【作者】 刘静；

【导师】 张祥宏；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2012， 博士

【摘要】 饮食及环境中真菌毒素的污染是人类面临的重要公共卫生问题，它与人类疾病的发生关系密切。2006年，在对我国胃癌高发区之一的河北省赞皇县（胃癌年均死亡率为77.67/10万）居民食用小麦检测时发现赭曲霉毒素A（Ochratoxin A，OTA）的检出率高达45.16%，当地居民OTA的日暴露量为1.17μg/kg，远远超过世界卫生组织/粮农组织联合专家委员会（Joint FAO/WHO Expert Committee on FoodAdditives，JECFA）暂定的每周允许摄入量100ng/kg。说明当地居民进食OTA污染粮食可能与胃癌的发生发展有密切关系。OTA是由曲霉菌属和青霉菌属的某些菌株产生的一种污染广泛的真菌毒素，普遍存在于粮食、饮料、饲料和动物组织中。OTA化学性质稳定，在血液中的半衰期是35天。流行病学调查数据显示OTA可能与巴尔干地方性肾病、慢性间质性肾病和泌尿系上皮肿瘤有关。体内、外实验研究发现，OTA具有肾毒性、肝毒性、神经毒性、免疫毒性和致畸性。因此，国际癌症研究中心（InternationalAgency for Research on Cancer，IARC）将OTA列为“B级可能人类致癌物”。研究发现许多致癌性真菌毒素早期毒性作用是导致细胞增殖抑制、细胞周期阻滞和凋亡。在针对OTA致胃癌可能机制的研究中，我们的前期体外实验发现OTA可以抑制人胃黏膜上皮细胞GES-1细胞增殖，通过MAPK（p38，ERK）信号通路和ATM（ATM-Chk2，ATM-p53-p21WAF1/CIP1）信号通路介导G2期阻滞，并且诱导细胞凋亡。实验揭示OTA诱导胃组织的靶细胞-胃黏膜上皮细胞细胞周期G2期阻滞可能在胃癌的发生发展过程中起重要作用。但是引起这一系列细胞级联反应的原因及其上游事件并不清楚。探讨OTA这一生物学效应可能对进一步认识其参与胃癌发生的可能机制具有重要意义。OTA的潜在致癌性在二十世纪七十年代被认可，但是其致癌机制至今并不十分清楚。JECFA等专家组织在一些高度可能的机制中，越来越聚焦于氧化应激机制。研究发现，很多致癌性真菌毒素都可以诱导细胞DNA氧化应激损伤，进而导致细胞周期阻滞或凋亡。已有文献报道OTA可以诱导人肝细胞和肾脏细胞DNA氧化应激损伤。活性氧（reactiveoxygen species，ROS）对生物大分子（特别是DNA）的氧化损伤被认为是启动和促进肿瘤发生的最重要因素。细胞周期检测点的激活和修复系统的启动是DNA损伤引发的细胞级联反应，这使得细胞在复制前或有丝分裂前有足够的时间修复受损DNA，修复成功的细胞脱离周期阻滞进入细胞周期；修复失败的细胞会发生凋亡，这对于维持基因组的稳定性有重要意义。线粒体是细胞进行电子传递、三羧酸循环和氧化磷酸化产生能量的细胞器。同时也是氧化应激损伤的靶细胞器，是产生ROS的主要部位，线粒体功能受损时ROS显著增加。所以，OTA诱导氧化应激损伤可能在OTA诱发胃黏膜上皮细胞细胞周期阻滞的毒理学效应中起重要作用。为验证这一假设，本研究在前期实验的基础上选取人胃黏膜上皮细胞株GES-1，从氧化应激角度入手，首先观察OTA对GES-1细胞内ROS含量的影响，评价OTA诱导GES-1细胞DNA损伤的性质及特征，基于抗氧化策略的DNA损伤靶向干预揭示OTA诱导的氧化应激与DNA损伤和G2期阻滞的关系；接着研究OTA对GES-1细胞线粒体损伤的影响，探讨OTA诱导GES-1细胞ROS产生的分子机制。肿瘤的发生发展是多因素综合作用的结果，在肿瘤的发展、转移过程中免疫功能抑制发挥重要作用，免疫功能降低可能导致肿瘤发生几率增加。致癌性霉菌毒素诱发机体的免疫毒性作用与肿瘤的发生具有密切关系。为了进一步验证OTA诱发氧化应激损伤介导机体免疫细胞周期阻滞参与其免疫毒性作用，本研究选取人外周血单个核细胞（human peripheralblood mononuclear cells，hPBMC）作为研究对象，检测OTA对hPBMC细胞的氧化应激损伤作用和对细胞周期的影响及其可能分子机制。本研究拟从OTA作用靶细胞和机体免疫细胞两个方面揭示OTA诱导氧化应激损伤介导细胞周期阻滞的毒理学特性及其可能的分子机制，丰富和加深了人们对OTA生物效应的认识，为OTA暴露的合理处置提供分子位点，这将有助于全面评价OTA在胃癌发生、发展中的可能作用。第一部分赭曲霉毒素A诱导GES-1细胞氧化应激损伤介导G2期阻滞目的：探讨赭曲霉毒素A诱导人胃黏膜上皮细胞GES-1氧化应激损伤作用，及ATM和MAPK信号通路在氧化应激损伤介导GES-1细胞G2期阻滞中的调控作用。方法：1采用荧光探针DCFH-DA和DHE检测5、10、20μM OTA处理GES-1细胞24h后，细胞内ROS含量。2采用超氧化物歧化酶（SOD）测试盒检测OTA对GES-1细胞SOD活性的影响。3采用高效液相-电化学检测技术、免疫荧光技术和Western blot方法分析OTA处理后GES-1细胞DNA的损伤情况。4在以上研究的基础上给予抗氧化剂N-乙酰半胱氨酸（NAC）4mM预处理1h，观察OTA处理对GES-1细胞ROS、SOD水平的影响及对DNA的损伤作用。5采用流式细胞术检测NAC预处理对OTA作用后细胞周期的影响。6采用Western blot方法检测NAC预处理对OTA作用后细胞周期关键调控分子Cdc25C、Cdc2和cyclinB1，信号通路ERK、p38MAPK、ATM蛋白表达及其磷酸化的变化情况。7采用免疫共沉淀技术检测NAC预处理对Cdc2-cyclinB1复合物形成的影响。结果：1.1OTA对GES-1细胞ROS水平的影响流式细胞术检测结果显示，5、10、20μM OTA处理组DCF、DHE平均荧光强度均明显高于对照组（P<0.05）；抗氧化剂NAC预处理1h，DCF、DHE平均荧光强度明显减低（P<0.05）。结果提示，OTA诱导GES-1细胞ROS生成增多，NAC缓解了OTA的促ROS升高作用。1.2OTA对GES-1细胞SOD活性的影响SOD活性检测结果表明，GES-1细胞经OTA（5、10、20μM）处理24h，SOD活性分别为285.77±39.81、402.79±18.20、737.80±44.73U/mgprotein，显著高于对照组157.51±12.49U/mg protein（P<0.05）；4mM NAC预处理+10μM OTA处理组SOD活性较单独10μM OTA处理组明显降低（202.35±10.51vs394.74±16.12U/mg protein，P<0.05）。1.3OTA对GES-1细胞DNA损伤的影响8-OHdG为氧化应激损伤标志物。高效液相-电化学检测结果显示，不同浓度OTA作用GES-1细胞24h，8-OHdG含量明显较对照组升高（P<0.05）。γ-H2AX为DNA双链断裂标志物，免疫荧光结果观察到OTA处理组GES-1细胞形成了典型的γ-H2AX焦点。Western blot结果显示，各个OTA处理组γ-H2AX蛋白表达上调（P<0.05）。为了进一步明确ROS升高导致GES-1细胞DNA损伤，Western blot方法检测了NAC预处理后γ-H2AX蛋白的变化情况。结果显示，NAC+OTA处理组γ-H2AX蛋白表达水平明显低于OTA处理组（P<0.05）。综合以上实验结果表明，OTA诱导GES-1细胞DNA发生氧化损伤。1.4NAC预处理对OTA作用后ATM表达的影响为了进一步探讨ROS诱导GES-1细胞DNA损伤参与了OTA激活ATM激酶，我们给予抗氧化剂NAC清除ROS。Western blot检测结果显示，NAC预处理组ATM磷酸化水平明显低于单独OTA处理组（P<0.05），ATM蛋白表达水平则明显高于OTA处理组（P<0.05），说明抗氧化剂拮抗了OTA对GES-1细胞ATM激酶的激活作用。1.5NAC预处理对OTA作用后ERK和p38MAPK信号通路的影响为了进一步明确ROS参与了OTA处理对ERK和p38信号通路的激活作用，给予抗氧化剂NAC预处理，应用Western blot方法检测了ERK、p38蛋白及其磷酸化水平的变化。结果显示，NAC预处理+OTA处理组ERK磷酸化水平和p38磷酸化水平显著低于OTA处理组（P<0.05），提示NAC拮抗了OTA对GES-1细胞ERK和p38MAPK信号通路的激活作用。1.6NAC预处理对OTA作用后GES-1细胞周期的影响为了进一步证明OTA诱导氧化应激损伤通过ATM和MAPK途径介导GES-1细胞发生G2期阻滞，在前面NAC拮抗了OTA对GES-1细胞ATM和MAPK（ERK和p38）通路激活的基础上，本研究在给予抗氧化剂NAC预处理后，观察了OTA对GES-1细胞周期及周期调控蛋白的影响。流式细胞术细胞周期检测结果显示，NAC预处理组G2/M期的细胞比例较10μM OTA处理组显著减少（P<0.05）。结果提示NAC预处理可以部分逆转OTA诱导的GES-1细胞G2期阻滞。Western blot结果显示，NAC预处理后，GES-1细胞Cdc25C、Cdc2和cyclinB1的蛋白表达水平及p-Cdc25C和p-Cdc2水平均较10μM OTA单独处理组明显升高（P<0.05）。结果表明NAC缓解了OTA对的G2期关键调节因子（Cdc25C、Cdc2和cyclinB1）的抑制作用。实验结果揭示OTA诱导氧化应激损伤可能通过ATM和MAPK途径介导GES-1细胞发生G2期阻滞。第二部分赭曲霉毒素A对GES-1细胞线粒体的损伤作用目的：线粒体是产生ROS的重要细胞器，其损伤启动细胞的氧化应激损伤，本部分探讨OTA对GES-1细胞线粒体DNA、功能的影响。方法：1采用超氧化物歧化酶（SOD）分型测试盒检测5、10、20μMOTA处理24h，GES-1细胞MnSOD活力变化情况。2高效液相-电化学检测线粒体DNA（mtDNA）中8-OHdG含量的变化。3Real-time PCR检测mtDNA编码的呼吸链上的13个亚基（ND1，ND2，ND3，ND4，ND4L，ND5，ND6，COXⅠ，COXⅡ，COXⅢ，Cytb，ATP6，ATP8）mRNA水平变化情况。4采用线粒体呼吸链复合体I活性比色法定量检测试剂盒检测呼吸链复合体I活性。5采用Oxygraph-2k液相氧电极测定透膜细胞中线粒体氧耗速率。6罗丹明123染色，流式细胞术检测线粒体膜电位的变化。7Annexin V-FITC染色，流式细胞术检测GES-1细胞凋亡情况。8Western blot检测MnSOD、碱基切除修复基因OGG1和凋亡因子Bax、Bcl-2、Bcl-xL、cytochrome c、caspase-9的表达情况。9给予GES-1细胞抗氧化剂4mM NAC预处理1h，检测OTA对GES-1细胞凋亡的影响。结果：2.1OTA对GES-1细胞MnSOD的影响MnSOD是线粒体中清除ROS的主要抗氧化酶。Western blot结果显示，各个OTA处理组MnSOD蛋白条带逐渐增强，明显高于对照组（P<0.05）。MnSOD酶活性检测结果发现，5、10、20μM OTA处理组MnSOD活性分别为115.26±9.83，162.63±13.18和285.04±3.10U/mgprotein，显著高于对照组64.73±4.42U/mg protein（P<0.05）。2.2OTA对GES-1细胞mtDNA8-OHdG水平的影响应用高效液相-电化学检测技术检测8-OHdG，用以分析OTA对mtDNA的氧化损伤。结果显示OTA处理组8-OHdG水平明显较对照组升高（P<0.05）。提示OTA诱导GES-1细胞mtDNA发生氧化损伤。2.3OTA对GES-1细胞线粒体中OGG1蛋白水平表达的影响Western blot结果显示，OTA5、10、20μM处理组线粒体中OGG1蛋白条带较对照组显著减弱（P<0.05）。结果提示，OTA抑制线粒体碱基切除修复途径。2.4OTA对线粒体基因表达的影响Real-time PCR检测了线粒体DNA编码的氧化呼吸链复合体的13个亚基mRNA水平的变化。5、10μM OTA处理组各亚基mRNA水平无明显变化（0.5＜Fold change＜1.5）；20μM OTA处理组复合体I亚基ND1、ND2、ND3、ND4L、ND5和ND6mRNA表达水平较对照组明显升高（Foldchange＞1.5），ND4mRNA变化不明显；复合体III亚基cytb和复合体IV亚基COXI、COXII、COXIII mRNA无明显变化（0.5＜Fold change＜1.5）；复合体V亚基ATP6mRNA水平明显升高（Fold change＞1.5），ATP8mRNA水平无明显变化（0.5＜Fold change＜1.5）。2.5OTA对线粒体呼吸链复合体I活性的影响Real-time PCR检测发现OTA主要诱导呼吸链复合体I亚基mRNA表达升高，为了进一步明确OTA对复合体I的影响，对其活性进行了检测。结果发现各OTA处理组（5、10、20μM）复合体I活性均明显低于对照组（P<0.05），提示OTA抑制了线粒体呼吸链复合体I活性。2.6OTA对线粒体呼吸的影响检测结果发现，5、10、20μM OTA处理组线粒体ST3呼吸速率均较对照组显著降低（P<0.05）；ST4呼吸速率无明显变化；10和20μM OTA处理组呼吸控制比（RCR）与对照组相比明显降低（P<0.05），提示OTA抑制了GES-1细胞线粒体呼吸功能。2.7OTA对GES-1细胞凋亡的影响因为线粒体损伤后会启动线粒体途径介导细胞凋亡，本实验进一步验证OTA致线粒体损伤诱导细胞氧化应激损伤导致细胞通过线粒体途径凋亡。2.7.1OTA对线粒体膜电位（ΔΨm）的影响线粒体膜电位降低是线粒体功能障碍的特点，是细胞凋亡的早期事件之一。流式细胞术检测结果显示，与对照组相比，OTA处理明显减低了罗丹明123的平均荧光强度（P<0.05）。为了探讨ROS是否参与了ΔΨm的变化，抗氧化剂NAC预处理细胞1h再给予10μM OTA孵育24h，罗丹明123平均荧光强度较单独10μM OTA处理组明显升高（P<0.05）。提示OTA诱导ROS生成增加介导了GES-1细胞线粒体膜电位降低。2.7.2NAC对OTA诱导GES-1细胞凋亡的影响为了进一步明确OTA通过氧化应激诱导GES-1细胞凋亡，实验采用抗氧化剂NAC预处理来减少ROS。Annexin V-PI双染法检测结果显示，10μM OTA处理组细胞凋亡率为12.67±2.12%，明显高于对照组3.03±0.77%；4mM NAC+10μM OTA处理组细胞凋亡率为5.56±1.80%，较10μM OTA处理组显著降低（P<0.05）。2.7.3OTA对凋亡相关蛋白的影响为了研究线粒体途径是否参与了OTA诱导的GES-1细胞凋亡，我们首先检测了促凋亡基因Bax和抑凋亡基因Bcl-2、Bcl-xL的蛋白水平变化。Western blot检测结果显示，OTA处理增加了Bax蛋白水平；相反，降低了Bcl-2和Bcl-xL的蛋白表达量（P<0.05）。NAC预处理1h， NAC+OTA处理组Bax蛋白表达较相应的OTA处理组明显降低，而Bcl-2和Bcl-xL的蛋白表达明显升高（P<0.05）。Cytochrome c从线粒体释放到细胞胞浆是线粒体凋亡途径级联反应的一个关键步骤，激活下游的caspase。Western blot分别检测了线粒体部分和胞浆部分cytochrome c的蛋白变化情况。OTA暴露24h，线粒体中cytochrome c蛋白表达量明显减少；同时，胞浆中cytochrome c表达量明显增加（P<0.05）。NAC抗氧化剂预处理后，和相应的单独OTA处理组相比，NAC+OTA处理组线粒体部分cytochrome c蛋白表达量明显升高，胞浆部分明显减少（P<0.05）。释放到胞浆的cytochrome c激活caspase-9。各种浓度OTA处理GES-1细胞后，非活性procaspase-9蛋白表达降低，而活性形式表达增强（P<0.05）。NAC明显逆转了OTA对caspase-9的激活作用（P<0.05）。综合以上实验结果，提示OTA通过氧化应激激活线粒体途径介导GES-1细胞凋亡。第三部分赭曲霉毒素A诱导人外周血单个核细胞DNA氧化损伤及其介导G1期阻滞目的：探讨OTA对人外周血单个核细胞（hPBMC）的氧化应激损伤作用及其毒理学效应。方法：1采用荧光探针DCFH-DA和DHE，应用流式细胞术检测OTA（5，10，20μM）处理24h对hPBMC细胞ROS含量的影响。2采用谷胱甘肽（GSH）检测试剂盒检测hPBMC细胞内GSH水平的变化。3采用高效液相-电化学方法、碱性彗星实验及Western blot技术检测hPBMC细胞DNA的损伤情况。3采用流式细胞术检测OTA对hPBMC细胞周期的影响。4采用Western blot方法检测OTA作用后细胞周期关键调控分子CDK4和cyclinD1在蛋白水平的表达变化。5应用流式细胞术、Hoechst33258荧光染色分析OTA对hPBMC细胞凋亡的影响。6给予抗氧化剂NAC预处理后重复上述实验，探讨氧化应激参与了OTA的生物效应。结果：3.1OTA对hPBMC细胞ROS的影响流式细胞术结果显示，5、10、20μM OTA处理24h后，DCF和DHE的平均荧光强度均明显增加（P<0.05）。给予4mM NAC预处理1h，20μMOTA+4mM NAC处理组DCF的平均荧光强度显著低于20μM OTA单独处理组；DHE的平均荧光强度较OTA单独处理组也显著降低（P<0.05）。提示OTA处理可以诱导ROS生成增加，此作用可以被抗氧化剂NAC缓解。3.2OTA对hPBMC细胞内GSH含量的影响给予不同浓度OTA处理24h后，hPBMC细胞内GSH含量明显较对照组降低（P<0.05）。NAC预处理组GSH水平较OTA处理组明显升高（P<0.05）。因为NAC是巯基化合物，既可以直接清除自由基，又能参与还原型谷胱甘肽的合成，本实验提示NAC可以保护hPBMC细胞，防止OTA导致GSH含量降低，进一步证明OTA对hPBMC的氧化应激作用。3.3OTA对hPBMC细胞DNA损伤的影响高效液相-电化学检测结果显示，各种浓度OTA处理组中8-OHdG的水平明显高于对照组（P<0.05）。荧光显微镜下观察碱性彗星实验结果发现，OTA处理组细胞电泳后出现了“彗星”现象，彗星拖尾明显；20μM OTA+4mM NAC处理组细胞彗星尾部小于OTA处理组。统计分析发现，与对照组相比，各处理组hPBMC细胞彗星的Tail DNA%、Tail Length及Olive Tail Moment值均明显高于溶剂对照组（P<0.05）。抗氧化剂NAC预处理1h，上述指标均显著降低（P<0.05）。Western blot结果显示，5、10、20μM OTA处理组γ-H2AX蛋白表达明显增加（P<0.05）。NAC预处理明显下调γ-H2AX表达（P<0.05）。上述结果表明OTA诱导hPBMC细胞DNA发生氧化损伤。3.4OTA对hPBMC细胞周期的影响流式细胞术检测结果发现，10、20μM OTA处理组G1期细胞比例分别为52.27±3.00%、52.67±1.45%，显著高于对照组31.03±7.88%（P<0.05）。NAC预处理后，NAC+OTA处理组G1期细胞比例较OTA处理组显著降低（37.07±2.48%vs48.30±2.07%，P<0.05）。Western blot结果显示，OTA处理可以降低hPBMC细胞cyclinD1和CDK4蛋白表达（P<0.05）。4mM NAC预处理+20μM OTA处理组cyclinD1和CDK4蛋白表达均较单独20μM OTA处理组明显提高（P<0.05）。综合实验结果表明OTA诱导hPBMC细胞发生G1期阻滞，抑制cyclinD1和CDK4蛋白表达；NAC可以逆转OTA对cyclinD1和CDK4蛋白表达的抑制作用，从而缓解G1期阻滞。3.5OTA对hPBMC细胞凋亡的影响流式细胞术细胞周期检测结果显示，hPBMC细胞经5、10、20μM OTA处理24h后，OTA各处理组细胞凋亡率分别为5.99±1.54%、7.07±1.30%和11.84±1.51%，均高于溶剂对照组3.77±0.38%（P<0.05）。NAC预处理，部分缓解了OTA的促凋亡作用（7.12±0.94%vs17.81±1.23%，P<0.05）。荧光显微镜下观察Hoechst33258荧光染色法凋亡细胞形态学改变，20μM OTA作用24h后，hPBMC细胞胞核出现核固缩、碎裂，产生凋亡小体，NAC预处理组没有观察到明显的细胞核改变。结果提示，OTA诱导细胞氧化应激损伤介导对hPBMC的促凋亡作用。结论：1OTA可以诱导GES-1细胞发生氧化应激损伤。2OTA诱导细胞氧化应激损伤通过ATM和MAPK通路参与其诱导GES-1细胞G2期阻滞的发生。3OTA诱导GES-1细胞线粒体DNA损伤，抑制碱基切除修复功能，呼吸功能降低；这可能是GES-1细胞ROS水平升高、发生氧化应激损伤的重要原因。4OTA激活线粒体途径诱导GES-1细胞发生凋亡。5OTA可以诱导hPBMC细胞ROS生成增多，发生DNA氧化损伤，进而介导hPBMC细胞G1期阻滞和凋亡。这可能参与了OTA的免疫抑制生物效应。6OTA通过诱发氧化应激损伤作用介导胃组织靶细胞和机体免疫细胞细胞周期阻滞和凋亡，这一毒理学效应可能与OTA参与胃癌的发生、发展具有密切关系。更多还原

【Abstract】 Mycotoxins are the secondary metabolites produced by different fungithat contaminate a large variety of grains and feedstuffs in the world, whichcan cause several health problems. Zanhuang County is one of the highincidence areas of gastric cancer in north China with an annual gastric cancermortality being77.67/100,000/year. Our previous study showed thatmycotoxin Ochratoxin A (OTA) in wheat samples reached to2.41μg/kg inthis area, which was significantly higher than that of provisional tolerableweekly intake allocated by the Joint FAO/WHO Expert Committee on FoodAdditives (JECFA).OTA is a mycotoxin considered of concern for human health. It is producedby a number of Aspergillus and Penicillium fungal species known to colonizea range of food commodities including cereals, wine, spices, dried fruits, grapejuice, as well as animal products. When ingested as a food contaminant, OTAis a persistent toxin with a blood half-life of35days following a single oraldose. Epidemiological studies have indicated that OTA might contribute to theetiology of some sporadic diseases such as the Balkan endemic nephropathy,chronic interstitial nephropathy, and urothelial tumors. Under experimentalconditions, OTA had a diverse range of toxicological effects, includingnephrotoxicity, teratogenicity, immunotoxicity, neurotoxicity andhepatotoxicity. Therefore, OTA was classified as a possible human carcinogen(group2B) by the International Agency for Research on Cancer.It has been generally accepted that the induced cell cycle arrest andapoptosis is the important bioeffects of many carcinogenic mycotoxins. Ourprevious study showed that OTA could induce G2phase arrest and apoptosisin immortalized human gastric epithelial cells (GES-1). We also found that theactivation of ERK, p38pathways and ATM pathway were involved in OTA-induced G2arrest. However, the detailed molecular mechanism of howOTA trigerring cell cycle arrest through ATM and MAPK signaling is stillunknown.It looks mostly accepted that oxidative damage is a critical event in theinitiation and development of carcinoma. Mutations and/or acquired defectsbrought about by DNA damage are thought to underlie the development andprogression of cancer. Oxidative stress is elicited by reactive oxygen species(ROS) generation. Oxidative stress can trigger cell damage by oxidizingbiomolecules including that of lipids, proteins and DNA, and modify theirbiological functions that ultimately cause cell cycle arrest and cell apoptosis.Mitochondria are the major source and at the same time are targets of ROS;this ‘vicious cycle’ leads to an accumulation of damages to several moleculesincluding the mitochondrial DNA (mtDNA). Several studies have shown thatoxidative DNA damage plays an important role in the toxins-induced cellcycle arrest. Amongst the mechanisms of OTA carcinogenic, oxidative stresshas been highlighted as one of the most probable by JECFA. A number ofstudies have demonstrated that OTA could result in oxidative stress associatedwith the production of ROS in different cells through various direct andindirect mechanisms. Thus, OTA-induced oxidative DNA damage mightcontributes to OTA-induced cells cycle arrest, which associated with thedevelopment of gastric cancer.Thus based on our previous study, the current study first evaluated theeffects of OTA on ROS production and DNA damage in GES-1cells，as wellas the role of oxidative stress in OTA-induced G2phase arrest through ATMand MAPK pathways in GES-1cells. Furthermore, the effects of OTAexposure on mitochondria damage in GES-1cells were investigated.The microenvironment, especially the immune system is playing animportant role in the development and metachoresis of carcinoma. Resentstudys indicated that mycotoxins could lead to immunosuppressive effects,which may be associated with an increased susceptibility to tumors.Finally, we explored the putative toxicological effects and related mechanism of OTA on human peripheral blood mononuclear cells (hPBMC).Our study may provide new data to elucidate its possible epigeneticmechanism of OTA hazard bioeffects and carcinogenicity. Our findings in thisreport provide new insights in the possible carcinogenic mechanism of OTAexposure in human gastric cancer.PartⅠOchratoxin A induced Oxidative stress involved in G2arrest inGES-1cells in vitroObjective: Based on our previous study, the current study systematicallyevaluated the role of ROS production, DNA damage, as well as oxidativestress-mediated ERK, p38and ATM activation on OTA-induced G2phasearrest in GES-1cells.Methods: GES-1cells were treated with5,10and20μM OTA orpre-treated with4mM NAC plus10μM OTA for24h.1ROS were detectedby staining the cells with DHE or DCFH-DA.2Total SOD activity wasdetermined by a SOD detection kits.38-OHdG was assayed by HPLC-ECD.We detected the generation of γ-H2AX foci by immunofluorescence stainingand assessed the expression of γ-H2AX using Western blot.4The percentageof cells in each phase of the cell cycle was determined using flow cytometry.5Effects of OTA on expression of Cdc25C, Cdc2, cyclin B1, ERK, p38andATM were analyzed by Western blot.6The effect of OTA on theCdc2-cyclinB1complex was detected by immunoprecipitation.Results:1.1Effect of OTA exposure on intracellular ROS levelThe results demonstrated that both of DCF and DHE mean fluorescenceintensity (MFI) were notably increased after OTA exposure for24h (P<0.05).Pretreatment of GES-1cells with NAC, a well-established antioxidant, greatlyinhibited OTA-induced increase in MFI of DCF and DHE (P<0.05). Inconclusion, OTA caused increases in intracellular steady-state levels of ROS(i.e., superoxide and hydroperoxides) in GES-1cells.1.2Effect of OTA exposure on SOD activitySOD activity was significantly increased in the OTA-exposed groups (P< 0.05). OTA-induced the increase of SOD was prevented by NAC pretreatment.1.3Effects of OTA exposure on oxidative DNA damageTo further determine OTA induced oxidative DNA damage in GES-1cells,we determined the levels of8-OHdG, as a sensitive marker of oxidative DNAdamage, in OTA treated cells. The results showed the levels of8-OHdG inOTA treatment groups were significantly higher than that of control group(P<0.05), which suggested that OTA could induce oxidative DNA damage inGES-1cells.Among different types of DNA damage, double-DNA breaks are arguablyone of the most deleterious lesions. γ-H2AX is a reliable and exquisitelysensitive marker for this lesion. We observed that OTA could induce theaccumulation of γ-H2AX foci in nucleus after20μM OTA treatment byimmunofluorescence staining. In addition, Western blot results showed thatOTA could significantly increase the expression of γ-H2AX in GES-1cells(P<0.05). It was in consistent with the result of immunofluorescence staining,further confirmed that OTA exposure causes double-DNA breaks in GES-1cells.To further confirm that OTA-induced oxidative stress trigerred the DNAdamage, NAC was applied prior to treatment with10μM OTA in GES-1cells.The result showed that pre-treatment with NAC resulted in a significantreduction in the expression of γ-H2AX (P<0.05).Taken together, these results confirmed that OTA could induce oxidativeDNA damage in GES-1cells.1.4Antioxidants NAC blocked OTA-induced activation of ATMHere, to dissect the role of oxidative stress in OTA-induced activation ofATM, GES-1cells were pre-incubated with NAC. Western blot result showedthat NAC resulted in a significant reduction in OTA-induced the up-regulationof ATM phosphorylation (P<0.05).1.5Antioxidants NAC blocked OTA-induced phosphorylation of ERKand p38MAPKOur previous study demonstrated that ERK and p38MAPK signaling pathways were involved in the regulation of OTA-induced G2arrest in GES-1cells. To investigate whether oxidative stress in response to OTA regulatedERK and p38MAPK activation, GES-1cells were pre-treated with NAC. Wefound that NAC markedly reduced OTA-induced ERK and p38MAPKphosphorylation (P<0.05). The results showed that OTA-induced oxidativedamage activated ERK and p38MAPK pathway in GES-1cells.1.6Antioxidants NAC abolishes OTA-induced G2arrest in GES-1cellsFor evaluate whether ROS-induced DNA damage may contribute toOTA-induced G2arrest through MAPK and ATM pathway, we pre-treatedGES-1cells with NAC to examine the role of OTA on G2arrest in GES-1cells.The result showed that pre-treatment with NAC was associated with areduction of cells arresting at the G2/M cell cycle phase (P<0.05). In addition,Western bolt results showed that OTA caused a significant down-regulation ofG2/M phase related proteins (Cdc25C/p-Cdc25C, Cdc2/p-Cdc2and cyclinB1)and the cyclinB1-Cdc2complex in GES-1cells, which were abolished byNAC (P<0.05).Taken together, the results indicated that OTA-induced oxidative damageregulated G2arrest in GES-1cells through ERK and p38MAPK and ATMsignaling pathway.PartⅡThe effects of OTA exposure on mitochondria damage in GES-1cellsObjective: To explore the effect of OTA on mitochondria DNA andmitochondrial function in GES-1cells.Methods:1MnSOD activity was determined by a SOD detection kits.28-OHdG was assayed by HPLC-ECD.3Mitochondrial genes encodedproteins mRNA expression was determined by Real-time quantitative PCR.4The activity of respiratory chain complex Ⅰ were assayed using adetection kits.5Mitochondrial respiratory function was measuredpolarographically at25℃using a Clark-type oxygen electrode.6The level ofmitochondrial membrane potential was determined using amitochondria-sensitive dye Rhodamine123by flow cytometry.7To quantify the OTA-induced apoptotic death of GES-1cells, Annexin V and PI stainingwas performed by flow cytometry.8The espression of MnSOD, OGG1, Bax,Bcl-2, Bcl-xL, cytochrome c and caspase-9were measured by Western blotanalysis.Results:2.1Effect of OTA on MnSOD in GES-1cellsMnSOD represents the first line of cell defence againstmitochondria-derived ROS. We measured directly the activity of the enzymein OTA-exposured cells and found OTA (5,10and20μM) induced significantincreases in MnSOD activity (115.26±9.83,162.63±13.18,285.04±3.10U/mgprotein vs64.73±4.42U/mg protein, P<0.05). Western blot analysis alsorevealed that OTA treatment for24h caused a significant increase in MnSODexpression in GES-1cells (P<0.05). Thus, the elevated of MnSOD activity inOTA groups might point to an adaptive reaction to oxidative stress.2.2Effect of OTA on8-OHdG in mtDNAConcerning oxidative damage to mtDNA, namely8-OHdG, significantincreases were observed in OTA groups compared with control group(P<0.05).2.3Effect of OTA on the expression of mitochondrial OGG1protein8-oxoguanine glycosylase1(OGG1), a key base excision repair enzyme,plays a key role in the removal of8-OHdG adducts. Western blot analysis,compared to control group, showed that OTA led to strong depression of theamount of mitochondrial OGG1protein in OTA-treated groups (P<0.05). Thisdata suggested that OTA inhibited the mitochondrial base excision repair.2.4Effect of OTA on mitochondrial gene expressionReal-time PCR studies detected mRNA transcription corresponding to13mitochondria genes encoded protein in complex Ⅰ, Ⅲ, Ⅳ, and Ⅴ ofmitochondrial respiratory chain. We observed significant increases intranscription level of ND1, ND2, ND3, ND4L, ND5, ND6and ATP6in20μMOTA group (Fold change＞1.5).2.5Effects of OTA on the activity of mitochondrial respiratory chain complex I.The enzymatic activity related to complex I was performed onmitochondrial fraction prepared from GES-1cells. Our findings documented asignificant decrease in the specific activity of complex I following5,10and20μM OTA treatment for24h (P<0.05).2.6Effect of OTA on mitochondrial respiratory functionOTA induced a significant decrease in state3respiration rate in5,10and20μM OTA groups compared with control group (P<0.05). Meanwhile,mitochondrial RCR values were significantly decline in10and20μM OTAgroups (P<0.05), suggesting OTA impairs mitochondrial respiratory function.2.7Effect of OTA on apoptosis of GES-1cellsOxidative stress, mitochondrial damage and disrupted mitochondrialrespiration have been found to promote cell death, functional failure, anddegeneration. Thus, we investigated whether mitochondrial damage isinvolved in apoptosis caused by OTA in GES-1cells.2.7.1Effect of OTA on mitochondrial membrane potential (ΔΨm)Mitochondrial dysfunction characterized by a loss of transmembranepotential is one of the earliest intracellular events leading to cell damage. Inthis study we evaluated mitochondrial ΔΨm as an indicator of mitochondrialhealth in cells treated with OTA at different concentrations for24h. OTAexposure significantly decreased Rhodamine123MFI compared to the control(P<0.05). In order to ascertain whether ROS were involved in the alteration ofΔΨm, the effects of OTA on ΔΨm were evaluated in presence or absence ofNAC. The result showed that NAC treatment counteracted the effect of OTA.2.7.2NAC protected OTA induced apoptosis of GES-1cellsTo investigate the possible role of ROS in OTA-induced apoptosis, theeffects of specific modifiers of ROS on apoptosis were determined. Theapoptotic rate in NAC pretreatment with OTA group was5.56±1.80%significantly lower than that in only OTA treatment group12.67±2.12%(P<0.05).2.7.3Effects of OTA on the regulatory factors of mitochondrial pathway, with NAC pretreatmentTo investigate the mitochondrial apoptotic events involved in OTA-inducedapoptosis, we first analyzed the changes in the levels of pro-apoptotic proteinsBax and anti-apoptotic proteins Bcl-2and Bcl-xL. Immunoblot analysisshowed that treatment of GES-1cells with OTA increased Bax protein levels.In contrast, OTA decreased Bcl-2and Bcl-xLlevels.To elucidate whether the release of cytochrome c from mitochondria wasinvolved in OTA-induced apoptosis, mitochondria and cytosolic fractionswere prepared from GES-1cells. We found the release of cytochrome c intocytosol was detected relative to gradual decrease in mitochondrial cytochromec.Caspase-9is activated in response to cytochrome c. Proteolytic cleavage ofprocaspase-9observed in OTA-treated cells.Bax increase, Bcl-2and Bcl-xLreduction in response to OTA were blockedby NAC pretreatment. It was also found that NAC has abrogated the OTAinduced cytochrome c release and caspase-9activation.All the results indicated that OTA induced the execution of apoptosisthrough activation of the mitochondrial pathway and ROS were probablyinvolved in OTA-induced apoptosis in GES-1cells.PartⅢ Ochratoxin A induces oxidative DNA damage and G1phase arrestin human peripheral blood mononuclear cells in vitroObjective: To explore the putative toxicological effects and relatedmechanism of OTA on hPBMC.Methods:1The level of intracellular ROS (e.g. superoxide andhydroperoxides) was estimated by oxidations of DCFH-DA and DHE. Themean fluorescence intensity (MFI) was detected by a FACS flow cytometer.2The intracellular content of glutathione (GSH) was assessed using a reducedGSH assay kit.3To investigate the possibility of types of DNA damage, weperformed a comprehensive analysis of OTA-induced DNA damage.8-OHdGwas assayed by HPLC-ECD. Alkaline Comet Assay was performed todetermine whether OTA induce DNA damage. After that, the property of the stand breaks was further analyzed by the detection of γ-H2AX protein byWestern blot.4Flow cytometry was used to analyze cell cycle and Westernbolt measured the expression of cyclinD1and CDK4protein.5The cells werestained with PI and analyzed by flow cytometry. Apoptosis was quantified asthe percentage of cells containing hypodiploid amounts of DNA (SubG1peak).In addition, cells were analyzed for apoptotic nuclei fluorescence staining withHoechst33258.6hPBMC were pre-treated with4mM NAC for1h, awell-established antioxidant, followed by20μM OTA for24h. Intracellularlevels of ROS and GSH, DNA damage, cell cycle and apoptosis inOTA-induced cells were assessed as previously described.Results:3.1OTA induced increased steady state levels of superoxide andhydroperoxides in hPBMCThe state levels of intracellular superoxide and hydroperoxides weremeasured in hPBMC after treated with different concentrations of OTA for24h using FCM assay. The mean fluorescence intensity of DCF was significantlyincreased with5,10and20μM OTA treatment (P<0.05). We also found thatOTA increased the MFI of DHE in hPBMC (P<0.05).To further confirm that OTA induces the increased ROS generation inhPBMC, NAC was applied as a blocker for the increased ROS. The data fromthe flow cytometry analysis showed the increased mean fluorescenceintensities of DCF and DHE in20μM OTA-treated hPBMC were significantlyattenuated by NAC pre-treatment (P<0.05). All these results showed thatincreased oxidation of DHE and DCFH suggesting increases in steady statelevels of superoxide and hydroperoxides in hPBMC by OTA.3.2OTA decreased intracellular GSH in hPBMCWe further measured the intracellular content of glutathione (GSH) inhPBMC after OTA exposure for24h. The results showed that a significantdecrease of intracellular reduced GSH content in OTA-treated in hPBMCcould be found (P<0.05). We also found that NAC effectively blocked thedecrease in GSH induced by OTA (P<0.05). 