| Summary: | 博士 === 國立臺灣大學 === 毒理學研究所 === 95 === Cisplatin (cis-diamminedichloroplatinum II), which is an alkylating agent and exerts cytotoxic effects through the formation of covalent DNA-adducts, is one of the most potent chemotherapeutic antitumor agents. Cisplatin has been demonstrated against a variety of neoplasms, particularly for head and neck, testicular, ovarian, bladder and small cell lung cancers. The major toxic effects of cisplatin include ototoxicity, nephrotoxicity and peripheral neuropathy. Although its nephrotoxicity can be decreased in severity through systemic hydration, ototoxicity is the dose-limiting side effect. In the past, studies concerning cisplatin ototoxicity were mainly limited to cochleotoxicity, based on the change of auditory function, instead of vestibulotoxicity. Vestibular function test is very difficult to perform in experimental animals because they have to be retrained in a special holder and keep awake. By means of vestibulo-ocular reflex testing system together with auditory brainstem response, we could sequentially compare the different responses between vestibular and cochlear toxicity induced by cisplatin in guinea pigs. In addition, our study will investigate the biochemical mechanisms leading to cisplatin-induced cochleotoxicity and vestibulotoxicity, which are rarely reported in the literatures.
Chemoprotection refers to protection from the side effects of chemotherapeutic agents without reducing their oncological efficacy. Most of chemoprotective agents, such as D-methionine, sodium thiosulfate, or N-acetyl-cysteine, are thiols (sulfur-containing compounds), electrophilic and thought to act as free radical scavengers or by covalent binding, or both. Because ototoxicity is still the dose-limiting side effect for cisplatin, administration of chemoprotective agents in the rescue from ototoxicity to increase the dose of cisplatin is necessary to improve its oncological effect and therapeutic efficacy. Previous studies indicated that chemoprotective agents could protect against cisplatin-induced hearing loss. However, it has never been reported whether these agents will also protect from cisplatin-induced vestibulotoxicity. To our best knowledge, this is the first study to evaluate if D-methionine might also protect from cisplatin-induced vestibulotoxicity in addition to its cochleotoxicity and their underlying biochemical mechanisms.
Noise is the greatest causative factor among the defined etiologies of hearing loss. Exposure to loud noise may cause sensorineural hearing loss temporarily for minutes, hours or days, or permanently, depending on the intensity and duration of the noise exposure and the animal’s susceptibility to noise exposure. Noise-induced temporary threshold shift (TTS) is a reversible elevation in hearing threshold that may occur following acoustic overstimulation. The remaining hearing loss after the exposure that does not fully recover to its preexposure level is known as permanent threshold shift (PTS). Conventionally, prevention of noise-induced hearing loss (NIHL) has been addressed by providing wearable hearing protection and reducing noise emissions. However, for personnel in certain military and industrial occupations, this has been insufficient, especially when noise levels exceed 130 decibels (dB). The efficacy of hearing-protection devices and hearing-protection measures could be augmented by pharmacological agents that might reduce NIHL more effectively. A common result after acoustic injury is the development of a TTS. With multiple, cumulative exposure events, significant irreversible hearing loss can occur that produces a PTS. Therefore, pharmacological agents that prevent TTS may be effective against PTS and have a potential clinical role in the prophylaxis of noise-induced hearing loss in the future.
Reactive oxygen species (ROS) have been shown to play a toxic role in the cochlea. Direct effects of ROS on cochlear outer hair cell shape in vitro, on cochlear explants and on cochlear function in vivo have been identified. ROS have recently been demonstrated to play an important role in NIHL. The role of antioxidants in protecting noise-induced cochlear injury has been widely studied in recent years. Our previous studies documented that reversible inactivation of enzyme activities (Na+, K+-ATPase and Ca2+-ATPase) and increased production of endogenous NO in the cochlear lateral wall might play a role in the pathogenesis of noise-induced TTS. D-methionine, functioning as a ROS and /or RNS scavenger, may thus prevent noise-induced cochlear dysfunction. Although antioxidants (such as glutathione, D-methionine, N-acetylcysteine) have been proven to provide excellent pharmacological prevention from noise-induced hearing loss, none of them were studied for the underlying biochemical mechanisms such as changes of enzyme activities, lipid peroxidation, and NO production.
1. Correlation of increased activities of Na+, K+-ATPase and Ca2+-ATPase with the reversal of cisplatin cochleotoxicity induced by D-methionine and the role of curcumin and quercetin in protection from cisplatin-induced cochleotoxicity in guinea pigs
Na+, K+-ATPase and Ca2+-ATPase in the cochlear lateral wall play an important role in maintaining ionic homeostasis and physiologic function of the cochlea. The present study was designed to test whether the changes of Na+, K+-ATPase and Ca2+-ATPase activities of the cochlear lateral wall and the brainstem of guinea pigs after receiving cisplatin for 7 consecutive days were correlated with the altered auditory brainstem responses (ABR). Furthermore, whether chemoprotective agents, D-methionine, curcumin or quercetin, reversed the increased ABR threshold induced by cisplatin accompanied with the increased ATPase activities was also evaluated. The results obtained showed that cisplatin exposure caused not only a significant increase of threshold but also altered various absolute wave and interwave latencies of ABR. In addition, cisplatin significantly decreased the Na+, K+-ATPase and Ca2+-ATPase activities in the cochlear lateral wall with a good dose-response relationship. Regression analysis indicated that an increase of ABR threshold was well correlated with a decrease of both Na+, K+-ATPase and Ca2+-ATPase activities in the cochlear lateral wall. Chemoprotectants, D-methionine, curcumin or quercetin, indeed reversed both abnormalities of ABR and ATPase activities in a well correlation function. The selectivity of these observed changes induced by cisplatin and D-methionine was revealed by the findings that cisplatin-treated guinea pigs had normal III-V interwave latency of ABR and no reduction of Na+, K+-ATPase and Ca2+-ATPase specific activities in the brainstem, which is in accordance with the nonpenetrable cisplatin across the blood brain barrier. Taken all together, the present findings suggest that biochemical damage and ionic disturbance may contribute to cisplatin-induced ototoxicity to some extent, which can be reversed by D-methionine, curcumin or quercetin.
2. D-methionine inhibited cisplatin-induced vestibulotoxicity through preventing the decrease of ATPase activities and attenuating oxidative stress in guinea-pigs
Cisplatin has been used as a chemotherapeutic agent to treat many kinds of malignancies. Its damage to the vestibulo-ocular reflex (VOR) system has been reported. However, the underlying biochemical change in the inner ear or central vestibular nervous system is not fully understood. In this study, we attempted to examine whether cisplatin-induced vestibulotoxicity and D-methionine protection were correlated with the changes of ATPase activities and oxidative stress of ampullary tissue of vestibules as well as cerebellar cortex (the inhibitory center of VOR system) of guinea pigs. By means of a caloric test coupled with electronystagmographic recordings, we found that cisplatin exposure caused a dose-dependent (1, 3, or 5 mg/kg) vestibular dysfunction as revealed by a decrease of slow phase velocity (SPV). In addition, cisplatin significantly inhibited the Na+, K+-ATPase and Ca2+-ATPase activities in the ampullary tissue with a good dose-response relationship, but not those of cerebellar cortex. Regression analysis indicated that a decrease of SPV was well correlated with the reduction of Na+, K+-ATPase and Ca2+-ATPase activities of the ampullary tissue. D-methionine (300 mg/kg) reduced both abnormalities of SPV and ATPase activities in a correlated manner. Moreover, cisplatin exposure led to a significant dose-dependent increase of lipid peroxidation and nitric oxide concentrations of the vestibules, which could be significantly suppressed by D-methionine. However, cisplatin did not alter the levels of lipid peroxidation and nitric oxide of the cerebellum. In conclusion, cisplatin inhibited ATPase activities and increased oxidative stress in guinea-pig vestibular labyrinths. D-methionine attenuated cisplatin-induced vestibulotoxicity associated with ionic disturbance through its antioxidative property.
3. Protection from noise-induced temporary threshold shift by D-methionine is associated with preservation of ATPase activities
The present study was designed to test whether noise-induced temporary threshold shift (TTS) could be attenuated by D-methionine and if this protection was correlated with the changes of ATPase activities and oxidative stress of cochlear lateral walls in experimental animals. Thirty-two normal-hearing male guinea pigs were randomly divided into saline-treated and D-methionine-treated (300 mg/kg) experimental groups. One hour after treatment, they were exposed to a continuous broadband white noise at 105±2 dB sound pressure level for 10 min, causing TTS. Each group was then divided into four subgroups based on the number of survival days after noise exposure (0, 1, 2, and 7 days). Each subgroup had four animals and eight ears included. By means of click-evoked auditory brainstem responses (ABR), auditory thresholds of guinea pigs were measured before noise exposure, immediately after noise exposure, and before sacrifice. After animals were sacrificed, cochlear lateral walls were immediately harvested and assayed for enzyme-specific activities of Na+, K+-ATPase and Ca2+-ATPase, lipid peroxidation (LPO) and nitric oxide (NO). A 15.31±3.80 dB threshold shift was found immediately after noise exposure in saline-pretreated guinea pigs. In contrast, ABR threshold shift was significantly attenuated to 4.06±2.35 dB in D-methionine-protected animals. Furthermore, D-methionine enhanced the restoration of ABR threshold to baseline level by one day. In addition, noise significantly decreased Na+, K+-ATPase and Ca2+-ATPase activities and increased LPO and NO levels of the cochlear lateral walls, all of which could be significantly suppressed by D-methionine. In conclusions, noise-induced TTS is associated with the inhibited ATPase activities and the increased oxidative stress in guinea-pig cochlear lateral walls; all of these changes could be attenuated by D-methionine through its antioxidative property.
In summary, our experimental data show that both cisplatin and noise cause the dose-dependent impairment of auditory function, and that cisplatin also leads to the dose-dependent injury of vestibular function in guinea pigs. Impairment of auditory or vestibular function has a significant positive correlation with a decrease of ATPase specific activities of cochlear lateral walls or vestibular labyrinth in cisplatin-exposed or noise-treated guinea pigs, suggesting that deterioration of ATPase activities in the inner ear may play important roles in the pathogenesis of ototoxicity. In addition, an inverse correlation between the decreased enzyme activities and increased production of LPO and NO implies that the pathogenesis of ototoxicity might be mediated by oxidative stress. Protection of D-methionine against ototoxicity induced by cisplatin or noise may relate to the reverse of the reduction of specific enzyme activities. D-methionine has a potential clinical role in the prophylaxis of ototoxicity resulted from cisplatin or noise in the future.
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