Ferulic acid-mediated protection against neomycin-induced hair cell loss in transgenic zebrafish

Journal of Functional Foods 28 (2017) 157–167

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Ferulic acid-mediated protection against neomycin-induced hair cell loss in transgenic zebrafish Ju Chang-Chien a,c, Yung-Chang Yen d,e, Shuan-Yow Li a, Tsai-Ching Hsu c,f,⇑⇑, Jiann-Jou Yang a,b,⇑ a

Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan Department of Medical Sciences, Chung Shan Medical University Hospital, Taichung, Taiwan c Institute of Biochemistry, Microbiology and Immunology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan d Department of Ophthalmology, CHi-Mei Medical Center, Liou-Ying, Tainan, Taiwan e Department of Nursing, Min Hwei College of Health Care Management, Tainan, Taiwan f Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan b

a r t i c l e

i n f o

Article history: Received 1 October 2016 Received in revised form 11 November 2016 Accepted 16 November 2016

Keywords: Transgenic zebrafish Neomycin Ototoxicity Ferulic acid

a b s t r a c t Ferulic acid (FA) derivatives have applied in American clinical trials for preventing and treating auditory dysfunctions. However, the effects of FA on neomycin-mediated sensorineural hearing loss are still unknown. We developed a transgenic zebrafish (pvalb3b:TagGFP) expressing green-colored hair cells in the inner ear and lateral line neuromasts by Tol2 system. Then, we determined the effects of FA on neomycin-induced ototoxicity in 4 dpf transgenic larvae. FA conferred significant protection against hair cell loss across a wide range of neomycin concentrations. In addition, FA significantly decreased intracellular ROS production and TUNAL reactions in larvae pretreated with FA prior to neomycin exposure. These findings suggest that FA has antioxidant effects and can attenuate neomycin-induced hair cell death in neuromasts. The green hair cells of transgenic zebrafish lateral line may provide a useful basis on which to investigate the cellular pathways related to the hair cell death and survival imparted by FA. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Sensory hair cells are mechanoreceptors within the inner ear that are involved in the mechanotransduction of sound and head movements into neural signals for auditory stream formation and balance (Gillespie & Muller, 2009). In addition to genetic mutation-mediated hearing loss (e.g. connexin and OPTN) (Su et al., 2012; Yen, Yang, Chou, & Li, 2008), hair cell death also results in deafness and balance disorders, causing severe disequilibrium, nystagmus, and ataxia, which significantly affect more than 600 million people worldwide (Schuknecht & Gacek, 1993). Progressive Abbreviations: ao, anterior otolith; hpf, hours post-fertilization; dpf, days postfertilization; FA, ferulic acid; GFP, green fluorescent protein; MI, middle neuromasts; O, otic neuromasts; OC, occipital neuromasts; pvalb3, parvalbumin 3; po, posterior otolith; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling. ⇑ Corresponding author at: Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan. ⇑⇑ Co-corresponding author at: Institute of Biochemistry, Microbiology and Immunology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan. E-mail addresses: [email protected] (T.-C. Hsu), [email protected] (J.-J. Yang). http://dx.doi.org/10.1016/j.jff.2016.11.019 1756-4646/Ó 2016 Elsevier Ltd. All rights reserved.

hair cell damage is commonly associated with various factors, including exposure to excessive noise, aging, and ototoxic drug administration. Aminoglycoside antibiotics and platinum-based chemotherapeutic drugs such as cisplatin and carboplatin are two major classes of clinically therapeutic agents that are recognized to have ototoxicity (Rybak & Ramkumar, 2007). Aminoglycosides such as kanamycin, streptomycin, gentamicin, and neomycin are bactericidal aminoglycosidic aminocyclitols, which are used to target aerobic gram-negative and some gram-positive bacterial infections as a powerful first-line therapy. Although aminoglycosides are well-known for their ototoxicity and nephrotoxicity in humans, they are still widely used for clinical treatment in developing countries due to their relatively low cost, high antimicrobial efficacy, and broad antibacterial spectrum and the fact that they do not cause allergic responses (Perletti et al., 2008). Several reports have indicated that free radical damage to hair cells upon aminoglycoside administration was linked to ototoxicity (Priuska & Schacht, 1995; Rybak & Ramkumar, 2007; Sha & Schacht, 2000). Following entry of aminoglycosides into outer hair cells through mechanoelectrical transducer channels, the aminoglycoside molecule has no toxicity by itself but may decompartmentalize and chelate metal ions from biomolecules so that it

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induces ototoxic effects (Priuska & Schacht, 1995; Sha & Schacht, 2000). In the inner ear, ROS-mediated hair cell death due to oxidative stress plays a crucial role in the mechanism of aminoglycoside ototoxicity that results in permanent sensorineural hearing loss. Interestingly, it has been shown that the outer hair cells at the base of the cochlea are more susceptible to ototoxic injury than the inner hair cells. Degeneration of nerve fibers, spiral ganglion neurons, and supporting cells generally occurs after hair cell damage (Guthrie, 2008). Apoptosis (also known as programmed cell death) occurs due to the sequential actions of caspases and intrinsic pathway involvement (Wong & Ryan, 2015). Intrinsic pathways initiated by a change in the permeability of the mitochondrial membrane can cause the release of ROS and cytochrome C, resulting in classical morphologic pathology (Rybak & Ramkumar, 2007). Overall, meaningful information regarding aminoglycosideinduced hearing loss had been purposed, including aminoglycoside uptake by hair cells, intracellular containment within lysosomes, ROS-mediated toxicity, and the apoptotic cell death pathway (Rizzi & Hirose, 2007). In contrast with the sensory hair cells in non-mammalian vertebrates including birds (Jagger, Nickel, & Forge, 2014), amphibians (Steyger, Burton, Hawkins, Schuff, & Baird, 1997) and fish (Liang et al., 2012), and in mammal mice (Maass et al., 2015), which can regenerate, the loss of sensory hair cells in humans and other mammals are irreparable after acoustic or ototoxic trauma and age-related degradation (Matsui, Mackenzie, & Raible, 2012). However, ototoxic effects are easily underestimated due to the ambiguity of symptoms (Matsui et al., 2012). Protection against mammalian hair cell death seems to be significant and important for the prevention of toxin-induced hearing loss. Identification of a preventive therapy would allow for safe use of efficacious antibiotics and chemotherapeutics without the devastating ototoxic side effects (Kovacic & Somanathan, 2008). FA (4-hydroxy-3-methoxycinnamic acid), an Angelica sinensisderived phenolic phytochemical, is a component of quisetum, angelica, and some Chinese herbal medicines. It is also ubiquitous in various fruits and vegetables, such as bananas, citrus fruits, whole grains, bamboo shoots, eggplants, cabbage, spinach, and broccoli. FA has been reported that it performs numerous physiological functions, including antioxidant, antimicrobial, antiinflammatory, antifibrosis, anticancer, and antidiabetic functions (Zhao & Moghadasian, 2008). Most important of all, an American patent (US20130324594 A1) has applied FA derivatives in clinical trials for preventing and treating auditory dysfunctions (e.g., hearing loss, hyperacusis, tinnitus, and related auditory disorders) that resulted from aging, exposure to loud sounds, and ototoxicity. Although the exact mechanism by which FA mediates these physiological processes remains to be elucidated. In guinea pig model, FA reduces both the oxidative stress and apoptotic cell death pathway; meanwhile, FA increases the activity of cytoprotective enzyme heme oxygenase-1 (HO-1) to protect hair cells in the organ of Corti from noise-induced auditory dysfunction (Fetoni et al., 2010). The small size, high fecundity, transparent embryos, and external development of zebrafish (Danio rerio) provide an excellent screening model system for ototoxicity, neurotoxicology, and pharmacological prevention of hair cell damage (Coffin et al., 2010). The lateral line, a series of sensory organs aligned along the head and body (trunk and tail) of fish, is mainly consisted of rosette-like structures called neuromasts that developmentally, morphologically, and physiologically resemble the sensory patches within the mammalian inner ear (Ghysen & Dambly-Chaudiere, 2004). The neuromasts are easily accessible for analysis and can be in direct contact with surrounding water that contains drugs (Esterberg, Hailey, Rubel, & Raible, 2014; Froehlicher et al., 2009). In addition, like the hair cells in the mammalian inner ear, the hair

cells in the zebrafish lateral line are sensitive to ototoxic agents such as aminoglycoside antibiotics and platinum derivatives. Thus, these hair cells can be used to screen for drugs that prevent aminoglycoside-induced hair cell death. (Coffin et al., 2010). Previous studies investigated the effects of drugs on neomycin-induced hair cell damage in the wild type zebrafish or a transgenic zebrafish line (Brn3C:EGFP). In the present study, we used multisite Gateway cloning technology to develop a novel transgenic zebrafish line (pvalb3b:TagGFP) with naturally expressed hair cellspecific green fluorescent protein (GFP) in the inner ear and lateral line. The purpose of this study was to evaluate the potential protective effects of FA on neomycin-mediated ototoxicity in a Tg (pvalb3b:TagGFP) transgenic line. 2. Materials and methods 2.1. Zebrafish strains and maintenance Zebrafish, including AB wild type and transgenic zebrafish line (pvalb3b:TagGFP), were conducted in this study. All zebrafish embryos were produced by mating paired adult fish maintained and raised at 28.5 °C in a continuous flow-through system with 14 h light and 10 h dark light-dark cycle. Embryos were reared in egg water at 28.5 °C and were staged according to hours post fertilization (hpf) as described (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995). Of all components in egg water, 0.06% sea salt is included on the basis of ‘‘The Zebrafish Book”. All of the animal experiments using protocol have been reviewed and were approved by the Institutional Animal Care and use Committee (IACUC) of Chung-Shan Medical University Experimental Animal Center (IACUC Approval No.1415). 2.2. Plasmid construction In this study, multisite Gateway cloning technology was used for establishment of transgenic zebrafish line according to manufacturer’s recommendations (Invitrogen). To generate a pvalb3b promoter entry clone, polymerase chain reaction (PCR) was used to amplify a 0.6 kb promoter region upstream of start codon for zebrafish parvalbumin 3b (pvalb3b) (McDermott et al., 2010) from whole-larvae genomic DNA. In addition, the full-length coding sequence of fluorescent protein TagGFP was also amplified using a specific primer pair from the indicated template plasmid, pTagGFP2-N (Clontech), to generate a TagGFP entry clone. PCRamplified products mentioned above are all flanked by attB or attBr sites for BP recombination. All forward and reverse primers containing an attB site for Gateway recombination cloning are showed in Table 1. Following the PCR amplification, flanked PCR products were mixed with pDONRP4-P1r (for pvalb3b promoter) and pDONR221 (for TagGFP fragments) respectively in a BP recombination reaction overnight at room temperature. The resulting entry clones are referred to p5E-pvalb3b and pME-TagGFP respectively. To obtain the expression plasmid for microinjection into zebrafish embryos, multisite gateway LR recombination reaction with our three entry clones (p5E-pvalb3b, pME-TagGFP and p3E-polyA) and a single Destination vector, pDestTol2pA, was performed overnight at room temperature. After BP or LR reaction, the entry or expression clones were chemically transformed into recA, endA E. coli strain DH10B, followed by appropriate antibody selection. 2.3. Microinjection and generation of germline transgenic zebrafish To synthesize capped mRNA for microinjection, the pCS2transposase plasmid encoding the Tol2 transposase was digested with NotI to generate the linearized plasmid, and subsequently

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J. Chang-Chien et al. / Journal of Functional Foods 28 (2017) 157–167 Table 1 Primers used for PCR amplification in this study. Primer name

