International Journal of Pediatric Otorhinolaryngology 76 (2012) 111–115 Contents lists available at SciVerse ScienceDirect International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl Inhalation of hydrogen gas attenuates cisplatin-induced ototoxicity via reducing oxidative stress Juan Qu a, Xu Li a, Juan Wang a, Wenjuan Mi a, Keliang Xie b,*, Jianhua Qiu a,** a b Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China Department of Anesthesiology, General Hospital of Tianjin Medical University, Tianjin 300052, China A R T I C L E I N F O A B S T R A C T Article history: Received 26 August 2011 Received in revised form 12 October 2011 Accepted 13 October 2011 Available online 3 November 2011 Objective: Cisplatin, an anticancer drug used extensively to treat a broad range of tumors, has strong ototoxic side effects induced by reactive oxygen species (ROS). Recently, it has been reported that hydrogen gas (H2) is a new antioxidant by selectively reducing hydroxyl radical, the most cytotoxic ROS. The present study was designed to investigate whether H2 treatment is beneﬁcial to cisplatin-induced ototoxicity via reducing oxidative stress. Methods: The animals were intraperitoneally given a 30 min infusion of 16 mg/kg cisplatin or the same volume of saline. H2 treatment was given twice with 2% H2 inhalation for 60 min starting at 1 h and 6 h after cisplatin or saline injection, respectively. The hearing status of all animals was evaluated by auditory brainstem responses (ABR). The hair cell damage was observed by phalloidin staining. In addition, the levels of oxidative products in serum and cochlear tissue were measured. Results: We found that H2 treatment signiﬁcantly attenuated cisplatin-induced hearing loss evaluated by click-evoked and tone burst ABR threshold. Furthermore, histological analysis revealed that 2% H2 treatment signiﬁcantly alleviated cisplatin-induced hair cell damage in the organ of corti. In addition, cisplatin signiﬁcantly increased the levels of malondialdehyde (MDA) and 8-iso-prostaglandin F2a (8iso-PGF2a) in serum and cochlear tissue, which was attenuated by H2 treatment. Conclusion: These results demonstrate that H2 is beneﬁcial to cisplatin-induced ototoxicity via reducing oxidative stress. Therefore, H2 has potential for improving the quality of life of patients during chemotherapy by efﬁciently mitigating the cisplatin ototoxicity. ß 2011 Elsevier Ireland Ltd. All rights reserved. Keywords: Cisplatin Ototoxicity Hearing loss Hydrogen gas (H2) Oxidative stress 1. Introduction Cisplatin (cis-diamine-dichloroplatinum II) is currently one of the most effective chemotherapeutic agents, which can be widely used in the treatment of a variety of tumors, such as those of head, neck, testis, ovary and breast [1,2]. Unfortunately, irreversible ototoxicity is a serious side effect of cisplatin therapy, which leads to hearing loss in approximately half a million new cancer patients annually in the United States [3]. The hearing loss is due, in part, to the increased generation of reactive oxygen species (ROS) in cochlea, leading to lipid peroxidation and damage or death of outer hair cells in the organ of corti [4,5]. Antioxidant therapy might be Abbreviations: 8-iso-PGF2a, 8-iso-prostaglandin F2a; ABR, auditory brainstem response; H2, hydrogen gas; MDA, malondialdehyde; OH, hydroxyl radicals; OHCs, outer hair cells; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SPL, sound pressure levels. * Corresponding author. Tel.: +86 22 60361519; fax: +86 22 27813550. ** Corresponding author. Tel.: +86 29 84773427; fax: +86 29 83224744. E-mail addresses: xiekeliang2009@hotmail.com (K. Xie), qiujh@fmmu.edu.cn, tougaozy2011@126.com (J. Qiu). 