赭麴毒素 A 與黃麴毒素 B1 對斑馬魚胚胎發育的影響

Abstract

赭麴毒素 A (Ochratoxin A, OTA) 為一種廣泛存在於受到真菌汙染食品和穀物當中的黴菌毒素，已知 OTA 具有腎臟毒性、致畸胎毒性、肝毒性、基因毒性以及致癌性等，目前對於 OTA 導致胚胎發育以及肝臟損傷的機制尚不明確，因此本研究將利用脊椎動物斑馬魚模式探討 OTA 對於胚胎發育和肝臟損傷的分子機轉，並且利用已知具有肝毒性的黃麴毒素 B1 (Aflatoxin B1, AFB1) 作為比較。 以 0.5 µM OTA 處理斑馬魚胚胎達 72 hpf (hours post fertilization) 後，其卵黃呈現深色以及心包膜腫大的現象，此外在胚胎頭部有出血的情形，且出血機率與 OTA 劑量成正比。將 6 hpf 斑馬魚胚胎以 0.5 µM OTA 處理至 48 hpf 後進行微陣列技術分析，發現多個凝血因子及由肝臟合成的蛋白質基因表現皆明顯下降。以半定量及定量聚合酶鏈鎖反應進一步確認顯示，凝血因子 VII ( F7)、VIIi (F7i)、IXb (F9b) 和纖維蛋白原 (FGB) 的 mRNA 表現量皆隨著 OTA 劑量的增加而顯著減少，並且呈現劑量效應關係，此外由肝臟專一合成的銅藍蛋白 (ceruloplasmin, CP) 及玻連蛋白 (vitronectin α, vtna) 的 mRNA 表現量也顯著減少。以 OTA 處理 6 hpf 肝臟螢光魚 Tg (L-FABP：EGFP) 達 96 hpf 時，肝臟螢光強度隨著毒素劑量增加而顯著下降，並且導致肝臟體積明顯縮小；若是以 OTA 處理 5 dpf (day post-fertilization) 的幼魚至8 dpf，則發現肝臟螢光強度並沒有統計差異，顯示 OTA 主要在斑馬魚的發育階段導致胚胎肝臟損傷。從組織切片的層面來看，1 µM OTA 處理 6 hpf 野生型胚胎達 96 hpf 時不會造成肝臟組織的異常，但是同時以 0.5 µM OTA 處理轉殖螢光魚胚胎則會造成肝臟面積減少；在頭部出血的組織切片則可以觀察到出血區域有異常的細胞堆積現象，頭部頂蓋出現大面積空洞。 在 0.25-0.5 µM AFB1 處理的組別中，AFB1 會導致轉殖螢光魚胚胎 (6-96 hpf) 和幼魚 (5-8 dpf) 的肝臟螢光量顯著降低，肝臟體積明顯縮小，結果顯示 AFB1 會同時影響胚胎肝臟發育或是發育完成的階段。但是 AFB1 並不會顯著影響 72 hpf 野生型胚胎中凝血因子及肝臟專一合成蛋白質的 mRNA 表現量，且在胚胎肝臟及頭部組織切片皆沒有顯著的組織損傷。以 OTA 或是 AFB1 處理斑馬魚肝臟細胞株 (ZFL)，皆會顯著降低 F7、FGB、CP 及 VTNA 的 mRNA 表現量，且呈現劑量效應關係，末端脫氧核苷酸轉移酶脫氧尿苷三磷酸切口末端標記螢光染色 (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL) 的結果則顯示 OTA 和 AFB1 皆會令 ZFL 進行細胞凋亡，然而抗氧化劑 NAC 和 quercetin (槲皮素) 的使用並無法回復 OTA 所造成的細胞死亡或是胚胎肝臟損傷。由上述實驗結果推論，OTA 可能是藉由導致斑馬魚胚胎肝臟損傷，進而造成凝血基因的表現異常，而此種損傷的現象可能由非氧化壓力所導致的細胞凋亡所造成。

Ochratoxin A (OTA) is a mycotoxin, which widely exists in food and grain because of fungal contamination. OTA shows nephrotoxicity, teratogenicity, hepatotoxicity, genotoxicity and carcinogenicity in various animal and cellular models. However, the molecular mechanisms of OTA causing developmental toxicities and hepatotoxicity to embryos remain unclear. Therefore, we used the zebrafish embryos as a vertebrate model and aflatoxin B1 (AFB1) with hepatotoxicity serves as a comparison in the present study. When the embryos at 6 hpf (hour post-fertilization) were exposed to 0.5 µM OTA, phenomena of darken yolk, cardiac edema and brain hemorrhage were found at 72 hpf；the percentage of brain hemorrhage was increased in a dose-dependent manner. The microarray analysis showed that 0.5 M OTA treatment of embryos led to the down-regulation of genes involved in the synthesis of coagulation factors and liver-specific protein. Semi-quantitative and quantitative polymerase chain reaction confirmed that the mRNA expression of VII (F7), VIIi (F7i), IXb (F9b), fibrinogen (FGB) , ceruloplasmin (CP) and vitronectin α (vtna) significantly decreased OTA treatment of Tg (L-FABP：EGFP), a transgenic line with fluorescent liver, resulted in, the decrease of fluorescence intensity and liver volume. However, OTA didn’t affect the liver fluorescence intensity of 5-8 dpf (day post-fertilization) larvae, suggesting that OTA induced liver damage mainly at the early developmental stage. From the viewpoint of histological examination, OTA had no effect on liver tissue of wild-type embryos, but significantly decreased the hepatic area of Tg (L-FABP：EGFP) embryos. Additionally, embryos exposed to 0.5 μM OTA demonstrated that abnormal accumulation of cells and cavities could be observed in the hemorrhage area of head tissue section. About AFB1 at the concentrations of 0.25 and 0.5 µM significantly decreased the fluorescence intensity and volume of liver in 6-96 hpf and 5-8 dpf Tg (L-FABP：EGFP) embryos. However, AFB1 didn’t affect the gene expression of coagulation factors and liver-specific- synthesis protein in 6-72 hpf wild-type embryos. Besides, when the zebrafish liver cell line (ZFL) was used as a model, both OTA and AFB1 could reduce the expression of F7, FGB, CP and VTNA dose dependently. The results of terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) showed both OTA and AFB1 could induce the apoptosis in ZFL cells. Liver injury of embryos and ZFL cell death mediated by OTA could not be restored by the presence of antioxidants N-acetylcysteine (NAC) and Quercetin. In conclusion, OTA might cause liver injury and thus decreased the expression of coagulating factorsgenes；however, liver impairment by OTA was not mediated by oxidative stress.