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Cutaneous photoprotection from ultraviolet injury by green tea polyphenols
REPORTS
Cutaneous photoprotection from ultraviolet injury by green tea polyphenols Craig A. Elmets, MD,a,b Divya Singh, MD,a Karen Tubesing, MS,a Mary Matsui, PhD,c Santosh Katiyar, MDa and Hasan Mukhtar, PhDa Cleveland, Ohio, Birmingham, Alabama, and Melville, New York Background: In animal models, extracts from green tea have been shown to be remarkably effective at reducing the severity of adverse human health effects of overexposure to ultraviolet (UV) radiation. Although sunscreens and other photoprotective measures have traditionally been used for this purpose, there is a need for additional measures and natural products are increasingly being explored for that purpose. Objective: Our purpose was to evaluate the effect of polyphenols from green tea on parameters associated with acute UV injury. Methods: Areas of skin of normal volunteers were treated with an extract of green tea or one of its constituents. Thirty minutes later, the treated sites were exposed to a 2 minimal erythema dose solar simulated radiation. UV-treated skin was examined clinically for UV-induced erythema, histologically for the presence of sunburn cells or Langerhans cell distributions, or biochemically for UV-induced DNA damage. Results: Application of green tea extracts resulted in a dose-dependent inhibition of the erythema response evoked by UV radiation. The (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) polyphenolic fractions were most efficient at inhibiting erythema, whereas (-)-epigallocatechin (EGC) and (-)-epicatechin (EC) had little effect. On histologic examination, skin treated with green tea extracts reduced the number of sunburn cells and protected epidermal Langerhans cells from UV damage. Green tea extracts also reduced the DNA damage that formed after UV radiation. Conclusion: Polyphenolic extracts of green tea are effective chemopreventive agents for many of the adverse effects of sunlight on human health and may thus serve as natural alternatives for photoprotection. (J Am Acad Dermatol 2001;44:425-32.)
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utaneous overexposure to the ultraviolet (UV) component of solar radiation (290-400 nm) has a variety of adverse effects on human health including sunburn,1 basal cell and squamous
From the Department of Dermatology, Case Western Reserve University, Clevelanda; the Department of Dermatology, University of Alabama at Birminghamb; and the Biological Research Division, Estee Lauder Companies, Inc, Research Park, Melville.c Supported by National Institutes of Health grants CA73096, CA79820 CA51802, and CA78809, National Cancer Institute Contract N01 CN-85083-57, American Institute for Cancer Research Grant 96B015, and by funds from Estee Lauder Companies, Inc. Accepted for publication Nov 8, 2000. Reprint requests: Craig A. Elmets, MD, EFH 414, 1530 Third Ave S, Birmingham, AL 35294-0009. E-mail: [email protected]. Copyright © 2001 by the American Academy of Dermatology. 0190-9622/2001/$35.00 + 0 16/1/112919 doi:10.1067/mjd.2001.112919
cell carcinoma,2,3 melanoma,2,4 cataracts,3,5 photoaging of the skin,6 and immune suppression.7 These consequences are attracting considerable attention because there has been an alarming increase in the incidence of sunlight-related skin and eye disorders. For example, between 1960 and 1986, there was a 240% increase in the incidence of cutaneous squamous cell carcinoma and a 400% increase in melanoma in certain areas of the United States.8 Changes in lifestyle have led to a significant augmentation in the amount of UV radiation that many people receive.9 This can be attributed primarily to greater amounts of time people spend in outdoor recreational activities.10 Depletion of stratospheric ozone3 and the expanded use of suntanning devices for cosmetic purposes11 may also contribute to the problem. The skin is endowed with endogenous methods of limiting the potential damage caused by solar UV radiation exposure. Light scattering by the stratum 425
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corneum, absorption of light by melanin, and repair of UV-damaged DNA by repair enzymes each can diminish the deleterious effects of UV radiation on the skin. However, for many people, these endogenous means of photoprotection are not fully capable of counteracting all the adverse effects of UV radiation overexposure. Consequently, there has been a concerted effort among physicians’ organizations and governmental agencies to educate the public about how to protect against the harmful effects of overexposure to UV radiation.12 These programs encourage (1) avoidance of sun exposure at times of peak intensity (10:00 AM-3:00 PM); (2) the use of protective clothing (long-sleeved shirts and pants), wide-brimmed hats, and sunglasses; and (3) the conscientious application of sunscreens with a sun protection factor 15 or higher. Chemoprevention is another potential option for protection against sunlight-related skin disorders.13 Chemoprevention refers to the prevention of disease through dietary manipulation or pharmacologic intervention. Among the agents that have been identified as having potential chemopreventive activities in humans are retinoids14,15 and low-fat diets.16 In addition, a polyphenolic fraction isolated from green tea has been shown to have multiple chemopreventive activities in animal models and in in vitro systems.17-19 Previous studies in mice have shown that they afford significant protection against the UVB-induced (290-320 nm) sunburn reaction, sunburn cell formation, immunosuppression, and skin cancer.20 However, their ability to reduce UV-induced photodamage in humans in vivo has not been addressed. This study was undertaken to determine whether green tea polyphenols (GTPs) and the individual constituents were able to limit UV-induced photodamage in human volunteers.
METHODS Subjects The protocol was approved by the Institutional Review Board of University Hospitals of Cleveland. Written consent was obtained from all participants in the study. Men and women whose ages ranged from 18 to 50 years were eligible for the studies. All were in good health with no evidence of acute or chronic disease. Subjects were excluded if they were pregnant or nursing, had a history of an abnormal response to sunlight, or were taking a medication with known photosensitizing properties. Persons were instructed not to take aspirin or nonsteroidal anti-inflammatory agents during the course of the study. For each aspect of the study, 5 or 6 volunteers were recruited.
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GTPs A defined green tea polyphenolic fraction was prepared in the laboratory according to methods described previously.20 Unless otherwise stated, a 5% solution was prepared by weighing 0.5 g of GTP and dissolving it in 10 mL of 1:1 ethanol/water vehicle. Study protocol Six visits per person were required. During the first two visits, each individual’s minimal erythema dose (MED) was determined by exposing the skin to graded doses of UV radiation from a solar simulator (Oriel Instruments, Stratford, Conn). The possible induction of erythema by vehicle and test samples in the absence of UV radiation was also determined. The lowest dose resulting in uniform erythema over the irradiation site was considered the MED. A 200µL solution of GTPs dissolved in an ethanol/water vehicle or one of its constituents was applied to a 5× 5-cm area on the back. Neither the vehicle nor any of the test constituents caused erythema visually or by chromameter (Minolta) measurements in any volunteer. On the third visit, an aliquot of GTP solution or one of its constituents was applied to the back 30 minutes before exposure with a 2-MED dose of solar simulated light. Baseline chromameter readings were performed at that time. Patients returned 24 (fourth visit), 48 (fifth visit) and 72 (sixth visit) hours later, at which time chromameter readings and biopsies were performed. Langerhans cells CD1a+ Langerhans cells were quantified en face according to previously described techniques.21 In some specimens, Langerhans cells were examined in vertical sections with immunoperoxidase techniques. Detection of UV-induced mutations in DNA UV-induced DNA damage was detected by a sensitive phosphorus 32 (32P)–postlabeling technique according to a method that we have described previously.22 Briefly, 5 µg of DNA was isolated from the epidermis. Samples were dissolved in water and then were phosphorylated by incubating the samples with (γ-32P) adenosine triphosphate and T4 polynucleotide kinase. Lesions were resolved in 3 dimensions on polyethyleneimine–thin-layer chromatography plates. DNA lesions were detected by overnight exposure to a PhosphoImager (Dynamic 400S PhosphoImager, Sunnydale, Calif). UVA exposure Subjects were treated identically to those receiving solar simulated light with the exception that a UVA light source (Bluelight 2002 combined with
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Fig 1. Effect of GTP on the erythema response evoked by 2-MED solar simulated radiation. Data represent the mean ± standard error of the mean erythema index at 24, 48, and 72 hours after irradiation with a solar simulator. Measurements were made with a chromameter on 6 volunteers. Areas of skin were pretreated with indicated concentration of an extract of green tea (GTE) 30 minutes before UV exposure.
plateglass, Dr Hönle) was used. Output was monitored with a research radiometer (model IL700, International Light, Newburyport, Mass) coupled to a UVA photodetector. Output in the UVB range was less than 5 mJ/cm2. Constituents of GTPs The GTP constituents (-)-epicatechin (EC), (-)epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin-3-gallate (EGCG) were more than 95% pure and were obtained as a gift from Mitsui Norin Company, Shizouka, Japan. Statistical analysis Mean and standard error of the mean were calculated for all data. The Student t test was used for comparison between groups with a P value less than .05 considered significant.
Fig 2. Clinical appearance of skin 24 hours after having been treated with GTP and solar simulated light. Skin was treated with GTP followed 30 minutes later by a 2-MED dose of solar simulated light. At left is skin treated with UV alone. Middle is skin treated with both GTP and UV. Right is skin treated with vehicle.
RESULTS To determine whether GTP could inhibit the UV radiation–induced erythema response, 0.2 mL of GTP in concentrations ranging from 1% to 10% was applied to the skin on the backs of 6 volunteers who were recruited into the study. Thirty minutes after the preparation was applied, subjects were exposed to a 2-MED dose of UV radiation from a solar simulator. Measurements of the erythema response were quantified with a chromameter 24, 48, and 72 hours after solar radiation exposure. There was a dose-dependent reduction in erythema
with a 10% solution producing almost complete protection at 48 and 72 hours. In all subjects, a 2.5% solution was found to provide excellent protection, and in some subjects even a 0.5% solution was able to produce a significant reduction in the sunburn response (Fig 1). This was a consistent finding because in all 6 patients, the sunburn response was inhibited by application of green tea before solar simulated light (P < .01). Visual inspection confirmed the findings of the chromameter readings
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A
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B
Fig 3. Histologic appearance of skin treated with GTP and solar simulated light. Skin was treated with GTP followed 30 minutes later by 2-MED of solar simulated light. Specimens were taken 48 hours after UV exposure. A, Untreated skin. B, UV-treated skin. C, Skin treated with GTP and UV.
(Fig 2). The effect of GTPs was most pronounced when they were applied immediately before solar simulated light, although protection was observed even when GTPs were applied as long as 4 hours before UV exposure (not shown). To exclude the possibility that green tea extracts were merely acting as a sunscreen, spectrophotometric analysis was performed on GTPs. It showed UV absorption maxima at 273 nm. It did not absorb wavelengths within the UVB range and therefore did not filter out the wavelengths of solar simulated light that were responsible for the erythema. These results together with the information that oral administration of GTP in the drinking water of animal models results in photoprotection strongly suggests that the photoprotective effects observed in human volunteers are not related to the sunscreen effects of GTP.23 Thus the reduction in UVinduced erythema was produced by a mechanism that appeared to be distinct from that of traditional sunscreens.
Histologic findings in skin treated by green tea extracts Biopsy specimens of skin that had been exposed to a 2-MED dose of UV radiation 24 hours previously were examined for sunburn cells to determine whether GTP had a protective effect on this parameter of acute UV injury. GTP applied 30 minutes before UV radiation reduced the number of sunburn cells by 66% (Fig 3 and Table I). UV radiation is also known to cause a reduction in the number of epidermal Langerhans cells.21,24 Experiments were therefore conducted to determine whether GTPs protected Langerhans cells from UV injury. The skin of volunteers was exposed to 2MED of UV radiation daily for 4 consecutive days. After the final UV exposure, suction blisters were taken and stained en face for CD1a+ reactivity. Langerhans cells are bone marrow–derived resident epidermal cells that initiate T-cell–mediated immune responses to antigenic substances encountered by the epidermis. Application of GTPs before UV expo-
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Fig 4. Effect of individual constituents present in GTP on the erythema response evoked by 2MED of solar simulated radiation. Data represent the mean ± standard error of the mean erythema index at 48 hours after irradiation with a solar simulator. Measurements were made with a chromameter on 6 volunteers. Areas of skin were pretreated with an equimolar concentration of the indicated constituent of GTP 30 minutes before UV exposure.
Table I. Inhibition of solar simulated DNA damage by topical application of GTP
Vehicle alone GTP alone Vehicle + 2 MED solar simulated radiation GTP + 2 MED solar simulated radiation
Sunburn cells*
Langerhans cells†
DNA damage‡
0.1 ± 0.1 0.1 ± 0.1 5.8 ± 0.7 1.9 ± 0.3 (68%§,¶)
589 ± 46 569 ± 38 88 ± 29 377 ± 28 (58%§,,¶)
250 ± 29 262 ± 31 1582 ± 162 846 ± 132 (55%§,,¶)
*Data represent the mean concentration of sunburn cells per linear millimeter of epidermis (± standard error of the mean) from 6 volunteers. †Data represent the mean concentration of Langerhans cells per square millimeter (± standard error of the mean) from 6 volunteers. The subjects tested for Langerhans cell were different from those tested for sunburn cells. ‡Data are the comparison of raw data values of 32P-labeled spots from densitometric readings from PhosphoImager analysis of DNA samples obtained from epidermal biopsy specimens of humans. Each value represents the mean ± standard error of the mean of 4 independent specimens where each assay was conducted in duplicate. §Percent reduction in UV-induced sunburn cell formation, Langerhans cell damage, or DNA damage by GTP. ||P < .01. ¶Percent reduction was calculated according to the formula: ([Vehicle + 2 MED Solar Simulated Radiation] – [GTP + 2 MED Solar Simulated Radiation])/([Vehicle + 2 MED Solar Simulated Radiation] – [Vehicle Alone]) × 100%.
sure resulted in a 58% reconstitution of this epidermal immune cell population (Table I). Effect of constituents of green tea on UVinduced erythema Purified polyphenol constituents isolated from green tea were next tested to determine which among them was responsible for its chemopreventive activities. Skin sites on the back were treated with
equimolar concentrations of EGCG, ECG, EC, EGC, and 5% GTP solution. The sites were then exposed to two times the MED of solar simulated light. The 5% GTP solution that contained a mixture of polyphenols was most efficient at protecting against erythema. Among the different polyphenolic fractions, EGCG and ECG, both of which contain a galloyl group at the 3 position, were most efficient at inhibiting erythema, whereas EGC and EC had little effect (Fig 4).
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Fig 5. Effect of GTP on the erythema response evoked by 135 J/cm2 UVA radiation. Data represent the mean ± standard error of the mean erythema index at 48 hours after irradiation with a UVA light source. Measurements were made with a chromameter on 6 volunteers. Areas of skin were pretreated with a 5% extract of GTP 30 minutes before UVA exposure.
Effect of green tea extracts on UVA-induced erythema Wavelengths within the UVB (290-320 nm) are the principal wavelengths that are responsible for the erythema that accompanies exposure to sunlight. However, there is an increasing awareness that wavelengths within the UVA (320-400 nm) also can affect the skin. The extent to which GTPs were able to inhibit UVA erythema was examined. GTPs were placed on the skin of volunteers and 30 minutes later, instead of exposing the skin to solar simulated radiation, it was exposed to 135 J/cm2 UVA radiation from a UVA light source. Erythema was measured at 24, 48, and 72 hours later. Similar to the effect on UVB-induced erythema, GTP significantly reduced the erythema response after UVA exposure (Fig 5). DNA damage One of the major adverse effects of UV radiation is damage to DNA. Damage to DNA initiates lesions that are necessary for UV carcinogenesis.25 Studies to assess the effect of GTP on DNA damage were conducted by treating skin with a 5% green tea extract and then exposing it to a 2-MED dose of solar simulated light. Biopsy specimens were taken and the extent of DNA damage was assessed by a 32Ppostlabeling technique. As shown in Table I, the extent of DNA damage was significantly reduced by application of GTP before UV exposure.
DISCUSSION The results of these studies indicate that GTPs exert a photoprotective effect on human skin. Areas of skin that were pretreated with GTP before UV exposure developed less erythema and were found, on microscopic examination, to have fewer sunburn cells. GTPs were also found to reduce the damaging effects of UV radiation on Langerhans cells, a subpopulation of epidermal cells known to play a key role in the development of cutaneous cell-mediated immune responses. EGCG and ECG, the two polyphenols that contain a galloyl group at the 3 position, were the two constituents that were most effective at limiting the adverse effects of UV radiation on the skin. This is important because it provides structural information that may pave the way for the synthesis of more effective chemopreventive molecules. Interestingly, GTPs were effective at reducing the erythema response both to UVB and to UVA radiation. Currently, sunscreens are the major pharmacologic agent used to protect against solar UV radiation exposure. Sunscreens were originally developed in the 1940s, but high-potency sunscreens have only been commercially available since the late 1970s.26 These topically applied compounds act as a physical barrier to either absorb or reflect incident UV radiation, thereby reducing the amount that reaches beyond the most superficial layers of the epidermis. Data from this and other studies indicate that GTP,
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EGCG, and ECG act in a manner that is quite different from that of sunscreens. Unlike sunscreens, these compounds do not appear to absorb significant amounts of light in the UV range. The implication of this finding is that GTPs, when combined with topical formulations that contain traditional sunscreens, may have additive or synergistic photoprotective effects when compared with either of these agents alone. GTPs may also have value in persons who are allergic to sunscreens or who for some other reason are unable to tolerate them. In this study, GTPs were shown to protect against the adverse effects of both UVB and UVA radiation. Although wavelengths within the UVB are more effective at causing sunburn and nonmelanoma skin cancer than UVA, there is considerable experimental evidence that wavelengths within the UVA also have deleterious effects on human health.27-30 The importance of devising strategies to protect against UVA radiation has only recently been recognized. Most sunscreens that are currently available are comparatively much more effective at limiting UVB penetration into the skin than UVA.10 Those persons who do wear sunscreens and stay outside for longer periods are likely to receive a greater total dose of UVA than persons who do not wear sunscreens. UVA can cause a sunburn reaction and has been implicated as an etiologic agent in photoaging and some forms of nonmelanoma skin cancer. Its role in melanoma is controversial. There is evidence in animal models and from epidemiologic studies suggesting that UVA may play a role in the development of this malignancy as well.31-33 The finding that GTPs effectively inhibit UVA-induced erythema means that their incorporation into topical formulations may thus provide photoprotection over a wider UV range. An obvious question that remains to be determined is the effect of GTPs on the prevention of nonmelanoma skin cancer. Experimental data from animal models strongly suggest that GTPs may in fact reduce the incidence of sunlight-related skin cancer.23 Although this study did not specifically address the issue of skin cancer in humans, the fact that they reduced lesions in DNA, which are necessary for the development of cancer suggests that this may be the case. In addition to its role in causing sunburn and skin cancer, UV radiation has a profound influence on the immune response.7 This is mediated at least in part by the damaging effects of UV radiation on epidermal Langerhans cells.21,24 Epidermal Langerhans cells are extremely sensitive to UV radiation.34 Pretreatment of skin with doses of UV radiation sufficient to diminish the number of identifiable Langerhans cells coincides with a reduction in immunization rates through the
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UV-irradiation skin site.35,36 The results presented herein indicate that GTPs abrogate the effects of UV radiation on Langerhans cells. In animal models and in cell culture systems, GTPs have been shown to be potent antioxidants.37 With respect to the skin, UV-induced oxidative stress has been implicated, among other things, in the production of prostaglandin E2 and in activation of the epidermal growth factor receptor.38,39 Both are involved in causing epidermal hyperproliferation and inflammation and both are known to be associated with UV injury. Other antioxidants such as genestein40 and a low-fat diet,16 which lowers lipid peroxidation in the skin, have also been shown to reduce UV-induced injury. In an era in which the incidence of the adverse effects of UV radiation is rising at an alarming rate, the introduction of GTPs and other natural chemopreventive agents may prove to be a new alternative for protection from exposure to UV radiation. REFERENCES 1. Soter NA. Acute effects of ultraviolet radiation on the skin. Semin Dermatol 1990;9:11-5. 2. Mukhtar H, Elmets CA. Photocarcinogenesis: mechanisms, models and human health implications. Photochem Photobiol 1996;63:355-447. 3. Longstreth J, de Gruijl FR, Kripke ML, Abseck S, Arnold F, Slaper HI, et al. Health risks. J Photochem Photobiol B 1998;46:20-39. 4. Koh HK, Kligler BE, Lew PA. Sunlight and cutaneous malignant melanoma: evidence for and against causation. Photochem Photobiol 1990;51:765-79. 5. West SK, Duncan DD, Munoz B, Rubin GS, Fried LP, BandeenRoche K, et al. Sunlight exposure and risk of lens opacities in a population-based study: the Salisbury Eye Evaluation project. JAMA 1998;280:714-8. 6. Gilchrest BA, Yaar M. Ageing and photoageing of the skin: observations at the cellular and molecular level. Br J Dermatol 1992;127(Suppl 41):25-30. 7. Krutmann JT, Elmets CA. Photoimmunology. Oxford: Blackwell Scientific; 1995. 8. Glass A, Hoover R. The emerging epidemic of melanoma and squamous cell skin cancer. JAMA 1989;262:2097-100. 9. Miller D,Weinstock M. Non-melanoma skin cancer in the United States: incidence. J Am Acad Dermatol 1994;30:774-8. 10. Elmets C, Mukhtar H. Ultraviolet radiation and skin cancer: progress in pathophysiologic mechanisms. Prog Dermatol 1996;30:1-16. 11. Swerdlow AJ, Weinstock MA. Do tanning lamps cause melanoma? An epidemiologic assessment. J Am Acad Dermatol 1998;38:89-98. 12. Chapman S, Marks R, King M.Trends in tans and skin protection in Australian fashion magazines, 1982 through 1991. Am J Public Health 1992;82:1677-80. 13. Greenwald P. Chemoprevention of cancer. Sci Am 1996;275:969. 14. Miller WH Jr. The emerging role of retinoids and retinoic acid metabolism blocking agents in the treatment of cancer. Cancer 1998;83:1471-82. 15. DiGiovanna JJ. Retinoid chemoprevention in the high-risk patient. J Am Acad Dermatol 1998;39(Suppl):S82-5.
