Otoprotection Mechanisms Against Oxidative Stress Caused by Cisplatin

THIEME Original Research Otoprotection Mechanisms Against Oxidative Stress Caused by Cisplatin Maiara Santos Gonçalves1 Aron Ferreira da Silveira2 Maria Rossato3 Miguel Angelo Hippolito3 1 Department of Phonoaudiology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil 2 Department of Morphology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil 3 Department of Ophthalmology, Otorhinolaryngology and Head and Neck Surgery, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Ribeirao Preto, SP, Brazil Adriana de Andrade Batista Murashima3 Address for correspondence Maiara Santos Gonçalves, Departamento de Fonoaudiologia, Universidade Federal de Santa Maria, Avenida Roraima, 1000, Santa Maria, RS, 97105900, Brazil (e-mail: ). Int Arch Otorhinolaryngol 2020;24(1):e47–e52. Abstract Keywords ► cisplatin ► toxicity ► free radicals ► cochlea ► oxidative stress Introduction Cisplatin damages the auditory system and is related to the generation of free radicals. Glutathione peroxidase is an endogenous free radicals remover. Objective To investigate the mechanisms involved in otoprotection by N-acetylcysteine through the expression of glutathione peroxidase in outer hair cells from rats treated with cisplatin. Methods Male Wistar rats were intraperitoneally injected with cisplatin (8 mg/Kg) and/or received oral administration by gavage of N-acetylcysteine (300 mg/Kg) for 3 consecutive days. On the 4th day, the animals were euthanized and beheaded. The tympanic bullae were removed and prepared for scanning electron microscopy and immunofluorescence. Results Among the groups exposed to ototoxic doses of cisplatin, there was an increase in glutathione peroxidase immunostaining in two groups, the one exposed to cisplatin alone, and the group exposed to both cisplatin and N-acetylcysteine. Conclusion The expression of glutathione peroxidase in the outer hair cells of rats exposed to cisplatin showed the synthesis of this enzyme under cellular toxicity conditions. Introduction The cisplatin-based regimen is considered the gold standard for the treatment of a variety of solid tumors.1 This antineoplastic agent acts in the cell by reducing the levels of endogenous antioxidant enzymes and activating the apoptotic cascade.2,3 Despite its proven efficacy, cisplatin should be administered with caution due to its side effects, which include nephrotoxicity, neurotoxicity, and ototoxicity.4 There are different mechanisms through which cisplatin causes injury and awakens the cell death pathway in the auditory system. The mechanism most discussed currently received July 4, 2019 accepted September 7, 2019 DOI https://doi.org/ 10.1055/s-0039-1698782. ISSN 1809-9777. relates to the generation of free radicals, specifically the reactive oxygen species (ROS), which cause injuries and can lead to cell death by apoptosis.5 Damages to the auditory system occur with both acute high doses and cumulative doses. The hearing loss is bilateral sensorineural, irreversible, and associated with tinnitus.5 Due to the tonotopic arrangement of the cochlea, high frequencies are affected first,5,6 since damages to the outer hair cells (OHCs) progress from the base to the cochlea apex and from the third to the first row of these cells; then, the damage affects the inner hair cells (IHCs).7 Still, the injuries are not limited to hair cells, they also affect supporting cells, the stria vascularis, and the spiral ganglion.8 Copyright © 2020 by Thieme Revinter Publicações Ltda, Rio de Janeiro, Brazil e47 e48 Otoprotection Mechanisms Against Oxidative Stress Gonçalves et al. In the cochlea, cisplatin accumulates in the tissue, integrates into the DNA and causes disorder in protein synthesis and antioxidant enzymes. As the cochlea is in an isolated anatomical position and is virtually a closed system, it becomes unable to expel the accumulated toxin at the same rate and speed at which it is generated. Thus, there is a ROS overload associated with an impaired antioxidant system.9 This condition causes lipid peroxidation increase, thus initiating events that culminate in hair cells, supporting cells, stria vascularis, and auditory nerve apoptosis.10 Facing irreversible ototoxicity, the challenge has been to discover and enable a substance with otoprotective action that does not interfere with the cytotoxic effect of cisplatin in tumor cells. Among these substances is N-acetylcysteine (NAC), a drug from the group of thiols with potential antioxidant effect that increases the levels of intracellular glutathione.11,12 Clinically, NAC is used as a mucolytic agent to clear air pathways and as an antidote to acetaminophen poisoning.13 This compound was selected for the study protocol by presenting antioxidant properties, having a free radicals direct scan action and an indirect action, serving as a precursor of intracellular free cysteine, which can be used to synthesize glutathione peroxidase. Glutathione peroxidase (GSH-Px) is an endogenous free radicals remover,14 and it is present in the cell cytoplasm and in the mitochondria.15 Thus, this study aimed to investigate the mechanisms involved in otoprotection by NAC through the expression of GSH-Px in the OHCs from rats treated with cisplatin. Methods The study population consisted of male Wistar rats subjected to a light-dark cycle of 12/12 hours (light period started at 7 AM), kept at room temperature (19–23°C) and humidity (55%  15%) controlled and also with water and food ad libitum. All procedures were performed according to the criteria of the Arouca Law (Law n° 11.794, of October 8, 2008) and the Brazilian Society of Laboratory Animal Science (Sociedade Brasileira de Ciências em Animais de Laboratório). The project was submitted for approval by the Ethics Committee on Animal Experimentation of the institution of origin. Animals weighing between 260 and 300 g, with Preyer16 reflex present and electrophysiological threshold of brainstem auditory evoked potential (BAEP) of 20 dB HL in both ears were included in the study. Animals that showed signs of otitis external or acute otitis media at examination of the external auditory canal, , those that had earwax that was difficult to remove, and those with very narrow canals that prevented placing the probe for BAEP testing were excluded from the sample. The drugs administered were cisplatin (Tecnoplatin - Eurofarma Laboratórios Ltda, São Paulo, SP, Brazil); acetyl-cysteine (Laboratório EMS, Santo André, SP, Brazil); ketamine hydrochloride 10% (Ketamin 50mg/ml - Laboratório Cristália, Itapira, SP, Brazil); xylazine hydrochloride (Dopaser 200mg - Laboratório Hertape Calier, Juatuba, MG, Brazil). For immunofluorescence, Rabbit polyclonal antibody and anti- GSH-Px 1 (100 µg presentation) were used; goat International Archives of Otorhinolaryngology Vol. 24 No. 1/2020 polyclonal secondary antibody anti-IgG - H&L ( Alexa Fluor 488–Thermo Fisher Scientific, Waltham, MS, USA); goat p (...truncated)