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Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish
G Model PEDOT 7137 No. of Pages 5
International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish Moo Kyun Park a , Gi Jung Im b , Jiwon Chang b , Sung Won Chae b , Jun Yoo b , Won-gue Han b , Gyu Ho Hwang b , Jong Yoon Jung b , Jungim Choi b , Hak Hyun Jung b , Ah-Young Chung c, Hae-Chul Park c , June Choi b, * a b c
Department of Otolaryngology – Head and Neck Surgery, Seoul National University College of Medicine, Seoul, South Korea Department of Otorhinolaryngology – Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea Laboratory of Neurodevelopmental Genetics, Graduate School of Medicine, Korea University, Ansan-City, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 December 2013 Received in revised form 12 May 2014 Accepted 14 May 2014 Available online xxx
Objective: Caffeic acid phenethyl ester (CAPE) is known to reduce the generation of oxygen-derived free radicals, which is a major mechanism of aminoglycoside-induced ototoxicity. The objective of the present study was to evaluate the effects of CAPE on neomycin-induced ototoxicity in zebrafish (Brn3c: EGFP). Methods: Five-day post-fertilization zebrafish larvae (n = 10) were exposed to 125 mM neomycin and one of the following CAPE concentrations for 1 h: 50, 100, 250, 500, or 1000 mM. Ultrastructural changes were evaluated using scanning electron microscopy (SEM). The terminal deoxynucleotidyl transferase (TdT)mediated dUTP-biotin nick-end labeling (TUNEL) assay and 2-[4-(dimethylamino)styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. Results: CAPE decreased neomycin-induced hair cell loss in the neuromasts (500 mM CAPE: 12.7 1.1 cells, 125 mM neomycin only: 6.3 1.1 cells; n = 10, P < 0.05). In the ultrastructural analysis, structures of mitochondria and hair cells were preserved when exposed to 125 mM neomycin and 500 mM CAPE. CAPE decreased apoptosis and mitochondrial damage. Conclusion: In the present study, CAPE attenuated neomycin-induced hair cell damage in zebrafish. The results of the current study suggest that neomycin induces apoptosis, and the apoptotic cell death can be prevented by treatment with CAPE in zebrafish. ã 2014 Published by Elsevier Ireland Ltd.
Keywords: Caffeic acid phenethyl ester Neomycin Ototoxicity Zebrafish
1. Introduction Aminoglycosides are clinically important antibiotics with a broad antibacterial spectrum. They induce no allergic response and are easy to use in an emergency situation. Although aminoglycosides are notorious for their ototoxicity and nephrotoxicity, they are still widely used for treatment of aerobic, gram-negative and some gram-positive bacterial infections. In addition, the use of aminoglycosides is recommended in cases of multidrug-resistant tuberculosis. They are more widely used in developing countries due to their low cost [1,2]. Hair cell death by aminoglycosides is due to both apoptosis and necrosis. The overproduction of reactive oxygen species (ROS) is an
* Corresponding author at: Department of Otorhinolaryngology – Head and Neck Surgery, Korea University Ansan Hospital, Korea University College of Medicine, 123 Jeokgeum-ro (Gojan-dong), Danwon-gu, Ansan-city, Gyeonggi-do 425-707, South Korea. Tel.: +82 31 412 5170; fax: +82 31 412 5174. E-mail address: [email protected] (J. Choi).
important toxic action of aminoglycosides toward hair cells [2,3]. Although the details of ROS formation differ depending on the circumstances, ROS overproduction is a mechanism of hair cell damage in noise, aging, and ototoxicity. Many antioxidants have shown a protective effect on hair cells in various models of damage both in vitro and in vivo [1–4]. Caffeic acid phenethyl ester (CAPE) is an active component of honey bee propolis extracts. It is a natural phenolic chemical compound and superior antioxidant. In addition, it has antiinflammatory, antimicrobial, immunomodulatory, and anticarcinogenic effects. It has been used in folk medicine for many years, and many experimental studies have recently shown its antioxidative effect [5,6]. These findings lead us to believe that CAPE may be a good candidate for prevention of ototoxicity by aminoglycosides. Thus, the aim of the current study was to investigate whether CAPE has protective effects on neomycin-induced hair cell damage in transgenic zebrafish.
http://dx.doi.org/10.1016/j.ijporl.2014.05.018 0165-5876/ ã 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018
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performed in accordance with the guidelines of the Animal Care Ethics Committee of Korea University Medical Center and National Institutes of Health (NIH) guidelines. 2.3. Chemical administration and zebrafish preparation Neomycin solutions were prepared by adding the neomycin powder to the embryo medium. Neomycin has been shown to decrease the viability of neuromasts in a dose-dependent manner. We previously reported that neuromasts treated with 125 mM neomycin for 1 h show a viability rate of 50% [9]. Thus, that concentration was chosen for the following study. For the current experiment, 10 fish were tested at each of the following concentrations of caffeic acid and repeated three times. The 5dpf zebrafish larvae were exposed to 125 mM neomycin and 50, 100, 250, 500, and 1000 mM CAPE for 1 h. The larvae were then washed with embryo medium three times. The fish were anesthetized with tricaine (3-aminobenzoic acid 0.4 g/ethyl ester; 100 mL; pH 7, adjusted using Tris buffer) for 5 min [10–12]. 2.4. Evaluation of zebrafish hair cells
Fig. 1. Quantitative assay of neuromasts in zebrafish. Hair cells from four neuromasts (SO1, SO2, O1, and OC1) were counted. Hair cell survival was calculated as a percentage of the control group. Treatment of the zebrafish with 125 mM neomycin (neo) for 1 h significantly decreased the number of hair cells in neuromasts. Caffeic acid phenethyl ester (CAPE) protected against neomycininduced hair cell loss of the neuromasts in zebrafish (n = 10, P < 0.05).
The zebrafish were placed with methylcellulose on a depression slide for evaluation of hair cell damage under a fluorescence microscope. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines on one side of each fish were analyzed [10–15]. The average numbers of hair cells of the SO1, SO2, O1, and OC1 neuromasts were evaluated in each zebrafish for all experimental and control conditions (n = 10) using fluorescence microscopy (AxioCam MRc5; Carl Zeiss).
2. Methods 2.1. Chemical materials Neomycin and caffeic acid phenethyl ester (CAPE) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Zebrafish model Zebrafish larvae (Brn3c: EGFP) were produced by pairwise matings, raised at 28.5 C in egg water or embryo medium (EM; 15 mM NaCl, 0.5 mM KCl,1 mM CaCl2,1 mM MgSO4, 0.15 mM KH2PO4, 0.05 mM NH2PO4, and 0.7 mM NaHCO3) [7], and staged according to days post-fertilization (dpf) and morphological criteria [8]. The larvae were collected and raised at 28.5 C. The current study was approved by the Korea University Institutional Animal Care and Use Committee (Approval No. KUIACUC-2012-116). All methods were
2.5. Terminal deoxynucleotidyl transferase (TdT)-Mediated dUTPbiotin nick end labeling (TUNEL) assay for apoptosis evaluation and 2[4-(dimethylamino)styryl]-N-ethylpyridiniumiodide (DASPEI) assay for mitochondria Apoptotic cells in zebrafish were identified by the TUNEL method using an in situ cell detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's protocol. The larvae were exposed to medium containing 125 mM neomycin and 500 mM CAPE for 1 h. The larvae were then washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde. Next, the larvae were incubated with 50 mL of TUNEL reaction mixture (TdT and fluorescein-dUTP) at 37 C for 60 min in a humid atmosphere. The 5-dpf zebrafish larvae were evaluated using a fluorescence microscope. In the current study, the fluorescent dye 2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide (DASPEI;
Fig. 2. Fluorescence microscopy images (OC1, 40). Zebrafish (5 dpf) were treated with 125 mM neomycin (neo) and 500 mM CAPE for 1 h. Treatment with neomycin resulted in a significant decrease in the number of hair cells of the neuromasts. CAPE attenuated the neomycin-induced hair cell damage. Bar = 10 mm.
Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018
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Fig. 3. Mitochondrial damage evaluation using DASPEI (SO2, 40). Zebrafish (5 dpf) were treated with 125 mM neomycin (neo) and 500 mM CAPE for 1 h. Treatment with neomycin resulted in significantly decreased DASPEI staining of mitochondria. Mitochondrial damage was ameliorated by CAPE. Bar = 10 mm.
Invitrogen, Carlsbad, CA, USA) was used as a vital dye to stain mitochondria within hair cells. The zebrafish larvae were incubated in an embryo medium containing 0.005% DASPEI for 15 min.
treated in a graded series of ethanol and t-butyl alcohol, dried in a freeze dryer (Hitachi, ES-2030; Tokyo, Japan), platinum-coated using an ion coater (Eiko,l IB-5; Katsuda, Japan), and observed by a field emission SEM (Hitachi, S-4700) [16].
2.6. Scanning electron microscopy 2.7. Statistical analysis For scanning electron microscopy (SEM), 5-dpf zebrafish larvae were exposed to 125 mM neomycin and 500 mM CAPE for 1 h, prefixed by immersion in 2% glutaraldehyde in 0.1 M PBS, and postfixed for 2 h in 1% osmic acid dissolved in PBS. The larvae were
All values are presented as means standard deviation (SD). For statistical comparisons, one-way analysis of variance was used for multiple comparisons using the SPSS software (version 11.0; SPSS,
Fig. 4. Results of TUNEL assay. Neomycin-induced apoptosis was confirmed by TUNEL assay. Apoptotic cells are indicated by light red dots in red-colored fish after the TUNEL reaction when observed using fluorescence microscopy (TUNEL-positive cells: arrow). The negative control showed no light red dots in red-colored fish. Comparison of the color intensity between the 125 mM neomycin (neo) group and the CAPE-treated group for 1 h; 500 mM CAPE significantly decreased the number of TUNEL-positive cells and protected against neomycin-induced apoptotic cell death of the hair cells of the neuromasts of zebrafish (green-colored fish of right column, control for TUNEL reaction; redcolored fish of middle column, TUNEL reaction; green- and red-colored fish of left column, merging of control and TUNEL reaction). Bar = 200 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018
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Fig. 5. Scanning electron microscopy images (SO2, 3k). Compared with the negative control neuromasts, neuromasts in zebrafish treated with 125 mM neomycin (neo) for 1 h showed severe morphological damage to stereocilia and kinocilia under SEM, such as loss or fusion. However, this damage was obviated by treatment with 500 mM CAPE. Bar (at the bottom of each figure, one space) = 10 mm.
