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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 vitro and 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 Korea Jeju Biodiversity Research Institute, Jeju Technopark, Jeju 699-943, Republic of Korea School of Marine Biomedical Sciences, Jeju National University, Jeju 690-756, Republic of Korea d Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan b c
a r t i c l e
i n f o
Article history: Received 22 February 2012 Received in revised form 22 May 2012 Accepted 28 May 2012 Available online 5 June 2012 Keywords: Ultraviolet (UV) B Quercitrin (QR) Oxidative stress Keratinocyte Photoaging Zebrafish
a b s t r a c t Chronic exposure of skin to ultraviolet (UV) B radiation induces oxidative stress, which in turn, plays a crucial role in the induction of skin aging. The search for strategies to reverse skin aging is being constantly pursued. Here, the cytoprotective effect of quercitrin (QR) on UVB-induced cell injury in HaCaT human 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 treatment with QR, and significantly so with QR at 50 lM. As a result, QR reduced UVB-induced cell death and apoptosis in HaCaT cells. QR similarly reduced UVB-induced ROS generation and cell death in live zebrafish. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Ultraviolet (UV) radiation is the major environmental cause of skin damage. Although only 0.5% of UVB radiation reaches the Earth, it is the main cause of sunburn and probably the most carcinogenic component of sunlight [1,2]. Exposure of mammalian skin to UV light impairs enzymatic and non-enzymatic antioxidant activity [3,4] and increases the cellular levels of reactive oxygen species (ROS), which, in turn, damage lipids, proteins, and nucleic acids in epidermal cells and are likely to contribute to the process of photocarcinogenesis and photoaging [5,6]. The increase in ROS is accompanied by the activation of many ROS-sensitive signaling pathways and induced gene transcription [7]. In addition, exposure of 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 fish that has emerged as a useful vertebrate model organism because of its small size, large clutches, transparency, low cost, and physi-
⇑ Corresponding authors. Tel.: +82 64 800 4930; fax: +82 64 805 7800. E-mail addresses: [email protected] (D. Kim), [email protected] (K.-N. Kim). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.05.020
ological similarity to mammals [9,10]. The administration of drugs and/or small molecules to zebrafish is uncomplicated because the early-stage embryo rapidly absorbs small molecular compounds diluted in the bathing media through the skin and gills. In addition, zebrafish has melanin pigments on the body surface, allowing simple observation of the pigmentation process without complicated experimental procedures [11]. Therefore, the zebrafish has been recently used as an in vivo model of oxidative stress for studying UV protection [12,13]. Flavonoids (FVs), one of the most diverse and widespread groups of natural compounds, are probably the most common natural phenolics [14,15]. FVs are efficient antioxidants that scavenge oxygen radicals and possess anticancer, hypolipidemic, anti-aging, and anti-inflammatory activities [16]. Among the broad variety of FVs, quercitrin (QR; quercetin-3-O-rhamnoside) has been employed previously as an antibacterial agent [17] and has been shown to inhibit the oxidation of low-density lipoproteins and prevent allergic reaction [18,19]. Furthermore, we previously demonstrated that QR exerts protective effects against H2O2-induced dysfunction in lung fibroblast cells [20]. However, the protective effects of QR against UV-induced cell death in vitro and in vivo have not been assessed thus far. Therefore, in this study, we examined the in vitro protective effects of QR against UVB-induced cell damage and adopted the zebrafish as an alternative in vivo experimental model.
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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-diphenyltetrazolium bromide (MTT), RNase A, propidium iodide (PI), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and Hoechst 33342 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin, and trypsin–ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Life Technologies, Burlington, ON, Canada). The other chemicals and reagents used were of analytical grade. 2.2. Cell culture HaCaT (human skin keratinocyte cells) cells were grown in DMEM supplemented with 10% (v/v) heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 lg/mL). The cells were then incubated in an atmosphere of 5% CO2 at 37 °C and were subcultured 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) according to the method describe by Heo et al. [21,22]. Optimum irradiation dose was evaluated at 50 mJ/cm2; therefore, the 50 mJ/cm2 of UVB was used in further experiments. 2.4. Intracellular reactive oxygen species (ROS) scavenging activity To detect intracellular ROS, the cells were seeded in 96-well plates at a concentration of 1.0 105 cells/mL. After 16 h, the cells were exposed to UVB (50 mJ/cm2) and the QR was treated with different concentrations (12.5, 25 and 50 lM) and incubated at 37 °C under a humidified atmosphere. The cells were incubated for an additional 2 h at 37 °C under a humidified atmosphere with 5% CO2. Finally DCHF-DA (5 lg/mL) was introduced to the cells, and 20 70 -dichlorodihydrofluorescein fluorescence was detected at an excitation wavelength of 485 nm and an emissions wavelength of 535 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 of 1.0 105 cells/mL. After 16 h, the cells were exposed to UVB (50 mJ/cm2) with various concentrations (12, 25, and 50 lM) of QR and incubated for 24 h at 37 °C. MTT stock solution (50 lL;
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2 mg/mL in PBS) was then added to each well to obtain a total reaction volume of 250 lL. After 4 h of incubation, the plate was centrifuged at 2000 rpm for 10 min, and the supernatant was aspirated. The formazan crystals in each well were dissolved in DMSO. The amount of purple formazan was determined by measuring the absorbance at 540 nm. 2.6. Nuclear double staining The nuclear morphology of cells was studied by using cell-permeable DNA dyes Hoechst 33342 and PI. Cells were seeded in a 24well 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) and incubated for 12 h at 37 °C. Then, Hoechst 33342 and PI were added to the culture medium at a final concentration of 10 and 5 lg/mL, and the plate was incubated for another 10 min at 37 °C. The stained cells were observed under a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera to examine the degree of nuclear condensation. Cells with homogeneously stained nuclei were considered to be viable, whereas the presence of chromatin condensation and/or fragmentation was considered indicative of apoptosis. 2.7. Cell-cycle analysis Cell-cycle analysis was performed to determine the proportion of apoptotic sub-G1 hypodiploid cells [23]. Cells were seeded in a 6-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). After 12 h of incubation, the cells were harvested at the indicated time and fixed in 1 ml of 70% ethanol for 30 min at 4 °C. They were then washed twice with PBS and incubated in the dark in 1 mL of PBS containing 100 lg PI and 100 lg RNase A for 30 min at 37 °C. Flow cytometric analysis was performed with a FACSCalibur flow cytometer. The effect of QR on the cell cycle was determined by changes in the percentage of cells in each phase of the cell cycle and assessed with histograms generated by software programs CellQuest 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 L acrylic tank at 28.5 °C with a 14/10 h light/dark cycle. Zebrafish were fed 3 times a day, 6 days/week, with Tetamin flake food supplemented with live Artemia salina. Embryos were obtained from natural 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 rate of 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 embryos were rinsed with fresh embryo medium. Then, zebrafish embryos were layered in a glass slide, covered with sufficient embryo medium, and exposed to 50 mJ/cm2 UVB. 2.10. Waterborne exposure of embryos to QR
Fig. 1. Chemical structure of QR.
Exposure of embryos to UVB and QR was done as a previously described method [12]. At approximately 2 days post-fertilization (dpf), embryos (n = 25) were transferred to individual wells of a 24-well plate and maintained in embryo medium containing 1 mL of vehicle (0.1% DMSO) or 50 lM QR for 1 h. Then, they were
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Generation of ROS (%)
250
using a Perkin-Elmer LS-5B spectrofluorometer. Stained embryos were observed under a fluorescence microscope.
A
200
150
*
100
50
0 12.5
QR (μM) UVB (50 mJ/cm2) 120
25
50
B
Cell viability (%)
100 80
* 60
2.12. Measurement of oxidative stress-induced cell death in zebrafish embryos Cell death was detected in live embryos using acridine orange staining, a nucleic acid-specific metachromatic dye that interacts with 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 embryos with UVB, the embryos were transferred into 96-well plate and treated with acridine orange solution (7 lg/mL), and the plates were incubated for 1 h in the dark at 28.5 °C. After incubation, the embryos were rinsed in embryo medium and anesthetized with phenoxyethanol before visualization as described above. Individual embryo fluorescence intensity was quantified using a Perkin-Elmer LS-5B spectrofluorometer. Stained embryos were observed under a fluorescence microscope. 2.13. Statistical analysis
40 20 0
QR (μM) UVB (50 mJ/cm2)
12.5
25
50
All data are presented as the mean ± SD of at least three replicates. Significant differences among the groups were determined by using the unpaired Student’s t-test. P < 0.05 was considered statistically significant. 3. Results and discussion
Fig. 2. Protective effect of QR against UVB-induced intracellular ROS generation and cytotoxicity in HaCaT cells. Cells were exposed to UVB (50 mJ/cm2) and treated with QR. After an additional 2 h, the intracellular ROS generated were detected by spectrofluorometry (A). The viability of HaCaT cells was determined by MTT assay (B). Significantly different from only UVB-exposed cells (p < 0.05).
irradiated with UVB (312 nm) alone or in combination with QR treatment. 2.11. Estimation of intracellular ROS generation and image analysis Generation of ROS production in zebrafish embryos was assessed using an oxidation-sensitive fluorescent probe dye, DCFH-DA [12]. 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 irradiating embryos with UVB, the embryos were transferred into a 96-well plate and treated with DCFH-DA (20 lg/mL), after which the plates were incubated for 1 h in the dark at 28.5 °C. After incubation, the embryos were rinsed in embryo medium and anesthetized with phenoxyethanol (1/20 dilution; 5 min) before visualization. Individual embryo fluorescence intensity was quantified
Control
UV irradiation is a potent inducer of ROS such as hydroxyl radicals (OH), superoxide radicals (O 2 ), and peroxyl radicals and their active precursors, namely singlet oxygen 1O2, hydrogen peroxide (H2O2), and ozone [25], all of which play a role in the modulation of apoptosis [26]. Several studies have shown UVB-induced ROS formation leading to apoptosis [27]. Therefore, the removal of excess ROS or the suppression of their generation by antioxidants may prove effective in preventing UVB-induced cell death. QR has been reported to be a protective agent against H2O2-induced cell injury in lung fibroblasts and osteoblastic cells. However, there is no information available on the neuroprotective ability of QR against UVB-induced oxidative stress in skin keratinocyte cells. Here, we show for the first time that QR protects against UVBinduced ROS production and cell damage in vitro and in vivo. 3.1. Effects of QR on UVB-induced intracellular ROS generation and cytotoxicity Generation of intracellular ROS can be detected by using DCFH-DA, which permeates the cell membrane freely. DCFH-DA is
UVB
UVB+QR
Fig. 3. Protective effect of QR against UVB-induced apoptosis in HaCaT cells. Cells were exposed to UVB (50 mJ/cm2) with QR and incubated for 12 h. The cells were double stained with Hoechst 33342 and PI solution and then observed under a fluorescence microscope using a blue filter. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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250
Generation of ROS (%)
hydrolyzed by intracellular esterase to convert to nonfluorescent DCFH. In the presence of ROS, DCFH is oxidized to highly fluorescent DCF [28], a measure of intracellular ROS scavenging activity. Our results are illustrated in Fig. 2A. The level of ROS was 192.7% in UVB-irradiated cells compared to non-irradiated control cells. However, the addition of QR to the cells after exposure to UVB reduced intracellular ROS accumulation, with levels of 170.1%, 162.9%, and 121.6% at QR concentrations of 12.5, 25, and 50 lM, respectively. This reduction was statistically significant for the 50-lM concentration (p < 0.05). Hence, QR inhibited UVB-induced ROS intracellular formation. Increases in ROS levels under physiological conditions are important but exert adverse effects under oxidative stress conditions. Therefore, ROS are generally believed to function as key mediators of cell death [29]. Because QR was found to exert a ROS scavenging effect, we further evaluated its protective effect against UVB-induced cell damage. The protective effects of QR on cell viability in UVB-induced HaCaT cells were measured by an MTT assay. In the absence of QR, UVB-irradiated cells showed marked cell death, whereas QR (at 12.5, 25, and 50 lM) prevented UVB-induced damage, restoring cell viability to levels of 38.6%, 42.3%, and 59.4%, respectively (Fig. 2B). In addition, QR did not show any cytotoxic effect it self in the tested cells line (data not shown). Our results demonstrate that QR protects against UVB-induced cell death via ROS scavenging effect. FVs such as QR are known to have powerful antioxidant properties, which are generally attributed to the presence of one or more phenolic hydroxyl groups within the FV structure [30].
A
200
∗
150
100
50
0 Control
UVB
UVB+QR
B
Control
UVB
UVB+QR
Fig. 4. Protective effect of QR against UVB-induced ROS generation in zebrafish. The embryos 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 by spectrofluorometry (A) and fluorescence microscopy (B). Significantly different from only UVB-exposed zebrafish (p < 0.05).
