Cisplatin is a widely used and effective drug for the treatment of solid tumors ranging from ovarian, lung, head, and neck to testicular cancer. Dose limiting side effects, such as ototoxicity, neurotoxicity, and nephrotoxicity, are generally encountered with a majority of patients treated with cisplatin-based chemotherapy. Methods to increase diuresis, such as hydration, have been shown to reduce nephrotoxicity. However, to date no effective FDA approved treatment for ototoxicity is available. Cisplatin-induced hearing loss is primarily in the high frequency range. It is bilateral and permanent and severely affects the quality of life for cancer patients. The incidence of cisplatin-induced hearing loss in children ranges from 22 to 77% (Knight et al., 2005; Kushner et al., 2006; Coradini et al., 2007). This range reflects hearing loss observed with the different doses and duration of cisplatin treatment in addition to the different age groups of the patients who were treated. Accordingly, children show greater risk for developing hearing loss following cisplatin treatment than adults (Knight et al., 2005; Kushner et al., 2006; Gurney et al., 2007). Hearing loss is especially difficult for children who are undergoing treatment for brain tumors, such as neuroblastoma. It could affect early speech development and hamper social integration. Development of effective therapies for treating hearing loss is therefore of primary importance.

Several approaches have been undertaken over the past two decades to treat cisplatin ototoxicity. These include the use of localized or systemic administration of antioxidants or drugs which activate endogenous antioxidant systems. Cisplatin induces apoptosis of hair cells through activation of mitochondrial pathway which can be targeted to inhibit cell death. Another approach involves using anti-inflammatory agents which target the pro-inflammatory mechanisms associated with cisplatin treatment in the cochlea. Recent insights into the entry processes of cisplatin in hair cells and other cells in the cochlea should stimulate the development of drugs which specifically block entry of drugs into these cells without affecting cisplatin entry into cancer cells. This report reviews the currently accepted mechanisms underlying cisplatin-induced damage or death to cochlear cells and highlights how these mechanisms could guide the future development of effective otoprotective agents.

The normal function of the cochlea requires its high metabolic activity in areas such as the stria vascularis, spiral ligament, and spiral prominence (Salt et al., 1987; Ryan, 1988). This high metabolic activity leads to leakage of electrons from the mitochondrial respiratory chain which can react with oxygen (O₂) to form superoxide (). Environmental stimuli which increase the metabolic activity of the cochlea, such as loud noise, are expected to increase oxidative stress in the cochlea. The metabolic demand on the cochlea renders it very sensitive to hypoxic events and ischemia-reperfusion injuries (Seidman et al., 1991). Ototoxic drugs, such as cisplatin have been shown to increase the generation of reactive oxygen species (ROS) (Clerici and Yang, 1996; Kopke et al., 1997) by either stimulating enzyme systems linked to this process or by inactivating antioxidant systems (Church et al., 1995; Rybak et al., 1995). Rats injected with cisplatin demonstrate reduced cochlear glutathione (GSH) and antioxidant enzyme activities (Ravi et al., 1995). A primary target of cisplatin for generation of ROS is the NOX3 NADPH oxidase system (Banfi et al., 2004). NOX3 is induced by cisplatin and knockdown of this enzyme by trans-tympanic delivery of siRNA protects against cisplatin-induced ototoxicity (Mukherjea et al., 2010). Since NOX3 is localized primarily to the cochlea, systemic administration of inhibitors of NOX3 could effectively reduce enzyme activity and treat hearing loss. Another active ROS generating system in the cochlea is xanthine oxidase. This enzyme converts hypoxanthine (a metabolite derived from the breakdown of adenosine by adenosine deaminase) to uric acid. Inhibition of this enzyme by allopurinol contributes to reductions in cisplatin-induced ototoxicity and nephrotoxicity when administered with ebselen, a glutathione peroxidase (GSH.Px) mimetic (Lynch et al., 2005).

