汞化合物長期低劑量暴露對鼷鼠及其子代神經毒性作用機轉之研究

Studies on mechanisms of neurotoxicological effects of mercurial compounds in mice and offsprings after long-term and low-dose exposure

黃俊發(Chun-Fa Huang)

指導教授 ： 蕭水銀

國立臺灣大學/醫學院/毒理學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.00076

摘要

汞化物基於其化學特性及生物效用，隨著農工業發達，民生品的需求如日光燈、汞電池、消毒劑及殺蟲劑等均是含汞製品。然而廣泛的使用加上不適當的管制而造成環境污染，人類經由食物鏈攝食含汞的食物，危害健康。因此有史以來汞化物已變成一種惡名昭彰的毒性重金屬，也是目前污染較為嚴重且較廣泛存在於環境中。它以有機汞、無機汞及元素汞的形式在環境中循環。目前對於汞化合物所產生的毒性作用雖已有相當多的文獻報導，但對於傳統中醫用藥--硃砂(主成分為硫化汞-HgS)的藥理作用與毒性反應的研究卻不多。根據古籍記載硃砂乃是一種礦物性中藥，其主要成分乃硫化汞。【神農本草經】記載，硃砂乃上品藥之首，有「養精神、安魂魄、益氣明目」之功效。【本草綱目】中更進一步認為，硃砂可「明目、安胎、解毒、發汗，隨佐使而見功，無所往而不可」，從此擴大了硃砂的應用範圍。但單獨或過量使用硃砂，自古以來一直列為禁忌，【本草從新】有“獨用多用，乏人呆悶”的敘述。【本草用法藥方】則記述“硃砂若經伏火及一切烹煉，則毒等砒霜能殺人”，此乃因硫化汞加熱分解，使汞游離出來所致。近年來在亞洲地區有臨床報告指出誤用硃砂所產生致死或相關的神經毒性反應，在台灣則有小兒八寶散含硃砂所造成嬰幼兒重金屬中毒的案例。現代藥理研究報告指出：硃砂具有降低大腦中樞神經興奮性並降低腦中serotonin(5-HT)含量而減低焦慮感，進而達到安眠鎮靜作用。亦可延長由strychnine 所引起之痙孿死亡時間。毒理學研究報告亦指出：給予實驗動物餵食高劑量的硃砂或是硫化汞，會引起神經毒性的產生，包含：學習記憶的喪失、自發性活動能力的改變、前庭-眼球反射功能失調、聽力系統的損傷及抑制混合型肌肉動作電位的不完全恢復。然而其詳細的作用機制到目前為止仍然不是相當的清楚，因此對於闡明硃砂的藥效與毒性反應間相關性與作用機制的研究乃是相當急迫且重要的課題，尤其是在低劑量且長時間的使用下更是值得我們去詳加探討。藉由本研究的實驗方法(包含：自發性活動能力及滾輪運動平衡測試、聽性腦幹反應、睡眠時間的延長、腦組織酵素及脂質過氧化分析)，能夠清楚界定在低劑量(中醫臨床治療劑量)使用硃砂後，其產生藥理作用時間及使用的安全期限，並闡明長時間的使用所造成毒性反應與相關的作用機轉。期望藉由我們一系列的實驗分析，能夠解開硃砂是藥亦是毒的謎題！
此外，由於環境中的汞化物常常藉由受污染魚類海產的食物進入人體中(尤其是在受到汞污染的地區，其魚類海鮮含汞量更是驚人)，使得懷孕的婦女及生長發育階段中的小孩暴露到汞，進而造成神經系統功能的缺陷。以往的研究已經證實實驗動物在懷孕初期或胎兒時期暴露到高劑量的甲基汞，其子代出生後會造成神經功能及行為的缺陷。而在流行病學的研究調查也發現：在受到甲基汞污染的地區，該地區的小孩或是青少年有很高的比率有著神經功能及認知方面的障礙。然而這些汞污染所造成子代產生神經毒性的作用機制到目前仍有待更進一步的研究，尤其是探討在懷孕前後及發育過程中暴露到較低劑量下汞化合物(在汞污染地區可能暴露到的劑量)，對於子代所造成的神經毒性反應及相關作用機轉更是刻不容緩的課題！因此本研究探求了在出生前後暴露到汞化物(甲機汞、氯化汞及硃砂)之鼷鼠子代，其神經行為及聽力的損傷是否與鈉鉀-腺三磷酸酵素改變及氧化性壓力的增加具有相關性。這些實驗結果可以提供出生前後暴露到汞化物對子代所產生神經毒性之生化機轉，並證明鈉鉀-腺三磷酸酵素活性的改變可以作為偵測暴露到低劑量汞化物所產生毒性之生物指標。
一、鼷鼠長時間低劑量使用硃砂所產生藥理作用與神經毒性反應的作用機轉:
本研究測試鼷鼠在每天給予低劑量(中醫臨床使用劑量)硃砂，11個星期後，腦組織鈉鉀-腺三磷酸酵素與一氧化氮改變及氧化性壓力的增加。與毒性反應的產生是否具有相關性。將雄性與雌性鼷鼠分為實驗組與對照組，實驗組每天給予低劑量硃砂(10 mg/kg/day,中醫臨床使用劑量)，對照組則給予去離子水。於不同時間(3到11個星期)分析實驗組鼷鼠所產生影響，包含: 自發性活動能力與滾輪運動平衡測試、睡眠時間延長的改變及血中一氧化氮。之後將鼷鼠犧牲，分離出腦組織，分別測量鈉鉀-腺三磷酸酵素活性、脂質過氧化產物的增加、一氧化氮改變與金屬汞的含量。結果發現在給予餵食低劑量硃砂後，其所含的金屬汞還是可以經由腸胃道吸收而轉移蓄積在腦組織中。在餵食硃砂三個星期後，發現對於自發活動行為能力有抑制的情形。而對於巴比妥鹽所引起睡眠時間的延長與滾輪運動平衡測試時間減少則是在餵食後第六週出現且隨著餵食時間的增加有逐漸嚴重的情形。在血液與腦組織生化酵素分析的實驗中發現腦組織鈉鉀-腺三磷酸酵素活性受到抑制、脂質過氧化產物與一氧化氮含量則是顯著的增加。此外，綜合這一系列的分析，我們也發現雄性與雌性鼷鼠對硃砂所引起神經毒性之影響有不同的反應產生。
二、長時間低劑量使用硃砂對鼷鼠所引發耳毒性反應與作用機轉-經由增加腦幹氧化性壓力與鈉鉀-腺三磷酸酵素活性改變訊息傳遞路徑:
硃砂(主要成分乃硫化汞)，在傳統礦物性中藥的使用上已超過了二千年。現今亞洲及中東地區的許多國家，硃砂仍然使用於嬰幼兒作為鎮靜安神的藥物。誠如甲基汞所引起嚴重的耳毒性，硃砂是否具有引起聽力損傷及產生耳毒性可能的作用機轉，到目前為止仍然是不清楚，尤其是在低劑量且長時間的使用下。本實驗測試鼷鼠在每天給予低劑量(10 mg/kg/day,中醫臨床使用劑量)硃砂，10個星期後，腦幹組織鈉鉀-腺三磷酸酵素與一氧化氮改變及氧化性壓力的增加與耳毒性反應的產生是否具有相關性。將雄性與雌性鼷鼠分為實驗組與對照組，實驗組每天給予低劑量硃砂，對照組則給予去離子水。於不同時間(2到10個星期)分析實驗組鼷鼠所產生聽覺腦幹反應的改變。之後將鼷鼠犧牲，分離出腦幹組織，分別測量鈉鉀-腺三磷酸酵素活性、脂質過氧化產物的增加、一氧化氮改變與金屬汞的含量。結果發現在給予餵食低劑量硃砂後，其所含的金屬汞還是可以經由腸胃道吸收而轉移蓄積在腦幹組織中，同時並伴隨著逐漸嚴重的不正常之聽覺腦幹反應，包含:聽性腦幹反應閾值之增加、改變其絕對波潛時及波與波(I-V, III-V)之間的潛時(在餵食後4至10星期)。此外，對於餵食硃砂所產生不正常之聽覺腦幹反應有著性別的差異。而雄性鼷鼠對於硃砂所引起聽力損傷所伴隨生化酵素的改變，包含: 腦幹組織及血漿中脂質過氧化產物的增加、腦幹組織鈉鉀-腺三磷酸酵素活性的改變及降低一氧化氮的含量、腦幹組織汞金屬的含量，也都比雌性鼷鼠的變化要來的敏感。這些研究結果提供了重要的訊息，就是：長時間低劑量(中醫臨床使用劑量)硃砂使用下仍然是會造成耳毒性的產生，而腦幹組織(中樞聽力調控系統)之`活性氧物種-鈉鉀-腺三磷酸酵素-一氧化氮`訊息傳遞路徑則可能在硃砂所引起的耳毒性傷害中扮演重要的角色。此外，對於硃砂所引起神經毒性影響之性別差異則是值得未來更進一步的研究。
三、長時間低劑量暴露甲機汞對鼷鼠所引起神經毒性反應與作用機轉-經由增加氧化性壓力與調控鈉鉀-腺三磷酸酵素活性改變訊息傳遞路徑:
甲基汞是一已知作用相當強的神經毒性物質，它能夠輕易的穿過血腦障壁並累積在腦組織中，進而造成嚴重且不可逆的毒性傷害。然而有關於甲基汞產生神經毒性的作用機制到目前為止仍然不是很清楚，尤其是在尤其是在低劑量且長時間的暴露下。本實驗則是試圖探求鼷鼠在每天給予低劑量甲機汞(0.05 mg/kg/day, 在甲機汞污染地區可能暴露到劑量) 7個星期，所造成神經毒性的作用與腦組織鈉鉀-腺三磷酸酵素與一氧化氮改變及氧化性壓力的增加與神經毒性反應的產生是否具有相關性。將雄性鼷鼠分為實驗組與對照組，實驗組每天給予低劑量甲機汞，對照組則給予去離子水。於不同時間分析實驗組鼷鼠所產生影響，包含: 聽性腦幹反應的改變、自發性活動能力與滾輪運動平衡測試並分析血中一氧化氮的變化。之後將鼷鼠犧牲，分離出腦組織，分別測量鈉鉀-腺三磷酸酵素活性、脂質過氧化產物的增加、一氧化氮改變與金屬汞的含量。結果發現在給予餵食甲機汞一個星期後，滾輪運動平衡功能喪失即出現。自發性活動能力改變則是在餵食三星期後出現。聽信腦幹反應的不正常則是在四週出現，且隨著餵食時間的增加聽力閾值有更明顯的增加。這些神經毒性的損傷與腦組織中金屬汞含量顯著的蓄積和生化酵素的改變有著非常大的相關性，包含：影響腦組織鈉鉀-腺三磷酸酵素活性與一氧化氮含量的改變、腦組織及血漿中脂質過氧化產物的增加。本研究的結果提供了相當重要的證據：對於了解低劑量長時間暴露甲機汞所產神經毒性的影響提供了毒理學上與作用機轉的研究基礎。
四、長時間暴露低劑量甲機汞或氯化汞對發育中鼷鼠子代所產生神經毒性之影響:
汞是一已知具有相當毒性的重金屬。它廣泛的散佈在環境中，且容易引起嚴重的神經功能缺失或混亂，尤其是在嬰幼兒或是小孩的身上。而過去研究也證實實驗動物在懷孕初期或胎兒時期暴露到高劑量的甲基汞，其子代出生後會造成神經功能及行為的缺陷，然而其詳細的作用機制到目前仍未被清楚的了解。因此本研究之目的在於探求在出生前後暴露到低劑量甲機汞或氯化汞(在汞污染地區可能暴露到劑量)所產生之神經毒性的影響並找出可能的作用機轉。首先，給予正常鼷鼠子代於離乳後暴露低劑量甲機汞或氯化汞7個星期(CM或CH組)，對照組則給予去離子水。於不同時間分析實驗組鼷鼠子代所產生影響，包含:自發性活動能力、滾輪運動平衡測試與聽性腦幹反應的改變。之後將鼷鼠犧牲，分離出腦組織，分別測量鈉鉀-腺三磷酸酵素活性、脂質過氧化產物的增加與腦中金屬汞的含量並分析血中一氧化氮的變化。此外，藉由餵食懷孕鼷鼠母鼠低劑量甲機汞或氯化汞，使其子代於出生前後及哺乳時期間接暴露到汞化物(MV或HV組)；或是將上出生前後及哺乳時期間接暴露到甲機汞或氯化汞之鼷鼠子代於離乳後繼續暴露低劑量汞化物7個星期(MM或HH組)。此二組之鼷鼠子代於不同的時間進行上述之一系列實驗。由實驗的結果發現腦組織中脂質過氧化產物在CM或CH組有明顯增加，而在MM或HH組則是有更顯著的增加。在腦組織鈉鉀-腺三磷酸酵素活性及血液中一氧化氮含量的分析也發現在CM或CH組低劑量暴露甲機汞或氯化汞7個星期有明顯改變，而在MM或HH組則是有更嚴重的變化。在CM或CH組神經行為與聽性腦幹反應的評估也指出低劑量甲機汞或氯化汞暴露會使得聽性腦幹反應閾值之升高、改變其絕對波潛時(第III或是第V波)及波與波之間的潛時(I-III, I-V, III-V)、改變自發性活動能力及滾輪運動平衡功能障礙的產生，而MM或HH組則是有更顯著不正常的改變。此外，在MV或HV組中則是發現在出生前後及哺乳時期間接暴露到低劑量汞化物會造成這些生化酵素或是神經行為不可逆的傷害或改變。而上述實驗結果則是與腦組織中大量蓄積的汞金屬有著相關聯性。綜合以上的實驗結果，本研究證實了在出生前後及哺乳時期暴露低劑量甲機汞或氯化汞會影響腦組織中`活性氧物種-鈉鉀-腺三磷酸酵素-一氧化氮`訊息傳遞路徑並伴隨著不可逆之神經功能及聽力系統之傷害。此外，鈉鉀-腺三磷酸酵素活性改變可能可以作為嬰幼兒及孩童(子代)暴露到低劑量甲機汞或氯化汞所引起神經毒性的一個重要且有用的生物指標！
五、長時間暴露低劑量硃砂對於發育中鼷鼠子代所產生神經毒性之影響:
本研究目的在研究出生前後暴露到低劑量硃砂(10 mg/kg/day,中醫臨床使用劑量)，對於不同子代所產生之神經毒性的影響並探求可能的作用機轉。首先，給予正常鼷鼠子代於離乳後暴露低劑量硃砂7個星期(F1-C-Cin組)，對照組則給予去離子水。於不同時間分析實驗組鼷鼠子代所產生影響，包含:自發性活動能力、滾輪運動平衡測試、睡眠時間延長的改變與聽性腦幹反應的改變。之後將鼷鼠犧牲，分離出腦組織，分別測量鈉鉀-腺三磷酸酵素活性與腦中金屬汞的含量並分析血中一氧化氮的變化。此外，藉由餵食不同世代之懷孕鼷鼠母鼠(F0 和F1)低劑量硃砂，使其子代於出生前後及哺乳時期間接暴露到硃砂(F1-和F2-Cin-V組)；或是將出生前後及哺乳時期間接暴露到硃砂之鼷鼠子代於離乳後繼續暴露低劑量汞硃砂7個星期(F1-和F2-Cin-Cin組)。此二組之鼷鼠子代於不同的時間進行上述之一系列實驗。由神經行為與聽性腦幹反應評估的實驗結果發現正常子代鼷鼠暴露低劑量硃砂組(F1-C-Cin組)會使自發性活動能力改變及滾輪運動平衡功能失調的產生，並且使聽性腦幹反應之聽力閾值升高、改變其絕對波潛時(第V波)及波與波之間的潛時(I-V, III-V)，而在出生前後及哺乳時期間接暴露到硃砂之鼷鼠子代於離乳後繼續暴露低劑量汞硃砂7個星期(F1-和F2-Cin-Cin組)則是有更明顯不正常的反應。對於巴比妥鹽所引起睡眠時間延長的實驗中也發現F1-和F2-Cin-Cin組，不是F1-和F2-Cin-V組，較正常子代鼷鼠暴露低劑量硃砂組(F1-C-Cin組)有顯著的延長。在腦組織鈉鉀-腺三磷酸酵素活性及血液中一氧化氮含量的分析也發現在F1-C-Cin組低劑量暴露硃砂7個星期有明顯改變，而F1-和F2-Cin-Cin組則是有更強烈的變化。而上述實驗結果則是與腦組織中有較高的汞金屬蓄積有著相關聯性。此外，在F1-和F2-Cin-V組中則是發現在出生前後及哺乳時期間接暴露到低劑量硃砂會造成這些生化酵素或是神經行為不可逆的傷害或改變。
