Quercitrin protects against ultraviolet B-induced cell death in vitro and in an in vivo zebrafish model

Journal of Photochemistry and Photobiology B: Biology 114 (2012) 126–131

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Journal of Photochemistry and Photobiology B: Biology

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

Quercitrin protects against ultraviolet B-induced cell death in vitroand in an in vivo zebrafish model

Hye-Mi Yang a, Young-Min Ham b, Weon-Jong Yoon b, Seong Woon Roh a, You-Jin Jeon c, Tatsuya Oda d,Sung-Myung Kang c, Min-Cheol Kang c, Eun-A Kim c, Daekyung Kim a,⇑, Kil-Nam Kim a,⇑a Marine Bio Research Team, Korea Basic Science Institute (KBSI), Jeju 690-140, Republic of Koreab Jeju Biodiversity Research Institute, Jeju Technopark, Jeju 699-943, Republic of Koreac School of Marine Biomedical Sciences, Jeju National University, Jeju 690-756, Republic of Koread Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan

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

Article history:Received 22 February 2012Received in revised form 22 May 2012Accepted 28 May 2012Available online 5 June 2012

Keywords:Ultraviolet (UV) BQuercitrin (QR)Oxidative stressKeratinocytePhotoagingZebrafish

1011-1344/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jphotobiol.2012.05.020

⇑ Corresponding authors. Tel.: +82 64 800 4930; faE-mail addresses: [email protected] (D. Kim), knkim

Chronic exposure of skin to ultraviolet (UV) B radiation induces oxidative stress, which in turn, plays acrucial role in the induction of skin aging. The search for strategies to reverse skin aging is being con-stantly pursued. Here, the cytoprotective effect of quercitrin (QR) on UVB-induced cell injury in HaCaThuman keratinocytes and in the zebrafish was investigated. Intracellular reactive oxygen species (ROS)generated by the exposure of HaCaT cells to UVB radiation were significantly decreased after treatmentwith QR, and significantly so with QR at 50 lM. As a result, QR reduced UVB-induced cell death and apop-tosis in HaCaT cells. QR similarly reduced UVB-induced ROS generation and cell death in live zebrafish.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

Ultraviolet (UV) radiation is the major environmental cause ofskin damage. Although only 0.5% of UVB radiation reaches theEarth, it is the main cause of sunburn and probably the most car-cinogenic component of sunlight [1,2]. Exposure of mammalianskin to UV light impairs enzymatic and non-enzymatic antioxidantactivity [3,4] and increases the cellular levels of reactive oxygenspecies (ROS), which, in turn, damage lipids, proteins, and nucleicacids in epidermal cells and are likely to contribute to the processof photocarcinogenesis and photoaging [5,6]. The increase in ROS isaccompanied by the activation of many ROS-sensitive signalingpathways and induced gene transcription [7]. In addition, exposureof cells to UVB radiation results in the loss of keratinocyte viability,increase in membrane blebbing, cytoskeletal molecular changes,and apoptosis [5,8].

The zebrafish (Danio rerio) is a small tropical freshwater fishthat has emerged as a useful vertebrate model organism becauseof its small size, large clutches, transparency, low cost, and physi-

ll rights reserved.

x: +82 64 805 [email protected] (K.-N. Kim).

ological similarity to mammals [9,10]. The administration of drugsand/or small molecules to zebrafish is uncomplicated because theearly-stage embryo rapidly absorbs small molecular compoundsdiluted in the bathing media through the skin and gills. In addition,zebrafish has melanin pigments on the body surface, allowing sim-ple observation of the pigmentation process without complicatedexperimental procedures [11]. Therefore, the zebrafish has beenrecently used as an in vivo model of oxidative stress for studyingUV protection [12,13].

Flavonoids (FVs), one of the most diverse and widespread groupsof natural compounds, are probably the most common naturalphenolics [14,15]. FVs are efficient antioxidants that scavenge oxy-gen radicals and possess anticancer, hypolipidemic, anti-aging, andanti-inflammatory activities [16]. Among the broad variety of FVs,quercitrin (QR; quercetin-3-O-rhamnoside) has been employedpreviously as an antibacterial agent [17] and has been shown to in-hibit the oxidation of low-density lipoproteins and prevent allergicreaction [18,19]. Furthermore, we previously demonstrated that QRexerts protective effects against H2O2-induced dysfunction in lungfibroblast cells [20]. However, the protective effects of QR againstUV-induced cell death in vitro and in vivo have not been assessedthus far. Therefore, in this study, we examined the in vitro protec-tive effects of QR against UVB-induced cell damage and adoptedthe zebrafish as an alternative in vivo experimental model.

