Accepted Manuscript Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line I.F. Almeida, A.S. Pinto, C. Monteiro, H. Monteiro, L. Belo, J. Fernandes, A.R. Bento, T.L. Duarte, J. Garrido, M.F. Bahia, J.M. Sousa Lobo, P.C. Costa PII: DOI: Reference:

S1011-1344(15)00022-6 http://dx.doi.org/10.1016/j.jphotobiol.2015.01.010 JPB 9930

To appear in:

Journal of Photochemistry and Photobiology B: Biology

Received Date: Revised Date: Accepted Date:

10 November 2014 5 January 2015 19 January 2015

Please cite this article as: I.F. Almeida, A.S. Pinto, C. Monteiro, H. Monteiro, L. Belo, J. Fernandes, A.R. Bento, T.L. Duarte, J. Garrido, M.F. Bahia, J.M. Sousa Lobo, P.C. Costa, Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line, Journal of Photochemistry and Photobiology B: Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.01.010

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Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line IF Almeida*1, AS Pinto1, C Monteiro1, H Monteiro1, L Belo2,3, J Fernandes3,4, AR Bento3, TL Duarte3, J Garrido5, MF Bahia1, JM Sousa Lobo1, PC Costa1 1

Laboratório de Tecnologia Farmacêutica, Departamento de Ciências do Medicamento

Faculdade de Farmácia da Universidade do Porto, Porto, Portugal 2

Laboratório de Bioquímica, Departamento de Ciências Biológicas, Faculdade de

Farmácia da Universidade do Porto, Porto, Portugal 3

Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto,

Portugal 4

Institute for Biomedical Imaging and Life Science (IBILI), Faculdade de Medicina,

Universidade de Coimbra, Coimbra, Portugal 5

CIQ-Departamento de Engenharia Química, Instituto Superior de Engenharia do Porto

(ISEP), Politécnico do Porto, Portugal

*Corresponding author Isabel F Almeida Laboratório de Tecnologia Farmacêutica, Departamento de Ciências do Medicamento Faculdade de Farmácia da Universidade do Porto Rua Jorge Viterbo Ferreira nº 228 4050-313 Porto - PORTUGAL Tel: + 351 22 0428621 e-mail: [email protected]

1

Abstract

Toxic effects of ultraviolet (UV) radiation on skin include protein and lipid oxidation, and DNA damage. The latter is known to play a major role in photocarcinogenesis and photoageing. Many plant extracts and natural compounds are emerging as photoprotective agents. Castanea sativa leaf extract is able to scavenge several reactive species that have been associated to UV-induced oxidative stress. The aim of this work was to analyze the protective effect of C. sativa extract (ECS) at different concentrations (0.001, 0.01, 0.05 and 0.1 µg/mL) against the UV mediated-DNA damage in a human keratinocyte cell line (HaCaT). For this purpose, the cytokinesis-block micronucleus assay was used. Elucidation of the protective mechanism was undertaken regarding UV absorption, influence on 1O2 mediated effects or NRF2 activation. ECS presented a concentration-dependent protective effect against UV-mediated DNA damage in HaCaT cells. The maximum protection afforded (66.4%) was achieved with the concentration of 0.1 µg/mL. This effect was found to be related to a direct antioxidant effect (involving 1O2) rather than activation of the endogenous antioxidant response coordinated by NRF2. Electrochemical studies showed that the good antioxidant capacity of the ECS can be ascribed to the presence of a pool of different phenolic antioxidants. No genotoxic or phototoxic effects were observed after incubation of HaCaT cells with ECS (up to 0.1 µg/mL). Taken together these results reinforce the putative application of this plant extract in the prevention/minimization of UV deleterious effects on skin.

2

1.

