Protective Effect of Mulberry (Morus alba L.) Extract against Benzo[a

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Protective Effect of Mulberry (Morus alba L.) Extract against Benzo[a]pyrene Induced Skin Damage through Inhibition of Aryl Hydrocarbon Receptor Signaling Hyunju Woo,† JungA Lee,† Deokhoon Park,† and Eunsun Jung*,† †

Biospectrum Life Science Institute, A-1805, U-Tower, 767, Sinsu-ro, Suji-gu, Yongin-si, Gyeonggi-do Republic of Korea ABSTRACT: Benzo[a]pyrene (B[a]P), a type of polycyclic aromatic hydrocarbon, is present in the atmosphere surrounding our environment. Although B[a]P is a procarcinogen, enzymatically metabolized benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) could intercalate into DNA to form bulky BPDE-DNA adducts as an ultimate carcinogenic product in human keratinocytes. The aim of this study was to evaluate the protective effect of mulberry extract, purified from the fruit of Morus Alba L., on B[a]P-induced cytotoxicity in human keratinocytes and its mechanisms of action. In this study, we confirmed that B[a]P induced nuclear translocation and the activation of aryl hydrocarbon receptor (AhR) were decreased by pretreatment of mulberry extract. Mulberry extract could decrease DNA damage through the suppression of B[a]P derived DNA adduct formation and restoration of cell cycle retardation at S phase in a dose-dependent manner. Additionally, cyanidin-3-glucoside (C3G), a major active compound of mulberry extract, showed biological activities to protect the cells from B[a]P exposure, similar to the effectivity of the mulberry extract. These results indicated that the inhibitory effect of C3G against B[a]P inducing skin cancer is attributable to repress the AhR signaling pathway. KEYWORDS: benzo[a]pyrene induced toxicity, Morus alba L., aryl hydrocarbon receptor, cyanidin-3-glucoside

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forming bulky DNA adducts.13,14 Several studies have demonstrated that human keratinocytes are capable of B[a]P metabolism into BPDE.15 The first event that occurred with the treatment of B[a]P was the binding of B[a]P to AhR, and the produced B[a]P-AhR complex was translocated into the nucleus as the exposure of the nuclear localization sequences of AhR. In the nucleus, B[a]P-AhR complex forms a heterodimer with aryl hydrocarbon receptor nuclear translocator, and this complex can interact with DNA by specific recognition sequence referred to xenobiotic responsive element (XRE) located upstream in the promoter region of some AhR target gene such as CYP1A1, 1A2, 1B1, and aryl hydrocarbon receptor repressor.16,17 More recent studies also demonstrated that AhR controlling cell cycle progression at the S phase diminishes the damaged DNA replication and cell proliferation.18 Morus alba L. has been widely used as a traditional herbal medicine in Asian countries to treat diabetes, hypertension, and hangover and also used for the purpose of beauty like antiaging and whitening.19−21 Pharmacological studies have demonstrated that the leaves, root bark, and fruits of M. alba L. possess anticancer, anti-inflammatory, hypoglycemic, antihypertension, antibacterial, antioxidant, neuroprotective, and hepatic and renal protective activities.20,22−25 Among these, the fruit extract of M. alba L., commonly called as mulberry, contains a number of bioactive components including cyanidin-3-glucoside, cyaniding-3-rutinoside, rutin, chlorogenic acid, protocatecuic acid, and gallic acid.26−28

INTRODUCTION Air pollution is one of the biggest threats to human health especially in sudden industrialization countries including China, Vietnam, and India in Asia.1 In several studies, polycyclic aromatic hydrocarbons (PAHs) known to cause genotoxic carcinogen, mutagenesis, and teratogenesis have been used for monitoring air, water, and soil pollution.2−5 PAHs consisting of multiple fused-ring aromatic compounds are produced by natural incomplete combustion of forest and brush fire or combustion of fossil fuel to automobile emission and cigarette smoke. As mentioned above, the facts that PAHs could affect the progression of lung, skin, bladder, liver, and stomach cancer could be identified by using well-established animal models, exposed to a mixture of PAHs.6,7 In the United States, the U.S. Environmental Protection Agency (US EPA) has classified 16 PAHs including naphthalene, benzo[a]anthracene, benzo[k]fluoranthene, and benzo[a]pyrene as priority pollutants based on the toxicity and adverse effects on human.6 Moreover, the International Agency for Research on Cancer also classified benzo[a]pyrene into Group 1 carcinogen possibly carcinogenic to humans.3 Benzo[a]pyrene (B[a]P) is the most extensively investigated marker of PAHs carcinogenic risk because of its cytotoxic, carcinogenic, and mutagenic effect on various animal models among the PAHs.8,9 The sources of B[a]P are automobile exhaust fumes, cigarette smoke, charbroiled food, residential wood burning, and volcanoes.10−12 Through multiple enzymatic metabolism including xenobiotic enzyme primarily the cytochrome P450-dependent monooxygenase family 1 (CYP1) and microsomal epoxide hydrolase (mEH), B[a]P could be converted into benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). These metabolites could intercalate in DNA by covalent binding to guanine nucleotide base at the N2 position © XXXX American Chemical Society

Received: August 30, 2017 Revised: November 22, 2017 Accepted: November 27, 2017

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DOI: 10.1021/acs.jafc.7b04044 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

DNA transfection reagent according to the manufacturer’s instructions. After transfection with plasmids for 4 h, the medium was replaced with mulberry extract containing medium and incubated for another 4 h. To induce the xenobiotic derived AhR nuclear translocation, transfected cells were treated with 5 μM of B[a]P. After 24 h incubation, the cells were washed with phosphate buffered saline solution and lysed with the addition of lysis buffer to prepare cellular extract. Luciferase reporter activities were determined by Firefly Luciferase Reporter Assay System according to the manufacturer’s instruction using a TECAN infinite 200 pro (TECAN Deutschland GmbH, Crailsheim, Germany). The βgalactosidase activity of each cell extract was determined to normalize transfection efficiencies. Preparation of Nuclear and Cytosolic Extract, and Western Blot Analysis. Nuclear and cytosolic extracts of HaCaT cells exposed to B[a]P with or without of mulberry extract for 6 h were prepared using an NE-PER Nuclear and Cytoplasmic Extraction Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Pittsburgh, PA, USA). The protein concentration was quantified using a Coomassie Plus (Bradford) Assay Kit (Thermo scientific, Pittsburgh, PA, USA). Twenty micrograms of cytosolic protein and 7 μg of nuclear protein were electrophoresed into ∼4−12% precast SDS gradient polyacrylamide gel with MOPS buffer. The proteins were electrophoresed to Immobilon-P membranes and probed with specific antibodies for AhR (1:1,000, Santa Cruz Biotechnology, Inc.), CYP1A1 (1:1,000, Santa Cruz Biotechnology, Inc.), and a secondary antibody conjugated to horseradish peroxidase (1:5000, Santa Cruz Biotechnology, Inc.). The specific antibodies for β-actin and PARP were used to confirm equal protein loading of cytosolic and nuclear fractions, respectively. Each protein was detected using a PicoEPD Western reagent kit (ELPIS-Biotech, Daejeon, Korea). Flow Cytometric Analysis. Flow cytometry was performed to analyze the cell cycle progression of HaCaT cells, following the treatment of B[a]P with or without mulberry extract. In brief, 1 × 106 cells were suspended in 100 μL of PBS, followed by adding 200 μL of 95% ethanol while vortexing for fixation of all the cells without clumping. After the fixation for 1 h at 4 °C, the cells were treated with 12.5 μg of RNase for another 30 min. Then DNA in the fixed cells was stained with 50 μg/mL of propidium iodide for 30 min at room temperature. The cell cycle distribution profiles were analyzed using a FACSCalibur flow cytometer for relative DNA content (Becton Dickinson, San Jose, CA, USA). BPDE-DNA Adduct Analysis. HaCaT cells were grown to 70− 80%. The cells were pretreated with the mulberry extract or active chemicals for 4 h and then the cells were exposed to 5 μM of B[a]P for another 24 h. The BPDE-DNA adduct detection was performed according to previously published methods.29 In brief, harvested cells were washed with PBS and fixed in 2% of paraformaldehyde. Sequentially, the fixed cells were washed with PBS and then resuspended in permeabilization solution containing 0.2% Triton X100 in 0.1% sodium citrate buffer for 10 min incubation on ice. After washing with PBS, the samples were incubated with 100 μg/mL of RNase A for 1 h at 37 °C, followed by treating with 10 μg/mL of proteinase K for additional 10 min at room temperature. DNA denaturation was performed with 2 N HCl incubation for 30 min. After samples were blocked with 5% normal goat serum, the samples were incubated with anti-BPDE-DNA antibody (1:25 dilution) raised in mouse against benzo[a]pyrene diol epoxide modified DNA (BPDEDNA) overnight at 4 °C. After washing with PBS, the samples were exposed to FITC-conjugated goat antimouse IgG (1:200 dilution) for 1 h at 37 °C. Fluorescence was measured by flow cytometry. Normal mouse IgG was used as the negative control. Ex Vivo Experiment. Eighteen human skin explants with an average diameter of 11 mm (±1 mm) were prepared from abdominoplasty performed on 59-years-old Caucasian women, and the explants were maintained in culture at 37 °C in a humid, 5% CO2 atmospheric condition in BEM culture medium for 5 days. Powder of mulberry extract (2 mg/cm2) was applied topically on the skin explants from day 0 to day 4. On day 4, heavy metals and hydrocarbon containing materials were topically treated to the skin explants with a 9