0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.10.014 clinically useful in preserving hearing, but there is evidence that antioxidants may interfere with the tumoricidal action of cisplatin [5]. Currently, there are no effective treatments against cisplatininduced ototoxicity in clinical use. In 2007, Ohsawa et al. has found that hydrogen gas (H2) exerts a therapeutic antioxidant activity by selectively reducing hydroxyl radicals (OH, the most cytotoxic ROS) without interfering with other physiological ROS [6]. Recent studies have shown that H2 has widely therapeutic roles in many diseases via reducing oxidative stress, inﬂammation and apoptosis, such as ischemia– reperfusion injury, sepsis, multiple organ dysfunction, acute pancreatitis, chronic allograft nephropathy, tumor and type 2 diabetes [6–15]. Furthermore, H2 can effectively protect auditory hair cell against the morphological and functional damage induced by ROS [16]. More importantly, pretreatment with H2rich water can facilitate the recovery of hair cell function and attenuate noise-induced temporary hearing loss [17]. In addition, H2 alleviates nephrotoxicity induced by cisplatin without compromising anti-tumor activity in mice [18]. These ﬁndings strongly indicate that H2 treatment may be beneﬁcial to cisplatininduced ototoxicity. 112 J. Qu et al. / International Journal of Pediatric Otorhinolaryngology 76 (2012) 111–115 In the present study, therefore, we tested the hypothesis that H2 could attenuate cisplatin-induced ototoxicity and hearing loss in rats via reducing oxidative stress. 2. Materials and methods 2.1. Animals Male Wistar rats of 250–300 g with normal Preyer’s reﬂex were used in the study. The animals were provided by the animal center of the Fourth Military Medical University in China. The procedures in this study were approved by the Animal Care and Use Committee at the Fourth Military Medical University. 2.2. Grouping The animals were randomized to one of four groups (n = 6 per group): saline, saline + H2, cisplatin and cisplatin + H2. The animals in the cisplatin and cisplatin + H2 groups were intraperitoneally given a 30 min infusion of 16 mg/kg cisplatin (Sigma–Aldrich Corp., St. Louis, MO, USA) [19]. The remaining two groups received saline (isotonic sodium chloride solution) injection in equal volume. Based on our previous studies and preliminary experiment [11,12,14,15], H2 treatment was given twice with 2% H2 inhalation for 60 min starting at 1 h and 6 h after cisplatin or saline injection, respectively. 2.5. Detection of hair cell damage To observe the hair cell damage [22], all animals were sacriﬁced by decapitation under deep anesthesia after the last ABR test (at 3 d after cisplatin or saline injection). The cochlea was quickly removed and ﬁxed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 24 h at room temperature. After ﬁxation, the cochleae were dissected. After permeabilization with 0.3% Triton X-100 in PBS for 10 min, the organ of corti was stained for ﬁlamentous actin with Alexa Fluor 488 conjugated to phalloidin (green ﬂuorescence) (Molecular Probes, Eugene, OR, USA) for 40 min to outline hair cells and their stereocilia for a quantitative assessment. Hair cell loss was evaluated at each cochlear turn with a ﬂuorescence microscope (Nikon Eclipse 80i, Japan). 2.6. Detection of oxidative products To investigate the mechanism, the levels of oxidative products in serum and cochlear tissue were measured after the last ABR test (at 3 d after cisplatin or saline injection). The levels of malondialdehyde (MDA) and 8-iso-prostaglandin F2a (8-isoPGF2a) were detected by commercial kits (Cayman Chemical Company, Ann Arbor, MI, USA) using a microplate reader (CA 94089, Molecular Devices, Sunnyvale, Canada). The concentration of tissue protein was determined by using a standard commercial kit (Sigma–Aldrich Corp., St. Louis, MO, USA). 2.3. H2 treatment 2.7. Statistical analysis The animals with H2 treatment were put in a sealed plexiglas chamber with inﬂow and outﬂow outlets. H2 was supplied through a gas ﬂowmeter, TF-I (YUTAKA Engineering Corp., Tokyo, Japan), and delivered by air into the chamber at a rate of 4 L/min. The concentration of H2 in the chamber was continuously monitored with a commercially available detector (Hy Alerta Handheld Detector Model 500, H2 Scan, Valencia, CA, USA). The concentration of oxygen in the chamber was maintained at 21% by using supplemental oxygen and continuously monitored with a gas analyzer (Medical Gas Analyzer LB-2, Model 40M, Beckman, USA). Carbon dioxide was removed with baralyme. The animals without H2 treatment were exposed to room air in this chamber [11,12,14,15]. 2.4. Auditory brainstem responses To investigate the effects of H2 on cisplatin-induced ototoxicity, the hearing status of all animals was evaluated at baseline just before and again 3 d after cisplatin or saline administration using auditory brainstem responses (ABR) as previously described [17,20]. Elevation of ABR threshold has been shown to provide an excellent, reliable indicator of the degree of cochlear hearing loss in experimental animals [21]. Animals were anesthetized with an intraperitoneal injection of 40 mg/kg pentobarbital sodium. The needle electrodes were placed subcutaneously beneath the pinna of measured ear (reference electrode), the apex of nose (ground), and the vertex (active electrode). The stimulus signal was generated through an Intelligent Hearing Systems device (Biologic Systems, USA). Click sounds at a rate of 57.7/s were given to evoke the ABRs. Tone burst sounds at 4, 8, 16 and 32 kHz (0.2-ms rise/fall time and 1-ms ﬂat segment) were generated to estimate frequency-speciﬁc thresholds. Responses of 1024 sweeps were averaged at each intensity level step. The stimulus intensity varied in 5 dB sound pressure levels (SPL) stepwise increments, and the threshold was deﬁned as the lowest intensity level at which a response was still observed. Rectal temperature was monitored and maintained at 37 8C by a warming pad during the experiment. All data are expressed as mean SEM. The data were analyzed by one-way ANOVA followed by LSD-t Test for multiple comparisons. The statistical analysis was performed with SPSS 16.0 software. In all tests, a P value of less than 0.05 was considered statistically signiﬁcant. 3. Results 3.1. H2 treatment attenuated cisplatin-induced hearing loss The hearing status was evaluated before and 3 d after cisplatin or saline injection by click-evoked and tone burst ABR threshold shift shown in Figs. 1 and 2. In the cisplatin group, there was one mouse died on day 2 and 83.3% of mice survived during the experiment. In contrast, all mice survived in the cisplatin + H2 group. Intraperitoneal injection of 16 mg/kg cisplatin signiﬁcantly increased the click-evoked and tone burst ABR threshold at 4, 8, 16 and 32 kHz when compared with saline injection group (P < 0.05, n = 6 per group), suggesting cisplatin caused signiﬁcant hearing loss. However, H2 treatment markedly attenuated the click-evoked and tone burst ABR threshold shift at 4, 8, 16 and 32 kHz in cisplatin-challenged rats (P < 0.05, n = 6 per group). In addition, H2 treatment had no effects on the click-evoked and tone burst ABR threshold in the rats with saline injection (P > 0.05, n = 6 per group). The results suggest that cisplatin causes hearing loss in rats, which can be signiﬁcantly attenuated by 2% H2 treatment. 3.2. H2 treatment attenuated cisplatin-induced damage of hair cells Furthermore, because cisplatin mainly damages outer hair cells (OHCs) [3,4], the representative micrographs of hair cell staining in basal turn were shown in Fig. 3. Cisplatin signiﬁcantly caused the damage of OHCs, which was improved by H2 treatment. In addition, the lost OHCs in the basal, second, third and apical turns were counted (Fig. 4). H2 treatment signiﬁcantly alleviated cisplatin-induced OHCs loss (P < 0.05, n = 6 per group). The results J. Qu et al. / International Journal of Pediatric Otorhinolaryngology 76 (2012) 111–115 Fig. 1. H2 treatment attenuated cisplatin-induced click-evoked auditory brainstem response (ABR) threshold shift. H2 treatment was given twice with 2% H2 inhalation for 60 min starting at 1 h and 6 h after cisplatin or saline injection, respectively. The click-evoked ABR threshold was measured at baseline just before and again 3 d after cisplatin or saline administration. The values are expressed as means SEM (n = 6 per group). *P < 0.05 vs. saline group; yP < 0.05 vs. cisplatin group. suggest that cisplatin causes hair cell damage in rats, which can be signiﬁcantly attenuated by 2% H2 treatment. 3.3. H2 treatment attenuated cisplatin-induced oxidative stress in serum and cochlear tissue To investigate the underlying mechanism, we detected the levels of MDA and 8-iso-PGF2a in serum and cochlear tissue after the last ABR test (at 3 d after cisplatin or saline injection) (Fig. 5). Cisplatin led to the increased levels of MDA and 8-iso-PGF2a in serum and cochlear tissue when compared to that of saline group (P < 0.05, n = 6 per group). However, H2 treatment signiﬁcantly decreased the levels of MDA and 8-iso-PGF2a in serum and cochlear tissue in cisplatin-challenged rats (P < 0.05, n = 6 per group). In addition, H2 treatment had no effects on the levels of MDA and 8-iso-PGF2a in serum and cochlear tissue in the rats with saline injection (P > 0.05, n = 6 per group). The results suggest that cisplatin results in signiﬁcant oxidative stress, which can be attenuated by 2% H2 treatment. 4. Discussion In the present study, we have found that 2% H2 treatment improved the click-evoked and tone burst ABR threshold shift induced by intraperitoneal injection of cisplatin. Furthermore, H2 treatment attenuated cisplatin-induced hair cell damage in rats. 113 Fig. 2. H2 treatment attenuated cisplatin-induced tone burst auditory brainstem response (ABR) threshold shift. The tone burst ABR threshold was measured at baseline just before and again 3 d after cisplatin or saline administration. The values are expressed as means SEM (n = 6 per group). *P < 0.05 vs. saline group; yP < 0.05 vs. cisplatin group. In addition, H2 treatment decreased the levels of MDA and 8-iso-PGF2a in serum and cochlear tissue in cisplatin-challenged rats. These results demonstrate that H2 treatment may be a useful therapeutic agent for cisplatin-induced ototoxicity via reducing oxidative stress. For systemic cisplatin administration, the Wistar rat is a well established model of ototoxicity [23]. Unlike the chinchilla, the Wistar rats have good renal clearance and cisplatin ototoxicity can be induced without high mortality [23]. When compared with multiple doses of cisplatin, a single high-dose administration resulted in a similar pattern of hearing loss that occurred more rapidly and remained stable for at least 6 d [23,24]. In this study, we found that intraperitoneal administration of 16 mg/kg cisplatin caused signiﬁcant hearing loss evaluated by ABR threshold shift and outer hair cell damage in Wistar rats, which is consistent with previous studies [23,24]. Administration of platinum containing drugs, such as cisplatin, causes signiﬁcant hearing loss, which is usually permanent and cumulative [4,5]. The organ of corti represents a major site for cisplatin-induced hearing loss, where the drug leads to the permanent loss of outer hair cells [3,4]. Several reports have concluded that the generation of ROS is linked to cisplatin ototoxicity [3–5]. Antioxidant therapy has proven to be beneﬁcial in animal models of cisplatin ototoxicity, but there is evidence that antioxidants may interfere with the tumoricidal action of cisplatin [5]. Therefore, new or improved methods are needed for Fig. 3. H2 treatment attenuated cisplatin-induced damage of hair cells. The hair cells were stained for ﬁlamentous actin with Alexa Fluor 488 conjugated to phalloidin (green ﬂuorescence) at 3 d after cisplatin or saline administration. The representative micrographs of basal turn are shown in (A) saline group; (B) cisplatin group; (C) cisplatin + H2 group. 