Primer sequence

attB site

Tol2-pvalb3b-attB1r

5 -ggggACTgCTTTTTTgTACAAACTTgCggAAgTAAgTgACATATTCAAACT-3

Tol2-pvalb3b-1-attB4

50 -ggggACAACTTTgTATAgAAAAgTTggCgAAgTgCAggCACATgTgCgC-30

attB4

Tol2-TagGFP-attB1

50 -ggggACAAgTTTgTACAAAAAAgCAggCTTAATgAgCgggggCgAggAgCTgTT-30

attB1

Tol2-TagGFP-attB2

50 -ggggACCACTTTgTACAAgAAAgCTgggTATTACCTgTACAgCTCgTCCATgC-30

attB2

0

0

attB1r

Above underline sequences are attB sites for BP recombination reaction.

conducted in vitro transcription reaction using SP6 polymerase (Message Machine, Ambion). The indicated Tol2-containing plasmid, pDestTol2pA:pvalb3b-TagGFP-polyA, was diluted to 30 ng/ll and mixed with diluted transposase mRNA (30 ng/ll) at a 1:1 ratio. Approximately 2.3 nl of DNA/RNA mixture was co-injected into one-cell stage embryos using a Nanoject II automatic injector (Drummond). For generation of germline transgenic zebrafish, injected embryos were raised to adulthood (F0). F0 adult fish were out-crossed with AB wild type to produce F1 embryos, which could be visualized under a fluorescent microscope (Zeiss Axioplam). The GFP-positive F1 embryos from different lines were individually raised to sexual maturity. Their progeny were maintained later from an incross or an outcross of heterozygous GFP carrier for germline transmission. 2.4. Vital labeling of hair cells in lateral line neuromasts The lateral line hair cells were labeled in live zebrafish larvae with a lipophilic styryl based probe, N-(3-triethylammonium-pro pyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM4-64; Invitrogen). For fluorescent staining, live larvae were immersed in a 50 lM solution of FM4-64 for 2 min at room temperature in the dark. They were then rinsed thoroughly with egg water and visualized under fluorescent light with the wavelengths of 508 nm (excitation) and 751 nm (emission). 2.5. Immunohistochemistry Immunohistochemistry (IHC) studies on whole tissues were performed using specific antibodies for target genes. For wholemount imaging of kinocilia of hair cells, mouse anti-acetylated tubulin (Sigma-Aldrich) (Chang-Chien et al., 2014) was used as primary antibody in this study. Five days post-fertilization (dpf) embryos were fixed in fresh 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 48 h at room temperature, rinsed four times for 5 min each in 0.1% PBST (0.1% Tween 20 in PBS), and then immersed in decolorizing solution (0.5% KOH: 15% H2O2) for 20 min at room temperature until melanin pigment removed. Decolorizing samples were permeabilized with pure acetone for 30 min at -20 °C and then re-fixed in buffered 2% PFA for 15 min at room temperature. After incubated in blocking solution [5%horseserumand2.5%DMSOin0.1%PBSTriton(0.1%TritonX-100inP BS)] at room temperature for 3 h, the embryos were incubated with a 1:400 dilution of primary antibody diluted in blocking solution overnight at 4 °C. Following four 60-min 0.1% PBSTriton washes, the primary antibody was detected with a 1:200 dilution of Alexa-Fluor-594-conjugated goat anti-mouse IgG (H+L) antibody (Invitrogen, Carlsbad, CA) to develop fluorescent signals. After secondary antibody incubation overnight at 4 °C, the embryos were rinsed three times for 45 min each in 0.1% PBSTriton, followed by signal detection under microscope. 2.6. TUNEL staining The TUNEL [terminal deoxynucleotidyl transferase (TdT)mediated deoxyuridine triphosphate (dUTP) nick end-labeling]

experiments were conducted to examine apoptotic cells in the neuromasts. For TUNEL analysis, embryos were staged, fixed, decolorized, permeabilized and re-fixed as for whole-mount IHC experiments. After re-fixation, embryos were rinsed in PBST and subsequently immersed in 50 ll of TUNEL reaction mixture according to the manufacturer’s instructions (Roche). Embryos were incubated with TdT and TMR-labeled dUTP on ice for 1 h, followed by 1 h incubation at 37 °C. The zebrafish embryos were then visualized using a fluorescent microscope. 2.7. Drug administration and evaluation of hair cells in transgenic zebrafish Neomycin is a common aminoglycoside antibody and has known to decrease the viability of hair cells in a dose-dependent manner (Ou et al., 2009). For the current study, neomycin administration experiments were independently examined in a transgenic zebrafish (pvalb3b:TagGFP). The serial concentrations of neomycin were prepared by diluting a 10 mg/ml stock solution of neomycin (Sigma-Aldrich) to egg water. The 4-dpf transgenic larvae were separately exposed to 50, 100, 150 and 200 lM of neomycin for 1 h. Following the incubation of neomycin, approximately all anterior and posterior neuromasts were examined per larva under a fluorescent microscope at
20 to
40 magnifications. To confirm the appropriate duration of neomycin, the time-course experiments were performed. Transgenic larvae were exposed to optimal concentration (with least toxicity) of neomycin for one of the following duration: 0.5, 1 and 1.5 h(s). Thus, the optimal exposure condition of neomycin (almost complete loss of hair cells in every neuromast with least toxicity) was decided as a satisfactory experimental condition for following methods (N = 20 larvae per treatment). Because the purpose of this study was to evaluate the beneficial effects of the ferulic acid (FA; Sigma-Aldrich) against neomycininduced hair cell damage, dose-response analyses were important to determine the effective dose range for FA and to determine the protective efficacy of an optimal dose of FA against a range of neomycin concentrations. To examine the optimal concentration of FA, the 4-dpf transgenic larvae were pretreated at each of the following concentrations of FA and repeated three times. Transgenic larvae were exposed to 50, 100, 150 and 200 lM of FA for 1 h, followed by neomycin treatment with the FA still present. Similar to time-response testing for neomycin, the temporal experiments for FA were conducted in transgenic larvae. The larvae were treated with the optimal concentration (greatest protection with least toxicity) of FA for one of the following duration previous to neomycin treatment: 0.5, 1, 2 and 3 h(s). To examine the efficacy of an optimal dose of FA against a range of neomycin, transgenic larvae were pretreated with FA under the optimal condition, followed by treatment with 0, 25, 50, 100, 150, or 200 lM neomycin. After the exposure of FA and neomycin, the treated larvae were rinsed thoroughly 3–4 times in egg water, anesthetized with tricane (3-aminobenzoic acid 0.4 g/ethyl ester; 100 ml; pH 7.0, adjusted with This buffer) and mounted in 0.5% low-melting-point LE agarose (Seakem) on a depression slide for estimation of hair cell damage under a fluorescence microscope (Zeiss Axioplam).

