【摘要】：前言：黃綠青霉毒素（citreoviridin, CIT）是主要由黃綠青霉菌（Penicilliumcitreoviridin）產(chǎn)生的具有毒性的次級代謝產(chǎn)物。CIT廣泛存在于霉變作物中，例如霉變的谷物，大米，玉米等。研究發(fā)現(xiàn)黃綠青霉菌在大米中生長速度最快，產(chǎn)毒數(shù)量也最高。溫度與環(huán)境中的PH值對黃綠青霉菌的生長及產(chǎn)毒有著密切影響。 CIT最早引起人們關(guān)注是在100多年前的日本。1891年，日本學(xué)者Sakaki發(fā)現(xiàn)用發(fā)霉的大米喂養(yǎng)實驗動物會導(dǎo)致其出現(xiàn)中樞神經(jīng)系統(tǒng)麻痹的癥狀。1920年，日本學(xué)者M(jìn)iyake和Takada首次報道了來自霉變食物的青霉菌屬（Penicilliumcommune）引起實驗動物兔和老鼠的病變。隨后Miyake等人從日本本土大米和臺灣大米中提取出了一種真菌并命名為黃綠青霉菌（P. citreonigrum）。該菌株可產(chǎn)生一種毒性很強的代謝產(chǎn)物即黃綠青霉毒素。 CIT可通過干擾受試?yán)鲜竽X部的糖代謝進(jìn)而導(dǎo)致中樞神經(jīng)系統(tǒng)功能障礙。研究認(rèn)為CIT的毒性效應(yīng)源于其導(dǎo)致的呼吸系統(tǒng)及心血管系統(tǒng)的損傷，如呼吸暫停、腦電圖異常、竇性心律失常、低血壓等，從而出現(xiàn)由于主動脈組織缺氧而導(dǎo)致的中樞神經(jīng)系統(tǒng)癥狀。CIT可致大鼠心肌原發(fā)性壞死，心肌細(xì)胞呈顆粒狀變性，心肌纖維凝集，溶解。CIT可結(jié)合并抑制線粒體中的三磷酸腺苷合成酶。研究認(rèn)為CIT與“黃變米中毒”，“心源性腳氣病”以及克山病有著密切關(guān)系。 CIT可致心肌細(xì)胞，人胃癌細(xì)胞(SGC-790)DNA損傷。但具體機制尚不清楚。本研究選用HepG2細(xì)胞，探討CIT的DNA損傷毒性及其可能機制。HepG2細(xì)胞來源于人類肝胚細(xì)胞瘤，分化程度較高，不僅保留了人類正常肝實質(zhì)細(xì)胞的許多功能和特點，還保留了較完整的生物轉(zhuǎn)化代謝Ⅰ相酶和Ⅱ相酶。被認(rèn)為是用來檢測外來化合物DNA損傷毒性的理想細(xì)胞系。CIT為親脂性毒素，吸收后在肝臟中濃度最高。這也就是我們采用HepG2細(xì)胞作為該試驗系統(tǒng)的原因之一。 本研究選用HepG2細(xì)胞，探討CIT的DNA損傷毒性及其可能機制，旨在為評估CIT對人類健康的潛在危害提供實驗室依據(jù)。 方法：本研究選用HepG2細(xì)胞作為試驗系統(tǒng)。通過單細(xì)胞凝膠電泳（SCGE）試驗檢測細(xì)胞DNA損傷情況，評價CIT的DNA損傷毒性。為探討其可能的DNA損傷毒性機制，以2＇，7＇—二氫二氯熒光素(DCFH)法測定細(xì)胞內(nèi)ROS水平，以苯二醛（OPT）測定細(xì)胞內(nèi)還原型谷胱甘肽（GSH）水平，以免疫組化方法檢測細(xì)胞內(nèi)8-羥基脫氧鳥苷（8-OHdG）的表達(dá)水平，用吖啶橙（Acridine orange）和羅丹明123（Rhodamine123）分別測定細(xì)胞內(nèi)溶酶體膜穩(wěn)定性和線粒體膜電位的改變水平。實驗結(jié)果用SPSS v11.5統(tǒng)計軟件包進(jìn)行統(tǒng)計分析。 結(jié)果：2.5-5μM的CIT作用于HepG2細(xì)胞1h后，引起細(xì)胞DNA鏈斷裂，細(xì)胞形成彗星樣拖尾，其尾DNA%明顯大于未用CIT處理的細(xì)胞。2.5-5μM的CIT染毒60min引起細(xì)胞內(nèi)ROS水平明顯增加，GSH水平顯著降低，與對照組相比差異具有統(tǒng)計學(xué)意義（P 0.01）；10mM的N-乙酰半胱氨酸(NAC)預(yù)處理60min后可降低CIT引起的HepG2細(xì)胞內(nèi)ROS水平增加，對CIT所致HepG2細(xì)胞DNA損傷具有保護(hù)作用。 1.25-5μM CIT作用于HepG2細(xì)胞3h后，可致8-OHdG的表達(dá)升高；1.25-5μM的CIT作用于HepG2細(xì)胞60min后，可引起細(xì)胞內(nèi)溶酶體膜穩(wěn)定性明顯改變；5μM的CIT作用60min引起細(xì)胞內(nèi)線粒體膜電位的改變，，與對照組比較，其差異有統(tǒng)計學(xué)意義（P＜0.05）。 結(jié)論：CIT可引起HepG2細(xì)胞DNA損傷，提示CIT對HepG2細(xì)胞具有DNA損傷毒性。CIT可誘發(fā)細(xì)胞內(nèi)ROS水平增高，GSH水平降低，提示CIT對HepG2細(xì)胞產(chǎn)生的DNA損傷毒性可能與氧化應(yīng)激機制有關(guān)。N-乙酰半胱氨酸(NAC)預(yù)處理60min后可降低CIT引起的HepG2細(xì)胞內(nèi)ROS水平增加，對CIT所致HepG2細(xì)胞DNA損傷具有保護(hù)作用。CIT作用于細(xì)胞后導(dǎo)致氧化性DNA損傷的標(biāo)志性產(chǎn)物8-OHdG形成增強，進(jìn)一步表明CIT引起HepG2細(xì)胞的DNA損傷為氧化性損傷。此外，CIT對HepG2細(xì)胞溶酶體膜穩(wěn)定性有顯著影響，并使得HepG2細(xì)胞線粒體膜電位顯著降低。
[Abstract]:Citroviridin (CIT) is a toxic secondary metabolite, which is mainly produced by Penicillium citriviridin. CIT is widely used in mildewed crops, such as mildewed grains, rice, corn, etc. The results showed that the growth rate of the fungus in the rice was the fastest in the rice, and the number of the production was the highest. The pH value in the temperature and the environment has a close effect on the growth and the production of the yellow-green-green mould. CIT first raised concerns over the 100-year-old Japan. In 1891, Japanese scholar Sakaki found that feeding experimental animals with musty rice could lead to the symptoms of central nervous system paralysis. In 0, the Japanese scholar, Miyake and Takada, reported for the first time that the Penicillium communis from mildewed food caused experimental animal rabbits and mice The lesion. Then Miyake et al. extracted a fungus from Japanese native rice and Taiwan rice and named P.citregoniru. m) the strain can produce a metabolite with strong toxicity, namely, the yellow-green-green mildew The toxin. CIT can result in the central nervous system by interfering with sugar metabolism in the brain of the tested mouse The study found that the toxic effect of CIT is due to the damage of the respiratory system and the cardiovascular system, such as apnea, abnormal electroencephalogram, ventricular arrhythmia, hypotension, and so on, so that the central God caused by the hypoxia of the aorta Through systemic symptoms, CIT can induce primary necrosis of the myocardium of the rat, and the cardiac muscle cells are in the form of granular degeneration, cardiac muscle fiber, Agglutination and dissolution. CIT can bind and inhibit the triphosphoric acid in the mitochondria Adenoid synthase. It is considered that CIT is associated with 鈥測ellow rice poisoning鈥

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