赭曲霉毒素A的产生、毒性机制与生物合成研究进展

微生物学通报

2023, Vol. 50

Issue (3): 1265−1280

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高婧, 刘惠卿, 张真真, 梁志宏

GAO Jing, LIU Huiqing, ZHANG Zhenzhen, LIANG Zhihong

赭曲霉毒素A的产生、毒性机制与生物合成研究进展

Production, toxicity and biosynthesis of ochratoxin A: a review

微生物学通报, 2023, 50(3): 1265-1280

Microbiology China, 2023, 50(3): 1265-1280

DOI: 10.13344/j.microbiol.china.220641

文章历史

收稿日期: 2022-07-10

接受日期: 2022-08-22

网络首发日期: 2022-10-24

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高婧, 刘惠卿, 张真真, 梁志宏. 赭曲霉毒素A的产生、毒性机制与生物合成研究进展[J]. 微生物学通报, 2023, 50(3): 1265-1280.

GAO Jing, LIU Huiqing, ZHANG Zhenzhen, LIANG Zhihong. Production, toxicity and biosynthesis of ochratoxin A: a review[J]. Microbiology China, 2023, 50(3): 1265-1280.

赭曲霉毒素A的产生、毒性机制与生物合成研究进展

高婧 , 刘惠卿 , 张真真 , 梁志宏

中国农业大学食品科学与营养工程学院北京食品营养与人类健康高精尖创新中心, 北京 100083

收稿日期: 2022-07-10; 接受日期: 2022-08-22; 网络首发日期: 2022-10-24

基金项目: 国家自然科学基金(32172170)；中国农业大学研究生自主创新研究基金(2022TC160)

摘要: 赭曲霉毒素A (ochratoxin A, OTA)是曲霉属和青霉属等真菌的次生代谢物，广泛污染谷物、葡萄、坚果等农产品和饲料，造成严重的经济损失。此外，OTA的肝肾毒性和三致作用(致畸、致癌、致突变)已经得到越来越多的数据支撑，证实其对人类健康存在巨大威胁。OTA及其衍生物的理化性质已经有较为全面的研究，但是其合成过程及调控机理尚不明确。本文整理了赭曲霉毒素A的理化性质及产毒菌株，总结了OTA污染及致病情况的最新研究进展和各国的限量标准，最后分析了OTA的合成机制，讨论了未来OTA理论及应用层面的研究方向，为赭曲霉毒素A的风险评估提供数据支持，同时为OTA生物合成和调控机制提供理论参考。

关键词: 赭曲霉毒素A 限量标准合成调控风险评估食品污染

Production, toxicity and biosynthesis of ochratoxin A: a review

GAO Jing , LIU Huiqing , ZHANG Zhenzhen , LIANG Zhihong

Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China

Received: 10-07-2022; Accepted: 22-08-2022; Published online: 24-10-2022

Foundation item: This work was supported by the National Natural Science Foundation of China (32172170) and the Graduate Independent Innovation Research Fund of China Agricultural University (2022TC160)

^*Corresponding author: LIANG Zhihong, E-mail: lzh105@cau.edu.cn.

Abstract: Ochratoxin A (OTA) is a secondary metabolite of fungi such as Aspergillus and Penicillium, which contaminated a variety of agricultural products and feeds such as grain, grapes, and nuts, causing serious economic losses. Moreover, increasing studies have demonstrated the liver and kidney toxicity as well as the teratogenicity, carcinogenicity, and mutagenesis of OTA, which poses a huge threat to human health. Although OTA and its derivatives have been studied comprehensively in terms of physicochemical properties, the synthesis process and regulatory mechanism of them remain unclear. This paper introduced the physicochemical properties and toxin-producing strains of OTA, reviewed the latest research progress in OTA contamination and pathogenic conditions, summarized the limit standards of different countries, expounded the synthesis mechanism of OTA, and finally discussed the future research directions. With this paper, we hope to provide data support for the risk assessment of OTA and provide theoretical reference for the research on the biosynthesis and regulatory mechanism of OTA.

Keywords: ochratoxin A limit standard regulation on synthesis risk assessment food contamination

真菌毒素(mycotoxins)是真菌(fungi)产生的一类次生代谢产物，据联合国粮食组织(Food and Agriculture Organization, FAO)估算，全世界每年大约有5%–7%的粮食、饲料等农产品受霉菌的侵害，25%的粮食作物受到霉菌毒素的污染^[1]。我国粮食中危害最严重的真菌毒素污染物是黄曲霉毒素(aflatoxin, AF)、玉米赤霉烯酮(zearalenone, ZEN)、脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)、赫曲霉毒素(ochratoxin, OT)和伏马毒素(fumonisin, FB)等，果蔬及其制品中是AF、OT、交链孢霉毒素和展青霉毒素(patulin, PAT)等。同时，真菌毒素对人或动物具有一定的毒性及致病性，某些毒素针对特定动物还有致癌性。真菌毒素大多被国际癌症研究机构(International Agency for Research on Cancer, IARC)和世界卫生组织(World Health Organization, WHO)列为A类或B类致癌物，可侵害肝脏、肾脏、造血器官、神经系统、皮肤等组织和器官^[2]。当人、畜、禽类食入被毒素污染的食物或饲料时，可能发生不同种类、不同程度的急性、亚急性或慢性的真菌毒素中毒症(mycotoxicosis)^[2]。

赭曲霉毒素A (ochratoxin A, OTA)是继黄曲霉毒素后又一种在世界范围内引起重点关注的霉菌毒素，与黄曲霉毒素同属异香豆素类化合物，二者具有相似的合成原料和催化酶，自然界中还存在既可以产生OT又可以产生AF的交叉产毒菌(如Aspergillus wentti)。另外，赭曲霉毒素毒性相比AF较小，可以作为模式毒素进行机理研究，在相对安全的情况下将OTA的研究成果推及更广泛的真菌污染及致病性的防治，可以保障研究者的安全。本文对赭曲霉毒素A的理化性质、产毒真菌进行综述，同时介绍国内外最新的赭曲霉毒素A的污染及致病性情况，最后对赭曲霉毒素A的生物合成机理进行归纳分析，以期为赭曲霉毒素A的风险评估提供支持，同时为OTA生物合成和调控机制提供理论参考。

1 赭曲霉毒素A的理化性质

赭曲霉毒素A化学名为N-[(3R)-(5-氯-8-羟基-3-甲基-1-氧代-7-苯并二氢异吡喃基)羰基]-l-苯丙氨酸，(N-[(3R)-(5-chloro-8-hydroxy- 3-methyl-1-oxo-3, 4-dihydro-1H-isochromen-7-yl) carbonyl]-L-phenylalanine)，化学分子式为C₂₀H₁₈ClNO₆，分子量为403.8，其chemical abstracts service (CAS)认证编号为303-47-9 (图 1A)^[3]，最早于1965年被van der Merwe等发现，1967年Steyn等解析出OTA的结构^[4]。赭曲霉毒素属于异香豆素类化合物，截至目前，科学家们共发现自然条件下或人体内经过生物代谢转化、基本骨架上的各位点由不同的官能团取代的20余种OTs结构类似物(图 1B，表 1)。OTA是一种呈现无色结晶状态的酸性分子，羧酸基团的pKa是4.4，苯环的pKa是7.1；OTA也是一种荧光物质，长波紫外光下显示绿色或黄绿色荧光，碱性条件下紫外检测为蓝色荧光，并且OTA含量和荧光强度成正比，荧光变化表现为可逆过程。其在苯溶液中的紫外特征吸收波长为333 nm，易溶于极性有机溶剂，如甲醇、乙醇、苯、乙腈、乙酸乙酯、三氯甲烷，也可以溶解在碱性水溶液中，不溶于己烷、石油醛、乙醛和酸性水溶液，酸水解后形成苯丙氨酸和OTα。OTA具有一定的热稳定性，二甲苯中的熔点为169 ℃，苯中的熔点为89‒95 ℃^[5]。

图 1　赭曲霉毒素A结构式(A)及赭曲霉毒素基本骨架(B) Figure 1　Structure of ochratoxin A (A) and ochratoxins basic skeleton (B).

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表 1　赭曲霉毒素衍生物的化学结构 Table 1　Chemical structures of ochratoxin derivatives

Metabolites	Abbreviations	Mw	R1	R2	R3	R4	R5	R6
Ochratoxin A	OTA	403	Phe	Cl	H	H	H	OH
Ochratoxin B	OTB	370	Phe	H	H	H	H	OH
Ochratoxin C	OTC	431	Phe ethyl ester	Cl	H	H	H	OH
Ochratoxin α	OTα	256	OH	Cl	H	H	H	OH
Ochratoxin β	OTβ	223	OH	H	H	H	H	OH
4R-hydroxy ochratoxin A	4R-OHOA	419	Phe	Cl	H	OH	H	OH
4S-hydroxy ochratoxin A	4S-OHOA	419	Phe	Cl	OH	H	H	OH
10-hydroxy ochratoxin A	10-OHOA	419	Phe	Cl	H	H	OH	OH
Ochratoxin A open lactone	OP-OA	421	Phe	Cl	H	H	−	OH
Ochratoxin B open lactone	OP-OB	388	Phe	H	H	H	−	OH
Ochratoxin α open lactone	OP-OTα	274	OH	Cl	H	H	−	OH
Ochratoxin β open lactone	OP-OTβ	241	OH	H	H	H	−	OH
Ochratoxin A quinone	OTQ	383	Phe	O	H	H	H	O
Ochratoxin A hydroquinone	OTHQ	385	Phe	OH	H	H	H	OH
OTHQ decarboxylated	DC-OTHQ	366	Decarboxylated Phe	OH	H	H	H	OH
Conjugate ochratoxin A quinone-glutathion	OTQ-glutathion	689	Phe	O	H	H	H	O
Conjugate ochratoxin A-acyl hexose	Acyl-hexose-OTA	565	Phe acyl hexose	Cl	H	H	H	OH
Conjugate ochratoxin A-acyl pentose	Acyl-pentose-OTA	535	Phe acyl pentose	Cl	H	H	H	OH
Ochratoxin A methyl ester	OTA-Me	417	Phe methyl ester	Cl	H	H	H	OH
Ochratoxin B methyl ester	OTB-Me	384	Phe methyl ester	H	H	H	H	OH
Ochratoxin B ethyl ester	OTB-Et	398	Phe ethyl ester	H	H	H	H	OH
4R-hydroxy ochratoxin A methyl ester	4R-OHOA-Me	433	Phe methyl ester	Cl	H	OH	H	OH
10-hydroxy ochratoxin A methyl ester	10-OHOA-Me	433	Phe methyl ester	Cl	H	H	OH	OH
Ethylamide ochratoxin A	OE-OA	430	Phe ethyl amide	Cl	H	H	H	OH
Ochratoxin A decarboxylated	DC-OA	359	Phe decarboxylated	Cl	H	H	H	OH
Ochratoxin A O-methyl	OM-OA	417	Phe	Cl	H	H	H	OCH₃
d-ochratoxin A	d-OA	403	d-Phe	Cl	H	H	H	OH
Ochratoxin α ester methyl	M-Oα	270	OCH₃	Cl	H	H	H	OH
Tyrosine ochratoxin A	OTA-tyrosine	419	Tyrosine	Cl	H	H	H	OH
−：该位置无官能团 −: No functional group at this position.

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2 产毒菌株

赭曲霉毒素A主要由曲霉属(Aspergillus)和青霉属(Penicillium)中部分菌株产生(表 2)，根据产毒菌所适应的气候条件不同，呈现出的污染分布存在差异，温带地区更多的是曲霉属，而青霉则更适应较为寒冷的环境^[6]。真菌毒素的产生与环境中产毒菌的密度密切相关^[7]，相较于高密度，低密度条件下真菌产生更多的真菌毒素，这是基于真菌的一种信号交流机制——群体感应(quorum sensing, QS)现象^[8]，OTA的产量受到环境中群体感应信号分子(quorum sensing molecules, QSM)氧脂素的影响，外源添加9-羟基十八碳二烯酸(9-hydroxyoctadecadienoic acid, 9-HODE)会促进OTA的产生，而外源添加13-HODE会抑制OTA的生成^[9-10]。以上结果提示，研究者对于产毒菌种类、数量及环境的控制也是防控OTA的关键。1965年，研究者在高粱穗上分离出赭曲霉(Aspergillus ochraceus) (section Circumdati)，并发现发霉谷物的毒性是由一种新的有毒代谢物即赭曲霉产生的OTA所引起的，20 kg的发霉谷物中共分离出了500 mg的OTA^[11]。在很长一段时间内，A. ochraceus都被认为是世界范围内主要的OTA产生菌，此后，许多曲霉和青霉被证实具有合成OTA的能力。1969年，首次发现了产OTA的青霉(Penicillium verrucosum)^[12]，截至目前，研究发现产OTA的曲霉属菌株主要归为曲霉属环绕曲霉群(section Circumdati or Aspergillus group ochraceus)和黑色曲霉群(section Black or Aspergillus group niger)^[13]。经过广泛的形态学、代谢组学和系统发育研究，Visagie等描述了Aspergillus section Circumdati中的一系列新菌种，其中的A. westerdijkiae和A. steynii是目前已知该属中最重要的OTA生产者^[14]。另外，一些之前被错误鉴定为A. ochraceus的分离株，包括最早发现产OTA的模式菌，经重新鉴定后归为A. westerdijkiae^[13]。Baba通过对曲霉属进行分类学评估，将section Circumdati分成3类：Circumdati、Sclerotiorum和Steyniorum^[15]。Aspergillus section Nigri也包含重要的OTA产毒菌株，如A. carbonarius、A. niger和A. welwitschiae；Aspergillus section Flavi中主要的产毒菌是A. alliaceus^[5]。但事实上不是所有的菌种都可以产生OTA，甚至由于环境或实验条件的差异，同一株菌是否产毒及产毒量也有不同的表现。如同样由西班牙烟熏红辣椒中分离出的A. ochraceus，菌株PDF2-1经HPLC测定产生OTA (12.1 μg/L)，而菌株PDF2和PDF3均未检测出产生OTA^[13]。除了A. westerdijkiae和A. steynii外，其他菌株产毒能力的研究尚无确切的结论。青霉属中重要的OTA产生菌有疣孢青霉(P. verrucosum)和日耳曼青霉(P. nordicum)，前者主要污染谷物，后者可以污染高盐、高蛋白食物如奶酪和咸肉，其他青霉属菌株OTA产生量往往较少，或者被错误判断^[6]。目前分离、鉴定的产毒菌株仍在增加，国外的研究开展得较多，表 2列出的产毒菌株仅有一株(A. birdgeri)是由中国科学院微生物研究所首先发现的^[16]。

表 2　部分产赭曲霉毒素A的菌株 Table 2　Species reported as OTA producers

Genera	Section	Species	Location	Source	Year	References
Aspergillus	Circumdati	Aspergillus ochraceus	South Africa	Sorghum	1965	[11]
		A. affinis	Italy	Decomposingleaves, fluvial mycobiota	−
		A. birdgeri	China	Preserved strain	1995	[16]
		A. carbonarius	−	Grapes	1996	[17]
		A. foetidus	−	Grapes	1996
		A. steynii	−	Coffee beans	2004	[18]
		A. neobridgeri
		A. flocculosus	India	Substratum
		A. pseudoelegans	Costa Rica	Soil in Gauguin
		A. westerdijkiae	South Africa	Sorghum
		A. cretensis	Israel	Citrus sp.
		A. roseoglobulosus	Bahamas	Decaying leaves of Mangrove (Rhizophora mangle)
		A. ostianus
		A. petrakii
		A. sclerotiorum			2002	[19]
		A. glaucus	Europe	Cereal grains	1987	[20]
		A. melleus	−	NRRL3519	1970	[21]
		A. sulphureus	Indian	NRRL4077
		A. fumigatus	−	Animal mixed feeds and raw material	1994	[22]
		A. versicolor	−
		A. lanosus	California, USA	Tree nuts	2000	[23]
		Neopetromyces muricatus	Australia	Peanuts	2000	[24]
		Petromyces alliaceus	Indonesia	Soybeans
	Nigri	A. lacticoffeatus	Venezuela, India	Coffee beans	2004	[25]
		A. sclerotioniger
		A. carbonarius	Kenya	Coffee beans
		A. tubingensis (received as A. miyakoensis, identified as A. awamori)	−	CBS 126.52
		A. niger	−	CBS 101699
		A. japonicus	Tropical soil	CBS 611.78
		A. awamori	−	NRRL 3112	2002	[26]
		A. foetidus	−	CBS 618.78	1996	[17]
		A. niger var. niger	−	A136	1994	[22]
		A. usamii	−	CBS 139.52	1995	[27]
		A. welwitschiae	Japan, Portugal, Spain, Italy, Greece	Koji, grapes
	Flavi	A. alliaceus	USA, Australia, Indonesia	Macrobasis albida, great barrier reef, Kemiri nut	1972	[28]
		A. albertensis	Canada	Ear swab	1996	[17]
Penicillium		Penicillium nordicum	Italy	Sausage	2001	[29]
		P. verrucosum	Canada	Wheat	1969	[12]
		P. brevicompactum	Hawaii, USA	Coffee	2006	[30]
		P. crustosum
		P. olsonii
		P. oxalicum
−：未报道内容 −: Unreported content.