綜合以上的實驗結果，本研究證實了不同世代之鼷鼠子代在出生前後及哺乳時期暴露低劑量硃砂會影響腦組織中`鈉鉀-腺三磷酸酵素`活性並伴隨著不可逆之神經功能及聽力系統之傷害，同時若持續的暴露下則是會造成更嚴重的傷害。此外，也提供了: 鈉鉀-腺三磷酸酵素活性改變可能可以作為嬰幼兒及孩童(子代)暴露到低劑量硃砂所引起神經毒性的一個重要且有用的生物指標！
綜合以上的結果，我們證實低劑量(10 mg/kg/day，中醫臨床使用劑量)硃砂餵食鼷鼠後，確實具有鎮靜安神的藥理功效，使用安全期限估計約六星期，在這期間，神經毒性如運動平衡失調及耳毒性尚未明顯出現。然而在更長時間的暴露下則會造成腦組織脂質過氧化產物的增加，也使得鈉鉀-腺三磷酸酵素活性受抑制及改變一氧化氮的含量，進一步造成神經毒性及耳毒性的產生並伴隨腦組織中較高含量汞金屬的蓄積。進一步研究硃砂的作用機轉，與其所造成神經毒性及耳毒性，我門發現與神經膜鈉鉀-腺三磷酸酵素活性受抑制、改變一氧化氮的含量及氧化壓力產生有關。此外，我們也比較研究三種不同汞化物，對鼷鼠及其子代所產生神經毒性(行為功能異常與聽力損傷)的影響，所使用的劑量與汞污染地區暴露到劑量類似，所得結果發現鼷鼠胎兒對三種汞化物敏感性特高，其所引起的耳毒性及活動量變化是不可逆性的，與腦組織中氧化壓力產生、改變鈉鉀-腺三磷酸酵素活性及一氧化氮含量的有關聯。因此，本研究結果對於硃砂的藥理作用與毒性反應提供臨床應用的重要的研究理論基礎。同時也可以作為醫藥衛生單位日後在訂定硃砂臨床使用上安全劑量及使用時間的重要參考依據。另一方面，低劑量長時間暴露汞化物對鼷鼠及其子代所產生之神經毒性的作用機轉亦可解釋在流行病學上所觀察到在汞污染地區之小孩及青少年神經功能不正常之表現。而鈉鉀-腺三磷酸酵素活性在未來可以用來做為偵測低劑量汞化物所引起神經毒性的一個重要且有用的生物指標！

關鍵字

汞化物 ； 神經毒性 ； 耳毒性 ； 鈉鉀-腺三磷酸酵素活性 ； 脂質過氧 ； 一氧化氮

並列摘要

Mercury is a useful heavy metal in industrial and agricultural applications, owing to its chemical characteristic and biological properties. It is frequently used in manufacturing fluorescent lamp, mercury battery, disinfectants and insecticides et al. However, the abuse of mercury has caused the environmental pollution and resulted in human intoxication from ingesting the mercury-contaminated food via food chain. Therefore, mercury is a notorious toxic heavy metal that is widespread in the environment, and its different chemical forms (organic, inorganic and matellic) account for the various degrees of toxicities. Many studies have been reported that it was significantly induced toxic effects by mercurial compounds. However, there are only a few reports about the pharmacological and toxicological effects of cinnabar (a Chinese mineral medicine, HgS). Chinese medicine as a sedative and hypnotic for more than 2000 years and is still widely used in Asian and the Middle East countries. The estimated human therapeutic dose of cinnabar in traditional medicine used is approximately 5-25 mg/kg/day or limited to 