【作者】 李增宁；

【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， 2007， 博士

【摘要】 赭曲霉毒素（Ochratoxins,OT）是由赭曲菌（Asperegillus alutacells）和纯绿青霉（Penicillium viridicatum）产生的有毒代谢产物,包括七种结构类似的化合物,其中以赭曲霉毒素A（Ochratoxin A, OA）为主,毒性也最大。OA的分子量为404,熔点169℃,为无色晶体,易溶于有机溶剂（三氯甲烷和甲醇）和稀碳酸氢钠溶液,微溶于水。在紫外线照射下OA呈绿色荧光,最大吸收峰为333nm。该化合物相当稳定,一般的烹调和加工方法只有部分破坏。动物实验表明,赭曲霉毒素A具有肾毒性、免疫毒性和致癌、致畸、致突变性。1993年国际癌症研究中心将赭曲霉毒素A列为可能的人类致癌物。一些国家已报道了从人全血、血清及人奶中检出赭曲霉毒素A。研究表明,粮食污染是人类赭曲霉毒素A暴露的主要途径。研究分析粮食赭曲霉毒素A污染情况对保障食品卫生和安全具有非常重要的意义。目前,国外文献中已有不少粮食赭曲霉毒素A污染情况的分析检测报道,不少国家和国际组织还根据赭曲霉毒素A的污染情况制定了人体赭曲霉毒素A摄入量的限制标准。迄今为止,国内有关粮食中赭曲霉毒素A的污染情况报道较少。目前,有关粮谷中赭曲霉毒素A的检测方法主要有薄层色谱法（TCL）、酶免疫检测法（ELISA）、高效液相色谱法（HPLC）等。我国谷物和大豆中赭曲霉毒素A的国家标准检测方法是TCL法,属于目测半定量法,最低检出量为10μg/kg,不适用于粮谷中赭曲霉毒素A的准确测定和居民赭曲霉毒素A的膳食摄入量的暴露评估研究。为准确分析我国粮食中OA的污染情况,进一步探讨OA污染的生物学意义,本研究对OA的检测方法进行了改进,建立了灵敏度高、重现性好、准确可靠的OA高效液相色谱荧光定量检测方法。对河北省不同地理环境的三个地区粮食中赭曲霉毒素A污染情况进行了定量检测。同时,以细胞培养、FCM等方法研究了OA对人外周血单个核细胞增殖和凋亡的影响,探讨了OA对人正常肾小管上皮细胞株（HKC）和人肺泡II型上皮细胞（AT-II）细胞系A549凋亡的诱导作用。在上述工作的基础上,采用Western Blotting和免疫组化方法等方法在蛋白水平上对JNK通路在OA诱导细胞损伤中作用及OA诱导细胞损伤的机制进行了研究。本研究共分为五部分:第一部分:赭曲霉毒素A检测方法的建立目的:建立SPE - C₁₈柱纯化和富集—高效液相色谱法荧光检测粮谷和酒类样品中赭曲霉毒素A的系列方法。方法:1用三氯甲烷提取玉米、小麦和大麦样品中的OA后,经C₁₈固相萃取柱净化处理,以乙腈:水:乙酸（99:99:2）作为流动相,用带有荧光检测器的反相高效液相色谱法（激发波长为333nm,发射波长为460nm）检测OA含量;2选用C₁₈固相萃取小柱（250mg/3ml）,用2ml乙腈和2ml水分别活化C₁₈小柱,30ml酒样通过C₁₈固相萃取小柱,控制流速30滴/分。再用2ml乙腈溶液洗脱,最后将C₁₈小柱抽干。45℃下氮气吹干,流动相溶液稀释至0.5ml,0.45μm微孔滤膜过滤后取20μL进高效液相色谱仪进行检测。结果:1.分别在小麦和大麦样品中加入3种不同量的OA标准物。小麦加标样品的回收率为78.60%⁹2.73%,变异系数为1.46%².98%,大麦加标样品的回收率为77.61%⁹3.21%,变异系数为1.74%³.45%;为考察该方法的准确度,对北京中检维康玉米加标盲样（OA浓度范围介于5.33⁹.91ng/g）进行了检测,结果为6.75ng/g,在盲样范围内,经t检验,在95%的置信水平上,测定平均值（n=3）与盲样给定值无显著性差异。2.本实验对干红葡萄酒,白葡萄酒和红葡萄酒分别进行了0.25ng/ml、0.5ng/ml、1.5ng/ml三个浓度的加标回收,对三种葡萄酒样品,每个加标水平平行3份,按上述方法提取测定,各种样品的加标回收率均在80.1¹09.8%。平行三次测定加标回收率的相对标准偏差在1.5⁵.2%之间。第二部分:河北省不同地区粮食中赭曲霉毒素A污染情况研究目的:分析河北省不同地理环境地区居民食用粮食OA污染情况,了解河北省居民OA的暴露量,揭示其对健康的可能影响,为减少OA污染及其影响奠定基础。方法:抽样检测河北省不同地理环境的三个代表性地区粮食OA污染情况,三个地区分别为河北省南部邯郸市磁县、河北省中部石家庄市赞皇县和河北省北部塞外地区张家口市赤城县。分别入户采取中南部地区居民日常食用主粮小麦和北部地区居民日常食用主粮大麦样品。采用反相—高效液相色谱法检测OA含量:用三氯甲烷提取小麦和大麦样品中的OA后,经C18固相萃取柱净化处理,以乙腈:水:乙酸（99:99:2）作为流动相,用带有荧光检测器的高效液相色谱仪（激发波长为333nm,发射波长为460nm）检测OA含量。结果:大麦样品OA经反相—高效液相色谱法检测结果表明,张家口市赤城县31份大麦样品中,11份样品检测到OA,污染率为35.48%,其平均含量为6.96μg/kg,最低者为0.21μg /kg,而最高可达35.36μg/kg;赞皇县居民食用小麦样品中赭曲霉毒素A的检出率为45.16%,平均含量2.41μg/kg,最高达14.25μg/kg。河北省磁县居民食用小麦中赭曲霉毒素A的检出率为33.33%,平均含量0.59μg/kg,最高为1.63μg/kg。根据居民人均食用粮食重量计算,赞皇县农村居民OA的日暴露量为1.17μg/ kg,磁县农村居民OA的日暴露量为0.31μg/kg,张家口赤城农村居民OA的日暴露量为3.38μg/kg,超过世界卫生组织/粮农组织联合专家委员会暂定的容许摄入量。可见河北省不同地理环境居民食用粮食中OA的污染均比较普遍,应当引起有关部门的重视。第三部分赭曲霉毒素A对体外培养HPBMCs增殖与凋亡的研究目的:探讨OA对体外培养HPBMCs增殖与凋亡的影响,揭示OA暴露对机体免疫功能的可能作用。方法:健康献血员抗凝静脉血200mL,采用聚蔗糖-泛影葡胺密度梯度离心法（ficoll-hypaque density gradient centrifug- ation）分离人外周静脉血单个核细胞,接种于含10%胎牛血清,105U/L青霉素,100mg/L链霉素的RPMI-1640培养液中,加入PHA并使其终浓度为300μg/mL,37℃、5%CO₂培养24h,更换不含PHA的新的培养液。HPBMCs随机分为4组:空白对照组、溶剂对照组、1μmol/L OA和5μmol/L OA实验组。实验组分别给予乙醇溶解的OA处理,使其终浓度分别为1μmol/L和5μmol/L,对照组和溶剂对照组分别给予等体积生理盐水和乙醇处理。细胞继续培养24h,常规收集细胞,采用流式细胞定量检测术（FCM）检测HPBMCs细胞凋亡率和增殖指数,以及细胞凋亡相关蛋白Caspase-3的表达。结果: FCM检测结果表明, OA处理24h,1μmol/L OA和5μmol/L OA组HPBMCs细胞凋亡率分别为10.90±0.85%,16.03±0.37%,均高于对照组（8.17±1.45%,P<0.05）。1μmol/L OA和5μmol/L OA组细胞增殖指数分别为35.77±4.74和36.89±7.63,与对照组比较差异无显著性（33.53±1.97,P>0.05）,说明OA处理24h可以促进HPBMCs凋亡,但对细胞增殖没有影响。FCM定量分析表明,OA作用24h后,1μmol/L OA和5μmol/L OA组HPBMCs细胞Caspase-3蛋白表达FI值明显高于对照组（1.13±0.02和1.14±0.06 vs 1.00±0.04,P < 0.05）。第四部分:赭曲霉毒素A对A-549和HKC细胞损伤的研究目的:探讨OA处理对A549和HKC细胞增殖与凋亡的影响及可能机制,并观察OA对体外分离的DNA致损伤作用。方法:取对数生长期A549和HKC细胞,用10%DMEM调整细胞浓度为（1²）×10⁸L^-1,接种于细胞培养板和细胞培养瓶中。细胞培养24h后,随机分为4组:空白对照组、溶剂对照组、1μmol/L OA和5μmol/L OA实验组。实验组分别给予OA 1μmol/L和5μmol/L处理,溶剂对照组和对照组分别给予DMSO(0.4mL·L^-1)和生理盐水处理,继续细胞培养24h。采用MTT法检测A549和HKC细胞存活率。采用FCM检测A549和HKC细胞凋亡率,以及细胞凋亡相关蛋白Caspase-3的表达表达情况;采用紫外以及荧光光谱法检测了OA与小牛胸腺和鲑鱼精DNA的相互作用形成的DNA加合物。结果:MTT检测结果表明,OA对A549和HKC细胞的生长均有一定抑制作用:1μM、5μM OA作用不同时间后,两株细胞存活率均较相应对照组降低,尤以24h更明显（P<0.05）。1μM、5μM OA处理24h后,A549细胞存活率分别为80.71%和70.85%,明显低于对照组（100%,P<0.05）;HKC细胞存活率分别为51.09%和54.69%,也低于对照组（100%,P<0.05）。提示OA对A549和HKC细胞具有明显的致损伤作用。FCM检测细胞凋亡率检测结果表明,A549细胞经1μM、5μM OA处理24h后,OA处理细胞凋亡率明显升高,5μM OA处理组凋亡率为2.68±0.55%,明显高于对照组（1.53±0.23%,P<0.05）。HKC细胞经OA处理24h后,1μM和5μM OA组细胞凋亡率分别为3.85±0.29%和4.86±0.51%,均明显高于对照组（1.23±0.05%,P<0.05）。表明OA对A549和HKC细胞具有明显的促凋亡作用。FCM检测组方图及定量分析表明,OA作用24h后,A549和HKC细胞Caspase-3蛋白表达量均明显高于对照组。紫外光谱法分析结果显示DNA与OA没有发生相互作用;荧光光谱法分析结果显示OA没有与DNA相结合。第五部分:JNK信号转导通路在赭曲霉毒素A诱导HKC凋亡中的作用目的:探讨JNK信号转导通路在赭曲霉毒素A诱导HKC凋亡中的作用,以期进一步探讨OA诱导凋亡的可能机制。方法:5.1 OA对HKC细胞JNK激酶活性和Caspase-3蛋白表达影响的检测实验分组与处理同论文第四部分有关OA作用HKC细胞24h的实验分组和处理。细胞处理24h后,离心收细胞用于相关指标的检测。采用免疫细胞化学方法和Western blot方法检测HKC细胞p-JNK水平,JNK和Caspase-3蛋白表达。5.2 JNK抑制剂SP600125预处理对OA诱导HKC细胞JNK激酶活性、凋亡率、Caspase-3蛋白表达和细胞损伤影响的检测选择对数生长期的细胞（传代24h后）,随机分为空白对照组、溶剂对照组、OA处理组和JNK阻断剂组。1μM OA处理组给予终浓度为1μM OA;JNK阻断剂组预先加入0.5μM SP600125孵育30min后再加入1μM OA ;空白对照组加入生理盐水;溶剂对照组加入乙醇（1:500）+DMSO（1:30000）。细胞培养24h后消化离心收集细胞。采用Western blot方法检测HKC细胞p-JNK水平;采用流式细胞术（FCM）检测细胞凋亡率和Caspase-3蛋白表达;采用MTT比色法检测HKC细胞的存活率。结果5.1OA对HKC细胞Caspase-3蛋白表达的影响免疫细胞化学染色,光镜下观察可见Caspase-3蛋白免疫阳性反应产物为棕黄色颗粒状,定位于HKC细胞胞浆内。1μMOA、5μM OA处理组HKC细胞Caspase-3阳性表达细胞比率分别为54.03±9.21%,60.75±9.95%,明显高于对照组（14.31±4.24%,P<0.05）。Western blot检测发现,给予不同剂量OA处理后,细胞Caspase-3蛋白表达明显高于对照组,进一步证实给予OA处理可以促进体外培养HKC细胞Caspase-3蛋白的表达。5.2OA对HKC细胞p-JNK水平的影响免疫细胞化学染色可见p-JNK阳性染色为棕黄色颗粒,定位于HKC细胞胞浆及胞核内。1μMOA和5μMOA处理组HKC细胞阳性表达细胞比率分别为53.31±11.01%和58.53±11.11% ,明显高于对照组（10.19±6.85%,P<0.05）。Western Blot检测发现,给予不同剂量OA处理后,HKC细胞p-JNK水平明显高于对照组（P<0.05）,实验结果表明,给予OA处理可以提高体外培养HKC细胞JNK磷酸化水平。5.3OA对HKC细胞JNK蛋白表达的影响免疫细胞化学方法和Western blot结果显示OA对HKC细胞JNK蛋白表达没有影响。5.4 SP600125预处理对OA作用后HKC细胞p-JNK激酶活性的影响免疫细胞化学染色可见,JNK阻断剂组HKC细胞p-JNK激酶阳性表达细胞比率为15.51±3.35%,明显低于1μM OA处理组（53.28±7.08%,P<0.05,）。Western blot检测发现,给予SP600125预处理HKC细胞p-JNK水平明显降低。实验结果表明,给予SP600125预处理可以降低HKC细胞p-JNK的表达,进一步说明OA处理可以激活JNK信号转导通路。5.5SP600125预处理对OA作用后HKC细胞凋亡率的影响FCM细胞凋亡率检测结果表明, HKC细胞经SP600125预处理30min后,JNK阻断剂组+OA组细胞凋亡率为2.44±0.38%,低于单纯OA处理组（4.24±0.38%, P<0.05）,但高于空白对照组的细胞凋亡率1.06±0.14%（P<0.05）。