Skin response to urban pollution in an ex vivo model

A vast range of pollutants (polycyclic aromatic hydrocarbons, heavy metals, particulate matters) are generated by increasing urbanization. The skin epidermis, as the outermost line of defense of the body, is continuously exposed to external aggressions, including environmental pollution. To study the effects of air pollutant exposure on skin physiology, the first challenge was to define a model that mimics as close as possible the in vivo skin exposure to atmospheric pollution. In the present study, the pollutant mixture was designed on the basis of the French non-profit organization ‘Air Parif’ database and nebulized on human skin explants using the Pollubox® device. Cellular and molecular modifications in skin explants were investigated from several donors exposed to a pollutant mix (P) or an organic solvent (OS). Transcriptomic analyses coupled to immunolabelling showed that P exposure induced the expression of detoxifying enzymes notably in the stratum granulosum. Interestingly, transmission electron microscopy observations demonstrated that, in our model, diesel particles smaller than 300 nm could penetrate the skin up to the granular layer. This may cause local alterations of the microenvironment which alter the skin physiology. Therefore, the response of the epidermis to pollutant exposure is likely to be layer-specific. The study was approved by an ethics committee and performed in accordance with the Declaration of Helsinki after the patient had given informed consent.Full-thickness human skin biopsies were obtained from abdomen of healthy female donors who had undergone plastic surgery. The hypodermis was removed from the skin and circular explants were excised using a sample punch. The samples were placed immediately in BIO-EC’s Explant Medium (BEM) for 5 days and were cultured under classical cell culture conditions (37°C in 5% CO2). On day 5 (D5), the explants of the concerned batches were placed in the exposure system Pollubox® and treated with a solution containing heavy metals, hydrocarbons and fine particulate matters for 1h30 min. Some of these compounds were solubilized in an organic solvent (OS) which was also evaluated on the skin explants. On D6 and D7 respectively 24 and 48 hours after pollutants exposure, skin explants were harvested and were cut in half for storage in RNAlater (Qiagen) or fixation in buffered formol. Total RNAs were extracted using the ReliaPrep Tissue Miniprep kit from Promega after disruption and homogenization using the TissueLyser Kit. RNAs (70ng) of each explant from 4 donors (V1 to V4) 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 V3 AMADID072363) which contains 60.000 probes, derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq. Microarray data were quantified (GeneExtraction Feature V10.7) and normalized with R tools (Bioconductor). The different experiments completed during our study on the human skin explants from various volunteers in the context of the URBASKIN project supported by french FUI (Fonds Unique Interministeriel).