H.-M. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 126–131 127

2. Materials and methods

2.1. Reagents

Quercitrine (QR, purity: >95%, Fig. 1) purchased from ChromaDex(Santa Ana, CA, USA), 20 70-dichlorodihydrofluorescein diacetate(DCFH2-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), RNase A, propidium iodide (PI), dimethyl sulfoxide(DMSO), phosphate-buffered saline (PBS), and Hoechst 33342 werepurchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’smodified Eagle’s medium (DMEM), fetal bovine serum (FBS), peni-cillin–streptomycin, and trypsin–ethylenediaminetetraacetic acid(EDTA) were purchased from Gibco BRL (Life Technologies, Burling-ton, ON, Canada). The other chemicals and reagents used were ofanalytical grade.

2.2. Cell culture

HaCaT (human skin keratinocyte cells) cells were grown inDMEM supplemented with 10% (v/v) heat-inactivated FBS, penicil-lin (100 U/mL), and streptomycin (100 lg/mL). The cells were thenincubated in an atmosphere of 5% CO2 at 37 �C and were subcul-tured every 3 day.

2.3. UVB radiation in vitro cells

Cells were exposed to UVB radiation range at a dose rate of 10–100 mJ/cm2 (UV Lamp, VL-6LM, Vilber Lourmat, France) accordingto the method describe by Heo et al. [21,22]. Optimum irradiationdose was evaluated at 50 mJ/cm2; therefore, the 50 mJ/cm2 of UVBwas used in further experiments.

2.4. Intracellular reactive oxygen species (ROS) scavenging activity

To detect intracellular ROS, the cells were seeded in 96-wellplates at a concentration of 1.0 � 105 cells/mL. After 16 h, the cellswere exposed to UVB (50 mJ/cm2) and the QR was treated with dif-ferent concentrations (12.5, 25 and 50 lM) and incubated at 37 �Cunder a humidified atmosphere. The cells were incubated for anadditional 2 h at 37 �C under a humidified atmosphere with 5%CO2. Finally DCHF-DA (5 lg/mL) was introduced to the cells, and2070-dichlorodihydrofluorescein fluorescence was detected at anexcitation wavelength of 485 nm and an emissions wavelength of535 nm, using a PerkinElmer LS-5B spectrofluorometer.

2.5. Assessment of cell viability

The cell viability was determined by a colorimetric MTT assay.Cells were seeded in a 96-well plate at a concentration of1.0 � 105 cells/mL. After 16 h, the cells were exposed to UVB(50 mJ/cm2) with various concentrations (12, 25, and 50 lM) ofQR and incubated for 24 h at 37 �C. MTT stock solution (50 lL;

Fig. 1. Chemical structure of QR.

2 mg/mL in PBS) was then added to each well to obtain a total reac-tion volume of 250 lL. After 4 h of incubation, the plate was centri-fuged at 2000 rpm for 10 min, and the supernatant was aspirated.The formazan crystals in each well were dissolved in DMSO. Theamount of purple formazan was determined by measuring theabsorbance at 540 nm.

2.6. Nuclear double staining

The nuclear morphology of cells was studied by using cell-per-meable DNA dyes Hoechst 33342 and PI. Cells were seeded in a 24-well plate at a concentration of 1.0 � 105 cells/mL. After 16 h, thecells were exposed to UVB (50 mJ/cm2) with QR (50 lM) and incu-bated for 12 h at 37 �C. Then, Hoechst 33342 and PI were added tothe culture medium at a final concentration of 10 and 5 lg/mL, andthe plate was incubated for another 10 min at 37 �C. The stainedcells were observed under a fluorescence microscope equippedwith a CoolSNAP-Pro color digital camera to examine the degreeof nuclear condensation. Cells with homogeneously stained nucleiwere considered to be viable, whereas the presence of chromatincondensation and/or fragmentation was considered indicative ofapoptosis.

2.7. Cell-cycle analysis

Cell-cycle analysis was performed to determine the proportionof apoptotic sub-G1 hypodiploid cells [23]. Cells were seeded in a6-well plate at a concentration of 1.0 � 105 cells/mL. After 16 h,the cells were exposed to UVB (50 mJ/cm2) with QR (50 lM). After12 h of incubation, the cells were harvested at the indicated timeand fixed in 1 ml of 70% ethanol for 30 min at 4 �C. They were thenwashed twice with PBS and incubated in the dark in 1 mL of PBScontaining 100 lg PI and 100 lg RNase A for 30 min at 37 �C. Flowcytometric analysis was performed with a FACSCalibur flowcytometer. The effect of QR on the cell cycle was determined bychanges in the percentage of cells in each phase of the cell cycleand assessed with histograms generated by software programsCellQuest and ModFit [24].