实验结果表明,SP600125预处理可以部分抑制体外培养人肾小管上皮细胞HKC的凋亡。5.6 SP600125预处理对OA作用后HKC细胞凋亡相关蛋白―Caspase-3蛋白表达的影响免疫细胞化学染色可见,JNK阻断剂组Caspase-3阳性表达细胞比率为31.42±4.67%,较1μMOA处理组明显降低（55.86±6.51%,P<0.05）,但高于对照组（13.97±1.59%,P<0.05）。Western blot检测结果证实给予SP600125预处理可以部分降低OA引起的HKC细胞Caspase-3蛋白的表达。推测OA给予SP600125预处理可以部分降低OA引起的HKC细胞Caspase-3蛋白的表达。OA通过激活JNK信号转导通路部分介导caspase-3蛋白活化。5.7SP600125预处理对OA作用后HKC细胞存活率的影响MTT检测结果发现给予特异性JNK阻断剂-SP600125预处理后,JNK阻断剂+OA组HKC细胞的存活率为82.09%,明显高于单独OA处理组（66.83%,P<0.05）;但是低于溶剂对照组（98.58%,P<0.05）,说明SP600125对OA致HKC细胞损伤具有部分阻断作用,OA通过JNK信号转导通路参与介导细胞死亡。结论:1.建立了反相—高效液相色谱法检测粮谷和酒类样品中赭曲霉毒素A的系列方法,本方法具有准确度高、精密度好和灵敏度高等优点。可定量检测粮谷和酒类样品中赭曲霉毒素A,检测结果准确可靠,提取净化过程简单等优点,为食品中赭曲霉毒素A污染情况调查和居民对赭曲霉毒素A的暴露评估提供了有力的技术支持和保障。2.通过对河北省不同地区居民食用主粮调查发现:河北省中南部地区居民食用小麦中OA的检出率达39.34%,北部地区居民食用的大麦中OA的检出率达到35.48%,均明显高于国内已有的研究报道。赞皇县农村居民OA的日暴露量为1.17μg/ kg,磁县农村居民OA的日暴露量为0.31μg/ kg,张家口赤城农村居民OA的日暴露量为3.38μg/ kg,超过世界卫生组织/粮农组织联合专家委员会暂定的容许摄入量。可见河北省不同地理环境居民食用粮食中OA的污染均比较普遍,应当引起重视。3. OA可以促进体外培养HPBMCs凋亡,上调Caspase-3蛋白表达,但对HPBMCs增殖没有明显影响。4. OA可抑制体外培养A549和HKC细胞增殖,上调Caspase-3蛋白的表达,促进体外培养A549和HKC细胞凋亡。OA可能对体外分离的DNA没有损伤作用。5. OA可以提高体外培养HKC细胞p-JNK水平,激活JNK途径。6. JNK抑制剂SP600125预处理降低OA诱导的细胞凋亡、Caspase-3蛋白上调及细胞损伤作用,提示JNK信号转导通路部分参与OA介导细胞损伤和凋亡过程。更多还原

【Abstract】 Ochratoxins （OT）is the toxic metabolites of Asperegillus alutacells, Penicillium viridicatum etc, including 7 structurally similar compounds （Ochratoxin A, B, C, D, etc）. Ochratoxin A （OA） is the most common and toxic one among the 7 Ochratoxins. Chemically, OA is a colorless crystal with molecular weight of 404, fusion point of 169℃, solvable in both organic solvents （chloroform and methanol） and NaHCO3 solution, while minimally solvable in water. Under ultraviolet exposure, OA shows green fluorescence with peak absorption wavelength at 333nm. Being quite stable in nature, OA could only be partly destroyed during cooking and grain processing. Animal experiment showed that OA was of nephrotoxicity, immunotoxicity and was carcinogenic, teratogenic and mutagenic. In 1993, International Cancer Research Center anounced OA was a possible human carcinogen. OA was detected in human blood and milk in some countries. Studies showed that the main route for human OA exposure was via grain contamination of OA. Thus, the evaluation of the contamination of OA in the grain is very important for food safety and human health. The detection and monitoring of OA contamination in the foodstuff and grains have been widely reported abroad. Several countries have set the maximal limitation for human OA intake. Up to now, few works have been done in the evaluation of OA contamination in grain and foodstuffs and the background of OA contamination in grains and human exposure is still not clear enough in our country. The methods used currently for OA analysis in grain and foodstuff include thin layer chromatography （TLC）, high-performance liquid chromatography （HPLC）, enzyme-linked immunosorbnent assay （ELISA）, etc. The authorized method in Examination Methods of Ochratoxin A（OA） in Grains and Beans （NSSn GB/T 13111-1991） issued by China authority in 1991 is thin layer chromatography（TLC）, which is a semi-quantitative method with relatively poor accuracy, the lowest detectable limit is only 10μg/kg. Thus,it is not a reliable analysis method for the accurate evaluation of human exposure to OA via food intake.In order to accurately evaluate the contamination of OA in the grain and other foodstuffs in our country and to further explore the putative biological significance of OA contamination, we conducted the following studies. The method for OA analysis was improved and a new sensitive, repeatable and quantitative HPLC analysis method of OA was established. The contamination of OA in different grains from three different geographic areas in Hebei province was analyzed with HPLC method. At the same time, the effects of OA on the proliferation and apoptosis of human peripheral blood mononuclear cells in vitro were studied with cell culture, FCM methods. The injury effects of OA on human kidney tubular epithelial cell line- HKC and human lung AT-II cell line -A549 was also studied. On the basis of the above study, the putative role of JNK pathway on the apoptosis of HKC induced by OA was explored at protein level with immunocytochemical method, Western Blot, etc.The study included five parts:1 Establishment of method for the analysis of ochratoxin AObjective: To establish a sensitive method of high-performance liquid chromatography （HPLC） for the analysis of ochratoxin A in grains and wines.Methods: The wheat and barley samples for determination of OA were first extracted with chloroform. OA was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection （excitation wavelength 333 nm, emission wavelength 460 nm） using acetonitrile-water-acetic acid （99:99:2） as mobile phase; 30 ml wine samples were recovered from activated C18 column with 2ml methy1 cyanide and 2ml water （250mg/3ml）. The flowing velocity was 30drops/min. OA was eluted with methanol, dryed by nitrogen gas at 45℃and quantified by HPLC with fluorometric detection using mobile phase with 20μl OA samples. 2 Detection of the contamination of OA in grains from three different geographic areas in Hebei provinceObjective: To analyze OA contamination in grains from three different geographic areas in Hebei province and to evaluate the OA exposure level of local residents in Hebei province.Method: Grain samples were randomly taken from household in the south, middle and north part of in Hebei province with different geographic feature. Wheat samples were taken from Cixian County in the south Hebei province and Zanhuang County in the middle part of Hebei province while the samples of barley were taken from Cicheng County in the north of Hebei province with quite