Journal of Photochemistry and Photobiology B: Biology 105 (2011) 133–142

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier .com/locate / jphotobiol

The effects of grape seeds polyphenols on SKH-1 mice skin irradiatedwith multiple doses of UV-B

Adriana Filip a,⇑, Doina Daicoviciu a, Simona Clichici a, Pompei Bolfa b, Cornel Catoi b, Ioana Baldea a,Laura Bolojan c, Diana Olteanu a, Adriana Muresan a, I.D. Postescu d

a Department of Physiology, ‘‘Iuliu Hatieganu’’ University of Medicine and Pharmacy, 1-3 Clinicilor Str., 400012 Cluj-Napoca, Romaniab Pathology Department, University of Agricultural Sciences and Veterinary Medicine, 3-5 Calea Manastur Str., 400372 Cluj-Napoca, Romaniac Faculties of Physics, University ‘‘Babes-Bolyai’’ Cluj-Napoca, 1 Mihail Kogalniceanu Str., 400084 Cluj-Napoca, Romaniad Department of Radiobiology and Tumor Biology, ‘‘Prof. I.Chiricuta’’ Oncologic Institute, 34-36 Republicii Str., 400015 Cluj-Napoca, Romania

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 April 2011Received in revised form 1 August 2011Accepted 6 August 2011Available online 26 August 2011

Keywords:Grape seedsOxidative stressCyclobutane pyrimidine dimersSunburn cellsUltraviolet

1011-1344/$ - see front matter � 2011 Elsevier B.V. Adoi:10.1016/j.jphotobiol.2011.08.002

⇑ Corresponding author. Tel.: +40 745 268704; fax:E-mail address: adrianafilip33@yahoo.com (A. Filip

The study investigated the protective activity of red grape seeds (Vitis vinifera L, Burgund Mare variety)(BM) extracts in vivo on multiple doses of ultraviolet radiation (UV)-B-induced deleterious effects inSKH-1 mice skin.

Eighty 8-weeks-old female SKH-1 mice were divided into 8 groups: control, vehicle, UV-B irradiated,vehicle + UV-B irradiated, BM 2.5 mg polyphenols (PF)/cm2 + UV-B irradiated, BM 4 mg PF/cm2 + UV-Birradiated, UV-B + BM 2.5 mg PF/cm2, UV-B + BM 4 mg PF/cm2. The extract was applied topically beforeor after each UV-B exposure (240 mJ/cm2), for 10 days consecutively. The antioxidant activity of BMextract is higher than gallic acid (kBM = 0.017, kgallic acid = 0.013). Multiple doses of UV-B generated the for-mation of cyclobutane pyrimidine dimers (CPDs) and sunburn cells, increased glutathione peroxidase(GPx) and catalase (CAT) activities respectively glutathione (GSH) and IL-1b levels in skin. In group trea-ted with 2.5 mg PF/cm2 before UV-B irradiation BM extract inhibited UV-B-induced sunburn cells,restored the superoxide dismutase (MnSOD) activity, increased insignificantly CAT and GPx activitiesand reduced IL-1b level. The BM 4.0 mg PF/cm2 treatment decreased GSH level and reduced the percent-age of CPDs positive cells in skin. Both doses of BM extract administered after UV-B irradiation increasedthe MnSOD and GPx activities and reduced the formation of sunburn cells in skin.

Our results suggest that BM extract might be a potential chemo-preventive candidate in reducing theoxidative stress and apoptosis induced by multiple doses of UV-B in skin.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction

Skin exposure to sunlight is known to cause the damage of struc-ture and function of skin by inducing a number of biological re-sponses, including development of erythema and edema, sunburncell formation, hyperplasia, photoaging and melanogenesis [1,2].While the erythema is a manifestation of an initial inflammatory re-sponse to UV irradiation, the chronic UV exposure can induce ede-ma formation, mutations in DNA, alteration of the immune systemand development of skin cancer [3]. The effects of chronic UV expo-sure are of lower intensity than those caused by acute irradiation, aphenomenon known as photoadaptation or tolerance [4,5]. Themechanisms are poorly understood and involve epidermal thicken-ing, tanning, vascularization and DNA repair.

It has been shown that UV-B (315–280 nm), the most damagingof solar wavelengths, directly affects the genetic material of

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exposed cells by forming thymine dimers [6]. Evidence stronglysuggests that DNA photoproducts formed at the sites of adjacentpyrimidine bases by the direct absorption of UV-B are the predom-inant premutagenic events responsible for the initiation of skinnonmelanoma cancer [7]. DNA damage leads to the altered expres-sion of various gene products involved in protective or reparativeprocesses such as apoptosis, genomic repair and cell growth arrest.Disregulated sunburn apoptosis together with the loss of normalfunction of the product of the p53 gene and immunosuppressionseems to play an important role in photocarcinogenesis [8]. Inaddition, UV-B radiation produces reactive oxygen species (ROS)that can also damage DNA molecules, lipid components and cellu-lar proteins contributing to the sunburn reaction and photocarci-nogenesis [9,10].

Skin possesses a variety of enzymatic as well as small molecularantioxidants that can inhibit oxidative damage [2]. Excessive ROSgeneration can overwhelm the antioxidant defense capacity ofthe skin, resulting in oxidative stress and consequently in oxidativephotodamaging of the main skin biomolecules [11].

134 A. Filip et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 133–142

Considering that UV induces oxidative stress-mediated adverseeffects in the skin, the regular intake or topical application of antiox-idants is suggested to be a useful way to reduce the harmful effectsof UV radiation [12]. Chemoprevention, defined as ‘‘the use of agentscapable of ameliorating the adverse effects of UV-B on the skin’’ bynatural compounds, represents a new concept in the attempt to con-trol the carcinogenesis process [13]. Among many photochemopro-tective agents, botanical origin antioxidants are showing promise.

Grapes ( Vitis Vinifera) are one of the most widely consumedfruits in the world. Grape seed are rich in polyphenols, flavan-3-ol derivatives (catechins) and oligomeric and polymeric proantho-cyanidins [13]. Several studies have shown that polyphenols fromgrape seeds have anti-inflammatory, antioxidant effects and inhi-bit the oxidative stress – mediated activation of MAPK and NF-kBinvolved in carcinogenesis pathways [14]. Previously, we haveshown that topical application of a grape seed extract (BurgundMare variety, BM) to mouse skin before a single UV-B exposure(240 mJ/cm2) markedly decreased UV-B-induced production of li-pid peroxides and nitric oxide and reduced caspase-3 activity[15]. Our preliminary date revealed that BM extract reduce UV-B-induced increase in cytokine (IL-6, TNF-a) levels and afforded pro-tection against UV-induced alteration in GPx and CAT activities(unpublished data). In addition, at 24 h following irradiation BMextract inhibited one dose UV-B-induced sunburn cells and cyclo-butane pyrimidine dimers (CPDs) formation (unpublished data).

Although the effects of a single UV exposure on oxidative stressand DNA damage are useful for examining the complex skin re-sponses to UV, multiple exposures are perhaps more relevant tothe sunlight exposure that is naturally received by humans. Thisstudy was designed to assess the effects of Vitis Vinifera BurgundMare variety on multiple doses of UV-B-induced responses in theskin of SKH-1 hairless mice. Specifically, we determined the effectsof BM extract on UV-B-mediated oxidative stress, on cyclobutanepyrimidine dimers and sunburn cell formation in skin.

2. Materials and methods

2.1. Chemicals

Trichloroacetic acid, o-phtalaldehyde, 2-thiobarbituric acid, t-butyl hydroperoxide, glutathione reductase 2,20-azino-bis(3-ethyl-benzothiazoline-6-sulfonate (ABTS), potassium cyanide, Bradfordand Folin Ciocalteu reagents were purchased from Sigma-AldrichChemicals GmbH (Germany). EDTA-Na2 was obtained from MerckKGaA Darmstadt (Germany) and absolute ethanol and n-butanolfrom Chimopar (Bucuresti). Gallic acid and Tempol (2,2,6,6-tetra-methyl-4-hydroxypiperidine-N-oxyl) were obtained from Fluka,Buchs (Switzerland). Monoclonal antibody to CPDs for IHC (MouseIgG2a, code number NMDND001) was purchased from Cosmo BioCo. Ltd. (Japan). A secondary antibody included in the kitLSAB + System HRP (Code K0679), Mayer’s Haematoxylin andhydrogen peroxide were from Dako (Denmark). Rhodamine (Goatpolyclonal to mouse Ig H&L, ab6786) was purchased from ABcam(UK) and DRAQ5 (Cat No. 4084) from Cell Signaling TechnologyInc. (Danvers, MA). All chemicals and reagents were of high gradepurity. ELISA tests to measure IL-1b was obtained from R&D Sys-tems (Minneapolis, MN, USA).

The hydroethanolic extract from grape seeds (Vitis vinifera L),variety Burgund Mare (Romania) was prepared as previously de-scribed from 1:10 w/v mixture of finely powdered dried seedsand water/ethanol 50/50 (v/v) [16]. The solution was concentrated,in vacuo, (10 fold), and the product was characterized by its totalpolyphenolic content (TPC), assessed by the Folin–Ciocalteu color-imetric reaction [17] and expressed as equivalents (Eq) galic acid(GA) per unit of volume. For topical use, the grape seed extractwas dried and re-suspended in 40% acetone.

2.2. BM extract antioxidant activity

2.2.1. Trolox-equivalent antioxidant capacity (TEAC)TEAC of BM extract was measured using ABTS test (unpublished

results). A concentration-response curve (% inhibition) of absor-bance at 734 nm of ABTS � + solution as a function of standard Trol-ox solutions was used to evaluate the antioxidant capacity. Resultsare expressed as eq. nmol Trolox/ml [18].

2.2.2. Electron paramagnetic resonance (EPR) measurements ofantioxidant activity

The antioxidant capacity was evaluated by EPR using Tempol(nitroxide radical 4-hydroxy-2,2,6,6-tetramethyl-4-hydroxypiperi-dine-N-oxyl) [19]. The homogenized solution composed of 2 mlextract and 1 mM water Tempol solution was injected with a Ham-ilton microsyringe into a quartz capillary (10 cm length and an inte-rior diameter about 1 mm). EPR measurements were performed ona Bruker EMX spectrometer operating at 9.4548 GHz with 100 kHzmodulation frequency, at room temperature. The reaction mixturewas transferred to a 20 ll capillary, which was then positioned inthe high sensitivity (HS) cavity (Bruker Instruments, ER 4102ST).The sample was scanned using the following parameters: centerfield, 3510 G; sweep width, 60 G; power, 2 mW; receiver gain,1 � 103; modulation amplitude, 2 G; time of conversion, 30 ms;time constant, 61.4 ms; number of scans, 1. The spectra were re-corded at different time intervals. The integral intensities of EPRspectra were obtained by evaluating their double integrals (DIEPR)using the WIN EPR program (Bruker Instruments).