114 J. Qu et al. / International Journal of Pediatric Otorhinolaryngology 76 (2012) 111–115 Fig. 4. H2 treatment attenuated cisplatin-induced loss of outer hair cells (OHCs). Hair cell loss was evaluated at each cochlear turn at 3 d after cisplatin administration. The values are expressed as means SEM (n = 6 per group). y P < 0.05 vs. cisplatin group. alleviating the cisplatin ototoxicity without compromising its anticancer effect. H2 can selectively alleviate hydroxyl radicals and peroxynitrite radical-induced cytotoxicity without affecting other ROS, such as superoxide, hydrogen peroxide, or nitric oxide [6]. It is advantageous in medical treatments because the use of hydrogen could not cause serious unwanted side effects. We and other researchers have found that H2 treatment can effectively protect against organ damage such as brain, spinal cord, heart, lung, liver and kidney through reducing oxidative stress, suggesting that H2 has a potential role in preventive and therapeutic applications for organ damage [6–15,25]. Furthermore, an in vitro study has also demonstrated the potential of hydrogen to protect both the inner hair cells and outer hair cells from oxidative damage induced by different concentrations of antimycin A [16]. Incubation with a hydrogen-saturated medium signiﬁcantly reduced ROS generation and subsequent lipid peroxidation in the auditory epithelia, leading to the increased survival of hair cells [16]. Recently, a study from our department has shown that pretreatment with H2rich water can prevent noise-induced hearing loss [17]. In addition, H2 has antitumor effects [9]. H2 treatment can alleviate nephrotoxic side effects induced by cisplatin without compromising its antitumor activity [18,26]. These studies supported that H2 may be a good choice for treating cisplatin-induced ototoxicity. In the present study, we found that 2% H2 treatment signiﬁcantly attenuated the cisplatin-induced hearing loss evaluated by clickevoked and tone burst ABR threshold. In addition, H2 treatment could signiﬁcantly mitigate the hair cell damage induced by cisplatin. These results demonstrate that H2 is beneﬁcial to cisplatin-induced ototoxicity. MDA is a commonly measured end point of free radical-induced lipid peroxidation, and the MDA level correlates with the extent of free radical-induced damage [14,27]. In addition, measurement of 8-iso-PGF2a, free radical-catalyzed products of arachidonic acid, can offer a reliable approach for quantitative measurement of oxidative stress status in vivo [28]. Thus, the detection of MDA and 8-iso-PGF2a has been widely used to estimate the overall status of oxidative stress. In the present study, we found that cisplatin signiﬁcantly increased the levels of MDA and 8-iso-PGF2a in serum and cochlear tissue, which was attenuated by H2 treatment. This suggests that the decrease of oxidative stress may contribute to the protection of H2 treatment, which is consistent with our previous studies [11,12,14,15]. In conclusion, the present study supports that H2 inhalation may be an effective therapeutic agent attenuating cisplatininduced ototoxicity. H2 is one of the most plentiful gases in the universe. It is neither explosive nor dangerous at a concentration of less than 4.7% in air and 4.1% in pure oxygen, respectively [10]. Besides hydrogen inhalation, there are other alternative ways to Fig. 5. H2 treatment attenuated cisplatin-induced oxidative stress in serum (A and C) and cochlear tissue (B and D). The levels of MDA and 8-iso-PGF2a in serum and cochlear tissue were detected at 3 d after cisplatin or saline administration. The values are expressed as means SEM (n = 6 per group). *P < 0.05 vs. saline group; yP < 0.05 vs. cisplatin group. J. Qu et al. / International Journal of Pediatric Otorhinolaryngology 76 (2012) 111–115 deliver hydrogen to the cochlea, including hydrogen rich water, hydrogen saturated saline, electrolysed-reduced water, and hydrogen generated from coral calcium hydride solution [29]. H2 has potential for improving the quality of life of patients during chemotherapy by efﬁciently mitigating the side effects of cisplatin. Conﬂict of interest The authors have declared that no conﬂict of interest exists. 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