Based on the profound antioxidative activity evidenced by the QR, we investigated the effects of QR against UVB-induced apoptosis. DNA damage is known to be one of the most sensitive biological markers for evaluating oxidative stress, representing the imbalance between ROS generation and the efficacy of the antioxidant system [31,32]. To assess DNA damage in dead cells, nuclei of HaCaT cells were double stained with Hoechst 33342 and PI. The microscopic photograph in Fig. 3 shows that control cells had intact nuclei, whereas UVB-irradiated cells exhibited significant fragmentation, nuclear condensation, destruction characteristic of apoptosis (bright blue color), and cell death (red color). However, the amount of fragmentation, nuclear condensation, and destruction of UVB-irradiated cells was dramatically reduced when the cells were treated with QR. Moreover, UVB exposure increased the portion of sub-G1 peaks to 39.3%, whereas QR-treated cells evidenced significant reductions in sub-G1 DNA contents (21.9%) (Table 1). DNA damage can be enhanced by exposure to various chemicals, environmental pollutants, steroid hormones, and radiation, leading to diseases such as cancer and heart disease [33–35]. The cells of the human body are continuously attacked by physical agents (like solar radiation) and a variety of chemical compounds
Table 1 Effect of QR on the cell cycle pattern and the apoptotic portion of HaCaT cells by flow cytometric analysis. QR (50 lM)
UVB (50 mJ/ cm2)
% Of cells Sub-G1
G0/G1
S
G2/M
+
+ +
5.4 ± 3.2 39.3 ± 2.3 21.9 ± 3.7*
34.4 ± 4.4 19.9 ± 3.5 27.9 ± 4.9*
28.2 ± 3.2 25.6 ± 2.4 29.8 ± 2.9
32.0 ± 2.7 14.0 ± 1.5 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 represents the mean ± SD of three independent experiments. * Significantly different from only UVB-exposed cells (p < 0.05).
300 250
Cell death (%)
3.2. Protective effect of QR on UVB-induced apoptosis
A
200
*
150 100 50 0
B
Control
UVB
UVB+QR
Control
UVB
UVB+QR
Fig. 5. Protective effect of QR against UVB-induced cell death in zebrafish. The embryos 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 by spectrofluorometry (A) and fluorescence microscopy (B). Significantly different from only UVB-exposed zebrafish (p < 0.05).
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and ROS, the latter of which arise as natural by-products of metabolism. These substances can induce DNA damage. If DNA lesions are not repaired, a cascade of adverse biological consequences can be initiated [36]. Hence, many natural and synthetic compounds have been investigated in the recent past for their efficacy to protect against oxidative stress in biological systems [21,22,37]. In the present study, we found that QR not only suppressed the generation of ROS but also protected against UVB irradiation-induced DNA damage.
3.3. Protective effect of QR against UVB-induced cell death in zebrafish The zebrafish has become a popular model in pharmacological studies for screening of chemical libraries, mode-of-action studies of gene function, predictive toxicology, teratogenicity, and pharmaco- and toxico-genomics. It was shown that zebrafish can be used as a suitable model in carcinogenesis studies, anticancer drug investigation, inflammatory processes, as well as for lipid metabolism, since the response to cholesterol blockers is similar to that in mammals [38]. The number of chemicals that need to be tested in the field of chemical toxicity and drug discovery is steadily increasing. Therefore, the need for high-throughput screening methods arises, where the use of zebrafish embryos was proposed because of their small size and thus suitability for studies in multi-well plates [12,39]. Not only are toxicity screening applications imaginable but also application for the clarification of toxicity mechanisms have been reported [40]. Here, we have investigated the protective efficacy of QR against UVB-induced oxidative stress in zebrafish as an alternative animal model system. ROS scavenging effect of QR in zebrafish is shown in Fig. 4. The level of ROS was 187.8% in UVB-irradiated zebrafish compared to non-irradiated control zebrafish. However, the addition of QR to the zebrafish after exposure to UVB significantly reduced ROS level to 134.8% at 50 lM (Fig. 4A). Fig. 4B is a typical fluorescence micrograph of ROS in the zebrafish. The negative control (no QR or UVB irradiation) generated a clear image, whereas the positive control, which was irradiated with UVB, showed a marked increase in the fluorescence signal, suggesting that ROS generation took place during UVB irradiation in the zebrafish. However, in zebrafish treated with QR prior to UVB irradiation, a dramatic reduction in the amount of ROS was observed. Furthermore, we determined UVB-induced cell death by measuring acridine orange fluorescence intensity in the body of the zebrafish (Fig. 5). The UVB irradiation-induced cell death in the zebrafish was 242.2% compared to the negative control. However, cell death was reduced (161.2%) by the addition of QR to UVB-irradiated zebrafish (Fig. 5A). In agreement, images of UVB-induced zebrafish showed a significant increase in red fluorescence compared to nonirradiated zebrafish. Red fluorescence, however, was decreased in UVB-irradiated zebrafish pre-treated with QR at 50 lM (Fig. 5B).
4. Conclusions In the present study, QR provided protection in a UVB-induced cell injury model through the inhibition of ROS generation. Furthermore, QR inhibited ROS generation and cell death induced by UVB irradiation in a zebrafish model. These results indicate that QR could be used as a potent skin damage protective agent in cosmeceutical products after further in vivo confirmation.
Acknowledgement This work was supported by KBSI Grant (K31092) to D. Kim.