0.1-0.5 g per dose, three times per day as indicated in Pharmacopoeia of China (2000). An overdose of cinnabar in drugs such as Ba Paul San, which is used as a sedative and for management of external infections in infants and children, has been reported to cause occasional intoxication in the Chinese population. The exposure to high-dose (1.0 g/kg/day, for 7 or 14 consecutive days) of cinnabar or HgS was caused neurotoxicity including: dysfunction of the vestibule-ocular reflex (VOR) system, learning memory deficit, hearing damage, impairment of spontaneous locomotor activity and suppression of compound muscle action potentials Although these studies demonstrated neurotixic effects of cinnabar at high dose, the pharmacological and toxicological effects, and possible toxic action mechanism of cinnabar exposure on the brain tissues have not been established, especially long-term and low-dose exposure. In this study, by means of various pharmacological effects (sedation, locomotor activities, prolongation of pentobarbital-induced sleeping time) and the toxic effects (auditory brainstem responses abnormalities, motor balancing performance dysfunction, increase of LPO levels, alteration of Na+-K+ ATPase activities and NOx levels) were concurrently monitored. The correlation of these effects with the tissue Hg contents will also be elucidated. The aim of this experimental design is to explore whether the pharmacological effects and the toxic effects of cinnabar can be differentiated in their time courses. The result obtained from this study is enable us to calculate the non-observable toxic dose of cinnabar as well as the dose ranges capable of producing effective sedation and that with the observable toxic effects. The importance of this study is to shed some light on the pharmacological and toxicological properties of the clinically used cinnabar which are subjected to fewer studies in the literatures.
The major source of mercurial compounds (MeHg, HgCl2 and cinnabar) is through biotransformation in food chain such as consumption of contaminated fish, seafood and aquatic mammals. In perinatal and weaning stages exposed to high-dose of mercurial compounds, which caused irreversible neurobehavioral and neuropsychological disorders has been found in humans and animals. Epidemiological researches indicated that the children and adolescents in Faroe Island where the population with relatively high consumption of MeHg-contaminated seafood was found to be higher risk for neurobehavioral disorders (such as motor dysfunction and problems of cognitive behavior). Following these reasons, it is important in studying neurotoxicological effects and understanding the potential mechanisms of toxic effects underlying low-dose and long-term exposure to mercurial compounds in animal models which representing a possible exposure dose in human. In this study, by means of the toxicological parameters (spontaneous locomotor activities and motor balancing performance abnormalities, auditory brainstem responses dysfunction) and biochemical changes (increase of LPO levels, alteration of Na+-K+ ATPase activities and NOx levels) were concurrently monitored. The correlation of these effects with the tissue Hg contents will also be elucidated. To our best knowledge, this is the first study to demonstrate that offsprings mice which were exposed to low-dose of mercurial compounds during perinatal and weaning stages result in influence of ROS/Na+-K+-ATPase/NO pathway with accompanying irreversible impairments of neurobehaviour and auditory system. It also provides evidence that Na+-K+-ATPase enzymic activities is an important and useful biomarker of the brain dysfunction in offsprings exposed to low-dose of mercurial compounds.
1. Neurotoxicological effects of cinnabar (a Chinese mineral medicine, HgS) in mice:
Cinnabar, a naturally occurring mercuric sulfide (HgS), has long been used in combination with traditional Chinese medicine as a sedative for more than 2000 years. Up to date, its pharmacological and toxicological effects are still unclear, especially in clinical low-dose and long-term used. In this study, we attempted to elucidate the effects of cinnabar on the time course of changes in locomotor activities, pentobarbital-induced sleeping time, motor equilibrium performance and neurobiochemical activities in mice during 3-11 weeks administration at a clinical dose of 10 mg/kg/day. The results showed that cinnabar was significantly absorbed by gastrointestinal (G-I) tract and transported to brain tissues. The spontaneous locomotor activities of male mice but not female mice were preferentially suppressed. Moreover, frequencies of jump and stereotype-1 episodes were progressively decreased after 3 weeks oral administration in male and female mice. Pentobarbital-induced sleeping time was prolonged and the retention time on a rotating rod (60 rpm) was reduced after treatment with cinnabar for 6 weeks and then progressively to a greater extent until 11 weeks experiment. In addition, the biochemical changes in blood and brain tissues were studied; the inhibition of Na+/K+-ATPase activities, increased production of lipid peroxidation (LPO) and nitric oxide (NO) were found with a greater extent in male mice than those in female mice, which were apparently correlated with their differences in the neurological responses observed. In conclusion, these findings, for the first time, provide evidence of the pharmacological and toxicological basis for understanding the sedative and neurotoxic effects of cinnabar used as a Chinese mineral medicine for more than 2000 years.
2. Ototoxicity induced by cinnabar (a naturally occurring HgS) in mice through oxidative stress and down-regulated Na+/K+-ATPase activities:
Cinnabar, a naturally occurring mercuric sulfide (HgS), has long been used in Chinese mineral medicine for more than 2000 years; currently it is still used as a sedative for infants in Asian and the Middle East countries. Since methylmercury is potently ototoxic, whether cinnabar also induces hearing impairment is awaited for delineation. In this study, we attempted to explore the toxic effects of cinnabar on the auditory brainstem response (ABR) system during 2-10 weeks administration at a clinical oral dosage of 10 mg/kg/day in mice. The results obtained showed that Hg contents of the brainstem were significantly increased accompanied with gradual progressive abnormality of ABR during 4-10 weeks administration of cinnabar. The progressive increase in hearing thresholds, prolonged absolute and interwave latencies of ABRs apparently exhibited a gender difference. The male mice were more sensitive to cinnabar in producing hearing impairment correlated with the biochemical alterations in plasma and brainstem e.g. while increase in lipid peroxidation (LPO), altered Na+/K+-ATPase activities and decrease of nitric oxide (NOx) levels. Moreover, a significant accumulation of Hg contents in the brainstem with a greater extent was found in male mice. These findings provide important information that the clinical dosage of cinnabar (10 mg/kg/day) still exhibited ototoxicity after continuous long-term exposure. A signaling pathway of ROS/Na+-K+-ATPase activities/NO of brainstem (a central auditory regulatory system) probably plays a role, at least in part, in cinnabar-induced ototoxicity. The gender difference in cinnabar-induced neurotoxicological effects merits further investigation.
3. Neurotoxicological mechanism of methylmercury induced by low-dose and long-term exposure in mice: oxidative stress and down-regulated Na+/K+-ATPase involved:
Methylmercury, a potent neurotoxicant, easily passes through the blood-brain barrier (BBB), accumulates in the brain regions and causes severe irreversible damages. However, the neurotoxic effects and action mechanisms of MeHg are still unclear, especially in low-dose and long-term exposure. In this study, we attempted to explore the toxic effects of low-dose of MeHg (0.05 mg/kg/day), which was the possible exposed dose by ingestion in MeHg-contaminated areas, on the time course of changes in locomotor activities and auditory brainstem response (ABR) system after administration for 7 consecutive weeks in mice. The results showed that the retention time on the rotating rod (60 rpm) was preferentially decreased after 1 week oral administration with MeHg. The locomotor activities parameters of ambulatory distances and stereotype-1 episodes were significantly increased and vertical-plane entries were progressively decreased after 3 consecutive weeks MeHg-exposure. The gradual progressive abnormality of ABR (increase in hearing thresholds, prolonged absolute and interwave latencies) was found during 4-6 weeks administration of MeHg. These impairments correlated with significant Hg accumulation and biochemical alterations in brain regions and/or other tissues, including the increase of lipid peroxidation (LPO) production, influence of Na+/K+-ATPase activities and nitric oxide (NO) levels were found. In conclusion, this study provides evidence that the action mechanisms (ROS/Na+,K+-ATPase/NO signaling) for understanding the neurotoxic effects of MeHg exposed to low-dose and long-term regimen. The Na+/K+-ATPase enzymic activity is considered to be a useful biomarker of MeHg-induced neurotoxicity.
4. Neurotoxicological effects of low-dose of methylmercury and mercuric chloride on developing offspring mice:
Mercury is a well-known toxic metal that widespread in the environment and easily to induce severe neurotoxicological disorders, especially in infants and children. The purpose of this study was attempted to explore the neurotoxic effects of low-dose of mercurial compounds (0.02 mg/kg/day MeHg (M) or 0.5mg/kg/day HgCl2 (H)) on developing mice as compared with normal offsprings mice (control, C). Thus, the exposure regimen was designed: (1) exposed only after weaning for 7 consecutive weeks (CM or CH groups) and (2) exposed during perinatal and weaning stages without (MV and HV groups) or with continuing exposure for 7 consecutive weeks (MM and HH groups). Assessments of exposure deterioration were performed in these six groups at the end of 7 weeks after weaning. The results obtained showed that CM and CH mice revealed neurobehavioral defects including increased locomotor activities, impairment in motor equilibrium performance and especially auditory dysfunction. These neurobehavioral abnormalities were apparently correlated with increased Hg accumulation as well as increased LPO levels and alteration of Na+/K+-ATPase activities in brain tissues and changes of NOx levels in whole blood. On the other hand, MV and HV mice still had higher Hg contents in brain tissues as compared with those in control mice (CV) and ranged to about one to tow tenth of those of the respective CM and CH mice. The most prominent defects in MV and HV mice were auditory impairment which appeared to be severer than those found in CM and CH mice such as delay of absolute wave III and interwave I-III latencies. Moreover, body weights of MV and HV mice significantly decreased. The spontaneous locomotor activities decreased in MV but increased in all of HV, CM and CH mice. The motor equilibrium performance remained normally in MV and HV mice, suggesting that this system of the fetus was either less susceptible or could reversibly repair. The extensive exposure in both prenatal and postnatal stages of MM and HH mice revealed severer in neurotoxicological effects including decreased body weight and depressed locomotor activities induced by MeHg, particular hearing impairment and altered Na+/K+-ATPase activities (increased in cerebellar cortex but decreased in both of cerebral cortex and brainstem) induced MeHg and HgCl2. These defects were attributed to their higher Hg accumulation found in brain tissues. However, dysfunction in motor equilibrium performance and increased brain LPO levels in MM and HH mice did not show further deteriorated as compared with those in MV and HV mice, suggesting possibility that prenatal exposure caused either adaptation or resistance to MeHg and HgCl2 to the additional postnatal exposure. In conclusion, all of these findings provide evidence that the fetuses were much more susceptible to MeHg and HgCl2 in inducing irreversible neurobehavioral and hearing impairment. By contrast, dysfunction of motor equilibrium performance could be attenuated by prenatal and weaning exposure to low-dose of mercurial compounds apparently presumably due to a certain degree of adaptation to free radical production. The major differences between organic MeHg and inorganic HgCl2 so far tested were those including marked decreased in body weight induced only by MeHg but not by HgCl2, and locomotor activities decreased by MeHg but increased by HgCl2.
5. Neurotoxicological effects of cinnabar (a naturally occurring HgS) on developing offspring mice:
The purpose of this study was to explore the toxic effects of low-dose of cinnabar (10 mg/kg/day) on developing mice as compared with normal offsprings (control-vehicle, F1-C-V group). Thus, the exposure regimen was designed: (1) exposed only after weaning for 7 consecutive weeks (F1-C-Cin), (2) exposure during perinatal and weaning stages without (F1-Cin-V group) or with continuing exposure for 7 consecutive weeks (F1-Cin-Cin group) and (3) F2 generation similarly defined as F1 (F2-Cin-V and F2-Cin-Cin groups). Assessments of exposure deterioration were performed in these five groups at the end of 7 weeks after weaning. The results obtained showed that F1-C-Cin mice revealed neurobehavioral defects including increased locomotor activities, impairment in motor equilibrium performance and auditory dysfunction. These neurobehavioral abnormalities were apparently correlated with increased Hg accumulation as well as increased Na+/K+-ATPase activities in brain tissues and NOx levels in whole blood. On the other hand, F1- and F2-Cin-V mice still had higher Hg contents in cerebral cortex and the most prominent defects in F1- and F2-Cin-V mice were auditory impairment which appeared to be severer than those found in control mice such as elevation of hearing thresholds, absolute wave III and interwave I-III latencies. Moreover, body weights of F1-V-Cin mice remained normally and those in F1- and F2-Cin-V mice decreased significantly, but not severer in F1- and F2-Cin-Cin mice. The spontaneous locomotor activities increased in all of F1-, F2-Cin-V and Cin-Cin mice. The motor equilibrium performance remained normally in F1- and F2-Cin-V mice, suggesting that this system of the fetus was either less susceptible or could reversibly repair to cinnabar-induced damage. The extensive exposure in both prenatal and postnatal stages of F1- and F2-Cin-Cin mice revealed severer in increased locomotor activities, dysfunction in motor equilibrium performance, prolongation of sleeping time by pentobarbital-induced, hearing impairment and further Na+/K+-ATPase activities increased in brain tissues induced by cinnabar, which were perhaps correlated with their higher Hg accumulation in brain tissues. In conclusion, this study demonstrates that offsprings mice which were exposed to low-dose of cinnabar during perinatal and weaning stages result in irreversible impairments of neurobehavioral and auditory system. By contrast, motor equilibrium performance and Na+/K+-ATPase activities of brain regions were more susceptible after prenatal, weaning exposure to low-dose of cinnabar. Furthermore, these results also provide evidence of the Na+/K+-ATPase enzymic activity may be considered to be a biomarker in offsprings exposure to low-dose of cinnabar-induced neurotoxicity.
In summary, our experimental data demonstrated that exposure to low-dosage of cinnabar in mice produced the pharmacological effects such as sedation and hypnosis after oral regime for not longer than 6 weeks. However, neuro- and oto-toxic effects of long-term exposure to low-dose of cinnabar were produced which were accompanied with significant mercury accumulation in brain regions. The action mechanisms of those effects were correlated with induced LPO production, inhibited Na+/K+-ATPase activities and altered NOx levels. Moreover, we also studied the neurotoxicological effects (ototoxicity and neurobehavioral dysfunction) of mice and offsprings induced by long-term and low-dose of three mercurial compounds (the dose used similar to exposed dose in mercury-contaminated areas). The results indicated that offsprings which exposed to mercurial compounds in perinatal and weaning stages were more sensitive and caused irreversible damages including ototoxicity and locomotor dysfunction. Those effects were also correlated with enhanced oxidative stress, changed Na+/K+ ATPase activities and altered NOx levels in brain tissues. In conclusion, these findings, for the first time, provide evidence of the pharmacological and toxicological basis for understanding the sedative and neurotoxic effects of cinnabar used as a Chinese mineral medicine for more than 2000 years. The importance of this study is to shed some light on the pharmacological and toxicologucal properties of the clinically used cinnabar which are subjected to fewer studies in the past decade. On the other hand, the action mechanisms (ROS/Na+,K+-ATPase/NO signaling) for understanding the neurotoxic effects of mercurial compounds at low-dose and long-term regimen simulated to those observed in children and adolescents where gestational and development mercury exposure was correlated with neurobehavioral and nerve system dysfunction. The Na+/K+-ATPase enzymic activity is considered to be an important and useful biomarker of neuronal dysfunctions induced by mercurial compounds.

並列關鍵字

Mercurial compounds ； Neurotoxicity ； Ototoxicity ； Na+/K+-ATPase activities ； lipid peroxidation ； nitric oxide

參考文獻

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