Different Oxidative Stress Response in Keratinocytes and Fibroblasts of Reconstructed Skin Exposed to Non Extreme Daily-Ultraviolet Radiation

Experiments characterizing the biological effects of sun exposure have usually involved solar simulators. However, they addressed the worst case scenario i.e. zenithal sun, rarely found in common outdoor activities. A non-extreme ultraviolet radiation (UV) spectrum referred as “daily UV radiation” (DUVR) with a higher UVA (320–400 nm) to UVB (280–320 nm) irradiance ratio has therefore been defined. In this study, the biological impact of an acute exposure to low physiological doses of DUVR (corresponding to 10 and 20% of the dose received per day in Paris mid-April) on a 3 dimensional reconstructed skin model, was analysed. In such conditions, epidermal and dermal morphological alterations could only be detected after the highest dose of DUVR. We then focused on oxidative stress response induced by DUVR, by analyzing the modulation of mRNA level of 24 markers in parallel in fibroblasts and keratinocytes. DUVR significantly modulated mRNA levels of these markers in both cell types. A cell type differential response was noticed: it was faster in fibroblasts, with a majority of inductions and high levels of modulation in contrast to keratinocyte response. Our results thus revealed a higher sensitivity in response to oxidative stress of dermal fibroblasts although located deeper in the skin, giving new insights into the skin biological events occurring in everyday UV exposure

Exposure to Non-Extreme Solar UV Daylight: Spectral Characterization, Effects on Skin and Photoprotection

The link between chronic sun exposure of human skin and harmful clinical consequences such as photo-aging and skin cancers is now indisputable. These effects are mostly due to ultraviolet (UV) rays (UVA, 320–400 nm and UVB, 280–320 nm). The UVA/UVB ratio can vary with latitude, season, hour, meteorology and ozone layer, leading to different exposure conditions. Zenithal sun exposure (for example on a beach around noon under a clear sky) can rapidly induce visible and well-characterized clinical consequences such as sunburn, predominantly induced by UVB. However, a limited part of the global population is exposed daily to such intense irradiance and until recently little attention has been paid to solar exposure that does not induce any short term clinical impact. This paper will review different studies on non-extreme daily UV exposures with: (1) the characterization and the definition of the standard UV daylight and its simulation in the laboratory; (2) description of the biological and clinical effects of such UV exposure in an in vitro reconstructed human skin model and in human skin in vivo, emphasizing the contribution of UVA rays and (3) analysis of photoprotection approaches dedicated to prevent the harmful impact of such UV exposure

The Damaging Effects of Long UVA (UVA1) Rays: A Major Challenge to Preserve Skin Health and Integrity

Within solar ultraviolet (UV) light, the longest UVA1 wavelengths, with significant and relatively constant levels all year round and large penetration properties, produce effects in all cutaneous layers. Their effects, mediated by numerous endogenous chromophores, primarily involve the generation of reactive oxygen species (ROS). The resulting oxidative stress is the major mode of action of UVA1, responsible for lipid peroxidation, protein carbonylation, DNA lesions and subsequent intracellular signaling cascades. These molecular changes lead to mutations, apoptosis, dermis remodeling, inflammatory reactions and abnormal immune responses. The altered biological functions contribute to clinical consequences such as hyperpigmentation, inflammation, photoimmunosuppression, sun allergies, photoaging and photocancers. Such harmful impacts have also been reported after the use of UVA1 phototherapy or tanning beds. Furthermore, other external aggressors, such as pollutants and visible light (Vis), were shown to induce independent, cumulative and synergistic effects with UVA1 rays. In this review, we synthetize the biological and clinical effects of UVA1 and the complementary effects of UVA1 with pollutants or Vis. The identified deleterious biological impact of UVA1 contributing to clinical consequences, combined with the predominance of UVA1 rays in solar UV radiation, constitute a solid rational for the need for a broad photoprotection, including UVA1 up to 400 nm

Diversity of Biological Effects Induced by Longwave UVA Rays (UVA1) in Reconstructed Skin

<div><p>Despite their preponderance amongst the ultraviolet (UV) range received on Earth, the biological impacts of longwave UVA1 rays (340–400 nm) upon human skin have not been investigated so thoroughly. Nevertheless, recent studies have proven their harmful effects and involvement in carcinogenesis and immunosuppression. In this work, an i<i>n vitro</i> reconstructed human skin model was used for exploring the effects of UVA1 at molecular, cellular and tissue levels. A biological impact of UVA1 throughout the whole reconstructed skin structure could be evidenced, from morphology to gene expression analysis. UVA1 induced immediate injuries such as generation of reactive oxygen species and thymine dimers DNA damage, accumulating preferentially in dermal fibroblasts and basal keratinocytes, followed by significant cellular alterations, such as fibroblast apoptosis and lipid peroxidation. The full genome transcriptomic study showed a clear UVA1 molecular signature with the modulation of expression of 461 and 480 genes in epidermal keratinocytes and dermal fibroblasts, respectively (fold change> = 1.5 and adjusted p value<0.001). Functional enrichment analysis using GO, KEGG pathways and bibliographic analysis revealed a real stress with up-regulation of genes encoding heat shock proteins or involved in oxidative stress response. UVA1 also affected a wide panel of pathways and functions including cancer, proliferation, apoptosis and development, extracellular matrix and metabolism of lipids and glucose. Strikingly, one quarter of modulated genes was related to innate immunity: genes involved in inflammation were strongly up-regulated while genes involved in antiviral defense were severely down-regulated. These transcriptomic data were confirmed in dose-response and time course experiments using quantitative PCR and protein quantification. Links between the evidenced UVA1-induced impacts and clinical consequences of UVA1 exposure such as photo-aging, photo-immunosuppression and cancer are discussed. These early molecular events support the contribution of UVA1 to long term harmful consequences of UV exposure and underline the need of an adequate UVA1 photoprotection.</p></div