2.3. Animals and experimental protocol

Eighty female mice SKH-1 (8 weeks old, weighing 25 ± 3 g), ob-tained from Charles River Laboratory, Germany, were used in thisstudy. All the experiments were performed according to the ap-proved animal-care protocols of the Ethical Committee on AnimalWelfare of the ‘‘Iuliu Hatieganu’’ University of Medicine. The ani-mals were fed with normocaloric standard diet (VRF 1) and waterad libitum. The animals were housed (5 animals/cage) at room tem-perature (24 ± 2 �C), with a 12/12 hrs light dark cycle. Eight groupsof 10 animals each, randomly divided, were treated as follows:group 1: control, received no treatment; group 2: vehicle (acetone40%); group 3: UV-B irradiated; group 4: vehicle + UV-B irradiated;group 5: BM (2.5 mg PF/cm2) + UV-B irradiated; group 6: BM (4 mgPF/cm2) + UV-B irradiated; group 7: UV-B + BM (2.5 mg PF/cm2);group 8: UV-B + BM (4 mg PF/cm2). Before treatment, the animalswere anaesthetized with an i. p. injection of ketamine xylazinecocktail (90 mg kg�1 b.w. ketamine, 10 mg kg�1 b.w. xylazine).The extract was applied 30 minutes before the UV-B (240 mJ/cm2) irradiation and exposures were made 10 consecutive days.In groups 7 and 8 the extract was administered 30 min after eachUV-B irradiation, also for 10 days.

UVB irradiation was done with a Waldmann UV 181 broadbandUV-B source, with 1.35 mW/cm2 intensity at 7 cm distance fromthe source. The UV-B emission was monitored before each expo-sure with a Variocontrol radiometer (Waldmann GmbH, Germany).At 24 h after the last treatment the animals were anaesthetizedand fragments of dorsal skin were excised from each mouse andused for biochemical and histopathological investigations.

2.4. Measurement of oxidative stress parameters

After the removal of subcutaneous tissue, skin tissue fragmentswere homogenized with a Polytron homogenizer (BrinkmanKinematica, Switzerland) for 3 min on ice in phosphate bufferedsaline (pH 7.4) added at a ratio of 1:4 (w/v). The suspension wascentrifuged for 5 min at 3000 g and 4 �C in order to prepare the

A. Filip et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 133–142 135

cytosolic fraction. The proteins content in homogenates was mea-sured with Bradford method [20].

Superoxide dismutase (SOD) activity was determined using cyto-chrome c reduction test with some adjustments [21]. Skin homog-enates were introduced in a cytochrome c solution (2 lM inphosphate buffer 50 mM, pH 7.8) containing xanthine (5 lM).The reaction was started by adding xanthine oxidase (0.2 U/ml in0.1 mM EDTA). The increasing absorbance at 550 nm, indicatingcytochrome c reduction was recorded for 5 min. MnSOD was dif-ferentiated from CuZnSOD by its resistance to 2 mM potassiumcyanide. One unit of SOD inhibits the rate of increase in absorbanceat 550 nm by 50% of those produced for a control sample withoutSOD under the conditions of the assay. Results were expressed inU/mg protein.

Catalase activity (CAT) was assayed according to Pippengermethod in a reaction mixture containing 10 mM hydrogen perox-ide in 50 mM kalium phosphate buffer, pH 7.4 [22]. The reductionin absorbance at 240 nm was recorded for 3 min. The enzymequantity which produced a 0.43 reduction in absorbance per min-ute at 25 �C was defined as one unit of catalase activity and ex-pressed as units/mg protein.

Glutathione peroxidase activity (GPx) was determined with Floheand Gunzler method, slightly modified [23]. The reaction mixtureconsisting in 1 mM GSH, 0.24 U/ml glutathione reductase and0.15 mM NADPH (final concentrations) in 50 mM phosphate buffer(pH 7.0) was incubated at 37 �C for 5 min with appropriateamounts of tissue homogenates. The assay was initiated with a(12 mM) t-butyl hydroperoxide solution. The decrease in absor-bance at 340 nm was recorded for 3 min. GPx activity was ex-pressed as lmoles of NADP produced/min/mg protein andcalculated using a molar absorptivity for NADPH of 6.2 � 10�6, at340 nm.

Reduced glutathione (GSH) was measured fluorimetrically usingo-phtalaldehyde [24]. Samples were treated with trichloroaceticacid (10%) and centrifuged. A solution of o-phtalaldehyde (1 mg/ml in methanol) was added to supernatants diluted with sodiumphosphate buffer 0.1 M/EDTA 5 mM, pH 8.0. After 15 min, the fluo-rescence was recorded (350 nm excitation and 420 nm emission).GSH concentration was determined using a standard curve and ex-pressed as nmoles/mg protein.

Malondialdehyde (MDA) was determined using the fluorimetricmethod with 2-thiobarbituric acid described by Conti et al. [25].The skin homogenate samples were mixed with a solution of10 mM 2-thiobarbituric acid in 75 mM K2HPO4, pH 3 solutionand heated in a boiling water bath for 1 hour. After cooling, thereaction products were extracted in n-butanol. The MDA wasdetermined spectrofluorimetrically in the organic phase using asynchronous technique with excitation at 534 nm and emissionat 548 nm. The MDA values are expressed as nmoles/mg protein.

2.5. Cytokine level

For the analysis of IL-1 b ELISA assays was used according to theproducer instructions. The IL-1 b was expressed as pg/mg protein.

2.6. Immunohistochemistry (IHC) of cyclobutane pyrimidine dimers

Direct UV-B-induced DNA damage was measured immunohis-tochemically by CPDs formation. Briefly, we used the followingIHC protocol: after paraformaldehyde fixation, the samples wereembedded in paraffin, then sectioned at 5 lm using a Leica RM2125 RT microtome and mounted on slides embedded with APES(amino-propyl-tri-ethoxy-silane). For a better adherence, sampleswere incubated at 37 �C for 24 h. Deparaffinization and rehydra-tion were followed by antigen retrieval in a sodium citrate solution(10 mM pH 6). Samples were brought to boiling temperature, kept

10 min at sub-boiling temperature and then cooled 30 min onbenchtop. Peroxidase blocking was done with 3% hydrogen perox-ide. We used a monoclonal anti-cyclobutane pyrimidine dimersprimary antibody (Cosmo Bio Co Ltd., Japan) and for the visualiza-tion a secondary antibody included in the kit LSAB + System HRP(Dako). Nuclei were counterstained with Mayer’s Haematoxylin.Tissue sections stained exclusively with the secondary antibodywere used as a negative control.

2.6.1. Analysis of CPDs positive cellsTo evaluate the inhibitory effect of BM extract on UV-B-induced

CPDs formation, CPDs positive cells from the epidermis of thestained skin sections were double blinded counted, on ten micro-scopic fields. We used a 400� magnification and an OlympusBX51 microscope equipped with Olympus cell B software. The re-sults were expressed as the mean percent of positive cells perfield ± SD.

2.7. Sunburn cells

For the apoptotic sunburn cells counting, the skin samples werefixed with formalin 10% for 24–48 hrs, dehydrated and embeddedin paraffin. The sections were haematoxylin–eosin stained andexamined. Apoptotic cells were morphologically distinct, withsmall, dense nuclei due to nuclear condensation and eosinophiliccytoplasm. A total of ten microscopic fields were counted, usinga double blind method. The results were expressed as the meanpercent of positive cells per field ± SD.

2.8. Statistical analysis

Experimental data were analyzed by one-way analysis of vari-ance (ANOVA) followed by the Tukeys multiple comparisons posttest using GraphPad Prism 5.0 software (GraphPad, San Diego,Ca.,SUA). The four groups treated with BM extract were compared withthe control group (untreated), treated with vehicle and UV-B irra-diated group, respectively. The effects of BM extract and UV-B andtheir interaction were considered statistically significant atp < 0.05. The data were expressed as means ± standard deviation(SD).

3. Results

3.1. Antioxidant activity of BM extract

The antioxidant capacity of BM extract evaluated by ABTS testwas 52.89 ± 0.02 eq. mM Trolox), in agreement with the results ob-tained previously using the 2,2-diphenyl-l-picryl-hydrazyl(DPHH�) scavenging assay [26]. The antioxidant activity of BM ex-tract was evaluated also by EPR technique, monitoring of the Tem-pol concentrations after the addition of BM extract or gallic acid[27]. The rate of reaction between antioxidant compounds andTempol was monitored by using normalized double integratedresidual EPR signal which is correlated with the number of para-magnetic species in time [28]. This dependence is represented inFig. 1. The best fit (r2 = 0.993) was obtained using the first orderexponential decay:

IðtÞ ¼ I0 þ I1 expð�ktÞ

where I0, and I1 are the fitation constants, and k is the kinetic con-stants of reaction corresponding to each type of extracts orantioxidants.

Comparing the antioxidant characteristics of BM extract withthose of the gallic acid, we can observe that BM extract has the

Fig. 1. The antioxidant capacity of BM extract evaluated by EPR using nitroxide radical 4-hydroxy-2,2,6,6-tetramethyl-4-hydroxypiperidine-N-oxyl. EPR spectra of Tempol atdifferent time of incubation in (a) BM extract and (b) gallic acid. (c) Normalized double integrated of EPR spectra for BM extract and gallic acid as function of time.

136 A. Filip et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 133–142

most significant antioxidant character (kBM = 0.017, kgallic acid =0.013) (Fig. 1a–c).

3.2. Antioxidant defence parameters SOD, CAT, GPx and GSH

In order to evaluate the ability of BM extract to reduce the UV-B-induced skin damage a successively 10 days irradiation wasperformed.

MnSOD activity at 24 h after the last UV-B irradiation decreasedsignificantly compared to control group (48% inhibition;14.9 ± 4.18 vs. 28.57 ± 10.86 U/mg protein; p < 0.05). BM pretreat-ment with 2.5 mg PF/cm2 restored the MnSOD activity near to con-trol leading to a recovery of 46% in enzyme activity (27.14 ± 9.12 U/mg protein) (Fig. 2a). High concentration in PF (4 mg/cm2) main-tained the reduced MnSOD activity in skin (18.53 ± 4.64 U/mg

protein). In both groups treated with BM extract after the UVB irra-diation the activities of MnSOD increased 2.0 fold (31.15 ± 7.62respectively 31.23 ± 8.96 U/mg protein; p < 0.01), suggesting theprotective activity of extract. CuZnSOD activity evaluated inhomogenates of whole skin in groups 7 and 8 revealed increasedvalues compared to UV-B irradiated group (35.23 ± 9.96 respec-tively 28.68 ± 19.21 U/mg protein vs. 24.08 ± 5.04 U/mg protein).Similar results were obtained in groups treated with BM extract be-fore UV-B irradiation.

CAT activity insignificantly increased (34%; 1.11 ± 0.54 vs.0.74 ± 0.12 U/mg protein; p > 0.05) after UV-B exposure and noalterations in CAT activity were evidenced in group pretreated withvehicle (p > 0.05) (Fig. 2b). A dose of 2.5 mg PF/cm2 administeredbefore each UV-B exposure increased insignificantly CAT activity(1.05 ± 0.18 vs. 1.11 ± 0.54 U/mg protein; p > 0.05). The pretreat-ment with 4 mg PF/cm2 maintained the activity of enzyme at levels

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Fig. 2. Parameters of antioxidant defence: MnSOD (a), CAT (b), GPx (c) and GSH (d) in skin at 24 h after the last UV-B irradiation. Data are means ± standard deviation.Statistical analysis was done by a one-way ANOVA, with Tukeys multiple comparisons post test. �p < 0.01; ��p < 0.001; ���p < 0.0001.

