D型甲硫胺酸降低順-雙氨雙氯鉑及噪音引發天竺鼠耳毒性之作用機轉

Abstract

順-雙氨雙氯鉑(cisplatin)是眾多抗癌藥物的一種，為目前最有效而被廣泛使用化學治療藥之一，尤其對於頭頸部腫瘤、睪丸腫瘤、卵巢腫瘤、膀胱腫瘤與小細胞肺癌有明顯的療效，然而，順-雙氨雙氯鉑的副作用很大，主要的毒性包括耳毒性、腎毒性、神經毒性、骨髓抑制等。其中腎毒性因充水治療已可降低其毒性，然而耳毒性仍無法有效預防，因而降低化療使用劑量而干擾癌症治療效果。以往順-雙氨雙氯鉑對於耳毒性只偏向對於耳蝸的聽覺生理研究，對於其生化機轉及前庭平衡系統的毒性則少有研究。主因實驗動物被麻醉後，平衡系統被抑制而無法客觀量度，比如前庭眼反射系統，實驗動物必須是清醒的且能配合，以免干擾電位的記錄。相對於順-雙氨雙氯鉑對於耳蝸的毒性，可藉由麻醉實驗動物後，聽性腦幹反應的變化而得到客觀的量度。藉由本研究的方法，吾人可以檢視清醒實驗動物的前庭眼反射系統功能，同時比較順-雙氨雙氯鉑對天竺鼠平衡及聽覺系統之傷害程度，分析這些生理功能變化與內耳酵素活性及氧化性壓力變化之關係，以闡明產生耳毒性之生化機轉。我們期望尋求一種或數種化療保護劑，可以減少順-雙氨雙氯鉑化療的副作用，但又不影響其抗癌作用，進而可以提高順-雙氨雙氯鉑的劑量，因而提高化療的療效。有報告指出，化療保護劑D型甲硫胺酸(D-methionine)可以減少順-雙氨雙氯鉑造成的聽力喪失又不至於減少其抗癌療效，但是對於其生化機轉及是否有減少前庭系統的毒性則未有報告。本研究擬藉由檢測前庭眼反射系統的功能，我們期望可以回答預先處理D型甲硫胺酸後，是否可以減少化療對於前庭系統的毒性，同時比較順-雙氨雙氯鉑對於兩者毒性的差異，分析其生理功能變化與內耳酵素活性及氧化壓力變化之關係，以釐清D型甲硫胺酸減輕耳毒性之生化機轉。 噪音是導致聽力障礙最主要的原因之一。目前預防噪音傳入內耳造成傷害的方法是以佩帶耳塞、耳罩等防護設備、減少噪音的產生及暴露，然而在許多職業與娛樂場所如此預防措施仍嫌不足，噪音依然會產生聽力障礙，因此用藥物進一步來預防噪音性聽力障礙相當重要。以往的研究已證實氧化性壓力的增加，在噪音性聽力障礙扮演著重要角色，然而有關噪音破壞耳蝸生理功能以及抗氧化劑可降低此傷害之生化機轉則罕被提出報告。因此本研究檢驗D型甲硫胺酸在減少噪音的耳蝸傷害，其聽力閾值的改變是否與鈉鉀-腺三磷酸酵素及鈣-腺三磷酸酵素活性與氧化性壓力的改變具有相關性。這些實驗結果可提出D型甲硫胺酸在預防天竺鼠噪音性聽力障礙之生化機轉，以及探討D型甲硫胺酸未來其在臨床上用來預防噪音性聽力障礙的潛力。 壹、D型甲硫胺酸逆轉天竺鼠順-雙氨雙氯鉑耳蝸毒性與增加耳蝸外側壁鈉鉀-腺三磷酸酵素及鈣-腺三磷酸酵素活性之相關性以及薑黃素與檞皮酮減緩順-雙氨雙氯鉑耳蝸毒性之角色 本研究測試天竺鼠在接受順-雙氨雙氯鉑7天後，耳蝸外側壁與腦幹之鈉鉀-腺三磷酸酵素與鈣-腺三磷酸酵素活性之改變，與聽性腦幹反應的改變是否具相關性。此外，化療保護劑D型甲硫胺酸、薑黃素或檞皮酮是否可逆轉順-雙氨雙氯鉑所引發聽性腦幹反應閾值之增加與內耳耳蝸腺三磷酸酵素活性之減少。實驗動物為天竺鼠，分為實驗組及對照組，均以腹腔注射給予順-雙氨雙氯鉑連續七天，實驗組在給予順-雙氨雙氯鉑前三十分鐘給予化療保護劑D型甲硫胺酸、薑黃素或檞皮酮，對照組則給予生理食鹽水。測量給藥後天竺鼠之聽性腦幹反應。之後將實驗動物犧牲，分離出腦幹及內耳耳蝸外側壁，分別測量其鈉鉀-腺三磷酸酵素與鈣-腺三磷酸酵素酵素活性。結果發現順-雙氨雙氯鉑不僅造成天竺鼠聽性腦幹反應閾值之增加，同時改變其絕對波潛時及波與波之間的潛時，但無改變III-V波之間的潛時，而且順-雙氨雙氯鉑有意義地減少耳蝸外側壁鈉鉀-腺三磷酸酵素與鈣-腺三磷酸酵素的活性，但無改變腦幹鈉鉀-腺三磷酸酵素與鈣-腺三磷酸酵素的活性。化療保護劑D型甲硫胺酸、薑黃素與檞皮酮的確逆轉了順-雙氨雙氯鉑所造成聽性腦幹反應及腺三磷酸酵素活性之異常。總結順-雙氨雙氯鉑並無改變III-V波的潛時與腦幹腺三磷酸酵素的活性，佐證了順-雙氨雙氯鉑無法通過血腦屏障。本研究顯示順-雙氨雙氯鉑所引發之耳蝸毒性部分歸因於耳蝸內生化的損害與離子的失衡，而這些傷害可被D型甲硫胺酸、薑黃素或檞皮酮所逆轉。 貳、經由改變前庭腺三磷酸酵素的活性與氧化壓力D型甲硫胺酸可減緩天竺鼠順-雙氨雙氯鉑前庭毒性 數十年來，化療藥物順-雙氨雙氯鉑已被用來治療許多癌症。雖然過去曾報導過它可能造成前庭眼反射系統的破壞，然而對其生化機轉仍不十分了解。本研究探討順-雙氨雙氯鉑是否造成天竺鼠前庭毒性及化療保護劑D型甲硫胺酸是否可逆轉此毒性。此外D型甲硫胺酸的保護作用與前庭腺三磷酸酵素活性與氧化壓力的改變是否具相關性。實驗動物為天竺鼠，分為實驗組及對照組，均以腹腔注射給予順-雙氨雙氯鉑連續七天，實驗組在給予順-雙氨雙氯鉑前三十分鐘給予化療保護劑D型甲硫胺酸，對照組則給予生理食鹽水。藉由眼振電圖，紀錄給藥七天後天竺鼠溫差測驗的結果。之後將實驗動物犧牲，分離出內耳前庭壺腹及小腦皮質組織，分別測量其鈉鉀-腺三磷酸酵素與鈣-腺三磷酸酵素酵素活性、脂質過氧化及一氧化氮的濃度。藉由溫差測驗眼振慢速相速度的變化得知順-雙氨雙氯鉑確可破壞天竺鼠的前庭功能，而且有意義地減少前庭壺腹鈉鉀-腺三磷酸酵素與鈣-腺三磷酸酵素的活性，但無改變小腦皮質鈉鉀-腺三磷酸酵素與鈣-腺三磷酸酵素的活性。D型甲硫胺酸的確逆轉了順-雙氨雙氯鉑所造成眼振慢速相速度變慢及前庭壺腹腺三磷酸酵素活性之異常。此外，順-雙氨雙氯鉑可造成前庭壺腹溫差測驗的增加，而這些變化可被D型甲硫胺酸所抑制。順-雙氨雙氯鉑可抑制天竺鼠內耳前庭壺腹腺三磷酸酵素的活性，增加其氧化壓力。順-雙氨雙氯鉑所引發之前庭毒性至少部分歸因於前庭內生化的損害與離子的失衡，而D型甲硫胺酸藉由其抗氧化的特性，可減輕順-雙氨雙氯鉑所造成的前庭毒性。 參、 D型甲硫胺酸可減緩噪音所引發之暫時聽閾值變動與腺三磷酸酵素相關 雖然噪音引發聽力障礙之機制至今仍未完全明瞭，活性氧化物已被證實在噪音性聽力障礙扮演一重要角色。儘管在動物實驗中，抗氧化劑可降低噪音性聽力障礙之發生，然而其機制鲜少被研究。還原性麩胱氨酸是存在於人類真核細胞中主要的抗氧化物之一，且有明確證據顯示麩胱氨酸可減少耳蝸因毒性或噪音因氧化性壓力增加所產生之傷害。在抗氧化物之中，D型甲硫胺酸是一種已經美國食品藥物管理局認可的含硫氨基酸，可用於臨床上使用。相較於其他多數可清除自由基的藥物，D型甲硫胺酸除了可以直接清除活性氧化物之外，亦可增加細胞內麩胱氨酸的濃度。活性氧化物會破壞細胞膜的構造及功能，包括酵素活性、離子通道、通透性、滲透性、接受器等。鈉鉀-腺三磷酸酵素及鈣-腺三磷酸酵素在維持內耳細胞離子的恆定與生理功能扮演著重要角色。我們先前的研究已經指出天竺鼠在經過噪音暴露後耳蝸外側壁此兩種酵素的活性顯著減少，且內生性一氧化氮的濃度顯著增加。基於這些事實，本研究進一步測試D型甲硫胺酸是否可減緩噪音所引發之暫時聽閾值變動，且此保護作用是否與耳蝸外側壁腺三磷酸酵素與氧化壓力的改變有關。本研究之實驗動物為天竺鼠，暴露於105±2分貝聲音音壓階的噪音持續10分鐘，暴露前1小時在其腹腔內分別注入D型甲硫胺酸(300 毫克/公斤)或生理食鹽水。聽性腦幹反應則分別在噪音暴露前、暴露後、暴露後1天、暴露後2天、或暴露後7天施行。之後分別將其犧牲，分離出內耳耳蝸外側壁，測量其鈉鉀-腺三磷酸酵素、鈣-腺三磷酸酵素活性，以及脂質過氧化物與一氧化氮的濃度。實驗結果顯示D型甲硫胺酸藉由其抗氧化的特性，可有效地減少噪音所引發之暫時聽閾值變動及加速聽性腦幹閾值的回復正常值，同時，減緩噪音所引發耳蝸外側壁腺三磷酸酵素的減少與氧化壓力的增加。 本研究證實順-雙氨雙氯鉑與噪音皆會抑制耳蝸外側壁鈉鉀-腺三磷酸酵素及鈣-腺三磷酸酵素的活性，同時順-雙氨雙氯鉑也會抑制前庭壺腹此酵素之活性，進而破壞耳蝸與前庭內細胞離子的恆定與生理功能。因此，耳蝸毒性與前庭毒性的產生可能與腺三磷酸酵素活性的減少有關。順-雙氨雙氯鉑與噪音皆會造成耳蝸外側壁與(或)前庭壺腹之氧化性壓力增加，如此可能和腺三磷酸酵素活性的減少有關，進一步影響聽覺與平衡系統的生理功能。D型甲硫胺酸不但可減輕順-雙氨雙氯鉑的耳蝸毒性、前庭毒性，亦可減少噪音引發的耳蝸傷害。因此，未來在臨床研究發展上，D型甲硫胺酸有潛力用來降低順-雙氨雙氯鉑的耳毒性，包括耳蝸毒性、前庭毒性，以提高其化療使用劑量而增加癌症治療效果；降低噪音所造成的耳蝸生化傷害，以預防噪音性聽力障礙。

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.