cold climate. The species of grain sample was selected on the bases of the amount of consumption. OA was exacted with chloroform from wheat and barley, eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection （excitation wavelength 333 nm, emission wavelength 460 nm） using acetonitrile-water-acetic acid （99:99:2） as mobile phase.Results: Reversed-phase HPLC analysis showed that of the 31 barley samples of Chicheng county, OA could be detected in 11 （35.5%） with the mean content 6.96ug/kg. The highest contamination level reached 35.36ug/kg. The positive detection rates of ochratoxin A, the mean content and highest contents in wheat samples from Zanhuang country and Cixian county were 45.16%, 2.41μg/kg, 14.25μg/kg and 33.33%, 0.59μg/kg, 1.63μg/kg, respectively.3 Studies of the effects of Ochratoxin A on the apoptosis and proliferation of human peripheral blood mononuclear cells in vitroObjective: To explore the effects of Ochratoxin A （OA） on the apoptosis and proliferation of human peripheral blood mononuclear cells in vitro and to evaluate the putative effects of OA exposure on human immune system.Methods: Human Peripheral blood mononuclear cells （HPBMc）, isolated from 200ml heparinic acid antiagglutinated fresh venous blood of healthy Donors by Ficolly-Hypaque density gradient centrifugation, were initially cultured in RPMI 1640 medium supplemented with 10% new-born calf serum （NBCS）, streptomycin （100μg/mL）, penicillin （100U/mL） and phytohemagglutinin （PHA, 30μg/mL） at 37℃, 5% CO₂ for 24h. After initial culture, the medium was replaced by new medium without PHA and then the flasks were randomly divided into 4 groups: control, solvent control, OA 1μmol/L and OA 5μmol/L. OA was added to the medium at final concentrations of 1μmol/L and OA 5μmol/L in OA treated groups, alcohol was added to the medium of solvent control group and saline was added to the medium of control group. The cells were cultured for 24 h after treatment and harvested for detection. The cultured cells were centrifuged and fixed in 70% ethanol for preparation of single cell suspension and used for flow cytometric analysis of apoptosis rates, proliferation index （PI） and the expression of Caspase-3.Results: FCM analysis showed that the apoptosis rates of HPBMc in 1μmol/L and 5μmol/L OA-treated groups were 10.90±0.85% and 16.03±0.37%, respectively, which were significantly higher than that in control group （8.17±1.45%,P<0.05）. No significant difference in PI value was seen between OA-treated groups and control group （35.77±4.74% and 36.89±7.63% vs 33.53±1.97%,P>0.05）. The results suggest OA may induce apoptosis of HPBMc, but have no effect on the proliferation of HPBMc.FI of Caspase-3 protein expression in HPBMc treated with 1μmol/L and 5μmol/L OA for 24 h was 1.13±0.02 and 1.14±0.06 respectively, which was significantly higher than that in control group （1.00±0.04,P < 0.05）. The results indicated that exposure of OA could increase the expression of Caspase-3 protein in HPBMc in vitro.4 Effects of Ochratoxin A on the cell injury of A549 and HKC cells in vitroObjective: To explore the effects and mechanism of Ochratoxin A （OA） on the apoptosis and proliferation of A549 and HKC cells and the effects on the injury of DNA induced by OA.Methods: A549 and HKC cells were harvested, centrifuged and resuspended in DMEM medium supplemented with 10% FCS at the concentration of （1²）×10⁸ cells·L^-1. Flasks of the two cell lines were both randomly divided into 4 groups: control, solvent control, OA 1μmol/L and OA 5μmol/L. A549 and HKC cells in OA groups were treated with OA at the final concentration of 1μmol/L and 5μmol/L, respectively, while these in solvent control and control group were incubated with DMSO and saline, respectively. The cells were cultured for 24h after treatment and harvested for detection. Cell viability for A549 cells was assessed with MTT assay. Then A549 and HKC cells were centrifuged and fixed in 70% ethanol for preparation of single cell suspension respectively and used for flow cytometric analysis of apoptosis rates, proliferation index （PI） and the expression of Caspase-3. The salmine DNA and calf thymus DNA was coadminstrated with different concentrations of OA, DNA-adduct was detected by the Ultraviolet spectra and Fluorescence spectra.Results: MTT assay showed that 24h after OA treatment, the survival rates of A549 cells in OA 1μmol/L and OA 5μmol/L groups were 80.71% and 70.85% ,respectively, significantly lower than that in control group （100%,P<0.05）, while the survival rates of HKC cells in OA 1μmol/L and OA 5μmol/L groups were also significantly lower than that in control group（ 51.09% and 54.69% vs 100%, P<0.05）.The results showed that OA could cause proliferation inhibition and injuries in A549 and HKC cells. FCM analysis showed that the apoptosis rate of A549 cells in 5μmol/L OA-treated groups was significantly higher than that in control group （2.68±0.55% vs 1.53±0.23%,P<0.05）. The apoptosis rates of HKC cells in 1μmol/L and 5μmol/L OA-treated groups were 3.85±0.29% and 4.86±0.51%, respectively, which were significantly higher than that in control group（1.23±0.05%,P<0.05）. The results showed that OA could induce apoptosis of A549 and HKC cells. A significant increase in Caspase-3 protein expression of A549 and HKC cells after OA treatment revealed that OA could increase the expression of Caspase-3 protein to modulate the apoptosis of A549 and HKC cells. NO significant DNA-adduct was found in the mixture of DNA and OA measured by the Ultraviolet spectra and Fluorescence spectra. 