The cochlea possesses an efficient antioxidant defense system. This includes antioxidants such as vitamin C, vitamin E, and low molecular weight thiols, such as GSH (Kopke et al., 1999). Studies in the guinea pig cochlea show that the highest levels of GSH are present in the basal and intermediate cells of the stria vascularis and in cells of the spiral ligament (Usami et al., 1996). This distribution matches well with the distribution of glutathione S-transferase (el Barbary et al., 1993), an enzyme which conjugates and detoxifies xenobiotics (such as cisplatin). Increased levels of glutathione S-transferase in cancer cells can aid in the inactivation of cisplatin and contribute to resistance to cisplatin in the cochlea. In addition, the cochlea expresses several antioxidant enzymes, which include superoxide dismutase (SOD), GSH.Px, and catalase (CAT). SOD catalyzes the conversion of to H₂O₂ and O₂ while CAT converts H₂O₂ to O₂ and H₂O. GSH.Px reduces H₂O₂ and possibly other peroxides. The enzyme GSH.Px also catalyzes the conversion of reduced GSH to its oxidized form (GSSG), in the process of detoxifying H₂O₂. In addition, glutathione reductase (GR) is important for the defense against ROS by aiding in the regeneration of GSH from GSSG (Figure 1). Two forms of SOD are expressed in the cochlea. A Cu/Zn isoform of SOD is localized in the cytosol, while a Mn-regulated isoform (Mn-SOD) is localized to the mitochondria (Yao and Rarey, 1996). Mn-SOD is localized to metabolically active sites in the cochlea such as stria vascularis, spiral ligament, spiral prominence, spiral limbus, and organ of Corti (Lai et al., 1996). Activities of SOD were higher in the subcellular fractions of stria vascularis and spiral ligament compared to the organ of Corti (Pierson and Gray, 1982; Yao and Rarey, 1996). High activities of other antioxidant enzymes were also observed in the lateral wall tissues compared to the rest of the cochlea, suggesting their roles in mitigating oxidative stress in these regions (Pierson and Gray, 1982). In the absence of this ROS detoxification system, ROS can produce cellular damage by lipid peroxidation, with increased levels of the lipid peroxide, malondialdehyde, and 4-hydroxynonenal.

Exposure of explants to ROS induces bleb formation and changes in length of outer hair cells which were attenuated by co-administration of the antioxidant, deferoxamine, or antioxidant enzymes (Clerici et al., 1995). Infusion of ROS into the guinea pig ear led to increased compound action potential (CAP) threshold amplitudes, which were reduced by co-administration of antioxidant or antioxidant enzymes (Clerici and Yang, 1996). Furthermore, it was observed that cochleae obtained from cisplatin-treated animals showed depletion of GSH and reduced activities of antioxidant enzymes, such as SOD, CAT, GSH.Px, and GR along with increased evidence of lipid peroxidation (Rybak et al., 2000). This reduction in antioxidant capacity in the cochlea could result from: (1) covalent binding of cisplatin to sulfhydryl groups within the antioxidant enzymes, causing enzyme inactivation; (2) loss of metal cofactors, such as copper and selenium, which are vital for activity of SOD and GSH.Px; (3) increased ROS which could exhaust antioxidant enzymes; and/or (4) depletion of cochlear antioxidant enzyme cofactors, such as GSH and NADPH, which are essential for GSH.Px and GR activities, respectively (for review see Rybak et al., 2000). The increased oxidative stress within the cochlea could increase lipid peroxidation of membranes, inactivate essential cellular enzymes and membrane transporters, and disrupt ion channel function. The ultimate effect of increased ROS generation is to promote apoptotic and necrotic cell death. These data suggest that ROS plays a critical role in cisplatin-induced ototoxicity and that its inhibition could ameliorate hearing loss.

As discussed below, one important result of increased oxidative stress is induction of inflammatory processes in the cochlea. The importance of managing ROS in mediating otoprotection is underscored by the observation that polymorphisms of glutathione S-transferase gene increase susceptibility to cisplatin hearing loss (Oldenburg et al., 2007). In this report, it was shown that patients inheriting the 105Ile/105Ile-GSTP1 or 105Val/105Ile-GSTP1 alleles had greater hearing loss than those inheriting the 105Val/105Val-GSTP1 alleles (Oldenburg et al., 2007).

The cochlea is endowed with a number of distinct cellular mechanisms which could contribute to otoprotection. These include various endogenous antioxidant enzymes and antioxidants (detailed above), heat shock proteins, kidney injury molecule-1, anti-apoptotic proteins, transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf2) and signal transducer and activator of transcription (STAT) proteins, and a number of hormone and G-protein-coupled receptors (Ford et al., 1997; Oh et al., 2000; Mukherjea et al., 2006; So et al., 2006; Borse et al., 2017). Therefore, it is very surprising that these mechanisms are not able to effectively protect against cisplatin ototoxicity. However, it is possible that over the course of cisplatin treatment in animal models and humans that these protective systems become overwhelmed and are no longer able to manage the toxicity. It is believed that exogenously administered antioxidants or other drugs can boost the local protective mechanism to ensure otoprotection.