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No significant differential sensitivity to aminoglycosides among neuromasts is reported in previous study (Harris et al., 2003). In accordance with the nomenclature of Raible and Kruse (2000) (Raible & Kruse, 2000), the three neuromasts within anterior lateral lines [otic (O1), occipital (OC1) and middle (MI1)] and three posterior trunk neuromasts (P1, P3 and P4) on one side of each zebrafish were directly analyzed. Total hair cell numbers were calculated by adding the hair cell counts from the O1, OC1, MI1, P1, P3 and P4 six neuromasts in each larval zebrafish for all control and experimental conditions. Hair cell survival as s percentage of the control was evaluated by dividing the total number of hair cells of each larva in the experimental group by the mean total number of hair cells in an undamaged control group (N = 20 larvae per treatment). 2.8. Twenty-four-hour survival analysis of hair cells The 4-dpf transgenic larvae were pretreated with an optimal dose of FA for 1 h, followed by exposure to the optimal exposure condition of neomycin with FA still present. Both drugs were removed and then rinsed thoroughly in egg water, and transgenic larvae were allowed to recover for 24 h. Recovered larvae were euthanized, mounted and imaged to estimate the hair cell counts. Hair cell survival in the FA-treated group was compared to undamaged controls (only with 0.2% DMSO exposure for 1 h, followed by 24 h of recovery) and damaged controls (with 0.2% DMSO exposure for 1 h, followed by neomycin and a 24 h recovery) (N = 20 larvae per group). 2.9. Washout analysis of FA The 4-dpf transgenic larvae were pretreated with an optimal dose of FA for 1 h, and then washed four times in egg water. After a washout of FA, larvae were sequentially exposed to neomycin for 0.5 h, euthanized, mounted and imaged. Hair cell survival in the FA-treated group was compared to undamaged controls treated only with 0.2% DMSO, as well as damaged controls treated with 0.2% DMSO followed by an identical exposure condition of neomycin (N = 20 larvae per group). 2.10. ROS analysis In order to confirm the antioxidant activity of ferulic acid (FA) against neomycin-induced neuromast damage, neomycininduced intracellular ROS accumulation was directly analyzed using a membrane permeable and oxidation sensitive fluorescent probe, DCF-DA (20 ,70 -dichlorofluorescein diacetate; SigmaAldrich). Due to optical transparency of the zebrafish larvae, fluorescent intensity could be detected to measure the ROS intensity. A set of ten transgenic zebrafish larvae (pvalb3b:TagGFP) were exposed to neomycin (50 lM) in the presence or absence of FA (150 lM). Then larvae were transferred into DCF-DA solution (20 lg/ml) to detect the intracellular ROS production in zebrafish larvae. After a 30-m incubation in the dark at 28 °C, larvae were rinsed thoroughly in egg water, followed by the measurement of individual larval fluorescence intensity at the wavelengths of 485 nm (excitation) and 530 nm (emission) using a SpectraMax M5 microplate reader (Molecular Devices). The mean fluorescence intensity of ten larvae was calculated for each group. To remove the GFP signal of stable transgenic larvae, the ROS generation as a percentage of the control was determined by dividing the mean fluorescent intensity of larvae in the experimental group by the mean fluorescent intensity of larvae in the negative control group (neomycin-, DMSO- and ferulic acid-free) (N = 200 larvae per group).

2.11. Statistical analysis All values were calculated and presented as mean ± standard error (mean ± SE) in this study. A statistical comparison between different treatment groups was performed via a one-way analysis of variance (ANOVA) by GraphPad Prism 5 Software, followed by posterior comparisons using Turkey’s test HSD (honestly significant difference). A P-value of less than 0.05 was considered to be statistically significant. Star code for statistical significance is illustrated as follow: ⁄⁄⁄P < 0.001, ⁄⁄P < 0.01 and ⁄P < 0.05. 3. Results 3.1. Establishment of transgenic zebrafish (pvalb3b:TagGFP) line Parvalbumin generally serves as a calcium-binding protein that is specifically expressed in the mammalian auditory and vestibular hair cells of the organ of Corti (McDermott et al., 2010; Steyger et al., 1997). In zebrafish, both parvalbumin 3a (pvalb3a) and parvalbumin 3b (pvalb3b) are co-orthologs to mammalian pvalb3 (Hsiao, Tsai, & Tsai, 2002). For the current study, we amplified a 0.6 kb promoter region upstream of start codon of pvalb3b and conducted the serial recombination reaction (BP and LR) to generate an indicated Tol2-containing plasmid, pDestTol2pA: pvalb3b-TagGFP-polyA (Fig. 1A). Then, the indicated plasmid and Tol2 transposase mRNA were co-injected into one-cell stage embryos to develop a stable transgenic zebrafish line (pvalb3b: TagGFP) (Fig. 1B and C). By 4-dpf, the spatiotemporal expression pattern of TagGFP was detected in the otic vesicles and the lateral line neuromasts. Of all sensory epithelia in the otic vesicles, strong TagGFP expression was observed in the anterior macula, posterior macula, and crista (Fig. 1D–G). We then proceeded to identify the cellular localization of TagGFP in sensory patches, so that transgenic larvae were immunostained with an acetylated tubulin antibody. Immunostaining patterns showed that a single, long kinocilium was above each TagGFP-positive cell, indicating that the pvalb3b promoter we cloned exactly drive TagGFP expression in the macular hair cells of otic vesicles (Fig. 1H–J). In addition, both anterior and posterior lateral line neuromasts were labeled with FM4-64 fluorescent dye, which selectively labels hair cells of neuromasts by entering the mechanotransduction channel (Fig. 1K–S). In this study, we had developed germline cells of a transgenic zebrafish which exhibits green-colored hair cells in the inner ear and lateral line. We will use this stable transgenic zebrafish line to determine whether FA can protect hair cells from neomycin-induced damage. 3.2. Neomycin ototoxicity of neuromasts hair cells in transgenic zebrafish (pvalb3b:TagGFP) line To determine the optimal exposure condition of neomycin in stable Tg(pvalb3b:TagGFP) transgenic line we developed, the dose-response analyses were performed by assessing neuromasts hair cells 1 h after exposure to different concentrations of neomycin ranging from 50 to 200 lM. Neomycin exposure led to loss of hair cells in neuromasts. Fluorescent images of O1 neuromast after FM4-64 staining were represented in Fig. 2A–F. Loss of hair cells in neuromasts hair cells (Fig. 2G) and larval survival (Fig. 2H) were dependent on neomycin dose. Hair cell survival was nearly 24.23 ± 1.797% of negative control levels in larvae treated with 50 lM neomycin; meanwhile, larva survival was approximately 68.86 ± 4.211%. Also, larval survival was significantly decreased to 33.65 ± 2.00% after 200 lM. Neomycin demonstrated significant hair cell loss and larval mortality with increasing neomycin concentration. Then, the time-response experiments were conducted

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Fig. 1. GFP expression in Tg(pvalb3b:TagGFP) transgenic larva at 4 days post fertilization (dpf). (A) All fluorescent images of transgenic larvae are lateral views, anterior are to left and dorsal are up. At 4 dpf, expression patterns of TagGFP in the otic vesicle and lateral line neuromasts were shown (B–C). In the otic vesicle (D), TagGFP was expressed in anterior macula (am) (E), posterior macula (pm) (F) and lateral crista (G). Then, the hair cells of anterior macula were immunostained with a specific antibody against acetylated tubulin (a hair cell indicator). The fluorescent images showed that TagGFP-labeled hair cells (green) were co-localized with kinocilia (red) (H–J). In addition, TagGFP (green) was also expressed in the neuromasts of anterior lateral line (ALL) (K–M) and posterior lateral line (PLL) (N–P), which were selectively labeled with FM4-64 fluorescent dye (red). The transgenic zebrafish line (pvalb3b:TagGFP) were used to obtain clearer confocal fluorescent images of the lateral line neuromast at 4 dpf (Q–S). ao, anterior otolith; po, posterior otolith; kc, Kinocilia of the crista hair cells. Scale bar represent 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

in transgenic larvae treated with 50 lM neomycin at different time of exposure (0.5, 1, and 1.5 h) and time-dependent hair cell loss was observed (Fig. 2I). After 0.5-h treatment with 50 lM neomycin, hair cell survival was nearly 23.77 ± 1.73% of negative control levels in larvae with high larval survival (99.31 ± 0.44%; p = 0.19) (Fig. 2J). The overall decrease in both hair cell survival and larval survival with increasing neomycin dose and duration of neomycin treatment was highly significant (⁄⁄⁄p < 0.001, one-way ANOVA; N = 20). These results demonstrated significant hair cell loss with least mortality in transgenic larvae treated with 50 lM neomycin for 0.5 h. Thus, that was chosen as a satisfactory experimental condition for following studies. 3.3. The beneficial effects of FA against different concentrations of neomycin In this study, a 50 lM concentration of neomycin for 0.5 h was an optimal experimental condition in vitro in larval zebrafish. To

determine the protective effects of FA, the dose- and timeresponse experiments were performed in 4-dpf transgenic larvae. Larvae were pretreated for 1 h with FA at 0, 50, 100, 150, and 200 lM concentrations, and then exposed to 50 lM of neomycin for 0.5 h with FA still present. Hair cell survival was then calculated as a percentage of the hair cell counts from vehicle controls (VC) treated identically with 0.2% DMSO without neomycin exposure (N = 20 larvae per group). Fluorescent images of O1 neuromast after drugs treatment and FM4-64 staining were shown in Fig. 3A–I. Up to 0.2% vehicle DMSO does not independently cause hair cell death. In addition, a 50 lM concentration of neomycin led to significant hair cell damage in the absence or presence of DMSO (Fig. 3J). While neomycin significantly decreased the number of hair cells (13.48 ± 0.75% of vehicle controls; ⁄⁄⁄P < 0.001), FA provided dose-dependent protection against neomycininduced hair cell death in the zebrafish lateral line. FA demonstrated significant protection at 50 lM higher pretreatment concentrations. Of all concentrations, neuromasts hair cells were

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Fig. 2. The dose- and time-response relationships between neuromast hair cell survival and neomycin treatment. Live neuromasts hair cells were labeled with fluorophore FM4-64 in untreated 4-dpf transgenic larvae (negative controls; NC) (A–C) and 4-dpf transgenic larvae after treatment with 50 lM neomycin for 0.5 h (D–F). Fluorescent images are close views of O1 neuromasts after FM4-64 staining (scale bar: 10 lm). Transgenic larvae exposed to various doses of neomycin for 1 h were examined for the viability of hair cells in neuromasts (G) and the viability of larvae (H). The doses used were 0, 50, 100, 150, and 200 lM neomycin in egg water. A decrease in hair cell survival and in larva survival with increasing neomycin concentration was highly significant. (I) Then, the time-response curve demonstrated significant hair cell death in larvae treated with 50 lM neomycin for 0.5 h, (J) whereas no significant change in the embryonic mortality was observed. Larva survival was approximately 99.31 ± 0.44% of negative control levels. For both graphs, summary data from 20 independent larvae were relative to negative controls (NC) (ns, not significant; ⁄⁄⁄P < 0.001; one-way ANOVA).

