- Email: [email protected]
UVR-induced oxidative stress in human skin in vivo: effects of oral vitamin C supplementation
Free Radical Biology & Medicine, Vol. 33, No. 10, pp. 1355–1362, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter
PII S0891-5849(02)01042-0
Original Contribution UVR-INDUCED OXIDATIVE STRESS IN HUMAN SKIN IN VIVO: EFFECTS OF ORAL VITAMIN C SUPPLEMENTATION F. MCARDLE, L. E. RHODES,1 R. PARSLEW, C. I. A. JACK, P. S. FRIEDMANN,2
and
M. J. JACKSON
Department of Medicine, University of Liverpool, Liverpool, UK (Received 25 July 2002; Revised 25 July 2002; Accepted 13 August 2002)
Abstract—Previous studies of cultured skin cells and murine skin in vivo have indicated that UVR-induced damage involves the generation of reactive oxygen species and depletion of endogenous antioxidant systems. In order to explore the relevance of this to UVR-induced damage to human skin, we have undertaken a detailed examination of the time-course of changes in markers of oxidative stress in human skin following exposure to physiological amounts of UVR in vivo. In addition, we have examined the skin bioavailability of a common nutritional antioxidant, vitamin C, and have assessed the effects of supplementation on markers of oxidative stress. Our hypothesis was that acute exposure of human skin to UVR in vivo would lead to oxidation of cellular biomolecules that could be prevented by prior vitamin C treatment. A UVR-challenge of 120 mJ/cm2 of broadband UVB (peak 310 nm, range 270 – 400 nm) was applied to buttock skin of 8 healthy volunteers. This caused a rapid and significant rise in activity of skin catalase at 1 h and an increase in the oxidized/total glutathione ratio at 6 h post-UVR. AP-1 DNA binding also peaked at 1– 6 h post-UVR, then declined rapidly to baseline levels. No significant changes were seen in skin malonaldehyde content. Oral vitamin C supplements (500 mg/day) were taken by 12 volunteers for 8 weeks resulting in significant rises in plasma and skin vitamin C content. Supplementation had no effect on the UVR-induced erythemal response. The skin malonaldehyde content was reduced by vitamin C supplementation, but surprisingly, reductions in the skin content of total glutathione and protein thiols were also seen. We speculate that this apparently paradoxical effect could be due to regulation of total reductant capacity by skin cells, such that vitamin C may have been replacing other reductants in these cells. No evidence was obtained for an effect of the supplementary vitamin C on the mild oxidative stress seen in human skin following UVR exposure. © 2002 Elsevier Science Inc. Keywords—Reactive oxygen species, Antioxidants, Transcription factors, Ascorbic acid, Free radicals
INTRODUCTION
considerable discussion concerning the potential role of oxidative stress in the deleterious effects of UVR on human skin. UVR is composed of UVA (320 – 400 nm), UVB (290 –320 nm) and UVC (289 –200 nm), but only UVA and UVB reach the earth’s surface and are of physiological significance [5,6]. A considerable amount of data has been published on the effects of UVA and UVB on oxidant generation in cultured skin cells [7–9] and murine skin in vivo [10 –13], but little has been reported on human skin in vivo. Most interest has concerned the effects of UVA in generating free radical and reactive oxygen species in skin cells [6,14], but data also indicate that, following exposure to UVB, cutaneous tissue, both in vitro and in vivo, generates free radicals and other reactive oxygen species (ROS) [15]. In cultured cells or murine skin, high doses of UVR have been reported to increase the hydrogen peroxide content asso-
Excessive exposure of skin to ultraviolet radiation (UVR) can result in acute and chronic damage [1,2] Short-term overexposure to UVR causes erythema (sunburn), whilst chronic overexposure leads to increased risk of skin cancer and premature photoageing [3]. Recently, concern has been expressed that humans are becoming exposed to higher doses of UVR due to depletion of ozone in the atmosphere [4]. There has been Address correspondence to: Professor M. J. Jackson, Department of Medicine, University of Liverpool, Duncan Building, Daulby Street, Liverpool L69 3GA, UK; Tel: ⫹44 151-706-4261; Fax: ⫹44 151-7065802; E-Mail: [email protected]. 1 Current address: Photobiology Unit, Dermatology Centre, University of Manchester, Manchester, UK. 2 Current address: Department of Dermatology, University of Southampton, Southampton, UK. 1355
1356
F. MCARDLE et al.
ciated with a decrease in cellular glutathione content and a decrease in the activity of protective enzymes, such as superoxide dismutase and catalase [7–13]. The relevance of these studies to human skin is contentious, since many utilize nonphysiological doses of UVR and the responses of murine and human skin differ in numerous respects. Nevertheless the few studies that have been undertaken on human skin in vivo confirm that hydrogen peroxide is produced following exposure to UVR [16] and that some changes in protective enzymes also occur [17]. Free radicals and reactive oxygen species may theoretically mediate many of the deleterious effects of UVR on skin by causing direct damage to cellular macromolecules or by modulating the expression of redox-sensitive genes and transcription factors to influence processes such as cell growth and proliferation [18]. The potential role of free radicals in UVR-induced skin damage has also led to considerable interest in whether supplementation with nutritional or pharmacological antioxidants can reduce the deleterious effects of UVR on skin [19,20]. Vitamin C is a powerful watersoluble antioxidant that has been shown to reduce UVBinduced oxidative damage in mouse keratinocytes in vitro [21] and to protect human keratinocytes from UVA-induced lipid peroxidation [22]. In mice, dietary vitamin C reduced the incidence of UVR-induced skin neoplasms [23], although in humans photoprotection against acute or chronic UVR effects by vitamin C supplementation alone has not been established. The clinical erythema endpoint is only one of a number of events in the skin triggered by UVR, and although it is frequently used as a guide to photoprotection, it cannot accurately reflect all aspects. We hypothesized that acute exposure of human skin to UVR would induce reproducible changes in indicators of free radical damage to skin and that these would be attenuated by prior supplementation with vitamin C. The aim of the work described here was, therefore, to examine the time course of changes in markers of oxidative damage following exposure of normal skin to UVR light and to examine the effects of supplementary oral vitamin C on the changes seen.
type II or III) were recruited. None had been exposed to excessive natural or artificial UV radiation in the 6 months prior to recruitment. A fixed UVR dose of 120 mJ/cm2 was given to three circular sites of 1 cm diameter on buttock skin. The irradiation source was a fluorescent broadband lamp (TL12, Phillips Lighting, Croydon, UK), which emitted UVR between 270 and 400 nm (peak emission 310 nm). Irradiance was measured with an IL1400A radiometer (International Light, Durham, UK). Skin punch biopsies (4 mm diameter) were taken from each of these sites at selected times after UVR exposure and from adjacent unexposed skin. The time points examined were 0, 1, 6, 24, 48, and 216 h postexposure, but for ethical reasons a maximum of only four biopsies could be taken from an individual. Immediately prior to biopsy, erythema at each site was assessed visually and by objective measurements (see erythema assessment below). All skin samples were rapidly frozen in liquid nitrogen and stored at ⫺70°C prior to analysis. Effect of vitamin C supplements Twelve healthy Caucasian volunteers (9 male, 3 female; mean age 23 years, range: 22–33 years, skin type II or III) were recruited. None had been exposed to excessive natural or artificial UV radiation in the 6 months prior to recruitment. A fixed UVR dose of 120 mJ/cm2 was given to two circular sites of 1 cm diameter on buttock skin as above. At 6 h following the UVR exposure, skin punch biopsies were taken from the UVRexposed site together with two biopsies of control nonexposed tissue from adjacent sites. The volunteers then took vitamin C supplements (500 mg ascorbic acid, Roche, Basel, Switzerland) for 8 weeks and the procedure of UVR exposure and skin biopsy at 6 h postexposure was repeated. In addition, at the beginning and end of the supplementation period, the minimal erythema dose (MED) of the gluteal skin was assessed (see below) and blood samples were obtained for analysis of vitamin C content. Erythema assessment
MATERIALS AND METHODS
Protocol Ethical permission for all studies was obtained from the local Research Ethics Committee and all subjects gave written informed consent prior to participation. Time course of changes in indicators of oxidative damage following UV exposure Eight healthy Caucasian volunteers (5 male, 3 female; mean age 32 years, range: 24 –50 years, skin
Pre- and postvitamin C supplementation, the MED was assessed after giving a geometric series (7– 80 mJ/ cm2) of UVR to buttock skin (Phillips TL12 lamp). After 24 h the lowest dose of UVR to result in a visually discernible erythema was recorded. Erythema was objectively measured at all sites in the time course and supplementation studies using a reflectance instrument (Diastron, Andover, UK). This gives a ratio of reflected red and green light, the erythema index. Measurements were taken in triplicate, and the reading from adjacent unexposed skin was subtracted.
UVR-induced oxidative stress in human skin in vivo
1357
Analysis of skin biopsy samples The skin biopsies provided approximately 20 mg wet weight of tissue, of which 10 –12 mg was epidermal/ dermal tissue. This was separated from the rest of the biopsy (subcutaneous fat) by dissection and divided prior to homogenization for the biochemical analyses. Catalase activity Catalase activity was measured by monitoring the enzymatic decomposition of hydrogen peroxide spectrophotometrically at 240 nm, using a method derived from Claiborne [24]. Malonaldehyde content The malonaldehyde (MDA) content of the sample was measured as an index of lipid peroxidation. The method of Fukunaga et al. [25] was adapted to use small volumes of sample. Total and oxidized glutathione and protein thiol content of samples The automated glutathione recycling method described by Anderson [26] was used to assess both the total and oxidized glutathione content of samples, using a 96 well plate reader (Benchmark, Bio-Rad, Hemel Hempstead, UK). The protein thiol content of samples was analyzed by the method of Di Monte et al. [27] adapted for use on a 96 well plate reader. Protein determination The protein content of samples was determined using a BCA protein assay kit (Sigma Chemical Co., Dorset, UK), based on the method of Smith et al. [28]. Fatty acid content Total lipids were extracted from the samples using the method of Folch et al. [29] and fatty acid methyl esters prepared prior to analysis by gas-liquid chromatography with flame ionization detection using the method of Metcalfe and Schmitz [30]. Vitamin E and vitamin C content Both vitamin E and vitamin C were measured using HPLC techniques modified to increase the sensitivity of the assay by the use of electrochemical detection. Vitamin C was measured using the method of Mohr and Stocker [31] with electrochemical detection at on oxidation potential of ⫹500 mV, and vitamin E was measured by the method of Catignani and Bieri [32] with electrochemical detection at an oxidation potential of ⫹650 mV.
Fig. 1. Time course of changes in erythema following UVR exposure. *p ⬍ .05 compared with value at baseline (time 0).
Activity of AP-1 transcription factor DNA-binding activity by AP-1 was measured by gel retardation assay using a commercial kit (Gel Shift Assay System, Promega, Madison, WI, USA). The extent of activation was quantified by densitometry of the autoradiographs. RESULTS
The dose of UVR used (120 mJ/cm2) induced an erythemal response in the skin of the control subjects, as shown in Fig. 1. The difference in erythema index (compared with unirradiated skin) reached statistical significance at 6 h postexposure (p ⬍ .05), returning towards control levels by 216 h (9 d later). Time course of changes in markers of oxidative stress The dose of UVR induced changes in some indicators of oxidation in the skin, although others were unchanged. A significant rise in the proportion of the cellular glutathione content in the oxidized form was observed at 6 h post-UVR exposure, p ⬍ .05 (Fig. 2B). This occurred in the absence of any significant change in the total skin glutathione (Fig. 2A) and had reversed by 24 h postexposure. There was a rapid and significant rise in the activity of catalase activity by 1 h that had reversed by 24 h postexposure (Fig. 3A). Activation of AP-1 binding activity was also detected very rapidly after exposure, peaking at 1– 6 h and then declining rapidly to baseline values. The AP-1 response showed high intersubject variability and only the mean data are shown for clarity. No significant changes were seen following UVR exposure in the malonaldehyde, vitamin E, or vitamin C contents of the skin samples (Table 1), or in the fatty acid composition of the skin (data not shown in detail).
1358
F. MCARDLE et al.
Fig. 2. Time course of changes in skin total glutathione (A) and oxidized glutathione (B) following UVR exposure. *p ⬍ .05 compared with value at baseline (time 0).
Fig. 3. Time course of changes in skin catalase activity (A) and AP-1 binding (B) following UVR exposure. *p ⬍ .05 compared with value at baseline (time 0).
Effect of vitamin C supplementation Vitamin C supplementation had no effect on the MED, with a median value of 36 (range 28 –36) mJ/cm2 at baseline and 36 (range 28 –36) mJ/cm2 after 2 months supplementation. Tissue parameters Vitamin C supplementation significantly increased the concentration of vitamin C in both the plasma (baseline: 3.8 ⫾ 0.27 g/ml compared with supplemented: 4.5 ⫾ 0.54 g/ml; p ⬍ .05) and skin (baseline nonexposed: 390 ⫾ 103 ng/mg compared with supplemented nonexposed 680 ⫾ 56 ng/mg; p ⬍ .05) of the subjects. UVR exposure had no significant effect on the skin vitamin C content (data not shown). The total glutathione content of the skin samples prior to UVR exposure was reduced by the vitamin C supplementation (Fig. 4A), although the proportion present in the oxidized form was unchanged (Fig. 4B). Vitamin C
supplementation had no significant effect on the oxidation of glutathione following UVR exposure. The oxidized glutathione was increased at 6 h post-UVR in both the supplemented and nonsupplemented samples (Fig. 4B) although there was no further change in the total skin content of glutathione (Fig. 4A). In order to help interpret the effect of vitamin C on cellular glutathione content, the total protein thiol content of the samples was also measured. These data show a smaller, but significant fall in total protein thiols following vitamin C supplementation in comparison with the changes in total glutathione (Fig. 5). No changes in protein thiol content were seen with UVR exposure. Vitamin C supplementation also decreased the malonaldehyde content of the skin prior to UVR exposure, indicating a reduction in the baseline lipid peroxidation in skin samples (Fig. 6). No statistically significant effect of UVR was seen on the malonaldehyde content of
UVR-induced oxidative stress in human skin in vivo
1359
Table 1. Malonaldehyde, Vitamin E, and Vitamin C Content of Skin Following UVR Exposure Time post-UV exposure (h) Malonaldehyde content (nmole/mg protein) Vitamin E (ng/mg tissue) Vitamin C (ng/mg tissue)
0
1
6
24
48
216
1.1 ⫾ 0.21 3.2 ⫾ 0.26 320 ⫾ 99
1.0 ⫾ 0.13 3.2 ⫾ 0.42 220 ⫾ 39
1.5 ⫾ 0.45 3.3 ⫾ 0.42 230 ⫾ 40
1.0 ⫾ 0.27 3.5 ⫾ 0.4 230 ⫾ 100
1.0 ⫾ 0.36 3.3 ⫾ 0.27 220 ⫾ 33
1.0 ⫾ 0.36 3.6 ⫾ 0.4 ND
ND ⫽ No sample was analyzed.
supplemented or nonsupplemented skin. Vitamin C had no significant effect on the catalase activity of either exposed or non-UVR exposed skin (data not shown in detail). DISCUSSION
The light source used in these studies emitted primarily UVB with a small UVA component in comparison with other studies of UVR-induced oxidative stress
[6,14]. The dose of UVR administered (120 mJ/cm2) was also much lower than that used in most published murine [10 –13] and cell culture [7–9] studies, but approximates to what may be experienced during an acute physiological exposure to UVR and is comparable to previous human studies [16,17]. This dose of UVR induced a significant erythemal response in the gluteal skin of the subjects that peaked within 6 –24 h and also induced a relatively mild oxidative stress in the skin. This was
Fig. 5. Protein thiol content of skin samples from subjects prior to, and following vitamin C supplementation and prior to and following UVR exposure. *p ⬍ .05 compared with non-UVR exposed skin prior to vitamin C supplementation.
Fig. 4. Total glutathione content (A) and proportion on the oxidized form (B) of skin samples from subjects prior to, and following vitamin C supplementation and prior to and following UVR exposure. *p ⬍ .05 compared with non-UVR exposed skin prior to vitamin C supplementation. **p ⬍ .05 compared with non-UVR exposed skin following vitamin C supplementation.
Fig. 6. Malonaldehyde content of skin samples from subjects prior to, and following vitamin C supplementation and prior to and following UVR exposure. *p ⬍ .05 compared with non-UVR exposed skin prior to vitamin C supplementation.
1360
F. MCARDLE et al.
manifested by an increase in the oxidation of intracellular glutathione at 6 h postexposure and an increase in the activity of catalase within 1 h of the exposure. Previous data from both cell culture models and murine studies in vivo have indicated that UVR reduces the glutathione content skin cells [9 –12] and leads to a loss of skin ascorbate and ␣-tocopherol content [10], with decreases in the activity of superoxide dismutase, catalase, and glutathione peroxidase activities [33]. Data from Katiyar and colleagues [17], together with that reported here, illustrate that such dramatic changes do not occur in human skin in response to more physiological doses of UVR. Katiyar et al. [17] report that higher doses of UVR than those reported here decreased skin glutathione by approximately 30% over 48 h, whereas no significant change in the total glutathione was seen in our study. In contrast, we observed a significant increase in oxidized glutathione with no change in the total glutathione content. Such differences are entirely compatible with the differing degree of exposure to UVR, since with increasing amounts of UVR exposure it is likely that the oxidized glutathione formed would be lost from the skin cells leading to the fall in total glutathione content seen in other studies. Katiyar and colleagues [17] also noted an increase in catalase activity in UVR-exposed human skin, in agreement with the data presented here, but in contrast to the changes seen in animal and cell culture models. The rapidity of the response with significant changes at 1 h postexposure suggests that the increased activity was due to posttranslational mechanisms. This type of regulation of catalase activity has previously been reported [34]. Our previous studies of exposure of fibroblasts to UVR have also demonstrated rapid adaptive changes in the activity of catalase as part of a coordinated response to reduce the subsequent risk of oxidative damage to the tissue [15]. The malonaldehyde content of skin samples tended to rise following UVR exposure, but this change was not significant. Analysis of the malonaldehyde is a widely utilized marker of oxidative stress in cells and tissues [35], which we have previously shown is elevated in cell cultures exposed to this type of UVR [15]. Similarly, the less specific indicator of lipid peroxidation, thiobarbituric acid-reactive substances, is elevated in human skin irradiated with the same light source [36]. However, the degree of oxidative stress induced in the present study did not appear to be sufficiently severe to induce a reproducible change in skin malonaldehyde due, in part, to a high degree of interindividual variability. Similarly, there was no change in the fatty acid composition, vitamin E content, or vitamin C content of the skin, further supporting the conclusion that the degree of oxidative
stress induced by this dose and spectrum of UVR was relatively mild. The dose of UVR also induced a variable activation of the transcription factor AP-1 in human skin in vivo (Fig. 3B). Our current data do not permit a full evaluation of the mechanisms by which this factor was activated by UVR exposure, and insufficient sample was obtained to allow analysis of this factor following vitamin C supplementation. These data are included here to illustrate the ability of this level of UVR exposure to activate redoxregulated transcription factors [37] in the skin in the absence of major oxidation of the tissue. Effects of vitamin C supplementation The level and duration of vitamin C supplementation was higher than could be achieved from dietary sources, but importantly, this was shown to be appropriate for elevation of tissue vitamin C content. Most previous studies of vitamin C effects have not examined for bioavailability, leading to concern that lack of observed effects of supplementation could be due to failure of uptake. Very high supplementation doses, e.g., 3 grams vitamin C daily, have been shown to result in significant uptake of the antioxidant into human buccal mucosa [20], but we have now confirmed incorporation into the skin with the lower dose (500 mg/day) used here. Vitamin C had seemingly paradoxical effects on markers of oxidation in the skin. In the basal, non-UVR exposed skin, malonaldehyde content was significantly reduced by vitamin C supplementation, indicating a reduction in baseline levels of lipid peroxidation. However, these changes were accompanied by a reduction in the glutathione content of the skin and in protein thiol content. One possible explanation for these data may be related to observations that the glutathione content of skin fibroblasts increases greatly over a delayed period in response to UV-induced oxidative stress [15], and a vitamin C-induced reduction in the basal levels of oxidative stress within the skin may have reduced the stimulus to maintain a high cellular glutathione level. Alternatively, it is possible that skin cells regulate their total thiol or reductant capacity (potentially to facilitate redoxsignaling pathways) and the increased vitamin C content may simply have replaced some other reducing agents, such as glutathione, within the cell. The increased vitamin C content had no significant effect on the oxidation of glutathione that occurred in response to UVR (Fig. 4B). Again this is somewhat surprising given the effect on baseline malonaldehyde content, but may be related to whether the increased ascorbate accumulates at the sites at which UVR generates oxidants in the cell. Theoretically, combinations of antioxidants might be expected to have a greater protective effect against UVR-
UVR-induced oxidative stress in human skin in vivo
induced oxidative stress than single antioxidant agents, since their modes of action may be complementary [38]. However, in our studies we wished to establish the potential properties of an individual agent. One murine study has shown protection against carcinogenesis by vitamin C alone [23], although similar effects have not been reported in humans. In a placebo-controlled trial, 10 healthy humans were supplemented with vitamin C (3 grams/day) alone for 50 d, but no change in the sunburn threshold was seen [39]. Similarly, our lower dose of vitamin C supplementation did not affect the macroscopic erythemal response, although changes in indicators of oxidative stress were observed at the tissue level. In contrast, two recent studies have reported protection against UVR-induced erythema in humans with ingestion of combined supplements of vitamins E and C [20,40]. In summary therefore, these data indicate that exposure of human skin to UVR followed by analysis of biopsies from the exposed areas is an appropriate model to study the effects of a mild oxidative stress on human skin. Vitamin C from oral supplements appears to accumulate in the skin, reducing the basal content of malonaldehyde and, surprisingly, glutathione. No evidence for an effect of the vitamin C on UVR-induced oxidative stress was seen. Acknowledgements — The authors would like to thank the U.K. Ministry of Agriculture, Fisheries and Food and the Food Standards Agency for financial support for this project, the late Professor Tony Diplock for his helpful comments, and Mr. Chris Price and Ms. Karen Brady for technical support.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19] [20]
REFERENCES [1] Pathak, M. A.; Fitzpatrick, T. B. Preventive treatment of sunburn, dermatoheliosis, and skin cancer with sun-protective agents. In: Fitzpatrick, T. B.; Eisen, A. Z.; Wolff, K.; Freedburg, I. M.; Austen, K. F., eds. Dermatology in general medicine, 4th ed. New York: McGraw-Hill, Inc.; 1993:1689 –1716. [2] Miyachi, Y. Reactive oxygen species in photodermatology. In: Hayashi, O.; Imamura, S.; Miyachi, Y., eds. The biological role of reactive oxygen species in skin. New York: Elsevier Science; 1987:37– 41. [3] Kligman, L. H.; Kligman, A. M. The nature of photoaging: its prevention and repair. Photodermatology 3:215–227; 1986. [4] Frederick, J. E. Ultraviolet sunlight reaching the earth’s surface: a review of recent research. Photochem. Photobiol. 57:175–178; 1993. [5] Black, H. S. Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage. Photochem. Photobiol. 46:213–221; 1987. [6] Tyrrell, R. M. Ultraviolet radiation and free radical damage to skin. Biochem. Soc. Trans. 61:47–53; 1996. [7] Meewes, C.; Brenneisen, P.; Wenk, J.; Kuhr, L.; Ma, W.; Alikoski, J.; Poswig, A.; Krieg, T.; Schaffetter-Kochanek, K. Adaptive antioxidant response protects dermal fibroblasts from UVA-induced phototoxicity. Free Radic. Biol. Med. 30:238 –247; 2001. [8] Soriani, M.; Hejmadi, V.; Tyrrell, R. M. Modulation of c-jun and c-fos transcription by UVB and UVA radiations in human dermal
[21]
[22]
[23]
[24]
[25]
[26]
[27]
1361
fibroblasts and KB cells. Photochem. Photobiol. 71:551–558; 2000. Moysan, A.; Marquis, I.; Gaboriau, F.; Santus, R.; Dubertret, L.; Morliere, P. Ultraviolet A-induced lipid peroxidation and antioxidant defense systems in cultured human skin fibroblasts. J. Invest. Dermatol. 100:692– 698; 1993. Shindo, Y.; Witt, E.; Han, D.; Packer, L. Dose-response effects of acute ultraviolet irradiation on antioxidants and molecular markers of oxidation in murine epidermis and dermis. J. Invest. Dermatol. 102:470 – 475; 1994. Lopez-Torres, M.; Thiele, J. J.; Shindo, Y.; Han, D.; Packer, L. Topical application of ␣-tocopherol modulates the antioxidant network and diminishes ultraviolet-induced oxidative damage in murine skin. Br. J. Dermatol. 138:207–215; 1998. Connor, M. J.; Wheeler, L. A. Depletion of cutaneous glutathione by ultraviolet radiation. Photochem. Photobiol. 46:239 –245; 1987. Gonzalez, S.; Pathak, M. A. Inhibition of ultraviolet-induced formation of reactive oxygen species, lipid peroxidation, erythema and skin photosensitization by Polypodium leucotomos. Photodermatol. Photoimmunol. Photomed. 12:45–56; 1996. Tyrell, R. M.; Pidoux, M. Correlation between endogenous glutathione content and the sensitivity of cultured human skin cells to radiation at defined wavelengths in the solar UV range. Photochem. Photobiol. 47:405– 412; 1988. Jones, S. A.; McArdle, F.; Jack, C. I. A.; Jackson, M. J. Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress. Redox Rep. 4:291–299; 1999. Schallreuter, K. A.; Wood, J. M. Thioredoxin reductase—its role in epidermal redox status. J. Photochem. Photobiol. B 64:179 – 184; 2001. Katiyar, S. K.; Afaq, F.; Pewrez, A.; Mukhtar, H. Green tea polyphenol (⫺)-epigallocatechin-3-gallate treatment of human skin inhibits ultraviolet radiation-induced oxidative stress. Carcinogenesis 22:287–294; 2001. Tyrell, R. M. Activation of mammalian genes by the UV component of sunlight—from models to reality. Bioessays 18:139 – 148; 1996. Nachbar, F.; Korting, H. C. The role of vitamin E in normal and damaged skin. J. Mol. Med. 73:7–17; 1995. Fuchs, J.; Kern, H. Modulation of UV-light induced skin inflammation by d-alpha-tocopherol and l-ascorbic acid: a clinical study using solar-simulated radiation. Free Radic. Biol. Med. 25:1006 – 1012; 1998. Stewart, M. S.; Cameron, G. S.; Pence, B. C. Antioxidant nutrients protect against UVB-induced oxidative damage to DNA of mouse keratinocytes in culture. J. Invest. Dermatol. 106:1976 – 1989; 1996. Tebbe, B.; Wu, S.; Geilen, C. C.; Eberle, J.; Kodelija, V.; Orfanos, C. E. L-ascorbic acid inhibits UVA-induced lipid peroxidation and secretion of IL-1␣ and IL-6 in cultured human keratinocytes in vitro. J. Invest. Dermatol. 108:302–306; 1997. Dunham, W. B.; Zuckerkandl, E.; Reynolds, R.; Willoughby, R.; Marcuson, R.; Barth, R.; Pauling, L. Effects of intake of Lascorbic acid (vitamin C) on the incidence of dermal neoplasms induced in mice by ultraviolet light. Proc. Natl. Acad. Sci. USA 79:7532–7536; 1982. Claiborne, A. Catalase activity. In: Greenwald, R. A., ed. CRC handbook of methods for oxygen radical research. Boca Raton, FL: CRC Press; 1985:283–284. Fukunaga, K.; Suzuki, T.; Takama, K. Highly sensitive highperformance liquid chromatography for the measurement of malondialdehyde in biological samples. J. Chromatogr. 621:77– 81; 1993. Anderson, M. E. Measurement of antioxidants: glutathione. In: Punchard, N. A.; Kelly, F. J., eds. Free radicals, a practical approach. Oxford: IRL Press; 1996:213–226. Di Monte, D.; Bellomo, G.; Thor, H.; Nicotera, P.; Orrenius, S. Menadione-induced cytotoxicity is associated with protein thiol
1362
[28]
[29] [30] [31]
[32] [33] [34]
F. MCARDLE et al. oxidation and alteration in intracellular Ca2⫹ homeostasis. Arch. Biochem. Biophys. 235:343–350; 1984. Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76 – 85; 1985. Folch, J.; Lees, M.; Stanley, G. S. H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497–503; 1957. Metcalf, L. D.; Schmitz, A. A. Boron-trifluoride method for the derivitisation of fatty acids. Anal. Chem. 33:363–370; 1961. Mohr, D.; Stocker, R. Selective and sensitive measurement of vitamin C, ubiquinol-10, and other low molecular weight antioxidants. In: Punchard, N. A.; Kelly, F. J., eds. Free radicals, a practical approach. Oxford: IRL Press; 1996:271–285. Catignani, G. L.; Bieri, J. G. Rat liver alpha-tocopherol binding protein. Biochim. Biophys. Acta 497:349 –357; 1983. Shindo, Y.; Packer, L. Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light. J. Invest. Dermatol. 100:260 –265; 1993. Reimer, D.; Bailley, J.; Singh, S. Complete cDNA and 5'genomic
[35] [36]
[37] [38]
[39]
[40]
sequence and multilevel regulation of the mouse catalase gene. Genomics 15:325–336; 1994. Halliwell, B.; Gutteridge, J. M. C. Free radicals in biology and medicine. Oxford, UK: Oxford University Press; 1989. Rhodes, L. E.; O’Farrell, S.; Jackson, M. J.; Friedmann, P. S. Dietary omega-3 fatty acids reduce UVB-induced erythema while increasing lipid peroxidation. J. Invest. Dermatol. 103:151–154; 1994. Jackson, M. J.; McArdle, A.; McArdle, F. Antioxidant micronutrients and gene expression. Proc. Nutr. Soc. 57:301–305; 1988. Packer, J. E.; Slater, T.; Wilson, R. L. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278:737–738; 1979. Fuchs, J. Potentials and limitations of the natural antioxidants RRR-alpha-tocopherol, L-ascorbic acid and b-carotene in cutaneous photoprotection. Free Radic. Biol. Med. 25:848 – 873; 1998. Eberlin-Konig, B.; Placzek, M.; Przybilla, B. Protective effect against sunburn of combined systemic ascorbic acid (vitamin C) and d-␣-tocopherol (vitamin E). J. Am. Acad. Dermatol. 38:45– 48; 1998.
PII S0891-5849(02)01042-0
Original Contribution UVR-INDUCED OXIDATIVE STRESS IN HUMAN SKIN IN VIVO: EFFECTS OF ORAL VITAMIN C SUPPLEMENTATION F. MCARDLE, L. E. RHODES,1 R. PARSLEW, C. I. A. JACK, P. S. FRIEDMANN,2
and
M. J. JACKSON
Department of Medicine, University of Liverpool, Liverpool, UK (Received 25 July 2002; Revised 25 July 2002; Accepted 13 August 2002)
Abstract—Previous studies of cultured skin cells and murine skin in vivo have indicated that UVR-induced damage involves the generation of reactive oxygen species and depletion of endogenous antioxidant systems. In order to explore the relevance of this to UVR-induced damage to human skin, we have undertaken a detailed examination of the time-course of changes in markers of oxidative stress in human skin following exposure to physiological amounts of UVR in vivo. In addition, we have examined the skin bioavailability of a common nutritional antioxidant, vitamin C, and have assessed the effects of supplementation on markers of oxidative stress. Our hypothesis was that acute exposure of human skin to UVR in vivo would lead to oxidation of cellular biomolecules that could be prevented by prior vitamin C treatment. A UVR-challenge of 120 mJ/cm2 of broadband UVB (peak 310 nm, range 270 – 400 nm) was applied to buttock skin of 8 healthy volunteers. This caused a rapid and significant rise in activity of skin catalase at 1 h and an increase in the oxidized/total glutathione ratio at 6 h post-UVR. AP-1 DNA binding also peaked at 1– 6 h post-UVR, then declined rapidly to baseline levels. No significant changes were seen in skin malonaldehyde content. Oral vitamin C supplements (500 mg/day) were taken by 12 volunteers for 8 weeks resulting in significant rises in plasma and skin vitamin C content. Supplementation had no effect on the UVR-induced erythemal response. The skin malonaldehyde content was reduced by vitamin C supplementation, but surprisingly, reductions in the skin content of total glutathione and protein thiols were also seen. We speculate that this apparently paradoxical effect could be due to regulation of total reductant capacity by skin cells, such that vitamin C may have been replacing other reductants in these cells. No evidence was obtained for an effect of the supplementary vitamin C on the mild oxidative stress seen in human skin following UVR exposure. © 2002 Elsevier Science Inc. Keywords—Reactive oxygen species, Antioxidants, Transcription factors, Ascorbic acid, Free radicals
INTRODUCTION
considerable discussion concerning the potential role of oxidative stress in the deleterious effects of UVR on human skin. UVR is composed of UVA (320 – 400 nm), UVB (290 –320 nm) and UVC (289 –200 nm), but only UVA and UVB reach the earth’s surface and are of physiological significance [5,6]. A considerable amount of data has been published on the effects of UVA and UVB on oxidant generation in cultured skin cells [7–9] and murine skin in vivo [10 –13], but little has been reported on human skin in vivo. Most interest has concerned the effects of UVA in generating free radical and reactive oxygen species in skin cells [6,14], but data also indicate that, following exposure to UVB, cutaneous tissue, both in vitro and in vivo, generates free radicals and other reactive oxygen species (ROS) [15]. In cultured cells or murine skin, high doses of UVR have been reported to increase the hydrogen peroxide content asso-
Excessive exposure of skin to ultraviolet radiation (UVR) can result in acute and chronic damage [1,2] Short-term overexposure to UVR causes erythema (sunburn), whilst chronic overexposure leads to increased risk of skin cancer and premature photoageing [3]. Recently, concern has been expressed that humans are becoming exposed to higher doses of UVR due to depletion of ozone in the atmosphere [4]. There has been Address correspondence to: Professor M. J. Jackson, Department of Medicine, University of Liverpool, Duncan Building, Daulby Street, Liverpool L69 3GA, UK; Tel: ⫹44 151-706-4261; Fax: ⫹44 151-7065802; E-Mail: [email protected]. 1 Current address: Photobiology Unit, Dermatology Centre, University of Manchester, Manchester, UK. 2 Current address: Department of Dermatology, University of Southampton, Southampton, UK. 1355
1356
F. MCARDLE et al.
ciated with a decrease in cellular glutathione content and a decrease in the activity of protective enzymes, such as superoxide dismutase and catalase [7–13]. The relevance of these studies to human skin is contentious, since many utilize nonphysiological doses of UVR and the responses of murine and human skin differ in numerous respects. Nevertheless the few studies that have been undertaken on human skin in vivo confirm that hydrogen peroxide is produced following exposure to UVR [16] and that some changes in protective enzymes also occur [17]. Free radicals and reactive oxygen species may theoretically mediate many of the deleterious effects of UVR on skin by causing direct damage to cellular macromolecules or by modulating the expression of redox-sensitive genes and transcription factors to influence processes such as cell growth and proliferation [18]. The potential role of free radicals in UVR-induced skin damage has also led to considerable interest in whether supplementation with nutritional or pharmacological antioxidants can reduce the deleterious effects of UVR on skin [19,20]. Vitamin C is a powerful watersoluble antioxidant that has been shown to reduce UVBinduced oxidative damage in mouse keratinocytes in vitro [21] and to protect human keratinocytes from UVA-induced lipid peroxidation [22]. In mice, dietary vitamin C reduced the incidence of UVR-induced skin neoplasms [23], although in humans photoprotection against acute or chronic UVR effects by vitamin C supplementation alone has not been established. The clinical erythema endpoint is only one of a number of events in the skin triggered by UVR, and although it is frequently used as a guide to photoprotection, it cannot accurately reflect all aspects. We hypothesized that acute exposure of human skin to UVR would induce reproducible changes in indicators of free radical damage to skin and that these would be attenuated by prior supplementation with vitamin C. The aim of the work described here was, therefore, to examine the time course of changes in markers of oxidative damage following exposure of normal skin to UVR light and to examine the effects of supplementary oral vitamin C on the changes seen.
type II or III) were recruited. None had been exposed to excessive natural or artificial UV radiation in the 6 months prior to recruitment. A fixed UVR dose of 120 mJ/cm2 was given to three circular sites of 1 cm diameter on buttock skin. The irradiation source was a fluorescent broadband lamp (TL12, Phillips Lighting, Croydon, UK), which emitted UVR between 270 and 400 nm (peak emission 310 nm). Irradiance was measured with an IL1400A radiometer (International Light, Durham, UK). Skin punch biopsies (4 mm diameter) were taken from each of these sites at selected times after UVR exposure and from adjacent unexposed skin. The time points examined were 0, 1, 6, 24, 48, and 216 h postexposure, but for ethical reasons a maximum of only four biopsies could be taken from an individual. Immediately prior to biopsy, erythema at each site was assessed visually and by objective measurements (see erythema assessment below). All skin samples were rapidly frozen in liquid nitrogen and stored at ⫺70°C prior to analysis. Effect of vitamin C supplements Twelve healthy Caucasian volunteers (9 male, 3 female; mean age 23 years, range: 22–33 years, skin type II or III) were recruited. None had been exposed to excessive natural or artificial UV radiation in the 6 months prior to recruitment. A fixed UVR dose of 120 mJ/cm2 was given to two circular sites of 1 cm diameter on buttock skin as above. At 6 h following the UVR exposure, skin punch biopsies were taken from the UVRexposed site together with two biopsies of control nonexposed tissue from adjacent sites. The volunteers then took vitamin C supplements (500 mg ascorbic acid, Roche, Basel, Switzerland) for 8 weeks and the procedure of UVR exposure and skin biopsy at 6 h postexposure was repeated. In addition, at the beginning and end of the supplementation period, the minimal erythema dose (MED) of the gluteal skin was assessed (see below) and blood samples were obtained for analysis of vitamin C content. Erythema assessment
MATERIALS AND METHODS
Protocol Ethical permission for all studies was obtained from the local Research Ethics Committee and all subjects gave written informed consent prior to participation. Time course of changes in indicators of oxidative damage following UV exposure Eight healthy Caucasian volunteers (5 male, 3 female; mean age 32 years, range: 24 –50 years, skin
Pre- and postvitamin C supplementation, the MED was assessed after giving a geometric series (7– 80 mJ/ cm2) of UVR to buttock skin (Phillips TL12 lamp). After 24 h the lowest dose of UVR to result in a visually discernible erythema was recorded. Erythema was objectively measured at all sites in the time course and supplementation studies using a reflectance instrument (Diastron, Andover, UK). This gives a ratio of reflected red and green light, the erythema index. Measurements were taken in triplicate, and the reading from adjacent unexposed skin was subtracted.
UVR-induced oxidative stress in human skin in vivo
1357
Analysis of skin biopsy samples The skin biopsies provided approximately 20 mg wet weight of tissue, of which 10 –12 mg was epidermal/ dermal tissue. This was separated from the rest of the biopsy (subcutaneous fat) by dissection and divided prior to homogenization for the biochemical analyses. Catalase activity Catalase activity was measured by monitoring the enzymatic decomposition of hydrogen peroxide spectrophotometrically at 240 nm, using a method derived from Claiborne [24]. Malonaldehyde content The malonaldehyde (MDA) content of the sample was measured as an index of lipid peroxidation. The method of Fukunaga et al. [25] was adapted to use small volumes of sample. Total and oxidized glutathione and protein thiol content of samples The automated glutathione recycling method described by Anderson [26] was used to assess both the total and oxidized glutathione content of samples, using a 96 well plate reader (Benchmark, Bio-Rad, Hemel Hempstead, UK). The protein thiol content of samples was analyzed by the method of Di Monte et al. [27] adapted for use on a 96 well plate reader. Protein determination The protein content of samples was determined using a BCA protein assay kit (Sigma Chemical Co., Dorset, UK), based on the method of Smith et al. [28]. Fatty acid content Total lipids were extracted from the samples using the method of Folch et al. [29] and fatty acid methyl esters prepared prior to analysis by gas-liquid chromatography with flame ionization detection using the method of Metcalfe and Schmitz [30]. Vitamin E and vitamin C content Both vitamin E and vitamin C were measured using HPLC techniques modified to increase the sensitivity of the assay by the use of electrochemical detection. Vitamin C was measured using the method of Mohr and Stocker [31] with electrochemical detection at on oxidation potential of ⫹500 mV, and vitamin E was measured by the method of Catignani and Bieri [32] with electrochemical detection at an oxidation potential of ⫹650 mV.
Fig. 1. Time course of changes in erythema following UVR exposure. *p ⬍ .05 compared with value at baseline (time 0).
Activity of AP-1 transcription factor DNA-binding activity by AP-1 was measured by gel retardation assay using a commercial kit (Gel Shift Assay System, Promega, Madison, WI, USA). The extent of activation was quantified by densitometry of the autoradiographs. RESULTS
The dose of UVR used (120 mJ/cm2) induced an erythemal response in the skin of the control subjects, as shown in Fig. 1. The difference in erythema index (compared with unirradiated skin) reached statistical significance at 6 h postexposure (p ⬍ .05), returning towards control levels by 216 h (9 d later). Time course of changes in markers of oxidative stress The dose of UVR induced changes in some indicators of oxidation in the skin, although others were unchanged. A significant rise in the proportion of the cellular glutathione content in the oxidized form was observed at 6 h post-UVR exposure, p ⬍ .05 (Fig. 2B). This occurred in the absence of any significant change in the total skin glutathione (Fig. 2A) and had reversed by 24 h postexposure. There was a rapid and significant rise in the activity of catalase activity by 1 h that had reversed by 24 h postexposure (Fig. 3A). Activation of AP-1 binding activity was also detected very rapidly after exposure, peaking at 1– 6 h and then declining rapidly to baseline values. The AP-1 response showed high intersubject variability and only the mean data are shown for clarity. No significant changes were seen following UVR exposure in the malonaldehyde, vitamin E, or vitamin C contents of the skin samples (Table 1), or in the fatty acid composition of the skin (data not shown in detail).
1358
F. MCARDLE et al.
Fig. 2. Time course of changes in skin total glutathione (A) and oxidized glutathione (B) following UVR exposure. *p ⬍ .05 compared with value at baseline (time 0).
Fig. 3. Time course of changes in skin catalase activity (A) and AP-1 binding (B) following UVR exposure. *p ⬍ .05 compared with value at baseline (time 0).
Effect of vitamin C supplementation Vitamin C supplementation had no effect on the MED, with a median value of 36 (range 28 –36) mJ/cm2 at baseline and 36 (range 28 –36) mJ/cm2 after 2 months supplementation. Tissue parameters Vitamin C supplementation significantly increased the concentration of vitamin C in both the plasma (baseline: 3.8 ⫾ 0.27 g/ml compared with supplemented: 4.5 ⫾ 0.54 g/ml; p ⬍ .05) and skin (baseline nonexposed: 390 ⫾ 103 ng/mg compared with supplemented nonexposed 680 ⫾ 56 ng/mg; p ⬍ .05) of the subjects. UVR exposure had no significant effect on the skin vitamin C content (data not shown). The total glutathione content of the skin samples prior to UVR exposure was reduced by the vitamin C supplementation (Fig. 4A), although the proportion present in the oxidized form was unchanged (Fig. 4B). Vitamin C
supplementation had no significant effect on the oxidation of glutathione following UVR exposure. The oxidized glutathione was increased at 6 h post-UVR in both the supplemented and nonsupplemented samples (Fig. 4B) although there was no further change in the total skin content of glutathione (Fig. 4A). In order to help interpret the effect of vitamin C on cellular glutathione content, the total protein thiol content of the samples was also measured. These data show a smaller, but significant fall in total protein thiols following vitamin C supplementation in comparison with the changes in total glutathione (Fig. 5). No changes in protein thiol content were seen with UVR exposure. Vitamin C supplementation also decreased the malonaldehyde content of the skin prior to UVR exposure, indicating a reduction in the baseline lipid peroxidation in skin samples (Fig. 6). No statistically significant effect of UVR was seen on the malonaldehyde content of
UVR-induced oxidative stress in human skin in vivo
1359
Table 1. Malonaldehyde, Vitamin E, and Vitamin C Content of Skin Following UVR Exposure Time post-UV exposure (h) Malonaldehyde content (nmole/mg protein) Vitamin E (ng/mg tissue) Vitamin C (ng/mg tissue)
0
1
6
24
48
216
1.1 ⫾ 0.21 3.2 ⫾ 0.26 320 ⫾ 99
1.0 ⫾ 0.13 3.2 ⫾ 0.42 220 ⫾ 39
1.5 ⫾ 0.45 3.3 ⫾ 0.42 230 ⫾ 40
1.0 ⫾ 0.27 3.5 ⫾ 0.4 230 ⫾ 100
1.0 ⫾ 0.36 3.3 ⫾ 0.27 220 ⫾ 33
1.0 ⫾ 0.36 3.6 ⫾ 0.4 ND
ND ⫽ No sample was analyzed.
supplemented or nonsupplemented skin. Vitamin C had no significant effect on the catalase activity of either exposed or non-UVR exposed skin (data not shown in detail). DISCUSSION
The light source used in these studies emitted primarily UVB with a small UVA component in comparison with other studies of UVR-induced oxidative stress
[6,14]. The dose of UVR administered (120 mJ/cm2) was also much lower than that used in most published murine [10 –13] and cell culture [7–9] studies, but approximates to what may be experienced during an acute physiological exposure to UVR and is comparable to previous human studies [16,17]. This dose of UVR induced a significant erythemal response in the gluteal skin of the subjects that peaked within 6 –24 h and also induced a relatively mild oxidative stress in the skin. This was
Fig. 5. Protein thiol content of skin samples from subjects prior to, and following vitamin C supplementation and prior to and following UVR exposure. *p ⬍ .05 compared with non-UVR exposed skin prior to vitamin C supplementation.
Fig. 4. Total glutathione content (A) and proportion on the oxidized form (B) of skin samples from subjects prior to, and following vitamin C supplementation and prior to and following UVR exposure. *p ⬍ .05 compared with non-UVR exposed skin prior to vitamin C supplementation. **p ⬍ .05 compared with non-UVR exposed skin following vitamin C supplementation.
Fig. 6. Malonaldehyde content of skin samples from subjects prior to, and following vitamin C supplementation and prior to and following UVR exposure. *p ⬍ .05 compared with non-UVR exposed skin prior to vitamin C supplementation.
1360
F. MCARDLE et al.
manifested by an increase in the oxidation of intracellular glutathione at 6 h postexposure and an increase in the activity of catalase within 1 h of the exposure. Previous data from both cell culture models and murine studies in vivo have indicated that UVR reduces the glutathione content skin cells [9 –12] and leads to a loss of skin ascorbate and ␣-tocopherol content [10], with decreases in the activity of superoxide dismutase, catalase, and glutathione peroxidase activities [33]. Data from Katiyar and colleagues [17], together with that reported here, illustrate that such dramatic changes do not occur in human skin in response to more physiological doses of UVR. Katiyar et al. [17] report that higher doses of UVR than those reported here decreased skin glutathione by approximately 30% over 48 h, whereas no significant change in the total glutathione was seen in our study. In contrast, we observed a significant increase in oxidized glutathione with no change in the total glutathione content. Such differences are entirely compatible with the differing degree of exposure to UVR, since with increasing amounts of UVR exposure it is likely that the oxidized glutathione formed would be lost from the skin cells leading to the fall in total glutathione content seen in other studies. Katiyar and colleagues [17] also noted an increase in catalase activity in UVR-exposed human skin, in agreement with the data presented here, but in contrast to the changes seen in animal and cell culture models. The rapidity of the response with significant changes at 1 h postexposure suggests that the increased activity was due to posttranslational mechanisms. This type of regulation of catalase activity has previously been reported [34]. Our previous studies of exposure of fibroblasts to UVR have also demonstrated rapid adaptive changes in the activity of catalase as part of a coordinated response to reduce the subsequent risk of oxidative damage to the tissue [15]. The malonaldehyde content of skin samples tended to rise following UVR exposure, but this change was not significant. Analysis of the malonaldehyde is a widely utilized marker of oxidative stress in cells and tissues [35], which we have previously shown is elevated in cell cultures exposed to this type of UVR [15]. Similarly, the less specific indicator of lipid peroxidation, thiobarbituric acid-reactive substances, is elevated in human skin irradiated with the same light source [36]. However, the degree of oxidative stress induced in the present study did not appear to be sufficiently severe to induce a reproducible change in skin malonaldehyde due, in part, to a high degree of interindividual variability. Similarly, there was no change in the fatty acid composition, vitamin E content, or vitamin C content of the skin, further supporting the conclusion that the degree of oxidative
stress induced by this dose and spectrum of UVR was relatively mild. The dose of UVR also induced a variable activation of the transcription factor AP-1 in human skin in vivo (Fig. 3B). Our current data do not permit a full evaluation of the mechanisms by which this factor was activated by UVR exposure, and insufficient sample was obtained to allow analysis of this factor following vitamin C supplementation. These data are included here to illustrate the ability of this level of UVR exposure to activate redoxregulated transcription factors [37] in the skin in the absence of major oxidation of the tissue. Effects of vitamin C supplementation The level and duration of vitamin C supplementation was higher than could be achieved from dietary sources, but importantly, this was shown to be appropriate for elevation of tissue vitamin C content. Most previous studies of vitamin C effects have not examined for bioavailability, leading to concern that lack of observed effects of supplementation could be due to failure of uptake. Very high supplementation doses, e.g., 3 grams vitamin C daily, have been shown to result in significant uptake of the antioxidant into human buccal mucosa [20], but we have now confirmed incorporation into the skin with the lower dose (500 mg/day) used here. Vitamin C had seemingly paradoxical effects on markers of oxidation in the skin. In the basal, non-UVR exposed skin, malonaldehyde content was significantly reduced by vitamin C supplementation, indicating a reduction in baseline levels of lipid peroxidation. However, these changes were accompanied by a reduction in the glutathione content of the skin and in protein thiol content. One possible explanation for these data may be related to observations that the glutathione content of skin fibroblasts increases greatly over a delayed period in response to UV-induced oxidative stress [15], and a vitamin C-induced reduction in the basal levels of oxidative stress within the skin may have reduced the stimulus to maintain a high cellular glutathione level. Alternatively, it is possible that skin cells regulate their total thiol or reductant capacity (potentially to facilitate redoxsignaling pathways) and the increased vitamin C content may simply have replaced some other reducing agents, such as glutathione, within the cell. The increased vitamin C content had no significant effect on the oxidation of glutathione that occurred in response to UVR (Fig. 4B). Again this is somewhat surprising given the effect on baseline malonaldehyde content, but may be related to whether the increased ascorbate accumulates at the sites at which UVR generates oxidants in the cell. Theoretically, combinations of antioxidants might be expected to have a greater protective effect against UVR-
UVR-induced oxidative stress in human skin in vivo
induced oxidative stress than single antioxidant agents, since their modes of action may be complementary [38]. However, in our studies we wished to establish the potential properties of an individual agent. One murine study has shown protection against carcinogenesis by vitamin C alone [23], although similar effects have not been reported in humans. In a placebo-controlled trial, 10 healthy humans were supplemented with vitamin C (3 grams/day) alone for 50 d, but no change in the sunburn threshold was seen [39]. Similarly, our lower dose of vitamin C supplementation did not affect the macroscopic erythemal response, although changes in indicators of oxidative stress were observed at the tissue level. In contrast, two recent studies have reported protection against UVR-induced erythema in humans with ingestion of combined supplements of vitamins E and C [20,40]. In summary therefore, these data indicate that exposure of human skin to UVR followed by analysis of biopsies from the exposed areas is an appropriate model to study the effects of a mild oxidative stress on human skin. Vitamin C from oral supplements appears to accumulate in the skin, reducing the basal content of malonaldehyde and, surprisingly, glutathione. No evidence for an effect of the vitamin C on UVR-induced oxidative stress was seen. Acknowledgements — The authors would like to thank the U.K. Ministry of Agriculture, Fisheries and Food and the Food Standards Agency for financial support for this project, the late Professor Tony Diplock for his helpful comments, and Mr. Chris Price and Ms. Karen Brady for technical support.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19] [20]
REFERENCES [1] Pathak, M. A.; Fitzpatrick, T. B. Preventive treatment of sunburn, dermatoheliosis, and skin cancer with sun-protective agents. In: Fitzpatrick, T. B.; Eisen, A. Z.; Wolff, K.; Freedburg, I. M.; Austen, K. F., eds. Dermatology in general medicine, 4th ed. New York: McGraw-Hill, Inc.; 1993:1689 –1716. [2] Miyachi, Y. Reactive oxygen species in photodermatology. In: Hayashi, O.; Imamura, S.; Miyachi, Y., eds. The biological role of reactive oxygen species in skin. New York: Elsevier Science; 1987:37– 41. [3] Kligman, L. H.; Kligman, A. M. The nature of photoaging: its prevention and repair. Photodermatology 3:215–227; 1986. [4] Frederick, J. E. Ultraviolet sunlight reaching the earth’s surface: a review of recent research. Photochem. Photobiol. 57:175–178; 1993. [5] Black, H. S. Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage. Photochem. Photobiol. 46:213–221; 1987. [6] Tyrrell, R. M. Ultraviolet radiation and free radical damage to skin. Biochem. Soc. Trans. 61:47–53; 1996. [7] Meewes, C.; Brenneisen, P.; Wenk, J.; Kuhr, L.; Ma, W.; Alikoski, J.; Poswig, A.; Krieg, T.; Schaffetter-Kochanek, K. Adaptive antioxidant response protects dermal fibroblasts from UVA-induced phototoxicity. Free Radic. Biol. Med. 30:238 –247; 2001. [8] Soriani, M.; Hejmadi, V.; Tyrrell, R. M. Modulation of c-jun and c-fos transcription by UVB and UVA radiations in human dermal
[21]
[22]
[23]
[24]
[25]
[26]
[27]
1361
fibroblasts and KB cells. Photochem. Photobiol. 71:551–558; 2000. Moysan, A.; Marquis, I.; Gaboriau, F.; Santus, R.; Dubertret, L.; Morliere, P. Ultraviolet A-induced lipid peroxidation and antioxidant defense systems in cultured human skin fibroblasts. J. Invest. Dermatol. 100:692– 698; 1993. Shindo, Y.; Witt, E.; Han, D.; Packer, L. Dose-response effects of acute ultraviolet irradiation on antioxidants and molecular markers of oxidation in murine epidermis and dermis. J. Invest. Dermatol. 102:470 – 475; 1994. Lopez-Torres, M.; Thiele, J. J.; Shindo, Y.; Han, D.; Packer, L. Topical application of ␣-tocopherol modulates the antioxidant network and diminishes ultraviolet-induced oxidative damage in murine skin. Br. J. Dermatol. 138:207–215; 1998. Connor, M. J.; Wheeler, L. A. Depletion of cutaneous glutathione by ultraviolet radiation. Photochem. Photobiol. 46:239 –245; 1987. Gonzalez, S.; Pathak, M. A. Inhibition of ultraviolet-induced formation of reactive oxygen species, lipid peroxidation, erythema and skin photosensitization by Polypodium leucotomos. Photodermatol. Photoimmunol. Photomed. 12:45–56; 1996. Tyrell, R. M.; Pidoux, M. Correlation between endogenous glutathione content and the sensitivity of cultured human skin cells to radiation at defined wavelengths in the solar UV range. Photochem. Photobiol. 47:405– 412; 1988. Jones, S. A.; McArdle, F.; Jack, C. I. A.; Jackson, M. J. Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress. Redox Rep. 4:291–299; 1999. Schallreuter, K. A.; Wood, J. M. Thioredoxin reductase—its role in epidermal redox status. J. Photochem. Photobiol. B 64:179 – 184; 2001. Katiyar, S. K.; Afaq, F.; Pewrez, A.; Mukhtar, H. Green tea polyphenol (⫺)-epigallocatechin-3-gallate treatment of human skin inhibits ultraviolet radiation-induced oxidative stress. Carcinogenesis 22:287–294; 2001. Tyrell, R. M. Activation of mammalian genes by the UV component of sunlight—from models to reality. Bioessays 18:139 – 148; 1996. Nachbar, F.; Korting, H. C. The role of vitamin E in normal and damaged skin. J. Mol. Med. 73:7–17; 1995. Fuchs, J.; Kern, H. Modulation of UV-light induced skin inflammation by d-alpha-tocopherol and l-ascorbic acid: a clinical study using solar-simulated radiation. Free Radic. Biol. Med. 25:1006 – 1012; 1998. Stewart, M. S.; Cameron, G. S.; Pence, B. C. Antioxidant nutrients protect against UVB-induced oxidative damage to DNA of mouse keratinocytes in culture. J. Invest. Dermatol. 106:1976 – 1989; 1996. Tebbe, B.; Wu, S.; Geilen, C. C.; Eberle, J.; Kodelija, V.; Orfanos, C. E. L-ascorbic acid inhibits UVA-induced lipid peroxidation and secretion of IL-1␣ and IL-6 in cultured human keratinocytes in vitro. J. Invest. Dermatol. 108:302–306; 1997. Dunham, W. B.; Zuckerkandl, E.; Reynolds, R.; Willoughby, R.; Marcuson, R.; Barth, R.; Pauling, L. Effects of intake of Lascorbic acid (vitamin C) on the incidence of dermal neoplasms induced in mice by ultraviolet light. Proc. Natl. Acad. Sci. USA 79:7532–7536; 1982. Claiborne, A. Catalase activity. In: Greenwald, R. A., ed. CRC handbook of methods for oxygen radical research. Boca Raton, FL: CRC Press; 1985:283–284. Fukunaga, K.; Suzuki, T.; Takama, K. Highly sensitive highperformance liquid chromatography for the measurement of malondialdehyde in biological samples. J. Chromatogr. 621:77– 81; 1993. Anderson, M. E. Measurement of antioxidants: glutathione. In: Punchard, N. A.; Kelly, F. J., eds. Free radicals, a practical approach. Oxford: IRL Press; 1996:213–226. Di Monte, D.; Bellomo, G.; Thor, H.; Nicotera, P.; Orrenius, S. Menadione-induced cytotoxicity is associated with protein thiol
1362
[28]
[29] [30] [31]
[32] [33] [34]
F. MCARDLE et al. oxidation and alteration in intracellular Ca2⫹ homeostasis. Arch. Biochem. Biophys. 235:343–350; 1984. Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76 – 85; 1985. Folch, J.; Lees, M.; Stanley, G. S. H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497–503; 1957. Metcalf, L. D.; Schmitz, A. A. Boron-trifluoride method for the derivitisation of fatty acids. Anal. Chem. 33:363–370; 1961. Mohr, D.; Stocker, R. Selective and sensitive measurement of vitamin C, ubiquinol-10, and other low molecular weight antioxidants. In: Punchard, N. A.; Kelly, F. J., eds. Free radicals, a practical approach. Oxford: IRL Press; 1996:271–285. Catignani, G. L.; Bieri, J. G. Rat liver alpha-tocopherol binding protein. Biochim. Biophys. Acta 497:349 –357; 1983. Shindo, Y.; Packer, L. Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light. J. Invest. Dermatol. 100:260 –265; 1993. Reimer, D.; Bailley, J.; Singh, S. Complete cDNA and 5'genomic
[35] [36]
[37] [38]
[39]
[40]
sequence and multilevel regulation of the mouse catalase gene. Genomics 15:325–336; 1994. Halliwell, B.; Gutteridge, J. M. C. Free radicals in biology and medicine. Oxford, UK: Oxford University Press; 1989. Rhodes, L. E.; O’Farrell, S.; Jackson, M. J.; Friedmann, P. S. Dietary omega-3 fatty acids reduce UVB-induced erythema while increasing lipid peroxidation. J. Invest. Dermatol. 103:151–154; 1994. Jackson, M. J.; McArdle, A.; McArdle, F. Antioxidant micronutrients and gene expression. Proc. Nutr. Soc. 57:301–305; 1988. Packer, J. E.; Slater, T.; Wilson, R. L. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278:737–738; 1979. Fuchs, J. Potentials and limitations of the natural antioxidants RRR-alpha-tocopherol, L-ascorbic acid and b-carotene in cutaneous photoprotection. Free Radic. Biol. Med. 25:848 – 873; 1998. Eberlin-Konig, B.; Placzek, M.; Przybilla, B. Protective effect against sunburn of combined systemic ascorbic acid (vitamin C) and d-␣-tocopherol (vitamin E). J. Am. Acad. Dermatol. 38:45– 48; 1998.