5 Effects of JNK signaling pathways on the apoptosis of HKC cells induced by OAObjective: To explore the effects of JNK signaling pathways on the apoptosis of HKC cells induced by OA.Methods:5.1 Determination of the effects on activation of JNK and the expression of Caspase-3 protein in HKC cells treated with OA for 24 hThe culture, grouping and treatment of HKC were the same as that described in part 4 of the study. The HKC cells in OA groups were treated with OA for 24h. The cells were cultured for 24h after treatment and harvested for further study. The phospho-JNK （p-JNK） levels and the Caspase-3 protein expression were determined by immunocytochemical staining and Western blot.5.2 The effects of JNK inhibitor, SP600125 on OA induced activation of JNK, apoptosis, proliferation inhibition of HKC cells in vitroHKC cells seeded at （1²）×10⁸ cells·L^-1 in culture flasks were randomly divided into 4 groups: control, solvent control, JNK inhibitor pretreatment and OA 1μM mg·L^-1. 24h later, the cells in JNK inhibitor group were pretreated for 30 min with 0.5μM SP600125. Then the cells in JNK inhibitor group and AFG1 group were treated with AFG₁ 1.0 mg·L^-1, while these in solvent control and control groups were incubated with alcohol and saline respectively. The cells were cultured for 24 h after treatment and harvested for further analysis. The phospho-JNK （p-JNK） levels were detected by Western blot. The apoptosis rates and the expression of Caspase-3 were determined by FCM. Cell viability for HKC cells was assessed with MTT assay.Results:5.1 Effects of OA on the expression of Caspase-3 protein in HKC cellsThe results of immunocytochemical staining showed the expression of Caspase-3 in 1μM OA and 5μM OA group was 54.03±9.21% and 60.75±9.95%, both were significantly higher than that in control group （14.31±4.24%,P<0.05）. Western blot results showed the expression of Caspase-3 in 1μM OA and 5μM OA group was increased in HKC cells treated with OA.5.2 Effects of OA on the phospho-JNK levels of HKC cellsImmunocytochemical staining results showed that p-JNK level in 1μM OA and 5μM OA group was significantly higher than that in control group （53.31±11.01% and 58.53±11.11% vs 10.19±6.85%,P<0.05）. Western blot results showed p-JNK level in 1μM OA and 5μM OA group was increased in HKC cells treated with OA. The results suggest that OA could cause the activation of JNK in HKC cells.5.3 Effects of OA on the expression of JNK protein in HKC cellsNo difference in the expression of JNK among OA treatment groups and the control group was found by Western blot and immunocytochemical staining.5.4 Effects of SP600125 pretreatment on OA induced increase of the p-JNK levels in HKC cellsImmunocytochemical staining and Western blot showed that pretreatment of OA cell with 0.5μM SP600125 could significantly decrease the relative p-JNK level as compared with that of OA treatment alone. The effect of activation of JNK in OA-treated cells was inhibited in the presence of SP600125.5.5 Effects of SP600125 pretreatment on OA induced