Levels of secreted proteins in culture medium of reconstructed skin exposed to UVA1.

<p>Culture media were taken at 48 hours post UVA1 exposure and used to measure the amount of extracellular matrix remodeling proteins (matrix metalloproteinases, MMPs and GDF15), pro-inflammatory proteins (IL-6, GM-CSF/CSF2, CCL20 and GDF15), the HGF growth factor and the CXCL10 ( = IP10) interferon inducible protein. Values of control samples were adjusted to the 1 value. Asterisks indicate a significant difference between mean protein amount of control samples and mean protein amount of UVA1 exposed samples (p<0.05, Student's t test). AU, arbitrary units.</p

Overall analysis of gene expression in reconstructed skin exposed to UVA1, using microarray.

<p>Triplicates of reconstructed skins were unexposed (control) or exposed to 40 J/cm² UVA1. Six hours later, a full genome transcriptomic study was conducted using Affymetrix microarray in fibroblasts (F) and keratinocytes (K), separately, for the 3 control reconstructed skins (samples F1-F3 and K1-K3) and for the 3 UVA1 exposed reconstructed skins (samples F4-F6 and K4-K6). <b>A</b>: Hierarchical clustering based on all probe set normalized expression data, using Ward's method and correlation distance. Y-axis of dendrogram represents the linkage distance that separates singletons or clusters. Height at which two clusters are merged in dendrogram reflects distance of the two clusters. <b>B</b>: Fold change comparison plot between fibroblasts and keratinocytes depicting number of significantly modulated probe sets 6 hours after exposure to 40J/cm² UVA1. Each probe set is plotted. On y-axis: log of fold change value in keratinocytes; on x-axis: log of fold change value in fibroblasts. In keratinocytes and in fibroblasts, 502 and 494 probe sets were differentially expressed between UVA1 exposed and control reconstructed skins (fold-change >1.5 or <0.67, Adjp<0.001), respectively. Blue circles (n = 107) represent probe sets modulated in both keratinocytes and fibroblasts. Green (n = 395) and red (n = 387) circles represent probe sets modulated only in keratinocytes and in fibroblasts, respectively. Grey circles (n = 2787): un-modulated probe sets. <b>C</b>: Heat map showing relative expression levels of the probe sets differentially expressed between UVA1-exposed and control samples (fold-change threshold >1.5 or <0.67 and Adjp value<0.001). Two-dimensional hierarchical clustering was carried out with the 502 and 494 probe sets differentially expressed between UVA1 exposed and control samples in keratinocytes and fibroblasts, respectively. Euclidean distance and Ward's method, based on normalized log2-transformed gene expression value relative to median value of each row were used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105263#pone.0105263-Eisen1" target="_blank">[85]</a>. Each row represents a probe set, each column represents one sample. Red, high expression. Black, median expression. Green, low expression.</p

Cellular effects in human reconstructed skin exposed to UVA1.

<p>Sham-exposed (control) and UV-exposed samples were taken for classical histology and for vimentin staining (vimentin: green labeling, nuclei counterstaining: red labeling) at 48 h post UVA1 exposure. Arrows indicate fibroblast disappearance in human dermal equivalent.</p

Distribution of UVA1 modulated genes in functional families.

<p>In order to perform an exhaustive bibliographic analysis including literature related to skin and dermatology, the list of UVA1 modulated genes was reduced by using a fold change threshold>2 or <0.5, and an Adjp<0.001. Under these criteria, 134 and 141 genes were found modulated in fibroblasts and keratinocytes respectively of UVA1 exposed reconstructed skins. In fibroblasts 24 genes out of 134 could not be classified because their functions were poorly described; 110 genes were distributed in functional families (<b>A</b>). In keratinocytes 30/141 genes could not be classified; 111 genes were distributed in functional families (<b>B</b>). Some genes could be classified in several functional families. Lists of gene names associated with gene bank accession number, fold change values and their distribution in functional families are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105263#pone.0105263.s011" target="_blank">Tables S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105263#pone.0105263.s012" target="_blank">S7</a>, for fibroblasts and keratinocytes respectively.</p