Introduction

DNA damage is one of the toxic effects of skin exposure to ultraviolet (UV) radiation and plays a major role in photocarcinogenesis and photoageing [1]. Direct absorption of UVB (and to a lower extent UVA) by cellular DNA leads to the formation of pyrimidine

base

lesions

including

cyclobutanepyrimidine

dimers

(CPDs),

6-4 photoproducts (6-4 PPs), and Dewar isomers (formed by photoisomerization of 6-4 PPs), which become covalently linked and distort the DNA helix. In addition, exposure of skin cells to UVA and near visible radiation leads to the formation of 1O2 (singlet oxygen) due to the excitation of cellular chromophores or photosensitizers (e.g. flavins, porphyrins) [2]. 1O2 was detected directly in human skin following UVA irradiation by measurement of its luminescence at 1270 nm [3]. Several other reactive oxygen species (ROS) have been identified in the skin after UV exposure using specific ROS quenchers, namely O2˙- (superoxide radical), ROO˙ (peroxyl radical) and HO˙ (hydroxyl radical) [4, 5]. UV-induced oxidative stress is responsible for several types of DNA damage, such as DNA strand breaks and the base lesions 7,8-dihydro-8-oxoguanine (8-oxoGua), thymine glycol, and 5-hydroxymethyluracil [6, 7]. Many plant extracts and natural compounds are emerging as candidates for minimization of the effects of UV radiation on skin. The UV-protective effects of these ingredients are versatile and include (i) direct absorption (UV filters); (ii) inhibition of chronic inflammation; (iii) modulation of immunosuppression; (iv) induction of apoptosis; (v) direct antioxidant (i.e., scavenging of reactive oxygen and nitrogen intermediates); and (vi) indirect antioxidant (i.e., induction of intrinsic cytoprotective responses through the KEAP1/NRF2/ARE system) [8]. Plant antioxidants such as green tea catechins, soya isoflavones, and resveratrol have recently been investigated with promising results [9, 10]. A leaf extract from Castanea sativa (ECS) was previously shown to exhibit in vitro scavenging activity against several reactive species that are detected in the skin after UV exposure, including 1O2 [11]. This botanical extract showed good skin compatibility [12] and was successfully incorporated in a stable semisolid formulation [13]. The extraction process used to prepare ECS is reproducible, which is a key parameter regarding industrial processing [14]. Taken together these data are favorable for application of this extract in skin care products. For a more comprehensive characterization of its activity on the prevention/minimization of UV-induced damaging

3

effects on the skin it is necessary to conduct studies in skin cells with biomolecules, such as DNA, which are known to be affected by UV radiation. The current study assessed the ECS protective effect towards UV-induced-DNA damage in HaCaT human keratinocytes. Elucidation of the protective mechanism was undertaken regarding UV absorption, influence on 1O2 mediated effects or NRF2 activation. Cytotoxicity and genotoxicity were also determined. Redox potentials of ECS and the major phenolic compounds present in its composition were evaluated.

2. 2.1

Materials and Methods Reagents

Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicilin/streptomycin, horseradish peroxidase-conjugated goat anti-rabbit IgG, TriZol, Turbo DNA-free kit and ThermoScript RT-PCR System were obtained from Life Technologies (Carlsblad, CA, USA). All other chemicals and reagents were purchased from Sigma–Aldrich (Munich, Germany) unless stated otherwise. 2.2

C. sativa leaf extract preparation

Dried leaves (4 g) were grounded, sieved (500 µm) and extracted five times (5 × 100 mL) with ethanol:water (7:3) at 40°C under magnetic stirring (10 min, 500 rpm). Each alcoholic extract was filtered using a glass filter G4 funnel (5–15 µm porosity, Schott, Mainz, Germany), and gathered in a kitasato flask. Ethanol was evaporated at 40°C under vacuum with a rotary evaporator (R-114, Büchi, Switzerland). Finally, the resulting aqueous mixtures were lyophilized for 48 h (Cryodos, Telstar, Spain), generating the final dry extract, which was kept in an amber flask at room temperature in a desiccator. 2.3

