樟芝對懷孕母鼠與胎鼠的致基因毒性與抗基因毒性研究

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Title:	樟芝對懷孕母鼠與胎鼠的致基因毒性與抗基因毒性研究 The Genotoxic and Anti-genotoxic Effects of Antrodia cinamomea Chang & Chow on Pregnant and Fetal Mice
Authors:	江素瑛;謝慶良;高尚德
Contributors:	中國醫藥大學中國藥學研究所
Keywords:	樟芝;牛樟菇;基因毒性;DNA鏈結物;基因突變率;微核;Antrodia cinnamomea Chang-genotoxic;micronuclei;mutant frequency
Date:	2004-07
Issue Date:	2010-06-30 14:44:43 (UTC+8)
Abstract:	樟芝為台灣所特有之一種食、藥兩用高等真菌，本計畫期望利用科學實驗方法來確定樟芝之安全性，並評估其是否有減少基因傷害的功效，而可作為增進民眾健康的健康食品。本計劃將研究妊娠期間若服用樟芝是否會誘發懷孕母鼠、胎鼠與新生鼠產生基因毒性，此基因毒性有潛在的危險性最後演變成母鼠腫瘤、子代畸形、遺傳疾病、嬰兒或兒童期腫瘤。計畫以樟芝每天餵食懷孕初期（孕期第六天）的老鼠，並於生產前（孕期第十六天）停止餵食。兩天後採尾血計數懷孕母鼠與新生鼠周邊血液嗜多染紅血球的微核 (micronuclei)生成率，另一批老鼠待生產完一個月後犧牲，以T-淋巴細胞cloning方法計算懷孕母鼠與新生鼠脾臟淋巴細胞hprt基因的突變機率，並觀察這兩個基因毒性指標是否有異常增高的現象，藉以評估口服樟芝誘發懷孕母鼠與新生鼠產生基因突變之可能性及其程度。此研究結果有助於進一步的評估樟芝安全性，並維護懷孕婦女與其後代的健康。基於化學預防DNA損傷與基因突變在減緩癌症發生的重要性，本計劃將利用本實驗室已建立的輻射老鼠體內基因突變測試模式，進一步的評估樟芝對輻射所誘發懷孕母鼠的基因傷害之拮抗作用。將以游離輻射誘發老鼠產生的遺傳損傷(微核、hprt基因突變、氧化DNA傷害、DNA鏈斷裂)為指標，投與樟芝進行體內測試，以T-淋巴細胞cloning方法計算老鼠脾臟淋巴細胞hprt基因的突變機率，使用高壓液相層析儀加電化學偵測器(HPLC/ECD)方法分析老鼠組織內氧化DNA傷害(8-hydroxy-2’-deoxyguanosine)的濃度，採用彗星試驗法測量DNA鏈斷裂情形，以微核試驗法計算老鼠周邊血液未成熟紅血球染色體變異程度，以評估樟芝抑制游離輻射誘發之DNA傷害與突變之效用。另將探討樟芝抗輻射作用的可能作用機轉，是否與去除輻射所誘發的氧自由基、脂質過氧化有相關。此結果將進一步的了解樟芝之抗輻射遺傳傷害效果，可能提供急性輻射傷害治療一個新的方向。 Antrodia cinnamomea Chang & Chou is a new, rare and expensive fungus first identified in 1994 in Taiwan. Our grant proposal plans to examine the safety and anti-genotoxic effects of Antrodia cinnamomea. We will determine the potential genotoxicity of Antrodia cinnamomea in female mice and their offspring using an established hprt gene mutation assay and micronuclei assay. After gavage ingestion of aqueous extracts of Antrodia cinnamomea from day 6 to day 18 of pregnancy, micornuclei in mouse polychromatic erythrocytes will be scored under fluorescent microscopy. The dams and pups will be sacrificed one month after delivery. The splenic lymphocytes will be isolated and cultured in the presence of the selective agents 6-thioguanine. The mutant colonies will be counted to determine hprt mutation frequency. These data will shed light on the genotoxicity potential of Antrodia cinnamomea and help us evaluate whether Antrodia cinnamomea is safe health food. Furthermore, the anti-genotoxic effects of Antrodia cinnamomea in mice will be examined by using the radiation-induced DNA damage and mutation animal model. The oxidative DNA damage, DNA strand break, micronuclei and hprt mutation frequency will be used as biomarkers to assess the protective effects of Antrodia cinnamomea administrated before or after irradiation. The oxidative DNA damage (8-hydroxy-2？H？H-deoxyguanosine) in the tissues will be analyzed using HPLC/ECD. The amounts of DNA strand breaks will be quantitated with alkaline COMET assay. Micornuclei in mouse polychromatic erythrocytes will be scored under fluorescent microscopy. The mononuclear cells from the mouse spleens will be isolated and the hprt mutation frequency in the T-lymphocytes will be examined in the presence of the selective agent 6-thioguanine. To further understand the underlying mechanism of the radio-protective effects of Antrodia cinnamomea, the level of oxygen free radicals in peripheral blood and the amounts of lipid peroxidation in mouse tissues will be analyzed by chemiluminescence detector and TBAR method, respectively. The data from this study will help us to elucidate whether the radioprotective effects of Antrodia cinnamomea is related to its protective effects against radiation-induced DNA damage, mutations, and lipid peroxidation.
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