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Chronic UVA1 Irradiation of Human Dermal Fibroblasts: Persistence of DNA Damage and Validation of a Cell Cultured–Based Model of Photoaging

2019; Elsevier BV; Volume: 139; Issue: 8 Linguagem: Inglês

10.1016/j.jid.2019.02.022

1523-1747

Alicia Montoni, Kelly M. George, Jérémie Sœur, Christian Tran, Laurent Marrot, Patrick J. Rochette,

Indoor Air Quality and Microbial Exposure

Solar UVA, representing 90–95% of solar UV, can be subdivided into UVA2 (315–340 nm) and UVA1 (340–400 nm). UVA1 represents 75% of total solar UV and can induce reactive oxygen species (Tewari et al., 2012Tewari A. Sarkany R.P. Young A.R. UVA1 induces cyclobutane pyrimidine dimers but not 6-4 photoproducts in human skin in vivo.J Invest Dermatol. 2012; 132: 394-400Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) and cyclobutane pyrimidine dimer (CPD) formation (Rochette et al., 2003Rochette P.J. Therrien J.P. Drouin R. Perdiz D. Bastien N. Drobetsky E.A. et al.UVA-induced cyclobutane pyrimidine dimers form predominantly at thymine-thymine dipyrimidines and correlate with the mutation spectrum in rodent cells.Nucleic Acids Res. 2003; 31: 2786-2794Crossref PubMed Scopus (185) Google Scholar). Penetrance of UVR in human skin is wavelength-dependent and UVA1 penetrate into the deeper layers of the skin (D'Orazio et al., 2013D'Orazio J. Jarrett S. Amaro-Ortiz A. Scott T. UV radiation and the skin.Int J Mol Sci. 2013; 14: 12222-12248Crossref PubMed Scopus (1031) Google Scholar). Exposure to UVA1 is exacerbated in indoor tanning users, where these rays are overrepresented (Balk et al., 2013Balk S.J. Fisher D.E. Geller A.C. Teens and indoor tanning: a cancer prevention opportunity for pediatricians.Pediatrics. 2013; 131: 772-785Crossref PubMed Scopus (22) Google Scholar). UVA1 are less influenced than UVB by environmental and geo-orbital parameters (Marionnet et al., 2014Marionnet C. Pierrard C. Golebiewski C. Bernerd F. Diversity of biological effects induced by longwave UVA rays (UVA1) in reconstructed skin.PLoS One. 2014; 9: e105263Crossref PubMed Scopus (71) Google Scholar), and are also the most difficult UV wavelengths to filter through sunscreens. Many changes related to photoaging can occur in skin. Among them, the photoaging-induced changes in dermal extracellular matrix are well defined. They include the aberrant overproduction of elastin (Bernstein and Uitto, 1996Bernstein E.F. Uitto J. The effect of photodamage on dermal extracellular matrix.Clin Dermatol. 1996; 14: 143-151Abstract Full Text PDF PubMed Scopus (54) Google Scholar) and a dysregulation of dermal collagens (Gniadecka et al., 1998Gniadecka M. Nielsen O.F. Wessel S. Heidenheim M. Christensen D.H. Wulf H.C. Water and protein structure in photoaged and chronically aged skin.J Invest Dermatol. 1998; 111: 1129-1133Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). There is strong evidence showing that photoaging leads to a decrease in collagen synthesis (Quan et al., 2004Quan T. He T. Kang S. Voorhees J.J. Fisher G.J. Solar ultraviolet irradiation reduces collagen in photoaged human skin by blocking transforming growth factor-beta type II receptor/Smad signaling.Am J Pathol. 2004; 165: 741-751Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Moreover, UV-induced reactive oxygen species stimulate gene expression of collagen degrading matrix metalloproteinase (MMP) (Quan et al., 2009Quan T. Qin Z. Xia W. Shao Y. Voorhees J.J. Fisher G.J. Matrix-degrading metalloproteinases in photoaging.J Investig Dermatol Symp Proc. 2009; 14: 20-24Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). These photoaging-related changes in the skin are primarily attributed to UVA wavelengths (Krutmann, 2000Krutmann J. Ultraviolet A radiation-induced biological effects in human skin: relevance for photoaging and photodermatosis.J Dermatol Sci. 2000; 23: S22-S26Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). In this study, we have developed a photoaging model by chronically irradiating fibroblasts with UVA1. We used residual CPDs, that we found both in the papillary dermis of sun-exposed skin and in chronically UVA1-exposed fibroblasts, as a marker of cumulative solar exposure. We used this model to determine transcriptomic changes that can occur in skin fibroblasts as a result of