In this study, we investigated the antipollution activity of mulberry extract for the first time and we found out that cyanidin-3-glucoside (C3G) was bioactive component in mulberry extracts against pollutant materials. Herein, we report that C3G possess a potent inhibitory activity against B [a] P induced cell cycle arrest at the S phase, BPDE-DNA adduct formation and AhR target gene expression through the blocking of AhR nuclear translocation.

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MATERIALS AND METHODS

Materials and Reagent. Dried fruits of mulberry were purchased from a local fruit market in Jeju, Korea. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Welgene (Daegu, Korea), and streptomycin and penicillin, fetal bovine serum, propidium iodide (PI) and kit for cytoplasmic and nuclear extraction (NE-PER nuclear and cytoplasmic extraction reagents) were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). Benzo[a]pyrene and MTT reagent were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Duchefa (Haarlem, Netherlands) respectively. Anti-BPDE-DNA, anti-CYP1A1, antipoly(ADP-ribose) polymerase (PARP), anti-β-actin and horse radish peroxide (HRP)-conjugated antimouse IgG and antirabbit IgG antibodies used in this study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and C3G was purchased from Extrasynthese (Genay, France). Proteins were detected using PicoEPD Western reagent (ELPIS-Biotech, Daejeon, Korea). pGL4.43[luc2P/ XRE/Hydro] vector, pSV-β-gal, and luciferase assay system were purchased from Promega (Madison, WI, USA). Extraction of Mulberry Extract. The dried fruits of mulberry powder (100 g) were extracted overnight with 10 L of 70% ethanol at room temperature, and the supernatant was collected by filtration. Ethanol was removed by rotatory evaporation (Heidolph, Schwabach, Germany), and the extract was lyophilized (50 g). In the present study, the moisture of lyophilized mulberry fruit extract was 14.65 ± 0.13%. HPLC Analysis of C3G. The content of C3G in the 70% ethanol extract of mulberry was analyzed by using a Shimadzu (Kyoto, Japan) HPLC system with a CBM-20A controller, SPD-M20A PDA detector, LC-20AD pump, and SIL-20A autosampler. Chromatographic separations were performed using a CAPCELL PAK C18 UG120 column (250 mm × 4.6 mm, 5 μm, Shiseido, Tokyo, Japan) with UV− visible detector at 254 nm. Gradient elution was performed using solvent A (Water, containing 0.1% trifluoroacetic acid) and solvent B (acetonitrile) as the mobile phase at a flow rate of 1 mL/min. C3G was identified and quantified at 517 nm by comparing the retention time with the pure standard. Cell Culture and Treatments. Human keratinocyte (HaCaT) cells were cultured in DMEM supplemented with 10% FBS and 100 μg/mL streptomycin and penicillin at 37 °C in humidified with 5% CO2 atm. When the cell has reached approximately 80% confluence, the cells were subcultured. To investigated the following experiments, HaCaT cells were seeded plastic dishes and grown to 70−80% confluence. The cells were treated with various concentration of mulberry extract from 25 to 75 μg/mL or C3G from 25 to 75 μM for 4 h before B[a]P induction. Cell Proliferation Assay. The cell proliferation retardation by B[a]P and the restoration of cell proliferation by C3G pretreatment were determined with MTT assay. For MTT assay, HaCaT cells were treated with or without C3G at various concentration before B[a]P induction in a 96-well plate. After the incubation for 72 h, 50 μL of MTT reagent (1 mg/mL) was added to each well and incubate another 4 h. Crystal formazan produced from MTT reagent was dissolved with dimethyl sulfoxide, and the absorbance was measured at 570 nm using spectrophotometer (Epoch microplate spectrophotometer, BioTek, Winooski, Vermont, USA). Transient Transfections and Luciferase Activity Assay. HaCaT cells were seeded in 60 mm diameter plastic dishes and grown to 70−80% confluence. The adherent cells on each dish were cotransfected with 1.5 μg of pGL4.43 luciferase plasmid containing three copies of a XRE and 0.5 μg of pSV-β-gal using as normalization control. Transfection was performed using the X-tremeGENE HP B