Cell culture conditions

The immortalized human keratinocyte cell line (HaCaT) was obtained from Cell Lines Service (CLS, Germany). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1% penicillin/streptomycin (growth medium). For the experiments, HaCaT cells were incubated with freshly prepared dilutions of the 4

ECS (dissolved in glycerin:water solution 1:1) in growth medium for the specified time and concentration range. Cell passages used were from 47 to 49. 2.3.1 Cytotoxicity Cytotoxicity was evaluated using the 3-[4,5-dimethyl-2-yl]-2,5-diphenyl tetrazolium

bromide (MTT) assay. HaCaT cells (3×104 cells/well, 96-well microplates) were incubated with different concentrations of ECS (0, 0.5, 5, 50, 100, 250, 500 µg/mL). After 24 h incubation, cells were washed with Hank's Balanced Salt Solution (HBSS) and MTT (0.5 mg/mL) was added. After 2 h incubation, dimethyl sulfoxide (DMSO) was added and absorbance was measured at 562 nm. As negative control, the assay was conducted with cells and the solvent. A previous assay demonstrated that ECS did not reduce MTT for the concentrations tested (data not shown). 2.3.2 Protective effect against DNA damage induced by UV radiation Irradiation conditions UV irradiation was conducted with an Ultra-Vitalux® lamp (Osram, Germany) which emits radiation similar to natural sunlight. In comparison to other solar simulators (like xenon lamps), it has the advantage of being more economic and releases less heat. To prevent contamination from other light sources, the assay was performed in a dark room.. A radiometer (UVM-7, Arimed, USA) was used to determine the UVA irradiation intensity. The distance between cells and the light source was set to 41 cm. Irradiation time was adjusted to obtain the desired UVA irradiation doses (approximately 3 to 5 min). Cytokinesis – block micronucleus assay (CBMN) DNA damage was induced by exposing HaCaT cells to UV radiation. HaCaTs (5×104 cells/well, 12-well microplates) were incubated with different concentrations of ECS (0, 0.001, 0.01, 0.05 or 0.1 µg/mL) or solvent (control) for 30 min prior to UV radiation (UVA 0.5 J/cm2). Cytochalasin-B (Cyt-B) was added (5 µg/mL) at 32 h (before completion of 2nd cell division) after irradiation. The irradiation dose used was previously established as being genotoxic but non-cytotoxic. Cells were fixed in methanol:glacial acetic acid (3:1) 55 h after irradiation, dropped onto clean microscopic slides, air-dried and stained with Wright colouration. For each sample, 1000 binucleated keratinocytes were blindly scored using an optical microscope. DNA damage was expressed as the number of micronucleus (MN) formed. DNA damage in 5

the presence of ECS in the non-irradiated samples was taken as measure of its genotoxicity. Cytotoxicity was also evaluated under these experimental conditions, using the trypan blue exclusion test. The same assays were conducted in the absence of irradiation. Protective effect was calculated with the following equation:

 MNs Protective effect (%) = 1 −  MNc

  × 100 

where MNs is the number of micronuclei counted in the presence of ECS and MNc is the number of micronuclei counted in the control. 2.3.3. Elucidation of the protective effect mechanism UV absorption The absorption spectrum of ECS in DMEM (0.1 µg/mL) was recorded between 290 nm and 400 nm in a UV-Vis spectrophotometer (V-650 Jasco, Japan) using water as a baseline. DMEM absorption spectrum was also obtained. Protective effect against 1O2 -induced DNA damage Oxidative DNA damage was induced by exposing cells to the polar photosensitizer Ro19-8022 in the presence of light. HaCaT cells (4×104 cells/well, 24-well microplates) were incubated with ECS (0.5 µg/mL) or solvent for 6 h. After washing with phosphatebuffered saline (PBS), cells were incubated with PBS containing 1 µM Ro19-8022 (obtained in kindness from Roche, Basel, Switzerland) and immediately irradiated on ice at 35 cm distance from a 500-W halogen lamp to induce oxidative modification of DNA via 1O2. After trypsinization, cells were suspended in 0.6% low-melting point agarose. Eighty microliters of the agarose gel (containing approximately 2×104 cells) were dispensed onto glass microscope slides previously coated with 1% normalmelting-point agarose. The agarose was allowed to set on ice under a coverslip and the slides were left overnight in ice-cold lysis buffer (100 mM disodium EDTA, 2.5 M NaCl, 10 mMTris–HCl, pH 10, containing 1% Triton X-100 added fresh). Slides were washed once with distilled water and immersed in two changes of enzyme digestion buffer [40 mM Hepes, 0.1 M KCl, 0.5 mM EDTA, and 0.2 mg/mL bovine serum albumin (pH 8.0)], for 5 min each time, at room temperature. Gels were covered with a coverslip and incubated with human 8-oxoguanine DNA glycosylase 1 (hOGG1, New England Biolabs, Hitchin, UK), diluted 1:500 in enzyme reaction buffer to a final concentration of 6

3.2 U/mL, or enzyme buffer (50 µL/gel) in a humidified chamber at 37°C for 45 min. The coverslips were removed and the slides were placed in a horizontal electrophoresis tank, covered with cold alkaline electrophoresis buffer (300 mM NaOH, 1 mM disodium EDTA, pH≥13) for 20 min and electrophoresed for 20 min at 0.66 V/cm, 300 mA. Slides were neutralized with PBS for 20 min, washed with distilled water, and then allowed to dry. All procedures were carried out under subdued light to minimize adventitious DNA damage. For staining, slides were re-hydrated in distilled water, incubated with a freshly made solution of 2.5 µg/mL propidium iodide for 20 min, washed again for 30 min and allowed to dry. Comets were visualized by fluorescence microscopy at ×200 magnification. Images were captured by an on-line CCD camera and analyzed with the Comet IV software version 4.3 (Perceptive Instruments, Suffolk, UK). A total of 100 cells were analyzed per sample, 50 per duplicate slide. DNA damage was expressed as the median of percentage of DNA in the comet tails. The level of hOGG1 sensitive sites was obtained as the difference in score between gels that had been incubated with enzyme or buffer. Activation of the endogenous antioxidant response HaCaT cells were incubated with extract (5 µg/mL) or solvent for 6 h. We assessed the levels of NRF2 protein in nuclear extracts and the steady-state mRNA expression levels of 5 genes with antioxidant functions: NAD(P)H dehydrogenase, quinone 1 (NQO1), glutamate-cysteine ligase, catalytic subunit (GCLC), heme oxygenase (decycling) 1 (HMOX1), superoxide dismutase 2, mitochondrial (SOD2) and catalase (CAT). For protein analysis, nuclear extracts from ECS- and control-treated HaCaT cells were prepared exactly as described by Oliveira et al. (2009) [15]. Total protein content was measured using the RC/DC Protein Assay (Bio-Rad, Hercules, CA) and 15 µg were resolved by electrophoresis on sodium dodecyl sulphate polyacrylamide gels. Proteins were transferred to nitrocellulose Hybond-C Extra membranes (Amersham Biosciences, Little Chalfont, UK). After blocking with 5% dry milk in Tris-buffered saline for 1 h, membranes were incubated with rabbit anti-NRF2 antibody (H-300, Santa Cruz Biotechnology) overnight at 4°C, followed by a horseradish peroxidase-conjugated goat anti-rabbit IgG (Invitrogen) for 1 h at room temperature. After washing with TBS-T, signal was developed with the SuperSignal West Pico (Pierce, Rockford, IL) chemiluminescence kit and the blots exposed to CL-X Posure films (Pierce, Rockford, IL). For normalization of protein loading, blots were stripped and reprobed with an antibody against Lamin B1 (S-20, Santa Cruz Biotechnology). 7

