Protective Effect of α-Tocopherol-6-O-Phosphate Against Ultraviolet B-Induced Damage in Cultured Mouse Skin  Satomi Nakayama, Shizuko Kobayashi, Ph.D.  Journal of Investigative Dermatology  Volume 121, Issue 2, Pages 406-411 (August 2003) DOI: 10.1046/j.1523-1747.2003.12351.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 (A) Structure of α-TP and absorbance of α-Toc (a), α-TP (b), and α-TA (c) at a concentration of 500 μM in methanol (left Y-axis) and the emission spectrum of the UVB-lamp (d; right Y-axis). Journal of Investigative Dermatology 2003 121, 406-411DOI: (10.1046/j.1523-1747.2003.12351.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The effect of α-TP and α-TA pretreatments on TBARS generation by UVB-irradiation. (A) Time course of TBARS generation after UVB-irradiation. Dorsal skins incubated in medium without DMSO (□) or DMSO for 3 h were exposed to UVB irradiation at a dose of 10 kJ per m2 (•):–DMSO; ○: +DMSO). At 0 to 24 h after the exposure, lipid peroxidation in the skin homogenates was measured by TBARS assay. (B) Dose–response of TBARS generation by UVB exposure. Fresh mouse skin was cultured for 3 h in the 0.5% α-TP or α-TA containing media. The tissues were then washed with phosphate-buffered saline and UVB irradiation was performed. After 24 h of exposure, TBARS were determined in the dorsal homogenates. □, nontreated skin cultured in media with DMSO; ○, nontreated skin cultured in media without DMSO; ▪, α-TA-treated skin cultured in media with DMSO; •, α-TP-treated skin cultured in media without DMSO. Each bar represents the mean±SD of 10 skin samples from five mice. #p<0.05 and ##p<0.01 relative to nontreated skin cultured in media with DMSO. *p<0.05 and **p<0.01 relative to nontreated skin cultured in media without DMSO. Journal of Investigative Dermatology 2003 121, 406-411DOI: (10.1046/j.1523-1747.2003.12351.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Photomicrographs of cultured mouse skin stained with 1% hematoxylin and eosin (A), and by the TUNEL method (B), 24 h after UVB irradiation at 20 kJ per m2. ▴, sunburn cells, *, TUNEL-positive nuclei. Journal of Investigative Dermatology 2003 121, 406-411DOI: (10.1046/j.1523-1747.2003.12351.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effects of α-TP and α-TA pretreatment on sunburn cell formation induced by UVB exposure. Twenty-four hours after UVB exposure, the number of sunburn cells was counted in 0.2 mm of epidermis at three sites along the cultured skin. These cells were characterized by their dense, irregular nuclei that were darker than the nuclei of neighboring keratinocytes. □, nontreated skin cultured in media with DMSO; ○, nontreated skin cultured in media without DMSO; ▪, α-TA-treated skin cultured in media with DMSO; •, α-TP-treated skin cultured in media without DMSO. Each bar represents the mean±SD of 10 skin samples from five mice. #p<0.05 relative to nontreated skin cultured in media with DMSO. **p<0.01 relative to nontreated skin in media without DMSO. Journal of Investigative Dermatology 2003 121, 406-411DOI: (10.1046/j.1523-1747.2003.12351.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effects of α-TP and α-TA pretreatment on the reduction in the concentration of α-Toc induced by UVB irradiation. Fresh mouse skin was cultured for 3 h in media containing either 0.5% α-TP or α-TA containing media. The tissues were then washed with phosphate-buffered saline and UVB irradiation was performed. The amount of α-Toc was determined by HPLC using a reverse-phase column 30 min and 24 h later. Each bar represents the mean±SD of 10 skin samples from five mice. ##p<0.01 relative to nontreated, nonirradiated cultured skin. **p<0.01 relative to nontreated, irradiated cultured skin. Journal of Investigative Dermatology 2003 121, 406-411DOI: (10.1046/j.1523-1747.2003.12351.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Dephosphorylation of α-TP and ester hydrolysis of α-TA in mouse skin homogenates. Skin from hairless mice was homogenized in HEPES buffer, at pH 7.2 (100 mg skin per mL). The homogenates were incubated with 1 mM α-TP (•) or α-TA (▪) for 1 to 120 min and their concentration of α-Toc was then determined by HPLC. The results shown represent the mean of two experiments. Journal of Investigative Dermatology 2003 121, 406-411DOI: (10.1046/j.1523-1747.2003.12351.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Incorporation of α-TP and its conversion to α-Toc in the human cultured skin model. The skin was cultured for 2 or 6 h in media containing 0.5% α-TP. The amount of α-Toc was determined by HPLC using a reverse-phase column. The results shown represent the mean of two experiments. In the present experiment, little amount of α-Toc was detected in nontreated, control human cultured skin. Each bar represents the mean±SD of two skin samples. Journal of Investigative Dermatology 2003 121, 406-411DOI: (10.1046/j.1523-1747.2003.12351.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions