探討長期共同暴露玉米赤黴烯酮與脫氧雪腐鐮刀菌 烯醇對秀麗隱桿線蟲生長與生殖毒性之交互影響

Abstract

真菌毒素為真菌產生之次級代謝物，常污染農作物及其製品，對人類與動物健康構成急性與慢性風險，已成為重要的食品安全與公共衛生議題。真菌毒素在自然環境與食品中多以混合形式存在，共同暴露可能導致毒性交互作用 (包括加成、協同及拮抗作用)，進而增加毒理風險，然而相關毒性評估與機制研究仍相對不足。 鐮刀菌屬 (Fusarium spp.) 真菌所產生之玉米赤黴烯酮 (Zearalenone, ZEN) 與脫氧雪腐鐮刀菌烯醇 (Deoxynivalenol, DON) 為穀物中常見且經常共存之真菌毒素。儘管相關研究逐漸增加，多數仍侷限於單一濃度條件，且缺乏系統性交互作用分析，導致其聯合毒性尚未完全釐清。因此，本研究以秀麗隱桿線蟲 (Caenorhabditis elegans) 為模式生物，探討ZEN與DON於不同濃度組合長期共同暴露下對生長與生殖毒性之影響，並分析毒物交互作用與長期低濃度共同暴露之潛在分子機制。試驗透過評估體長、生殖能力、體內氧化壓力、粒線體型態，再利用Bliss獨立性模型與Chou-Talalay模型評估ZEN與DON之交互作用，並進一步解析相關基因表現，以釐清可能之調控機制。 研究結果顯示，ZEN與DON共同暴露皆會抑制C. elegans生長並降低子宮內卵數。交互作用分析發現，生長毒性僅於ZEN 1.25 μM + DON 2.5 μM濃度組合呈現輕微協同作用，其餘濃度 (ZEN + DON = 2.5 + 5、5 + 10、25 + 50、75 + 150與125 + 250 μM) 皆呈現拮抗作用；而生殖毒性則於低濃度組合 (ZEN + DON = 1.25 + 2.5、2.5 + 5、5 + 10及25 + 50 μM) 呈現較明顯的協同作用，高濃度組合 (ZEN + DON = 75 + 150與125 + 250 μM) 則為拮抗作用，且共同暴露之交互作用具有濃度依賴性，由低濃度組合之協同作用逐漸轉變為高濃度組合之拮抗作用。進一步以ZEN 1.25 μM + DON 2.5 μM濃度組合探討其作用機制，結果顯示共同暴露下可協同促進活性氧物質 (Reactive oxygen species, ROS) 累積與粒線體碎裂，並協同活化細胞凋亡相關基因 egl-1、ced-4及drp-1與抑制抗凋亡基因ced-9之表現，同時協同下調卵黃蛋白生成相關基因vit-2及vit-6，以及MAPK/JNK訊號傳遞相關基因egl-15與jnk-1等之表達量，顯示ZEN與DON共同暴露可能透過促進ROS累積、粒線體功能失衡、細胞凋亡活化、卵黃蛋白生成受抑，以及MAPK/JNK訊號傳遞失調等多重機制，共同促成其協同生殖毒性。 綜合而言，本研究證實ZEN與DON在低濃度共同暴露下會加劇對C. elegans生長發育與生殖功能之毒性影響，並可能透過生殖調控相關傳遞路徑之協同作用而導致。本研究結果有助於補足多重真菌毒素共同暴露之毒理資料，並提供食品安全毒性評估之重要科學依據。

Mycotoxins are secondary metabolites produced by fungi that frequently contaminate agricultural commodities and their derived products, posing both acute and chronic health risks to humans and animals. They have become a major concern in food safety and public health. In natural environments and food matrices, mycotoxins commonly occur as mixtures, and co-exposure may result in toxicological interactions, including additive, synergistic, or antagonistic effects, thereby increasing overall toxicological risk. However, studies investigating the combined toxicity and underlying mechanisms of mycotoxin mixtures remain limited. Among Fusarium mycotoxins, zearalenone (ZEN) and deoxynivalenol (DON) are two of the most prevalent contaminants in cereal grains and frequently co-occur in food and feed. Although increasing attention has been given to their combined toxicity, most previous studies have focused on a single concentration or limited exposure conditions, and systematic evaluations of their toxicological interactions remain insufficient. Therefore, this study employed Caenorhabditis elegans as an in vivo model to investigate the effects of long-term co-exposure to ZEN and DON at different concentration combinations on growth and reproductive toxicity, as well as to characterize their toxicological interactions and explore the underlying molecular mechanisms under long-term low-dose co-exposure. Body length, reproductive capacity, intracellular oxidative stress, and mitochondrial morphology were evaluated. The Bliss independence model and the Chou-Talalay model were applied to determine the interaction between ZEN and DON, followed by gene expression analysis to elucidate the potential regulatory mechanisms. The results demonstrated that co-exposure to ZEN and DON significantly inhibited the growth of C. elegans and reduced the number of uterus eggs. Interaction analysis revealed that growth toxicity exhibited slight synergism only at the lowest concentration combination (ZEN 1.25 μM + DON 2.5 μM), whereas all higher concentration combinations (ZEN + DON = 2.5 + 5、5 + 10、25 + 50、75 + 150 and 125 + 250 μM) showed antagonistic effects. In contrast, reproductive toxicity exhibited pronounced synergism at the lower concentration combinations (ZEN + DON = 1.25 + 2.5, 2.5 + 5, 5 + 10, and 25 + 50 μM) but antagonism at the higher concentration combinations (ZEN + DON = 75 + 150 and 125 + 250 μM). Overall, the interaction between ZEN and DON was concentration-dependent, shifting from synergism at lower concentrations to antagonism at higher concentrations. Further mechanistic investigation using the synergistic combination of ZEN 1.25 μM + DON 2.5 μM showed that co-exposure synergistically promoted reactive oxygen species (ROS) accumulation and mitochondrial fragmentation. It also synergistically upregulated the expression of the apoptosis-related genes egl-1, ced-4, and drp-1, while downregulating the anti-apoptotic gene ced-9. In addition, the expression of the vitellogenin synthesis-related genes vit-2 and vit-6, as well as the MAPK/JNK signaling-related genes egl-15 and jnk-1, was synergistically suppressed. These findings suggest that the synergistic reproductive toxicity induced by ZEN and DON may be mediated through multiple mechanisms, including enhanced ROS accumulation, mitochondrial dysfunction, activation of apoptosis, inhibition of vitellogenin synthesis, and dysregulation of the MAPK/JNK signaling pathway. In conclusion, this study demonstrates that long-term low-dose co-exposure to ZEN and DON exacerbates growth and reproductive toxicity in C. elegans, possibly through the synergistic disruption of reproductive regulatory pathways. The findings of this study help fill the existing knowledge gap regarding the toxicological effects of combined exposure to multiple mycotoxins and provide important scientific evidence for food safety risk assessment.