Early studies were directed at examining the effects of drugs which reduce oxidative stress for treating hearing loss. Studies performed in animals indicated that antioxidants protected against cisplatin-induced hearing loss. Many of these antioxidants are thiol compounds which have high affinities for platinum. This forms the basis for their protection against cisplatin toxicity but also could account for the antitumor interference with cisplatin. Studies showed that N-acetyl cysteine (NAC) protected against cisplatin-ototoxicity in rats (Dickey et al., 2008) and guinea pigs (Choe et al., 2004). Sodium thiosulfate (STS) was also effective against cisplatin-induced hearing loss (Otto et al., 1988). However, systemic administration of this drug led to formation of cisplatin–STS complex, which reduced the levels of cisplatin in circulation needed for effective antitumor therapy (Wimmer et al., 2004). Thus, local application of STS into the cochlea would be required to provide effective otoprotection (Wimmer et al., 2004), as was shown previously (Wang et al., 2003). Several studies have shown that D-methionine, another sulfur-containing compound, protects against cisplatin-induced hearing loss when administered by both the systemic and local routes (Campbell et al., 1996; Korver et al., 2002). At the cellular level, the otoprotection mediated by this compound was associated with its ability to increase the activity of intrinsic antioxidant enzymes (Campbell et al., 2003). Other antioxidants which show promise against cisplatin-induced hearing loss include ebselen, lipoic acid, diethyldithiocarbamate, and 4-methylthiobenzoic acid (Rybak et al., 1999). Similarly, high doses of amifostine provided otoprotection in hamsters but its use was associated with neurotoxicity (Church et al., 2004).

Contrary to the pre-clinical findings, studies performed in humans showed that STS protected against cisplatin-induced nephrotoxicity (Goel et al., 1989) but was ineffective against cisplatin-induced hearing loss (Zuur et al., 2007). Amifostine was also ineffective as an otoprotectant in patients with metastatic melanoma (Ekborn et al., 2002) and in children suffering from neuroblastoma or germ cell tumors and were on a chemotherapeutic regimen including cisplatin (Marina et al., 2005; Sastry and Kellie, 2005). However, later studies showed that higher doses of amifostine were able to provide significant otoprotection (Fouladi et al., 2008).

Several G-protein-coupled receptors have been characterized in the cochlea, activation of which confer protection against cisplatin-induced hearing loss. Immunolabeling studies show distribution of the A₁ adenosine receptors (A₁ARs) in the stria vascularis, spiral ganglion cells, and organ of Corti (Vlajkovic et al., 2009). In the organ of Corti, the greatest distribution of A₁AR is in the inner hair cells, Deiter’s cells and lower levels in the outer hair cells. Expression of A₁AR was also observed in mouse organ of Corti-derived cell lines including the UB/OC-1 cells (Kaur et al., 2016) and HEI-OC1 cells (unpublished data). Localized application of A₁AR agonist resulted in an increase in the activities of the antioxidative enzymes GSH.Px and SOD (Ford et al., 1997). Furthermore, A₁AR agonist also reduced the cisplatin-mediated increase in malondialdehyde levels in the cochlea resulting in protection against cisplatin-induced hair cell damage and hearing loss (Whitworth et al., 2004). No significant benefits was observed following activation of the A_2aAR and A₃AR (Whitworth et al., 2004) which are also distributed in the cochlea (Vlajkovic et al., 2009). Systemic administration of the adenosine amine congener (ADAC) was shown to protect against cisplatin ototoxicity, presumably by activating the A₁AR (Gunewardene et al., 2013). Recent data support an anti-inflammatory role of A₁AR activation in the cochlea mediated by suppression of the NOX3 isoform of NADPH oxidase and suppression of STAT1-mediated inflammatory pathway (Kaur et al., 2016). STAT1 activation plays an integral role in cisplatin ototoxicity, as inhibition or knockdown of this transcription factor reduced cisplatin-induced hearing loss (Schmitt et al., 2009; Kaur et al., 2011; Mukherjea et al., 2011). A recent study from our laboratory has further documented an essential role of STAT1 in mediating cisplatin-induced hearing loss, as inhibition of this factor by a green tea extract and a known STAT1 inhibitor, epigallocatechin-3-gallate (EGCG), provided otoprotection (Borse et al., 2017). In a rodent model, EGCG protected against cisplatin-induced hearing loss without compromising cisplatin antitumor efficacy (Borse et al., 2017) (see model depicted in Figure 2). In vitro studies performed in HEI-OC1 cells demonstrate that cannabinoid 2 receptor (CB2) agonists reduce cisplatin-induced cell killing (Jeong et al., 2007). CB2 are also expressed in the stria vascularis, inner hair cells and spiral ganglion cells of the cochlea from adult albino rats (Martin-Saldana et al., 2016). Recent studies from our laboratories support an otoprotective role of CB2 activation in the cochlea, which is mediated at least in part, through inhibition of STAT1 (Ghosh et al., 2016; unpublished data). Thus, the protective action of CB2 could share a similar mechanism as observed by A₁AR, namely inhibition of STAT1.

Additional studies from our laboratory implicated transient receptor potential vanilloid 1 (TRPV1) channels in cisplatin-mediated ototoxicity (Mukherjea et al., 2008). In a rat model, we showed knockdown of these channels by trans-tympanic administration of short interfering (si) RNA protected against cisplatin-induced hearing loss and damage to the outer hair cells (Mukherjea et al., 2008) (Figure 3). Protection was likely mediated by reducing the expression of a downstream target of TRPV1, such as NOX3, activation of which promotes ROS generation and STAT1 activation, as indicated above. STAT1 can promote both the inflammatory and pro-apoptotic actions of cisplatin in the cochlea (Kaur et al., 2011). This study suggests that inhibition of TRPV1 or NOX3 could serve as useful approaches for reducing cisplatin ototoxicity.

Characterization of cisplatin-induced cell death in HEI-OC1 cells showed induction of apoptosis by increased lipid peroxidation and altered mitochondrial permeability transition. It was shown that the calcium-channel blocker, flunarizine, attenuated cisplatin-induced cell death (So et al., 2006). The mechanism underlying the otoprotective action of flunarizine appears to involve activation of Nrf2 and increased expression of hemeoxygenase-1 (HO-1) (So et al., 2006). Flunarizine also exhibited an anti-inflammatory role, as evidenced from its ability to inhibit the ERK1/2 MAP kinase-nuclear factor (NF)-κB-dependent pathway (So et al., 2008).

Previous studies have shown that platinated DNA accumulates in the nuclei of outer hair cells, supporting cells, marginal cells of the stria vascularis and cells in the spiral ligament (van Ruijven et al., 2005). Other studies have also shown DNA adduct in spiral ganglion neurons following cisplatin administration. The cochlear cells express DNA repair enzymes which can reduce the level of DNA adduct formed over time. These repair enzymes, known as nucleotide excision repair (NER) enzymes, are classified as transcriptional-coupled repair (TCR) enzymes or global DNA repair (GDR) enzymes which differ in their targets. Defective TCR function is observed in Cockayne syndrome which is characterized by progressive hearing loss. Outer hair cells from Cockayne syndrome group A (Csa^-/^-) and group B (Csb^-/^-) mice were hypersensitive to cisplatin. In contrast, Xpc^-/^- mice which were deficient in global genome repair enzymes showed normal sensitivity to cisplatin (Rainey et al., 2016). This study implicates DNA damage as one mechanism of cisplatin-induced loss of outer hair cells and hearing loss and suggests that TCR plays a primary role in protecting hair cells from cisplatin-induced hearing loss. The effects of eight single-nucleotide polymorphisms in excision repair cross-complementing group 1, 2, 4, and 5 (ERCC1, ERCC2, ERCC4, and ERCC5) genes and xeroderma pigmentosum complementary group C and A (XPC and XPA) genes were assessed in patients with osteosarcoma who were treated with cisplatin. These studies showed that polymorphisms in DNA repair gene, XPC, was associated with increased cisplatin-induced ototoxicity in cancer patients. Increased ototoxicity was associated with the CC genotype of XPC Lys939Gln (Caronia et al., 2009).

One of the major entry ports for cisplatin in the cochlea is the mammalian copper transport 1 (Ctr1). Ctr1 is highly expressed in the cochlea where it is localized to outer hair cells, inner hair cells, stria vascularis, and spiral ganglion neurons and contributes to drug entry and cell apoptosis. Decreasing cisplatin entry by intra-tympanic administration of copper sulfate, a substrate of Ctr1, protects against hearing loss induced by cisplatin (More et al., 2010). Cisplatin entry into cochlear cells is also mediated by organic cation transporter (OCT). Three isoforms of this protein exists, OCT1-3, which are present mainly in the kidneys and liver. Expression of OCT2 has also been detected in the organ of Corti and stria vascularis (Ciarimboli et al., 2010). Inhibition of these transporters with cimetidine protects against cisplatin-induced nephrotoxicity and ototoxicity. OCT knockout mice exhibit reduced toxicity to cisplatin. Single-nucleotide polymorphism in OCT-2 gene protects against ototoxicity in children (Lanvers-Kaminsky et al., 2015). Several studies have reported that the entry of aminoglycosides into cochlear hair cells is mediated by mechanotransducer (MET) channels (Gale et al., 2001; Marcotti et al., 2005; Dai et al., 2006; Wang and Steyger, 2009; Alharazneh et al., 2011). In fact, changes in the structure of aminoglycosides which limits their entry through MET channels are less ototoxic (Huth et al., 2015). Cisplatin has also shown to block MET channels in chick cochlear hair cells (Kimitsuki et al., 1993). Recent studies have indicated that cisplatin-induced damage to hair cells in the zebrafish is dependent on functional MET channels (Thomas et al., 2013). These investigators showed that inhibition of mechanotransduction channels by quinine or EGTA protected against cisplatin-induced hair cell death. Furthermore, these investigators showed that zebrafish mutants which lacked mechanotransduction channels were also resistant to cisplatin-induced hair cell death. These studies suggest that mechanotransduction channels are a major contributor to the entry of cisplatin into hair cells, at least in the zebrafish. Interestingly, chemical inhibition of Ctr1 and OCT-2 did not provide significant protection against killing of lateral line hair cells in zebrafish (Thomas et al., 2013). Preliminary data from our lab indicate a similar pathway of entry of transplatin, the inactive isomer of cisplatin, which protects against cisplatin ototoxicity (Dhukhwa et al., 2017). These studies suggest that MET channels could serve as an additional entry ports for cisplatin into cochlear hair cells. Several other ion channels might also contribute to aminoglycoside uptake into hair cells. In addition to TRPV1, several TRP channels including TRPV4, TRPA1, TRPC3, and TRPML3 are expressed in the cochlea (Cuajungco et al., 2007; Asai et al., 2010). These channels have been shown to allow entry of aminoglycoside into kidney cells (Myrdal and Steyger, 2005). The potential role of these channels in mediating the entry of aminoglycosides and cisplatin into hair cells has not yet been established.

Megalin is a low-density lipoprotein which is highly expressed in the kidney and in the stria vascularis of the cochlea which accumulate high levels of platinum DNA adducts. Patients who showed cisplatin-induced hearing impairment demonstrate a higher frequency of megalin gene polymorphism compared to those with no hearing loss after cisplatin therapy. These findings implicate megalin gene polymorphisms in susceptibility to cisplatin ototoxicity (Riedemann et al., 2008). In the kidneys, megalin has been shown to bind β₂-microglobulin, cytochrome c, retinal binding proteins, and polybasic antibiotics (such as gentamicin). These investigators show that the accumulation of cisplatin in the renal proximal tubules is mediated by binding of cisplatin-metallothionein complex to megalin (Klassen et al., 2004). The expression of megalin in the stria vascularis might similarly allow the accumulation of cisplatin into the stria vascularis.

Studies described above have identified a number of different mechanisms which mediated cisplatin ototoxicity and provide the basis for rational drug design to treat this debilitating condition. While most of the drugs which target these mechanisms have shown promise, efficacy studies are still in the experimental animal stage (see Table 1 for list of drugs/drug targets). These studies need to be extended in human clinical trials for final validation. The routes of drug administration would be a major issue, as systemic administration of these drugs could compromise cisplatin chemotherapeutic efficacy. In this regard, the use of a number of cisplatin transporters (namely Ctr1 and OCT2) and antioxidants would have the greatest likelihood of interfering with cisplatin antitumor efficacy (as discussed above). Localized delivery of these drugs into the cochlea would eliminate potential systemic toxicities but would require an additional minor surgical procedure to accomplish this goal. This would allow the use of a majority of the agents listed in this table for protection against hearing loss. Several of these drugs are currently in clinical use or in clinical trials for other indications. These include TNF-α antagonists which are used for the treatment of chronic inflammatory diseases (Aggarwal et al., 2012) and EGCG which is in various clinical trials for the treatment of cancer (Singh et al., 2011). These agents which are effective systemically could serve as initial candidates for treating cisplatin-induced hearing loss and other forms of hearing loss. Other drugs which could be advanced quickly into clinical use are MET channel blockers, such as bulky aminoglycoside antibiotics, which demonstrate low potential for ototoxicity (Huth et al., 2015). Continued research in this area would uncover new mechanisms underlying cisplatin-induced hearing loss and validate novel targets and drugs to treat this condition.

SS and VR conceived and together outlined this review. SS and VR wrote the manuscript. DM and LR critiqued and revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.