Chicago, IL, USA). A post hoc analysis was performed using Tukey's honestly significant difference (HSD) test. A value of P < 0.05 was considered to indicate statistical significance.
stereocilia and kinocilia. CAPE attenuated neomycin-induced hair cell damage compared with treatment with neomycin alone. 4. Discussion
3. Results 3.1. Effect of caffeic acid phenethyl ester (CAPE) on hair cells To assess the effects of neomycin, 5-dpf zebrafish larvae were exposed to various concentrations of neomycin (0, 10, 50, 100, 125, 150, 200, 300, and 500 mM) for 1 h. The presence of neomycin decreased the number of hair cells in a dose-dependent manner. Hair cells from four neuromasts (SO1, SO2, O1, and OC1) were counted to investigate the changes in the neuromasts after treatment with CAPE and neomycin. Hair cell survival was calculated as the average number of hair cells in the control group that were not exposed to neomycin. Treatment of the zebrafish with 125 mM neomycin for 1 h significantly decreased the number of hair cells in the neuromasts. CAPE decreased neomycin-induced hair cell loss of the neuromasts in the zebrafish (negative control: 13.2 0.9 cells; neomycin only: 6.3 1.1 cells; 50 mM CAPE: 7.8 0.4 cells; 100 mM CAPE: 8.0 0.8 cells; 250 mM CAPE: 9.8 1.2 cells; 500 mM CAPE: 12.7 1.1 cells; 1000 mM CAPE: 7.7 0.7 cells; n = 10, P < 0.05) (Figs. 1 and 2). 3.2. Effect of caffeic acid phenethyl ester on mitochondria and apoptosis A caffeic acid phenethyl ester concentration of 500 mM was most effective and was thus used in the DASPEI and TUNEL assays. The DASPEI assay was performed to evaluate mitochondrial damage. Mitochondria within hair cells were protected by CAPE treatment (Fig. 3). The TUNEL reaction was performed to determine neomycin-induced death of neuromasts by apoptosis and the protective influence of CAPE treatment. Apoptotic cells were marked as light red dots after TUNEL reaction when observed by fluorescence microscopy. As shown in Fig. 4, comparison of the color intensity between the 125 mM neomycin group and the 500 mM CAPE group showed a significantly lower number of TUNEL-positive cells, and CAPE attenuated the neomycin-induced apoptotic cell death in zebrafish. 3.3. Effect of caffeic acid phenethyl ester on ultrastructure Fig. 5 shows SEM images of stereocilia and kinocilia from neomycin- and CAPE-treated zebrafish. Compared with control larvae, exposure to 125 mM neomycin for 1 h resulted in severe morphological damage to hair cells, including loss or fusion of
The purpose of this study was to investigate the protective effects of CAPE on neomycin-induced hair cell loss in transgenic zebrafish. In this study, CAPE showed a protective effect on hair cells in the cochlea with antiapoptotic and antioxidative activities. The increase in ROS and depletion of antioxidant protective molecules facilitate lipid peroxidation, which can increase calcium influx and apoptosis in cells of the cochlea. Antioxidants interfere with ROS pathways, protect against hair cell loss, and reduce aminoglycoside-induced hearing loss. Thus, antioxidants can be an effective treatment for aminoglycoside-induced ototoxicity. CAPE is superior natural antioxidants and cause no serious side effects. These antitumoral, anti-inflammatory, and antioxidant effects have been studied worldwide. CAPE inhibit production of ROS, lipid peroxidation, and the xanthine oxidase system in various organs including the liver, heart, lung, and cochlea [6,17–20]. In addition, CAPE is a potent inhibitor of NF-kB, which plays an important role in inducing the expression of many genes, including those coding for immune and inflammatory cytokines. CAPE showed a protective effect on endotoxin-induced cardiac stress by superinduction of heme oxygenase-1 [6,20]. CAPE also showed a protective effect on chemotherapy- and radiotherapy-induced toxicities by inhibition of the 5-lipoxygenase-catalyzed oxygenation of linoleic acid and arachidonic acid in vivo and in vitro [5]. CAPE showed a protective effect in streptomycin- and cisplatininduced ototoxicity in rats. In addition, CAPE inhibited H2O2-induced oxidative and inflammatory responses and lipopolysaccharideinduced inflammation in human middle ear epithelial cells [18,19,21]. Bakir et al. and