chronic UVA1 exposure. All experiments performed in this study were conducted in accordance with our institution's guidelines and the Declaration of Helsinki. The research protocols received approval by the Centre Hospitalier Universitaire de Québec institutional committee for the protection of human subjects with written informed patient consent. We compared the accumulation of CPDs in human skin from breast reductions (unexposed) and facelifts (sun exposed) (Figure 1a, 1b ), as well as in skin of dorsal (unexposed) and inner (sun exposed) forearm from nine individuals (Figure 1c, 1d). No CPD was detected in the epidermis, most likely due to damage dilution through the renewal of the epidermis. In dermis, where cells are non-dividing, we observed an accumulation of residual CPDs. This observation was possible in the most anterior, thus the most exposed to UVA, portion of the dermis, the papillary dermis. The observation of CPD accumulation in fibroblasts of the papillary dermis does not imply that they are absent in the reticular dermis, they are indeed difficult to quantify due to the high autofluorescence of the collagen present in reticular dermis. UVB-induced CPDs in dermis is unlikely, due to UVB absorption in epidermis (Mallet et al., 2016Mallet J.D. Dorr M.M. Drigeard Desgarnier M.C. Bastien N. Gendron S.P. Rochette P.J. Faster DNA repair of ultraviolet-induced cyclobutane pyrimidine dimers and lower sensitivity to apoptosis in human corneal epithelial cells than in epidermal keratinocytes.PLoS One. 2016; 11: e0162212Crossref PubMed Scopus (12) Google Scholar, Tewari et al., 2012Tewari A. Sarkany R.P. Young A.R. UVA1 induces cyclobutane pyrimidine dimers but not 6-4 photoproducts in human skin in vivo.J Invest Dermatol. 2012; 132: 394-400Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) and UVA induce CPDs preferentially in dermis (Tewari et al., 2012Tewari A. Sarkany R.P. Young A.R. UVA1 induces cyclobutane pyrimidine dimers but not 6-4 photoproducts in human skin in vivo.J Invest Dermatol. 2012; 132: 394-400Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Even if it cannot be excluded that some of the CPDs found in the papillary dermis are from UVB component of solar UV, these evidence strongly suggest that they are the result of exposure to the UVA component of solar UV. UV-induced CPDs can be eliminated by DNA damage repair and dilution through DNA replication. We have shown that, in fibroblasts, the absence of repair in certain chromosomic regions results in chronic UVB-induced CPD accumulation (Berube et al., 2018Berube R. Drigeard Desgarnier M.C. Douki T. Lechasseur A. Rochette P.J. Persistence and tolerance of DNA damage induced by chronic UVB irradiation of the human genome.J Invest Dermatol. 2018; 138: 405-412Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In vivo, the mitotic index of fibroblasts is very low in the dermis and declines with age (Gunin et al., 2011Gunin A.G. Kornilova N.K. Vasilieva O.V. Petrov V.V. Age-related changes in proliferation, the numbers of mast cells, eosinophils, and cd45-positive cells in human dermis.J Gerontol A Biol Sci Med Sci. 2011; 66: 385-392Crossref PubMed Scopus (71) Google Scholar). The elimination of DNA damage through replicative dilution should therefore be minimal. We believe that CPDs found in fibroblasts of papillary dermis are the result of chronic UVA irradiation during life, coupled with a lack of repair in certain areas of the DNA. We questioned the impact of residual UVA1-induced CPD on the functionality of dermal fibroblasts in connection with photoaging. To do so, we have developed an in cellulo photoaging model by chronically irradiating fibroblasts with 20 kJ/m2 UVA1 (lamp spectrum in Supplementary Figure S1 online), two times a day, 4 days a week for 5 weeks. This chronic UVA irradiation protocol caused the accumulation of CPDs in fibroblasts (Figure 1e, 1f). This accumulation of CPDs is proportional to what is found in papillary dermis of exposed regions (Figure 1a–1d), suggesting that our chronic UVA1 irradiation protocol mimics, at least in part, the cumulative solar exposure received by the dermis and can therefore be used as a model for skin photoaging. To our knowledge, this finding represents a previously unreported demonstration that chronic UVA1 irradiation can induce residual CPDs, and suggests that the residual CPDs found in the papillary dermis are the result of cumulative UVA exposure. We have then analyzed transcriptomic modifications related to the chronic UVA1 irradiation and consequently, to dermal photoaging (Supplementary Figure S2 online). Predominant collagens found in dermis (collagen I and III) have been found previously downregulated in photoaging (Quan et al., 2004Quan T. He T. Kang S. Voorhees J.J. Fisher G.J. Solar ultraviolet irradiation reduces collagen in photoaged human skin by blocking transforming growth factor-beta type II receptor/Smad signaling.Am J Pathol. 