長期暴露棒麴毒素誘導秀麗隱桿線蟲神經毒性之評估：以血清素神經元調控做探討

Abstract

棒麴毒素 (Patulin, PAT) 是一種主要由Penicillium expansum產生的黴菌毒素，常見於發黴的水果、蔬菜與穀類中，尤其是在蘋果及其加工製品中。由於其具有耐熱及耐酸的特性，人類經常透過果汁與嬰兒食品而暴露於PAT。已知PAT會引起多種毒性效應，包括急性噁心、嘔吐及腸胃不適，長期暴露則可能損害腎臟、肝臟與免疫系統。然而，關於其潛在神經毒性的研究仍相對有限，其作用機制亦尚未明確。因此，本研究以Caenorhabditis elegans為模式生物，探討長期暴露PAT是否會誘發神經傳導物質調控相關之生理行為神經毒性，以及其可能的毒性機制。 本研究評估了C. elegans在不同PAT暴露濃度下的發育、運動及攝食行為相關指標。初步實驗結果顯示，PAT在50-90 μM時顯著抑制C. elegans生長；在運動行為方面，PAT暴露未明顯影響身體彎曲 (Body bending) 頻率，但在0.1-90 μM時顯著降低頭部擺動 (Head thrashing) 頻率，顯示PAT對運動行為之影響可能具有選擇性，而非單純造成整體運動能力下降。此外，暴露10-90 μM PAT時明顯降低咽喉收縮 (Pharyngeal pumping) 速率及產卵速率 (Egg-laying rate)，並呈現劑量依賴性影響，顯示其可能干擾攝食及生殖相關行為表現。在C. elegans中，由於頭部擺動、咽喉收縮及產卵行為皆與血清素神經調控密切相關，本研究進一步利用轉基因株 (GR1366) 觀察血清素生合成相關之tph-1::GFP螢光表現。結果顯示，PAT暴露後可顯著降低頭部區域tph-1::GFP螢光強度，顯示PAT可能影響tph-1啟動子活性或血清素合成相關神經訊號。此外，PAT亦顯著提升體內活性氧物質 (Reactive oxygen species, ROS) 含量與上調代謝ROS之相關基因sod-3、gst-4，並顯著影響C. elegans體內多個血清素調控相關基因之表達，包含tph-1、mod-1、ser-1、cat-1、cat-4及mod-5。 為進一步驗證血清素訊號於PAT誘發行為異常中之角色，本研究亦進行外源性血清素補充試驗及tph-1突變株 (MT15434) 分析。結果顯示，外源性血清素可顯著改善PAT造成之血清素相關行為缺陷，而在tph-1突變株中，PAT對頭部擺動、咽喉收縮及產卵行為所造成之毒性較野生型N2減緩，支持PAT誘發之行為毒性可能與tph-1及血清素神經調控路徑有關。 綜合上述結果，本研究顯示長期暴露PAT可誘發C. elegans生長發育毒性，並影響頭部擺動、咽喉收縮及產卵速率等血清素相關行為表現，並發現PAT可能透過誘導氧化壓力與干擾血清素神經調控系統，進而導致神經行為毒性。本研究提供PAT誘發體內神經毒性之實驗證據，並補足目前PAT於生物體神經傳導物質調控相關研究之不足，可作為後續探討PAT神經毒性作用機制之參考基礎。

Patulin (PAT) is a mycotoxin mainly produced by Penicillium expansum and is commonly found in mold-contaminated fruits, vegetables, and cereals, particularly apples and apple-derived products. Due to its heat- and acid-resistant properties, humans are frequently exposed to PAT through fruit juices and infant foods. PAT has been reported to induce various toxic effects, including acute nausea, vomiting, and gastrointestinal discomfort, while long-term exposure may cause damage to the kidney, liver, and immune system. However, studies regarding its potential neurotoxicity remain relatively limited, and the underlying mechanisms are still unclear. Therefore, this study used Caenorhabditis elegans as a model organism to investigate whether long-term PAT exposure induces physiological and behavioral neurotoxicity associated with neurotransmitter regulation, as well as its possible toxic mechanisms. This study evaluated developmental, locomotor, and feeding-related behavioral endpoints in C. elegans exposed to different concentrations of PAT. Preliminary results showed that PAT significantly inhibited the growth of C. elegans at 50-90 μM. In terms of locomotor behavior, PAT exposure did not markedly affect the frequency of body bending, but significantly decreased the frequency of head thrashing at 0.1-90 μM, suggesting that the effects of PAT on locomotor behavior may be selective rather than simply causing an overall decline in motor ability. In addition, exposure to 10-90 μM PAT significantly reduced the pharyngeal pumping rate and egg-laying rate in a dose-dependent manner, indicating that PAT may interfere with feeding- and reproduction-related behavioral performance. Since head thrashing, pharyngeal pumping, and egg-laying behaviors in C. elegans are closely associated with serotonergic neuronal regulation, this study further used the transgenic strain GR1366 to observe the fluorescence expression of tph-1::GFP, which is related to serotonin biosynthesis. The results showed that PAT exposure significantly reduced tph-1::GFP fluorescence intensity in the head region, suggesting that PAT may affect tph-1 promoter activity or serotonin synthesis-related neuronal signaling. Furthermore, PAT significantly increased intracellular reactive oxygen species (ROS) levels and upregulated the ROS-metabolism-related genes sod-3 and gst-4. PAT also significantly altered the expression of multiple serotonin regulation-related genes in C. elegans, including tph-1, mod-1, ser-1, cat-1, cat-4, and mod-5. To further verify the role of serotonin signaling in PAT-induced behavioral abnormalities, this study also performed exogenous serotonin supplementation experiments and analysis using the tph-1 mutant strain MT15434. The results showed that exogenous serotonin significantly improved PAT-induced defects in serotonin-related behaviors. In the tph-1 mutant strain, the toxic effects of PAT on head thrashing, pharyngeal pumping, and egg-laying behaviors were attenuated compared with those observed in wild-type N2 worms, supporting that PAT-induced behavioral toxicity may be associated with tph-1 and the serotonergic neuronal regulatory pathway. Taken together, this study demonstrates that long-term PAT exposure induces developmental toxicity in C. elegans and affects serotonin-related behavioral performance, including head thrashing, pharyngeal pumping, and egg-laying rate. These findings further suggest that PAT may induce neurobehavioral toxicity by promoting oxidative stress and disrupting the serotonergic neuronal regulatory system. This study provides experimental evidence that PAT induces neurotoxicity in vivo and helps address the current lack of knowledge regarding the effects of PAT on the regulation of neurotransmitters in living organisms. These findings provide a valuable foundation for future investigations into the mechanisms underlying PAT-induced neurotoxicity.