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3 赭曲霉毒素A的污染情况

1965年，van der Merwe在高粱上分离到赭曲霉(A. ochraceus)，实验室培养条件下发现其能产生一种新的次生代谢产物，鉴定结构并将这种物质命名为赭曲霉毒素^[11]。1969年，Shotwell等首次在自然界的玉米中发现了这种OTA成分^[31]。1982年报道的大规模火鸡赭曲霉毒素A中毒事件使人们开始意识到OTA的危害^[32]。1991年，在60个对真菌毒素做出法律限制的国家中，却只有11个国家(巴西、捷克斯洛伐克、丹麦、法国、希腊、匈牙利、以色列、荷兰、罗马尼亚、瑞典和英国)有针对OTA的限量^[33]。到了2003年，FAO调查显示法律限量OTA的国家达到了37个，但相较于黄曲霉毒素的76个，OTA仍未受到特别重视。表 3列举了部分国家对OTA的限量标准，可以看出，可能考虑到加工环节、食物链富集、消化吸收能力等问题，不同食品的OTA限量差异较大，如饲料中OTA最大含量在100−300 μg/kg之间，而婴儿食品中则控制在1 μg/kg以下。

表 3　部分国家食品中OTA限量标准 Table 3　Maximum levels for OTA in food

Country	Foodstuffs	OTA (μg/kg)
Austria	Wheat, rye, durum wheat	5.0
Brazil	Rice, barley, beans, corn	50.0
Canada	Whole feed for poultry, whole feed for pigs	2.0
China	Cereals, legumes, nuts & seeds	5.0
	Wine	2.0
	Ground coffee	5.0
	Instant coffee	10.0
	Spices	15.0
China Taiwan	Rice, wheat, roasted coffee, ground coffee	5.0
Czech	Food	20.0
	Children food	5.0
	Baby food	1.0
	Cereals	5.0
French	Cereals	5.0
Germany	Pig kidney	25.0
Greek	Green coffee	20.0
Israel	Cereals products, beans	50.0
	Feed grains	300.0
Japan	Cereals	5.0
Korea	Wheat, barley, rye, coffee beans, roasted coffee	5.0
	Instant coffee	10.0
Romania	Food and Feed	5.0
Swedish	Whole feed for poultry	200.0
	Whole feed for pigs	100.0
Swiss	Cereals products	2.0
European Union	Cereals products	3.0
	Cereals	5.0
	Wine, grape juice	2.0
	Spice, pepper	15.0
	Roasted coffee	5.0
	Instant coffee	10.0
	Licorice	20.0
	Licorice extract	80.0
	Baby food	0.5

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OTA在不同环境中以不同的离子形式存在，包括非离子、负一价阴离子(OTA⁻)及负二价阴离子(OTA²⁻)，其性质稳定且不易降解，污染范围及种类十分广泛，可以通过食物链在动物体内蓄积，导致长期慢性的影响。近三年(2019−2021)欧盟食品与饲料快速预警系统(Rapid Alert System for Food and Feed, RASFF)通报的97例真菌毒素预警中，OTA占8%，仅次于AF (https://food.ec.europa.eu/safety/rasff-food-and-feed-safety-alerts_en)。OTA最主要的污染来源是粮食及其制品^[34]，之后依次是葡萄及其制品^[35]、咖啡豆及其制品^[36]、可可及其制品^[37]、豆类及其制品^[38]，还发现在牛奶及其制品^[39]、中草药^[40]、调味品^[41]、肉制品^[42]、干果类^[43]、水果及制品^[44]、婴儿食品^[45]中都检测到OTA。意大利检测的50份母乳中也发现了9份含有该毒素，范围在1.2−6.6 ng/mL之间，甚至连储藏室内的尘埃样本里也检测出了OTA^[46]，足以看出OTA的污染很广泛，目前的研究则开始重视塑料制品、食用色素及香辛料中OTA的存在。食品法典委员会(Codex Alimentarius Commission, CAC)依据2001年调查的21 367种样本的统计数据分析，发布OTA污染食品对人类威胁程度依次为：谷物 > 葡萄及其制品 > 咖啡及其制品 > 酒类 > 其他^[47]。我国公民饮食习惯属于典型的东方膳食体系，与欧美为代表的西方膳食体系和地中海膳食体系相比谷物摄入量更高，约为西方的4倍，因此OTA对我国公民的威胁更严峻^[48]。

OTA是全球食品和饲料的普遍污染物，大田或采后的谷物里一般都存在OTA。为此，世界卫生组织/粮农组织食品添加剂联合专家委员会(Joint FAO/WHO Expert Committee on Food Additives, JECFA)在第44届会议上提出OTA的每周容许摄入量(provisional tolerable weekly intake, PTWI)为100 (μg/kg-bw)；2001年的第56次会议上提出推算OTA所致肾毒性的最大无作用剂量(maximal no-effective dose, NOEL)的安全系数为1 500^[5]。2007年7月在罗马召开的第30届食品法典委员会中，修改并通过了婴儿配方食品、蛋和蛋制品及葡萄酒等若干食品安全和质量标准，尤其是对防止或减少葡萄酒中的OTA污染的所有措施进行了讨论。由此推测，OTA的防控与脱除已经在除谷物外的非主食酒类生产方面得到了足够的关注，OTA的研究在食品安全领域正在成为继黄曲霉毒素之后的又一新热点。