2.8. Origin and maintenance of parental zebrafish

Adult zebrafish were purchased from a commercial dealer(Seoul Aquarium, Seoul, Korea), and 10 fish were kept in a 3 Lacrylic tank at 28.5 �C with a 14/10 h light/dark cycle. Zebrafishwere fed 3 times a day, 6 days/week, with Tetamin flake food sup-plemented with live Artemia salina. Embryos were obtained fromnatural spawning, induced in the morning by turning on the light.Collection of embryos was completed within 30 min.

2.9. UVB radiation of zebrafish embryos

Zebrafish embryos were exposed to UVB radiation at a dose rateof 10–100 mJ/cm2. The optimal irradiation dose was determined(50 mJ/cm2) and used in all subsequent experiments [12]. Briefly,the incubation medium was removed, and the zebrafish embryoswere rinsed with fresh embryo medium. Then, zebrafish embryoswere layered in a glass slide, covered with sufficient embryo med-ium, and exposed to 50 mJ/cm2 UVB.

2.10. Waterborne exposure of embryos to QR

Exposure of embryos to UVB and QR was done as a previouslydescribed method [12]. At approximately 2 days post-fertilization(dpf), embryos (n = 25) were transferred to individual wells of a24-well plate and maintained in embryo medium containing1 mL of vehicle (0.1% DMSO) or 50 lM QR for 1 h. Then, they were

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Fig. 2. Protective effect of QR against UVB-induced intracellular ROS generation andcytotoxicity in HaCaT cells. Cells were exposed to UVB (50 mJ/cm2) and treated withQR. After an additional 2 h, the intracellular ROS generated were detected byspectrofluorometry (A). The viability of HaCaT cells was determined by MTT assay(B). �Significantly different from only UVB-exposed cells (p < 0.05).

128 H.-M. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 126–131

irradiated with UVB (312 nm) alone or in combination with QRtreatment.

2.11. Estimation of intracellular ROS generation and image analysis

Generation of ROS production in zebrafish embryos wasassessed using an oxidation-sensitive fluorescent probe dye,DCFH-DA [12]. At 2 dpf, the embryos were treated with 50 lMQR, and 1 h later, the plate was irradiated with UVB (50 mJ/cm2).After irradiating embryos with UVB, the embryos were transferredinto a 96-well plate and treated with DCFH-DA (20 lg/mL), afterwhich the plates were incubated for 1 h in the dark at 28.5 �C. Afterincubation, the embryos were rinsed in embryo medium and anes-thetized with phenoxyethanol (1/20 dilution; 5 min) before visual-ization. Individual embryo fluorescence intensity was quantified

Control UVB

Fig. 3. Protective effect of QR against UVB-induced apoptosis in HaCaT cells. Cells werestained with Hoechst 33342 and PI solution and then observed under a fluorescence micrlegend, the reader is referred to the web version of this article.)

using a Perkin-Elmer LS-5B spectrofluorometer. Stained embryoswere observed under a fluorescence microscope.

2.12. Measurement of oxidative stress-induced cell death in zebrafishembryos

Cell death was detected in live embryos using acridine orangestaining, a nucleic acid-specific metachromatic dye that interactswith DNA and RNA by intercalation or electrostatic attraction[12]. Acridine orange stains necrotic or very late apoptotic cells.At 2 dpf, the embryos were treated with 50 lM QR, and 1 h later,the plate was irradiated with UVB (50 mJ/cm2). After radiating em-bryos with UVB, the embryos were transferred into 96-well plateand treated with acridine orange solution (7 lg/mL), and the plateswere incubated for 1 h in the dark at 28.5 �C. After incubation, theembryos were rinsed in embryo medium and anesthetized withphenoxyethanol before visualization as described above. Individualembryo fluorescence intensity was quantified using a Perkin-ElmerLS-5B spectrofluorometer. Stained embryos were observed under afluorescence microscope.

2.13. Statistical analysis

All data are presented as the mean ± SD of at least three repli-cates. Significant differences among the groups were determinedby using the unpaired Student’s t-test. P < 0.05 was considered sta-tistically significant.

