Chem. Res. Toxicol. 1999, 12, 187-191
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UV-B-Induced Photooxidation of Vitamin E in Mouse Skin Kimberly Kramer-Stickland, Ed S. Krol, and Daniel C. Liebler* Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, P.O. Box 210207, Tucson, Arizona 85721 Received August 31, 1998
Topically applied R-tocopherol (R-TH, vitamin E) inhibits UV-B (290-320 nm) photocarcinogenesis, yet R-TH undergoes rapid photooxidation by UV-B in vitro. To examine the effect of UV-B on R-TH in vivo, we studied the fate of R-TH in UV-B-irradiated mouse skin. [14C]R-TH was applied to mouse skin at various times prior to UV-B irradiation. UV-B irradiation for 1 h at a dose rate of 2.6-2.9 J m-2 s-1 resulted in consumption of 40-60% of the applied dose and formation of oxidation products. The major product fraction formed in UV-B-irradiated mice treated topically with R-TH contained an R-TH dihydroxy dimer and its two-electron oxidation product, a spirodimer. Products previously identified as being derived from photochemical or peroxyl radical scavenging reactions of R-TH were also observed, including R-tocopherolquinone (R-TQ), R-tocopherolquinone 2,3-epoxide (R-TQE 1), R-tocopherolquinone 5,6-epoxide (R-TQE 2), and 8a-(hydroperoxy)epoxytocopherones. These results indicate that topically applied R-TH is extensively oxidized in skin and suggest that R-TH photoproducts may be involved in the observed effects of topically applied vitamin E in UV-B-irradiated skin.
Introduction The ability of R-tocopherol (R-TH,1 vitamin E) (1, Figure 1) to act as an antioxidant and absorb UV-B radiation makes it a potentially ideal topical chemopreventive agent against photocarcinogenesis. Studies in which the chemopreventive actions of R-TH in nonmelanoma skin cancer have been investigated have centered on evaluating the effects of topically applied vitamin E on the induction of UV-induced tumors in animals (1, 2). Topical R-TH inhibits UV-B-induced tumors, lipid peroxidation, and other indicators of UVB-induced oxidative damage in the skin, but the exact mechanism of cancer chemoprevention by R-TH is unknown. Antioxidant actions of R-TH are thought to be involved (3, 4), and Packer and colleagues recently measured R-tocopherol depletion as an indicator of antioxidant activity (5). The effects of UV radiation on skin levels of endogenous R-TH have been measured previously (6, 7), and the extent of depletion of topically applied tocopherols by UV-B has also been measured (5). Our previous studies indicate that R-TH turnover in UV-B-irradiated skin involves direct consumption of R-TH by UV-B in addition to antioxidant reactions of R-TH (8). Antioxidant consumption of R-TH results in the scavenging of peroxyl radicals and the formation of R-TH oxidation products. Photochemical consumption of R-TH results from the absorption of UV-B radiation, cleavage of the chromanol O-H bond, and formation of R-TH dimers or products resulting from R-TH autoxidation. Antioxidant consumption may be distinguished from Figure 1. Structures referred to in the text. * To whom correspondence should be addressed. Phone: (520) 6264488. Fax: (520) 626-2466. E-mail:
[email protected]. 1 Abbreviations: R-TH, R-tocopherol; R-T•, tocopheroxyl radical; R-TQ, R-tocopherolquinone; R-TQE 1, R-tocopherolquinone 2,3-epoxide; R-TQE 2, R-tocopherolquinone 5,6-epoxide; APCI, atmospheric pressure chemical ionization; BHT, butylated hydroxytoluene; MS-MS, tandem mass spectrometry; SDS, sodium dodecyl sulfate.
photochemical consumption of R-TH on the basis of some of the oxidation products formed. R-Tocopherolquinone (R-TQ, 3), R-tocopherolquinone epoxides (R-TQEs 4 and 5), and epoxytocopherones 7 and 8 may result from either antioxidant or photochemical turnover of R-TH via
10.1021/tx980204h CCC: $18.00 © 1999 American Chemical Society Published on Web 01/22/1999
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competing reactions of the tocopheroxyl radical 2. Photochemical reactions of R-TH in vitro form novel dimer products not observed in peroxyl radical scavenging reactions. We previously reported the formation of dihydroxy dimer 9 in UV-B photooxidation of R-TH (8). Here we report that multiple dimer products are formed by UV-B irradiation. Our investigation of the antioxidant properties and photochemistry of R-TH in vitro (8) provided a simple model system for examining the fate of UV-B-generated tocopheroxyl radicals. The present studies focus on determining the fate of topically applied R-TH in UV-Birradiated skin so we can better understand the mechanism of prevention of photocarcinogenesis by R-TH.
Experimental Procedures Chemicals and Instrumentation. R-TH (RRR-R-TH) was a gift from Henkel Fine Chemicals (La Grange, IL). d3- and d6R-TH were synthesized by reduction of their respective acetate esters (9), which were obtained from the Natural Source Vitamin E Association (Kingsport, TN). [14C]-R-TH (0.25 µCi/µmol) was synthesized by reductive alkylation of γ-TH with [14C]formaldehyde as previously described (10). Tocopherol dimer standards were purified by reverse-phase HPLC, using a 250 mm × 4.6 mm ODS-2, 5µ column with 75:25 (v/v) methanol/ethyl acetate as a mobile phase at a flow rate of 1.5 mL/min with the UV detector set at 300 nm. The identities of both the spirodimer and the dihydroxy dimer were confirmed by APCI-MS and MSMS and by comparison of the UV-vis spectra with literature values (10, 11). R-TH spirodimer 6 was prepared by the method of Nelan et al. (11), with the product eluting at 15 min under the above HPLC conditions. Dihydroxy dimer 9 was prepared from 6 using the method of Skinner et al. (12), with 9 eluting at 10 min. Other reagents were obtained commercially from standard sources and used without purification. UV irradiations were performed with Westinghouse FS-20 UV-B lamps (National Biological Corp., Twinsburg, OH) and a UVX digital radiometer with a UVX-31 sensor (Ultraviolet Products, Inc., San Gabriel, CA). Approximately 80% of the lamp output was in the UV-B range (290-320 nm), less than 1% in the UV-C range (