4 赭曲霉毒素A的毒性研究

赭曲霉毒素A已经被证明对动物具有多种毒性作用，其半数致死量(median lethal dose, LD₅₀)具有物种特异性，依给药途径、动物种类和品系不同而异，属于剧毒化学品，其LD₅₀ < 1 (mg/kg-bw)，从LD₅₀的结果可以看出：狗、猪对OTA最敏感，其次是大鼠和小鼠，而且幼鼠较成鼠敏感^[3]。动物实验结果显示，长时间摄入中、低浓度的OTA (8 μg/kg小鼠体重)也可能危害健康^[49]。1987年国际癌症研究机构(International Agency for Research on Cancer, IARC)将OTA定义为Ⅲ类致癌物，但随着动物实验中OTA致癌证据的增加，1993年IARC将其重新定义为ⅡB类致癌物(人类可能致癌物)^[3]。OTA是人体循环中停留时间最长的霉菌毒素，其对血清白蛋白有较强的亲和力，高达99%，非电离形式的OTA会经被动吸收进入细胞，显著延长其在血液中的半衰期^[50]，并且白蛋白与OTA的结合会限制其从血液到肝脏、肾脏的转移，影响OTA的消除^[51]，其足够的累积量和长期慢性毒性效应在一定程度上会导致人类肿瘤的发生^[52]。OTA对人类的危害尚无直接证实，但一系列证据表明其可能是巴尔干地方性肾病(Balkan endemic nephropathy, BEN)^[53]、地区性慢性间质性肾炎(chronic interstitial nephritis, CIN)^[54]、尿道肿瘤(urinary tract tumors, UTT)^[55]等人类疾病的原因，有学者怀疑糖尿病(diabetes)也与赭曲毒素A有一定的联系^[50]。

OTA的主要靶器官是肾脏，研究发现OTA对所有单胃哺乳动物均具有肾毒性，这是由于丙磺胺作用中止了OTA的转移和清除，导致器官积累的OTA超出负荷；最早表现为上皮细胞受损，慢性长期接触后肾脏变小，细胞间质出现纤维化；后期肾小球滤过率降低、近端肾小管功能障碍，肾功能受损，导致尿量减少与浓度降低、出现酶尿(如γ-谷氨酰转移酶、碱性磷酸酶、乳酸脱氢酶)、蛋白尿、血尿素氮升高、尿糖、血清肌酐升高等症状，最终导致肾脏完整性病变，病患同时并伴有舌头红肿、口渴、味苦等表象，部分患者出现头痛、腰痛、虚弱和缺铁性贫血等症状^[56-57]。OTA的其次靶器官是肝脏，禽类中OTA主要残留于肝脏，引起肝脏细胞的细胞膜增厚、内质网减少、线粒体肿胀溶解、肝细胞溶解等现象，导致肝糖原积聚，解化率降低，病死率增高^[58-59]。OTA吸收主要发生在单胃动物的胃肠道中，吸收率具有物种特异性，人体中的生物利用率为93%，猪约为60%，啮齿类则更低^[60]，但OTA影响宿主的肠道屏障和吸收功能，具有肠道毒性^[61-62]。反刍动物胃肠道中存在的羧肽酶、蛋白水解酶等可以将OTA降解为OTα等无毒产物，这种脱毒酶也可在细菌中表达^[63-64]。低剂量的OTA (0.20‒1.66 mg/kg)不会对牛产生毒性影响，但是高剂量的OTA (13 mg/kg)仍会使牛出现临床症状(如厌食、腹泻、产奶量难以上升和快速下降等)^[65]。OTA具有免疫毒性，可以抑制外周T细胞和B淋巴细胞的增殖，损害巨噬细胞的吞噬能力，降低白细胞介素-2及其受体的含量，减少免疫球蛋白G (immunoglobulin G, IgG)和免疫球蛋白M (immunoglobulin M, IgM)，以及降低杀伤细胞的活性等^[66]。当OTA以带电离子形式存在时，可以在有机阴离子转运体4 (organic anion-transporter 4, OAT4)的帮助下，通过主动转运穿透胎盘屏障并蓄积在胎儿组织内，也可以通过母乳影响婴儿^[67-68]。由于后代中的解毒效率较慢，因此婴儿血液中的OTA负荷量高于母体^[69]。另有研究显示，OTA会限制啮齿类动物睾丸间质细胞的睾酮分泌^[70]。另外，OTA抑制大鼠细胞系的蛋白合成与DNA复制，对神经元细胞的影响显著大于对星形细胞的作用，推测其可能影响脑部的特殊结构^[71-72]。图 2显示了动物实验中OTA效应的分子机制^[5]。

