苯并[a]芘致神经细胞凋亡的机制研究
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摘要
第一部分AhR/ARNT-CYP1A1途径在B[a]P致神经细胞凋亡中的作用
B[a]P在体内的代谢活化和致癌性是通过激活AhR/ARNT-CYP1A1途径实现的。而AhR与B[a]P的特异结合是AhR/ARNT-CYP1A1途径激活的前提条件。因此,AhR基因多态性变异可能影响个体对B[a]P神经毒性和细胞凋亡的易感性。研究表明,B[a]P可以引起AhR基因表达增高,B[a]P诱导的肝细胞凋亡中AhR、ARNT和CYP1A1 mRNA表达呈时间和剂量依赖性的上升。B[a]P必须经激活AhR,诱导CYP1A1代谢活化后才诱导肝细胞凋亡。B[a]P诱导的人宫颈癌细胞凋亡与CYP1A1激活有关,是由CYP1A1介导的。有报道鱼类暴露于B[a]P,CYP1A1蛋白水平和反应CYP1A1蛋白酶活力的EROD水平显著升高,二者高度相关。B[a]P诱导的神经细胞凋亡与CYP1A1 mRNA和蛋白的表达升高有关。由此可见,AhR/ARNT-CYP1A1途径可能介导B[a]P诱导的神经细胞凋亡。本部分内容通过人群研究分析AhR基因多态性与B[a]P神经毒性和致细胞凋亡的关系,通过动物体内外试验探讨AhR/ARNT-CYP1A1途径在B[a]P致神经细胞凋亡中的可能作用。
第一章AhR基因多态性与B[a]P神经毒性及外周血淋巴细胞凋亡的关系
我们选取了214名职业性接触B[a]P的男性健康焦炉工为接触组,81名原材料库的库工为对照组。对照组除了没有职业性接触B[a]P和其他神经毒物外,其他方面尽量与接触组相一致。采用世界卫生组织推荐的神经行为功能核心测试组合(NCTB/WHO)和Ewing DJ推荐的自主神经功能测试分别进行神经行为功能和自主神经功能测定;采集空腹肘静脉血,分离淋巴细胞和提取全血基因组DNA,用Annexin V-FITC/PI双染法测定淋巴细胞的凋亡率,用等位基因特异性PCR法和限制性片段长度多态性-PCR法检测了AhR基因第10号外显子C1549T(Pro517Ser)、G1661A(Arg554Lys)和G1708A(Val570Ile)3个位点的基因变异,分析这3种基因多态性与神经行为功能、自主神经功能和外周血淋巴细胞凋亡之间的关系。结果如下:
1.基因多态性分析结果:295名研究对象的基因多态性分析结果显示C1549T和G1708A这2个位点的基因在太原地区人群中为野生型纯合子,突变型基因的检测结果为0。G1661A位点基因可分为野生型纯合子GG、突变型杂合子GA和突变型纯合子AA三种基因型。GG、GA和AA三种基因型分布在接触组与对照组分别为52.8%、27.6%、19.6%和67.9%、19.8%、12.3%,差异没有统计学意义(P＞0.05);接触组和对照组G、A等位基因频率分别为77.8%、22.2%和66.6%、33.4%,差异没有统计学意义(P＞0.05);等位基因频率分布符合Hardy-Weinberg平衡定律。因此我们只分析接触组AhR基因多态性与神经毒性和淋巴细胞凋亡的关系。
2.AhR G1661A基因多态性与B[a]P神经行为毒性的关系:焦炉工总数字跨度得分、顺序数字跨度得分和数字译码得分明显低于对照组,差异有统计学意义(P＜0.05);其余神经行为功能和情感状态得分在接触组与对照组之间差异没有统计学意义(P＞0.05)。接触组按照G1661A3种不同的基因型分组后的分析结果表明,AA突变纯合子基因型携带者困惑-迷茫的得分显著高于GG野生型纯合子基因型携带者和GA杂合子基因型携带者,差异有统计学意义(P＜0.05);愤怒-敌意、忧郁-沮丧、疲惫-隋性、紧张-焦虑和有力-好动得分在三种基因型之间差异没有统计学意义(P＞0.05)。在神经行为功能方面,数字跨度得分、倒序和视觉记忆得分呈现AA＜GA＜GG基因型趋势,但是差异没有统计学意义(P＞0.05)。说明AhR基因多态性影响焦炉工的情感状态得分,AA突变纯合子基因型的携带者困惑-迷茫得分明显增加。
3.AhR G1661A基因多态性与B[a]P自主神经毒性的关系:与对照组相比,反应焦炉工副交感神经调节功能的HR-V值明显下降,差异有统计学意义(P＜0.05),其余指标的改变没有统计学意义。通过对焦炉工AhR G1661A位点三种基因型与自主神经功能的分析,结果显示GG、GA、AA三种基因型的副交感神经调节指标HR-V、HR-DB、RR30:15、RRmax:min和交感神经调节指标BP-IS方面,差异都没有统计学意义(P＞0.05)。说明AhR G1661A位点的基因多态性与焦炉工的自主神经功能无关。
4.AhR G1661A基因多态性与外周血淋巴细胞凋亡的关系:与对照组相比,焦炉工外周血淋巴细胞的早期凋亡率、晚期凋亡率和总凋亡率升高,但是差异没有统计学意义(P＞0.05)。接触组AhR基因多态性G1661A与外周血淋巴细胞凋亡的关系分析结果显示,焦炉工外周血淋巴细胞早期凋亡率、晚期凋亡率和总凋亡率呈现GG＜GA＜AA趋势,但是差异没有统计学意义(P＞0.05)。本研究未发现AhR G1661A基因多态性与外周血淋巴细胞凋亡有关。
第二章AhR/ARNT-CYP1A1途径在B[a]P致神经细胞凋亡中的作用研究
第一节B[a]P致神经细胞凋亡的体内研究
40只健康成年雄性SD大鼠,脑室插管一周后按体重随机分为4组:橄榄油组(溶剂对照组)、B[a]P低浓度组(0.625 mmol/L)、B[a]P中浓度组(1.25 mmol/L)、B[a]P低浓度组(2.5mmol/L)。高、中、低浓度组分别注入相应浓度的B[a]P溶液10μl,溶剂对照组注射10μl橄榄油。一周染毒一次,连续染毒4次。染毒结束后断头处死,分离脑组织,用光镜和电镜观察脑组织病理和超微结构的改变,用流式细胞仪检测神经细胞的凋亡率。
(1)病理形态改变光镜观察到,对照组大鼠海马神经细胞排列整齐,形态正常;随着B[a]P染毒剂量的增高,海马神经细胞出现体积缩小,细胞数目逐渐减少,排列紊乱,细胞核的皱缩、消失,染色质凝集、边聚,甚至有细胞核的溶解消失等现象。但是在皮质、小脑、延髓和脑桥部分未见明显改变。
(2)超微结构的改变透射电镜观察结果显示,对照组海马细胞核细胞核形态规则,对称分布,染色质分布均匀:线粒体形态规则,嵴排列整齐。随着B[a]P剂量的增高,细胞核皱缩,染色质边聚,核膜破裂,线粒体肿胀,甚至呈气球样变而且外膜破裂,并伴有线粒体嵴减少、变短、断裂甚至消失。
(3)神经细胞凋亡率的测定结果随着B[a]P染毒剂量的增加,大鼠神经细胞的早期凋亡率呈剂量反应性的上升。与对照组相比,各剂量组神经细胞的早期凋亡率显著上升,差异有统计学意义(P＜0.01);高剂量组的晚期凋亡率显著高于中、低剂量组和对照组,差异有统计学意义(P＜0.05),而中、低剂量组与对照组差异没有统计学意义(P＞0.05)。以上结果显示海马神经细胞凋亡可能是B[a]P神经毒性的主要机制之一。
第二节神经细胞凋亡与AhR、ARNT和CYP1A1基因和蛋白关系的体内研究
(1)实时荧光定量PCR结果显示:与对照组相比,各剂量组AhR和ARNT基因的表达差异没有统计学意义(P＞0.05);而CYP1A1的基因表达随着染毒剂量的增加而增加,与对照组相比,各剂量组的差异有统计学意义(P＜0.05);与低剂量组相比,高剂量组CYP1A1基因表达显著增加,差异有统计学意义(P＜0.05)。
(2)免疫组化结果显示:AhR、ARNT这2种蛋白质在海马汉神经细胞的胞浆和胞核都表达,而且表达量随着染毒剂量的增加而增加。与对照组相比,各染毒组AhR蛋白表达随着染毒剂量的增加都明显增加,差异有统计学意义(P＜0.05);ARNT蛋白的表达也随着染毒剂量的增加而增加,中、高剂量组的差异有统计学意义(P＜0.05),而低剂量组的差异没有统计学意义(P＞0.05)。
(3) Wesern-blot结果显示:AhR、ARNT和CYP1A1蛋白表达随着染毒剂量的增加而升高。与对照组相比,中、高剂量组AhR、ARNT蛋白的表达差异有统计学意义(P＜0.05);各剂量组CYP1A1的表达也显著高于对照组(P＜0.05),中、高剂量组CYP1A1蛋白表达显著增加高于低剂量组,差异有统计学意义(P＜0.05)。
(4)相关分析表明:神经细胞凋亡率与CYP1A1 mRNA表达呈正相关(r=0.804,P＜0.05),与AhR蛋白表达呈正相关(r=0.845,P＜0.01),与ARNT蛋白表达呈正相关(r=0.781,P＜0.05),与CYP1A1蛋白表达呈正相关(r=0.807,P＜0.01);AhR蛋白与CYP1A1 mRNA之间呈正相关(r=0.826,P＜0.05),与CYP1A1蛋白之间呈正相关(r=0.774,P＜0.05)。AhR和ARNT在mRNA和蛋白表达水平的差别可能与较长的染毒时间内,基因转录水平的变化已经完成并且已经翻译为蛋白质有关。以上结果表明AhR、ARNT和CYP1A1很可能参与了B[a]P致神经细胞的凋亡过程。
第三节B[a]P致神经细胞凋亡的体外研究
无菌条件下分离新生1～3d SD大乳鼠的皮质,将细胞浓度调至1×10~5个细胞/ml,于37℃、5%CO_2培养箱中培养,24 h后更换新鲜的DMEM培养液并加入0.5 mg/ml阿糖胞苷(终浓度10μM)培养液以抑制胶质细胞增殖。采用神经胶质细胞的特异标志物(GFAP)进行神经元的纯度检测。在细胞培养第5天左右,选取生长良好的同批次神经细胞,分为对照组、低、中和高剂量4个组,每组至少3孔,分别按照培养液总体积的0.%和0.3%加入B[a]P和S9,使B[a]P终浓度分别为0、10μM、20μM,40μM。继续培养40 h,观察细胞的生长状况和形态学改变,Annexin-V/PI双染法检测神经细胞的凋亡。
(1)神经细胞的形态学改变光学显微镜下可见,对照组神经细胞细胞核大而清晰,轴突长且比较均一,树突较多,细胞连接丰富而紧密。随着染毒剂量的增加,神经细胞的轴突逐渐变短,树突数量减少,细胞连接减少,严重者出现细胞体积收缩、变圆,部分树突融解、消失,轴突也严重受损,细胞连接明显减少。AO/EB荧光染色能将活细胞为亮绿色,死细胞染为红色,凋亡细胞染为桔色。根据细胞的颜色就可以区别细胞的生存状况。随着B[a]P染毒剂量的增加,被染成桔色的细胞比例逐渐增多,细胞轴突和树突数量减少,细胞间的连接也减少,有些细胞轴突变短、甚至消失,胞体变圆,胞核皱缩、边聚。
(2) Annexin-V/PI双染法检测到中、高剂量组细胞凋亡率显著高于对照组,差异有统计学意义(P＜0.05),但是低剂量组差异没有统计学意义(P＞0.05)。说明B[a]P能诱导原代培养神经元的凋亡。
第四节神经细胞凋亡与AhR、ARNT和CYP1A1基因和蛋白关系的体外研究
原代培养神经细胞的实时荧光定量PCR结果显示各剂量组AhR、ARNT和CYP1A1基因表达明显高于对照组,差异有统计学意义(P＜0.05);高剂量组CYP1A1基因表达显著高于低剂量组,差异有统计学意义(P＜0.05)。Western-blot检测结果也显示AhR、ARNT和CYP1A1蛋白表达随着染毒剂量的增加逐渐增高。与对照组相比,中、高剂量组AhR、ARNT蛋白的表达差异有统计学意义(P＜0.05);各剂量组CYP1A1的表达也显著高于对照组(P＜0.05),中、高剂量组CYP1A1蛋白表达显著高于低剂量组,差异有统计学意义(P＜0.05)。说明B[a]P诱导的神经细胞凋亡与AhR、ARNT和CYP1A1 mRNA和蛋白表达增加有关。
第二部分p53-bcl-2/bax线粒体途径在B[a]P致神经细胞凋亡中的作用
