黃曲霉毒素生物合成及代謝轉(zhuǎn)換的研究進(jìn)展

熊江林，周華林，丁斌鷹，劉建新

(1.武漢輕工大學(xué) 動(dòng)物科學(xué)與營(yíng)養(yǎng)工程學(xué)院， 湖北 武漢 430023； 2.浙江大學(xué) 奶業(yè)科學(xué)研究所，浙江 杭州 310058；3.襄陽(yáng)職業(yè)技術(shù)學(xué)院 生物工程學(xué)院，襄陽(yáng)市動(dòng)物醫(yī)學(xué)工程技術(shù)中心，湖北 襄陽(yáng) 441100)

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黃曲霉毒素生物合成及代謝轉(zhuǎn)換的研究進(jìn)展

熊江林1, 2，周華林3，丁斌鷹1，劉建新2*

(1.武漢輕工大學(xué) 動(dòng)物科學(xué)與營(yíng)養(yǎng)工程學(xué)院， 湖北 武漢 430023； 2.浙江大學(xué) 奶業(yè)科學(xué)研究所，浙江 杭州 310058；3.襄陽(yáng)職業(yè)技術(shù)學(xué)院 生物工程學(xué)院，襄陽(yáng)市動(dòng)物醫(yī)學(xué)工程技術(shù)中心，湖北 襄陽(yáng) 441100)

黃曲霉毒素(Aflatoxins，AFs)是真菌產(chǎn)生的有機(jī)代謝產(chǎn)物，具有強(qiáng)烈的生物毒性，廣泛存在于飼料原料和各類飼料之中。本文綜述了AFs的產(chǎn)生條件、合成途徑以及AFB1在動(dòng)物體內(nèi)的代謝轉(zhuǎn)化，旨為黃曲霉毒素的防控和動(dòng)物健康養(yǎng)殖提供參考。

黃曲霉毒素；合成途徑；代謝轉(zhuǎn)化；研究進(jìn)展

黃曲霉毒素(Aflatoxins，AFs)名稱源于該毒素的主要生產(chǎn)真菌-黃曲霉菌 (Aspergillusflavus)。有關(guān)AFs的最早報(bào)道是在1960年，英國(guó)東南部地區(qū)農(nóng)場(chǎng)給火雞飼喂巴西和非洲生產(chǎn)的花生粕后，導(dǎo)致10萬(wàn)只火雞突然死亡。最初因病因不明，該病被命名為火雞"X"病，后來(lái)被證實(shí)為黃曲霉菌產(chǎn)生的AFs嚴(yán)重污染飼料所致[1]。50多年來(lái)，人們廣泛開(kāi)展了AFs的生成、代謝、毒性和防控技術(shù)研究。

1 AFs的種類和產(chǎn)生菌株

AFs的CAS號(hào)1402-68-2，是一組結(jié)構(gòu)類似的二呋喃香豆素衍生物，其基本結(jié)構(gòu)為1個(gè)二呋喃環(huán)和1個(gè)氧雜萘鄰?fù)?(香豆素)組成，前者為基本毒性結(jié)構(gòu)而后者與致癌有關(guān)。自然界中至少存在14 種不同類型的AFs，主要有AFB1、AFB2、AFG1和AFG2等。其中，AFB1在體內(nèi)可羥化生成AFM1[2]，AFM1可見(jiàn)于動(dòng)物的肉、蛋、奶、腎臟和肝臟等動(dòng)物產(chǎn)品及內(nèi)臟之中。

AFs主要由曲霉屬中的黃曲霉 (Aspergillusflavus)和寄生曲霉 (A.parasiticus)污染谷物和谷物產(chǎn)品后所產(chǎn)生的一組次生代謝物[3-4]。其他一些品種，諸如特異曲霉 (A.nomius)、家蠶曲霉 (A.bombycis)、假溜曲霉菌 (A.pseudotamari)和赭曲霉(A.ochraceoroseus)也可產(chǎn)生AFs，但這些霉菌在自然界中出現(xiàn)的頻率很低[5-7]。黃曲霉分離菌株在產(chǎn)生AFs能力方面存在重大差異。據(jù)報(bào)道，上述真菌中的50%菌株可以產(chǎn)生AFs，而一些分離菌株可以產(chǎn)生高達(dá)106 μg/kg的AFs[8-9]。

2 AFs生成的外在條件

AFs形成和污染程度除了上述產(chǎn)毒真菌外，還需要適當(dāng)?shù)漠a(chǎn)毒外部環(huán)境條件包括：溫度、濕度、水活力、物理性損傷和其它儲(chǔ)存條件[10]以及飼料種類。產(chǎn)生的毒素量將取決于生物學(xué)因素 (植物品種、應(yīng)激、昆蟲(chóng)和霉菌孢子量)和物理因素 (水分、相對(duì)濕度、溫度和谷物的物理?yè)p傷)[11]。比如，黃曲霉和寄生曲霉等在谷物具有一定水分含量 (≥15%)、適當(dāng)?shù)臏囟?(20～30 ℃)和相對(duì)濕度 (≥80%)條件下即可生長(zhǎng)，并生成AFs，其中以AFB1比例最大[12-13]。

產(chǎn)毒真菌在收獲前侵染谷物需要的水分含量 (200～250 g/kg)通常比真菌在存儲(chǔ)間增殖所需水分含量 (130～180 g/kg)要高些。因此，水分含量在130 g/kg以上的大多數(shù)飼料是容易導(dǎo)致霉菌生長(zhǎng)和霉菌毒素的形成[14]。另外，谷物在30%～32%水分條件下成熟，水分下降時(shí)，谷粒容易發(fā)生機(jī)械性破損，這些破損的谷粒就會(huì)成為有利于產(chǎn)毒真菌生長(zhǎng)的基質(zhì)。大宗谷物在水分不均勻，且不同批次谷物進(jìn)行混合會(huì)有利于AFs產(chǎn)毒真菌的生長(zhǎng)，使得谷物受AFs污染情況更加嚴(yán)重，所以維持谷物低且均勻的水分十分重要[12]。

飼料種類不同，營(yíng)養(yǎng)基質(zhì)狀況差異很大，也會(huì)在很大程度上影響產(chǎn)毒真菌的生長(zhǎng)和產(chǎn)毒量，比如花生粕、玉米、棉籽粕和青貯飼料等飼料原料非常適合AFs產(chǎn)毒真菌生長(zhǎng)和AFs的形成，是導(dǎo)致飼料黃曲霉毒素污染的主要來(lái)源[15-17]。

3 AFs的生物合成途徑

AFs生物合成共需21個(gè)酶促步驟，并且這些酶的基因已經(jīng)得到克隆。aflR和aflJ基因編碼大多數(shù)結(jié)構(gòu)基因轉(zhuǎn)錄激活的蛋白質(zhì)，也是基因簇的一部分[18-19]。黃曲霉和寄生曲霉DNA的粘粒和λ噬菌體庫(kù)的限制性酶圖顯示所有的基因成簇聚集在真菌基因組70-kb區(qū)域[20]。其他曲霉 (如構(gòu)巢曲霉)生成AFs前體物，諸如雜色曲霉素 (ST)。ST生物合成途徑與黃曲霉和寄生曲霉類似，但是染色體上基因順序不同[21]。AFs的合成是通過(guò)一系列高度協(xié)調(diào)的氧化還原反應(yīng)實(shí)現(xiàn)[22]，主要合成途徑和相關(guān)基因見(jiàn)圖1(以黃曲霉合成AFs為例)。AFs的生物合成起始類似于脂肪酸的生物合成，即乙酰CoA作為起始單位，而丙二酸單酰作為延長(zhǎng)單位，在聚酮化合物合成酶(PKSA)催化作用下形成AFs的聚酮骨架，該過(guò)程由兩個(gè)脂肪酸合酶基因 (fas-1和fas-2)和聚酮合酶基因 (pksA)編碼產(chǎn)物催化[18-19]。無(wú)特定的酶催化noranthrone轉(zhuǎn)變?yōu)镹OR (可以分離的第一個(gè)穩(wěn)定代謝物)，但轉(zhuǎn)化可能涉及單加氧酶 (可能cypA編碼)和脫氫酶 (可能norB編碼)[19-22，24]。

圖1 黃曲霉和寄生曲霉合成AFs的途徑及相關(guān)基因簇和酶[23]

NOR轉(zhuǎn)化為AVN涉及脫氫酶，該酶由nor-1基因編碼[25]，但也能夠由norA編碼的脫氫酶催化[26]。奧弗尼紅素 (AVR)轉(zhuǎn)化為雜色曲霉B (VERB)的一些催化步驟仍未與基因簇上特定基因建立聯(lián)系。三個(gè)基因 (cypX、moxY和avfA)可能涉及個(gè)別步驟；兩個(gè)基因ver1 (編碼酮還原酶)和verA (編碼細(xì)胞色素P-450單加氧酶)是雜色曲霉A (VERA)轉(zhuǎn)化為脫甲基雜色曲霉素 (DMST)所必需的[27-28]。AFs形成的最后步驟是O-甲基雜色曲霉素(OMST)或二氫-O-甲基雜色曲霉素 (DHOMST)轉(zhuǎn)化為AFB1、B2、G1和G2，該反應(yīng)需要ordA基因編碼的NADPH依賴單加氧酶[29]。AFG毒素形成還可能與ordB編碼的酶有關(guān)[29]。其他基因，如aflT。編碼ABC轉(zhuǎn)運(yùn)蛋白，該蛋白可能是AFs從細(xì)胞轉(zhuǎn)運(yùn)出來(lái)所必需。