Kizilay et al. demonstrated that CAPE attenuated hair cell injury using either DPOAE or histopathological studies in rats. However, they did not show a dose-response curve due to the small number of determinations [21]. In the current study, we found some inhibitory effect of CAPE at above 1000 mM. CAPE showed inhibitory effect in colon cancer cell line (HT-29). In that study, CAPE exhibited significant toxicity at above 2500 mM concentration. In the present study, although the lack of direct information about changing ROS levels, we showed a dose-response curve of CAPE using the zebrafish model. The zebrafish is widely used for drug screening and evaluation of ototoxicity [13,22]. Although cells of the lateral line lack a stria vascularis and are morphologically more similar to vestibular hair cells than cochlear hair cells, zebrafish are less expensive than other animal models and are less time-consuming. In addition, it is easy to create a dose-response
Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018
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curve. We especially used the transgenic zebrafish line (Brn3c: EGFP) to evaluate the protective effect of CAPE on the ototoxicity of aminoglycosides. This transgenic line has naturally occurring green-colored neuromasts that are visible using fluorescence microscopy without any staining. The neuromasts in this transgenic line are similar to those of the wild-type zebrafish [10,13]. Mitochondria are a main site of ROS production under normal conditions. In addition, we evaluated aminoglycosideinduced mitochondrial damage in hair cells using DASPEI staining. This damage was alleviated by CAPE treatment. However, limitations of this study included the lack of specific information about comparing another molecule with CAPE. 5. Conclusion In conclusion, CAPE attenuates neomycin-induced hair cell damage and ultrastructural changes in zebrafish by reducing free radical levels. These results suggest that CAPE may enable otoprotection against neomycin-induced ototoxicity. Conflict of interest All authors have no financial or personal relationships with other people or organizations that could inappropriately influence or bias their work. Acknowledgements This research was supported by a Korea University Grant; by the Soo ENT Clinic and Communication Disorders Center, Korea University, and by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A1013003). References [1] L.P. Rybak, A.E. Talaska, J. Schacht, Drug-induced hearing loss, in: J. Schacht, A. N. Popper, R.R. Fay (Eds.), Auditory Trauma, Protection, and Repair, Springer, Chicago, 2008, pp. 219–256. [2] J. Xie, A.E. Talaska, J. Schacht, New developments in aminoglycoside therapy and ototoxicity, Hear. Res. 281 (2011) 28–37. [3] M.E. Huth, A.J. Ricci, A.G. Cheng, Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection, Int. J. Otolaryngol. 2011 (2011) 937861.
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[4] A.L. Poirrier, J. Pincemail, P. Van Den Ackerveken, P.P. Lefebvre, B. Malgrange, Oxidative stress in the cochlea: an update, Curr. Med. Chem. 17 (2010) 3591–3604. [5] S. Akyol, Z. Ginis, F. Armutcu, G. Ozturk, M.R. Yigitoglu, O. Akyol, The potential usage of caffeic acid phenethyl ester (CAPE) against chemotherapyinduced and radiotherapy-induced toxicity, Cell Biochem. Funct. 30 (2012) 438–443. [6] S.K. Jaganathan, M. Mandal, Antiproliferative effects of honey and of its polyphenols: a review, J. Biomed. Biotechnol. 2009 (2009) 830616. [7] M. Westerfield, The Zebrafish Book: A Guide for the Laboratory Use of Xebrafish (Danio Rerio), University of Oregon Press, Eugene, 2000. [8] C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn. 203 (1995) 253–310. [9] J. Choi, J. Chang, H.J. Jun, G.J. Im, S.W. Chae, S.H. Lee, et al., Protective role of edaravone against neomycin-induced ototoxicity in zebrafish, J. Appl. Toxicol. 34 (2014) 554–561. [10] H.C. Ou, D.W. Raible, E.W. Rubel, Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line, Hear. Res. 233 (2007) 46–53. [11] C. Ton, C. Parng, The use of zebrafish for assessing ototoxic and otoprotective agents, Hear. Res. 208 (2005) 79–88. [12] J. Choi, G.J. Im, J. Chang, S.W. Chae, S.H. Lee, S.Y. Kwon, et al., Protective effects of apocynin on cisplatin-induced ototoxicity in an auditory cell line and in zebrafish, J. Appl. Toxicol.: JAT 33 (2013) 125–133. [13] L.L. Chiu, L.L. Cunningham, D.W. Raible, E.W. Rubel, H.C. Ou, Using the zebrafish lateral line to screen for ototoxicity, J. Assoc. Res. Otolaryngol. 