A. Filip et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 133–142 137

of control and vehicle groups (0.72 ± 0.21 U/mg protein). The sameresults were obtained when the both doses of extracts were ap-plied after UV-B irradiation (0.79 ± 7.62 respectively 0.68 ±0.17 U/mg protein). The activities of CAT and MnSOD were directlycorrelated at 4 mg PF/cm2 dose administered after UV-B exposure(r2 = 0.97; p < 0.02).

GPx activity increased 2.0 fold in UV-B exposure group as com-pared to control (p < 0.02). Further, the GPx activity increased ingroup treated with vehicle and UV-B as compared to vehicle group(0.14 ± 0.02 vs. 0.07 ± 0.01 U/mg protein; p < 0.02). In BM treatedgroups the rise of GPx activity was insignificant comparatively toUV-B treated group (p > 0.05) (Fig. 2c). At 4 mg PF/cm2 of BM ex-tract administered before UV-B irradiation the GPx and CAT activ-ities were correlated (r2 = �0.55; p = 0.06).

GSH levels increased significantly (3.11 fold) in the irradiatedskin compared to the non UV-B exposed skin (20.25 ± 5.69 vs.6.51 ± 1.57 nmoles/mg protein; p < 0.0001) (Fig. 2d) whereaspretreatment with vehicle did not change the GSH levels in skincompared to UV-B group (p < 0.0001). At 2.5 mg PF/cm2 appliedbefore UV-B irradiation, the GSH generation decreased signifi-cantly (2.4 fold; 8.28 ± 2.17 vs. 20.25 ± 5.69 nmoles/mg protein;p < 0.0001). Similar results were obtained in the treatments with4.0 mg PF/cm2 (3.31 fold; 6.10 ± 1.50 nmoles/mg protein;p < 0.0001). It is noteworthy that the activity of GPx was directlycorrelated with GSH levels at 2.5 PF/cm2 dose applied beforeUV-B exposure (r2 = 0.91; p < 0.03). The two doses of extractsadministered after UV-B irradiation maintained the same low lev-els of GSH in skin (5.60 ± 0.62 respectively 3.93 ± 0.81 nmoles/mgprotein; p < 0.0001). The differences between GSH levels in PF trea-ted groups were not significant.

3.3. Cytokine level

IL-1b protein quantified by ELISA (pg/mg protein) showed thatmultiple UV-B exposures stimulated IL-1b protein synthesis in skincells (19.07 ± 8.73) vs. the control group (7.18 ± 4.05; 2.65 fold) orgroup treated with vehicle (6.58 ± 3.48; 2.89 fold) (Fig. 3a). Skintreatment with 2.5 mg PF/cm2 BM extract before UV-B exposureresulted in a significantly reduced IL-1b level in skin (7.27 ± 1.37;p < 0.05) compared with UV-B alone (62% inhibition). The level ofIL-1b was correlated with MnSOD activity in group 5 (r2 = �0.46;p < 0.08). When applied after UV-B irradiation the BM extract in-creased the IL-1b level both in group 7 (45.95 ± 30.90; 2.40 fold)as well as in group 8 (34.62 ± 24.10; 1.78 fold).

3.4. Malondialdehyde level

MDA is a useful marker for photooxidative damage. After multi-ple UV-B exposures a 30% decrease in MDA levels was evidenced ascompared to untreated and unirradiated skin. However, the differ-ence was not significant (0.50 ± 0.034 vs. 0.71 ± 0.29) (Fig. 3b).MDA levels were strongly correlated with CAT activity in UV-Bgroup (r2 = �0.68; p < 0.005) and with MnSOD activity in UV-B + BM 2.5 group (r2 = 0.96; p < 0.03). Both doses of BM adminis-tered before or after UV-B treatment did not change the MDA levelcompared to UV-B group (p > 0.05).

3.5. Effects of a multiple doses of UV-B on photoproduct induction

It is known that CPDs are formed in epidermal DNA after UV-Birradiation and are considered as important biomarkers of DNA

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Fig. 3. IL-1b (a) and MDA (b) levels in SKH-1 skin mice at 24 h after the last UV-B exposure. (a) IL-1 b secretion increased significantly after multiple doses of UV-B irradiation(240 mJ/cm2). Skin treatment with 2.5 mg PF/cm2 BM extract resulted in a significantly reduced of IL-1b level in skin. Values are mean ± SE, �p < 0.05 vs. control and BM 2.5 mgPF/cm2 treated groups. (b) MDA level in skin at 24 h after the last UV-B irradiation. Data are means ± standard deviation. Statistical analysis was done by a one-way ANOVA,with Tukeys multiple comparisons post test.

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Fig. 4. Immunohistochemical distribution of CPDs + cells in mice skin exposed to multiple doses of UV-B (240 mJ/cm2). (a) Paraffin embedded sections were subjected toimmunoperoxidase staining for CPDs + cells detection which are shown as dark brown. CPDs + cells are not detectable in the control (no UV-B exposure) skin group. Exposureto UV-B resulted in significantly increased CPDs levels in the skin of all irradiated groups, as compared to the both unirradiated groups. Original magnification 400� (Scalebar = 50 lm). Topical application of BM extract 4 mg PF/cm2 showed reduced immunostaining for UV-B-caused thymine dimer positive cells in skin. (b) The results wereexpressed as the mean percent of positive cells per field ± SD.