4 AFB1的吸收與代謝轉(zhuǎn)化

由于AFB1是脂溶性和低分子量，一旦被攝入后就會(huì)很快通過(guò)被動(dòng)擴(kuò)散機(jī)制被瘤胃壁和腸道吸收[30]。實(shí)驗(yàn)動(dòng)物和靈長(zhǎng)類動(dòng)物的有關(guān)試驗(yàn)顯示AFB1的吸收迅速、完全[31-32]。根據(jù)對(duì)大鼠的藥代動(dòng)力學(xué)研究，AFB1主要在十二指腸段吸收，并且在小腸的2個(gè)位點(diǎn)分2個(gè)階段吸收。AFB1在奶牛消化道迅速被吸收，使得AFM1很快遷移到乳中[33]。血液中約90%的AFB1存在于血漿中[34]。AFB1與蛋白質(zhì)結(jié)合的部分主要連接到白蛋白上，白蛋白賴氨酸和AFB1某種衍生物間形成的席夫堿共價(jià)鍵涉及到血蛋白中加合物形成。

腸道上皮組織、肝臟和腎臟是許多化合物發(fā)生生物轉(zhuǎn)化的部位，這些生物轉(zhuǎn)化包括兩個(gè)階段的反應(yīng)。第一階段由還原、氧化和水解反應(yīng)組成。微粒體細(xì)胞色素P-450、含有核黃素的單氧合酶、前列腺素類合成酶、氨基氧化酶和乙醇脫氫酶等是參與氧化反應(yīng)的主要酶類，然而還原反應(yīng)受到環(huán)氧化物水解酶、醛糖還原酶或酮還原酶控制。另外，哺乳動(dòng)物組織和體液含有大量能水解異生物質(zhì)分子的非特異性酯酶和酰胺酶[35-36]。第二階段由合成反應(yīng)組成，作用于第一階段形成的分子。這些反應(yīng)會(huì)減少毒性、增加AFs分子的水溶性以便通過(guò)尿 (奶)排除，以保護(hù)動(dòng)物[38]。主要的加成反應(yīng)酶是微粒體葡糖醛酸基轉(zhuǎn)移酶、S-谷胱甘肽轉(zhuǎn)移酶和N-乙?；D(zhuǎn)移酶[35]。AFB1在肝臟中發(fā)生的一系列生物轉(zhuǎn)化和和排泄途徑見(jiàn)圖2，奶?？蓪⑹橙氲腁FB1轉(zhuǎn)化為AFM1，然后分泌到牛奶中[39-40]，蛋禽也可將AFM1分泌到蛋中[41-42]。除了肝臟外，氧化系統(tǒng)還會(huì)在其他組織中發(fā)揮作用，比如骨髓、肥大細(xì)胞、皮膚、卵巢、睪丸和白細(xì)胞等[47]。

圖2 吸收的AFB1在肝臟中的生物轉(zhuǎn)化途徑[39]

5 結(jié) 語(yǔ)

AFs的產(chǎn)毒真菌、產(chǎn)毒條件和易感谷物等的研究可提供降低飼料和食品中AFs的方法。隨著真菌合成AFs的相關(guān)基因相繼被解析，應(yīng)用基因修飾方法可得到不產(chǎn)毒的曲霉真菌，再接種谷物等植株上，通過(guò)與野生的產(chǎn)毒曲霉真菌的生物生存競(jìng)爭(zhēng)，達(dá)到減少AFs對(duì)谷物等的污染。而AFs的代謝轉(zhuǎn)化途徑中一些關(guān)鍵酶和代謝中間產(chǎn)物相繼被研究楚，將為AFB1解毒藥物的開(kāi)發(fā)提供啟示。

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Review of Biosynthesis and Metabolic Turnover of Aflatoxins

XIONG Jiang-lin1,2, ZHOU Hua-lin3, DING Bin-ying1, LIU Jian-xin2

(1.SchoolofAnimalScience&NutritionalEngineering,WuhanPolytechnicUniversity,Wuhan, 430023,China. 2.InstituteofDairyScience,ZhejiangUniversity,Hangzhou310058,China; 3.XiangyangEngineeringResearchCenterofAnimalMedicine,XiangyangVocationalandTechnicalCollege,Xiangyang441100,China)

Aflatoxins are mebolites produced by fungus and widely exist in feedstuff and all kinds of feed. It has strong biological toxicity, and thus endanger feed production and the healthy development of animal husbandry. In order to assist the prevention and control and animal feeding practice, discussions were made on generating condition and denovo synthesis of aflatoxins and metabolic transformation of aflatoxin B1 in animals.

afltoxins; production; metabolic transformation

2014-12-15，

2015-01-05

現(xiàn)代奶牛產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)資金資助(CARS-37)

熊江林(1977-)，男，湖北鄂州人，講師，博士，研究方向?yàn)閯?dòng)物營(yíng)養(yǎng)與免疫。E-mail:xiongjianglin@126.com

*[通訊作者] 劉建新(1958-)，男，浙江杭州人，博導(dǎo)，教授，主要從事反芻動(dòng)物營(yíng)養(yǎng)研究。E-mail:liujx@zju.edu.cn

S811.6

A

1005-5228(2015)04-0085-05