图 2　赭曲霉毒素A毒性动力学的分子机制 Figure 2　General outline of the molecular mechanism involved in the toxicokinetics of the ochratoxin. 细胞色素P450 (cytochrome P450, CYP450)酶系的作用激发OTA的生物转化，其中低毒性残基被排出体外，高毒性代谢物诱导氧化应激，产生的一系列效应引起细胞毒性，导致可能的癌症反应 Action of CYP450 cohorts of enzymes instigated the biotransformation of OTA, whereby the low toxic residues are excreted, and the high toxic metabolites induce oxidative stress leading to a cascade of events which interfere the cellular mechanisms leading to carcinogenesis and cytotoxicity.

图选项

赭曲霉毒素A的病理学及毒理学研究侧重于动物试验的结果，对受试动物具有致畸、致癌的作用，致突变的影响结果不确定。根据真核生物、原核生物、哺乳动物细胞实验结果推测OTA毒性作用机理：首先，OTA通过影响线粒体呼吸途径抑制ATP生成，最终因为ATP的耗竭导致无法正常供能；其次，OTA主要通过与苯丙氨酸竞争苯丙氨酸-tRNA结合位点的方式抑制苯丙氨酸-tRNA转移酶，致使肽链的延长被终止，此外，OTA会降低苯丙氨酸羟化酶的活性，抑制DNA、RNA和蛋白质的合成；最后，OTA会影响超氧化物歧化酶和过氧化氢酶的活性，促进细胞膜脂的过氧化，使脂质过氧化物含量增加，产生活性氧(reactive oxygen species, ROS)造成氧化损伤^[73-76]。

了解OTA的毒性机理更有利于瞄定整个毒性作用的关键环节设计药物靶标，如急性OTA中毒出现呼吸系统紊乱时可紧急补充ATP、降低OTA的竞争性抑制效应、清除活性氧以减少氧化损伤等。虽然目前对于OTA的危害仍然处于“防控”大于“降低”的阶段，尚无具体针对OTA中毒后的解毒药物，但对OTA所造成的肝、肾等损伤的治疗将持续作为医学上的研究重点。

5 OTA生物合成途径

OTA是聚酮化合物的衍生物，由l-苯丙氨酸通过酰胺键连接异香豆素组成，二氢异香豆素环上的氯原子为OTA的毒性基团，脱去氯的OTB毒性仅为OTA的十分之一，其他结构类似物，如OTC、OTα、OTβ等均不具有显著毒性^[77]。最早根据化学结构推测其生物合成过程，利用放射标记实验确定了乙酸钠和丙二酸作为OTA生物合成的原料，乙酸钠既被转化为苯丙氨酸部分，又作为异香豆素部分前体，而丙氨酸只参与异香豆素部分的合成^[78-79]。OTA首先由五分子的乙酰CoA形成二氢异香豆素骨架，S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)为二氢异香豆素环提供C-7位的一碳单位——甲基, 后被氧化为羧基，随后与苯丙氨酸的氨基进行缩合形成酰胺键而合成OTA。OTA合成过程中存在着中间产物，OTα是在C-7处带有羧基的二氢异香豆素衍生物，连接苯丙氨酸合成OTA，OTβ和OTB是对应的脱氯类似物^[4]。蜂蜜曲霉素和4-羟基蜂蜜曲霉素在结构上类似于OTA和OTB的二氢异香豆素部分，随着生物信息学和分子生物学的发展，已经能从基因水平上推测OTA的合成机理^[80-82]。从1970年至今，陆续发现OTA生物合成相关基因分别编码聚酮合酶(polyketide synthase, PKS)家族、非核糖体肽酶(nonribosomal peptide synthetase, NRPS)、卤化酶(halogenase, HAL)、单加氧酶(cytochrome P450 monooxygenase, P450)、环化酶(cyclase, CYC)、碱性亮氨酸拉链结构调控因子(bZIP)和锌指结构辅助调控因子(Zn₂Cys₆)，但OTA的生物合成过程一直未被具体阐明，仍在不断地验证^[83]。Bennett等的研究认为，乙酸缩合成聚酮化合物蜂蜜曲霉素随后被转化为OTβ，引入氯原子形成OTα，OTα再通过与苯丙氨酸乙酯结合转化为OTC以保护中间体，通过去酯化将OTC转化为OTA^[84]。然而，Harris等根据前体添加实验认为OTC并非OTA生物合成的前体，苯丙氨酸直接连到潜在优势途径中，实现从OTβ到OTα再到OTA的生物转化，他们提出的另一种途径是OTβ与苯丙氨酸连接，然后转化为重要的中间体OTB，最后经过氯化步骤生成OTA^[85]。Gallo等假设的OTA生物合成途径类似于Harris提出的第二种途径，而OTα是OTA水解的副产物^[86]。Wang等最新研究中推测聚酮合酶PKS (OtaA)作为OTA合成的启动酶，利用乙酰辅酶A和丙二酰辅酶A合成7-甲基蜂蜜曲菌素，进而被单加氧酶P450 (OtaC)催化氧化成OTβ，OTβ和l-β-苯基丙氨酸在非核糖体肽酶NRPS (OtaB)催化下合成酰胺键形成OTB，最后，OTB在卤代酶HAL (OtaD)催化下形成OTA^[82]。otaR1为途径特异性调控基因，编码产生簇特异性调控因子bZIP (OtaR1)，otaR2编码辅助锌指结构调控因子Zn₂Cys₆ (OtaR2)，共同控制4个合成基因(otaABCD)的表达，调控OTA合成^[82]。图 3中整合了自1970年来对于OTA合成路径的推论演变。