3.3OTA induced DNA damage in hPBMCWe further evaluated whether the accumulated ROS in hPBMC by OTAtreatment could induce DNA damage.8-OHdG has been established as animportant biomarker of oxidative DNA damage. HPLC-ECD results indicatedthat the level of8-OHdG was significantly higher in OTA treatment groupsthan that in control group (P<0.05).The comet assay was performed under alkaline conditions for the detectionof a broad spectrum of DNA lesions. Treatment with OTA caused a significantincrease in%Tail DNA, Tail length and Olive tail moment (P<0.05). Next, wedetected γ-H2AX protein expression, a maker of DNA double-strand breaks.Treatment of hPBMC with5,10and20μM OTA resulted in the up-regulationof γ-H2AX protein in a dose-effect manner (r=0.998, P<0.05). Pre-treatmentwith antioxidant reagent NAC resulted in a significant reduction in DNAdamage as well as γ-H2AX protein expression in20μM OTA-treated group(P<0.05). All these results confirmed that OTA-induced ROS contributed tooxidative DNA damage in hPBMC.3.4OTA induced cell cycle arrested at G1phase in hPBMCAs we know that DNA damage is often accompanied by arrest in cell cycle,so we detected cell cycle arrest in hPBMC using flow cytometry analysis. Incomparison with the control group, the proportion of cells in G1phase wasaccumulated markedly after treated with10and20μM OTA for24h (P<0.05).To estimate the molecular mechanism accounting for cell cycle arrest in G1phase, G1-associated regulatory proteins (CDK4and cyclinD1) were furtherexamined. Western blot analysis demonstrated that cyclinD1and CDK4protein expression were both markedly decreased in hPBMC treated withdifferent concentrations of OTA (P<0.05).To determine whether ROS-induced DNA damage may contribute toOTA-induced G1arrest, hPBMC were pre-treated with the antioxidant NAC.We observed NAC inhibited OTA-induced G1arrest, which demonstrated thatOTA induced G1phase arrest is in part mediated through ROS-accumulationoxidative DNA damage (P<0.05). 3.5OTA induced apoptosis in hPBMCIn order to quantify the extent of apoptosis, the content of DNA in cells wasmeasured by flow cytometry. We found hPBMC exposed to5,10and20μMOTA for24h showed significant increase in cells in SubG1phase (P<0.05). Aremarkable feature of apoptosis is the condensation and fragmentation ofnuclear chromatin, which can be observed under fluorescence microscopeafter staining with Hoechst33258. Within24h of treatment with20μM OTA,hPBMC exhibited significant morphological changes and chromosomalcondensation, which was indicative of apoptotic cell. Furthermore,pre-treatment with NAC partly protected OTA-induced apoptosis in hPBMC(P<0.05).Conclusions:1OTA induced oxidative stress damage in GES-1cells.2OTA-induced oxidative damage regulated G2arrest in GES-1cellsthrough ERK and p38MAPK and ATM signaling pathway.3OTA induced oxidative mtDNA damage, inhibited the mitochondrial baseexcision repair and impaired mitochondrial function, which acted as thetrigger in OTA induced oxidative stress in GES-1cells.4Mitochondrial damage is involved in apoptosis caused by OTA in GES-1cells.5OTA-induced oxidative DNA damage caused G1phase arrest andapoptosis in hPBMC, which indicate that oxidative stress is involved inOTA-induced human immunotoxicity.6OTA-induced oxidative damage mediated cell cycle arrest and apoptosis ingastric epithelium cells and hPBMC, which might contribute to a possiblecarcinogenic mechanism of OTA exposure in human gastric cancer.更多还原