For the analysis of antioxidant gene expression, total RNA was extracted using TriZol, followed by DNase treatment. First-strand cDNA was prepared using the ThermoScript RT-PCR System according to the manufacturer's instructions. Relative gene expression levels were quantified using an iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Primer sequences were designed with Primer 3 software [16] and are listed in Table 1. All reactions were performed in a total volume of 20 µL with iQ SYBR Green Supermix (Bio-Rad). The amplification protocol consisted of denaturation at 95°C for 4 min and 40 cycles of 94°C for 30 s, annealing temperature for 45 s, and 72°C for 30 s. The quantity of each transcript was estimated against the respective standard curve and normalized against the quantity of the endogenous control gene Hypoxanthine phosphoribosyltransferase 1 (HPRT1).

8

CAT

AGCCTTCGACCCAAGCAACATGCC

AGGCGATGGCGGTGAGTGTCAGGAT

59 °C

GCLC

GCAGTGGTGGATGGTTGTGGCAAG

CCTTCCTTCCCATTGATGATGGTGT

59 °C

HMOX1a

GCAGTCAGGCAGAGGGTGATAGAAG

TGGTCCTTGGTGTCATGGGTCAG

57 °C

HPRT1

GCAGACTTTGCTTTCCTTGGTCAG

GTCTGGCTTATATCCAACACTTCGTG

57 °C

NQO1

AGATGCTGACTGGCACTGGTGGTT

AATTGCAGTGAAGATGAAGGCAACA

57 °C

SOD2

AAATTGCTGCTTGTCCAAATCAGGA

AGTAAGCGTGCTCCCACACATCAA

59 °C

9

Autolab, Netherlands). The working electrode used was a glassy carbon electrode

Annealing temperature

three-electrode cell in an Autolab PGSTAT 12 potentiostat/galvanostat (Metrohm

Reverse primer sequence (5'-3')

Differential pulse voltammetry (DPV) experiments were performed with a conventional

Forward primer sequence (5'-3')

2.4

Gene ID

Electrochemical methods

Table 1. Primer sequences

(GCE, d = 2 mm), the counter electrode was a platinum wire, with a saturated Ag/AgCl reference electrode completing the circuit. All experiments were conducted at room temperature (25 ± 1°C). Stock solutions of the phenolic compounds (ellagic acid, chlorogenic acid and rutin) were prepared at a concentration of 10 mM in methanol immediately prior to the analyses. From these solutions, an appropriate aliquot was removed and mixed with 10 mL of 0.1 M phosphate buffer solution (pH 7.4), resulting in a final concentration of 0.1 mM. For ECS methanolic solutions, a stock solution (0.6% m/V) was prepared in methanol and diluted in 10 mL of the supporting electrolyte (phosphate buffer), resulting in a concentration of 60 µg/mL in the electrochemical cell. Redox potentials were also determined using the cell culture medium. 2.5

Statistical analysis

The protective effect of ECS towards UV induced DNA damage was analyzed by linear regression. Statistical evaluation of cytotoxicity results was performed using one-sample student's t-test with test value 100. Gene expression differences between ECS and solvent were determined by independent samples student's t-test. Differences among multiple groups were compared with one-way analysis of variance followed by Tukey's multiple comparison test. Significance level was 0.05. Statistical analysis was conducted with IBM® SPSS® Statistics, version 22 (IBM Corp., Armonk, NY, USA).

3. 3.1

Results and Discussion Cytotoxicity

Cell viability was approximately 100% for the ECS concentrations of 0.5 and 5 µg/mL. For the concentration of 50 µg/mL, cell viability was 85.3% (significantly different from 100, p=0.004) (Fig 1). Higher concentrations markedly reduced the viability of HaCaT cells. Following these results ECS concentrations up to 5 µg/mL can be considered non cytotoxic for HaCaT cells.