3. Results and discussion

UV irradiation is a potent inducer of ROS such as hydroxyl rad-icals (OH�), superoxide radicals (O�2 ), and peroxyl radicals and theiractive precursors, namely singlet oxygen 1O2, hydrogen peroxide(H2O2), and ozone [25], all of which play a role in the modulationof apoptosis [26]. Several studies have shown UVB-induced ROSformation leading to apoptosis [27]. Therefore, the removal ofexcess ROS or the suppression of their generation by antioxidantsmay prove effective in preventing UVB-induced cell death. QRhas been reported to be a protective agent against H2O2-inducedcell injury in lung fibroblasts and osteoblastic cells. However, thereis no information available on the neuroprotective ability of QRagainst UVB-induced oxidative stress in skin keratinocyte cells.Here, we show for the first time that QR protects against UVB-induced ROS production and cell damage in vitro and in vivo.

3.1. Effects of QR on UVB-induced intracellular ROS generation andcytotoxicity

Generation of intracellular ROS can be detected by usingDCFH-DA, which permeates the cell membrane freely. DCFH-DA is

UVB+QR

exposed to UVB (50 mJ/cm2) with QR and incubated for 12 h. The cells were doubleoscope using a blue filter. (For interpretation of the references to color in this figure

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H.-M. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 126–131 129

hydrolyzed by intracellular esterase to convert to nonfluorescentDCFH. In the presence of ROS, DCFH is oxidized to highly fluorescentDCF [28], a measure of intracellular ROS scavenging activity. Ourresults are illustrated in Fig. 2A. The level of ROS was 192.7% inUVB-irradiated cells compared to non-irradiated control cells. How-ever, the addition of QR to the cells after exposure to UVB reducedintracellular ROS accumulation, with levels of 170.1%, 162.9%, and121.6% at QR concentrations of 12.5, 25, and 50 lM, respectively.This reduction was statistically significant for the 50-lM concentra-tion (p < 0.05). Hence, QR inhibited UVB-induced ROS intracellularformation. Increases in ROS levels under physiological conditionsare important but exert adverse effects under oxidative stress con-ditions. Therefore, ROS are generally believed to function as keymediators of cell death [29]. Because QR was found to exert a ROSscavenging effect, we further evaluated its protective effect againstUVB-induced cell damage. The protective effects of QR on cell viabil-ity in UVB-induced HaCaT cells were measured by an MTT assay. Inthe absence of QR, UVB-irradiated cells showed marked cell death,whereas QR (at 12.5, 25, and 50 lM) prevented UVB-induced dam-age, restoring cell viability to levels of 38.6%, 42.3%, and 59.4%,respectively (Fig. 2B). In addition, QR did not show any cytotoxiceffect it self in the tested cells line (data not shown). Our resultsdemonstrate that QR protects against UVB-induced cell death viaROS scavenging effect. FVs such as QR are known to have powerfulantioxidant properties, which are generally attributed to the pres-ence of one or more phenolic hydroxyl groups within the FV struc-ture [30].

Fig. 4. Protective effect of QR against UVB-induced ROS generation in zebrafish. Theembryos were exposed to UVB (50 mJ/cm2) and treated with QR. After incubation,the embryos were stained with DCFH-DA and intracellular ROS were detected byspectrofluorometry (A) and fluorescence microscopy (B). �Significantly differentfrom only UVB-exposed zebrafish (p < 0.05).

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3.2. Protective effect of QR on UVB-induced apoptosis

Based on the profound antioxidative activity evidenced by theQR, we investigated the effects of QR against UVB-induced apopto-sis. DNA damage is known to be one of the most sensitive biolog-ical markers for evaluating oxidative stress, representing theimbalance between ROS generation and the efficacy of the antiox-idant system [31,32]. To assess DNA damage in dead cells, nuclei ofHaCaT cells were double stained with Hoechst 33342 and PI. Themicroscopic photograph in Fig. 3 shows that control cells had in-tact nuclei, whereas UVB-irradiated cells exhibited significant frag-mentation, nuclear condensation, destruction characteristic ofapoptosis (bright blue color), and cell death (red color). However,the amount of fragmentation, nuclear condensation, and destruc-tion of UVB-irradiated cells was dramatically reduced when thecells were treated with QR. Moreover, UVB exposure increasedthe portion of sub-G1 peaks to 39.3%, whereas QR-treated cellsevidenced significant reductions in sub-G1 DNA contents (21.9%)(Table 1). DNA damage can be enhanced by exposure to variouschemicals, environmental pollutants, steroid hormones, and radia-tion, leading to diseases such as cancer and heart disease [33–35].The cells of the human body are continuously attacked by physicalagents (like solar radiation) and a variety of chemical compounds

Table 1Effect of QR on the cell cycle pattern and the apoptotic portion of HaCaT cells by flowcytometric analysis.

