Topical N-Acetyl Cysteine and Genistein Prevent Ultraviolet-Light-Induced Signaling That Leads to Photoaging in Human Skin in vivo Sewon Kang, Jin Ho.

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Topical N-Acetyl Cysteine and Genistein Prevent Ultraviolet-Light-Induced Signaling That Leads to Photoaging in Human Skin in vivo Sewon Kang, Jin Ho Chung, Joo Heung Lee, Gary J. Fisher, Yian Sheng Wan, Elizabeth A. Duell, John J. Voorhees Journal of Investigative Dermatology Volume 120, Issue 5, Pages 835-841 (May 2003) DOI: 10.1046/j.1523-1747.2003.12122.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 UV irradiation increases H2O2 levels in human skin in vivo. Subjects were irradiated on buttock skin with 2 MED UV. Ten and 20 min after UV, irradiated and nonirradiated skin were obtained and analyzed for H2O2 levels as described in Methods. Results are means ± SEM. *p<0.01 vs nonirradiated skin; †p<0.02 vs nonirradiated skin; n=9. Journal of Investigative Dermatology 2003 120, 835-841DOI: (10.1046/j.1523-1747.2003.12122.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Neither NAC nor genistein act as sunscreens to reduce UV-induced skin reddening. Subjects’ buttock skin was treated on separate sites topically with 20% NAC, 1% genistein, and their vehicles and sham irradiated (one site of each vehicle) or exposed to 2 MED UV (second site of each vehicle, NAC, and genistein sites). Skin reddening 24 h after UV irradiation was quantified with a Minolta chromameter using the *a scale. Values are expressed as the difference between *a value of nonirradiated vehicle-treated skin and irradiated skin. Results are means ± SEM, n=6. Journal of Investigative Dermatology 2003 120, 835-841DOI: (10.1046/j.1523-1747.2003.12122.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 UV-irradiation-induced EGF-R phosphorylation is blocked by genistein in human skin in vivo. Buttock skin was treated with vehicle (VEH) or 1% genistein (GEN) for 24 h prior to 2 MED UV exposure. Skin was obtained 30 min after UV irradiation. Skin samples were extracted and EGF-R was immunoprecipitated. Total and tyrosine-phosphorylated EGF-R in the immunoprecipitate was determined by Western analysis. Bar heights are means ± SEM. *p<0.05 vs vehicle+UV, n=5. Inset shows representative Western blot. Journal of Investigative Dermatology 2003 120, 835-841DOI: (10.1046/j.1523-1747.2003.12122.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Topical NAC treatment increases reduced glutathione and eliminates oxidized glutathione in human skin in vivo. Subjects were treated on buttock skin with vehicle (VEH) or NAC under occlusion. Skin was obtained 8, 16, 24, and 48 h after NAC treatment. Skin samples were extracted and reduced glutathione (GSH) (left) and oxidized glutathione (GS-SG) (right) levels were measured by high performance liquid chromatography. Results are means ± SEM. *p<0.05 vs vehicle-treated skin, n=6. Journal of Investigative Dermatology 2003 120, 835-841DOI: (10.1046/j.1523-1747.2003.12122.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 NAC and genistein inhibit UV stimulation of ERK MAP kinases in human skin in vivo. Buttock skin was treated with vehicle (VEH), NAC (20%), or genistein (1%, GEN) for 24 h prior to UV irradiation (2 MED). Skin was obtained 4 h after UV irradiation. Skin samples were homogenized, and ERK1/2 was immunoprecipitated from the soluble fraction. Bar heights are means ± SEM. *p<0.05 vs VEH+UV, n=9. Journal of Investigative Dermatology 2003 120, 835-841DOI: (10.1046/j.1523-1747.2003.12122.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Genistein but not NAC inhibits UV stimulation of JNK MAP kinase in human skin in vivo. Buttock skin was treated with vehicle (VEH), NAC (20%), or genistein (1%, GEN) for 24 h prior to UV exposure. Skin was obtained 4 h after UV irradiation (2 MED UV). Skin samples were homogenized and JNK was immunoprecipitated from the soluble fraction. JNK activity was measured in the immunoprecipitate as described in Methods. Bar heights are means ± SEM. *p<0.05 vs VEH+UV, n=8. Journal of Investigative Dermatology 2003 120, 835-841DOI: (10.1046/j.1523-1747.2003.12122.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Genistein and NAC inhibit UV induction of cJun protein in human skin in vivo. Buttock skin was treated with (a) vehicle (VEH) and genistein (1%, GEN) or (b) vehicle and NAC (20%) for 24 h prior to UV irradiation (2 MED). Skin was obtained 4 h after UV exposure. cJun levels in the nuclear extracts were determined by Western analysis. Insets show representative Western blots. Results are means ± SEM. *p<0.05 vs vehicle+UV; n=5 for genistein, n=12 for NAC. Journal of Investigative Dermatology 2003 120, 835-841DOI: (10.1046/j.1523-1747.2003.12122.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Genistein and NAC inhibt UV induction of MMP-1 mRNA in human skin in vivo. Buttock skin was treated with (a) vehicle (VEH) and genistein (1%, GEN) or (b) vehicle and NAC for 24 h prior to UV irradiation (2 MED). Skin samples were obtained 24 h after 2 MED UV irradiation. Total RNA was extracted and MMP-1 and 36B4 (control) mRNA was quantified by real-time RT-PCR. MMP-1 mRNA levels were normalized to 36B4 mRNA levels and are expressed relative to no UV control. Bar heights are means ± SEM. *p<0.05 vs VEH+UV; n=5 for GEN, n=4 for NAC. Journal of Investigative Dermatology 2003 120, 835-841DOI: (10.1046/j.1523-1747.2003.12122.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 A model for the pathophysiology of photoaging. Exposure of skin cells to UV results in the activation of growth factor and cytokine receptors that leads to activation of MAP kinases ERK and JNK. UV irradiation also results in rapid production of ROS, which are critical for MAP kinase activation. The MAP kinase signal transduction pathway leads to increased expression of the transcription factor AP-1 (cFos/cJun), which in turn upregulates MMP gene expression. MMPs (e.g., collagenase) degrade the extracellular matrix of the dermis. This matrix damage is followed by repair, which cannot be perfect, resulting in a deficit in the dermal structure. The residual deficit is an invisible solar scar. With repeated intermittent UV exposures, the solar scars will accumulate, eventually resulting in visible skin wrinkling or photoaging. Journal of Investigative Dermatology 2003 120, 835-841DOI: (10.1046/j.1523-1747.2003.12122.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions