Effects of UV light and tumor promoters on endogenous vitamin E status in mouse skin
Daniel C. Liebler1 and
Jeanne A. Burr
Department of Pharmacology and Toxicology and Southwest Environmental Health Sciences Center, College of Pharmacy, The University of Arizona, PO Box 210207, Tucson, AZ 85721-0207, USA
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Abstract
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Recent reports indicate that both orally administered and topically applied
-tocopherol (vitamin E, TH) prevent UVB-induced skin carcinogenesis in mice. Because UVB exposure causes the formation of oxidants associated with tumor promotion, epidermal TH status may be an important determinant of susceptibility to photocarcinogenesis. To test this hypothesis, we studied the status of epidermal TH in C3H mice following exposure to single and repeated UVB exposures at doses typical of chronic photocarcinogenesis protocols. Exposure of mice to a single 13 kJ/m2 dose over 60 min resulted in no acute depletion of epidermal TH and a modest increase in TH within 612 h. Daily exposure to 6.5 kJ/m2 over 30 min resulted in a gradual increase in epidermal TH, which reached 5-fold after five daily exposures. The increase in epidermal TH was accompanied by an increase in the TH oxidation products
-tocopherolquinone (TQ) and
-tocopherolhydroquinone (THQ). We also studied the effect of the prooxidant chemical tumor promoter benzoyl peroxide and the prooxidant azo initiators azobis(amidinopropane HCl) and azobis(2,4-dimethylvaleronitrile). Topical application of these prooxidant chemicals acutely oxidized epidermal TH to TQ and THQ. Topical treatments with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased epidermal TH levels without producing a significant accumulation of TH oxidation products. The results indicate that UVB and tumor promoting chemicals all exert qualitatively different effects on epidermal TH status and that UVB and TPA trigger an adaptive response involving epidermal TH accumulation.
Abbreviations: AAPH, azobis(amidinopropane HCl); AMVN, azobis(2,4-dimethylvaleronitrile); TH,
-tocopherol; THQ,
-tocopherolhydroquinone; T(H)Q, sum of measured TQ and THQ; TPA, 12-O-tetradecanoylphorbol-13-acetate; TQ,
-tocopherolquinone; TQE1, 5,6-epoxy-
-tocopherolquinone; TQE2, 2,3-epoxy-
-tocopherolquinone.
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Introduction
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Ultraviolet (UV) light is the most common cause of skin malignancies in humans. Several effects of UVB (290320 nm) are thought to contribute to skin carcinogenesis in vivo. First, the induction of DNA photodamage, such as pyrimidine cyclobutane dimers and pyrimidine 64 pyrimidone photoproducts, leads to mutations in critical genes (13). Second, UVB-induced activation of intracellular signaling pathways results in the activation of transcription factor AP-1, which is thought to contribute to tumor promotion (47). Third, UVB causes the generation of free radicals and related oxidants, which contribute to carcinogenesis by directly damaging cellular macromolecules, including DNA (reviewed in ref. 8). Finally, UVB induces the production of tumor necrosis factor-
and other cytokines by epidermal cells (9,10) and leads to an inflammatory response characterized by leukocyte infiltration and production of leukocyte-derived oxidants (11).
Topical application of
-tocopherol (TH) (Figure 1
), the most biopotent form of vitamin E, prevents UVB-induced carcinogenesis in C3H mouse skin (12). Dietary supplementation also prevented UVB photocarcinogenesis in C3H mice (13). Supplemental TH is hypothesized to strengthen epidermal antioxidant defenses against UVB-induced oxidative damage. UVB exposure consumes TH in mouse skin in vitro (14) and Shindo et al. reported that UVB modestly depleted TH in Sim HRS/hr hr mice in vivo (15). These results are consistent with the view that UVB causes production of free radicals, which then react with and deplete TH. However, these studies differ from those in which topical TH inhibited photocarcinogenesis in two significant ways. First, the UVB dose used in the in vivo studies of Shindo et al. was 2.5x105 J/m2 (15), which is ~14 times the dose used in chronic photocarcinogenesis studies (12). Second, Shindo et al. examined the effect of a single UVB exposure, whereas chronic photocarcinogenicity studies involve repeated exposures.
To further explore the significance of oxidative stress and TH antioxidant actions in UVB photocarcinogenesis, we have examined TH turnover and oxidative damage in response to UVB doses used in chronic photocarcinogenesis studies. We have applied highly sensitive and specific analytical methods to measure the consumption of TH by antioxidant reactions. Our data indicate that, at doses used in chronic photocarcinogenesis studies, UVB causes no discernible TH oxidation in single exposures. In contrast, multiple exposures of UVB induce a gradual increase in TH content accompanied by an increase in levels of TH oxidation products. Experiments with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the prooxidant chemicals indicate that these agents all produce distinctly different effects on epidermal TH. The data suggest that acute UVB causes a relatively subtle oxidative stress in the epidermis and that mild prooxidant tumor promoters such as UVB and TPA may induce adaptive antioxidant responses.
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Materials and methods
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Animals and UVB exposures in vivo
Female C3H/HeN Tac (H-2k, MTV) mice (Taconic, Germantown, NY) were used at 710 weeks of age in all experiments. Mice were fed a semi-synthetic (AIN 76) diet containing vitamin E as all-rac-TH acetate. The dorsal skin of the mice was shaved prior to irradiation. A bank of eight parallel Westinghouse FS-20 lamps was used for irradiation. Lamps were used without supplementary optical filters. According to the manufacturer's specifications, 85% of the lamp output was in the UVB (290320 nm), <1% in the UVC (<290 nm), 4% in the UVA (320400 nm) and the remainder was in the visible spectrum. Mice were placed in a uniform field of irradiation 26 cm from the lamp surface. During exposures, mice were housed four per cage in cages covered with wire mesh and fitted with clear plexiglass dividers to prevent animal interaction. Dose rates for exposures were measured with mesh and dividers in place and were 3.63.7 J/m2/s. UVB dose rates were measured with a UVX radiometer equipped with a UVX-31 sensor. Mice were killed by carbon dioxide asphyxiation and dorsal epidermis was isolated for biochemical analyses by scraping as described previously (16).
Exposures to TPA and oxidants in vivo
Mice were treated with 10 nmol TPA (Sigma, St Louis, MO) applied in 200 µl acetone, with 20 mg (83 µmol) benzoyl peroxide (Aldrich, Milwaukee, WI) in 200 µl acetone, with 20 mg (74 µmol) azobis(amidinopropane HCl) (AAPH) (Polysciences, Warrington, PA) in 200 µl acetone, with 20 mg (81 µmol) azobis(2,4-dimethylvaleronitrile) (AMVN) in 200 µl acetone or with 200 µl acetone only (vehicle controls). At the times indicated, mice were killed as described above and epidermis was isolated for biochemical analyses.
Analyses of TH and oxidation products
TH and its oxidation products were analyzed by gas chromatographymass spectrometry, as described previously (17). For analysis of epidermal samples from in vivo experiments, the dorsal skin was treated for 5 min with a depilatory agent (Nair with Baby OilTM) immediately after death to remove hair stubble. The excess depilatory agent was then rinsed off and 20 µg butylated hydroxytoluene was applied in methanol to prevent TH oxidation during work-up. The full-thickness skin was then taken and the epidermis was isolated by scraping. The epidermal scrapings (~35 mg) were weighed accurately, incubated with 0.5 ml 0.25% trypsin/EDTA (Sigma) for 30 min at 37°C and then diluted with 3.5 ml phosphate-buffered saline, pH 7.2. From this mixture, three aliquots, each containing ~3 mg protein, were drawn. To each aliquot was added 100 pmol d6-TH, d6-
-tocopherolquinone, d6-5,6-epoxy-
-tocopherolquinone, d6-2,3-epoxy-
-tocopherolquinone and d3-
-tocopherolhydroquinone as internal standards, followed by 1ml 1 M SDS, 2 ml ethanol and 2 ml hexane. The samples were then extracted, derivatized and analyzed for TH and its oxidation products as described. Because of some uncertainty regarding the ability of this method to accurately measure the ratio of
-tocopherolquinone (TQ) to
-tocopherolhydroquinone (THQ), which undergo redox interconversion, the sum of the measured amounts of these products is expressed as T(H)Q (17).
Statistical analyses
For each mouse analyzed, the measurements were correlated. The mixed linear model was used, in which the mouse was treated as a random effect nested within a group and the group was treated as a fixed effect (18). Significant differences between groups (P < 0.05) are indicated.
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Results
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Effects of UVB exposures on epidermal TH
We investigated the effects of single and multiple exposures on epidermal vitamin E status. The total UVB dose and dose rate are comparable to those used in previous photocarcinogenesis studies with C3H mice (12,13). In epidermis of unexposed mice, TH was present at a level of 3.52 ± 0.56 pmol/mg (Figure 2
). Immediately after a single UVB irradiation, the TH level was essentially unchanged. After a second 30 min irradiation 24 h after the first, TH was significantly increased to 8.61 ± 0.65 pmol/mg. The increase in epidermal TH continued in mice treated with four or five daily 30 min UVB exposures, such that after five exposures, the epidermal TH content had increased ~5-fold. Along with the increase in epidermal TH, we observed an increase in the epidermal levels of TH oxidation products. Both TQ and THQ were present at measurable levels, beginning immediately after the second UVB irradiation. Neither 5,6-epoxy-
-tocopherolquinone (TQE1) nor 2,3-epoxy-
-tocopherolquinone (TQE2) were detected.

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Fig. 2. Effect of repeated UVB exposures on epidermal TH (filled bars) and T(H)Q (open bars) in C3H mice. Mice were exposed to 6.5 kJ/m2 over 30 min daily and skin was harvested immediately following the last exposure. Epidermal TH and T(H)Q were then immediately extracted and analyzed as described in Materials and methods. Values marked * are significantly different from controls (P < 0.01).
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Time course of TH status after a single 1 h UV exposure
We examined the effect of a 1 h exposure to UVB at the same fluence used in the above experiment. Mice exposed to 13 kJ/m2 over 60 min exhibited no significant change in epidermal TH immediately after irradiation (Figure 3
). At 1 h post-irradiation, a slight decrease in TH was not statistically significant. By 3 and 6 h, a modest, but statistically significant, increase in epidermal TH was measured. Levels of TH increased at both time points by ~1.7-fold. TH levels then declined at 12 and 24 h. No significant increases in T(H)Q, TQE1 or TQE2 were detected.

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Fig. 3. Effect of a single UVB exposure on epidermal TH levels. Mice were exposed to 13 kJ/m2 over 60 min and skin was harvested at the times indicated. Epidermal TH was immediately extracted and analyzed as described in Materials and methods. Values marked * are significantly different from controls (P < 0.01).
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Effects of prooxidant tumor promoters on epidermal TH
Because single UVB exposures did not deplete TH and because repeated exposures actually increased epidermal TH, we examined the impact of other prooxidant tumor promoters on mouse epidermal TH. The compounds selected for study were: (i) benzoyl peroxide, a labile acyl peroxide; (ii) AMVN, a lipid-soluble azo initiator; (iii) AAPH, a water-soluble azo initiator. Benzoyl peroxide readily generates benzoyloxyl radicals by homolysis of the peroxide bond, whereas the azo compounds undergo thermolysis to carbon-centered radicals that readily add oxygen to form peroxyl radicals (19).
Application of benzoyl peroxide to mouse skin at the dose used here was shown previously to induce mouse skin tumor promotion and progression (20,21). This dose resulted in depletion of TH to ~50% of control levels and formation of T(H)Q (Figure 4
). Formation of T(H)Q appeared to account for essentially all the TH consumed; traces of TQE1/2 were detected, but the amounts were below our limits of quantitation. AAPH similarly depleted epidermal TH and formed T(H)Q, which appeared to account for essentially all the TH oxidized. AMVN modestly depleted TH and formed T(H)Q, however, only the increase in T(H)Q was statistically significant. The results thus indicate that prooxidant chemicals directly oxidize TH in mouse skin.

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Fig. 4. Effects of prooxidant chemicals on epidermal TH (filled bars) and T(H)Q (open bars) levels. Each prooxidant chemical (20 mg) was applied in acetone and epidermal TH and T(H)Q were analyzed 24 h after application as described in Materials and methods.
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Effect of phorbol ester application on epidermal TH status
TPA is a phorbol ester tumor promoter that induces a chronic inflammatory response and prooxidant states in epidermis in vivo (22). TPA is not itself an oxidant, but stimulates the production of superoxide, hydrogen peroxide, nitric oxide and other prooxidants in dermal and epidermal cells (2325). Daily application of TPA in acetone at 10 nmol (6.1 µg)/mouse is typically used in two-stage mouse skin carcinogenesis protocols.
Application of TPA resulted in an increase in epidermal TH measured 72 h later (Table I
). T(H)Q was not significantly increased. Application of TPA once at 0 and once at 24 h followed by measurement 72 h after the first application again indicated a significant elevation of TH without significant formation of TH oxidation products. The increase in TH was not observed in animals treated only with the acetone vehicle (data not shown). As in the other experiments, no significant formation of TQE1 nor TQE2 was detected.
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Discussion
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Exposure of mice to UV, prooxidants or phorbol esters results in tumor promotion in mouse models of skin carcinogenesis. All of these tumor promoter regimens are associated with the induction of prooxidant states and oxidative damage in skin. Moreover, topical application of vitamin E prevents UV-induced photocarcinogenesis. Because TH is widely considered a critical component of cellular antioxidant defense, we examined the impact of these diverse prooxidant stimuli on the status of epidermal TH. Despite the ability of UV, prooxidant chemicals and TPA all to induce oxidative stress in conjunction with their tumor promotion activities, each exerted a qualitatively different effect on epidermal TH status. These differences suggest different contributions of prooxidant mechanisms for tumor promotion by each class and reveal differences in the response of epidermal TH to different prooxidants.
UVB exposure has been shown previously to consume TH in mouse skin in vitro (14) and Shindo et al. reported that UVB depletes TH in Sim HRS/hr hr mice in vivo (15). These results are consistent with the view that UVB induces production of free radicals, which then react with and deplete TH. Nevertheless, UV irradiation at the doses used in our experiments did not cause acute TH depletion. This is in contrast to the results reported by Shindo et al., who reported that UVB depleted TH (15). However, Shindo et al. employed a UVB dose of ~250 kJ/m2, which is ~1014 times the dose used in our studies and in chronic photocarcinogenesis studies. The dose required to deplete mouse epidermal TH in the study by Shindo et al. was ~10 times the minimal erythemal dose (15).
We have found that a single UVB dose equivalent to that used in chronic photocarcinogenesis protocols (6.5 kJ/m2 administered over 30 min) did not deplete endogenous epidermal TH nor did a 13 kJ/m2 dose administered over 60 min. Following the 13 kJ/m2 dose, a modest elevation of epidermal TH was observed, which returned to approximately pre-irradiation levels within 24 h. Thus, a single UVB exposure at the dose used in chronic photocarcinogenicity studies caused only a mild, temporary elevation of epidermal TH, rather than TH depletion. On the other hand, repeated daily doses of 6.5 kJ/m2 resulted in a time-dependent increase in epidermal TH. This was accompanied by an increase in the levels of TQ and THQ, which are products of antioxidant reactions of TH. Thus, it appears that TH is recruited to the epidermis in response to repeated UVB exposures and that some of the epidermal TH is consumed by reactions with oxidants.
In contrast to UVB, topical application of prooxidant chemicals, including the tumor promoter benzoyl peroxide, resulted in epidermal TH oxidation. This is consistent with the view that prooxidant chemicals generate radical oxidants that are trapped by TH acting as a cellular protectant. A single treatment with benzoyl peroxide at the dose used in tumor promotion protocols resulted in oxidation of over half the epidermal TH. The principal products of TH depletion were T(H)Q. The water-soluble azo initiator AAPH has not, to our knowledge, been evaluated as a tumor promoter. However, topical application of a dose equivalent on a molar basis to the benzoyl peroxide dose used caused a similar degree of epidermal TH oxidation. In contrast, the lipid-soluble azo initiator AMVN did not significantly deplete TH and caused only a slight increase in T(H)Q. We cannot presently explain why AMVN was less effective in depleting TH than AAPH, as they exhibit similar kinetics of radical formation in solution (19). However, AMVN may penetrate the stratum corneum less effectively than AAPH and thus may be less able to generate radicals in basal keratinocytes, which contain most of the epidermal TH.
The phorbol ester tumor promoter TPA is associated with induction of prooxidant responses in the epidermis, but is not itself an oxidant. In our experiments, TPA produced unique effects on epidermal TH. At 72 h following a single 10 nmol application, epidermal TH was significantly increased, but no significant increase in TH oxidation products was detected. The same result was observed 48 h following a second TPA application. Thus, like UVB, TPA induced an adaptive response in which additional TH accumulated in the epidermis. In contrast to multiple UV exposures, multiple TPA treatments did not induce the oxidation of epidermal TH. This is particularly interesting in view of the fact that both UV and TPA induce an inflammatory response in the dermis, which is thought to contribute to oxidative damage in the epidermis.
