以斑馬魚為模式調查環境酸化與鉑類化療藥物對毛細胞與離子細胞的影響

Abstract

淡水生態系統的酸化已被認為是全球性的環境問題，並導致魚類行為的變化。然而，酸性環境是否造成淡水魚側線系統的功能改變仍然是未知的。此外，許多研究揭露藥物汙染水環境的事實，其中包括抗癌化療藥物。然而這些藥物造成的環境作用、環境影響的程度與廣度鮮為人知。本研究目的在於檢視斑馬魚胚胎暴露於酸性或鹼性淡水時，是否改變了神經丘毛細胞的表達和功能。並藉由決定鉑類化療藥物(順鉑)對於表皮離子細胞與毛細胞產生影響之最小濃度，了解順鉑之亞致死效應，以應用於鉑類藥物造成水環境影響之早期風險評估。將斑馬魚胚胎暴露於不同的酸鹼pH值環境中，分析長期暴露(受精後0~96小時)與短期暴露(受精後48~96小時)對胚胎形態與側線毛細胞功能之影響。另外，將斑馬魚胚胎受精後長期暴露於不同濃度的順鉑後，分析胚胎形態、存活率、體長、及魚體的離子(鈉、氯、鈣)與鉑含量。功能分析方面，利用掃描式離子選擇電極，分析側線毛細胞功能(機械傳導通道鈣離子流)與皮膚離子細胞排酸功能。結果顯示，長期暴露於pH5條件下，側線毛細胞數量與功能皆下降。短期暴露於pH5條件下，僅毛細胞功能下降，然而毛細胞數目不變。進一步利用morpholino oligonucleotides進行基因減弱，降低H+-ATPase與 gcm2 表現，使得調節酸鹼平衡機制受損後，發現胚胎毛細胞數量與功能皆下降。此外，胚胎長期暴露於濃度由低至高的順鉑中，毛細胞功能最先受損(濃度1 µM)、隨後是毛細胞數與魚體氯離子含量下降(濃度10 µM)、離子細胞排酸能力下降及魚體鈉與鈣離子含量下降(濃度50 µM)、體長與離子細胞密度下降(100 µM)、最後是存活率下降(濃度500 µM)。研究結果顯示酸性環境會導致神經丘毛細胞功能受損。對於順鉑引起之毒性，毛細胞明顯比離子細胞更敏感。本研究利用掃描式離子選擇電極偵測毛細胞與離子細胞功能之改變，決定順鉑最低影響濃度，此法有高達500倍相較於偵測存活率變化之敏感度，未來可應用於偵測鉑類藥物對於水環境造成之早期風險評估。Acidification of freshwater ecosystems has been recognized as a global environmental problem and causes changes in fish behavior. However, whether environmental acidification causes functional alterations in the freshwater fish lateral line system is still unknown. Moreover, many studies demonstrated pharmaceutical contaminants in aquatic systems, including antineoplastic drugs. Yet, the environmental behaviors, effects, and fates of these drugs are little known. The aims of this study were to investigate if exposure to acidic or basic freshwater altered the expression and function of neuromast hair cells in zebrafish embryos, and to assess the early aquatic risk of a platinum compound (cisplatin) by revealing the sublethal effects of cisplatin on skin ionocytes and hair cells. Zebrafish embryos were incubated in different pH [at 0~96 h post-fertilization (hpf), defined as long exposure; and 48~96 hpf, short exposure], and different concentrations of cisplatin (at 0~96 hpf). The survival rate, body length, and whole-body ion (Na+, Cl-, and Ca2+) and platinum contents were determined. Using a scanning ion-selective electrode technique, the function of hair cells [mechanotransducer (MET)-channel-mediated Ca2+ influx at the stereocilia of hair cells] and ionocyte ([H+] gradients) was measured in intact zebrafish larvae. The result shows that the cell number and function of neuromast hair cells are reduced after 0-96 hpf pH5 exposure. Only MET channel-mediated Ca2+ influx is decreased in the larvae which exposed to 48-96 hpf pH5. Gene knockdown by using morpholino oligonucleotides (MO) decreased H+-ATPase and gcm2 expression and impaired systemic acid-base balance in zebrafish larvae. The cell number and function of neuromast hair cells are significantly decreased in both MO knockdown larvae. The effects of cisplatin on zebrafish embryos were demonstrated as first impairing hair cell function (at 1 µM of cisplatin), the hair cell number, and body ion content of Cl- (at 10 µM of cisplatin), then decreasing ionocyte acid secretion and overall body ion contents of Na+ and Ca2+ (at 50 µM of cisplatin). The body length and ionocyte density decreased at 100 µM of cisplatin, and survival decreased at 500 µM of cisplatin. In conclusion, neuromast hair cell is sensitive to the acidic environment. Hair cells are significantly more susceptible to cisplatin toxicity than ionocytes. This study using zebrafish hair cells/ionocytes to detect cisplatin toxicity, demonstrating a 500-fold greater sensitivity than by detecting changes in survival, for early aquatic risk assessments of platinum-based chemotherapeutic contaminants.