图 3　OTA合成机理研究进展 Figure 3　Study on OTA synthesis mechanism. AC(adenylyl cyclase)腺苷酸环化酶；PKA(protein kinase A)蛋白激酶A；bZIP (basic region-leucine zipper)碱性亮氨酸拉链结构转录因子；OtaR2锌指结构转录因子；

表示当时尚未确证的酶系 AC: adenylyl cyclase; PKA: protein kinase A; bZIP (basic region-leucine zipper): basic leucine zipper transcription factor; OtaR2: zinc finger structure transcription factor;

represents enzyme lines that were not identified at the time.

图选项

相较于黄曲霉毒素与杂色曲霉素合成极其详尽的研究，OTA合成受多种因素及相关调控网络的调控^[87]，但细节仍不清楚，如同属香豆素族的AF合成聚酮化合物中，除PKS外还发现脂肪酸合成酶基因(aflA, aflB)，还有氧化还原酶基因、单加氧酶基因等，是否在OTA合成中也会有类似发现尚待探索。在曲霉属和青霉属中均有高度保守的OTA合成基因簇，包括pks、nrps、hal/chl、p450和bzip等，均存在bZIP结合的回文序列ATGACGTGTA或TACACGTCAT^[88]。辅助转录因子OtaR2与bZIP一样通过与簇内基因启动子结合，共同作为OTA合成的调控元件，其调控表达的氧化还原酶OtaE具有P450类似的功能^[89]，但该合成簇外是否有基因参与OTA合成未见报道。

6 展望

目前，赭曲霉毒素A被认为是农业中最重要的五大真菌毒素之一，因此，必须对赭曲霉毒素特别是OTA进行持续性的重点关注，最终目的是保护公众健康并且防止因OTA污染造成的经济损失。结合近年来的研究与报道，未来对于OTA的研究方向包括以下方面：(1) 利用分子生物学和生物信息学手段，丰富产毒菌的全基因组数据库，更准确地鉴定产毒菌的种属归类；(2) 扩充动物实验及人类病理学数据，深入研究OTA毒性作用机制；(3) 鉴定OTA生物合成基因簇及关键基因，推测具体的合成流程，以便施行更精准的靶向调控；(4) 观察环境因素对真菌毒素产生的影响，分析调控机制，推导防控关键环节；(5) 健全毒素产后脱除的物理化学等普适方法，同时大力研发推广绿色且高效的生物脱毒技术；(6) 提高群众对OTA的重视，加大真菌毒素危害的科普及预防、脱除手段的普及；(7) 完善更多样食品中OTA的限量标准，研发更高效、经济、多样性的快检技术。

简而言之，由于真菌无处不在和适应力极强的生物特性，而且真菌处于重要的生态位，对于生态系统的平衡起着至关重要的作用，这使得根除真菌及毒素是不可能的事情，但是可以通过全面、深入、创新的研究规避其威胁的同时弘扬有利的一面，实现人类与微生物和平共处。

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