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Figure 1. Cell proliferation under treatment of B[a]P. (A) Effect of B[a]P on the cell viability of HaCaT cells in a dose-dependent manner. To measure the proliferation of HaCaT cells treated with various concentration of B[a]P, the cells were incubated with medium containing from 1.25− 10 μM of B[a]P for 72 h, and more incubated with MTT reagent for another 3 h. The analysis was mean of triplicated measurements ± SD of four separate experiments. (B) Cell cycle distribution determination after the treatment of B[a]P for 24 h was performed using flow cytometry. In this experiment, the cells stained DNA with propidium iodide. (C) Quantification of cell cycle distribution in B[a]P treated HaCaT cells. Values are shown as percentages of the control. ∗P < 0.05, ∗∗P < 0.01 versus controls.

Figure 2. Inhibitory effect of mulberry extract on B[a]P induced nuclear translocation of AhR and binding activity of AhR on XRE. (A) XREdependent luciferase gene transcription was determined using luciferase activity assay. When HaCaT cells were confluent at 80%, cells were transfected with 1.5 μg of pGL4.43[Luc2P/XRE/Hygro] firefly luciferase reporter plasmid. After transfection, cells were exposed to vehicle or various concentration of mulberry extract for 4 h, followed by treatment with 5 μM of B[a]P. Reporter activity was measured after the treatment of B[a]P for 24 h with cellular extract. (B) Nuclear and cytosolic extracts were prepared from HaCaT cells treated with either B[a]P only or B[a]P plus mulberry extract (25, 50, 75 μg/mL) for 24 h. Translocation of AhR from cytosol to nuclear and the expression of CYP1A1 protein in cytoplasmic fraction was determined by Western blot analysis with specific antibodies. β-actin and PARP were used as loading control of cytosolic and nuclear extracts, respectively. ∗P < 0.05 compared with the B[a]P treated groups. Statistical Analysis. All experiments were conducted at least three times, and the statistical significance in the data was evaluated using a Student’s t test using Statview software (Abacus Concepts, Piscataway, NJ, USA). Statistical significance was indicated when p < 0.05.30

mm paper disk for 24 h. On day 0, the three explants from the control group were collected and on day 5, three explants from each group were collected after 24 h with or without the application of the pollutant mix; and the collected samples were fixed in buffered formalin. After 24 h of fixation, the samples were dehydrated and embedded in paraffin using Leica TP1010 dehydration automat (Leica, Rueil-Malmaison, France). Seven micrometer thick sections of paraffin-embedded samples were cut for light microscopy and mounted on the Superfrost plus silanized glass slides. To observe general morphological change, the paraffinized sections were stained with Masson’s trichrome, Goldner variant. The microscopical observations were performed using an Olympus BX43 microscope (Olympus Life Science, Hamburg, Germany) with an attached DP72 Olympus camera.

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RESULTS Inhibitory Effect of B[a]P on Proliferation of HaCaT Cells. The cytotoxic effect of B[a]P on HaCaT cells was determined by MTT assay. The cells treated with various concentration of B[a]P for 72 h showed reduced cell proliferation rate in a dose dependent manner. As shown in Figure 1, the cell number of HaCaT cells significantly decreased to 58.4% following the treatment of B[a]P at 5 μM C


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Figure 3. Inhibitory effect of mulberry extract on the formation of bulky BPDE-DNA adduct and synthesis of damaged DNA. (A) BPDE-DNA adducts were measured by using specific antibody for flow cytometry in HaCaT cells grown for 24 h with B[a]P presence or absence of mulberry extract. The formation of BPDE-DNA adducts in untreated (black line), B[a]P treated (red line) and pretreated with indicated concentration of mulberry extract before B[a]P induction (blue line) were analyzed by flow cytometry. (B) Evaluation the change of cell cycle kinetics in HaCaT cells following treatment of B[a]P and mulberry extract for 24 h was determined by flow cytometry after staining of DNA with propidium iodide.