B[a]P诱导的多种细胞凋亡都与p53相关信号途径的激活有关。P53通过诱导bax蛋白表达增加,降低bcl-2/bax的比例诱导细胞凋亡。bcl-2和bax是参与调节细胞凋亡的的一对功能相反的基因。bax通过形成二聚体,促使线粒体释放细胞色素C,激活caspase家族诱导细胞凋亡;bcl-2通过与bax形成异二聚体,减少bax二聚体的形成抑制细胞凋亡。因此bcl-2/bax的比例决定着细胞的存亡。本研究将对B[a]P诱导神经细胞凋亡的p53-bcl-2/bax线粒体机制进行研究。
第一节神经细胞凋亡与p53-bcl-2/bax基因和蛋白关系的体内研究
(1)实时荧光定量PCR结果显示:p53 mRNA表达随着染毒剂量的增加而上升,与对照组相比,各剂量组的差异有统计学意义(P＜0.05),高剂量组p53 mRNA水平明显高于低剂量组,差异有统计学意义(P＜0.05);各剂量组bcl-2 mRNA表达显著低于对照组,差异有统计学意义(P＜0.01),高剂量组bcl-2 mRNA水平明显低于低剂量组,差异有统计学意义(P＜0.05);而bax mRNA表达在各组间差异没有统计学意义(P＞0.05)。
(2)免疫组化结果显示随着B[a]P染毒剂量的增加,p53和bax蛋白的表达逐渐增加,bcl-2蛋白逐渐下降。与对照组相比,各剂量组p53蛋白显著增加,bcl-2蛋白显著下降,差异有统计学意义(P＜0.01),高剂量组bcl-2蛋白显著低于低剂量组,差异有统计学意义(P＜0.05);与对照组相比,中、高剂量组bax蛋白显著升高,差异有统计学意义(P＜0.05),但是低剂量组差异没有统计学意义(P＞0.05)。与对照组相比,各剂量组bcl-2/bax比值显著下降,差异有统计学意义(P＜0.05)。
(3) Wesern-blot检测p53蛋白随着染毒剂量的增加而增加,与对照组相比,中、高剂量组p53蛋白水平显著增高,差异有统计学意义(P＜0.05)。相关分析结果显示,细胞凋亡率与p53 mRNA表达量之间呈明显的正相关(r=0.864,P＜0.05),与p53蛋白水平之间也呈明显的正相关(r=0.757,P＜0.01);海马神经细胞凋亡率与bcl-2 mRNA表达呈负相关(r=-0.924,P＜0.01),与Bcl-2蛋白呈负相关(r=-0.893,P＜0.01),与Bax蛋白呈正相关(r=0.872,P＜0.01),与bcl-2/bax比值呈负相关(r=-0.876,P＜0.01)。以上说明p53-Bcl-2/bax途径也参与了B[a]P诱导的神经细胞凋亡。
第二节神经细胞凋亡与线粒体途径细胞色素C和caspase酶关系的体内研究
透射电镜下观察到B[a]P主要使神经细胞线粒体肿胀变形,伴有嵴缩短、断裂甚至缺失等现象。线粒体平均荧光强度随着染毒剂量的增加逐渐升高,膜电位逐渐下降。高剂量组平均荧光强度明显高于对照组、低剂量组和中剂量,差异有统计学意义(P＜0.05);高剂量组膜电位下降百分率较对照组和低剂量组显著下降,差异有统计学意义(P＜0.05)。Western-blot结果显示线粒体释放到胞浆中的细胞色素C含量随着染毒剂量的增加逐渐增加,与对照组相比,各剂量组差异有统计学意义(P＜0.05)。caspase-3,8,9蛋白酶活力测定结果显示中、高剂量组的caspase-3比活力显著高于对照组,差异有统计学意义(P＜0.05),而低剂量组差异没有统计学意义(P＞0.05);各剂量组caspase-9的比活性都显著高于对照组,差异有统计学意义(P＜0.05);但是caspase-8的活力在对照组与各剂量组之间差异没有统计学意义(P＞0.05)。海马神经细胞凋亡率与线粒体膜电位呈负相关(r=-0.820,P＜0.01),与胞浆细胞色素C水平和caspase以及caspase-9活力呈正相关,相关系数分别为0.883(P＜0.01),0.805(P＜0.01)和0.756(P＜0.01),表明B[a]P诱导的神经细胞凋亡依赖于caspase酶的激活,与线粒体膜电位的下降和细胞色素C释放有关。