maximally protected against neomycin at the 150 lM pretreatment concentration of FA (96.10 ± 1.88%; p = 0.12) (Fig. 3K). However, at a 200 lM dose of FA, larval mortality was significantly increased (5.00 ± 4.08%; ⁄P < 0.05) relative to vehicle control larvae, whereas other experimental groups had no significant differences (data not shown). Following the dose-response assay, timeresponse relationships between hair cell survival and duration of FA pretreatment were performed (N = 20 larvae per group). The 4-dpf transgenic larvae were pretreated with 150 lM FA at different time of exposure (0.5, 1, 2 and 3 h) prior to treatment with neomycin, and a time-dependent change in the viability of neuromasts hair cells was observed (Fig. 3L). Although hair cell survival has a dramatic decrease more that 1 h of FA treatment, larval mortality was not significantly different from the vehicle controls (data not shown). Thus, the lowest nontoxic pretreatment dose and duration affording maximal protection against neomycin-induced hair cell death was demonstrated to be 150 lM concentration of FA for 1 h (96.10 ± 1.88%; p = 0.12). To characterize if FA maintained its protection across a wide range of neomycin concentrations, the 150 lM concentration of FA was conducted against different concentrations of neomycin. Hair cell survival in transgenic larvae pretreated with FA for 1 h,

and then exposed to 0, 25, 50, 100, 150, or 200 lM neomycin with FA still present are compared with larvae treated with identical doses of neomycin. FA could confer significant protection from both lower and higher concentrations of neomycin (⁄⁄⁄p < 0.001, one-way ANOVA). Also, a 150 lM concentration of FA is not ototoxic by itself. Hair cell survival demonstrated no significant differences in larvae pretreated with FA alone compared to vehicle controls (103.8 ± 1.95%; p = 0.18) (Fig. 3M). 3.4. Twenty-four-hour survival of neuromasts hair cells It is well known the zebrafish mechanosensory hair cells have the potential to regenerate following neomycin-induced damage (Moon et al., 2011). To determine if neomycin-induced toxicity can be reversed after recovery, hair cells were allowed to recover and estimated 24 h after drugs treatment. As shown in Fig. 4, low levels of hair cell survival were detected in neomycin-treated group (38.35 ± 1.43%), and were significantly different from hair cell survival after 1 h of neomycin. For FA-pretreated group, hair cell survival (85.04 ± 2.21%; ⁄⁄⁄p < 0.001, one-way ANOVA) was significantly increased relative to unprotected, neomycin-treated group. These results demonstrated neomycin-induced hair cell loss

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Fig. 3. FA demonstrates significant protection against neomycin-induced hair cell damage in the Tg(pvalb3b:TagGFP) transgenic line. Live neuromasts hair cells were labeled with fluorophore FM4-64 in the 4-dpf transgenic larvae. Fluorescent images of O1 neuromasts of vehicle controls (VC) (A–C), neomycin-treated group (vehicle + neomycin) (D–F) and FA-treated group (FA + neomycin) (G–I) were shown after FM4-64 staining (scale bar: 10 lm). Quantification of hair cell survival in each group was then calculated and represented as the mean ± SE (⁄⁄⁄P < 0.001, one-way ANOVA; N = 20). (J) 0.2% vehicle DMSO does not independently cause hair cell death. (K) Larvae pretreated with various concentration of FA for 1 h and then exposed with 50 lM neomycin demonstrated significant dose-dependent protection by FA. (L) The time-response curve showed the maximal protective effects of 150 lM FA was the duration of 1 h. (M) FA pretreatment led to significant protection against a wide range of neomycin. Solid line symbolized transgenic larvae pretreated with FA prior to neomycin. Dotted line symbolized transgenic larvae with no FA pretreatment prior to neomycin.

could not be reversed extensively after a 24 h recovery even if zebrafish had the high regeneration capacity. Hair cells alive in larvae were indeed protected by treatment with FA. 3.5. The protective effects of FA after pre-neomycin washout To characterize whether protective effects still persist after washout and removal of FA prior to neomycin exposure, 4-dpf transgenic larvae were pretreated with the concentration of 150 lM of FA for 1 h, and then washed extensively before exposure to neomycin. As shown in Fig. 5, hair cell survival was evidently decreased after neomycin exposure, 18.15 ± 1.23% of vehicle controls in larvae was observed. However, even if FA was removed prior to treatment with neomycin, hair cell survival was approximately 79.28 ± 2.55% of vehicle controls in larvae pretreated with FA. FA exhibited significant hair cell protection relative to neomycin-treated group (N = 20, ⁄⁄⁄p < 0.001 by one-way ANOVA). This result suggests that once FA are taken up by hair cells or bound to its targets, the duration of FA activation is not disappeared at a significant rate. FA may affect intracellular pathways to protect neomycin-induced hair cell death.