plasmid. B[a]P could activate AhR through the translocation of AhR from the cytosol to nucleus in the HaCaT cells. However, the pretreatment of mulberry extract significantly decreased AhR activation induced by B[a]P treatment in a dose dependent manner with the inhibition of nuclear translocation of AhR concomitantly with the decreased expression of CYP1A1 protein (Figure 2B). In this study, mulberry extract was found to modulate the AhR activation by suppressing the AhR nuclear translocation, which is crucial for the transformation of ligand binding AhR to DNA binding form to transcribe the downstream genes. Mulberry Extract Suppressing Formation of BPDEDNA Adducts in B[a]P Activated HaCaT Cells. B[a]P is a type of PAHs and well-known to cause cancer through the formation of BPDE-DNA adducts by many experimental studies. The analysis of DNA adducts by flow cytometry was performed after immunostaining with anti-BPDE-DNA adduct antibody. When the cells were exposed to 5 μM of B[a]P for 24 h, the produced BPDE-DNA adduct levels significantly increased compared to those of the control cells (Figure 3A). However, the treatment of mulberry extract prior to the B[a]P exposure could suppress the formation of BPDE-DNA adducts. Mulberry Extract Regulates Cell Cycle Progression. Previously, we observed a significant decrease in the cell proliferation by the treatment of B[a]P. To examine whether the inhibitory effects of B[a]P on cell proliferation were caused by the suppression of cell cycle progression or induction of cell death, the cell cycle distribution was determined by flow cytometric analysis of propidium iodide stained cell. The cells after the serum deprivation induced a significant enrichment in the S phase by the treatment of B[a]P within 24 h compared to the control. The accumulation rate in the S phase was identical to the decreased rate in G0/G1 phase (Figure 3B). However,

concentration as compared to the control. However, in this condition with the short-term B[a]P exposure, the apoptotic cells could not be observed. These results suggest that B[a]P induced cell proliferation inhibition occurred by cell cycle arrest instead of apoptosis. Mulberry Extract Regulating the Binding Activity of AhR on XRE Sequences in HaCaT Cells Exposed to B[a]P. To investigate whether mulberry extract was effective for downregulation of B[a]P induced gene expression through an AhR dependent mechanism, transient cotransfection in HaCaT cells were carried out using XRE derived luciferase reporter vector and pSV-β-gal vector. The ligand-activated AhR translocated from cytosol to nucleus and bound to the XRE sequence in the promoter of many genes containing CYP1A1 and CYP1B1. This binding activity upregulates the transcription. As shown in Figure 2A, the luciferase activity results showed that the cells transfected XRE-luciferase vector alone could increase the luciferase activity compared to the control. Moreover, the activated HaCaT cells by B[a]P remarkably enhanced the luciferase activity by 20-fold in the XRE luciferase vector transfected cells. However, depending on the dosage of mulberry extract, the luciferase activity in B[a]P induced HaCaT cells was significantly suppressed. When the HaCaT cells were treated with the mulberry extract at the concentrations of 25, 50, and 75 μg/mL before the activation of B[a]P, the luciferase activity decreased to 81.7%, 69.1%, and 61.9% compared to B[a]P induced HaCaT cells. Mulberry Extract Suppressing AhR Translocation to the Nucleus in B[a]P Activated HaCaT Cells. AhR, ligandactivated transcription factor, binds to the sequence of XRE in the promoter region of CYP1A1 or CYP1B1 genes in response to the B[a]P. The treatment of B[a]P significantly induces the luciferase activity in the transfected cells with XRE containing D


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Figure 4. Protective effect of mulberry extract on the pyknotic cells induced by pollutant mixture in the human skin explants. (A) Day 0 of untreated batch, (B) day 5 of untreated batch, (C) day 5 of mulberry extract treated batch, (D) day 5 of pollutant mixture treated batch, (E) day 5 of mulberry extract + pollutant mixture treated batch. Black arrow indicate pyknotic nuclei containing cell.

