Materials and methods

Mice and diets

Twenty-five female SKH-1 mice, 6 to 8 wk of age, were purchased from Charles River Laboratories (Wilmington, Massachusetts). Mice were housed in our animal housing facility under controlled temperature and humidity, and alternating 12 h light and dark cycles for a period of 2 wk before use. Mice received drinking water ad libitum. They were housed into groups of four animals per cage and put into three different groups according to the diet administered. Mice of group I (n=9) were fed with the standard diet of Purina Rodent Chow 5001; mice of group II (n=8) were fed with Purina Rodent Chow 5001 enriched with 0.04% lutein and zeaxanthin and mice of group III (n=8) received Purina Rodent Chow 5001 enriched with 0.4% lutein and zeaxanthin. Crystalline FloraGLO brand lutein was provided in a stable form by Kemin Foods, L.C. (DesMoines, Iowa). This material contains lutein and zeaxanthin in a ratio of approximately 20:1. FloraGLO brand lutein is a purified source of lutein and zeaxanthin, which is manufactured and sold commercially according to a patented process. (US patent no. 5382 714) and is the only pure product available for oral supplementation, and was used in our studies without further separation. The diets were prepared by Research Diets Inc. (New Brunswick, New Jersey) and packed under vacuum in evacuated and sealed plastic bags, each containing 100 g of diet in pellet form. The dietary bags enriched with 0.4% and 0.04% lutein and zeaxanthin were refrigerated at 4°C to preserve the carotenoids. These concentrations were recommended by nutritional experts (Professor Curran-Celentano, personal communication). Mice were fed on alternate days for a total of 14 d prior to UVB exposure. The study aimed to evaluate the photo protective effects of dietary lutein supplementation was approved by our Institutional Animal Care and Ethic Committee and carried out in observance of the NIH Guidelines for the care and use of laboratory Animals.

UVB exposure

Mice were irradiated using a Spot Light Source Radiation (Model L2859-07, Hamamatsu Photonics K. K., Japan), equipped with a cut-off filter at 295 nm. UV radiation was delivered through a bundle of quartz optical fibers with a spot size of 1 cm in diameter. The emitted UVB radiation was measured with a spectroradiometer (model 742, Optronic Laboratories Inc., Orlando, Florida). The spectral output of this lamp was maximal at 310 nm with the majority of the UVB energy emitted below 320 nm. The filtered UV radiation was calibrated and measured with the aid of an IL1700 research radiometer equipped with an SED 240 UVB detector probe (International Light, Newburyport, Massachusetts).

Two weeks before UVB exposure, all mice were tattooed on lateral skin of the dorsum surface for carefully locating the irradiation exposure sites and for obtaining skin biopsies of the exposed skin sites. The mice were then exposed at duplicate sites to single doses of 40, 80, and 160 mJ per cm² of UVB radiation at the previously marked skin areas of the dorsal surface. Of note, 1 minimum erythema dose for skin phototype III is about 30 mJ per cm².

Skin edema

To assess the effect of p.o. administered lutein and zeaxanthin on UVB-induced skin inflammation and edema, ears of mice were exposed to 40 and 160 mJ per cm² of UVB radiation and the increases in skin-bifold thickness of the irradiated ears were measured at 24 h in a blinded manner. In doing this, the ear skin double fold thickness was determined using a Vernier Caliper micrometer (Scienceware, Switzerland). The increase in ear-bifold thickness of ears was calculated by subtracting the baseline values obtained prior to UV exposure.

Isolation of skin samples

From each experimental group, half of the experimental animals receiving UVB radiation were killed at 24 h and the remaining were killed at 48 h after UVB irradiation using IACUC (Institutional Animal Care and Use Committee) approved techniques of euthanasia. In each animal, 6 mm skin biopsies were obtained from the UVB-exposed skin sites and from nonirradiated skin. One skin specimen obtained from each exposure dose was immediately fixed in 10% buffered formaldehyde for paraffin-embedded sections, and the other skin specimen was immediately stored at –80°C for western blot studies.

Terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay

UVB-irradiated skin specimens were examined for the presence of apoptotic cells by the TUNEL assay. This assay identifies apoptotic cells and allows to quantify reliably the number of TUNEL-positive cells. We used ApopTag Fluorescein In Situ Apoptosis detection kit (S7110, Intergenco, Inc., Purchase, New York; http://www.intergenco.com). This kit labels digoxigenin-labeled 3'-OH ends of the genomic DNA. Briefly, 5 m sections were deparaffinized with xylene, treated with series of graded alcohols, washed once in phosphate-buffered saline and subsequently treated with proteinase K (Sigma, St Louis, Missouri; http://www.sigma-aldrich.com) (20 g per mL) for 15 min at 37°C. After proteinase digestion, sections were incubated with 75 L equilibration buffer for 10 s. Sections were then incubated in a humidified chamber with terminal deoxynucleotidyl transferase enzyme for 1 h at 37°C. Incubation of one section with phosphate-buffered saline was used as a negative control. The sections were then soaked in working strength stop/wash buffer, agitated for 15 s and rinsed in three changes with phosphate-buffered saline. After rinsing, sections were incubated with anti-digoxigenin fluorescein for 30 min at room temperature. Finally, sections were rinsed with three washes of phosphate-buffered saline and counterstained with propidium iodide/anti-fade. The slides were viewed under a fluorescence microscope equipped with a =380–420 nm emission filter (Axiophot, Göttingen, Germany). TUNEL-positive cells were counted using a 40objective lens on five randomly selected fields per section. A total of three sections per sample were analyzed.

Immunohistochemistry staining for PCNA

Immunohistochemical assays were performed using mouse anti-human PCNA antibody clone PC10 (cat. no. M 0879, DAKO, Glostrup, Denmark) and a M.O.M. kit staining procedure (Vector Laboratories, Burlingame, California; http://www.vectorlabs.com). Briefly, 5 m thick sections were deparaffinized and hydrated slowly with xylene and graded alcohol series and subsequently rinsed for 5 min in water. After appropriate antigen unmasking by using standard techniques, the endogenous peroxidase activity was blocked by incubating the skin sections with 3% hydrogen peroxide in water for 5 min. Then avidin/biotin blocking was applied and sections were incubated for 1 h in working solution of M.O.M. mouse immunoglobulin blocking reagent. Next, sections were incubated with mouse anti-human PCNA monoclonal antibody diluted 1:1000 in M.O.M. diluent for 4 h at room temperature. Slides were rinsed with phosphate-buffered saline, and subsequently incubated with working solution of M.O.M. biotinylated anti-mouse IgG reagent for 10 min at room temperature. ABC reagent (Vectastain, Vector Laboratories) was added to sections for 5 min 3,3'-diaminobenzidine (Sigma) peroxidase substrate solution was used as a chromogen followed by a light counterstaining with Mayer's hematoxylin (Vector Laboratories) for 1 min, cleared with xylene, mounted with a coverslip and examined under a light microscope. Cells showing a brown nuclear reaction indicated proliferating cells and were considered PCNA positive. The slides were examined and scored under a light microscope. PCNA-positive and PCNA-negative cells in every randomly selected field were counted using a 20objective lens. Labeling index was calculated as the percentage of PCNA-positive keratinocytes specifically in the basal cell layer. A total of five fields per slide and three slides per sample were evaluated.

BrdU incorporation into DNA

BrdU, a thymidine analog that is incorporated into proliferating cells mainly during the S-phase of DNA synthesis, was detected by a biotinylated mouse monoclonal anti-BrdU antibody (^{Olsen and Kirkhus, 1989}) (Clone BU 33, product no. B2531, Sigma) and a M.O.M. kit staining procedure (Vector Laboratories). Briefly, all animals were intraperitoneally injected with BrdU (Sigma) in normal saline (50 mg per kg) and killed 1 h after injection. Sections from all skin samples were prepared as described above for PCNA immuno-histochemistry. Endogenous peroxidase was blocked by incubating the sections in 3% hydrogen peroxide in methanol for 10 min at room temperature. DNA denaturation was achieved by incubating sections in 2 M HCl for 30 min at 37°C. Slides were rinsed thoroughly in phosphate-buffered saline and incubated in a moist chamber with 0.1% trypsin for 10 min at 37°C; they were then incubated with mouse monoclonal anti-BrdU antibody. Subsequent steps were performed similar to PCNA staining described above. The slides were examined and scored under a light microscope using a 20objective lens. The BrdU labeling index (LI) was calculated from the number of stained BrdU-positive cells per 100 basal cells counted in each field. A total of five fields per section, and three sections per sample were analyzed.