3.6. The effects of FA on neomycin-induced ROS production in zebrafish larvae Previous reports have indicated that aminoglycoside antibiotics are associated with the generation of ROS (Rizzi & Hirose, 2007). In this study, the intracellular ROS scavenging effects of FA were analyzed in transgenic zebrafish larvae using an oxidation sensitive indicator, DCF-DA, as a substrate. The scavenging function of FA on the intracellular ROS induced by neomycin administration was shown in Fig. 6A (N = 200 larvae per group; ⁄⁄⁄p < 0.001 by one-way ANOVA). The ROS level in the neomycin-treated larvae was significantly increased to 216.7 ± 9.94% (50 lM neomycin exposure) and 217.1 ± 18.11% (50 lM neomycin plus 0.2% DMSO exposure) as compared to the non-treated larvae (negative controls) and the DMSO-treated larvae (vehicle controls; 104.2 ± 2.98%) respectively. However, on the addition of FA (150 lM) to the transgenic larvae before exposure of neomycin, a dramatic reduction in intracellular ROS accumulation was observed (118.8 ± 5.61%). There were no significant differences in ROS level of FA-treated larvae relative to the vehicle controls, indicating that FA can effectively attenuate the neomycin-induced ROS

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generation in zebrafish. In addition, 0.2% DMSO does not independently induce production of intracellular ROS. 3.7. The effects of FA on apoptosis after neomycin treatment We assume that FA reduces the cell death of neuromast hair cell as a result of its beneficial effects of attenuating the neomycininduced apoptosis as previously reported (Song et al., 2014). Thus, the whole-mount TUNEL labeling was processed to detect apoptotic cells in 4-dpf transgenic zebrafish larvae treated with neomycin in the absence or presence of FA. Apoptotic cells were shown as bright red dots after TUNEL staining when observed by fluorescent microscope (TUNEL-positive cells: white arrow) (Fig. 6B–U). 0.2% DMSO does not independently induce TUNEL-reaction. DMSOtreated larvae (vehicle controls) showed no bright red dots in lateral line neuromasts as non-treated larvae (negative controls), whereas TUNEL-positive cells were apparent in neuromasts hair cells (O1, OC1 and MI1) of damaged groups (neomycin treatment with or without vehicle). However, in comparison with the neomycin-treated larvae, the 150 lM FA decreased significantly the TUNEL-reaction, and exactly protect hair cells from neomycin-induced apoptotic cell death in transgenic larvae. 4. Discussion Fig. 4. Twenty-four hour hair cell survival after neomycin treatment. (A) The 4-dpf transgenic zebrafish were pretreated with 150 lM FA for 1 h, followed by treatment with 50 lM neomycin for 0.5 h. Then, larvae recovered for 24 h, and were mounted for hair cell counts (O1, OC1, MI1, P1, P2 and P3). Fluorescent images are close views of O1 neuromasts (scale bar: 10 lm). (B) FA indeed had significant protection 24 h after the neomycin exposure relative to damaged controls (vehicle and neomycin treatment) (⁄⁄⁄P < 0.001, Turkey’s test; N = 20).

Fig. 5. Protective effects of FA after pre-neomycin washout. (A) The 4-dpf transgenic zebrafish were pretreated with 150 lM FA for 1 h, and then washed with multiple rinses in egg water to remove FA. Larvae were then treated with 50 lM neomycin for 0.5 h, followed by hair cell counts (O1, OC1, MI1, P1, P2 and P3). Fluorescent images are close views of O1 neuromasts (scale bar: 10 lm). (B) Quantification of hair cell survival from 20 independent experiments (⁄⁄⁄P < 0.001, Turkey’s test) compared to damaged controls (vehicle and neomycin treatment). FA demonstrated significant protection after pre-neomycin washout.

The lateral line, a sensory system found in fish and amphibians, has been widely used for toxicology, pharmacological screening, and neurotoxicology studies (Coffin et al., 2010). Many reports demonstrated that the rosette-like hair cells in the lateral line neuromasts of zebrafish are developmentally, structurally, and functionally similar to the sensory hair cells in mammalian inner ears (Coffin et al., 2010; Ghysen & Dambly-Chaudiere, 2004). Also, parvalbumin is a well-known calcium-binding protein that is specifically expressed in the mammalian auditory and vestibular hair cells of the organ of Corti. It performs several important inner ear functions, such as moderation of mechanosensory transduction and release of afferent neurotransmitters (McDermott et al., 2010; Steyger et al., 1997). In zebrafish, pvalb3a and pvalb3b are coorthologs to mammalian pvalb3 (Hsiao et al., 2002). Until know, in addition to brn3c and myosin 6b promoters (Obholzer et al., 2008; Xiao, Roeser, Staub, & Baier, 2005), pvalb3 promoter have been isolated to drive the expression of fluorescent reporters in hair cells of the inner ear and lateral line neuromasts, especially prior to hair bundle formation (McDermott et al., 2010). McDermott et al. (2010) generated two transgenic lines with promoter sequences extending 4.8 kb and 2.9 kb upstream of the start codon for the pvalb3b gene using the meganuclase-mediated and standard methods. However, those promoters drive nonspecific expression in other tissues except sensory cells (McDermott et al., 2010). In this study, we used a 0.6 kb fragment upstream of the start codon for pvalb3b as a promoter to develop a stable transgenic line using the MultiSite Gateway cloning method. By 4 dpf, TagGFP reporter was robustly expressed both in the hair cells of the inner ear (including the anterior maculae, posterior maculae, and crista) and in the lateral line neuromasts. Rare nonspecific TagGFP expression was observed in other tissues, such as the yolk syncytial layer, pronephric ducts, peridermal cells, and somites. TagGFP expression in transgenic zebrafish we developed was more specific than other transgenic lines. This transgenic zebrafish line provided direct insight into the hair cell neuromasts without staining under a fluorescent microscope and was applied to investigate neomycin-induced ototoxicity in this study. Neomycin treatment increased cellular apoptosis and hair cell loss in lateral line neuromasts of zebrafish. And that, neomycin also induced cellular changes in hair cells of zebrafish, including the