the cells treated with various concentrations of mulberry extract before the B[a]P activation recovered from the S phase arrest induced by B[a]P to normal phase progression. Therefore, mulberry extract could suppress the retardation of cell cycle progression after the treatment of B[a]P. Mulberry Extract Suppressing Alteration of General Morphology by B[a]P in ex Vivo Experiments. The control group, unexposed to heavy metals and hydrocarbon containing materials, showed normal morphology with 3−4 cell layers of epidermis with a good morphology. Moreover, the relief of the dermal−epidermal junction was moderated compared to the batch of day 0. The collagen network showed weakly dense papillary dermis. After the treatment of pollutant mix, the epidermis showed 3−4 cell layers with a definitely altered morphology characterized by a high number of pyknosis cells and slight detachment of the dermal−epidermal junction. However, the pretreatment of mulberry extract showed absolute reduction in the pyknotic cells and prevention of the detachment of dermal−epidermal junction induced by the pollutants (Figure 4). HPLC Analysis of C3G. Cyaniding-3-glucoside (C3G) is the key component of anthocyanin, which is responsible for the color pigment in ripe mulberry, and is known to have antioxidative and protective potential against harmful damages. We used the ripened mulberries, which were deep purple in color. Therefore, C3G was determined, which was considered as main active component in mulberry extract. The chemical components in the extract of mulberry were analyzed by HPLC. As shown in Figure 5A, C3G was shown in one of the main peak in UV chromatograms recorded at 254 nm. Quantitative analysis of C3G was carried out at 517 nm, C3G was detected as the major peak, tR at 25.628 min, and its content was calculated as >0.25% (w/w) (Figure 5B). C3G from Mulberry Extract Suppress DNA Damage Caused by B[a]P. In this study, we investigated the effects of mulberry extract on B[a]P induced DNA damage by determining the formation of BPDE-DNA adducts and change in the cell cycle distribution. As shown in Figure 6, C3G could recover the luciferase activity of B[a]P induced HaCaT cells in a dose dependent manner. In addition, it suppressed the formation of BPDE-DNA adducts caused by B[a]P treatment at 75 μM concentration. Moreover, other characteristics of DNA damage caused by B[a]P, cell cycle phase retardation, and inhibition of proliferation were recovered by the pretreatment of C3G. This result is similar to that of the mulberry extract treatment.

Figure 5. (A) HPLC chromatograms of mulberry extract at 254 nm. (B) Overlapped HPLC chromatograms of C3G standard and mulberry extract at 517 nm.

keratinocyte cells. Although B[a]P is less persistent than other halogenated substances, it can be transported and deposited to sediment or water environmental compartment for a long time until degraded. B[a]P is metabolized to benzo[a]pyrene-7,8diol-9,10-epoxide, which is one of the carcinogenic compounds of B[a]P, by cytochrome p450s (CYPs) dependent monooxygenase system. Many studies have shown that the incidence of cancer in various organ including lung and liver, the most important organ for detoxification was associated with the inhalation and oral administration of B[a]P. However, B[a]P toxicity and its mechanism in the human skin have not been fully understood. Skin, the largest organ of the human body, acts as first line of protective system of the body against numerous environmental stimuli such as chemicals, ultraviolet radiation, and invading bacterial pathogens. Previous research demonstrated that drug metabolism occurs in human skin cells by regulating the transcriptional expression of drug-metabolizing enzyme.31 In this study, we investigated the effect of B[a]P

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DISCUSSION In this study, mulberry extract and its active constituent, C3G, have a significant cytoprotective effect on B[a]P induced DNA damage by regulation of AhR signaling pathway in human E


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Figure 6. Suppressive effect of C3G from mulberry extract on various form of DNA damage caused by B[a]P. (A) Chemical structure of C3G. (B) HaCaT cells were transfected with XRE-containing luciferase plasmid, after transfection, C3G were treated with 5 μM of B[a]P. Reporter assay was performed by using firefly luciferase. α-naphthoflavone (NF) was used as a positive control. (C) BPDE-DNA adducts were measured in HaCaT cells grown for 24 h with B[a]P presence or absence of C3G. (D) Determination of the cell cycle distribution in HaCaT cells after the treatment of B[a]P and C3G for 24 h by using flow cytometry. (E) Effect of C3G pretreatment on the cell proliferation of B[a]P exposed HaCaT cells. For cell proliferation analysis, HaCaT cells were cultured in the various concentration of C3G for 4 h prior to B[a]P exposure. After 72 h incubation, the cells were treated with MTT reagent for additional 3 h. The analysis was mean of triplicated measurements ± SD of four separate experiments.∗P < 0.05 compared with the B[a]P treated groups.