138 A. Filip et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 133–142

damage. Therefore, we assessed the effect of BM extract on UV-B-mediated DNA damage in mouse epidermis by immunohistochem-ical analysis. As shown in Fig. 4, UV-B exposure of mouse skin 10consecutive days with/without vehicle induced dramatically in-creased number of thymine dimers in the nuclear DNA, both in epi-dermis and dermis. Immunohistochemical analysis performed onnon UV-B irradiated and vehicle treated skin did not reveal any

detectable CPDs formation, suggesting that DNA was unaffected.Exposure to UV-B resulted in significantly increased CPDs levelsin the skin of all irradiated groups, as compared to the both unirra-diated groups. The percentage of epidermal CPDs + cells in UV-B-alone-exposed sites was 52.87 ± 8.49% (p < 0.0001). This incrementin positively stained CPDs + cells (32.23 fold) indicated the in-creased severity in DNA damage. Topical application of BM extract

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a

b

Fig. 5. Induction of sunburn cells (white arrows) and epidermal hyperplasia in unirradiated and UV-irradiated mouse skin at 24 h after the last UV-B irradiation. (a) BMextract was administered topically (2.5 and 4 mg PF/cm2), 30 min before and after the UV-B irradiation and exposures were made 10 consecutive days. Animals weresacrificed at 24 h after the last UV-B exposure; skin biopsies were sampled, preserved in 10% buffered formalin and processed for histopatological investigations.Representative photomicrographs of Trichrome-Masson and Haematoxylin–eosin stain from each treatment group with an original magnification 400� (Scale bar = 50 lm).(b) A total of ten microscopic fields were counted. The results were expressed as the mean percent of positive cells per field ± SD.

A. Filip et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 133–142 139

4 mg PF/cm2 before UV-B irradiation showed reduced immuno-staining for UV-B-caused thymine dimer positive cells in skin(35.80 ± 2.27%; 33% inhibition; p < 0.01) (Fig. 4a and b). Quantita-tively the pretreatment with 2.5 mg PF/cm2 BM extract resultedin an insignificant decreased number of thymine dimer positivecells (41.13 ± 15.87%) accounting for 33% inhibition (p > 0.05).CPDs generation in skin in 2.5 PF/cm2 treated group was correlatedwith GSH level (r2 = 0.88; p < 0.04). Both doses of BM extractadministered after UV-B exposure maintained the percentage ofepidermal CPDs + cells at level of UV-B group (56.99 ± 10.46 %respectively 57.01 ± 10.36%). In group 7 the number of CPDs + cellswere indirectly correlated with GSH level (r2 = �0.97; p < 0.02) andCAT activity (r2 = �0.94; p < 0.06).

3.6. Induction of sunburn cells in multiple doses UV-B-irradiatedmouse skin

UV-B-irradiated mouse skin was examined for the presence ofsunburn cells 24 h after the last UV-B irradiation. Time coursestudies revealed that sunburn cells are detectable as early as 8 hafter UV exposure, are maximally expressed after 24–48 h and dis-appear after 60–72 h [29]. Histological sections of haematoxylin-and-eosin-stained mouse skin following 10 consecutive days ofirradiation were examined and representative photomicrographsare illustrated in Fig. 5. In the skin of irradiated mice, we noticedthe presence of sunburn cells, exhibiting the classic feature ofapoptosis, pyknotic nuclei. As previously described, some sunburncells displayed contracted nuclei and clear cytoplasm; others had

contracted nuclei and pink cytoplasm, probably directly linked toUV induced damage [30]. These cells were mostly localized inthe basal layer of the epidermis. For the unirradiated groups, onlya few cells undergoing normal cell death were observed, with arandom distribution (0.07 ± 0.15% respectively 0.74 ± 0.51%). Inthe UV-irradiated skin, the number of sunburn cells increased sig-nificantly (2.26 ± 1.15%; p < 0.01). Formation of sunburn cells afterUV-B irradiation is primarily a consequence of DNA damage. Sun-burn cells are keratinocytes undergoing apoptosis after receiving ahigh UV-B dose that has severely and irreversibly damaged theirDNA. Pretreatment with BM extract (2.5 mg PF/cm2) reduced thenumber of sunburn cells (1.34 ± 1.17%; 41% inhibition) (Fig. 5aand b). The same results were obtained with both doses of BM ex-tract applied after UV-B irradiation (1.21 ± 0.29; 47% inhibitionrespectively 1.07 ± 0.32; 53% inhibition).

4. Discussions

Skin cancer is a significant problem associated with mortalityand morbidity; therefore concerted efforts are needed to developnovel strategies for the prevention of UV-mediated damages. Pho-tochemoprevention has been appreciated as a viable concept to re-duce the occurrence of skin cancer and, in recent years, the use ofbotanical antioxidants, have gained considerable attention as pho-tochemopreventive agents for human use.

There is ample evidence that UV-B induces reactive oxygen spe-cies which have been implicated in the pathogenesis of skin cancer[31–33]. Several studies in vitro and in vivo have shown that the

140 A. Filip et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 133–142

clinical and histological changes of irradiated skin can be inhibitedby prior application of free radicals scavenging agents [9,34].

The present study examined the photochemoprotective effectsof a natural extract obtained from Vitis Vinifera grape seeds againstthe photodamaging action of a multiple exposures of UV-B. On onehand we analyzed the adaptive modifications involving oxidativestress, the antioxidant defense, inflammation and DNA lesions in-duced by multiple UVB irradiations and on the other hand we ana-lyzed the way the topical treatment with BM extract in differentdoses applied before irradiation or 30 min after influences theseparameters. Our results demonstrated that BM extract adminis-tered before each irradiation could inhibit multiple exposuresUV-B-induced skin damage by modulating the antioxidant defenceand inflammation and by reducing the apoptotic and DNA lesions.When applied 30 min after irradiation for 10 days the effects aresimilar regarding the antioxidant enzymes activities and percent-age of sunburn cells but without conferring an efficient protectionof DNA lesions.

In vitro, the grape seed extract exhibited appreciable antioxidantactivity demonstrated by ABTS test and EPR measurements. There-fore it might be suggested that BM has a greater efficiency in block-ing the chain reactions produced by ROS in skin as a consequenceto UV-B exposure.

