Ototoxicity is a health problem appearing after powerful treatments in serious health conditions.1–3 It is sometimes inevitable when treatment of the serious disease is required; such as in cancer survivors. For cancer patients, Cisplatin is a common antineoplastic agent that was investigated previously to reveal increased nitrogen and reactive oxygen radicals that damages hair cells resulting in ototoxicity.3–8

There are a few agents, including sodium thiosulfate, amifostine, D-methionin, vitamin E, dexamethasone, salicilates, neurotropins, flunarisine, lipoic acid, ebselen, diethyldithiocarbamate and 4-methylthiobensoic acid that claim to prevent ototoxicity.9–15 N-acetylcysteine (NAC), previously shown to decrease ototoxicity caused by different agents, is known to be a powerful in vitro antioxidant.16–18

Ototoxicity is mediated through reactive oxygen species, which results in cell death.1–3 In contrast, N-acetylcysteine is an antioxidant, which originally is a mucolytic for pulmonary treatment. However, it is also used for the diseases of the lungs, liver, heart and kidney in order to treat their toxic and ischemic injuries.19 For example, NAC improves renal hemodynamics in rats with cisplatin-induced nephrotoxicity.20 Probably NAC, in addition to its antioxidant effect, blocks a cascade where reactive oxygen species result in apoptosis in the cochlea.21

Permanent ototoxicity is a disabling condition that could further isolate the patient from the environment, in addition to the devastating effects of primary disease. Detection by Otoacoustic Emissions (OAEs) and Auditory Brainstem Responses (ABRs) and prevention of permanency by NAC would be very beneficial for the patient who is already dealing with the primary disease.

The purpose of this study is to evaluate the possible protective properties of a potent antioxidant NAC against cisplatin ototoxicity which occurs through free radicals. NAC may contribute to the literature to prevent ototoxicity in cisplatin chemotherapy because NAC can be used in humans.

The study was accepted by Erciyes University Local Ethics Committee for Animal Experiments (HADYEK) (n° 16/147). Experiments were performed in Animal Laboratory. This is a prospective, controlled animal study about cisplatin ototoxicity.

A total of 21 male Wistar Albino 5 month-old rats with an average weight of 300–350g, which were given a standard laboratory diet in the experiments, were studied. All rats were kept in cages in the same room and under the same environmental conditions, namely in a room which was illuminated and darkened for 12/12 h cycles at a temperature of 22°C±3 with a background noise level of under 50dB. The rats were fed ad libitum.

Initially, each rat was anesthetized with intraperitoneal (i.p.) ketamine (40mg/kg) and xylazine (5mg/kg). Following the anesthesia, the ear canals and tympanic membranes of each rat were examined with otomicroscopic inspection and no pathological finding were noted. Distortion Product Otoacoustic Emissions (DPOAEs) and ABRs were performed for both ears of each animal for baseline hearing threshold evaluation. During the experiments, three rats belonging to the control group, cisplatin group and NAC group and two rats belonging to the cisplatin+NAC group did not recover from anesthesia and were excluded from the study; therefore, new rats were included instead of dead animals in the study. In conclusion, 42 functionally normal ears of 21 rats were included in the study.

Different aspects of ototoxicity and preventive agents have been previously studied.9–15 Although there are other methods for detection of ototoxicity including postmortem histopathology, surveillance with OAEs and ABRs is a reliable way. Decrement in OAE produced by outer hair cells is the evidence of ototoxicity which was previously studied.5,7,17

NAC decreases hydrogen peroxide and increases cellular glutathione, thus known to reduce cisplatin ototoxicity.22,23 NAC might exert osteogenic activity via increased glutathione synthesis.24 No osteogenesis was observed after the use of NAC in this study. But there is a debate that NAC can also decrease the antitumor effect since it is known to interact with cisplatin molecule. To prevent this attenuation in antitumor effect NAC was introduced via a totally different route, i.e. transtympani, which insured that two molecules do not interact.25 Transtympanic introduction of different protective molecules, e.g. sodium thiosulfate was also tried by others.26 To solve the interaction problem, Muldoon et al.27 introduced NAC 4h after chemotherapy, and claimed that NAC chemoprotection did not alter cisplatin therapy, if delayed until 4h after chemotherapy. They also claimed that this kind of protocol prevents ototoxicity. To test this in our current study, we also introduced the protective NAC 4h after cisplatin injection, which turned out to reduce ototoxic effect cisplatin. While OAEs and ABRs were decreased in cisplatin group, NAC given 4h after cisplatin did not alter OAE and ABR. We were able to clearly detect ototoxicity with these measurements in vivo. The cisplatin group revealed the worst results in OAE and ABR tests. OAE and ABR were not altered in the group that received NAC, meaning NAC did not affect hearing by itself. Low et al.18 evaluated NAC administered 72h after radiation and claimed to see less oxygen radicals in the inner ear, which results in less apoptosis cochlea. This is similar to the mentality that we tested in the current study. In animal fertility studies, no adverse affects were reported at doses up to 250mg/kg NAC and no teratogenic effects were observed at doses as high as 2000mg/kg NAC.28 Duan et al.29 observed that a cumulative dose of NAC (1750mg/kg), administered before and after impulse noise trauma, resulted in a greater permanent threshold shift and more inner hair cells loss compared to control animals. Instead, a cumulative dose of 1050mg/kg, over 5 days, resulted in significant protection against impulse noise. In another experiment, two groups of animals were treated with the two different cumulative doses and were not exposed to acoustic trauma: these animals did not show any threshold shift. Thus, although NAC alone is not toxic for the cochlea, dosage of the drug is critical to elicit its protective effect. Fetoni et al.30 used a dose of 500mg/kg i.p. administered immediately after noise exposure and then during the following two days (cumulative dose of 1500mg/kg). So we used a dose of 500mg/kg i.p. NAC. We did not observe a toxic effect of NAC.

As Okur et al.22 revealed that carboplatin ototoxicity increased nitric oxide levels and N-acetylcysteine prevented NO production, there may be different mechanisms about ototoxicity and prevention.

Regarding otopreservation toward antineoplastic drugs, particularly cisplatin, Church et al.31 found an electrophysiological practice of encephalic-induced potential in hamsters, preservation by sodium thiosulfate and diethyldimethylthiocarbamate, and they did not find influential protection against amifostine and fosfomycin. Kaltenbach et al.32 investigated the same drugs, now associated with structural evaluation by electron microscopy and brainstem induced potential. They determined 91% renovation of the outer hair cells with sodium thiosulfate, 68% with diethyldimethylcarbamate, 52% with fosfomycin and 45% with amifostine.

Fetoni et al.30 revealed that outer hair cells disappeared and disarrayed outer hair cell stereocilia bundles were observed. In contrast, noise exposure plus NAC showed only a moderate outer hair cell loss in the same regions and stereocilia were normal.

To our knowledge, combined audiological and histopathological findings regarding cisplatin ototoxicity and protection by NAC have not been previously published. We have shown that the ABR and OAE values and the numbers of damaged cells did not markedly change in the group receiving cisplatin and NAC, in which those receiving cisplatin alone markedly changed. Histopathological findings in the cochlea were also similar to the audiological findings. Electron microscopy showed outer hair cell loss in cisplatin ototoxicity. We concluded that cisplatin ototoxicity might be prevented using NAC in rats.

In conclusion, there was an increased length of hair cells from the basement to the apex in the cochlear structure. Based on these findings, the length of the inner and outer hair cells were unable to be compared between the experimental group and the control group. Therefore, we conclude that hearing impairment may develop as a result of problems in K+ flow caused by irregularities in tight junctions and desmosomes in stereocilia or blockade of channels by cisplatin, considering the fact that the sensory information is transported from the outer hair cells to the auditory canals.

Considering its effective dose, timing, and method of administration, NAC may serve as a valuable antioxidant agent for minimizing the ototoxicity of not only cisplatin, but also other substances. Other similar studies are necessary to support the delayed introduction of the protective agent.

NAC does not exert any adverse effects on the inner ear when used alone. In our experimental model, cisplatin successfully triggered ototoxicity, which is evident from the decreases in DPOAE and ABR results and morphological findings; and NAC shows evident signals protection against cisplatin ototoxicity on the seventh day after application. NAC given 4h after cisplatin injection prevented negative histopathological findings and the attenuation of OAE and ABR thresholds caused by cisplatin.

The authors declare no conflicts of interest.