10

Figure 1 Effect of pre-incubation with ECS for 24 h on HaCaT viability analyzed with the MTT assay. Each data point represents the mean ± standard deviation (n≥3). 3.2

Protective effect against DNA damage induced by UV radiation

UV radiation is responsible for damaging DNA in skin cells, which can lead to several deleterious effects including skin cancer and photoaging. In the current study, the ability of ECS to protect HaCaT cells against the genotoxic effects of UV light was evaluated. Cells were incubated with ECS or solvent prior to UV irradiation (UVA - 0.5 J/cm2) and the MN formed were counted. MN are small particles consisting of either acentric fragments or entire chromosomes that hold back at anaphase of cell division. MN can only be expressed in cells that complete nuclear division, so a special technique was established that identifies such cells by their binucleated appearance when cytokinesis is blocked by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block MN assay enables better precision as the data acquired are not confounded with altered cell division kinetics caused by cytotoxicity of the agents tested or by sub-optimal cell culture conditions. This methodology has been used to assess the UV-induced DNA damage in keratinocytes [17]. The number of MN formed in the presence of increasing concentrations of the ECS was lower than observed for control (45.4 ± 6.42). ECS presented a concentration-dependent 11

protective effect in a linear fashion (R2= 0.906 p=0.048, Fig. 2). The maximum protection afforded (66.4%) was achieved with the concentration of 0.1 µg/mL. The number of MN was also counted in non-irradiated (NR) samples (control: 5.20 ± 1.48 and 0.1 µg/mL of ECS: 6.25 ±1 .71).

Figure 2 Effect of pre-incubation with ECS on DNA damage induced by UV irradiation on HaCaT cells. HaCaTs were pre-incubated with the extract at different concentrations for 30 min prior to UV radiation (0.5 J/cm2 -UVA). Each datapoint represents the mean ± standard deviation of 2 independent experiments, each corresponding to mean number of micronucleus in 1000 binucleated keratinocytes.

The similarity between both counts shows that the extract itself is not genotoxic. Cell viability was approximately 100% for the all the concentrations tested (Fig. 3). Similar results were obtained following UV radiation, which suggests that ECS is not phototoxic at the UV doses used (Fig.3). CBMN assay has emerged as one of the favorite methods for evaluating DNA damage since it allows both chromosome loss and chromosome breakage to be measured reliably and has been used to quantify DNA 12

damage in skin cells [17]. Noteworthy, since the European Union regulation nº 1223/2009 has come into force, animal testing of cosmetic and ingredients was banned and the study of their efficacy and safety has move towards in vitro methodologies. In this context, CBMN assay can be a valuable tool to assess the protective activity of cosmetic ingredients against genotoxic agents as well as to characterize their genotoxicity.

Figure 3 Effect of pre-incubation with ECS on HaCaT cell viability analyzed with the trypan blue assay, with and without exposure to UV radiation (0.5 J/cm2 –UVA). Each datapoint represents the mean ± standard deviation (n=3). 3.3

Elucidation of the protective mechanisms

Several experiments were subsequently conducted with the aim of elucidating the protective action of the ECS against UV-induced DNA damage. To exclude the possibility that the ECS absorbed UV radiation, the absorption spectrum of DMEM in the presence or absence of ECS was compared. No differences were observed between the two spectra (data not shown) which suggests that the UV-protective effect of ECS is related to a mechanism other than direct UV light absorption. We have previously reported the ability of the ECS to scavenge in vitro several ROS that are detected in the skin after UV exposure, including 1O2 [10]. This ROS is one of the most established players in the UV-induced oxidative stress in skin [2, 3, 18]. In the present study, we aimed to evaluate the biological significance of this direct antioxidant effect of the extract in HaCaTs. Cells were incubated with ECS or solvent prior to treatment with the polar photosensitizer Ro19-8022 in the presence of light. The 13