Western blot analysis

Epidermal sheets were separated by incubating skin biopsies in 6 M NaBr solution for 1 to 2 h. Only separated epidermis was processed for homogenization and subsequent western blot analysis. Next, proteins were extracted with a lysis buffer consisting of 0.25 M Tris–HCl (pH 7.5), 0.375 M NaCl, 2.5% sodium deoxycholate, 1% Triton X-100, 25 mM MgCl₂, 1 mM phenylmethyl sulfonyl fluoride, and 0.1 mg aprotinin per mL as described by^{Yaar et al (1991)}. Equal amounts of protein extract (50 g), quantified by Bradford method were processed for western blot analysis (^{Goukassian et al 2000}). Antibody reactions were performed with 1:200 dilutions of affinity-purified mouse monoclonal and rabbit polyclonal antibodies. Anti-PCNA (Ab-2) (Oncogene Science Inc., Cambridge, Massachusetts) was used as primary antibody. The secondary rabbit and donkey anti-mouse antibodies (Santa Cruz Biotechnology, Inc, Santa Cruz, California) were used at 1:2000 dilution. Equal protein loading in each lane was confirmed by hybridization with a 1:2000 dilution of b-actin antibody (Santa Cruz Biotechnology, Inc.). Antibody binding was detected by ECL detection kit (Amersham, Piscataway, New Jersey), followed by autoradiography (Kodak X-Omatic AR, Rochester, New Jersey). Autoradiographs of western blots were scanned (Microtek Scan Maker II, Taiwan) and band intensity was quantified after background subtraction by using densitometric program "Sigma gel" (Jandel Scientific, Corte Madera, California). Normalization for loading of the lanes was determined by Coomassie blue staining using multiple lanes.

Statistical analyses

Results are expressed as the meanSD. Data were evaluated by two-tailed unpaired Student's t test and analysis of variance using the StarView statistical program. The level of statistical significance was taken as p<0.05.

Results

Oral administration of lutein and zeaxanthin suppresses UVB-induced skin inflammation

To evaluate the protective effect of oral administration of lutein and zeaxanthin against UVB-induced inflammation we measured ear skin bifold-thickness. Animals fed with diet enriched with lutein and zeaxanthin at 0.4% (striped bars) were found to have a significantly suppressed edematous response induced by UVB radiation (p<0.001). Values of ear thickness increase in millimeters after UVB exposure of untreated and lutein and zeaxanthin-treated mice are shown in Figure 1.

Figure 1.

Effects of dietary 0.4% and 0.04% lutein and zeaxanthin supplementation against UVB-induced ear swelling as outlined inMaterials and Methods. The clear bar corresponds to control animals of group I, fed with normal diet, the black bar corresponds to animals of group II receiving dietary lutein and zeaxanthin in a concentration of 0.04% and the striped bar correlates to group III receiving a dietary supplementation of 0.4% lutein and zeaxanthin. X-axis shows UVB fluences in mJ per cm², Y-axis indicates the increase in ear bifold thickness in mm (^***p<0.001 vs control, irradiated mice of group I, fed with normal diet).