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Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Quercitrin protects against ultraviolet B-induced cell death in vitro and 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 Korea Jeju Biodiversity Research Institute, Jeju Technopark, Jeju 699-943, Republic of Korea School of Marine Biomedical Sciences, Jeju National University, Jeju 690-756, Republic of Korea d Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan b c
a r t i c l e
i n f o
Article history: Received 22 February 2012 Received in revised form 22 May 2012 Accepted 28 May 2012 Available online 5 June 2012 Keywords: Ultraviolet (UV) B Quercitrin (QR) Oxidative stress Keratinocyte Photoaging Zebrafish
a b s t r a c t Chronic exposure of skin to ultraviolet (UV) B radiation induces oxidative stress, which in turn, plays a crucial role in the induction of skin aging. The search for strategies to reverse skin aging is being constantly pursued. Here, the cytoprotective effect of quercitrin (QR) on UVB-induced cell injury in HaCaT human 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 treatment with QR, and significantly so with QR at 50 lM. As a result, QR reduced UVB-induced cell death and apoptosis in HaCaT cells. QR similarly reduced UVB-induced ROS generation and cell death in live zebrafish. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Ultraviolet (UV) radiation is the major environmental cause of skin damage. Although only 0.5% of UVB radiation reaches the Earth, it is the main cause of sunburn and probably the most carcinogenic component of sunlight [1,2]. Exposure of mammalian skin to UV light impairs enzymatic and non-enzymatic antioxidant activity [3,4] and increases the cellular levels of reactive oxygen species (ROS), which, in turn, damage lipids, proteins, and nucleic acids in epidermal cells and are likely to contribute to the process of photocarcinogenesis and photoaging [5,6]. The increase in ROS is accompanied by the activation of many ROS-sensitive signaling pathways and induced gene transcription [7]. In addition, exposure of 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 fish that has emerged as a useful vertebrate model organism because of its small size, large clutches, transparency, low cost, and physi-
⇑ Corresponding authors. Tel.: +82 64 800 4930; fax: +82 64 805 7800. E-mail addresses: [email protected] (D. Kim), [email protected] (K.-N. Kim). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.05.020
ological similarity to mammals [9,10]. The administration of drugs and/or small molecules to zebrafish is uncomplicated because the early-stage embryo rapidly absorbs small molecular compounds diluted in the bathing media through the skin and gills. In addition, zebrafish has melanin pigments on the body surface, allowing simple observation of the pigmentation process without complicated experimental procedures [11]. Therefore, the zebrafish has been recently used as an in vivo model of oxidative stress for studying UV protection [12,13]. Flavonoids (FVs), one of the most diverse and widespread groups of natural compounds, are probably the most common natural phenolics [14,15]. FVs are efficient antioxidants that scavenge oxygen radicals and possess anticancer, hypolipidemic, anti-aging, and anti-inflammatory activities [16]. Among the broad variety of FVs, quercitrin (QR; quercetin-3-O-rhamnoside) has been employed previously as an antibacterial agent [17] and has been shown to inhibit the oxidation of low-density lipoproteins and prevent allergic reaction [18,19]. Furthermore, we previously demonstrated that QR exerts protective effects against H2O2-induced dysfunction in lung fibroblast cells [20]. However, the protective effects of QR against UV-induced cell death in vitro and in vivo have not been assessed thus far. Therefore, in this study, we examined the in vitro protective effects of QR against UVB-induced cell damage and adopted the zebrafish as an alternative in vivo experimental model.