2004; 165: 741-751Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). In this study, we have found that chronic UVA1 irradiation causes an important gene expression decrease of type I collagen by a factor of 1.8 to 6.5 (1A2 and 1A1, respectively) (Figure 2a, 2b ) and of type III collagen (5.4-fold). These results are consistent with a phenotype of photoaging. Type V collagen, representing 5% of dermal collagen in vivo, has been found downregulated by 4.9 and 6.7 times (5A1 and 5A3, respectively). We found a reduction of 25-fold of collagen 4A4, which is known as a major component of basement membrane. We find 28% of collagens (12/43), the expression of which is significantly reduced following chronic irradiation. Those results indicate that chronic UVA1 irradiation affects the capacity of fibroblasts to generate collagen. Overexpression of MMP1, 3, and 9 is associated to photoaging (reviewed in Quan et al., 2009Quan T. Qin Z. Xia W. Shao Y. Voorhees J.J. Fisher G.J. Matrix-degrading metalloproteinases in photoaging.J Investig Dermatol Symp Proc. 2009; 14: 20-24Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). We observe non-significant variations of MMP1 and MMP9 gene expression, but a 2.8-fold increase in MMP3 expression level. This increase correlates with a photoaging phenotype following chronic UVA1 irradiation. Of all the MMPs analyzed, 26% are deregulated, all positively, following a chronic UVA1 irradiation. Tissue inhibitors of metalloproteinase, MMP antagonists, show no significant deregulation. Versican, biglycan, and decorin are the three main proteoglycans found in the skin (reviewed in Lee et al., 2016Lee D.H. Oh J.H. Chung J.H. Glycosaminoglycan and proteoglycan in skin aging.J Dermatol Sci. 2016; 83: 174-181Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Our photoaging model shows a 2.1-fold decrease in versican expression, but no change in biglycan and decorin (Figure 2e, 2f), which correlates with previous observations made on photoaged skin (Lee et al., 2016Lee D.H. Oh J.H. Chung J.H. Glycosaminoglycan and proteoglycan in skin aging.J Dermatol Sci. 2016; 83: 174-181Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We observed a 6.9-fold reduction in the elastin gene expression level, which is in contradiction with the generally reported elastin increase following sunlight irradiation, but consistent with the previously observed decrease of elastin expression following a 1-week chronic UVA1 irradiation in fibroblasts (Zheng et al., 2018Zheng Y. Xu Q. Chen H. Chen Q. Gong Z. Lai W. Transcriptome analysis of ultraviolet A-induced photoaging cells with deep sequencing.J Dermatol. 2018; 45: 175-181Crossref PubMed Scopus (10) Google Scholar). All those photoaging-related gene expression changes observed using narrow spectrum UVA1 highlight their importance in skin photoaging and the importance of extending the absorption spectrum of sunscreens formulas to wavelengths >340 nm via new ingredients or agents. The data sets (microarray data) generated and analyzed during the current study are available in the Gene Expression Omnibus National Center for Biotechnology Information database for public access. The GSE accession number is GSE125429. Alicia Montoni: http://orcid.org/0000-0002-8713-8103 Kelly M. George: http://orcid.org/0000-0001-5520-9897 Jérémie Soeur: http://orcid.org/0000-0001-6838-4299 Christian Tran: http://orcid.org/0000-0003-2244-3796 Laurent Marrot: http://orcid.org/0000-0001-5353-192X Patrick J. Rochette: http://orcid.org/0000-0002-0678-8869 KMG, JS, CT, and LM are employees of a company with a commercial interest in UV protection. AM and PJR state no conflict of interest. This work is supported by L'Oreal. PJR is