Photoaging is Associated with Protein Oxidation in Human Skin In Vivo

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Photoaging is Associated with Protein Oxidation in Human Skin In Vivo Christina S. Sander, Hong Chang, Susann Salzmann, Cornelia S.L. Müller, Swarna Ekanayake-Mudiyanselage, Peter Elsner, Jens J. Thiele Journal of Investigative Dermatology Volume 118, Issue 4, Pages 618-625 (April 2002) DOI: 10.1046/j.1523-1747.2002.01708.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunohistochemical localization of antioxidant enzymes and protein oxidation in human skin. Immunostaining of CuZnSOD (a-c), MnSOD (d-f), and CAT (g-i) in frozen human skin sections. Identification of protein oxidation (j-l) by incubating the sections with DNPH and, subsequently, with an anti-dinitrophenylhydrazone antibody. Sections were prepared from young control skin (a, d, g, j), intrinsically aged skin (b, e, h, k), and photoaged skin (c, f, i, l). Scale bar: 50 μm. Journal of Investigative Dermatology 2002 118, 618-625DOI: (10.1046/j.1523-1747.2002.01708.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Depleted antioxidant enzyme expression and accumulated protein oxidation in photoaged human skin. Densitometric analysis of staining intensity by image analysis software. Scanning of sections stained for CuZnSOD (a), MnSOD (b), CAT (c), and protein oxidation (d). Black bars, young control skin; grey bars, intrinsically aged skin; white bars, photoaged skin. *p < 0.05, **p < 0.01, ***p < 0.001. Mean ± SEM, n = 12. Journal of Investigative Dermatology 2002 118, 618-625DOI: (10.1046/j.1523-1747.2002.01708.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunohistochemical localization of CAT and protein oxidation in human skin upon repetitive solar simulated UV exposures. Immunostaining of CAT (a, b) in frozen human skin sections. Identification of protein oxidation (c, d) by incubating the sections with DNPH and, subsequently, with an anti-dinitrophenylhydrazone antibody. Sections were prepared from buttock skin of human subjects (a, c) before UV exposure and (b, d) after a 10 d repetitive UV irradiation protocol using a solar simulator (“dermalight vario” with filter h2, Dr Hoehnle). Scale bar: 50 μm. Journal of Investigative Dermatology 2002 118, 618-625DOI: (10.1046/j.1523-1747.2002.01708.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Depleted CAT expression and accumulated protein oxidation in human skin upon repetitive solar simulated UV exposures. Densitometric analysis of staining intensity by image analysis software. Scanning of sections stained for CAT (a) and protein oxidation (b). Black bars, before UV exposure; white bars, after 10 d UV exposure. *p < 0.05, **p < 0.01, ***p < 0.001. Mean ± SEM, n = 12. Journal of Investigative Dermatology 2002 118, 618-625DOI: (10.1046/j.1523-1747.2002.01708.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Increased protein oxidation in photoaged human skin. lmmunoblot analysis of carbonyl residues in protein extracts from epidermal (ED) and dermal (D) tissue of young control skin, intrinsically aged skin, and photaged skin (a). 10 μg of protein extracts were incubated with DNPH, subsequently electrophoresed by SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with polyclonal rabbit anti-dinitrophenylhydrazone antibody. Densitometric analysis of total carbonylated proteins was performed (b). Black bars, young control skin; grey bars, intrinsically aged skin; white bars, photoaged skin. **p < 0.01. Mean ± SEM, n= 4. Journal of Investigative Dermatology 2002 118, 618-625DOI: (10.1046/j.1523-1747.2002.01708.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 UVA irradiation and H2O2 treatment increased protein oxidation in human fibroblasts. lmmunoblot analysis of carbonyl residues in protein extracts from (a) UVA-irradiated (spectrum 340-440 nm, UVA1 sunbank “dermalight ultra A1”, Dr Hoehnle) and (b) hydrogen-peroxide-treated (1 mM for 12 h in serum-free medium) human fibroblasts (human embryogenic skin fibroblasts, American Type Culture Collection, CRL 1502). 10 μg of protein extracts were incubated with DNPH, subsequently electrophoresed by SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with polyclonal rabbit anti-dinitrophenylhydrazone antibody. Journal of Investigative Dermatology 2002 118, 618-625DOI: (10.1046/j.1523-1747.2002.01708.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 UVB irradiation and H2O2 treatment increased protein oxidation in human keratinocytes. lmmunoblot analysis of carbonyl residues in protein extracts from (a) UVB-irradiated (UVB medical bulb, absorption maximum 313 nm; Philips-Medicine) and (b) hydrogen-peroxide-treated (1 mM for 12 h in serum-free medium) human immortalized keratinocytes (HaCaT). 10 μg of protein extracts were incubated with DNPH, subsequently electrophoresed by SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with polyclonal rabbit anti-dinitrophenylhydrazone antibody. Journal of Investigative Dermatology 2002 118, 618-625DOI: (10.1046/j.1523-1747.2002.01708.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions