Green tea polyphenol ()-epigallocatechin-3-gallate treatment of human skin inhibits ultraviolet radiation-induced oxidative stress
Santosh K. Katiyar1,,
Farrukh Afaq,
Anaibelith Perez and
Hasan Mukhtar
Department of Dermatology, Volker Hall 501, 1530 3rd Ave S, The University of Alabama at Birmingham, Birmingham, AL 35294-0019, USA
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Abstract
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The use of naturally occurring botanicals with substantial antioxidant activity to afford protection to human skin against UV damage is receiving increasing attention. The green tea constituent ()-epigallocatechin-3-gallate (EGCG) is a potent antioxidant and has shown remarkable preventive effects against photocarcinogenesis and phototoxicity in mouse models. In this study we have investigated the effects of topical application of EGCG, the major polyphenol present in green tea, to human skin before UV irradiation on UV-induced markers of oxidative stress and antioxidant enzymes. Using immunohistochemistry and analytical enzyme assays, we found that application of EGCG (mg/cm2 skin) before a single UV exposure of 4x minimal erythema dose (MED) markedly decreases UV-induced production of hydrogen peroxide (6890%, P < 0.0250.005) and nitric oxide (30100%, P < 0.0250.005) in both epidermis and dermis in a time-dependent manner. EGCG pretreatment also inhibits UV-induced infiltration of inflammatory leukocytes, particularly CD11b+ cells (a surface marker of monocytes/macrophages and neutrophils), into the skin, which are considered to be the major producers of reactive oxygen species. EGCG treatment was also found to inhibit UV-induced epidermal lipid peroxidation at each time point studied (4184%, P < 0.05). A single UV exposure of 4x MED to human skin was found to increase catalase activity (109145%) and decrease glutathione peroxidase (GPx) activity (3654%) and total glutathione (GSH) level (1336%) at different time points studied. Pretreatment with EGCG was found to restore the UV-induced decrease in GSH level and afforded protection to the antioxidant enzyme GPx. Further studies are warranted to study the preventive effects of EGCG against multiple exposures to UV light of human skin.
Abbreviations: DAB, diaminobenzidine; DHR, dihydrorhodamine 123; EGCG, ()-epigallocatechin-3-gallate; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; GTP, green tea polyphenols; LPO, lipid peroxidation; MDA, malondialdehyde; MED, minimal erythema dose; PBS, phosphate-buffered saline; ROS, reactive oxygen species.
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Introduction
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Chronic exposure to solar UV radiation of mammalian skin induces a number of biological responses, including erythema, edema, sunburn cell formation, hyperplastic responses, photoaging and skin cancer development (14). UV exposure, particularly UVB (290320 nm), causes the generation of free radicals and related reactive oxygen species (ROS), which contribute to carcinogenesis by directly damaging cellular macromolecules, including DNA (reviewed in refs 1,5). ROS generated as a consequence of UV exposure produce oxidative stress in the skin when formation exceeds the antioxidant defense ability of the target cell. The skin possesses an elaborate antioxidant defense system to deal with UV-induced oxidative stress. However, excessive exposure to UV can overwhelm the cutaneous antioxidant capacity, leading to oxidative damage and ultimately skin cancer, immunosuppression and premature skin aging (14). Considering that UV induces oxidative stress-mediated adverse effects in the skin, regular intake of dietary antioxidants or treatment of the skin with creams and lotions containing antioxidant ingredients has been suggested as a useful preventive strategy against the mutagenic and carcinogenic effects of UV radiation. Consistent with this chemopreventive concept, we and others have demonstrated the preventive effects of polyphenols from green tea, which is a commonly consumed beverage in Oriental countries, including China, Japan and India, against chemical tumor promoters, and UV radiation-induced skin inflammation and tumorigenesis (612).
Green tea is a popular beverage consumed in some parts of the world and is a rich source of polyphenols, which are antioxidants in nature (6,7). In the last decade several investigators have concentrated their attention on the cancer chemopreventive potential of green tea polyphenols (GTP) in skin and other animal tumor bioassay systems (6,7). It was found that a polyphenolic constituent, ()-epigallocatechin-3-gallate (EGCG), is the major and most effective chemopreventive agent in green tea (6,7). We and others have demonstrated that oral administration of water extracts of green tea, GTP or EGCG inhibits carcinogen-induced tumors in various organs of animals, including the forestomach (13,14), lung (1316), duodenum (17), esophagus (18,19) and colon (20,21). Topical application of GTP to SENCAR mice was also found to afford protection against 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumorigenesis (22). In addition, topical application of GTP was also found to inhibit malignant conversion of chemically induced benign skin papillomas to squamous cell carcinomas in mice (23).
With relevance to the preventive effects of green tea against UV effects, it has been demonstrated that oral administration of a GTP fraction as the sole source of fluid to SKH-1 mice prevented UVB-induced skin tumorigenesis (8,9). Further, oral administration of tea constituents was found to inhibit the growth of established chemical carcinogen- as well as UV radiation-induced papillomas in mouse skin (10). In attempting to demonstrate the anticarcinogenic mechanism of action of GTP against UVB irradiation, we found that topical application of GTP to mouse skin protects against UVB-induced depletion of antioxidant enzymes and induction of epidermal cyclooxygenase activity (24) and lipid peroxidation (25). Recently, using an in vitro system, Wei et al. (26) have shown that an aqueous extract of green tea and black tea scavenges H2O2 and inhibits UV light-induced oxidative DNA damage. Zhao et al. (27) showed that topical application of GTP to mouse and reconstituted human skin affords protection against psoralen + UVA-induced photochemical damage to the skin.
The relevance of these animal data to the human situation has not been determined. In our attempts at translating the abundant information on the protective potential of GTP against UV-induced effects in murine models to the human population, we recently showed that EGCG affords protection against UV-induced erythema, myeloperoxidase activity and prostaglandin metabolite synthesis in human skin (28). In the present study we show a protective effect of topical application of EGCG (~1 mg/cm2 skin) to human skin before a single exposure to a 4x minimal erythema dose (MED) of UV irradiation on UV-induced markers of oxidative stress. These markers are H2O2 and nitric oxide production, lipid peroxidation and infiltration of inflammatory leukocytes (CD11b+ cells, a marker of monocytes/macrophages and neutrophils). In addition, we also show the effects of EGCG on UV-induced changes in the antioxidant defense enzymes glutathione peroxidase (GPx) and catalase and in reduced glutathione (GSH) levels in the skin.
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Materials and methods
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Chemicals and antibodies
The diaminobenzidine (DAB) reagent set and peroxidase-labeled streptavidin were purchased from Kirkgaard and Perry (Gaithersburg, MD). The monoclonal antibody to CD11b (anti-Mac-1, IgG1) was purchased from Immunotech (Westbrook, ME). The nitric oxide assay kit was purchased from Oxford Biomedical Research (Oxford, MI) and dispase was obtained from Collaborative Research (Bedford, MA). All other chemicals employed in this study were of analytical grade and purchased from Sigma Chemical Co. (St Louis, MO).
Human individuals and skin punch and keratome biopsies
Experiments on human skin were performed in accordance with approved institutional protocols. All individuals gave written informed consent to be included in this study/protocol, which was approved by the Institutional Review Board of Case Western Reserve University, Cleveland, and University Hospitals of Cleveland, Cleveland. Because UV radiation effects are of greater concern for Caucasian individuals, only Caucasians, six in number, both male and female, ranging from 25 to 56 years old, were recruited for this study. All participants were screened to ensure that they were free of health problems, including skin conditions such as atopic dermatitis and eczema. All UV irradiations were delivered through Westinghouse FS20 bulbs (Westinghouse, Pittsburgh, PA) that emit a spectrum with high irradiance in the UVB region and a peak at 313 nm. First, MED was determined in each recruited individual. To determine MED, for each volunteer five to eight skin sites (1x2 cm) in a row were exposed to UV radiation at gradually increasing exposure times with an increment of 30 s. The lowest UV dose that induced a just perceptible pinkness at the exposure site after 24 h was identified as the MED. In all experiments in this study solar UV-protected buttock skin sites were used because it is easier to obtain skin punch and keratome biopsies from the covered area of the body of human volunteers. Our experience indicates that volunteers are not receptive to providing skin samples from other parts of the body. After determining the MED in individual volunteers, in a follow-up visit the buttock skin area was exposed to the predetermined 4x MED dose of UV irradiation. This UV dose was selected based upon our previous observations and studies in human skin (28,29). Skin punch biopsies (4 mm diameter, 0.8 mm deep) for immunostaining purposes were taken and snap frozen in OCT liquid embedding medium under liquid nitrogen immediately after removal and stored at 80°C until use. Keratome biopsies (1.5x5.0 cm, 0.6 mm deep) were also taken from the individuals to prepare cytosolic or microsomal fractions for determination of antioxidant enzymes or lipid peroxidation (LPO) and to prepare epidermal and dermal single cell suspensions for the estimation of UV-induced intracellular production of H2O2. Skin biopsies were taken either from normal buttock skin (non-UV exposed) or at 6, 24 and 48 h after 4x MED UV irradiation with or without topical treatment with EGCG. According to the protocol approved by the Institutional Review Board for Human Investigation, to avoid inconvenience to human subjects we should not take more than six skin punch biopsies or four keratome biopsies from a single human subject. However, each data point is derived from at least three to four individuals treated at different time points. The dose of EGCG (~1 mg/cm2 skin in 50 µl acetone) was selected based upon our prior studies and experience in mouse skin as well as in human skin (28) and was topically applied using acetone as a vehicle 20 min before UV exposure. To maintain a similar treatment regimen, vehicle was also topically applied to control (non-UV exposed) and UV-alone exposed skin sites.
Immunohistochemistry
Immunostaining was performed on at least three to four different individuals of each group as described below.
Immunostaining of hydrogen peroxide
Immunohistochemical detection of H2O2 in normal as well as UV-irradiated skin was performed following the procedure as described earlier (30). Briefly, 6 µm thick frozen skin sections were incubated with 0.1 M TrisHCl buffer, pH 7.5, containing 1 mg/ml glucose and 1 mg/ml DAB for 56 h at 37°C. Sections were then washed in distilled water and counterstained with methyl green (2% for 60 min). The DABperoxidase reaction gave a brown reaction product and methyl green a blue nuclear counterstain.
Immunostaining of CD11b (
M ß2 integrin)-positive cells
CD11b staining was used as a surface marker to identify monocytes/macrophages and neutrophils, which are the major sources of H2O2 production after stimulation. Six micrometer thick frozen skin sections were fixed in cold acetone for 10 min and non-specific antibody binding was blocked using goat serum [10% in phosphate-buffered saline (PBS)]. Thereafter, sections were incubated with anti-human CD11b (anti-Mac-1) or its isotype control (IgG1). Bound anti-human CD11b was detected by incubation with biotinylated goat anti-mouse IgG1 followed by peroxidase-labeled streptavidin. Cells were incubated with DAB as substrate and counterstained with methyl green (2%, for 60 min), cleared and mounted. The DABperoxidase reaction gave a brown reaction product and methyl green a blue nuclear counterstain.
Microscopy and photography
Images from immunostained slides were obtained using a Zeiss Axiophot microscope (Thornwood, NY) and Kodak Ektachrome 160T film (Rochester, NJ). These were scanned using SprintScan software and formatted as tiff images in Adobe Photoshop and Microsoft Powerpoint in order to make composite figures.
Preparation of cytosol and microsomal fractions
The epidermal and dermal skin layers were separated using the enzyme dispase as described earlier (31). Briefly, after removal of subcutaneous tissues, the skin keratome biopsies were placed into a solution of 50 U/ml dispase overnight at 4°C. Thereafter, the epidermal layer was easily separated from the dermal layer using forceps. Both the epidermal and dermal layers were washed with PBS separately, weighed and homogenized with a Polytron homogenizer in PBS containing potassium chloride and centrifuged at 18 000 g for 15 min at 4°C to prepare cytosolic and microsomal fractions (10% w/v) as described previously (8).
Quantitative assay of intracellular H2O2 by fluorocytometry
Intracellular levels of H2O2 were determined using dihydrorhodamine 123 (DHR) as a specific fluorescent dye probe as described by Peus et al. (32) with some modifications. Epidermal and dermal single cell suspensions from the skin keratome biopsies obtained from different treatment groups were prepared as described previously (31). For estimation of intracellular H2O2, 1 000 000 epidermal or dermal cells from different treatment groups (UV-exposed and non-UV-exposed) were placed in each well of a 24-well tissue culture plate in triplicate. These cells were treated with DHR (5 µM) for 45 min. Reduced DHR is irreversibly oxidized and converted to the red fluorescent compound rhodamine 123 (32) by UVB-induced intracellular release of H2O2. Plates were read on a Cytofluor II fluorescence plate reader with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
Quantitative assay of nitric oxide
In aqueous solution nitric oxide (NO) rapidly degrades to nitrate and nitrite. In this procedure NO formation is determined spectrophotometrically by measuring its stable degradation product nitrite. For accurate assessment of the total nitric oxide generated we considered it essential to monitor both nitrate and nitrite. In this procedure nitrate is enzymatically converted into nitrite by the enzyme nitrate reductase, followed by quantitation of nitrite using Griess reagent. Thus nitric oxide was estimated in the cytosolic fraction of both epidermis and dermis in the form of total nitrite formed using a colorimetric nitric oxide assay kit (Oxford Biomedical Research) following the manufacturer's protocol. The detection limit of the nitric oxide assay kit is 1 pmol/µl (~1 µM) NO produced in aqueous solution.
Assay of LPO
The LPO assay was performed in epidermal microsomal fractions obtained from the different treatment groups. The generation of malondialdehyde (MDA) was employed as a marker of LPO and estimated by the method of Wright et al. (33) as described earlier, with some modifications (25). Briefly, epidermal microsomal protein (2.0 mg) was incubated for 1 h at 37°C in the presence of ferric ions (1 mM) and ADP (5 mM) in Ca2+-free 0.1 M phosphate buffer, pH 7.4, containing 0.1 mM MgCl2. The reaction was terminated by addition of 0.6 ml of 10% (w/v) trichloroacetic acid followed by 1.2 ml of 0.5% (w/v) 2-thiobarbituric acid. The mixture was heated for 20 min at 90°C in a water bath. After cooling, the MDA levels were measured in the clear supernatant by recording absorbance at 532 nm. The final concentration of MDA generated during the reaction was calculated using a molar extinction coefficient of 1.56x105/M/cm.
Assays of GPx and catalase activities and GSH levels
For enzymic assays skin was treated with or without EGCG (1 mg/cm2 skin) 1520 min before a 4x MED UV exposure. Skin keratome biopsies were obtained at the desired time points after UV exposure.
GPx assay
GPx activity (EC 1.11.1.9) was assayed according to the method of Mohandas et al. (34). The assay mixture consisted of 0.05 M phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide, 3 U glutathione reductase, 1 mM GSH, 0.2 mM NADPH, 0.25 mM H2O2 and 2030 µg epidermal or dermal cytosolic fractions as an enzyme source in a final volume of 1.5 ml. Oxidation of NADPH was recorded at 340 nm at 15 s intervals for 2 min and the enzyme activity was expressed as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22x103/M/cm.
Catalase assay
Catalase (EC 1.11.1.6) activity was assayed by the method of Claiborne (35). The assay mixture consisted of 0.05 M phosphate buffer (pH 7.0), 0.019 M H2O2 and 2030 µg epidermal or dermal cytosolic fraction as an enzyme source in a final volume of 1.5 ml. Change in absorbance was recorded at 280 nm at 15 s intervals for 2 min. Catalase activity was calculated in terms of nmol H2O2 consumed/min/mg protein using a molar extinction coefficient of 0.0041/M/cm for H2O2.
GSH levels
GSH level in epidermal cytosolic fractions was estimated by the traditional method of Sedlack and Lindsay (36). The assay mixture consisted of 2030 µg cytosolic fraction, 0.9 ml of EDTA (1 g/l) and 1.5 ml of precipitating agent (1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 ml of distilled water). After filtering the solution, 1.0 ml of the filtrate was mixed with 2 ml of 0.1 M disodium hydrogen phosphate buffer (pH 8.0) and 0.5 ml of 5,5'-dithiobis[2-nitrobenzoic acid] reagent. The color intensity was immediately read at 412 nm in a spectrophotometer. The results are expressed as nmol GSH/mg protein.
Statistical analysis
All experiments were performed on at least three or four individuals with each assay conducted in duplicate or triplicate. The results are expressed as means ± SD. Statistical analysis of all data between the UV exposure alone and EGCG-treated + UV exposure groups was performed by Student's t-test. A P value <0.05 was considered statistically significant.
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Results
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EGCG inhibits UV-induced H2O2 production
We found that skin exposure to UV induces significant intracellular release of H2O2 in the epidermis when measured at 6, 24 and 48 h post-irradiation (Figure 1A
). At the 6 and 24 h time points the increases were 230 and 150%, respectively, and little further increase in H2O2 generation was observed at 48 h after UV exposure (Figure 1A
). As shown in Figure 1B
, a significant increase in release of H2O2 was found at 48 h after UV exposure in dermis. The significant increase in H2O2 in dermis (275% of control) at 48 h after UV exposure may be due to UV-induced infiltration of inflammatory leukocytes, which are a major source of H2O2 production. As shown by immunostaining (Figure 2
), 48 h after UV exposure numerous infiltrating cells were found to be H2O2+, particularly in the dermis. Some of these H2O2+ infiltrating cells were also found in the epidermis. Quantitative analysis of H2O2+ cells in the dermis was performed on a Zeiss Axiophot microscope using an ocular micrometer grid corresponding to an area of 0.0625 mm2. H2O2+ cells were counted from multiple fields of different slides from three individuals. Normal skin sections did not show H2O2+ cells, whereas 48 h UV exposure alone and EGCG-treated + UV-exposed skin sites showed 39 ± 11 and 14 ± 8% H2O2+ cells in the dermis, respectively. These data clearly indicate that pretreatment with EGCG of human skin inhibits UV-induced H2O2 production. Because UV-induced H2O2-producing infiltrating cells reached up to the epidermis, the amount of H2O2 is also increased therein 48 h post-UV irradiation. This confirms the notion that infiltrating cells are the major source of H2O2 production. This suggests that UV-induced infiltrating leukocytes may result in secondary injury to UV-exposed epidermis. In this scenario primary skin injury/damage is caused by direct UV exposure of the epidermis and secondary injury/damage is caused by infiltrating leukocytes. This injury may result in further damage to keratinocytes and a breakdown in the structure of UV-exposed epidermis. EGCG treatment before UV exposure of the skin significantly inhibited UV-induced infiltration of leukocytes and H2O2-producing cells (Figure 2
), which resulted in a significant inhibition of UV-induced H2O2 production in both the epidermis (6890%, P < 0.0250.005) and dermis (70% at 48 h, P < 0.005) at the different time points studied (Figure 1A and B
). Thus, inhibition of UV-induced production of H2O2 by EGCG suggests an antioxidant potential of EGCG.

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Fig. 1. Inhibition of time-dependent UV-induced H2O2 production by EGCG in the epidermis (A) and dermis (B) of human skin. Epidermal and dermal single cell suspensions were prepared from the different treatment groups. One million cells from each treatment group were loaded into each well of a 24-well culture plate in triplicate. Details of the method for determination of UV-induced intracellular production of H2O2 using DHR as a fluorescent dye probe are described in Materials and methods. Data are presented as means ± SD of values from four different individuals where each sample was assayed in duplicate or triplicate. Significant inhibition of H2O2 production by EGCG + UV versus UV exposure alone was observed in both epidermis and dermis, **P < 0.005, ¶P < 0.025 and *P < 0.01.
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Fig. 2. Inhibition of UV-induced H2O2 production by EGCG treatment in human skin cells. Immunohistochemical detection of H2O2-producing cells was performed using the DABperoxidase reaction as detailed in Materials and methods. Normal skin (non-UV exposed) did not show H2O2-producing cells (A). UV-exposed skin showed the presence of H2O2-producing cells at 24 (B) and 48 h (C) after UV exposure. H2O2+ cells are detected in both epidermis and dermis 48 h after UV exposure (C). EGCG treatment prior to UV exposure inhibits UV-induced production of H2O2 (D). Representative data are shown from similar experiments performed in three different individuals with identical results. Scale bars: (A), (C) and (D), 100 µm; (B), 50 µm.
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Further, it is important to mention that EGCG treatment most likely inhibits UV-induced oxidative stress in two ways, directly and indirectly. Direct effects include inhibition of UV-induced oxidative stress before induction of infiltration of leukocytes and is measured at 6 h after UV exposure. The indirect effect is due to UV-induced infiltration of leukocytes, which are also the major source of ROS, and is measured at 24 and 48 h after UV exposure.
EGCG inhibits UV-induced infiltration of CD11b+ (
M ß2 integrin) cells (monocytes/macrophages and neutrophils)
Forty-eight hours after UV exposure of skin, infiltrating leukocytes were found in higher numbers, particularly in the dermis (Figure 3B
), compared with control, non-UV-exposed skin (Figure 3A
). To identify UV-induced infiltrating leukocyte cell types, whether primarily monocytes/macrophages and neutrophils, the surface marker CD11b was employed. These CD11b+ cells are phagocytic in nature. Although these cells are responsible for damage to and removal of infectious microorganisms through generation of ROS, excess production of ROS by these cells under the influence of UV exposure is considered to produce oxidative stress. In normal human skin monocytes/macrophages and neutrophils are clearly stained with the anti-CD11b antibody (Figure 3A
). Forty-eight hours after UV exposure of skin, CD11b+ cells were found in markedly higher numbers (Figure 3B
) compared to normal, non-UV-exposed skin (Figure 3A
). Topical application of EGCG before UV exposure significantly reduced the numbers of UV-induced infiltrating CD11b+ cells in the skin (Figure 3C
), whereas treatment of human skin with EGCG alone did not seem to alter the constitutive pattern of the cells when compared with normal human skin and also did not induce infiltration (data not shown). Quantitative analysis of CD11b+ cells in different treatment groups was performed by selecting multiple fields of different slides from different human individuals. The CD11b+ cells on different slides or in different treatment groups were counted at random using an ocular micrometer grid under a Zeiss Axiophot microscope with 200x magnification corresponding to an area of 0.0625 mm2. CD11b+ cells in epidermis accounted for 0 ± 0, 7 ± 3 and 1 ± 1 cells, respectively, in interfollicular regions of control, UV-exposed and EGCG-treated + UV-exposed skin. Thus, in UV-irradiated skin infiltrating CD11b+ cells are higher in number, in both epidermis and dermis (Figure 3B
), and are responsible for the higher production of ROS, as is evident from immunostaining (Figure 2B,C
) as well as from quantitative estimation using DHR (Figure 1
), in which UV exposure produced significantly higher amounts of H2O2. Higher production of H2O2 may generate higher levels of singlet oxygen and hydroxyl radicals and thus may produce oxidative stress. Inhibition of UV-induced oxidative stress by EGCG treatment may therefore ameliorate UV-induced skin disorders.

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Fig. 3. Inhibition of UV-induced infiltration of CD11b+ cells by EGCG treatment in human skin. Immunostaining of CD11b+ cells was performed in frozen skin sections using immunoperoxidase staining as detailed in Materials and methods. A brown color indicates the presence of CD11b+ cells. CD11b+ cells are observed in the dermis region of normal skin (A). Skin exposure to UV induces infiltration of leukocytes into epidermis as well as into dermis and also higher numbers of CD11b+ cells (B) as compared with normal skin (A). EGCG treatment before UV exposure inhibits UV-induced infiltration of CD11b+ cells into both epidermis and dermis (C) as compared with UV exposure alone (B). Representative data are shown from similar experiments performed in four different individuals with identical results. Scale bar: 50 µm.
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EGCG inhibits UV-induced NO production
Although all three forms of nitric oxide synthase (nNOS, iNOS and eNOS) catalyze the production of NO by the same biochemical pathway, they vary in their tissue expression and activational requirements. Activated macrophages are an important source of iNOS expression and the production of NO is central to the function of macrophages in infection and inflammation. We found that UV exposure of skin induces the production of NO, particularly in the cytosolic fraction of the epidermis (Figure 4A
). The generation of NO at sites exposed to UV at 6, 24 and 48 h was 109 ± 12, 208 ± 14 and 275 ± 21 µM, respectively, as compared with 16 ± 4 in the control (non-UV-exposed) sites (Figure 4A
). Compared with control sites, a greater increase in NO (485%) was observed in the dermis at 48 h after UV exposure (Figure 4B
). These data indicate that a significant increase at 48 h after UV exposure may be due to an additional flux of monocytes/macrophages into the skin. Treatment with EGCG before UV exposure was found to significantly inhibit UV-induced production of NO at 6, 24 and 48 h by 53 (P < 0.01), 43 (P < 0.005) and 30% (P < 0.025), respectively, in the epidermis. EGCG treatment was also found to inhibit UV-induced NO production in the dermis, as shown in Figure 4B
.

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Fig. 4. Inhibition of time-dependent UV-induced nitric oxide production by EGCG in the epidermis (A) and dermis (B) of human skin. Nitric oxide production was measured in the form of its stable degradation product, nitrite, in the cytosolic fraction of epidermis and dermis separately. Details of the method employed are provided in Materials and methods. Data are presented as means ± SD of values from four different individuals where each sample was assayed in duplicate or triplicate. Significant inhibition of nitric oxide production by EGCG + UV treatment versus UV alone was observed in both epidermis and dermis, **P < 0.01, *P < 0.025 and ¶P < 0.005.
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EGCG inhibits UV-induced epidermal LPO
ROS are highly reactive and can oxidize nucleic acids, proteins and lipid-rich cellular membranes and may lead to genetic alterations (35). LPO is thus used as a marker of oxidative damage. In our present study we found that UV exposure of skin increases the level of epidermal LPO at all time points studied when measured in the form of MDA production (Figure 5
). EGCG treatment prior to UV exposure was found to result in decreased levels of epidermal MDA formation and thus inhibited UV-induced epidermal LPO by 41, 67 and 84% at 6, 24 and 48 h, respectively, after UV exposure, as shown in Figure 5
.

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Fig. 5. Inhibition of time-dependent UV-induced epidermal LPO by EGCG in human skin. LPO was measured in the microsomal fraction of the epidermis. Generation of MDA was used as a marker of LPO. Details of the methods are provided in Materials and methods. Data are presented as means ± SD of values from four different individuals where each sample was assayed in triplicate. Significant inhibition of LPO by EGCG + UV treatment versus UV-alone was observed in epidermis, *P < 0.05.
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EGCG modulates the kinetics of UV-induced alterations in antioxidant enzymes
GPx is responsible for catalyzing the conversion of H2O2 into water and oxygen. During this conversion GSH is converted to its oxidized form (GSSG), with a concomitant decrease in GSH levels. GPx is also depleted. In this situation it is believed that GSH spontaneously oxidizes to GSSG utilizing H2O2 molecules. UV exposure of human skin was found to result in a lowering of GSH levels examined at 648 h after UV exposure (Table I
). Catalase, which is another antioxidant enzyme also responsible for catalyzing the conversion of UV-induced H2O2 to water and oxygen, is slightly elevated at 24 and 48 h after a single UV exposure. The elevated level of catalase after a single UV exposure also indicates conversion of the enhanced H2O2 to water and oxygen. In the case of antioxidant enzymes, EGCG treatment before UV exposure was found to reverse the UV-induced activation or depletion of these enzymes. These enzymic analyses were also performed in dermal cytosolic fractions of different treatment groups but did not provide a consistent pattern for any enzyme.
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Table I. Time-dependent effect of UV exposure on GPx and catalase activities and GSH level in epidermis of human skin and effect of pretreatment with EGCG on UV-induced effects on these parametersa
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Discussion
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Our data demonstrate that a single exposure of human skin to UV (4x MED) radiation is capable of generating ROS when measured using markers such as H2O2 and NO production in both epidermis and dermis. Changes in the levels of these parameters in the dermis also demonstrate the depth of penetration of UV radiation, as well as its damaging potential in deeper skin cells. At all time points studied UV exposure of the skin enhanced the levels of H2O2 and NO production (Figures 1 and 4
). Topical treatment with EGCG before UV exposure was found to inhibit UV-induced H2O2 and NO production in both the epidermis and dermis, thus clearly demonstrating the protective potential of this antioxidant from green tea in human skin. EGCG treatment was also found to block UV-induced infiltration of leukocytes (Figure 3
), which were shown to be the major source of ROS (Figure 2
). Earlier, we also showed that topical treatment with EGCG before UV exposure of both human and mouse skin blocked myeloperoxidase activity (28,31) and infiltration of leukocytes and CD11b+ cells into mouse skin (31). UV-induced production of H2O2 and NO is injurious and cytotoxic to target cells. Further, NO contains an unpaired electron and is paramagnetic, and thus may rapidly react with superoxide anions to form peroxynitrite anions in high yield. Peroxynitrite decomposition generates a strong oxidant with a reactivity similar to hydroxyl radicals. Inhibition of both UV-induced H2O2 and NO formation by EGCG is thus an additional antioxidant potential of this chemopreventive candidate.
Although under certain circumstances ROS help the host system to destroy invading microorganisms (37), they also damage host tissues and create suitable conditions for various disease states (37,38). The accumulation of UV-induced infiltrating cells is a characteristic feature of skin inflammation and further generation of ROS. In the present communication we have shown in human skin, as before in mouse skin (31), that EGCG has the ability to block UV-induced infiltration of specific cell types, such as CD11b+ cells, and is thus able to inhibit the further production of H2O2 and NO by these cells. It is important to mention that EGCG shows a peak of absorption near 270 nm in the UV range, which indicates that EGCG possibly does not absorb wavelengths within the UVB or UVA range.
In our efforts to translate the chemopreventive effect of EGCG from murine skin to human skin we have found that treatment of human skin with EGCG before UV exposure significantly inhibited UV-induced epidermal LPO at every time point studied, i.e. 6, 24 and 48 h after UV exposure (Figure 5
). LPO in biological membranes is a free radical-mediated event and is regulated by the availability of substrates in the form of polyunsaturated fatty acids, prooxidants which promote peroxidation and antioxidant defences such as
-tocopherol, GSH, ß-carotene and superoxide dismutase (3840). LPO is highly detrimental to cell membrane structure and function. Elevated levels of LPO have been linked to injurious effects such as loss of fluidity, inactivation of membrane enzymes and receptors, increased permeability to ions and, eventually, rupture of the cell membrane leading to release of cell organelles (38,41,42). Peroxidation products can also result in damage to crucial biomolecules, including DNA (43,44). Thus inhibition of UV-induced elevated LPO levels by EGCG in human skin should reduce the risk factors related to the UV-induced, ROS-mediated tumor promoting effects of UV radiation in cutaneous inflammatory responses and malignancies. In previous studies we have shown that topical application of EGCG to human skin before UV irradiation inhibits UV-induced epidermal prostaglandin synthesis through inhibition of cyclooxygenase activity (28). It has been shown that enhanced cycloxygenase and lipoxygenase activity can trigger LPO in biological systems (43). Indirectly, inhibition of UV induction of cyclooxygenase may reduce oxidative stress through inhibition of LPO.
The cellular concentration of ROS is determined by their rates of generation and detoxification. Antioxidant enzymes function cooperatively and any change in one of them may affect the equilibrium state, leading to excessive production of ROS, which may cause cellular damage and other biochemical alterations, such as inflammation, lipid peroxidation, DNA damage and enzyme activation or inactivation (45,48). It is interesting to find that a single UV exposure of human skin to 4x MED enhances catalase activity, which demonstrates that skin cellular targets activated their defense system to protect the cells from UV-induced adverse effects.
In summary, we have demonstrated a potent inhibitory effect of EGCG against UV-induced ROS production and antioxidant enzyme expression in human skin. Other studies have verified that EGCG possesses several-fold higher antioxidant activity than vitamin C and
-tocopherol (49). These effects of EGCG could be, at least in part, the mechanisms of action by which EGCG affords protection against UV-induced carcinogenesis. Moreover, based on the extensive laboratory evidence supporting the antioxidant and anticarcinogenic properties of green tea, many skin care products supplemented with polyphenols from green tea are sold to consumers (reviewed in ref. 50). It is unlikely that these skin care products have been tested in controlled clinical trials and the concentration of GTP within these preparations is not uniform. Therefore, on the basis of available data concerning the antioxidant and anticarcinogenic potential of green tea, appropriate clinical trials should be designed to translate animal data to human subjects.
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Notes
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1 To whom correspondence should be addressed Email: sxk32{at}po.cwru.edu 
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Acknowledgments
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Financial support for this work by the Cancer Research Foundation of America and Ohio Cancer Research Associates is gratefully acknowledged (S.K.K.). Anaibelith Perez was supported by the Medical Scientists Training Program (MSTP) of Case Western Reserve University (Cleveland, OH). This work was also supported by USPHS grant RO1 CA78809 and P-30 grant AR39750.
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References
|
---|
-
Mukhtar,H. and Elmets,C.A. (1996) Photocarcinogenesis: mechanisms, models and human health implications. Photochem. Photobiol., 63, 355447.[ISI]
-
Naylor,M.F. (1997) Erythema, skin cancer risk and sunscreens. Arch. Dermatol., 133, 373375.[Abstract]
-
Goihman-Yahr,M. (1996) Skin aging and photoaging: an outlook. Clin. Dermatol., 14, 153160.[ISI][Medline]
-
Young,A.R. (1990) Cumulative effects of ultraviolet radiation on the skin: cancer and photoaging. Semin. Dermatol., 9, 2531.[ISI][Medline]
-
Griffiths,H.R., Mistry,P., Herbert,K.E. and Lunec,J. (1998) Molecular and cellular effects of ultraviolet light-induced genotoxicity. Crit. Rev. Clin. Lab. Sci., 35, 189237.[ISI][Medline]
-
Katiyar,S.K. and Mukhtar,H. (1996) Tea in chemoprevention of cancer: epidemiological and experimental studies (review). Int. J. Oncol., 8, 221238.[ISI]
-
Yang,C.S. and Wang,Z.Y. (1993) Tea and cancer. J. Natl Cancer Inst., 85, 10381049.[Abstract]
-
Wang,Z.Y., Agarwal,R., Bickers,D.R. and Mukhtar,H. (1991) Protection against ultraviolet B radiation-induced photocarcinogenesis in hairless mice by green tea polyphenols. Carcinogenesis, 12, 15271530.[Abstract]
-
Wang,Z.Y., Huang,M.-T., Ferraro,T., Wong,C.-Q., Lou,Y.-R., Reuhl,K., Iatropoulos,M., Yang,C.S. and Conney,A.H. (1992) Inhibitory effect of green tea in the drinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol-13-acetate in the skin of SKH-1 mice. Cancer Res., 52, 11621170.[Abstract]
-
Wang,Z.Y., Huang,M.-T., Ho,C.-T., Chang,R., Ma,W., Ferraro,T., Reuhl,K.R., Yang,C.S. and Conney,A.H. (1992) Inhibitory effect of green tea on the growth of established skin papillomas in mice. Cancer Res., 52, 66576665.[Abstract]
-
Huang,M.T., Ho,C.T., Wang,Z.Y., Ferraro,T., Finnegan-Olive,T., Lou,Y.R., Mitchell,J.M., Laskin,J.D., Newmark,H., Yang,C.S. and Conney,A.H. (1992) Inhibitory effect of topical application of a green tea polyphenol fraction on tumor initiation and promotion in mouse skin. Carcinogenesis, 13, 947954.[Abstract]
-
Gensler,H.L., Timmermann,B.N., Valcic,S., Wachter,G.A., Dorr,R., Dvorakova,K. and Alberts,D.S. (1996) Prevention of photocarcinogenesis by topical administration of pure epigallocatechin gallate isolated from green tea. Nutr. Cancer, 26, 325335.[ISI][Medline]
-
Wang,Z.Y., Agarwal,R., Khan,W.A. and Mukhtar,H. (1992) Protection against benzo[a]pyrene and N-nitrosodiethylamine-induced lung and forestomach tumorigenesis in A/J mice by water extracts of green tea and licorice. Carcinogenesis, 13, 14911494.[ISI][Medline]
-
Katiyar,S.K., Agarwal,R., Zaim,M.T. and Mukhtar,H. (1993) Protection against N-nitrosodiethylamine and benzo[a]pyrene-induced forestomach and lung tumorigenesis in A/J mice by green tea. Carcinogenesis, 14, 849855.[Abstract]
-
Katiyar,S.K., Agarwal,R. and Mukhtar,H. (1993) Protective effects of green tea polyphenols administered by oral intubation against chemical carcinogen-induced forestomach and pulmonary neoplasia in A/J mice. Cancer Lett., 73, 167172.[ISI][Medline]
-
Xu,Y., Ho,C.-T., Amin,S.G., Han,C. and Chung,F.-L. (1992) Inhibition of tobacco-specific nitrosamine-induced lung tumorigenesis in A/J mice by green tea and its major polyphenol as antioxidants. Cancer Res., 52, 38753879.[Abstract]
-
Fujita,Y., Yamane,T., Tanaka,M., Kuwata,K., Okuzumi,J., Takahashi,T., Fujiki,H. and Okuda,T. (1989) Inhibitory effect of ()-epigallocatechin gallate on carcinogenesis with N-ethyl-N'-nitro-N-nitrosoguanidine in mouse duodenum. Jpn. J. Cancer Res., 80, 503505.[ISI][Medline]
-
Han,C. and Xu,Y. (1990) The effect of Chinese tea on the occurrence of esophageal tumors induced by N-nitrosomethylbenzylamine in rats. Biomed. Environ. Sci., 3, 3542.[Medline]
-
Xu,Y. and Han,C. (1990) The effect of Chinese tea on the occurrence of esophageal tumors induced by N-nitrosomethylbenzylamine formed in vivo. Biomed. Environ. Sci., 3, 406412.[Medline]
-
Yamane,T., Hagiwara,N., Tateishi,M., Akachi,S., Kim,M., Okuzumi,J., Kitao,Y., Inagake,M., Kuwata,K. and Takahashi,T. (1991) Inhibition of azoxymethane-induced colon carcinogenesis in rats by green tea polyphenol fraction. Jpn. J. Cancer Res., 82, 13361339.[ISI][Medline]
-
Narisawa,T. and Fukaura,Y. (1993) A very low dose of green tea polyphenols in drinking water prevents N-methyl-N-nitrosourea-induced colon carcinogensis in F344 rats. Jpn. J. Cancer Res., 84, 10071009.[ISI][Medline]
-
Katiyar,S.K., Agarwal,R., Wood,G.S. and Mukhtar,H. (1992) Inhibition of 12-O-tetradecanoylphorbol-13-acetate-caused tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated SENCAR mouse skin by a polyphenolic fraction isolated from green tea. Cancer Res., 52, 68906897.[Abstract]
-
Katiyar,S.K., Agarwal,R. and Mukhtar,H. (1993) Protection against malignant conversion of chemically induced benign skin papillomas to squamous cell carcinomas in SENCAR mice by a polyphenolic fraction isolated from green tea. Cancer Res., 53, 54095412.[Abstract]
-