QR(50 lM)

UVB (50 mJ/cm2)

% Of cells

Sub-G1 G0/G1 S G2/M

� � 5.4 ± 3.2 34.4 ± 4.4 28.2 ± 3.2 32.0 ± 2.7� + 39.3 ± 2.3 19.9 ± 3.5 25.6 ± 2.4 14.0 ± 1.5+ + 21.9 ± 3.7* 27.9 ± 4.9* 29.8 ± 2.9 20.4 ± 1.2*

HaCaT cells were seeded at 1 � 105 cells/mL and treated with the 50 lM for 12 h.The cells were stained with PI and analyzed by flow cytometry, Each point repre-sents the mean ± SD of three independent experiments.* Significantly different from only UVB-exposed cells (p < 0.05).

UVB

UVB+QR

Fig. 5. Protective effect of QR against UVB-induced cell death in zebrafish. Theembryos were exposed to UVB (50 mJ/cm2) and treated with QR. After incubation,the embryos were stained with acridine orange and cell death was detected byspectrofluorometry (A) and fluorescence microscopy (B). �Significantly differentfrom only UVB-exposed zebrafish (p < 0.05).

130 H.-M. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 126–131

and ROS, the latter of which arise as natural by-products ofmetabolism. These substances can induce DNA damage. If DNAlesions are not repaired, a cascade of adverse biological conse-quences can be initiated [36]. Hence, many natural and syntheticcompounds have been investigated in the recent past for theirefficacy to protect against oxidative stress in biological systems[21,22,37]. In the present study, we found that QR not onlysuppressed the generation of ROS but also protected against UVBirradiation-induced DNA damage.

3.3. Protective effect of QR against UVB-induced cell death in zebrafish

The zebrafish has become a popular model in pharmacologicalstudies for screening of chemical libraries, mode-of-action studiesof gene function, predictive toxicology, teratogenicity, and phar-maco- and toxico-genomics. It was shown that zebrafish can beused as a suitable model in carcinogenesis studies, anticancer druginvestigation, inflammatory processes, as well as for lipid metabo-lism, since the response to cholesterol blockers is similar to that inmammals [38]. The number of chemicals that need to be tested inthe field of chemical toxicity and drug discovery is steadily increas-ing. Therefore, the need for high-throughput screening methodsarises, where the use of zebrafish embryos was proposed becauseof their small size and thus suitability for studies in multi-wellplates [12,39]. Not only are toxicity screening applications imagin-able but also application for the clarification of toxicity mecha-nisms have been reported [40]. Here, we have investigated theprotective efficacy of QR against UVB-induced oxidative stress inzebrafish as an alternative animal model system. ROS scavengingeffect of QR in zebrafish is shown in Fig. 4. The level of ROS was187.8% in UVB-irradiated zebrafish compared to non-irradiatedcontrol zebrafish. However, the addition of QR to the zebrafishafter exposure to UVB significantly reduced ROS level to 134.8%at 50 lM (Fig. 4A). Fig. 4B is a typical fluorescence micrograph ofROS in the zebrafish. The negative control (no QR or UVB irradia-tion) generated a clear image, whereas the positive control, whichwas irradiated with UVB, showed a marked increase in the fluores-cence signal, suggesting that ROS generation took place during UVBirradiation in the zebrafish. However, in zebrafish treated with QRprior to UVB irradiation, a dramatic reduction in the amount of ROSwas observed.

Furthermore, we determined UVB-induced cell death by mea-suring acridine orange fluorescence intensity in the body of thezebrafish (Fig. 5). The UVB irradiation-induced cell death in the zeb-rafish was 242.2% compared to the negative control. However, celldeath was reduced (161.2%) by the addition of QR to UVB-irradiatedzebrafish (Fig. 5A). In agreement, images of UVB-induced zebrafishshowed a significant increase in red fluorescence compared to non-irradiated zebrafish. Red fluorescence, however, was decreased inUVB-irradiated zebrafish pre-treated with QR at 50 lM (Fig. 5B).

4. Conclusions

In the present study, QR provided protection in a UVB-inducedcell injury model through the inhibition of ROS generation. Fur-thermore, QR inhibited ROS generation and cell death induced byUVB irradiation in a zebrafish model. These results indicate thatQR could be used as a potent skin damage protective agent in cos-meceutical products after further in vivo confirmation.

Acknowledgement

This work was supported by KBSI Grant (K31092) to D. Kim.

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