9 (2008) 178–190. [14] D.W. Raible, G.J. Kruse, Organization of the lateral line system in embryonic zebrafish, J. Comp. Neurol. 421 (2000) 189–198. [15] J.J. Song, J. Chang, J. Choi, G.J. Im, S.W. Chae, S.H. Lee, et al., Protective role of NecroX-5 against neomycin-induced hair cell damage in zebrafish, Arch. Toxicol. 88 (2014) 435–441. [16] T.Y. Kim, B. Choi, C.H. Park, A study of structure of the sucker of Common freshwater goby (Rhinogobius brunneus) and Triden goby (Tridentiger brevispinis), Korean J. Electron. Microsc. 32 (2002) 57–66. [17] M. Iraz, E. Ozerol, M. Gulec, S. Tasdemir, N. Idiz, E. Fadillioglu, et al., Protective effect of caffeic acid phenethyl ester (CAPE) administration on cisplatininduced oxidative damage to liver in rat, Cell Biochem. Funct. 24 (2006) 357–361. [18] A. Kizilay, M.T. Kalcioglu, E. Ozerol, M. Iraz, M. Gulec, O. Akyol, et al., Caffeic acid phenethyl ester ameliorated ototoxicity induced by cisplatin in rats, J. Chemother. 16 (2004) 381–387. [19] J.J. Song, H.W. Lim, K. Kim, K.M. Kim, S. Cho, S.W. Chae, Effect of caffeic acid phenethyl ester (CAPE) on H2O2 induced oxidative and inflammatory responses in human middle ear epithelial cells, Int. J. Pediatr. Otorhinolaryngol. 76 (2012) 675–679. [20] T.K. Motawi, H.A. Darwish, A.M. Abd El Tawab, Effects of caffeic acid phenethyl ester on endotoxin-induced cardiac stress in rats: a possible mechanism of protection, J. Biochem. Mol. Toxicol. 25 (2011) 84–94. [21] S. Bakir, M. Ozbay, R. Gun, E. Yorgancilar, V. Kinis, A. Keles, et al., The protective role of caffeic acid phenethyl ester against streptomycin ototoxicity, Am. J. Otolaryngol. 34 (2013) 16–21. [22] G. Kari, U. Rodeck, A.P. Dicker, Zebrafish: an emerging model system for human disease and drug discovery, Clin. Pharmacol. Ther. 82 (2007) 70–80.
Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018
International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish Moo Kyun Park a , Gi Jung Im b , Jiwon Chang b , Sung Won Chae b , Jun Yoo b , Won-gue Han b , Gyu Ho Hwang b , Jong Yoon Jung b , Jungim Choi b , Hak Hyun Jung b , Ah-Young Chung c, Hae-Chul Park c , June Choi b, * a b c
Department of Otolaryngology – Head and Neck Surgery, Seoul National University College of Medicine, Seoul, South Korea Department of Otorhinolaryngology – Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea Laboratory of Neurodevelopmental Genetics, Graduate School of Medicine, Korea University, Ansan-City, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 December 2013 Received in revised form 12 May 2014 Accepted 14 May 2014 Available online xxx
Objective: Caffeic acid phenethyl ester (CAPE) is known to reduce the generation of oxygen-derived free radicals, which is a major mechanism of aminoglycoside-induced ototoxicity. The objective of the present study was to evaluate the effects of CAPE on neomycin-induced ototoxicity in zebrafish (Brn3c: EGFP). Methods: Five-day post-fertilization zebrafish larvae (n = 10) were exposed to 125 mM neomycin and one of the following CAPE concentrations for 1 h: 50, 100, 250, 500, or 1000 mM. Ultrastructural changes were evaluated using scanning electron microscopy (SEM). The terminal deoxynucleotidyl transferase (TdT)mediated dUTP-biotin nick-end labeling (TUNEL) assay and 2-[4-(dimethylamino)styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. Results: CAPE decreased neomycin-induced hair cell loss in the neuromasts (500 mM CAPE: 12.7 1.1 cells, 125 mM neomycin only: 6.3 1.1 cells; n = 10, P < 0.05). In the ultrastructural analysis, structures of mitochondria and hair cells were preserved when exposed to 125 mM neomycin and 500 mM CAPE. CAPE decreased apoptosis and mitochondrial damage. Conclusion: In the present study, CAPE attenuated neomycin-induced hair cell damage in zebrafish. The results of the current study suggest that neomycin induces apoptosis, and the apoptotic cell death can be prevented by treatment with CAPE in zebrafish. ã 2014 Published by Elsevier Ireland Ltd.
