA was extracted from keratinocyte using RNeasy Mini Plus Kits (Qiagen). Total RNA were used for reverse transcription, amplification and Cy3 labeling, using the Low Input Labeling kit, one-color (Agilent Technologies). All cRNAs were hybridized to human whole genome oligo microarrays (Agilent Technologies v2 (AMADID 039494), which contains 60.000 probes, derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq. Microarray data were quantified (GeneExtraction Feature V11.0,1) and normalized with R tools (Bioconductor).