Full figure and legend (14K)

Oral administration of lutein and zeaxanthin decreases the number of UVB-induced apoptotic keratinocytes

The presence of apoptotic keratinocytes was assessed in skin biopsies obtained at 24 and 48 h after UVB irradiation by using the TUNEL assay. Immunofluorescence microphotographs of representative control irradiated skin (Figure 2a1) and irradiated skin from mice fed with 0.04% (Figure 2a2) and 0.4% (Figure 2a3) lutein and zeaxanthin supplemented diets are shown. When a dose of either 80 or 160 mJ per cm² of UVB irradiation was used, almost all the cells in epidermis became TUNEL positive (data not shown) and the number of apoptotic cells per field in the epidermis of control (clear bars, Figure 2b) irradiated animals increased in a dose-dependent manner. In mice fed with lutein and zeaxanthin at 0.04% (black bars, Figure 2b) for 2 wk before UVB irradiation, the number of TUNEL-positive cells induced by 40 mJ per cm² of UVB radiation was significantly reduced (p<0.01). This reduction was even greater (by 210%, p<0.001) when mice were fed with lutein and zeaxanthin at 0.4% (striped bars Figure 2b). This reduction in the number of apoptotic cells was similar in specimens obtained at 24 and 48 h (Figure 2b).

Figure 2.

Effects of oral administration of 0.4% and 0.04% lutein and zeaxanthin on UVB-induced increase in TUNEL-positive cells. Female SKh-1 mice were fed with either normal diet or diet supplemented with lutein and zeaxanthin and killed at 24 h or 48 h after UVB irradiation. (a) TUNEL-stained sections from skin sites of unirradiated (a1) and irradiated control (a2) animals, and animals treated with 0.04% (a3) and 0.4% (a4) lutein and zeaxanthin after exposure to 40 mJ per cm² of UVB radiation. Original magnification200. (b) Graphic representation of the percentage of TUNEL-positive cells observed at 24 h (left panel) and 48 h (right panel) after exposing skin sites to 40 mJ per cm². Clear bar represents data of group I, who received no supplementation with lutein and zeaxanthin, the black bar corresponds to group II receiving dietary lutein and zeaxanthin in a concentration of 0.04% and the striped bar corresponds to group III with a dietary supplementation of 0.4% lutein and zeaxanthin. X-axis represents UVB doses at both 24 and 48 h time points after the UVB exposure and Y-axis represents the number of TUNEL-positive cells per 40X field of view. Each bar represents the mean value of six miceSD (^*p<0.05)

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Oral administration of lutein and zeaxanthin decreases the number of UVB-induced PCNA-positive cells in murine skin

To determine whether dietary administration of lutein and zeaxanthin affects epidermal cell proliferation after UVB irradiation, skin biopsies from irradiated and nonirradiated skin of mice were examined for PCNA expression by immuno-histochemical and western blot analyses. In nonirradiated skin of mice receiving a normal diet, the PCNA immunoreactivity was limited to the basal layer (Figure 3a1). Strong nuclear immunostaining was detected throughout the entire epidermis of UV irradiated control (Figure 3a2) and mice fed with 0.04% (Figure 3a3) and 0.4% (Figure 3a4) lutein and zeaxanthin. Significantly higher numbers of PCNA-positive cells, however, were observed in control versus lutein and zeaxanthin fed animals (Figure 3a2,a3,a4) consistent with UV-induced hyperproliferative response. To estimate the PCNA labeling index in our studies we counted only the basal PCNA-positive keratinocytes. At 24 and 48 h after exposure to UVB radiation, there was a UVB dose-dependent increase in PCNA labeling index (Figure 3b). Oral administration of diets enriched with lutein and zeaxanthin at 0.04% (black bars) or 0.4% (striped bars) for 2 wk before irradiation with 40 mJ per cm² reduced the percentage of basal PCNA-positive cells by 35% and 14%, respectively. Compared with control fed animals (clear bars), PCNA labeling index was significantly reduced for all UVB doses at both time points (p<0.05) (Figure 3b). PCNA protein expression was also examined by western blot analysis. (Figure 4b) There was a statistically significant decrease compared with control in PCNA protein levels in 0.4% (striped bars) lutein and zeaxanthin fed animals irradiated with 40 and 80 mJ per cm² UVB. At lower (40 mJ per cm²) UVB dose mice fed with 0.04% (black bars) lutein and zeaxanthin also showed an approximately 29% decrease in PCNA protein levels, which was not statistically significant (Figure 4b, left panel). Lutein and zeaxanthin supplementation did not have any effect on PCNA protein levels after the highest (160 mJ per cm²) UVB dose (Figure 4b, right panel). Of note, slightly different results of PCNA expression in immunohistochemical and western blot analysis, are most likely due to our methodologic approach of counting PCNA-positive cells only in the basal layer (immunochemistry), whereas the entire epidermal sheets were processed for western blot analysis.