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Fig. 6. FA inhibits neomycin-induced ROS accumulation and decreases the numbers of TUNEL-labeled cells in zebrafish. The 4-dpf transgenic zebrafish larvae were pretreated with ferulic acid (FA) for 1 h, followed by 30-min neomycin exposure. Intracellular ROS generation and TUNEL reaction were detected. (A) ROS values are expressed as the mean ± SE of duplicate determinations (⁄⁄⁄P < 0.001, one-way ANOVA; N = 200). Statistical evaluation was performed to compare the control groups (negative controls and vehicle controls), neomycin-treated groups and FA-treated groups. Also, TUNEL reaction was performed and lateral views of non-treated larvae (negative controls) (B–E) with 50 lM neomycin exposure (F–I), with 150 lM FA and neomycin exposure (J–M), with 0.2% DMSO exposure (vehicle controls) (N–Q) and with DMSO and neomycin exposure (R–U) were shown after TUNEL assay staining. Apoptotic cells are marked as bright red dots in transgenic zebrafish under a fluorescent microscope (TUNEL-positive cells: white arrow). ov, otic vesicle; O1, otic neuromasts; OC1, occipital neuromasts; MI1, middle neuromasts. Scale bars represent 50 lm.

disruption of kinocilia and stereocilia bundles, and fused stereocilia (Song et al., 2014). It is more important to identify a preventive therapy, so that we safely use aminoglycosides without the devastating ototoxic side effects. Although many agents that protect against neomycin-induced ototoxicity have been identified in zebrafish model, none has been approved for clinical use, indicating a disparity between the results obtained with laboratory animals and in clinical trials (Coffin et al., 2010). Until now, FA has been used clinically in America for preventing and treating auditory dysfunctions. In the present study, we investigated the effect of FA on neomycin-induced ototoxicity in 4-dpf Tg (pvalb3b:TagGFP) larvae. Our results identified that FA demonstrated significant protection against neomycin-induced hair cell loss. Although 200 lM concentration of FA had cytotoxic effects on hair cell survival and larval survival, this concentration may only be excessive to fish rather than human. Most importantly, the efficiency of FA in the zebrafish could protect hair cells over a wide range of neomycin dosages. The protective agents against a narrow range of neomycin concentra-

tions may limit their clinical benefits. The ideal protective agents confer protection from both low and high concentrations of neomycin (Ou et al., 2009). In addition, neomycin-induced ototoxicity was only observed in lateral line neuromasts, while the hair cells of the inner ear were not been destroyed. We suggest that hair cells in the inner ear are isolated within extracellular fluid spaces and cannot directly contact surrounding water that contains neomycin (Hammond & Whitfield, 2006). Because of the closed structure is similar to that of other vertebrates, we may attempt to directly inject neomycin into the larval otic vesicle to acquire a high local concentration of the ototoxic molecule, and then analyze the ototoxic effect of neomycin on hair cells of the inner ear in further studies. Aminoglycosides can interact with iron to form redox-active aminoglycoside–iron complexes, which correlate closely with ROS production and ROS-induced oxidative stress (Huth, Ricci, & Cheng, 2011). In our studies, FA was a free-radical scavenger and could retard both intracellular ROS generation and TUNAL

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reactions to prevent hair cell death. Also, FA demonstrated protection against neomycin-induced hair cell death after pre-neomycin washout, suggesting FA may be taken up by hair cells or bound to its targets and then affects intracellular pathways to protect hair cells. Accordingly, the formation of aminoglycoside–iron complexes is catalyze by unsaturated fatty acids, which may enable FA to remove superoxide anions, hydroxyl radicals, and hydrogen peroxide by binding phosphatidyl ethanolamine and to prevent intracellular biomacromolecules (e.g. nucleic acids, proteins, and membrane lipids) from being attacked by free radicals (Graf, 1992; Si et al., 2013). In addition, a recent report identified that aminoglycosides induce cytoplasmic ROS production in hair cells by disordering calcium homeostasis between endoplasmic reticulum and mitochondria (Esterberg et al., 2014). Excess ROS can break the balance between oxidation and the antioxidant defense system, resulting in an irreversible oxidative damage at the molecular (e.g., nucleic acids and proteins), cellular (e.g., enzymes, cell membranes, and ion channels), and tissue levels. In the in vivo antioxidant defense system, antioxidant enzymes, including glutathione reductase, glutathione peroxidase, catalase (CAT) and superoxide dismutase (SOD), play important roles in the prevention of free radical-induced cell damage (Choi et al., 2013; Si et al., 2013). In the zebrafish model, Si et al. have identified that FA is able to retard irradiation-induced oxidative injury marked by significant changes in the antioxidant enzyme activities (such as CAT and SOD), the content of glutathione (GSH) and malondialdehyde (MDA), and the mRNA expression levels of mitochondrial inner membrane proteins related to ROS production (Si et al., 2013). In addition to affecting intracellular pathways, some chemical agents, such as carvedilol, phenoxybenzamine and quinoline ring derivatives, can interfere with the integrity of the transduction channel to reduce or eliminate neuromast hair cell sensitivity to neomycin. They may block the uptake of aminoglycosides into neuromasts hair cells and subsequently protect against aminoglycoside-induced hair cell death (Ou et al., 2009, 2012). 5. Conclusions FA is an abundant natural phenolic phytochemical found in various fruits and vegetables, including citrus fruits, cabbage, and broccoli. In this study, we found that FA conferred its great protection across a wide range of neomycin concentrations in the transgenic line. And then, FA could retard the intracellular ROS generation and TUNAL reactions to prevent hair cell death. Surprisingly, we also observed the protection of FA after pre-neomycin washout, but there is a lake of specific information about the uptake of neomycin and cellular pathway. These findings could be valuable for further works that aim to avoid the side effects of neomycin. Further studies are needed to elucidate the mechanism of FA on neuromast hair cell function in the transgenic zebrafish (pvalb3b:TagGFP), including the expression levels of antioxidant components and the regulation of signaling pathways. Those will provide greater insight into the roles of aminoglycoside-induced toxicity and allow us to find potential synergistic drugs for FA. Formatting of funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements We thank Professor Chung-Der Hsiao, Department of Bioscience Technology, Chung Yuan Christian University, for kind providing plasmids of the multisite gateway cloning system, including

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