a research scholar from the Fonds de Recherche du Québec, Santé. Conceptualization: AM, KMG, JS, CT, LM, PJR. Data curation: AM, KMG, JS, CT, LM, PJR. Formal analysis: AM, CT, LM, PJR. Funding acquisition: KMG, JS, LM, PJR. Investigation: AM, KMG, JS, CT, LM, PJR. Methodology: AM, CT. Project administration: KMG, LM, PJR. Resources: KMG, JS, CT, LM, PJR. Supervision: KMG, LM, PJR, Validation: AM, KMG, JS, CT, LM, PJR. Visualization: AM, KMG, LM, PJR. Writing–original draft: AM, PJR. Writing–review and editing: AM, KMG, JS, CT, LM, PJR. All experiments performed in this study were conducted in accordance with our institution's guidelines and the Declaration of Helsinki. The research protocols received approval by the Centre Hospitalier Universitaire de Québec institutional committee for the protection of human subjects. Two different series of skin samples were used in this project. (i) Skin from plastic surgery—breast reduction from four patients and facelift from six patients—were used as sun-protected and sun-exposed, respectively. The patients were all women, with a median age of 51 years old. Skin samples were collected within 1 hour post-surgery, embedded in water-based Tissue-Tek O.C.T. compound (Sakura, Torrance, CA) and kept at −80°C until further analysis. Frozen tissues were cut in 5-μm slices and mounted onto slides. (ii) Skin from a study conducted at the Clinical Research Center for Hair and Skin Science (Department of Dermatology and Allergy Charité-Universitätsmedizin, Berlin, Germany) from nine women aged between 55 and 75 years. In this study design, each subject was its own control: inner forearm was considered as a solar unexposed area, while dorsal forearm was considered as photo-exposed. Solar exposure prior to CPD assessment was carefully controlled in this study. Subjects were instructed to "completely avoid using sunbeds or sun exposure on the forearms and face within the 4 weeks before inclusion to the study and having planned UV sessions or sun exposure of the forearms and face during the study period." Subjects were also instructed to "keep covered the investigational sites of biopsies until the last assessments (wear long armed clothes to protect the study site during the whole study." Two-mm punch biopsies were performed on both inner and dorsal forearms. Skin samples were fixed with 10% phosphate buffered formaldehyde and embedded in paraffin. Section were cut in 5-μm slices, mounted onto slides, de-waxed in xylene, and rehydrated in a graded ethanol series. Normal human diploid fibroblasts were obtained by skin biopsy (mastectomy) and provided from four different healthy patients aged between 18 and 38 years. Cells were cultured in DMEM (cellgro; Corning, Tewksbury, MA) complemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Wisent, Saint-Jean-Baptiste, Quebec, Canada) at 37°C, 5% CO2. Fully confluent fibroblasts were irradiated with a UVA1 lamp emitting UVA1 wavelengths (340–400 nm) with 2-fold positively or negatively deregulated genes between the two conditions are represented by black dots. (b) Heatmap depicting the >2-fold positively or negatively deregulated genes in chronically UVA1-irradiated fibroblasts and unirradiated controls. The heatmap shows that the changes in gene expression caused by the chronic UVA1-irradiation are reproducible. The color scale used to display the log2 expression level values was determined by the hierarchical clustering algorithm of the Euclidian metric distance between genes. Genes indicated in dark blue correspond to those with an expression that is very low, whereas highly expressed genes are shown in orange/red. n = 4. (c) Heatmap depicting the most deregulated 42 genes by chronic UVA1 irradiation. Three control genes (GOLGA1, TUBB, and B2M) are used as experimental controls (bottom three genes of the heatmap). The transcription level of those genes is stable, independent of cell type and condition (Lee et al., 2007Lee S. Jo M. Lee J. Koh S.S. Kim S. Identification of novel universal housekeeping genes by statistical analysis of microarray data.J Biochem Mol Biol. 2007; 40: 226-231Crossref PubMed Google Scholar). Among the most deregulated genes, we find a 25-fold reduction in the gene encoding the α-4 subunit of the type IV collagen (col4a1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)