The mechanisms by which UVB and TPA result in increased epidermal TH remain to be investigated. Because both UVB and TPA induce multiple genes, it is possible that up-regulation of a transport system for TH may occur. TH is introduced to cells primarily via low density lipoprotein (LDL) through cell surface receptors (26). Whether the status of this system is subject to regulation by UVB or TPA response elements is not known. UVB causes the release of several cytokines in the epidermis and in the dermis, which is accompanied by changes in vascular permeability (11,27,28). These changes may permit the accumulation of TH bound to serum LDL. Whether the accumulated TH in the epidermis is actually incorporated into epidermal keratinocytes remains to be determined. However, this would seem to be the case, as the accumulated TH appears highly resistant to photooxidation (see below).
A particularly interesting aspect of this study is the resistance of endogenous epidermal TH to UVB-induced depletion. This is in sharp contrast to topically applied TH, which is readily photooxidized to T(H)Q, TQE1/2, dimer and trimer products by UVB in C3H mice at the fluences used in our experiments (29). TH photooxidation proceeds via the tocopheroxyl radical, which is then further consumed by divergent reaction pathways. Although epidermal TH in basal keratinocytes may be somewhat shielded from UVB by the stratum corneum, UVB photons nevertheless penetrate the basal layer and will certainly reach cellular TH. We propose that tocopheroxyl radicals formed from epidermal TH by UVB are rapidly reduced by other cellular reductants. The lack of access of topically applied TH to intracellular reductants would explain the greater susceptibility of topically applied TH to photooxidation in vivo. Oxidation of epidermal TH by the prooxidant chemicals benzoyl peroxide and AAPH evidently reflects the concomitant oxidation of other cellular reductants, which would then be unable to sustain TH. Accumulation of the TH oxidation products T(H)Q with repeated UVB exposures probably reflects the consumption of TH by antioxidant reactions accompanying chronic inflammation in the skin. This would also be expected to occur in TPA-treated skin, as inflammation accompanies chronic TPA treatment. However, the lack of increase in TH oxidation products may reflect either our use of only two TPA treatments or some difference in the production of oxidants in TPA- and UVB-treated skin.
Another interesting aspect of TH oxidation in skin in these experiments is the apparent lack of formation of TQE1/2. The failure to observe TQE1/2 as products of TH oxidation is somewhat surprising, as these epoxyquinones are significant products of TH antioxidant chemistry in biomimetic models and in biological systems (3033). Although we detected traces of these products in our gas chromatographymass spectrometry analyses of UVB-exposed skin, the levels were below our limits of quantitation. Topical application of TQE1 and TQE2 to mice followed by analysis of the epidermis indicated that the epoxyquinones are stable in the epidermis (data not shown). Moreover, recovery of deuterated TQE1/2 standards was equivalent to that for TH, TQ and THQ, which were detected. Thus, we are confident that the absence of TQE1/2 in our analyses reflects a lack of formation of the epoxyquinones, rather than some flaw in our analytical methods.
Another point worth considering is the potential contribution of UVA to oxidant formation in the epidermis. The lamps used in our experiments emit ~80% of their output in the UVB and 4% in the UVA. Thus, our data reflect the combined effects of UVB and some UVA. Although UVA is associated with the production of oxidants, possibly including singlet oxygen in skin (34,35), UVA does not directly induce TH photooxidation due to a lack of absorbance of tocopherols in the UVA wavelength range. The possibility that UVA itself indirectly induces TH oxidation via the induction of cellular oxidant production cannot be ruled out and would be reasonable to address in a future study.
In summary, our results indicate that epidermal TH status responds differently to UVB, prooxidant chemicals and phorbol ester tumor promoters. Epidermal TH is resistant to UVB-induced photooxidation, although repeated exposures to UVB or TPA result in an adaptive response in which epidermal TH is increased. This increase in TH is accompanied by some formation of TH oxidation products in UVB-treated skin, but not in TPA-treated skin. In contrast, prooxidant chemicals induced acute oxidation of TH and the formation of T(H)Q. Differences in the responsiveness of TH to UVB and other tumor promoters may underscore differing contributions of oxidants and oxidative stress to tumor promotion in mouse skin in vivo.
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Acknowledgments
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This work was supported by NIH grants CA 59585 and ES 06694.
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Notes
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1 To whom correspondence should be addressed Email: liebler{at}pharmacy.arizona.edu

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Received June 15, 1999;
revised September 29, 1999;
accepted October 8, 1999.