system is a well-established generator of singlet oxygen, leading to the formation of oxidized purines, predominantly 8-oxoGua [19]. Oxidative DNA damage was then measured with the alkaline comet assay in conjunction with the hOGG1 repair glycosylase [20]. hOGG1 removes oxidation products of guanine from DNA, mainly 8-oxoGua, with great specificity [21]. In the method, hOGG1 acts at the sites of oxidized bases, leaving apurinic/apyrimidinic sites that are converted into breaks in the assay. As expected, exposure of HaCaTs to the photosensitizer plus light resulted in a significant increase in the number of hOGG1-sensitive sites (Figs. 4 A and B). Incubation of HaCaTs with ECS up to a concentration of 0.5 µg/mL caused no significant changes in the basal DNA damage, but it significantly reduced the number of lesions generated by the singlet oxygen generating system (Figs. 4 A and B).

Figure 4 Effect of pre-incubation with ECS on DNA damage induced by 1O2 in HaCaT cells. HaCaTs were pre-incubated with the extract (0.5 µg/mL) for 6h prior to sham irradiation or exposure to Ro19-8022 + light. Oxidatively modified DNA was measured using the alkaline comet assay as hOGG1-sensitive sites. A, Representative micrographs of hOGG1-treated samples, where the percentage of the DNA in the ‘tail’ of the comet is proportional to the damage. B, Quantification of hOGG1-sensitive DNA lesions in 3 independent experiments. Results are shown as individual data points and the mean ± standard deviation (n=3).

Whilst the previous results suggest that the UV-protective effect of ECS is due to its direct antioxidant action, we cannot exclude the contribution of an indirect antioxidant effect such as the activation of endogenous antioxidant defenses by the ECS. NRF2 (nuclear factor-erythroid related factor-2) is a redox sensitive transcription factor and a 14

master regulator of the transcriptional activation of genes encoding cytoprotective proteins. Many chemopreventive phytochemicals are known to activate NRF2 either by oxidative or covalent modification of its cytosolic repressor Kelch-like ECH-associated protein (KEAP1) or by phosphorylation of NRF2. Upon activation, NRF2 translocates to the nucleus and binds to the antioxidant response element (ARE) located in the promoter regions of genes encoding cytoprotective proteins [22]. The ECS composition includes several polyphenolic compounds, e.g. ellagic acid [12]. Recently, Hseu et al. [23] reported that this cinnamic acid protects HaCaT cells against UVA induced-DNA damage and attributed the protective effect to augmented nuclear translocation and transcriptional activation of NRF2. We have thus investigated the ability of ECS to activate NRF2 signalling in HaCaTs. Since the content of ellagic acid present in ECS is low (0.42%), we have incubated HaCaTs with the highest non-cytotoxic concentration of ECS (5 µg/mL) for 6 h. We assessed the levels of NRF2 protein in nuclear extracts and the steady-state mRNA expression levels of 5 genes with antioxidant functions. As depicted in Fig. 5, we found no evidence of nuclear accumulation of NRF2 in HaCaTs incubated with ECS. Likewise, the incubation of HaCaTs with the ECS resulted in no significant changes in the expression of NRF2 target genes (NQO1, GCLC, HMOX1, SOD2 and CAT).

15

Figure 5 Analysis of NRF2 activation in HaCaT cells incubated with ECS. HaCaTs were incubated with the extract or solvent (5 µg/ml) for 6 h. A, The nuclear fraction was prepared and used for Western blot analysis of NRF2 and Lamin B1. The immunoblot is representative of 3 experiments. B, The expression of antioxidant genes was determined by real-time RT-PCR. Each column represents the mean and error bars represents the standard deviation (n = 3). 16