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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-diphenyltetrazolium bromide (MTT), RNase A, propidium iodide (PI), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and Hoechst 33342 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin, and trypsin–ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Life Technologies, Burlington, ON, Canada). The other chemicals and reagents used were of analytical grade. 2.2. Cell culture HaCaT (human skin keratinocyte cells) cells were grown in DMEM supplemented with 10% (v/v) heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 lg/mL). The cells were then incubated in an atmosphere of 5% CO2 at 37 °C and were subcultured 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) according to the method describe by Heo et al. [21,22]. Optimum irradiation dose was evaluated at 50 mJ/cm2; therefore, the 50 mJ/cm2 of UVB was used in further experiments. 2.4. Intracellular reactive oxygen species (ROS) scavenging activity To detect intracellular ROS, the cells were seeded in 96-well plates at a concentration of 1.0 105 cells/mL. After 16 h, the cells were exposed to UVB (50 mJ/cm2) and the QR was treated with different concentrations (12.5, 25 and 50 lM) and incubated at 37 °C under a humidified atmosphere. The cells were incubated for an additional 2 h at 37 °C under a humidified atmosphere with 5% CO2. Finally DCHF-DA (5 lg/mL) was introduced to the cells, and 20 70 -dichlorodihydrofluorescein fluorescence was detected at an excitation wavelength of 485 nm and an emissions wavelength of 535 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 of 1.0 105 cells/mL. After 16 h, the cells were exposed to UVB (50 mJ/cm2) with various concentrations (12, 25, and 50 lM) of QR and incubated for 24 h at 37 °C. MTT stock solution (50 lL;
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2 mg/mL in PBS) was then added to each well to obtain a total reaction volume of 250 lL. After 4 h of incubation, the plate was centrifuged at 2000 rpm for 10 min, and the supernatant was aspirated. The formazan crystals in each well were dissolved in DMSO. The amount of purple formazan was determined by measuring the absorbance at 540 nm. 2.6. Nuclear double staining The nuclear morphology of cells was studied by using cell-permeable DNA dyes Hoechst 33342 and PI. Cells were seeded in a 24well 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) and incubated for 12 h at 37 °C. Then, Hoechst 33342 and PI were added to the culture medium at a final concentration of 10 and 5 lg/mL, and the plate was incubated for another 10 min at 37 °C. The stained cells were observed under a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera to examine the degree of nuclear condensation. Cells with homogeneously stained nuclei were considered to be viable, whereas the presence of chromatin condensation and/or fragmentation was considered indicative of apoptosis. 2.7. Cell-cycle analysis Cell-cycle analysis was performed to determine the proportion of apoptotic sub-G1 hypodiploid cells [23]. Cells were seeded in a 6-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). After 12 h of incubation, the cells were harvested at the indicated time and fixed in 1 ml of 70% ethanol for 30 min at 4 °C. They were then washed twice with PBS and incubated in the dark in 1 mL of PBS containing 100 lg PI and 100 lg RNase A for 30 min at 37 °C. Flow cytometric analysis was performed with a FACSCalibur flow cytometer. The effect of QR on the cell cycle was determined by changes in the percentage of cells in each phase of the cell cycle and assessed with histograms generated by software programs CellQuest 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 L acrylic tank at 28.5 °C with a 14/10 h light/dark cycle. Zebrafish were fed 3 times a day, 6 days/week, with Tetamin flake food supplemented with live Artemia salina. Embryos were obtained from natural 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 rate of 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 embryos were rinsed with fresh embryo medium. Then, zebrafish embryos were layered in a glass slide, covered with sufficient embryo medium, and exposed to 50 mJ/cm2 UVB. 2.10. Waterborne exposure of embryos to QR
Fig. 1. Chemical structure of QR.
Exposure of embryos to UVB and QR was done as a previously described method [12]. At approximately 2 days post-fertilization (dpf), embryos (n = 25) were transferred to individual wells of a 24-well plate and maintained in embryo medium containing 1 mL of vehicle (0.1% DMSO) or 50 lM QR for 1 h. Then, they were
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Generation of ROS (%)
250
using a Perkin-Elmer LS-5B spectrofluorometer. Stained embryos were observed under a fluorescence microscope.
A
200
150
*
100
50
0 12.5
QR (μM) UVB (50 mJ/cm2) 120
25
50
B
Cell viability (%)
100 80
* 60
2.12. Measurement of oxidative stress-induced cell death in zebrafish embryos Cell death was detected in live embryos using acridine orange staining, a nucleic acid-specific metachromatic dye that interacts with 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 embryos with UVB, the embryos were transferred into 96-well plate and treated with acridine orange solution (7 lg/mL), and the plates were incubated for 1 h in the dark at 28.5 °C. After incubation, the embryos were rinsed in embryo medium and anesthetized with phenoxyethanol before visualization as described above. Individual embryo fluorescence intensity was quantified using a Perkin-Elmer LS-5B spectrofluorometer. Stained embryos were observed under a fluorescence microscope. 2.13. Statistical analysis
40 20 0
QR (μM) UVB (50 mJ/cm2)
12.5
25
50
All data are presented as the mean ± SD of at least three replicates. Significant differences among the groups were determined by using the unpaired Student’s t-test. P < 0.05 was considered statistically significant. 3. Results and discussion
Fig. 2. Protective effect of QR against UVB-induced intracellular ROS generation and cytotoxicity in HaCaT cells. Cells were exposed to UVB (50 mJ/cm2) and treated with QR. After an additional 2 h, the intracellular ROS generated were detected by spectrofluorometry (A). The viability of HaCaT cells was determined by MTT assay (B). Significantly different from only UVB-exposed cells (p < 0.05).