第三节神经细胞凋亡与p53-bcl-2/bax基因和蛋白关系的体外研究
实时荧光定量PCR结果显示神经元p53 mRNA的表达随着染毒剂量的增加而上升。与对照组相比,中、高剂量组的差异有统计学意义(P＜0.05),而低剂量组的差异没有统计学意义(P＞0.05);bcl-2和bax mRNA的表达随着染毒剂量的增加呈下降趋势,与对照组相比,中、高剂量组bcl-2和bax mRNA的差异有统计学意义(P＜0.01),而低剂量组bcl-2和bax的差异没有统计学意义(P＞0.05);进一步分析发现bcl-2 mRNA下降较baxmRNA明显,bcl-2/bax mRNA比值随着染毒剂量的增加而下降。免疫组化结果显示bcl-2随着染毒剂量的增加而下降,bax随着染毒剂量的增加而升高。Western-blot结果也显示与对照组相比,中、高剂量组p53蛋白表达显著增加,bcl-2明显下降,差异有统计学意义(P＜0.05),但是低剂量组差异没有统计学意义。中、高剂量组bax蛋白表达明显高于对照组,差异有统计学意义(P＜0.05)。说明B[a]P诱导的神经细胞凋亡与p53-bcl-2/bax途径有关。
第四节神经细胞凋亡与线粒体、细胞色素C和caspase酶关系的体外研究
透射电镜观察到体外原代培养神经元线粒体肿胀,嵴变短、断裂甚至消失。线粒体膜电位随着染毒剂量的增加逐渐下降,与对照组相比,各剂量组差异有统计学意义(P＜0.05);胞浆细胞色素C含量逐渐增加,与对照组相比,各剂量组差异有统计学意义(P＜0.05);caspase-3,9的酶活力随着染毒剂量的增加而逐渐上升,差异有统计学意义(P＜0.05),但是caspase-8的活力变化在组间没有统计学意义(P＞0.05)。
第三部分拮抗AhR或抑制p53后细胞凋亡及凋亡相关基因的改变
前两部分的研究结果提示AhR/ARNT-CYP1A1和p53-bcl-2/bax-caspase途径共同参与B[a]P诱导的神经细胞凋亡过程。由于AhR/ARNT-CYP1A1途径主要介导B[a]P的代谢活化,p53-bcl-2/bax-caspase途径直接参与凋亡的发生。那么这两条信号途径一定存在着某种内在的联系。我们的猜想是:B[a]P通过激活AhR/ARNT-CYP1A1途径,诱导p53-bcl-2/bax-caspase机制活化诱导神经细胞的凋亡。为此我们采用原代培养的神经细胞,用AhR拮抗剂α-萘黄酮(ANF)或p53抑制剂pifithrin-α(PFT-α)来证实这一猜想。
由于ANF和PFT-α可能对CYP1A1有潜在的诱导性或非特异干扰效应,我们又采用特异性基因干扰技术沉默CYP1A1后,研究拮抗AhR或抑制p53后B[a]P毒性的改变,阐明B[a]P致神经细胞凋亡的可能机制。由于原代培养神经元生命周期短暂,不能自我分裂,很难达到较高的转染效率,我们干扰人神经母细胞瘤细胞株SH-SY5Y细胞的CYP1A1基因后进一步研究B[a]P诱导神经细胞凋亡的机制。
第一节ANF拮抗AhR后神经元凋亡与凋亡相关基因的改变
B[a]P染毒前2小时加入AhR拮抗剂ANF,使终浓度为50 nM,其余同前。凋亡测定结果表明B[a]P诱导的神经细胞早期凋亡率、晚期凋亡率和总凋亡率在对照组与各染毒组间差异没有统计学意义(P＞0.05)。ANF拮抗AhR后,B[a]P对神经细胞AhR、ARNT以及CYP1A1mRNA的表达仍有一定的诱导作用,但是组间差异没有统计学意义(P＞0.05)。ANF能使神经细胞p53 mRNA的表达在低、中剂量组迅速降低,但是ANF不能降低高剂量B[a]P对p53的诱导;ANF能使神经细胞bcl-2 mRNA的表达增高,bax mRNA的表达降低,bcl-2/bax比值升高。说明ANF阻止B[a]P诱导的神经细胞凋亡,其主要机制可能与ANF通过阻断B[a]P对CYP1A1的转录调控和诱导,与降低B[a]P的代谢活化有关。