This is possibly explained by the fact that the amount of ellagic acid present at the ECS concentration tested is several fold lower than the one reported by Hseu et al. [23]. Overall, our data suggest that it is unlikely that the protective effect of ECS against UV exposure observed in the present study could be attributed to the activation of the cellular antioxidant response coordinated by NRF2. However, we have found that the mechanism underlying this protective effect involves the reactive species 1O2. This finding is in accordance with previous results that showed that this extract was able to scavenge 1O2 [11]. The phenolic compounds identified in ECS composition, namely phenolic acids (chlorogenic acid and ellagic acid) and flavonoids (rutin, isoquercitrin and hyperoside) have all been described as scavengers of reactive species [24-27] and thus their putative contribution to the free radical scavenging activity of the whole extract might be inferred. Noteworthy, the literature is rather scarce regarding their effect in the prevention of UV-induced DNA damage, with the exception of ellagic acid, mentioned above. 3.4

Electrochemical analysis

Electrochemical techniques provide rapid, simple and sensitive methods for the analysis of bioactive compounds associated with the scavenging of the radicals as well as for the determination of antioxidant capacity. Electrochemical methods for antioxidant assays are based on the fact that one of the modes of action of antioxidants is their ability to donate electrons to reactive species and their prior oxidation in detriment of biologically important ones [28, 29] Thus, the oxidative behavior of ECS was studied, at phosphate buffer solution (pH 7.4) and in cell culture medium, by differential pulse voltammetry (DPV), using a glassy carbon working electrode. To clarify the oxidative profile of ECS, the electrochemical study of some of the major phenolic compounds identified in C. sativa leaf extract (rutin, ellagic acid and chlorogenic acid [12]), was also carried out. The differential pulse voltammetric study of ECS revealed the presence of a well-defined anodic wave at Ep = + 0.207 V (Fig. 6). Standard phenolic compounds were also studied by DPV at physiological pH 7.4 in GCE (Fig. 6). Differential pulse voltammograms showed a first anodic peak at Ep = + 0.215 V, Ep = + 0.227 V and Ep = + 0.191 V, for rutin, ellagic acid and chlorogenic acid, respectively. The oxidation

17

potentials obtained for all studied compounds in cell culture medium were similar to those found using phosphate buffer solution (pH 7.4).

Figure 6 Differential pulse voltammograms for 0.1 mM solutions of (▬) Castanea sativa extract, (----) chlorogenic acid, (●●●) rutin and (-●-●) ellagic acid, in physiological pH 7.4 supporting electrolyte.

The results obtained clearly indicate that the reducing properties observed for ECS are indeed associated with the presence in the extract of a pool of different antioxidants namely rutin, ellagic acid and chlorogenic acid. Considering that the compounds that exert strong scavenging capabilities are those presenting the lowest reducing potential [29, 30] it may be concluded that ECS can present a moderate to high antioxidant activity.

4.

Conclusions 18

C. sativa leaf extract presented a concentration-dependent protective effect against UV - mediated DNA damage in HaCaT cells. This effect was found to be related to a direct antioxidant effect (involving 1O2) rather than activation of the endogenous antioxidant response coordinated by NRF2. The oxidative behavior of ECS was studied and we showed that its good antioxidant capacity can be ascribed to the presence of a pool of different phenolic antioxidants. No genotoxic or phototoxic effects were observed after incubation of HaCaT cells with ECS. Taken together these results reinforce the putative usefulness of the application of this plant extract in the prevention/minimization of UV deleterious effects on skin. Further studies are necessary to assess the potential of C. sativa leaf extract as an antiaging or chemopreventive topical ingredient in human volunteers, after UV radiation exposure. These in vivo studies can be accomplished using the topical formulation (incorporating ECS) that has already been developed and characterized [13].

19

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Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line

Highlights



C. sativa extract showed a protective effect against UV mediated-DNA damage



This effect was found to be related to a direct antioxidant effect involving 1O2



The extract doesn’t activate the endogenous antioxidant defences involving NRF2



Reducing properties are associated with the presence of a pool of antioxidants

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Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line.

Toxic effects of ultraviolet (UV) radiation on skin include protein and lipid oxidation, and DNA damage. The latter is known to play a major role in p...
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