Keywords: Caffeic acid phenethyl ester Neomycin Ototoxicity Zebrafish
1. Introduction Aminoglycosides are clinically important antibiotics with a broad antibacterial spectrum. They induce no allergic response and are easy to use in an emergency situation. Although aminoglycosides are notorious for their ototoxicity and nephrotoxicity, they are still widely used for treatment of aerobic, gram-negative and some gram-positive bacterial infections. In addition, the use of aminoglycosides is recommended in cases of multidrug-resistant tuberculosis. They are more widely used in developing countries due to their low cost [1,2]. Hair cell death by aminoglycosides is due to both apoptosis and necrosis. The overproduction of reactive oxygen species (ROS) is an
* Corresponding author at: Department of Otorhinolaryngology – Head and Neck Surgery, Korea University Ansan Hospital, Korea University College of Medicine, 123 Jeokgeum-ro (Gojan-dong), Danwon-gu, Ansan-city, Gyeonggi-do 425-707, South Korea. Tel.: +82 31 412 5170; fax: +82 31 412 5174. E-mail address: [email protected] (J. Choi).
important toxic action of aminoglycosides toward hair cells [2,3]. Although the details of ROS formation differ depending on the circumstances, ROS overproduction is a mechanism of hair cell damage in noise, aging, and ototoxicity. Many antioxidants have shown a protective effect on hair cells in various models of damage both in vitro and in vivo [1–4]. Caffeic acid phenethyl ester (CAPE) is an active component of honey bee propolis extracts. It is a natural phenolic chemical compound and superior antioxidant. In addition, it has antiinflammatory, antimicrobial, immunomodulatory, and anticarcinogenic effects. It has been used in folk medicine for many years, and many experimental studies have recently shown its antioxidative effect [5,6]. These findings lead us to believe that CAPE may be a good candidate for prevention of ototoxicity by aminoglycosides. Thus, the aim of the current study was to investigate whether CAPE has protective effects on neomycin-induced hair cell damage in transgenic zebrafish.
http://dx.doi.org/10.1016/j.ijporl.2014.05.018 0165-5876/ ã 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018
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M.K. Park et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
performed in accordance with the guidelines of the Animal Care Ethics Committee of Korea University Medical Center and National Institutes of Health (NIH) guidelines. 2.3. Chemical administration and zebrafish preparation Neomycin solutions were prepared by adding the neomycin powder to the embryo medium. Neomycin has been shown to decrease the viability of neuromasts in a dose-dependent manner. We previously reported that neuromasts treated with 125 mM neomycin for 1 h show a viability rate of 50% [9]. Thus, that concentration was chosen for the following study. For the current experiment, 10 fish were tested at each of the following concentrations of caffeic acid and repeated three times. The 5dpf zebrafish larvae were exposed to 125 mM neomycin and 50, 100, 250, 500, and 1000 mM CAPE for 1 h. The larvae were then washed with embryo medium three times. The fish were anesthetized with tricaine (3-aminobenzoic acid 0.4 g/ethyl ester; 100 mL; pH 7, adjusted using Tris buffer) for 5 min [10–12]. 2.4. Evaluation of zebrafish hair cells
Fig. 1. Quantitative assay of neuromasts in zebrafish. Hair cells from four neuromasts (SO1, SO2, O1, and OC1) were counted. Hair cell survival was calculated as a percentage of the control group. Treatment of the zebrafish with 125 mM neomycin (neo) for 1 h significantly decreased the number of hair cells in neuromasts. Caffeic acid phenethyl ester (CAPE) protected against neomycininduced hair cell loss of the neuromasts in zebrafish (n = 10, P < 0.05).
The zebrafish were placed with methylcellulose on a depression slide for evaluation of hair cell damage under a fluorescence microscope. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines on one side of each fish were analyzed [10–15]. The average numbers of hair cells of the SO1, SO2, O1, and OC1 neuromasts were evaluated in each zebrafish for all experimental and control conditions (n = 10) using fluorescence microscopy (AxioCam MRc5; Carl Zeiss).
2. Methods 2.1. Chemical materials Neomycin and caffeic acid phenethyl ester (CAPE) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Zebrafish model Zebrafish larvae (Brn3c: EGFP) were produced by pairwise matings, raised at 28.5 C in egg water or embryo medium (EM; 15 mM NaCl, 0.5 mM KCl,1 mM CaCl2,1 mM MgSO4, 0.15 mM KH2PO4, 0.05 mM NH2PO4, and 0.7 mM NaHCO3) [7], and staged according to days post-fertilization (dpf) and morphological criteria [8]. The larvae were collected and raised at 28.5 C. The current study was approved by the Korea University Institutional Animal Care and Use Committee (Approval No. KUIACUC-2012-116). All methods were
2.5. Terminal deoxynucleotidyl transferase (TdT)-Mediated dUTPbiotin nick end labeling (TUNEL) assay for apoptosis evaluation and 2[4-(dimethylamino)styryl]-N-ethylpyridiniumiodide (DASPEI) assay for mitochondria Apoptotic cells in zebrafish were identified by the TUNEL method using an in situ cell detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's protocol. The larvae were exposed to medium containing 125 mM neomycin and 500 mM CAPE for 1 h. The larvae were then washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde. Next, the larvae were incubated with 50 mL of TUNEL reaction mixture (TdT and fluorescein-dUTP) at 37 C for 60 min in a humid atmosphere. The 5-dpf zebrafish larvae were evaluated using a fluorescence microscope. In the current study, the fluorescent dye 2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide (DASPEI;
Fig. 2. Fluorescence microscopy images (OC1, 40). Zebrafish (5 dpf) were treated with 125 mM neomycin (neo) and 500 mM CAPE for 1 h. Treatment with neomycin resulted in a significant decrease in the number of hair cells of the neuromasts. CAPE attenuated the neomycin-induced hair cell damage. Bar = 10 mm.
Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018
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Fig. 3. Mitochondrial damage evaluation using DASPEI (SO2, 40). Zebrafish (5 dpf) were treated with 125 mM neomycin (neo) and 500 mM CAPE for 1 h. Treatment with neomycin resulted in significantly decreased DASPEI staining of mitochondria. Mitochondrial damage was ameliorated by CAPE. Bar = 10 mm.
Invitrogen, Carlsbad, CA, USA) was used as a vital dye to stain mitochondria within hair cells. The zebrafish larvae were incubated in an embryo medium containing 0.005% DASPEI for 15 min.
treated in a graded series of ethanol and t-butyl alcohol, dried in a freeze dryer (Hitachi, ES-2030; Tokyo, Japan), platinum-coated using an ion coater (Eiko,l IB-5; Katsuda, Japan), and observed by a field emission SEM (Hitachi, S-4700) [16].
2.6. Scanning electron microscopy 2.7. Statistical analysis For scanning electron microscopy (SEM), 5-dpf zebrafish larvae were exposed to 125 mM neomycin and 500 mM CAPE for 1 h, prefixed by immersion in 2% glutaraldehyde in 0.1 M PBS, and postfixed for 2 h in 1% osmic acid dissolved in PBS. The larvae were
All values are presented as means standard deviation (SD). For statistical comparisons, one-way analysis of variance was used for multiple comparisons using the SPSS software (version 11.0; SPSS,
Fig. 4. Results of TUNEL assay. Neomycin-induced apoptosis was confirmed by TUNEL assay. Apoptotic cells are indicated by light red dots in red-colored fish after the TUNEL reaction when observed using fluorescence microscopy (TUNEL-positive cells: arrow). The negative control showed no light red dots in red-colored fish. Comparison of the color intensity between the 125 mM neomycin (neo) group and the CAPE-treated group for 1 h; 500 mM CAPE significantly decreased the number of TUNEL-positive cells and protected against neomycin-induced apoptotic cell death of the hair cells of the neuromasts of zebrafish (green-colored fish of right column, control for TUNEL reaction; redcolored fish of middle column, TUNEL reaction; green- and red-colored fish of left column, merging of control and TUNEL reaction). Bar = 200 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018
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Fig. 5. Scanning electron microscopy images (SO2, 3k). Compared with the negative control neuromasts, neuromasts in zebrafish treated with 125 mM neomycin (neo) for 1 h showed severe morphological damage to stereocilia and kinocilia under SEM, such as loss or fusion. However, this damage was obviated by treatment with 500 mM CAPE. Bar (at the bottom of each figure, one space) = 10 mm.
Chicago, IL, USA). A post hoc analysis was performed using Tukey's honestly significant difference (HSD) test. A value of P < 0.05 was considered to indicate statistical significance.
stereocilia and kinocilia. CAPE attenuated neomycin-induced hair cell damage compared with treatment with neomycin alone. 4. Discussion
3. Results 3.1. Effect of caffeic acid phenethyl ester (CAPE) on hair cells To assess the effects of neomycin, 5-dpf zebrafish larvae were exposed to various concentrations of neomycin (0, 10, 50, 100, 125, 150, 200, 300, and 500 mM) for 1 h. The presence of neomycin decreased the number of hair cells in a dose-dependent manner. Hair cells from four neuromasts (SO1, SO2, O1, and OC1) were counted to investigate the changes in the neuromasts after treatment with CAPE and neomycin. Hair cell survival was calculated as the average number of hair cells in the control group that were not exposed to neomycin. Treatment of the zebrafish with 125 mM neomycin for 1 h significantly decreased the number of hair cells in the neuromasts. CAPE decreased neomycin-induced hair cell loss of the neuromasts in the zebrafish (negative control: 13.2 0.9 cells; neomycin only: 6.3 1.1 cells; 50 mM CAPE: 7.8 0.4 cells; 100 mM CAPE: 8.0 0.8 cells; 250 mM CAPE: 9.8 1.2 cells; 500 mM CAPE: 12.7 1.1 cells; 1000 mM CAPE: 7.7 0.7 cells; n = 10, P < 0.05) (Figs. 1 and 2). 3.2. Effect of caffeic acid phenethyl ester on mitochondria and apoptosis A caffeic acid phenethyl ester concentration of 500 mM was most effective and was thus used in the DASPEI and TUNEL assays. The DASPEI assay was performed to evaluate mitochondrial damage. Mitochondria within hair cells were protected by CAPE treatment (Fig. 3). The TUNEL reaction was performed to determine neomycin-induced death of neuromasts by apoptosis and the protective influence of CAPE treatment. Apoptotic cells were marked as light red dots after TUNEL reaction when observed by fluorescence microscopy. As shown in Fig. 4, comparison of the color intensity between the 125 mM neomycin group and the 500 mM CAPE group showed a significantly lower number of TUNEL-positive cells, and CAPE attenuated the neomycin-induced apoptotic cell death in zebrafish. 3.3. Effect of caffeic acid phenethyl ester on ultrastructure Fig. 5 shows SEM images of stereocilia and kinocilia from neomycin- and CAPE-treated zebrafish. Compared with control larvae, exposure to 125 mM neomycin for 1 h resulted in severe morphological damage to hair cells, including loss or fusion of
