鉈誘發斑馬魚神經與心臟毒性：適應性未摺疊蛋白反應與DNA修復機制的保護作用

Abstract

鉈（Thallium, Tl）廣泛應用於半導體、成像系統、光纖玻璃與高溫超導材料等領域，近年來於全球多處地表水中被檢測，濃度最高可達1520 μg/L。鉈在水體中以穩定的一價形式（Tl⁺）存在，並展現超過10,000的生物濃縮係數。魚類與蔬菜中均可檢測出高濃度鉈殘留，顯示其於食物鏈中高度累積。作為食物鏈頂層的消費者，人類面臨鉈暴露的高度風險。即使在低劑量下，鉈亦與早產與發育異常相關，對兒童及孕婦等脆弱族群尤具威脅。儘管鉈的毒理機制尚未完全釐清，已有研究顯示其可誘發內質網壓力（ER stress），上調未摺疊蛋白反應（UPR）相關標誌如ATF6、IRE1，並促進XBP1剪接進入細胞核，導致細胞毒性。鑒於適應性UPR可能參與調節鉈所致毒性，本研究以斑馬魚為水生脊椎動物模式，探討鉈對胚胎發育之毒性機轉，以及選擇性XBP1活化劑IXA4之保護潛力，並與化學伴護劑TUDCA進行比較。 本研究建立鉈（Tl）誘導之斑馬魚毒性模型，結果顯示，隨鉈濃度上升，仔魚於120 hpf時的死亡率顯著增加，孵化率則明顯下降。形態學分析指出，在100–400 μg/L下魚鰾面積明顯縮小，且於200–400 μg/L濃度間觀察到心包腫脹與腹部異常，導致整體形態評分隨濃度提升而下降。qPCR分析顯示，鉈暴露顯著上調UPR標誌基因表現，反映出明確的內質網壓力反應。進一步以20 μM IXA4或TUDCA處理後，斑馬魚胚胎於6–144 hpf 期間之死亡率與對照組無顯著差異，顯示兩者本身無發育毒性。但在鉈處理下，單獨鉈暴露組的120 hpf存活率僅為53%，聯合IXA4或TUDCA處理則可顯著提升至79%與73%，並有效改善孵化率。組織學分析進一步發現，鉈會導致心包腫脹與腦部發育異常，而IXA4可顯著改善心臟與神經組織結構，展現器官層級的保護作用。轉錄體與生物資訊分析顯示，鉈暴露顯著抑制多條與心臟與神經發育相關之基因表現路徑，IXA4可部分恢復其轉錄活性，並上調DNA修復路徑，特別是核苷酸切除修復（NER）相關基因，其變化亦經qPCR驗證支持。 本研究顯示，選擇性sXBP1活化劑IXA4可透過促進DNA修復機制、調控心臟與神經發育相關基因表現，以及降低內質網壓力，顯著緩解鉈所誘導之發育毒性與器官損傷，為揭示鉈毒性機轉與未來治療策略的發展提供關鍵見解。

Thallium (Tl) is widely used in semiconductors, imaging systems, fiber optic glass, and high-temperature superconducting materials. In recent years, it has been detected in surface water across many regions of the world, with concentrations reaching up to 1520 μg/L. In aquatic environments, thallium exists in a stable monovalent form (Tl⁺) and exhibits a bioconcentration factor exceeding 10,000. High levels of thallium residues have been detected in both fish and vegetables, indicating its high accumulation within the food chain. As apex consumers, humans face a high risk of thallium exposure. Even at low doses, thallium has been associated with preterm birth and developmental abnormalities, posing a particular threat to vulnerable populations such as children and pregnant women. Although the toxicological mechanisms of thallium are not yet fully understood, studies have shown that it can induce endoplasmic reticulum (ER) stress, upregulate unfolded protein response (UPR) markers such as ATF6 and IRE1, and promote XBP1 splicing and nuclear translocation, leading to cytotoxicity. Given that adaptive UPR may be involved in regulating thallium-induced toxicity, this study used zebrafish as an aquatic vertebrate model to investigate the developmental toxicity mechanisms of thallium and the protective potential of the selective XBP1 activator IXA4, in comparison with the chemical chaperone TUDCA. In this study, a zebrafish toxicity model induced by thallium (Tl) was established. Results showed that with increasing thallium concentration, the mortality rate of larvae at 120 hpf significantly increased, while the hatching rate markedly decreased. Morphological analysis revealed that at concentrations of 100–400 μg/L, the swim bladder area was significantly reduced, and pericardial edema and abdominal abnormalities were observed at 200–400 μg/L, resulting in a concentration-dependent decline in overall morphological scores. qPCR analysis showed that thallium exposure significantly upregulated UPR marker gene expression, indicating a clear ER stress response. Further treatment with 20 μM IXA4 or TUDCA during the 6–144 hpf period showed no significant differences in mortality compared to the control group, indicating no developmental toxicity from either compound alone. However, under thallium exposure, the 120 hpf survival rate in the thallium-only group was only 53%, while co-treatment with IXA4 or TUDCA significantly increased survival to 79% and 73%, respectively, and effectively improved the hatching rate. Histological analysis further revealed that thallium caused pericardial edema and abnormal brain development, while IXA4 significantly improved the structure of cardiac and neural tissues, demonstrating organ-level protective effects. Transcriptomic and bioinformatic analyses showed that thallium exposure significantly suppressed multiple gene expression pathways related to cardiac and neural development. IXA4 partially restored transcriptional activity and upregulated DNA repair pathways, particularly nucleotide excision repair (NER)-related genes, which was also supported by qPCR validation. This study demonstrates that the selective sXBP1 activator IXA4 can significantly alleviate thallium-induced developmental toxicity and organ damage by enhancing DNA repair mechanisms, regulating gene expression related to cardiac and neural development, and reducing ER stress. These findings provide critical insights into the mechanisms of thallium toxicity and the development of future therapeutic strategies.