irradiated with UVB (312 nm) alone or in combination with QR treatment. 2.11. Estimation of intracellular ROS generation and image analysis Generation of ROS production in zebrafish embryos was assessed using an oxidation-sensitive fluorescent probe dye, DCFH-DA [12]. 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 irradiating embryos with UVB, the embryos were transferred into a 96-well plate and treated with DCFH-DA (20 lg/mL), after which the plates were incubated for 1 h in the dark at 28.5 °C. After incubation, the embryos were rinsed in embryo medium and anesthetized with phenoxyethanol (1/20 dilution; 5 min) before visualization. Individual embryo fluorescence intensity was quantified
Control
UV irradiation is a potent inducer of ROS such as hydroxyl radicals (OH), superoxide radicals (O 2 ), and peroxyl radicals and their active precursors, namely singlet oxygen 1O2, hydrogen peroxide (H2O2), and ozone [25], all of which play a role in the modulation of apoptosis [26]. Several studies have shown UVB-induced ROS formation leading to apoptosis [27]. Therefore, the removal of excess ROS or the suppression of their generation by antioxidants may prove effective in preventing UVB-induced cell death. QR has been reported to be a protective agent against H2O2-induced cell injury in lung fibroblasts and osteoblastic cells. However, there is no information available on the neuroprotective ability of QR against UVB-induced oxidative stress in skin keratinocyte cells. Here, we show for the first time that QR protects against UVBinduced ROS production and cell damage in vitro and in vivo. 3.1. Effects of QR on UVB-induced intracellular ROS generation and cytotoxicity Generation of intracellular ROS can be detected by using DCFH-DA, which permeates the cell membrane freely. DCFH-DA is
UVB
UVB+QR
Fig. 3. Protective effect of QR against UVB-induced apoptosis in HaCaT cells. Cells were exposed to UVB (50 mJ/cm2) with QR and incubated for 12 h. The cells were double stained with Hoechst 33342 and PI solution and then observed under a fluorescence microscope using a blue filter. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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250
Generation of ROS (%)
hydrolyzed by intracellular esterase to convert to nonfluorescent DCFH. In the presence of ROS, DCFH is oxidized to highly fluorescent DCF [28], a measure of intracellular ROS scavenging activity. Our results are illustrated in Fig. 2A. The level of ROS was 192.7% in UVB-irradiated cells compared to non-irradiated control cells. However, the addition of QR to the cells after exposure to UVB reduced intracellular ROS accumulation, with levels of 170.1%, 162.9%, and 121.6% at QR concentrations of 12.5, 25, and 50 lM, respectively. This reduction was statistically significant for the 50-lM concentration (p < 0.05). Hence, QR inhibited UVB-induced ROS intracellular formation. Increases in ROS levels under physiological conditions are important but exert adverse effects under oxidative stress conditions. Therefore, ROS are generally believed to function as key mediators of cell death [29]. Because QR was found to exert a ROS scavenging effect, we further evaluated its protective effect against UVB-induced cell damage. The protective effects of QR on cell viability in UVB-induced HaCaT cells were measured by an MTT assay. In the absence of QR, UVB-irradiated cells showed marked cell death, whereas QR (at 12.5, 25, and 50 lM) prevented UVB-induced damage, restoring cell viability to levels of 38.6%, 42.3%, and 59.4%, respectively (Fig. 2B). In addition, QR did not show any cytotoxic effect it self in the tested cells line (data not shown). Our results demonstrate that QR protects against UVB-induced cell death via ROS scavenging effect. FVs such as QR are known to have powerful antioxidant properties, which are generally attributed to the presence of one or more phenolic hydroxyl groups within the FV structure [30].
A
200
∗
150
100
50
0 Control
UVB
UVB+QR
B
Control
UVB
UVB+QR
Fig. 4. Protective effect of QR against UVB-induced ROS generation in zebrafish. The embryos 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 by spectrofluorometry (A) and fluorescence microscopy (B). Significantly different from only UVB-exposed zebrafish (p < 0.05).