Figure 3.

Effects of oral administration of lutein and zeaxanthin on UVB-induced increase in PCNA-positive cells. (a) Mice were killed at 24 h or 48 h after irradiation. Skin biopsies were taken immediately after exposure for PCNA immunohistochemical staining. PCNA-stained sections from unirradiated (a1) and irradiated (a2) control animals and from irradiated animals treated with 0.04% (a3) and 0.4% (a4) lutein and zeaxanthin (24 h after 40 mJ per cm² of UVB radiation). Original magnification 200. (b) Graphical representation of the immunohistochemical analysis of PCNA protein expression in UVB-irradiated mouse skin fed with either normal diet or 0.4% and 0.04% lutein and zexanthin enriched diet. Clear bar represents data of group I, who received no supplementation of diet with lutein and zeaxanthin, the black bar corresponds to group II receiving dietary lutein and zeaxanthin in a concentration of 0.04% and the striped bar correlates to group III with a dietary supplementation of 0.4% lutein and zeaxanthin. X-axis represents different UVB fluences at both 24 h (left panel) and 48 h (right panel) after the UVB exposure and Y-axis represents PCNA labeling indices. Each bar represents the mean of six mice, bars, SD (^*p<0.05, ^**p<0.01, ^***p<0.001).

Full figure and legend (95K)

Figure 4.

(a) Western blot analysis of PCNA and b-actin protein expression in unirradiated and UVB irradiated skin of mice fed with either normal diet or lutein and zeaxanthin enriched diet at various irradiation doses and at different time points. (b) Western blot analysis of autoradiographs from each of the three experiments were subjected to densitometric analysis of band intensity. X-axis represents the different UVB fluences, Y-axis indicates the PCNA protein level. Loading was adjusted for b-actin protein expression. Clear bars represent mice of group I without dietary supplementation, black bars show the results of group II with 0.04% lutein and zeaxanthin and the striped bars represents animals of group III with 0.4% dietary lutein and zeaxanthin. Each bar represents the mean of three animals, bars, SD (^*p<0.05).

Full figure and legend (80K)

Oral administration of lutein and zeaxanthin reduces UVB-induced increase in BrdU incorporation into basal epidermal keratinocytes

To evaluate the number of proliferating basal keratinocytes that are predominantly in the S-phase of cell cycle we measured the BrdU incorporation into the DNA of basal keratinocytes.

In unirradiated mice skin, only a fraction of basal keratinocytes was found in the S-phase, as shown by BrdU incorporation (Figure 5a1). UVB exposure alone increased BrdU incorporation into epidermal DNA in a dose-dependent manner and these increases were 2- to 4-fold the baseline values (Figure 5a2). Significantly lower numbers of BrdU-positive cells were observed in animals fed with 0.04% (Figure 5a3) and 0.4% (Figure 5a4) lutein and zeaxanthin at all given UVB doses. To calculate the BrdU incorporation index only the fraction of basal BrdU-positive cells were counted. The percentage of BrdU-positive basal cells was reduced in samples obtained from lutein and zeaxanthin fed mice 24 and 48 h after UVB irradiation (Figure 5b). This decrease in the percentage of BrdU-positive basal keratinocytes was lutein and zeaxanthin concentration dependent. At 24 h (left panel) and 48 h (right panel) after UVB exposure, the increase in the percentage of BrdU-positive cells was significantly reduced for all UVB doses tested in mice receiving 0.04% (black bars) and 0.4% (striped bars) lutein and zeaxanthin (p<0.05).

Figure 5.