apoptosis in HKC cellsFCM analysis showed apoptosis rate of JNK inhibitor group was significantly lower than that of OA group （2.44±0.38% vs 4.24±0.38%, P<0.05） and higher than that of control group （2.44±0.38% vs 1.06±0.14%, P<0.05）. The results showed that SP600125 pre-treatment might partly inhibit the apoptosis of cultured HKC induced by OA in vitro.5.6 Effects of SP600125 pretreatment on OA induced changes in the expression of Caspase-3 at protein level in HKC cellsImmunocytochemical staining results showed the expression of Caspase-3 in JNK inhibitor group was lower than that in 1μM OA group （31.42±4.67% vs 55.86±6.51%, P<0.05）, and higher than that in control group （31.42±4.67% vs13.97±1.59%, P<0.05）. Similar to the results with immunocytochemical methods, Western blot also confirmed activation of the JNK pathway in OA-treated cells was involved in the modulation of Caspase-3.5.7 Effects of SP600125 pretreatment on OA induced survival rate inhibition of HKC cellsThe survival rates of OA treated HKC cells pretreated with 0.5μM SP600 125 were 82.09%, which significantly higher than that in only OA treated group （66.83%,P<0.05）, but lower than that in control group （98.58%,P<0.05）. The results suggest the pretreatment with low concentration of SP600125 may partly reduce the cell injury of OA and the activation of JNK pathway in OA-treated cells may partly modulate the OA related cell injury.Conclusion:1 A sensitive, repeatable and quantitative reversed-phrase HPLC method for the analysis of OA in grain was established. The method was successfully used in the detection of OA contamination in wine and grains.2 Analysis of the contamination of OA in the consumption grains in three different geographic areas of Hebei province showed that OA contamination rate in the wheat samples from south and middle part of Hebei province reached 39.34% and that of barley from north of Hebei province was 35.48%, which were all significantly higher that reported in our country. Bases on the contents of OA contamination, the daily OA exposure level for the residents in south, middle and north part of Hebei province was 0.31μg/ kg, 1.17μg/ kg and 3.38μg/ kg respectively, significantly over the permitted intake limit by FAO/WHO JECFA.3 OA could induce apoptosis and increase the expression of Caspase-3 protein of HPBMc in vitro, but have no effect on the proliferation of the cells.4 OA could cause proliferation inhibition and injuries in A549 and HKC cells, and increase the expression of Caspase-3 protein and induce apoptosis of A549 and HKC cells.5 OA could increase the level of p-JNK and cause the activation of JNK.6 JNK inhibitor, SP600125 pretreatment could significantly decrease the apoptosis, up-regulation of Caspase-3 and cell injury caused by OA. The results revealed that JNK pathway may partly be involved in the injury of HKC by OA.更多还原