Multiple doses of UV-B (240 mJ/cm2) administered 10 consecu-tive days induced some changes compared with the control andvehicle treated groups for the same duration. Generally, exposureof skin to UV-B radiation results in increased ROS generation whichcontributes to several pathological conditions, including photocar-cinogenesis and photoaging [35]. To defend against damaging freeradical-mediated reactions, cells possess enzymes and smallmolecular weight molecules with antioxidant activity [36]. Severalreports have studied the effects of multiple doses of UV-B on ROSrelated biomarkers in animal skin but the results are different,sometimes difficult to be interpreting probably due to the adaptivemodifications that arise in the skin and the different experimentalparameters. Further more, the antioxidant enzymes in skin are notalways regulated coordinative in the chronic exposure to UV-B.

Two foremost antioxidant defense enzymes in the skin are SODand CAT. Superoxide dismutase belongs to major antioxidant en-zymes that contribute to the homeostasis of oxygen radicals inthe skin. The dismutation of superoxide by SOD results in the pro-duction of hydrogen peroxide, which is subsequently converted towater and oxygen through a reaction that is catalyzed by CAT. Inour experiment, MnSOD activity decreased after multiple UV-Bexposures and increased in pretreatment with both doses of BMextract, to a value close to control and vehicle groups. The increasein MnSOD activity is maintained in the groups that received BM30 min after irradiation. The lower MnSOD activity observed afterUV-B irradiation was most likely due to the mitochondrial damage[37,38]. Data in literature concerning this antioxidant enzymesactivity following UV irradiation are rather contradictory. Hachtro-udi et al. showed that irradiation of the epidermal cells with UV-Binduced a release of soluble factors (IL-1a, IL-1b, TNF-a) thatamplified MnSOD activity in fibroblasts via a paracrine mechanism[39]. We found that SOD activity decreased after multiple doses ofUV-B in correlation with increased IL-1b level in skin. In otherstudies, UV-B irradiation of human keratinocytes induced a signif-icant increase in SOD activity and protein level. This increase inSOD was attributed to CuZnSOD [40]. Our data confirm these re-sults. Some authors showed that the CuZnSOD activity was notinfluenced by UVB irradiation but the activity of MnSOD was in-creased by small doses of UVB and reduced after irradiation in highdoses [41].

We found that CAT had increased activity at 24 h after last UV-Bexposure in correlation with decreased MDA level in skin. Sharmaet al. reported a decrease in CAT activity after a single UV exposure

which was doubled after multiple irradiations [40]. Similarly,Svobodova et al. confirm a significant decrease in CAT activity inwhole skin following UVB (20 J/cm2, 24 h) [41]. In vitro experi-ments have revealed that the dose-dependent decline in catalaseactivity after UV exposure is mainly due to the direct photode-struction [2,42]. Iizawa et al. noticed that chronic UV-B irradiationhad no effect on CAT activity in mice [43]. In our experiment, only adose of 4 mg PF/cm2 of BM extract applied before each UVB expo-sure restore the CAT activity in skin close to control or vehicle val-ues. The same effects were noticed in groups treated with BMextract applied after UV-B exposure. Our results are in agreementwith those of Jeon et al. They showed that oral intake of catechinbefore UV-B irradiation increased CAT activity compared to UV-Bgroup [44].

Glutathione peroxidase is considered to be the most importantantioxidant defence system in the skin [2]. GPx is a selenoproteinthat catalyses the conversion of UV-induced hydrogen peroxideinto water and molecular oxygen using GSH as unique hydrogendonor. In our experiment, the multiple UV-B exposures inducedactivation of GPx. These data are consistent with studies showingthat GPx-1 expression increased in undifferentiated and differenti-ated keratinocytes after UV-B irradiation [45]. In hairless rats, GPxactivity was increased following UV-B exposure (1540–2410 mJ/cm2) [46]. We assigned the GPx activity and GSH levels enhance-ment after UV-B irradiation of skin as being due to the accumula-tion of lipid peroxides and hydrogen peroxide in cells. Severalstudies revealed that the GPx activity is not strongly affected byUV [47]. Some authors showed that either single or multiple UV-B irradiations of skin reduced significantly the GPx activity, respec-tively; by 31% and 44% comparative to control group [48]. In paral-lel, the GSH level decreased after single exposure (27%), as well asafter multiple exposures (39%), compared to non-UV-B irradiatedgroup. BM (2.5 mg PF/cm2) extract applied before UV-B exposureslightly increased the enzyme activity but its activity was not influ-enced by the other therapeutic regimes.

GSH is a major non-protein thiol in living organisms, whichplays a central role in coordinating the body’s antioxidant defenseprocess. The results obtained showed that the GSH level was signif-icantly increased in skin homogenates after multiple doses of UV-B. It is known that in UV-B exposure the transcriptional regulatorNF-E2-related factor 2 (Nrf2) is involved. It activates the expres-sion of a battery of ROS-detoxifying enzymes and stimulates theproduction of antioxidants, including the glutathione [49]. Anotherstudy showed that in vitro human skin fibroblast cells respond tomultiple doses of UV-B by a transient rapid up-regulation of theactivity of the antioxidant enzymes SOD and CAT, and a slowerbut more marked increase in cellular GSH content [50]. It seemsthat the mechanisms underlying these adaptive responses are cur-rently unclear. Moreover, due to the use of different UV-B lightsources and various experimental designs, the time interval whenthe evaluation is made, the results are conflicting and difficult togeneralize from or compare. In our study the GPx activity doesnot evolve in parallel to the level of GSH in the skin. It is possiblethat methods used to detect GSH could underestimate the peptideamount in our experimental conditions. The treatment with BM ex-tract decreased the GSH levels in skin in a dose related manner.Similar results were obtained in previous experiments when usedBM extract in association with one single UV-B exposure [15]. Allthe therapeutic regimens studied restored GSH levels close to nor-mal levels.