Effects of oral administration of lutein and zeaxanthin on UVB-induced increase in BrdU-positive cells. Female SKh-1 mice were fed with either normal diet or diet supplemented with 0.4% and 0.04% lutein and zeaxanthin. (a) BrdU IP injection was performed 1 h before killing the animals. Mice were killed at 24 h or 48 h after irradiation and skin biopsies were obtained for immunohistochemical staining as described in Materials and Methods. BrdU-stained sections from unirradiated (a1) and irradiated (a2) control animals and from irradiated animals treated with 0.04% (a3) and 0.4% (a4) lutein and zeaxanthin (24 h after 40 mJ per cm² of UVB radiation). Original magnification200. (b) Graphical representation of the percentage of BrdU-positive basal keratinocytes of mice fed with lutein and zeaxanthin. The percentage of BrdU-positive cells was significantly decreased in animals receiving dietary lutein and zeaxanthin. X-axis represents different UVB fluences at both 24 h (left panel) and 48 h (right panel) after the UVB exposure and Y-axis represents BrdU labeling indices. Clear bar represents data of group I, who received no supplementation with lutein and zeaxanthin, the black bar represents data of group II receiving dietary lutein and zeaxanthin in a concentration of 0.04% and the striped bar corresponds to group III with a dietary supplementation of 0.4% lutein and zeaxanthin. Each bar represents the mean value of six mice. bars, SD (^*p<0.05, ^**p<0.01, ^***p<0.001).

Full figure and legend (96K)

Discussion

To our knowledge, this is one of the first in vivo studies evaluating at cutaneous cellular level the protective effects of oral intake of lutein and zeaxanthin against epidermal hyperproliferation and inflammation induced by single exposure doses of UVB radiation. An acute exposure of human or murine skin to UVB radiation from natural sunlight or artificial light sources results in various biologic responses, through the direct formation of cyclobutane pyrimidine dimers and (6–4)pyrimidine pyrimidone dimers in DNA (^{Cadet and Vigny, 1990;Cadet et al, 1992}). UVB exposure also results in the formation of free radicals and ROS that damage DNA and non-DNA cellular targets. Among the latter, lipid-rich cellular membranes are particularly vulnerable to the deleterious effects of ROS. These biologic changes include cell cycle arrest, apoptosis, cell proliferation, and reparative hyperplasia during the early adaptive response and are considered to be involved in the pathophysiology of the UV-induced erythema, premature skin aging, and skin cancer (^{Norris et al, 1993;Young, 1993}). In recent years, our research interest has been focusing on minimizing the adverse acute and chronic UV responses using natural antioxidants known to act as radical scavengers and radical quenchers in vivo (^{González and Pathak, 1996;González et al, 1997}). In this study, we showed that supplementation of diets with 0.4% and 0.04% lutein and zeaxanthin for a period of 2 wk reduced UV-induced increases in the number of apoptotic cells and proliferating cells in the epidermis of the hairless albino (SKh-1) mouse model when exposed to graded doses of UVB radiation.

Only few studies in the past several years have reported the beneficial antioxidant properties of certain carotenoids in photoprotection of skin against UV radiation (^{Mathews-Roth et al, 1970,1972;Stahl et al, 2000}). Recently, Granstein and his coworkers¹ reported an anti-inflammatory effect of lutein in the skin of mice exposed to UV radiation. Mice were fed diets containing 0.04% and 0.4% lutein for 2 wk; ears were then exposed to UVB radiation to induce inflammation. The thickness of ear skin of mice before and 24 h after UV radiation was evaluated. The ear swelling in response to UV radiation was significantly inhibited in the mice fed lutein diet relative to the controls receiving no dietary supplementation of lutein and zeaxanthin. In our studies reported here we have confirmed these observations.^{Stahl et al (2000)} examined the effect of carotenoids (mainly -carotene) and carotenoids plus vitamin E on the skin of human volunteers on the development of erythema after exposure to UV radiation. The erythematous response was significantly reduced after 8 wk of supplementation with a carotenoid mixture that included -carotene and a small fraction of lutein. The ability of -carotene, lutein, and astaxanthin to protect against UVA-induced oxidative stress in cultured rat kidney fibroblasts was also recently reported by^{O'Connor and O'Brien (1998)}. Activities of antioxidant enzymes, such as catalase and superoxide dismutase, as well as thiobarbituric reactive substance were evaluated to ascertain enzymatic changes resulting from oxidative stress induced by UVA radiation. -carotene, lutein, and astaxanthin were found to protect against enzymatic changes with astaxanthin exhibiting superior photoprotective properties (^{O'Connor and O'Brien, 1998}).