The purpose of this study was to investigate the protective effects of CAPE on neomycin-induced hair cell loss in transgenic zebrafish. In this study, CAPE showed a protective effect on hair cells in the cochlea with antiapoptotic and antioxidative activities. The increase in ROS and depletion of antioxidant protective molecules facilitate lipid peroxidation, which can increase calcium influx and apoptosis in cells of the cochlea. Antioxidants interfere with ROS pathways, protect against hair cell loss, and reduce aminoglycoside-induced hearing loss. Thus, antioxidants can be an effective treatment for aminoglycoside-induced ototoxicity. CAPE is superior natural antioxidants and cause no serious side effects. These antitumoral, anti-inflammatory, and antioxidant effects have been studied worldwide. CAPE inhibit production of ROS, lipid peroxidation, and the xanthine oxidase system in various organs including the liver, heart, lung, and cochlea [6,17–20]. In addition, CAPE is a potent inhibitor of NF-kB, which plays an important role in inducing the expression of many genes, including those coding for immune and inflammatory cytokines. CAPE showed a protective effect on endotoxin-induced cardiac stress by superinduction of heme oxygenase-1 [6,20]. CAPE also showed a protective effect on chemotherapy- and radiotherapy-induced toxicities by inhibition of the 5-lipoxygenase-catalyzed oxygenation of linoleic acid and arachidonic acid in vivo and in vitro [5]. CAPE showed a protective effect in streptomycin- and cisplatininduced ototoxicity in rats. In addition, CAPE inhibited H2O2-induced oxidative and inflammatory responses and lipopolysaccharideinduced inflammation in human middle ear epithelial cells [18,19,21]. Bakir et al. and Kizilay et al. demonstrated that CAPE attenuated hair cell injury using either DPOAE or histopathological studies in rats. However, they did not show a dose-response curve due to the small number of determinations [21]. In the current study, we found some inhibitory effect of CAPE at above 1000 mM. CAPE showed inhibitory effect in colon cancer cell line (HT-29). In that study, CAPE exhibited significant toxicity at above 2500 mM concentration. In the present study, although the lack of direct information about changing ROS levels, we showed a dose-response curve of CAPE using the zebrafish model. The zebrafish is widely used for drug screening and evaluation of ototoxicity [13,22]. Although cells of the lateral line lack a stria vascularis and are morphologically more similar to vestibular hair cells than cochlear hair cells, zebrafish are less expensive than other animal models and are less time-consuming. In addition, it is easy to create a dose-response
Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018
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curve. We especially used the transgenic zebrafish line (Brn3c: EGFP) to evaluate the protective effect of CAPE on the ototoxicity of aminoglycosides. This transgenic line has naturally occurring green-colored neuromasts that are visible using fluorescence microscopy without any staining. The neuromasts in this transgenic line are similar to those of the wild-type zebrafish [10,13]. Mitochondria are a main site of ROS production under normal conditions. In addition, we evaluated aminoglycosideinduced mitochondrial damage in hair cells using DASPEI staining. This damage was alleviated by CAPE treatment. However, limitations of this study included the lack of specific information about comparing another molecule with CAPE. 5. Conclusion In conclusion, CAPE attenuates neomycin-induced hair cell damage and ultrastructural changes in zebrafish by reducing free radical levels. These results suggest that CAPE may enable otoprotection against neomycin-induced ototoxicity. Conflict of interest All authors have no financial or personal relationships with other people or organizations that could inappropriately influence or bias their work. Acknowledgements This research was supported by a Korea University Grant; by the Soo ENT Clinic and Communication Disorders Center, Korea University, and by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A1013003). References [1] L.P. Rybak, A.E. Talaska, J. Schacht, Drug-induced hearing loss, in: J. Schacht, A. N. Popper, R.R. Fay (Eds.), Auditory Trauma, Protection, and Repair, Springer, Chicago, 2008, pp. 219–256. [2] J. Xie, A.E. Talaska, J. Schacht, New developments in aminoglycoside therapy and ototoxicity, Hear. Res. 281 (2011) 28–37. [3] M.E. Huth, A.J. Ricci, A.G. Cheng, Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection, Int. J. Otolaryngol. 2011 (2011) 937861.
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Please cite this article in press as: M.K. Park, et al., Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.05.018