探討黴菌毒素橘黴素的心臟和神經毒性機轉並評估其健康風險

Abstract

橘黴素(Citrinin, CTN)是一種由青黴菌屬、麴菌屬及紅麴菌屬真菌產生並廣泛污染各類穀物的黴菌毒素(Mycotoxin)。世界各地的玉米、小麥、大麥、堅果、香料等食品中橘黴素的殘留量可達0.08–25,000 μg/kg，尤其是由紅麴發酵所製成的紅麴產品或保健食品中橘黴素的殘留量可以高達70–189,000 μg/kg、檢出率也高達33%–78%，可能對人類健康產生潛在的危害與風險。目前已知橘黴素具有腎毒性與生殖毒性，其作用機制為誘導氧化壓力、造成粒線體損傷、細胞凋亡，以及染色體不穩定、干擾微管與細胞週期停滯等。然而有關橘黴素的心臟毒性、神經毒性、致癌性的作用與機制尚不清楚，目前也缺乏較新的橘黴素健康風險評估報告，因此本研究將分成四大部分逐一探討上述議題。 首先在【橘黴素的心臟毒性作用與機制】方面，微管與粒線體分別作為維持心肌型態與心肌的重要能量來源，有必要探討橘黴素的潛在心臟毒性及其作用機制。本研究將大鼠心肌母細胞H9c2處理橘黴素後，發現橘黴素(25–75 μM)會產生與秋水仙素相似的微管骨架破壞作用，而解體的微管會使依附其上的粒線體分布異常、錯位、功能失調，並且這些損傷的粒線體會在細胞中堆積。同時，橘黴素也阻礙了H9c2細胞內自噬作用的進行，並造成溶酶體和泛素化蛋白的累積，使受損胞器或蛋白質廢物無法清除。除此之外，橘黴素(2–50 μM)會降低許多肌節基因的表現，並干擾分化中與分化後心肌細胞的型態與肌節蛋白表現；而胚胎斑馬魚模型也顯示暴露橘黴素(25–50 μM)後會顯著減弱心臟跳動與心率，並使心臟收縮功能指標下降。綜上所述，本研究鑑定並闡述了橘黴素的體外心臟毒性作用機制以及體內心臟收縮不良的現象，除了揭開橘黴素同時對細胞內微管與粒線體的作用，也更加證實了橘黴素的心臟毒性危害。 第二部分【橘黴素的神經毒性作用與機制】中，由於粒線體也在神經細胞中扮演重要的功能性角色，因此本研究使用人類神經母細胞SH-SY5Y作為體外神經細胞模型來鑑定橘黴素的神經毒性。在處理橘黴素(10–20 μM)後，其轉錄體數據發現橘黴素下調了神經軸突發育、神經投射引導、神經分化等相關路徑，並上調氧化壓力、電子傳遞鏈等路徑。橘黴素(2–20 μM)顯著降低了神經分化與神經投射引導相關基因的表現量，且抑制了SH-SY5Y細胞的分化與神經軸突的延展與生長，而氧化壓力相關基因、粒線體氧化壓力與粒線體功能僅在高濃度處理下(50 μM)才有影響，這說明神經軸突生長與分化比粒線體失能對橘黴素的暴露更為敏感。這些結果首次揭露了橘黴素的體外神經毒性作用與機制，拓展了我們對橘黴素的基本了解。 第三部分是探索有關【橘黴素的潛在基因毒性與致癌分子特徵】。有鑑於染色體不穩定性是致癌性的標誌之一，本研究將HEK293以短期或長期處理橘黴素後探討其潛在的染色體不穩定性與潛在致癌機制。其中短期處理包含3天的橘黴素暴露加27天的正常培養(無暴露)；而長期處理則是連續30天的橘黴素暴露。結果發現，橘黴素短期與長期處理(10–20 μM)皆會造成有絲分裂時的紡錘絲型態異常，顯示其具有染色體不穩定性；而轉錄體分析顯示細胞週期、RTK/KRAS/RAF/MAPK訊號路徑的富集，並且轉錄體變化與資料庫中許多癌症相關基因表現變化樣態高度吻合，且發現許多關鍵致癌分子特徵，並且橘黴素的致癌跡象是不可逆的。這些結果初步提供了橘黴素的潛在基因毒性與致癌跡象。 最後本研究以基於貝氏機率的方法執行了【橘黴素的機率健康風險評估】。因為過去橘黴素的毒性起始劑量具有許多不確定性，本研究以貝式機率方法為橘黴素推估了新的毒性起始劑量(45.73 μg/kg bw/day)並訂定了新的人類健康參考劑量(0.08 μg/kg bw/day)，減低了許多不確定性。此外也利用橘黴素殘留量資料、各類食品攝食量資料以及臺灣族群的人口學數據進行機率性暴露評估。初步的試算結果顯示，在最壞情境下對於頻繁食用紅麴食品的族群來說，可能會有高達約40%的人將面臨健康風險。然而由於缺乏紅麴的攝食量資料，本研究在暴露評估上有許多不確定性與其限制，因此這些結果將無法作為建議，期望未來有更精準的紅麴攝食風險評估以釐清臺灣食用紅麴族群中攝入橘黴素的健康風險。 總結來說，本研究(1)廣泛地闡述了橘黴素的心臟毒性機制，包含微管、粒線體與受損胞器/蛋白質清除的損害或受阻，以及肌節結構的干擾、造成心臟收縮功能衰弱；(2)首次鑑定了橘黴素的神經毒性作用與機制，並且神經分化與投射引導的干擾作用比粒線體毒性與氧化壓力更為敏感；(3)初步探索橘黴素的潛在致癌跡象；(4)精準且完整地訂定了橘黴素的毒性起始劑量與人類健康參考劑量。這些研究成果將拓展橘黴素的基礎毒理學認識，並為後續橘黴素的相關毒性研究提供重要資訊，期望能維護大眾的健康福祉。

Citrinin (CTN) is a mycotoxin produced from Penicillium, Aspergillus, and Monascus fungi. CTN widely and frequently contaminates a variety of grains including corn, wheat, barley, nuts, and spices, leading to global residual levels ranging from 0.08 to 25,000 μg/kg in foods. Notably, the residual levels in Monascus-fermented red yeast rice foods and health supplements can reach 70 to 189,000 μg/kg, with detection rates of 33% to 78%. These observations suggest that CTN may pose potential hazards and risks to human health. CTN is well known for its nephrotoxicity and reproductive toxicity by inducing oxidative stress, mitochondrial damage, apoptosis, chromosomal instability, microtubule disruption, and cell cycle arrest. However, the cardiotoxicity, neurotoxicity, and genotoxicity and carcinogenic characteristics of CTN along with their underlying mechanisms remain unclear. Additionally, an up-to-date health risk assessment for CTN is currently lacking. This study is structured into four sections to address these issues as follows. The first section focuses on the [Cardiotoxic Effects and Mechanisms of CTN]. Based on the critical roles of microtubules and mitochondria in maintaining cardiac morphology and energy supply, it is essential to investigate the potential cardiotoxicity and its underlying mechanisms of CTN. In this study, rat cardiomyoblast H9c2 was used as an in vitro model. The results showed that CTN (25–75 μM) disrupted microtubule cytoskeleton assembly as colchicine did, and the disassembled microtubules thus resulted in mitochondrial misalignment and dysfunction, with those damaged mitochondria accumulated in the cells. Additionally, CTN impaired autophagy in H9c2 cells and triggered the accumulation of lysosomes and ubiquitinated proteins, hindering the clearance of damaged organelles and protein aggregates. Furthermore, CTN (2–50 μM) downregulated the expression of sarcomere genes and interfered the morphology and sarcomere protein expression during and after the H9c2 differentiation. Exposure of CTN (25–50 μM) to an embryonic zebrafish model demonstrated significantly weakened heart contraction and cardiac function indices of the embryonic hearts. Collectively, these data identify and elucidate the in vitro cardiotoxic mechanisms of CTN which involves both microtubules and mitochondria, and CTN-triggered cardiac malfunction was also observed in vivo. The second section includes the [Neurotoxic Effects and Mechanisms of CTN]. Given the essential role of mitochondria in neuronal functioning, this study employed human neuroblastoma cell SH-SY5Y as an in vitro model to assess CTN's neurotoxicity. The transcriptomic profile of CTN (10–20 μM)-treated SH-SY5Y cells revealed downregulation of axon development, neuron projection guidance, and neuron differentiation, as well as upregulation of oxidative stress and electron transport chain. Further experiments confirmed that CTN (2–20 μM) significantly downregulated genes associated with neural differentiation and projection guidance, and inhibited the differentiation and neurite outgrowth of SH-SY5Y cells. Notably, mitochondrial oxidative stress and mitochondrial function were only affected at higher concentration (50 μM), suggesting that neurite outgrowth and differentiation is more sensitive to CTN exposure than mitochondrial dysfunction. These findings present novel evidence of in vitro neurotoxicity of CTN, which expands our fundamental understanding of CTN. The third section explores the [Potential Genotoxicity and Carcinogenic Molecular Characteristics of CTN]. As chromosomal instability is a hallmark of carcinogenesis, this study explored the potential in vitro chromosomal instability and carcinogenic characteristics of CTN by using HEK293 cells that were subjected to either short-term or long-term treatment of CTN, where short-term means 3-day exposure to CTN followed by 27 days of normal culture while long-term denotes continuous 30-day exposure to CTN. The results showed that both short- and long-term treatments (10–20 μM) triggered the formation of abnormal mitotic spindles, a sign of chromosomal instability. RNA sequencing analyses showed enrichment of cell cycle and RTK/KRAS/RAF/MAPK signaling, and the transcriptomic profile closely aligned to various cancer-related gene expression patterns and matched several key molecular characteristics of carcinogenicity. Notably, this genotoxic and carcinogenic potential of CTN was irreversible. These findings provide preliminary evidence for the potential signs of genotoxicity and carcinogenicity of CTN. Finally, this study performed a [Probabilistic Health Risk Assessment of CTN] using a Bayesian-based probabilistic approach. Since high uncertainties existed during the then determination of point of departure for CTN, this study set a new Bayesian-based probabilistic point of departure (45.73 μg/kg bw/day) and human reference dose (0.08 μg/kg bw/day) for CTN with reduced uncertainties. In addition, the probabilistic exposure assessment was conducted using food residual data, food consumption data, and demographic data in Taiwan. The preliminary results showed that up to 40% of the Taiwanese people who frequently consume red yeast rice may face unacceptable risk in a worst-case scenario. However, due to the lack of red yeast rice consumption data, large uncertainties and limitations existed in the exposure assessment of this study so that the risk characterization results cannot be used for recommendations. It is hoped that a more precise risk assessment will be performed in the future to clarify the health risks for Taiwanese people who consume CTN-contaminated red yeast rice. In summary, this study (1) comprehensively elucidates the cardiotoxic effects and mechanisms of CTN which involve the damage of microtubules and mitochondria, the hindered clearance of damaged organelles/proteins, and the impaired cardiac contractile function; (2) identifies for the first time the neurotoxic effects and mechanisms of CTN by demonstrating that neural differentiation and projection guidance are more sensitive to CTN exposure than mitochondrial toxicity and oxidative stress; (3) preliminarily explores the potential genotoxic and carcinogenic characteristics of CTN; and (4) precisely establishes a new probabilistic point of departure and human reference dose for CTN. These findings broaden the foundational understanding of CTN toxicity and offer valuable information for future toxicological studies on citrinin, with the aim of safeguarding public health and welfare.