Temporal Transcriptomic Analysis of Human Primary Keratinocytes Exposed to β-naphthoflavone Highlights the Protective Efficacy of Skin to Environmental Pollutants

The skin covers almost the entire body and plays an important role in detoxification and elimination of xenobiotics. These processes are initiated following the binding of xenobiotics to the aryl hydrocarbon receptor (AhR), which leads to the expression of several detoxification enzymes. To gain some insights on their impacts on skin cells over time, a temporal transcriptional analysis using gene expression arrays was performed in human primary epidermal keratinocyte (HEK) cells exposed for 6, 24 and 48h to β-naphthoflavone (βNF), a potent agonist of AhR. Our results demonstrated that expression of genes related to xenobiotic, inflammation, and extracellular matrix remodeling was increased upon βNF treatment from 6h onwards. In contrast, the anti-oxidative response was seen mainly starting at 24h. While some of the genes controlled by the epidermal differentiation complex was induced as soon as 6h, expression of most of the S100 related genes located within the same chromosomal locus and keratin genes was increased at later times (24 and 48h). Altogether our transcriptomic data highlight that following βNF exposure, HEK cells elicited a protective xenobiotic response together with the activation of inflammation and keratinocyte regeneration. Later on these processes were followed by the stimulation of anti-oxidant activity and terminal differentiation. 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).