Lutein and zeaxanthin, nonprovitamin A carotenoids, are the predominant carotenoids found in human retina and their concentration is greatest in the fovea centralis of the macula lutea of human retina where they constitute the macular pigment and serve to decrease the risk of age-related macular degeneration (^{Beatty et al, 1999;Pratt, 1999}) and to protect the retinal pigment epithelium against a photooxidative damage initiated in part by light absorption (^{Snodderly, 1995;Broekmans et al, 2002}). There is good evidence that this protection is due mainly to their antioxidant properties (^{Handleman and Dratz, 1986;Zhang et al, 1991;Taylor et al, 1993;Sujak et al, 1999}). In vitro lutein and zeaxanthin has been shown to be more effective than -carotene in inhibiting auto-oxidation of cellular lipids (^{Zhang et al, 1991}) and in further protecting against oxidant-induced cell damage (^{Martin et al, 1996}).

It is becoming more apparent that many of the maladies that affect human skin are the result of direct and indirect environmental interaction invariably caused by UV radiation. Excessive and often uncontrolled exposure to terrestrial solar radiation is harmful to human skin and causes acute sunburn, damage to epidermal and dermal cells, induction of cell death, immune suppression, and eventually skin cancer (^{Young, 1993}). Currently the most widely accepted method of photoprotection against UV radiation is the use of two or more topical sunscreens containing UVB and UVA absorbing chemicals that are nonphotolabile (e.g., octyl methoxy cinnamate, octyl dimethyl amino benzoate, benzophenones, or avobenzone (parsol 1789), etc.) with or without admixture of UV scattering and reflecting chemicals (e.g., ZnO, TiO₂ in micronized form) incorporated in a water-resistant base. Other approaches, including chemicals with antioxidant properties such as vitamin C, vitamin E, -carotene, etc., are also advocated (^{Thompson et al, 1993;Naylor et al, 1995;Green et al, 1999;Pathak et al, 1999;Stahl et al, 2000}). The in vivo presence of effective antioxidants such as lutein and zeaxanthin in the skin prior to UVB irradiation may reduce the deleterious effect of irradiation, most likely by decreasing the generation of UV-induced ROS (^{Athar et al, 1992;Dalle Carbonare and Pathak, 1992}). Although -carotene has been examined in our laboratories in the past (^{Mathews-Roth et al, 1970;1972;Greenberg et al, 1990}), we feel that the antioxidant lutein is less labile and thus better than -carotene in minimizing the photooxidative changes in the skin. Comparative studies using lutein and -carotene, however, have to be done to verify the better photoprotective properties of lutein and zeaxanthin. Because UV-induced inflammation and hyperproliferation (^{Pentland et al, 1999;An et al, 2002}) has been strongly involved in UVB carcinogenesis, inhibitory effects of dietary lutein and zeaxanthin on acute inflammation, and epidermal hyperproliferation may have a potential implication for the prevention of carcinogenesis in chronically UV-exposed skin.

Notes

¹ Granstein RD, Faulhaber D, Ding W: Lutein inhibits UVB radiation-induced tissue swelling and suppression of the induction of contact hypersensitivity (CHS) in the mouse. J Invest Dermatol 117(2):497, 2001 (Abstr.)

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Acknowledgments

The authors want to thank Martine Sifakis and William Farinelli for excellent technical assistance and Kemin Foods L.C. (DesMoines, IA) for funding this study through a research grant from The Skin Cancer Foundation, New York.