Effect of Ultraviolet Radiation and Benzo[a]pyrene Co-Exposure on Skin Biology: Autophagy as a Potential Target

Abstract

The skin is the outermost protective barrier of the human body. Its role is to protect against different physical, chemical, biological and environmental stressors. The vast majority of studies have focused on investigating the effects of single environmental stressors on skin homeostasis and the induction of several skin disorders, such as cancer or ageing. On the other hand, much fewer studies have explored the consequences of the co-exposure of skin cells to two or more stressors simultaneously, which is much more realistic. In the present study, we investigated, using mass-spectrometry-based proteomic analysis, the dysregulated biological functions in skin explants after their co-exposure to ultraviolet radiation (UV) and benzo[a]pyrene (BaP). We observed that several biological processes were dysregulated, among which autophagy appeared to be significantly downregulated. Furthermore, immunohistochemistry analysis was carried out to validate the downregulation of the autophagy process further. Altogether, the output of this study provides an insight into the biological responses of skin to combined exposure to UV + BaP and highlights autophagy as a potential target that might be considered in the future as a novel candidate for pharmacological intervention under such stress conditions.

Keywords: autophagy; benzo[a]pyrene; proteomics; skin; ultraviolet radiation.

Figure 1

Dysregulation of proteins from the cytoplasmic (A), cell membrane (B) and nuclear (C) extracts of human skin explants exposed to UV light or UV light and BaP, compared to untreated cells (Crtl). Each heatmap represents the expression profile of dysregulated proteins upon UV exposure with or without a co-exposure to BaP, based on the z-score of the protein’s normalized peak area. Rows represent dysregulated proteins and columns represent the samples at different exposure conditions. The red color represents an overexpression while the blue color represents a down-regulation of the protein compared to the mean value of a protein from all samples. The z-score is a normalization score of each row and has the same range of color values. Heatmaps were built using the Funrich tool, version 3.1.3. For each Venn diagram, the number of proteins over-expressed (p < 0.05; ≥1.5 fold) or under-expressed (p < 0.05; ≤0.67 fold) is indicated for each sub-localization of cells.

Figure 2

The major upregulated and downregulated biological processes following UV + BaP co-exposure. The heat map displays the major dysregulated biological process and the distribution of the identified proteins either upregulated (green color) or downregulated (red color), among each particular process. Some deregulated pathways solely harbor proteins that were all either downregulated (such as autophagy) or upregulated (such as cell proliferation) in the proteomics data, while other pathways have some of the proteins involved in these processes upregulated and others are downregulated (such as cell–cell adhesion).

Figure 3

Immunohistochemistry of skin explants for autophagy markers LC3-II and SQSTM. Seven days post the different treatments, either sham treated or exposed to UV and/or Bap, the staining of autophagy markers was conducted on the explants. (A) LC3-II staining; an increase in LC3-II puncta was detected upon the exposure of the samples to either UV or Bap compared to the untreated samples. Moreover, an increase was also detected for samples treated with both UV and Bap, but this was less prominent when compared to each treatment alone. (B) SQSTM staining; all of the different treatments displayed an increase in SQSTM staining, signifying a failure to eliminate autophagosomal aggregates. Examples of puncta are pointed out by arrows. To: explant on Day 0, tnt: untreated, Bap: Benzo[a]pyrene exposed, UV: UV irradiated, UV/Bap: UV irradiated and Benzo[a]pyrene exposed explant, *: p < 0.05, ***: p < 0.001, ns: non significant, 400× magnification (Obj. 40×).