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16. Black HS, Herd JA, Goldberg LH,Wolf JE Jr,Thornby JI, Rosen T, et al. Effect of a low-fat diet on the incidence of actinic keratosis. N Engl J Med 1994;330:1272-5. 17. Katiyar SK, Mukhtar H. Tea in chemoprevention of cancer: epidemiologic and experimental studies. Int J Oncol 1996;8:22138. 18. Yang CS, Wang ZY. Tea and cancer. J Natl Cancer Inst 1993;85: 1038-49. 19. Huang MT, Xie JG,Wang ZY, Ho CT, Lou YR,Wang CX, et al. Effects of tea, decaffeinated tea, and caffeine on UVB light-induced complete carcinogenesis in SKH-1 mice: demonstration of caffeine as a biologically important constituent of tea. Cancer Res 1997;57:2623-9. 20. Katiyar SK, Elmets CA, Agarwal R, Mukhtar H. Protection against ultraviolet-B radiation-induced local and systemic suppression of contact hypersensitivity and edema responses in C3H/HeN mice by green tea polyphenols. Photochem Photobiol 1995;62: 855-61. 21. Elmets CA, Vargas A, Oresajo C. Photoprotective effects of sunscreens in cosmetics on sunburn and Langerhans cell photodamage. Photodermatol Photoimmunol Photomed 1992;9:1320. 22. Chatterjee ML, Agarwal R, Mukhtar H. Ultraviolet B radiationinduced DNA lesions in mouse epidermis: an assessment using a novel 32p-postlabelling technique. Biochem Biophys Res Comm 1996;229:590-5. 23. Katiyar SK, Rupp CO, Korman NJ, Agarwal R, Mukhtar H. Inhibition of 12-O-tetradecanoylphorbol-13-acetate and other skin tumor-promoter-caused induction of epidermal interleukin-1 alpha mRNA and protein expression in SENCAR mice by green tea polyphenols. J Invest Dermatol 1995;105:394-8. 24. Toews GB, Bergstresser PR, Streilein JW, Sullivan S. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol 1980;124:445-53. 25. Elmets C. Cutaneous photocarcinogenesis. In: Mukhtar H, editor. Pharmacology of the skin. Boca Raton (FL): CRC Press; 1992. p. 389-416. 26. Anderson CY, Elmets CA. Sunscreens and photocarcinogenesis: an objective assessment. Photochem Photobiol 1996;63:43540. 27. Strickland PT. Photocarcinogenesis by near-ultraviolet (UVA) radiation in Sencar mice. J Invest Dermatol 1986;87:272-5. 28. Staberg B, Wulf HC, Klemp P, Poulsen T, Brodthagen H. The carcinogenic effect of UVA irradiation. J Invest Dermatol 1983;81: 517-9.
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29. Matsui M, DeLeo VA. Longwave ultraviolet radiation and promotion of skin cancer. Cancer Cells 1991;3:8-12. 30. Moan J, Dahlback A, Setlow RB. Epidemiological support for an hypothesis for melanoma induction indicating a role for UVA radiation. Photochem Photobiol 1999;70:243-7. 31. Swerdlow AJ, English JSC, MacKie RM, O’Doherty CJ, Hunter JA, Clark J, et al. Fluorescent lights, ultraviolet lamps, and risk of cutaneous melanoma. Br Med J 1988;297:647-50. 32. Walter SD, Marrett LD, From L, Hertzman C, Shannon HS, Roy P. The association of cutaneous malignant melanoma with the use of sunbeds and sunlamps. Am J Epidemiol 1990;131:23243. 33. Setlow RB, Grist E, Thompson K, Woodhead AD. Wavelengths effective in induction of malignant melanoma. Proc Natl Acad Sci U S A 1993;90:6666-70. 34. Rae V,Yoshikawa T, Bruins-Slot W, Streilein JW,Taylor JR. An ultraviolet B radiation protocol for complete depletion of human epidermal Langerhans cells. J Dermatol Surg Oncol 1989;15: 1199-202. 35. Yoshikawa T, Rae V, Bruins-Slot W, van der Berg J-W, Taylor JR, Streilein JW. Susceptibility to effects of UVB radiation on induction of contact hypersensitivity as a risk factor for skin cancer in man. J Invest Dermatol 1990;95:530-6. 36. Cooper KD, Oberhelman L, Hamilton TA, Baadsgaard O, Terhune M, LeVee G, et al. UV exposure reduces immunization rates and promotes tolerance to epicutaneous antigens in humans, relationship to dose, CDla-DR+ epidermal macrophage induction, and Langerhans cell depletion. Proc Natl Acad Sci U S A 1992;89:8497-501. 37. Khan SG, Katiyar SK, Agarwal R, Mukhtar H. Enhancement of antioxidant and phase II enzymes by oral feeding of green tea polyphenols in drinking water to SKH-1 hairless mice: possible role in cancer chemoprevention. Cancer Res 1992;52:4050-2. 38. Matsui MS, DeLeo VA. Induction of protein kinase C activity by ultraviolet radiation. Carcinogenesis 1990;11:229-34. 39. Miller CC, Hale P, Pentland AP. Ultraviolet B injury increases prostaglandin synthesis through a tyrosine kinase-dependent pathway: evidence for UVB-induced epidermal growth factor receptor activation. J Biol Chem 1994;269:3529-33. 40. Wei H. Photoprotective action of isoflavone genistein: models, mechanisms, and relevance to clinical dermatology. J Am Acad Dermatol 1998;39:271-2.
Cutaneous photoprotection from ultraviolet injury by green tea polyphenols Craig A. Elmets, MD,a,b Divya Singh, MD,a Karen Tubesing, MS,a Mary Matsui, PhD,c Santosh Katiyar, MDa and Hasan Mukhtar, PhDa Cleveland, Ohio, Birmingham, Alabama, and Melville, New York Background: In animal models, extracts from green tea have been shown to be remarkably effective at reducing the severity of adverse human health effects of overexposure to ultraviolet (UV) radiation. Although sunscreens and other photoprotective measures have traditionally been used for this purpose, there is a need for additional measures and natural products are increasingly being explored for that purpose. Objective: Our purpose was to evaluate the effect of polyphenols from green tea on parameters associated with acute UV injury. Methods: Areas of skin of normal volunteers were treated with an extract of green tea or one of its constituents. Thirty minutes later, the treated sites were exposed to a 2 minimal erythema dose solar simulated radiation. UV-treated skin was examined clinically for UV-induced erythema, histologically for the presence of sunburn cells or Langerhans cell distributions, or biochemically for UV-induced DNA damage. Results: Application of green tea extracts resulted in a dose-dependent inhibition of the erythema response evoked by UV radiation. The (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) polyphenolic fractions were most efficient at inhibiting erythema, whereas (-)-epigallocatechin (EGC) and (-)-epicatechin (EC) had little effect. On histologic examination, skin treated with green tea extracts reduced the number of sunburn cells and protected epidermal Langerhans cells from UV damage. Green tea extracts also reduced the DNA damage that formed after UV radiation. Conclusion: Polyphenolic extracts of green tea are effective chemopreventive agents for many of the adverse effects of sunlight on human health and may thus serve as natural alternatives for photoprotection. (J Am Acad Dermatol 2001;44:425-32.)
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utaneous overexposure to the ultraviolet (UV) component of solar radiation (290-400 nm) has a variety of adverse effects on human health including sunburn,1 basal cell and squamous
From the Department of Dermatology, Case Western Reserve University, Clevelanda; the Department of Dermatology, University of Alabama at Birminghamb; and the Biological Research Division, Estee Lauder Companies, Inc, Research Park, Melville.c Supported by National Institutes of Health grants CA73096, CA79820 CA51802, and CA78809, National Cancer Institute Contract N01 CN-85083-57, American Institute for Cancer Research Grant 96B015, and by funds from Estee Lauder Companies, Inc. Accepted for publication Nov 8, 2000. Reprint requests: Craig A. Elmets, MD, EFH 414, 1530 Third Ave S, Birmingham, AL 35294-0009. E-mail: [email protected]. Copyright © 2001 by the American Academy of Dermatology. 0190-9622/2001/$35.00 + 0 16/1/112919 doi:10.1067/mjd.2001.112919
cell carcinoma,2,3 melanoma,2,4 cataracts,3,5 photoaging of the skin,6 and immune suppression.7 These consequences are attracting considerable attention because there has been an alarming increase in the incidence of sunlight-related skin and eye disorders. For example, between 1960 and 1986, there was a 240% increase in the incidence of cutaneous squamous cell carcinoma and a 400% increase in melanoma in certain areas of the United States.8 Changes in lifestyle have led to a significant augmentation in the amount of UV radiation that many people receive.9 This can be attributed primarily to greater amounts of time people spend in outdoor recreational activities.10 Depletion of stratospheric ozone3 and the expanded use of suntanning devices for cosmetic purposes11 may also contribute to the problem. The skin is endowed with endogenous methods of limiting the potential damage caused by solar UV radiation exposure. Light scattering by the stratum 425
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corneum, absorption of light by melanin, and repair of UV-damaged DNA by repair enzymes each can diminish the deleterious effects of UV radiation on the skin. However, for many people, these endogenous means of photoprotection are not fully capable of counteracting all the adverse effects of UV radiation overexposure. Consequently, there has been a concerted effort among physicians’ organizations and governmental agencies to educate the public about how to protect against the harmful effects of overexposure to UV radiation.12 These programs encourage (1) avoidance of sun exposure at times of peak intensity (10:00 AM-3:00 PM); (2) the use of protective clothing (long-sleeved shirts and pants), wide-brimmed hats, and sunglasses; and (3) the conscientious application of sunscreens with a sun protection factor 15 or higher. Chemoprevention is another potential option for protection against sunlight-related skin disorders.13 Chemoprevention refers to the prevention of disease through dietary manipulation or pharmacologic intervention. Among the agents that have been identified as having potential chemopreventive activities in humans are retinoids14,15 and low-fat diets.16 In addition, a polyphenolic fraction isolated from green tea has been shown to have multiple chemopreventive activities in animal models and in in vitro systems.17-19 Previous studies in mice have shown that they afford significant protection against the UVB-induced (290-320 nm) sunburn reaction, sunburn cell formation, immunosuppression, and skin cancer.20 However, their ability to reduce UV-induced photodamage in humans in vivo has not been addressed. This study was undertaken to determine whether green tea polyphenols (GTPs) and the individual constituents were able to limit UV-induced photodamage in human volunteers.
METHODS Subjects The protocol was approved by the Institutional Review Board of University Hospitals of Cleveland. Written consent was obtained from all participants in the study. Men and women whose ages ranged from 18 to 50 years were eligible for the studies. All were in good health with no evidence of acute or chronic disease. Subjects were excluded if they were pregnant or nursing, had a history of an abnormal response to sunlight, or were taking a medication with known photosensitizing properties. Persons were instructed not to take aspirin or nonsteroidal anti-inflammatory agents during the course of the study. For each aspect of the study, 5 or 6 volunteers were recruited.
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GTPs A defined green tea polyphenolic fraction was prepared in the laboratory according to methods described previously.20 Unless otherwise stated, a 5% solution was prepared by weighing 0.5 g of GTP and dissolving it in 10 mL of 1:1 ethanol/water vehicle. Study protocol Six visits per person were required. During the first two visits, each individual’s minimal erythema dose (MED) was determined by exposing the skin to graded doses of UV radiation from a solar simulator (Oriel Instruments, Stratford, Conn). The possible induction of erythema by vehicle and test samples in the absence of UV radiation was also determined. The lowest dose resulting in uniform erythema over the irradiation site was considered the MED. A 200µL solution of GTPs dissolved in an ethanol/water vehicle or one of its constituents was applied to a 5× 5-cm area on the back. Neither the vehicle nor any of the test constituents caused erythema visually or by chromameter (Minolta) measurements in any volunteer. On the third visit, an aliquot of GTP solution or one of its constituents was applied to the back 30 minutes before exposure with a 2-MED dose of solar simulated light. Baseline chromameter readings were performed at that time. Patients returned 24 (fourth visit), 48 (fifth visit) and 72 (sixth visit) hours later, at which time chromameter readings and biopsies were performed. Langerhans cells CD1a+ Langerhans cells were quantified en face according to previously described techniques.21 In some specimens, Langerhans cells were examined in vertical sections with immunoperoxidase techniques. Detection of UV-induced mutations in DNA UV-induced DNA damage was detected by a sensitive phosphorus 32 (32P)–postlabeling technique according to a method that we have described previously.22 Briefly, 5 µg of DNA was isolated from the epidermis. Samples were dissolved in water and then were phosphorylated by incubating the samples with (γ-32P) adenosine triphosphate and T4 polynucleotide kinase. Lesions were resolved in 3 dimensions on polyethyleneimine–thin-layer chromatography plates. DNA lesions were detected by overnight exposure to a PhosphoImager (Dynamic 400S PhosphoImager, Sunnydale, Calif). UVA exposure Subjects were treated identically to those receiving solar simulated light with the exception that a UVA light source (Bluelight 2002 combined with
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Fig 1. Effect of GTP on the erythema response evoked by 2-MED solar simulated radiation. Data represent the mean ± standard error of the mean erythema index at 24, 48, and 72 hours after irradiation with a solar simulator. Measurements were made with a chromameter on 6 volunteers. Areas of skin were pretreated with indicated concentration of an extract of green tea (GTE) 30 minutes before UV exposure.
plateglass, Dr Hönle) was used. Output was monitored with a research radiometer (model IL700, International Light, Newburyport, Mass) coupled to a UVA photodetector. Output in the UVB range was less than 5 mJ/cm2. Constituents of GTPs The GTP constituents (-)-epicatechin (EC), (-)epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin-3-gallate (EGCG) were more than 95% pure and were obtained as a gift from Mitsui Norin Company, Shizouka, Japan. Statistical analysis Mean and standard error of the mean were calculated for all data. The Student t test was used for comparison between groups with a P value less than .05 considered significant.
Fig 2. Clinical appearance of skin 24 hours after having been treated with GTP and solar simulated light. Skin was treated with GTP followed 30 minutes later by a 2-MED dose of solar simulated light. At left is skin treated with UV alone. Middle is skin treated with both GTP and UV. Right is skin treated with vehicle.
RESULTS To determine whether GTP could inhibit the UV radiation–induced erythema response, 0.2 mL of GTP in concentrations ranging from 1% to 10% was applied to the skin on the backs of 6 volunteers who were recruited into the study. Thirty minutes after the preparation was applied, subjects were exposed to a 2-MED dose of UV radiation from a solar simulator. Measurements of the erythema response were quantified with a chromameter 24, 48, and 72 hours after solar radiation exposure. There was a dose-dependent reduction in erythema
with a 10% solution producing almost complete protection at 48 and 72 hours. In all subjects, a 2.5% solution was found to provide excellent protection, and in some subjects even a 0.5% solution was able to produce a significant reduction in the sunburn response (Fig 1). This was a consistent finding because in all 6 patients, the sunburn response was inhibited by application of green tea before solar simulated light (P < .01). Visual inspection confirmed the findings of the chromameter readings
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A
C
B
Fig 3. Histologic appearance of skin treated with GTP and solar simulated light. Skin was treated with GTP followed 30 minutes later by 2-MED of solar simulated light. Specimens were taken 48 hours after UV exposure. A, Untreated skin. B, UV-treated skin. C, Skin treated with GTP and UV.
(Fig 2). The effect of GTPs was most pronounced when they were applied immediately before solar simulated light, although protection was observed even when GTPs were applied as long as 4 hours before UV exposure (not shown). To exclude the possibility that green tea extracts were merely acting as a sunscreen, spectrophotometric analysis was performed on GTPs. It showed UV absorption maxima at 273 nm. It did not absorb wavelengths within the UVB range and therefore did not filter out the wavelengths of solar simulated light that were responsible for the erythema. These results together with the information that oral administration of GTP in the drinking water of animal models results in photoprotection strongly suggests that the photoprotective effects observed in human volunteers are not related to the sunscreen effects of GTP.23 Thus the reduction in UVinduced erythema was produced by a mechanism that appeared to be distinct from that of traditional sunscreens.
Histologic findings in skin treated by green tea extracts Biopsy specimens of skin that had been exposed to a 2-MED dose of UV radiation 24 hours previously were examined for sunburn cells to determine whether GTP had a protective effect on this parameter of acute UV injury. GTP applied 30 minutes before UV radiation reduced the number of sunburn cells by 66% (Fig 3 and Table I). UV radiation is also known to cause a reduction in the number of epidermal Langerhans cells.21,24 Experiments were therefore conducted to determine whether GTPs protected Langerhans cells from UV injury. The skin of volunteers was exposed to 2MED of UV radiation daily for 4 consecutive days. After the final UV exposure, suction blisters were taken and stained en face for CD1a+ reactivity. Langerhans cells are bone marrow–derived resident epidermal cells that initiate T-cell–mediated immune responses to antigenic substances encountered by the epidermis. Application of GTPs before UV expo-
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Fig 4. Effect of individual constituents present in GTP on the erythema response evoked by 2MED of solar simulated radiation. Data represent the mean ± standard error of the mean erythema index at 48 hours after irradiation with a solar simulator. Measurements were made with a chromameter on 6 volunteers. Areas of skin were pretreated with an equimolar concentration of the indicated constituent of GTP 30 minutes before UV exposure.
Table I. Inhibition of solar simulated DNA damage by topical application of GTP
Vehicle alone GTP alone Vehicle + 2 MED solar simulated radiation GTP + 2 MED solar simulated radiation
Sunburn cells*
Langerhans cells†
DNA damage‡
0.1 ± 0.1 0.1 ± 0.1 5.8 ± 0.7 1.9 ± 0.3 (68%§,¶)
589 ± 46 569 ± 38 88 ± 29 377 ± 28 (58%§,,¶)
250 ± 29 262 ± 31 1582 ± 162 846 ± 132 (55%§,,¶)
*Data represent the mean concentration of sunburn cells per linear millimeter of epidermis (± standard error of the mean) from 6 volunteers. †Data represent the mean concentration of Langerhans cells per square millimeter (± standard error of the mean) from 6 volunteers. The subjects tested for Langerhans cell were different from those tested for sunburn cells. ‡Data are the comparison of raw data values of 32P-labeled spots from densitometric readings from PhosphoImager analysis of DNA samples obtained from epidermal biopsy specimens of humans. Each value represents the mean ± standard error of the mean of 4 independent specimens where each assay was conducted in duplicate. §Percent reduction in UV-induced sunburn cell formation, Langerhans cell damage, or DNA damage by GTP. ||P < .01. ¶Percent reduction was calculated according to the formula: ([Vehicle + 2 MED Solar Simulated Radiation] – [GTP + 2 MED Solar Simulated Radiation])/([Vehicle + 2 MED Solar Simulated Radiation] – [Vehicle Alone]) × 100%.
sure resulted in a 58% reconstitution of this epidermal immune cell population (Table I). Effect of constituents of green tea on UVinduced erythema Purified polyphenol constituents isolated from green tea were next tested to determine which among them was responsible for its chemopreventive activities. Skin sites on the back were treated with
equimolar concentrations of EGCG, ECG, EC, EGC, and 5% GTP solution. The sites were then exposed to two times the MED of solar simulated light. The 5% GTP solution that contained a mixture of polyphenols was most efficient at protecting against erythema. Among the different polyphenolic fractions, EGCG and ECG, both of which contain a galloyl group at the 3 position, were most efficient at inhibiting erythema, whereas EGC and EC had little effect (Fig 4).
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Fig 5. Effect of GTP on the erythema response evoked by 135 J/cm2 UVA radiation. Data represent the mean ± standard error of the mean erythema index at 48 hours after irradiation with a UVA light source. Measurements were made with a chromameter on 6 volunteers. Areas of skin were pretreated with a 5% extract of GTP 30 minutes before UVA exposure.
Effect of green tea extracts on UVA-induced erythema Wavelengths within the UVB (290-320 nm) are the principal wavelengths that are responsible for the erythema that accompanies exposure to sunlight. However, there is an increasing awareness that wavelengths within the UVA (320-400 nm) also can affect the skin. The extent to which GTPs were able to inhibit UVA erythema was examined. GTPs were placed on the skin of volunteers and 30 minutes later, instead of exposing the skin to solar simulated radiation, it was exposed to 135 J/cm2 UVA radiation from a UVA light source. Erythema was measured at 24, 48, and 72 hours later. Similar to the effect on UVB-induced erythema, GTP significantly reduced the erythema response after UVA exposure (Fig 5). DNA damage One of the major adverse effects of UV radiation is damage to DNA. Damage to DNA initiates lesions that are necessary for UV carcinogenesis.25 Studies to assess the effect of GTP on DNA damage were conducted by treating skin with a 5% green tea extract and then exposing it to a 2-MED dose of solar simulated light. Biopsy specimens were taken and the extent of DNA damage was assessed by a 32Ppostlabeling technique. As shown in Table I, the extent of DNA damage was significantly reduced by application of GTP before UV exposure.
DISCUSSION The results of these studies indicate that GTPs exert a photoprotective effect on human skin. Areas of skin that were pretreated with GTP before UV exposure developed less erythema and were found, on microscopic examination, to have fewer sunburn cells. GTPs were also found to reduce the damaging effects of UV radiation on Langerhans cells, a subpopulation of epidermal cells known to play a key role in the development of cutaneous cell-mediated immune responses. EGCG and ECG, the two polyphenols that contain a galloyl group at the 3 position, were the two constituents that were most effective at limiting the adverse effects of UV radiation on the skin. This is important because it provides structural information that may pave the way for the synthesis of more effective chemopreventive molecules. Interestingly, GTPs were effective at reducing the erythema response both to UVB and to UVA radiation. Currently, sunscreens are the major pharmacologic agent used to protect against solar UV radiation exposure. Sunscreens were originally developed in the 1940s, but high-potency sunscreens have only been commercially available since the late 1970s.26 These topically applied compounds act as a physical barrier to either absorb or reflect incident UV radiation, thereby reducing the amount that reaches beyond the most superficial layers of the epidermis. Data from this and other studies indicate that GTP,
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EGCG, and ECG act in a manner that is quite different from that of sunscreens. Unlike sunscreens, these compounds do not appear to absorb significant amounts of light in the UV range. The implication of this finding is that GTPs, when combined with topical formulations that contain traditional sunscreens, may have additive or synergistic photoprotective effects when compared with either of these agents alone. GTPs may also have value in persons who are allergic to sunscreens or who for some other reason are unable to tolerate them. In this study, GTPs were shown to protect against the adverse effects of both UVB and UVA radiation. Although wavelengths within the UVB are more effective at causing sunburn and nonmelanoma skin cancer than UVA, there is considerable experimental evidence that wavelengths within the UVA also have deleterious effects on human health.27-30 The importance of devising strategies to protect against UVA radiation has only recently been recognized. Most sunscreens that are currently available are comparatively much more effective at limiting UVB penetration into the skin than UVA.10 Those persons who do wear sunscreens and stay outside for longer periods are likely to receive a greater total dose of UVA than persons who do not wear sunscreens. UVA can cause a sunburn reaction and has been implicated as an etiologic agent in photoaging and some forms of nonmelanoma skin cancer. Its role in melanoma is controversial. There is evidence in animal models and from epidemiologic studies suggesting that UVA may play a role in the development of this malignancy as well.31-33 The finding that GTPs effectively inhibit UVA-induced erythema means that their incorporation into topical formulations may thus provide photoprotection over a wider UV range. An obvious question that remains to be determined is the effect of GTPs on the prevention of nonmelanoma skin cancer. Experimental data from animal models strongly suggest that GTPs may in fact reduce the incidence of sunlight-related skin cancer.23 Although this study did not specifically address the issue of skin cancer in humans, the fact that they reduced lesions in DNA, which are necessary for the development of cancer suggests that this may be the case. In addition to its role in causing sunburn and skin cancer, UV radiation has a profound influence on the immune response.7 This is mediated at least in part by the damaging effects of UV radiation on epidermal Langerhans cells.21,24 Epidermal Langerhans cells are extremely sensitive to UV radiation.34 Pretreatment of skin with doses of UV radiation sufficient to diminish the number of identifiable Langerhans cells coincides with a reduction in immunization rates through the
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UV-irradiation skin site.35,36 The results presented herein indicate that GTPs abrogate the effects of UV radiation on Langerhans cells. In animal models and in cell culture systems, GTPs have been shown to be potent antioxidants.37 With respect to the skin, UV-induced oxidative stress has been implicated, among other things, in the production of prostaglandin E2 and in activation of the epidermal growth factor receptor.38,39 Both are involved in causing epidermal hyperproliferation and inflammation and both are known to be associated with UV injury. Other antioxidants such as genestein40 and a low-fat diet,16 which lowers lipid peroxidation in the skin, have also been shown to reduce UV-induced injury. In an era in which the incidence of the adverse effects of UV radiation is rising at an alarming rate, the introduction of GTPs and other natural chemopreventive agents may prove to be a new alternative for protection from exposure to UV radiation. REFERENCES 1. Soter NA. Acute effects of ultraviolet radiation on the skin. Semin Dermatol 1990;9:11-5. 2. Mukhtar H, Elmets CA. Photocarcinogenesis: mechanisms, models and human health implications. Photochem Photobiol 1996;63:355-447. 3. Longstreth J, de Gruijl FR, Kripke ML, Abseck S, Arnold F, Slaper HI, et al. Health risks. J Photochem Photobiol B 1998;46:20-39. 4. Koh HK, Kligler BE, Lew PA. Sunlight and cutaneous malignant melanoma: evidence for and against causation. Photochem Photobiol 1990;51:765-79. 5. West SK, Duncan DD, Munoz B, Rubin GS, Fried LP, BandeenRoche K, et al. Sunlight exposure and risk of lens opacities in a population-based study: the Salisbury Eye Evaluation project. JAMA 1998;280:714-8. 6. Gilchrest BA, Yaar M. Ageing and photoageing of the skin: observations at the cellular and molecular level. Br J Dermatol 1992;127(Suppl 41):25-30. 7. Krutmann JT, Elmets CA. Photoimmunology. Oxford: Blackwell Scientific; 1995. 8. Glass A, Hoover R. The emerging epidemic of melanoma and squamous cell skin cancer. JAMA 1989;262:2097-100. 9. Miller D,Weinstock M. Non-melanoma skin cancer in the United States: incidence. J Am Acad Dermatol 1994;30:774-8. 10. Elmets C, Mukhtar H. Ultraviolet radiation and skin cancer: progress in pathophysiologic mechanisms. Prog Dermatol 1996;30:1-16. 11. Swerdlow AJ, Weinstock MA. Do tanning lamps cause melanoma? An epidemiologic assessment. J Am Acad Dermatol 1998;38:89-98. 12. Chapman S, Marks R, King M.Trends in tans and skin protection in Australian fashion magazines, 1982 through 1991. Am J Public Health 1992;82:1677-80. 13. Greenwald P. Chemoprevention of cancer. Sci Am 1996;275:969. 14. Miller WH Jr. The emerging role of retinoids and retinoic acid metabolism blocking agents in the treatment of cancer. Cancer 1998;83:1471-82. 15. DiGiovanna JJ. Retinoid chemoprevention in the high-risk patient. J Am Acad Dermatol 1998;39(Suppl):S82-5.
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16. Black HS, Herd JA, Goldberg LH,Wolf JE Jr,Thornby JI, Rosen T, et al. Effect of a low-fat diet on the incidence of actinic keratosis. N Engl J Med 1994;330:1272-5. 17. Katiyar SK, Mukhtar H. Tea in chemoprevention of cancer: epidemiologic and experimental studies. Int J Oncol 1996;8:22138. 18. Yang CS, Wang ZY. Tea and cancer. J Natl Cancer Inst 1993;85: 1038-49. 19. Huang MT, Xie JG,Wang ZY, Ho CT, Lou YR,Wang CX, et al. Effects of tea, decaffeinated tea, and caffeine on UVB light-induced complete carcinogenesis in SKH-1 mice: demonstration of caffeine as a biologically important constituent of tea. Cancer Res 1997;57:2623-9. 20. Katiyar SK, Elmets CA, Agarwal R, Mukhtar H. Protection against ultraviolet-B radiation-induced local and systemic suppression of contact hypersensitivity and edema responses in C3H/HeN mice by green tea polyphenols. Photochem Photobiol 1995;62: 855-61. 21. Elmets CA, Vargas A, Oresajo C. Photoprotective effects of sunscreens in cosmetics on sunburn and Langerhans cell photodamage. Photodermatol Photoimmunol Photomed 1992;9:1320. 22. Chatterjee ML, Agarwal R, Mukhtar H. Ultraviolet B radiationinduced DNA lesions in mouse epidermis: an assessment using a novel 32p-postlabelling technique. Biochem Biophys Res Comm 1996;229:590-5. 23. Katiyar SK, Rupp CO, Korman NJ, Agarwal R, Mukhtar H. Inhibition of 12-O-tetradecanoylphorbol-13-acetate and other skin tumor-promoter-caused induction of epidermal interleukin-1 alpha mRNA and protein expression in SENCAR mice by green tea polyphenols. J Invest Dermatol 1995;105:394-8. 24. Toews GB, Bergstresser PR, Streilein JW, Sullivan S. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol 1980;124:445-53. 25. Elmets C. Cutaneous photocarcinogenesis. In: Mukhtar H, editor. Pharmacology of the skin. Boca Raton (FL): CRC Press; 1992. p. 389-416. 26. Anderson CY, Elmets CA. Sunscreens and photocarcinogenesis: an objective assessment. Photochem Photobiol 1996;63:43540. 27. Strickland PT. Photocarcinogenesis by near-ultraviolet (UVA) radiation in Sencar mice. J Invest Dermatol 1986;87:272-5. 28. Staberg B, Wulf HC, Klemp P, Poulsen T, Brodthagen H. The carcinogenic effect of UVA irradiation. J Invest Dermatol 1983;81: 517-9.
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29. Matsui M, DeLeo VA. Longwave ultraviolet radiation and promotion of skin cancer. Cancer Cells 1991;3:8-12. 30. Moan J, Dahlback A, Setlow RB. Epidemiological support for an hypothesis for melanoma induction indicating a role for UVA radiation. Photochem Photobiol 1999;70:243-7. 31. Swerdlow AJ, English JSC, MacKie RM, O’Doherty CJ, Hunter JA, Clark J, et al. Fluorescent lights, ultraviolet lamps, and risk of cutaneous melanoma. Br Med J 1988;297:647-50. 32. Walter SD, Marrett LD, From L, Hertzman C, Shannon HS, Roy P. The association of cutaneous malignant melanoma with the use of sunbeds and sunlamps. Am J Epidemiol 1990;131:23243. 33. Setlow RB, Grist E, Thompson K, Woodhead AD. Wavelengths effective in induction of malignant melanoma. Proc Natl Acad Sci U S A 1993;90:6666-70. 34. Rae V,Yoshikawa T, Bruins-Slot W, Streilein JW,Taylor JR. An ultraviolet B radiation protocol for complete depletion of human epidermal Langerhans cells. J Dermatol Surg Oncol 1989;15: 1199-202. 35. Yoshikawa T, Rae V, Bruins-Slot W, van der Berg J-W, Taylor JR, Streilein JW. Susceptibility to effects of UVB radiation on induction of contact hypersensitivity as a risk factor for skin cancer in man. J Invest Dermatol 1990;95:530-6. 36. Cooper KD, Oberhelman L, Hamilton TA, Baadsgaard O, Terhune M, LeVee G, et al. UV exposure reduces immunization rates and promotes tolerance to epicutaneous antigens in humans, relationship to dose, CDla-DR+ epidermal macrophage induction, and Langerhans cell depletion. Proc Natl Acad Sci U S A 1992;89:8497-501. 37. Khan SG, Katiyar SK, Agarwal R, Mukhtar H. Enhancement of antioxidant and phase II enzymes by oral feeding of green tea polyphenols in drinking water to SKH-1 hairless mice: possible role in cancer chemoprevention. Cancer Res 1992;52:4050-2. 38. Matsui MS, DeLeo VA. Induction of protein kinase C activity by ultraviolet radiation. Carcinogenesis 1990;11:229-34. 39. Miller CC, Hale P, Pentland AP. Ultraviolet B injury increases prostaglandin synthesis through a tyrosine kinase-dependent pathway: evidence for UVB-induced epidermal growth factor receptor activation. J Biol Chem 1994;269:3529-33. 40. Wei H. Photoprotective action of isoflavone genistein: models, mechanisms, and relevance to clinical dermatology. J Am Acad Dermatol 1998;39:271-2.