We evaluated also the effect of BM extract on UV-B-mediated li-pid peroxidation, which is a well accepted marker of oxidativestress. Multiple doses of UV-B irradiation had no significantly effecton MDA levels in mice skin. This effect could be explained by coun-teraction of an increased CAT activity. At 2.5 mg PF/cm2 the MDAlevel in skin decreased while the SOD and GPx activities increased.

A. Filip et al. / Journal of Photochemistry and Photobiology B: Biology 105 (2011) 133–142 141

The pretreatment of skin with 4 mg PF/cm2 increased MDA levelclose to control group probably due to on prooxidant effect. In pre-vious experiment, one single UV-B irradiation induced a 3.0 fold in-crease in MDA levels in skin at 24 h. Iizawa et al. reported anincrease of lipid peroxide levels in skin after acute UV-B irradiationand decrease in chronic irradiation after 1 week, and then return tobaseline after 3 weeks [43]. Our results are in agreement with Ari-cioglu et al. [51]. They confirm that the lipid peroxides level inmultiple exposures to UV-B increased in skin only in first daysand then they decreased gradually at normal levels, probably dueto an adaptive mobilization of the antioxidant defense systems[43,51].

Histological, beside the sunburn cells in the skin of UV-irradi-ated mouse, in some cases, inflammatory cells were present. Theyvaried from just a few mononuclear cells in the dermis (BM treatedgroups) to an abundant infiltrate, composed of mononuclear cellsand polymorphous neutrophils, accompanied by an increasednumber of mast cells (triggers of inflammation) [52]. Inflammatoryinfiltrate was mainly present around the blood vessels and wasmore severe in groups vehicle-irradiated and irradiated withoutprotection. In the same way, the IL-1b level increased after multi-ple UV-B exposures while the pretreatment with 2.5 mg PF/cm2 ofBM extract reduced the release of this cytokine from skin cells(p < 0.05). Additionally, the increasing of SOD activity was corre-lated with reducing of IL-1b level in skin treated with 2.5 mg PF/cm2. In groups treated with BM extract applied after UV-B irradia-tion IL-1b level increased due to releasing from inflammatory cellsrecruited into the skin.

These changes were accompanied by epidermal hyperplasia inthe UV-B-irradiated skin of all groups. Similar to inflammation,epidermal hyperplasia was more severe in the unprotected UV-irradiated groups (untreated or treated with vehicle). In somecases, we observed cells in focal areas of the epidermis that wereswollen and had fine granular cytoplasm, with a mainly basal dis-tribution (data not shown). In the UV irradiated groups, epidermalspongiosis of different intensities was also observed. This is an-other lesion directly linked to the effects of UV radiation on theskin [53]. In topical application before UV-B irradiation, PF contain-ing products reduce cyclobutane – pirimidine dimers formation inskin [54]. The treatment with 4 mg PF/cm2 inhibited UV-B-inducedDNA damage as detected by CPDs quantification (p < 0.01). Thisprotective effect of BM could be due to an interference with UVabsorption in epidermis – sunscreen effect (both physical andchemical) and interaction at molecular level in skin. The compo-nents of grape seeds extract being flavonoids have been shownto absorb radiation from the entire UV-B spectrum and when ap-plied topically they can provide additional protection against radi-ation penetration [55,56]. Topical BM treatment before a singledose of UV-B (240 mJ/cm2) did not inhibit CPDs formation at 1 hafter irradiation but strong protected and inhibited CPDs formationat 24 h (50% inhibition) [15]. When the BM extract was appliedafter UV-B exposure the protective effects decreased in correlationwith decreased GSH levels and CAT activity in skin. In addition, it ispossible that the DNA lesions once produced due to multiple dosesof UV-B may not be repaired in due time by the extract doses used.

Several studies demonstrated that human keratinocytes can un-dergo apoptosis in response to DNA damage-UV-B induced. In pre-vious acute experimental model, one single dose of UV-B inducedapoptosis in skin cells, an event reversibly restored after topicalapplication of the BM extract and (�)-epigallocatechin gallate solu-tion [15]. After multiple doses of UV-B exposure with/withoutvehicle the number of sunburn cells increased (p < 0.01). This sug-gests that the UV-B-induced DNA damage exceeded the cells’capacity to repair the DNA lesions leading to the activation of theapoptotic pathway. Kulms and Schwarz postulated that UV-dam-aged keratinocytes that have failed to repair their DNA damage will

die as sunburn cells, thus escaping the risk of becoming malignant[57]. When applied 2.5 mg PF/cm2 the number of sunburn cells de-creased (41% inhibition) demonstrating that BM extract protectedthe skin. The effect on sunburn cells formation is no dose-depen-dent suggesting that BM was protective due the interaction atmolecular level in skin. This effect is confirmed by the low percent-age of sunburn cells in the groups treated with both doses of BMextract after UV-B irradiation (47% respectively 53% inhibitions).

In summary, our data demonstrated that multiple UV-B irradi-ations disturbs the oxidant-antioxidant balance in SKH-1 hairlessskin by adaptative increasing of GPx activity and GSH level, byreleasing of inflammatory mediators and producing genotoxicand apoptotic lesions. The pretreatment with BM variety grapeseeds extract, reduced DNA lesions, modulated the inflammatoryand apoptotic responses to UV-B and increased the antioxidant en-zymes activities. The protective effects, especially on oxidativestress and apoptosis, were maintained when the extract wasadministered after UV-B irradiation. Further research is neededto clarify the mechanisms involved in effect of BM extract on geno-toxic lesions and inflammation generated in skin exposed to multi-ple doses of UV-B. Moreover, is very important to determine theprecise role of DNA photoproducts vs. oxidative stress or inflam-mation in initiating the carcinogenetic process.

Acknowledgements

This work was supported by the Ministry of Education, Researchand Youth by PN II Program (42-104/2008).

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