探討白僵菌素之心臟毒性及DNA複製壓力所扮演的角色

Abstract

白僵菌素 (Beauvericin, BEA) 與恩鏈黴素B (Enniatin B, ENNB) 為鐮孢菌屬 (Fusarium) 真菌所產生的新興黴菌毒素，常污染飼料與穀物等食品原料，其對人類與動物健康的潛在威脅日益受到重視。由於白僵菌素與恩鏈黴素B 皆具陽離子載體 (Ionophore) 特性，本研究假設心臟可能為其主要毒性靶器官，並以心臟毒性作為探討重點。在體內模式中，1–4 μM 白僵菌素可明顯促進野生型斑馬魚胚胎之心跳速率與血液輸出量，反映白僵菌素對心臟收縮功能及血液動力學具有急性促進效應。白僵菌素亦造成心臟螢光 Tg(BMP4:EGFP)as1 斑馬魚胚胎靜脈竇至動脈球的直線距離顯著增加，呈現白僵菌素會造成心臟折疊結構異常；但恩鏈黴素B 處理斑馬魚胚胎則未觀察到心率與心臟折疊結構的顯著變化。在體外模型方面，使用大鼠 H9c2 心肌母細胞株探討白僵菌素的心臟毒性機制。使用 MTT 分析細胞存活率，呈現出 H9c2 細胞株暴露於 1–4 μM 白僵菌素達 72 小時後，其存活率與相同濃度之 24 小時相比並無顯著差異；此外，經流式細胞儀分析顯示，白僵菌素使細胞週期顯著停滯於 G1 期。轉錄體學分析與 RT-qPCR 驗證結果指出，白僵菌素處理後在 DNA 複製相關基因 (Cdc6、Mcm2–7) 表達量顯著下降。進一步以 PCNA 蛋白質表達量及 EdU/BrdU 核苷相似物分析 DNA 複製能力，發現白僵菌素長時間處理可顯著抑制 H9c2 細胞株的 DNA 複製活性。在細胞增生評估中，分析 48 小時即時影像觀察、pHH3 (Ser10) 蛋白質表達與有絲分裂指數等指標，結果顯示 2–4 μM 白僵菌素可顯著抑制 H9c2 細胞株之細胞增生；同時，在 DNA 損傷指標方面，白僵菌素處理後微核數目與 DNA 斷裂現象顯著上升。在分子層次上，以 4 μM 白僵菌素短時間 (1 小時) 處理 H9c2 細胞株顯著抑制 DNA 複製相關蛋白質表達量 (CDC6, MCM2 與 PCNA)；此外，在 DNA 修復與損傷蛋白質表達方面，白僵菌素處理後並未顯著改變 pRPA (Ser33) 蛋白質表達量，但卻造成 pCHK1 (Ser345) 與 γH2AX (Ser139) 的表達量顯著提升，顯示白僵菌素的短時間處理可能會抑制 DNA 複製並造成 DNA 損傷與修復機轉活化；將細胞週期同步於 G1 後期，暴露於 4 μM 白僵菌素 3 小時，再將毒素移除 1 小時與 3 小時後，結果發現 PCNA 蛋白質與 γH2AX (Ser139) 蛋白質表達量呈高度正相關；再利用 BrdU 標定的 DNA 複製區域與 γH2AX (Ser139) DNA 損傷指標在細胞核內高度重疊，顯示白僵菌素會誘導 DNA 複製與損傷事件相伴而生之現象。綜合以上結果，本研究證實白僵菌素處理斑馬魚胚胎令其產生心臟毒性，並可誘發大鼠 H9c2 心肌母細胞株中 DNA 複製壓力及相關 DNA 損傷現象，為白僵菌素毒性機制提供初步分子層級證據。

Beauvericin (BEA) and Enniatin B (ENNB) are emerging mycotoxins produced by Fusarium species, commonly contaminating feed and grain-based food products; thus, their potential threat to human and animal health has received increasing attention. Based on the ionophore property of BEA and ENNB, this study hypothesized that the heart may serve as a primary target organ for their toxicity. In the in vivo model, exposure to 1–4 μM BEA significantly increased heart rate and cardiac output in wild-type zebrafish embryos, indicating an acute stimulatory effect on cardiac contractility and hemodynamics. BEA treatment also caused a marked increase in the linear distance between the sinus venosus and the bulbus arteriosus in Tg(BMP4:EGFP)as1 cardiac fluorescent zebrafish embryos, reflecting impaired cardiac looping. In contrast, ENNB exposure did not induce significant changes in heart rate or cardiac folding structure. In the in vitro model, the rat cardiomyoblast cell line H9c2 was used to investigate the cardiotoxic mechanisms of BEA. According to the MTT assay, exposure of H9c2 cells to 1–4 μM BEA for 72 hours did not result in a significant difference in cell viability compared with the same concentrations at 24 hours. Flow cytometry analysis indicated that BEA induced a significant G1 phase arrest. Transcriptomic analysis and RT-qPCR validation further showed a significant downregulation of DNA replication–related genes, including Cdc6 and Mcm2–7. Consistently, PCNA protein expression and EdU/BrdU incorporation assays demonstrated that BEA markedly suppressed DNA replication activity in H9c2 cells. Cell proliferation analysis—including 48-hour time-lapse imaging, pHH3 (Ser10) protein detection, and mitotic index quantification—revealed that 2–4 μM BEA significantly inhibited cell proliferation. Meanwhile, micronucleus formation and comet assay results indicated increased DNA damage upon BEA exposure. At the molecular level, short-term treatment (1 hour) of H9c2 cells with 4 μM BEA significantly suppressed the expression of DNA replication-related proteins (CDC6, MCM2, and PCNA). In contrast, the expression levels of pCHK1 (Ser345) and γH2AX (Ser139) were markedly increased, whereas pRPA (Ser33) remained unchanged, suggesting that BEA rapidly induces DNA damage and activates DNA damage response pathways. Furthermore, when cells were synchronized in late G1 and exposed to 4 μM BEA for 3 hours, followed by toxin removal for 1 and 3 hours, PCNA expression showed a strong positive correlation with γH2AX (Ser139) levels. Fluorescent co-localization analysis further confirmed that BrdU-labeled replication signals and γH2AX (Ser139) DNA damage foci extensively overlapped in nuclei, indicating that DNA replication and damage events are coupled. Taken together, the present study demonstrates that BEA treatment induces cardiotoxicity in zebrafish embryos and triggers replication stress–associated DNA damage in rat H9c2 cardiomyoblasts. These findings provide preliminary molecular-level evidence for the underlying mechanisms of BEA toxicity.