as environmental air pollutants on human keratinocytes and identified the antagonistic compounds from the mulberry extract against AhR activation. In this study, HaCaT cells were treated with B[a]P at 5 μM, and this concentration is considered as environmentally and occupationally relevant concentration equivalent to ∼0.2 μg/m3 airborne B[a]P concentration significantly lower than the threshold limit value for B[a]P suggested by the Occupational Safety and Health Administration.32,33 Furthermore, the concentration of B[a]P applied in this experiment is widely used to induce toxicity in many types of human cell lines and animal studies and observed similar results such as inhibition of cell proliferation and cell cycle retardation without apoptosis consistent with other.34,35 Because the toxic effect of B[a]P was mainly mediated by AhR activation, finding the antagonistic compound against AhR could be a meaningful treatment for the detoxification of PAHs. Although many studies have reported that mulberry extract has a protective effect on various diseases including memory deficit, liver fibrosis, and diabetes, the protective effects of the mulberry fruit extract on DNA damage caused by B[a]P have not yet sufficiently investigated. Herein, we demonstrated that mulberry extract could suppress the B[a]P induced target gene expression containing XRE sequence in the promoter region by inhibiting the nuclear translocation of AhR in human keratinocyte. To investigate whether pretreatment of mulberry extract could considerably reduce the formation of BPDE-DNA adducts and the following retardation of cell cycle distribution, the BPDE-DNA adducts formation was determined with specific antibody. The mulberry extract and its major constituent, C3G, significantly attenuated the formation of BPDE-DNA adducts, and this is the first report demonstrating the protective effect of mulberry extract on short-term exposure of B[a]P. B[a]P induced prolonged S phase arrest is probably

due to the activation of S checkpoint, which could recognize and repair severe DNA damage to block entry into mitosis of the cells harboring damaged DNA.36 Therefore, we presumed that mulberry extract could suppress the formation of BPDEDNA adduct and then inhibits DNA damage. To evaluate the protective effect of mulberry extract on the living human organism against environmental pollutants, human skin explants with closet constituents to the in vivo condition were used. The result of histological analysis indicated that the mulberry extract could suppress the number of pyknotic cells, which are the cells with irreversible condensation of chromatin attributable to necrosis or apoptosis, induced by the treatment of pollutant mixture. Furthermore, we found out that the protective active compound in the mulberry extract against DNA damage caused by B[a]P was C3G. Other polyphenolic compounds such as curcumin, resveratrol, hesperetin, and vitamin E could directly or indirectly ameliorate the PAHs induced cytotoxicity of AhR.37,38,32 However, the effect of C3G, a major active ingredient of mulberry extract, on B[a]P induced human keratinocyte has not been fully investigated. C3G, a polyphenolic anthocyanin, has been widely detected in the foods including some fruits (mulberry, blackcurrant, red raspberry, and plump) and pigmented cereals (black rice and black soybean seed coat). In this study, we demonstrated that C3G has a potent protective activity against B[a]P induced cell cycle arrest at the S phase, BPDE-DNA adduct formation, and AhR activation similar to those of mulberry extract. In conclusion, mulberry extract and its major compound C3G exhibited protective effects against B[a]P induced cytotoxicity, DNA damage, and cell cycle arrest at S phase through the inhibition of AhR activation in the human keratinocyte cells. This study manifests the mulberry extract to be used as a cytoprotective agent for the treatment of B[a]P; however, further investigation including clinical trials is needed F


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to elucidate the molecular mechanism and effectiveness of mulberry extract as a drug against environmental pollutant in human.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +82-31-698-3122. Fax: +82-31-698-3123. ORCID

Hyunju Woo: 0000-0002-1672-2556 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a grant from the Ministry of Trade, Industry and Energy, Republic of Korea (R0002895).

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ABBREVIATIONS USED B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; AhR, aryl hydrocarbon receptor; C3G, cyanidin-3-glucoside; PAHs, polycyclic aromatic hydrocarbons; CYP1, cytochrome P450-dependent monooxygenase family 1; XRE, xenobiotic responsive element; HaCaT, human keratinocyte; MTT, 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; FITC, fluorescein isothiocyanate

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REFERENCES

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Protective Effect of Mulberry (Morus alba L.) Extract against Benzo[a

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