二氧化鍺影響大鼠中樞神經傳導功能及造成神經細胞毒性之機轉探討

Germanium Dioxide Impairs the Central Neural Conduction in Rat and Its Molecular Mechanisms of Neurotoxicity

林壯澔(Chuang-Hao Lin)

指導教授 ： 陳順勝 ； 陳贊如

高雄醫學大學/醫學院/醫學研究所/博士(2008年)

https://doi.org/10.6832/KMU.2008.00032

摘要

近年來鍺元素 (germanium, Ge) 已被廣泛應用於半導體工業。由於鍺元素具有促進紅血球生成、抑制微生物生長、防止類澱粉蛋白形成、免疫調節作用、以及抗腫瘤作用等多種生物活性, 因此, 鍺元素亦被用來做為健康食品或醫療用途以促進健康。不過, 因食用大量鍺元素而發生腎毒性, 神經毒性, 及肌肉毒性的案例則隨後被報導。在自然界, 鍺元素一般以無機鍺或有機鍺的型態少量存在於土壤、岩石、金屬礦及動植物內。無機鍺多以二氧化鍺 (GeO2) 的型態存在 (De Ruijter & Van Sande 1993)。為了暸解鍺的組織累積量與發生鍺毒性之關連性, 本論文以動物實驗進行二氧化鍺的動力學及組織分佈分析。結果發現二氧化鍺的吸收半衰期為0.7?b0.1小時, 排除半衰期為2.3?b0.5小時。組織分佈實驗顯示二氧化鍺會累積在體內某些重要器官或組織, 包括腦部、週邊神經及腎臟等。雖然組織學證據顯示二氧化鍺會對腦幹神經核造成傷害, 但目前對於二氧化鍺在中樞神經系統的影響程度仍不清楚。因此本論文分別利用腦幹聽覺誘發電位 (BAEPs) 及全細胞膜片鉗制法 (whole cell patch clamp) 探討此一問題。結果發現給予二氧化鍺後會造成大鼠的聽覺閾值上升, 以及中樞聽覺神經路徑傳導時間延長。全細胞膜片鉗制法則顯示二氧化鍺會抑制電壓感應式鈉離子通道活性而降低鈉離子內流電流, 顯示二氧化鍺會促使神經細胞興奮性降低, 此結果可能與中樞聽覺傳導異常有關。此外, 組織學證據顯示二氧化鍺引發之腎毒性及肌肉毒性時常可見粒線體形態發生改變及功能喪失, 因此本研究針對二氧化鍺引發之神經毒性與粒線體功能間之關係進行探討。實驗結果顯示給予二氧化鍺會降低Neuro-2A細胞之存活率, 此一現象具有劑量相關性及時間相關性。二氧化鍺亦會導致Neuro-2A細胞之細胞核出現DNA斷裂等細胞凋亡的形態。此外, 二氧化鍺藉由引發Bax轉位及Bcl-2表現量降低導致粒線體膜電位喪失, 並釋出cytochrome c導致細胞凋亡。由此可知, 二氧化鍺藉由粒線體媒介引發之細胞凋亡為造成細胞毒性的主要原因。綜合以上所述, 本論文發現組織內二氧化鍺的累積造成之粒線體毒性及對電壓感應式鈉離子通道的抑制作用是影響神經系統正常功能的兩個可能原因。

關鍵字

二氧化鍺 ； 神經傳導 ； 神經毒性 ； 細胞凋亡

並列摘要

Germanium (Ge) is commonly used in the semiconductor industry. Biologically, germanium possesses erythropoietic, antimicrobial, anti-tumor, anti-amyloidosis and immunomodulative effects. Because of its biological activity, germanium also was used to promote health and to act as medicine. However, toxic effects of Ge-containing compounds on kidney, muscle, neuronal cells and nerves have been reported. To understand the relationship between GeO2-induced toxicity and its tissue accumulation effect, the kinetics of germanium dioxide (GeO2) in single dose and repeated exposures were investigated in male Wistar rats. The results revealed that the absorption half-life was 0.7 ± 0.1 hours and the elimination half-life was 2.3 ± 0.5 hours after Wistar rat was administrated 100 mg/kg GeO2. The tissue distribution study also showed that GeO2 was accumulated in some important organs or tissues in the body, including brain, peripheral nerves and kidney. This accumulation effect might explain the GeO2-induced toxicity on above organs. Furthermore, the brainstem auditory evoked potentials (BAEPs) was performed to investigate the effect of GeO2 on nervous system function in rat. The result also revealed that GeO2 administration dose-dependently elevated the BAEPs threshold and prolonged BAEPs latencies and interpeak latencies (IPLs) in these GeO2-induced hearing loss rats, but the amplitudes did not obviously changeed. In addition, the whole cell patch clamp data also revealed that GeO2 could inhibit the voltage-gated sodium channel activation. These results suggested that GeO2 was toxic to the function of auditory system. The mitochondrial toxicity and inhibitory effect of GeO2 on voltage-gated sodium channel might be a part of the mechanism underlying abnormal BAEPs after GeO2 administration. Mitochondrial dysfunction was found to be involved in the pathogenesis of GeO2-induced nephropathy and myopathy. Since it is well known that mitochondria play a major role in apoptosis triggered by many stimuli, an effort was made to examine whether the Ge-induced neurotoxicity occurs through mitochondria-mediated apoptosis. The cell viability of Neuro-2A cells was inhibited in a dose- and time-dependent manner after treatmeny with various concentrations of GeO2. Further analysis showed that aside from the changes in the nuclear morphology responsible for apoptosis, the release of cytochrome c, the loss of mitochondrial membrane potential, the translocation of Bax, and the reduction of Bcl-2 expression were also observed in Neuro-2A cells after GeO2 treatment. These results indicate that the mitochondria-mediated apoptosis is involved in this in vitro model of GeO2-induced neurotoxicity

並列關鍵字

germanium dioxide ； nerve conduction ； neurotoxicity ； apoptosis

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