環境荷爾蒙殺菌劑免克寧影響端足蟲 (Hyalella azteca) 基因表現差異之研究

Abstract

本研究探討內分泌干擾物質殺菌劑免克寧對於端足蟲 (Hyalella azteca) 基因表現之影響，以建立具篩選內分泌干擾物質物質潛力之生物標誌 (Biomarker)。免克寧 (Vinclozolin)是dicarboximide類型的殺菌劑，具有抗雄性激素之作用，為內分泌干擾物質。免克寧及其代謝物在生物體內會競爭雄性激素受體，造成雄性相關基因表現量下降。本研究中分別以抑制性扣減雜交 (Suppression subtractive hybridization, SSH) 及次世代定序 (Next generation sequencing, NGS) 篩選端足蟲的差異表現基因後，以核酸點漬法 (DNA dot blotting) 及定量即時聚合酶鏈鎖反應 (Quantitative real-time PCR) 進行確認，建立扣減cDNA基因庫後，將其序列進行比對及功能的確認，並與NGS之結果相互比較，以建立具篩選內分泌干擾物質物質潛力之生物標誌 (Biomarker)。 端足蟲72小時急毒性部分，依普同之半致死濃度 (LC50) 為 1.80 mg L-1、撲滅寧之半致死濃度為7.49 mg L-1。利用抑制性扣減雜交共篩選出481個差異表現基因，以核酸點漬法共篩選出73個顯著差異表現之基因，利用次世代定序共篩選出880個顯著差異表現之基因，並顯示免克寧會影響端足蟲荷爾蒙、呼吸及防禦相關基因之表達。將二部分結果對照後，選出9個基因進行後續靈敏度等測試，包含卵黃前質素1 (VTG1)、卵黃前質素2 (VTG2)、類卵黃前質素 (VTG-like)、血藍素、細胞色素c氧化酶及四個假說蛋白。由抑制性扣減雜交實驗結果顯示，端足蟲暴露於未致死濃度免克寧下，會對防禦及呼吸相關基因表達造成影響。其中類卵黃前質素在低濃度免克寧 (0.5-2 mg L-1) 處理下，有較佳之劑量效應，與卵黃前質素1及卵黃前質素2相比類卵黃前質素較具發展為生物標誌之潛力，在依普同及撲滅寧則所有處理表達量均受到抑制。假說蛋白 (L_8077) 暴露於依普同及撲滅寧時，有較佳之劑量效應，具發展為環境污染物監測之生物標誌潛力。端足蟲暴露於免克寧的基因表達趨勢與依普同及撲滅寧不同的原因，可能是免克寧具有表觀遺傳效應。三種藥劑於未致死濃度下，均會影響非目標生物端足蟲體內荷爾蒙相關基因表達，因此未來需更審慎評估此類藥劑對非目標生物之影響。 在抗氧化酵素活性測定上，則顯示免克寧會顯著增加GST及CAT之酵素活性，及免克寧對端足蟲 (H. azteca) 確實會造成氧化逆境。 本論文之研究結果，提供新興水體環境指標生物端足蟲 (H. azteca) 在基因層級的重要生物資訊。

In this study, we examined the expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin and isolated potential biomarkers, which can identify antiandrogen environmental hormones. Vinclozolin, a dicarboximide fungicide, is also an endocrine disrupter compound that competes with androgen. Vinclozolin and its metabolites compete with androgen receptor, which attribute to the decline of male gene related expression. After we identified the expression profile of a comprehensive set of genes in amphipod by suppression subtractive hybridization (SSH) and next generation sequencing (NGS), we used DNA dot blotting and quantitative real-time PCR (qPCR) to confirm the results. Building up the database of subtractive cDNA, we matched the sequences and functions, and compared it with the results of NGS, in order to isolate the potential biomarker which can identify environmental hormones. In the part of acute toxicity, iprodione lethal concentration 50% (LC50) was 1.80 mg L-1 and procymidone was 7.49 mg L-1. In the result of SSH we detected 481 differentially expressed genes, and DNA dot blotting revealed 55 genes with significant differential expression. In the result of NGS we detected 880 genes with significant differential expression (p value <0.05). Compared the results of SSH and NGS, we selected 9 genes to execute quantitative real-time PCR (qPCR), which include vitellogenin1, vitellogenin1, vitellogenin-like, hemocyanin, cytochrome c oxidase, and four hypothetical protein. The result showed that vitellogenin-like genes have better dose-response under low concentration vinclozolin treatment (0.5-2 mg L-1). Compared with to vitellogenin 1 and vitellogenin 2, vitellogenin-like has more potential to become a biomarker. One of hypothetical protein (ID : L_8077) was also have dose-response when H. azteca exposure in iprodione and procymidone with exposure concertation increased, and the expression of hypothetical protein were downregulated. Even though the function of this gene is unclear, it still has the potential to be biomarker due to the ability of dose-response. In regard to the results, we can find out even if under non-lethal concentration of vinclozolin, iprodione and procymidone, it still affects amphipod on gene level. The result of antioxidant enzyme activity showed vinclozolin increased activity of GST and CAT and also made an oxidative stress to H. azteca. This study offers data for further study of xenobiotics effects in amphipods.