Agarwal,R., Katiyar,S.K., Khan,S.G. and Mukhtar,H. (1993) Protection against ultraviolet B radiation-induced effects in the skin of SKH-1 hairless mice by a polyphenolic fraction isolated from green tea. Photochem. Photobiol., 58, 695700.[ISI][Medline]
-
Katiyar,S.K., Agarwal,R. and Mukhtar,H. (1994) Inhibition of spontaneous and photo-enhanced lipid peroxidation in mouse epidermal microsomes by epicatechin derivatives from green tea. Cancer Lett., 79, 6166.[ISI][Medline]
-
Wei,H., Zhang,X., Zhao,J.-F., Wang,Z.-Y., Bickers,D.R. and Lebwohl,M. (1999) Scavenging of hydrogen peroxide and inhibition of ultraviolet light-induced oxidative DNA damage by aqueous extracts from green and black teas. Free Radic. Biol. Med., 26, 14271435.[ISI][Medline]
-
Zhao,J.F., Zhang,Y.J., Jin,X.H., Athar,M., Santella,R.M., Bickers,D.R. and Wang,Z.Y. (1999) Green tea protects against psoralen plus ultraviolet A-induced photochemical damage to skin. J. Invest. Dermatol., 113, 10701075.[Abstract/Free Full Text]
-
Katiyar,S.K., Matsui,M.S., Elmets,C.A. and Mukhtar,H. (1999) Polyphenolic antioxidant ()-epigallocatechin-3-gallate from green tea reduces UVB-induced inflammatory responses and infiltration of leukocytes in human skin. Photochem. Photobiol., 69, 148153.[ISI][Medline]
-
Katiyar,S.K., Matsui,M.S. and Mukhtar,H. (2000) Ultraviolet-B exposure of human skin induces cytochromes P450 1A1 and 1B1. J. Invest. Dermatol., 114, 328333.[Abstract/Free Full Text]
-
Dannenberg,A.M.Jr, Schofield,B.H., Rao,J.B., Dinh,T.T., Lee,K., Boulay,M., Abe,Y., Tsuruta,J. and Steinbeck,M.J. (1994) Histochemical demonstration of hydrogen peroxide production by leukocytes in fixed-frozen tissue sections of inflammatory lesions. J. Leukoc. Biol., 56, 436443.[Abstract]
-
Katiyar,S.K., Challa,A., McCormick,T.S., Cooper,K.D. and Mukhtar,H. (1999) Prevention of UVB-induced immunosuppression in mice by the green tea polyphenol ()-epigallocatechin-3-gallate may be associated with alterations in IL-10 and IL-12 production. Carcinogenesis, 20, 21172124.[Abstract/Free Full Text]
-
Peus,D., Vasa,R.A., Meves,A., Pott,M., Beyerle,A., Squillace,K. and Pittelkow,M.R. (1998) H2O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes. J. Invest. Dermatol., 110, 966971.[Abstract]
-
Wright,J.R., Colby,H.D. and Miles,P.R. (1981) Cytosolic factors which affect microsomal lipid peroxidation in lung and liver. Arch. Biochem. Biophys., 206, 296304.[ISI][Medline]
-
Mohandas,J., Marshall,J.J., Duggins,G.G., Horvath,J.S. and Tiller,D.D. (1984) Differential distribution of glutathione and glutathione related enzymes in rabbit kidney: possible implications in analgesic neuropathy. Cancer Res., 44, 50865091.[Abstract]
-
Claiborne,A. (1985) Catalase activity. In Greenwald,R.A. (ed.), Handbook of Methods of Oxygen Radical Research. CRC Press, Boca Raton, FL, pp. 283284.
-
Sedlack,V. and Lindsay,R.H. (1968) Estimation of total protein-bound and nonprotein sulphhydryl groups in tissue with Ellman's reagent. Anal. Biochem., 25, 192205.[ISI][Medline]
-
Kehrer,J.P. (1993) Free radicals as mediators of tissue injury and disease. Crit. Rev. Toxicol., 23, 2148.[ISI][Medline]
-
Klebanoff,S.J. (1988) Phagocytic cells: products of oxygen metabolism. In Gallin,J.I., Goldstein,I.M. and Snyderman,R. (eds), Inflammation: Basic Principles and Clinical Correlates. Raven Press, New York, NY, pp. 391444.
-
Halliwell,B., Gutteridge,J.M.C. and Cross,C.E. (1992) Free radicals, antioxidants and human disease: where are we now? J. Lab. Clin. Med., 119, 598620.[ISI][Medline]
-
Bus,J.S. and Gibson,J.E. (1979) Lipid peroxidation and its role in toxicology. Rev. Biochem. Toxicol., 1, 125149.
-
Devasagayam,T.P.A. (1986) Low level of lipid peroxidation in newborn ratspossible factors for resistance in hepatic microsomes. FEBS Lett., 199, 203207.[ISI][Medline]
-
Sies,H. (1986) Biochemistry of oxidative stress. Angew. Chem. Int. Ed. Engl., 25, 10581071.[ISI]
-
Girotti,A.W. (1990) Photodynamic lipid peroxidation in biological systems. Photochem. Photobiol., 51, 497509.[ISI][Medline]
-
Halliwell,B. and Gutteridge,J.M.C. (1990) Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol., 186, 185.[Medline]
-
Girotti,A.W. (1985) Mechanisms of lipid peroxidation. Free Radic. Biol. Med., 1, 8795.
-
Park,J.W. and Floyd,R.A. (1992) Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA. Free Radic. Biol. Med., 12, 245250.[ISI][Medline]
-
Cerutti,P.A. and Trump,B.F. (1991) Inflammation and oxidative stress in carcinogenesis. Cancer Cell, 3, 17.[ISI]
-
Sun,Y. (1990) Free radical antioxidant enzymes and carcinogenesis. Free Radic. Biol. Med., 8, 583599.[ISI][Medline]
-
Rice-Evans,C. (1999) Implications of the mechanism of action of tea polyphenols as antioxidants in vitro for chemoprevention in humans. Proc. Soc. Exp. Biol. Med., 220, 262266.[Medline]
-
Katiyar,S.K., Ahmad,N. and Mukhtar,H. (2000) Green tea and skin. Arch. Dermatol., 136, 989994.[Abstract/Free Full Text]
Received July 28, 2000;
revised October 11, 2000;
accepted October 16, 2000.