Based on the profound antioxidative activity evidenced by the QR, we investigated the effects of QR against UVB-induced apoptosis. DNA damage is known to be one of the most sensitive biological markers for evaluating oxidative stress, representing the imbalance between ROS generation and the efficacy of the antioxidant system [31,32]. To assess DNA damage in dead cells, nuclei of HaCaT cells were double stained with Hoechst 33342 and PI. The microscopic photograph in Fig. 3 shows that control cells had intact nuclei, whereas UVB-irradiated cells exhibited significant fragmentation, nuclear condensation, destruction characteristic of apoptosis (bright blue color), and cell death (red color). However, the amount of fragmentation, nuclear condensation, and destruction of UVB-irradiated cells was dramatically reduced when the cells were treated with QR. Moreover, UVB exposure increased the portion of sub-G1 peaks to 39.3%, whereas QR-treated cells evidenced significant reductions in sub-G1 DNA contents (21.9%) (Table 1). DNA damage can be enhanced by exposure to various chemicals, environmental pollutants, steroid hormones, and radiation, leading to diseases such as cancer and heart disease [33–35]. The cells of the human body are continuously attacked by physical agents (like solar radiation) and a variety of chemical compounds
Table 1 Effect of QR on the cell cycle pattern and the apoptotic portion of HaCaT cells by flow cytometric analysis. QR (50 lM)
UVB (50 mJ/ cm2)
% Of cells Sub-G1
G0/G1
S
G2/M
+
+ +
5.4 ± 3.2 39.3 ± 2.3 21.9 ± 3.7*
34.4 ± 4.4 19.9 ± 3.5 27.9 ± 4.9*
28.2 ± 3.2 25.6 ± 2.4 29.8 ± 2.9
32.0 ± 2.7 14.0 ± 1.5 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 represents the mean ± SD of three independent experiments. * Significantly different from only UVB-exposed cells (p < 0.05).
300 250
Cell death (%)
3.2. Protective effect of QR on UVB-induced apoptosis
A
200
*
150 100 50 0
B
Control
UVB
UVB+QR
Control
UVB
UVB+QR
Fig. 5. Protective effect of QR against UVB-induced cell death in zebrafish. The embryos 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 by spectrofluorometry (A) and fluorescence microscopy (B). Significantly different from only UVB-exposed zebrafish (p < 0.05).
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and ROS, the latter of which arise as natural by-products of metabolism. These substances can induce DNA damage. If DNA lesions are not repaired, a cascade of adverse biological consequences can be initiated [36]. Hence, many natural and synthetic compounds have been investigated in the recent past for their efficacy to protect against oxidative stress in biological systems [21,22,37]. In the present study, we found that QR not only suppressed the generation of ROS but also protected against UVB irradiation-induced DNA damage.
3.3. Protective effect of QR against UVB-induced cell death in zebrafish The zebrafish has become a popular model in pharmacological studies for screening of chemical libraries, mode-of-action studies of gene function, predictive toxicology, teratogenicity, and pharmaco- and toxico-genomics. It was shown that zebrafish can be used as a suitable model in carcinogenesis studies, anticancer drug investigation, inflammatory processes, as well as for lipid metabolism, since the response to cholesterol blockers is similar to that in mammals [38]. The number of chemicals that need to be tested in the field of chemical toxicity and drug discovery is steadily increasing. Therefore, the need for high-throughput screening methods arises, where the use of zebrafish embryos was proposed because of their small size and thus suitability for studies in multi-well plates [12,39]. Not only are toxicity screening applications imaginable but also application for the clarification of toxicity mechanisms have been reported [40]. Here, we have investigated the protective efficacy of QR against UVB-induced oxidative stress in zebrafish as an alternative animal model system. ROS scavenging effect of QR in zebrafish is shown in Fig. 4. The level of ROS was 187.8% in UVB-irradiated zebrafish compared to non-irradiated control zebrafish. However, the addition of QR to the zebrafish after exposure to UVB significantly reduced ROS level to 134.8% at 50 lM (Fig. 4A). Fig. 4B is a typical fluorescence micrograph of ROS in the zebrafish. The negative control (no QR or UVB irradiation) generated a clear image, whereas the positive control, which was irradiated with UVB, showed a marked increase in the fluorescence signal, suggesting that ROS generation took place during UVB irradiation in the zebrafish. However, in zebrafish treated with QR prior to UVB irradiation, a dramatic reduction in the amount of ROS was observed. Furthermore, we determined UVB-induced cell death by measuring acridine orange fluorescence intensity in the body of the zebrafish (Fig. 5). The UVB irradiation-induced cell death in the zebrafish was 242.2% compared to the negative control. However, cell death was reduced (161.2%) by the addition of QR to UVB-irradiated zebrafish (Fig. 5A). In agreement, images of UVB-induced zebrafish showed a significant increase in red fluorescence compared to nonirradiated zebrafish. Red fluorescence, however, was decreased in UVB-irradiated zebrafish pre-treated with QR at 50 lM (Fig. 5B).
4. Conclusions In the present study, QR provided protection in a UVB-induced cell injury model through the inhibition of ROS generation. Furthermore, QR inhibited ROS generation and cell death induced by UVB irradiation in a zebrafish model. These results indicate that QR could be used as a potent skin damage protective agent in cosmeceutical products after further in vivo confirmation.
Acknowledgement This work was supported by KBSI Grant (K31092) to D. Kim.
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