第二节PFT-α抑制后神经元凋亡与凋亡相关基因的改变
B[a]P染毒前2小时加入p53抑制剂PFT-α,使终浓度为25μM,其余同前。凋亡测定结果显示B[a]P诱导的神经细胞早期凋亡率、晚期凋亡率和总凋亡率在对照组与各染毒组间差异没有统计学意义(P＞0.05)。实时荧光定量PCR结果显示AhR、ARNT以及CYP1A1 mRNA表达量随着S[a]P染毒剂量增加而升高,差异有统计学意义(P＜0.05)。各组间p53 mRNA水平差异没有统计学意义(P＞0.05),说明PFT-α不能抑制B[a]P对神经细胞AhR、ARNT以及CYP1A1 mRNA的诱导,但是能抑制B[a]P对p53 mRNA诱导,使bcl-2 mRNA表达增高,使bcl-2/bax比值升高。说明PFT-α抑制B[a]P诱导的神经细胞凋亡的主要机制可能是:降低p53 mRNA水平和对bcl-2家族的转录调控能力,使抑凋亡基因bcl-2表达上升,促凋亡基因bax表达下调,提高bcl-2/bax比值。
第三节B[a]P对人SH-SY5Y细胞的毒性以及AhR和p53的作用
人SH-SY5Y细胞用RPMI1640培养液培养(含15%胎牛血清,2 mM L-谷氨酰胺,10mM HEPES,80单位/ml青霉素和100μg/ml链霉素),于37℃、5%CO2培养箱中培养。每3～4天传代一次。传代24h后选择处于对数生长期的同一代细胞,分为对照组、低剂量组、中剂量组、高剂量组进行染毒。染毒时换用含3%胎牛血清的RPMI1640培养液,分别按照培养液总体积的0.3%和0.1%加入S9混合液和B[a]P,混匀,使B[a]P终浓度分别为0、2.5 nM、5.0 nM和10.0 nM。所有的化合物都用DMSO溶解,保证DMSO不超过培养液总体积的0.3%。抑制物或者拮抗剂在染毒前2小时加入。染毒后继续培养48 h后进行检测用荧光倒置显微镜观察细胞的形态学改变,用CCK-8法测定细胞活力,通过检测磷脂酰丝氨酸的Annexin V/PI方法和检测亚二倍体凋亡峰的PI染色法测定细胞凋亡。
对照组细胞状态良好,大小均匀,贴壁好,细胞间连接较多,细胞树突和轴突长而多;低剂量组有少部分死亡细胞,呈漂浮状,树突和轴突减少变短;中剂量组细胞数量明显减少,漂浮的细胞较多,部分细胞皱缩,突起明显缩短;高剂量组细胞数量进一步减少,突起进一步缩短甚至消失,细胞皱缩呈圆形,细胞间连接异常松散甚至消失。hoechst 33258是一种特异性与DNA结合的染料,用于观察细胞核形态的变化。正常细胞核呈圆形,着色均匀,凋亡细胞核皱缩,DNA高度凝聚使着色深,有的出现核的崩解、破裂。但是hoechst33258染色没有观察到典型的凋亡细胞核的变化。AO/EB染色将活细胞染成亮绿色,死细胞染成红色,而凋亡细胞染成桔色。结果显示随着剂量的增加,染成红色的坏死细胞有所增加,但是被染成橘黄色的凋亡细胞数量很少。细胞活力测定结果显示随着B[a]P染毒剂量的增加,细胞活力逐渐降低,与对照组相比,高剂量细胞活力下降差异有统计学意义(P＜0.05);Annexin V/PI法没有检测到细胞早期凋亡率,PI法检测到亚二倍体的DNA,有少量细胞凋亡,但是差异没有统计学意义(P＞0.05)。沉默CYP1A1基因后,ANF和PFT-α能够阻止B[a]P诱导的细胞活力下降。与对照组相比,ANT拮抗后中剂量组细胞活力明显增高,差异有统计学意义(P＜0.05);PFT-α抑制后低剂量组细胞活力明显增高,差异有统计学意义(P＜0.05)。结合前面的研究结果,我们认为AhR/ARNT-CYP1A1途径和p53-bcl-2/bax线粒体途径共同参与了B[a]P诱导的神经细胞凋亡。
结论:
1.AhR基因多态性与B[a]P神经毒性有关。
2.AhR/ARNT-CYP1A1途径和p53-bcl-2/bax-caspase途径共同介导和参与了B[a]P诱导的神经细胞凋亡。
3.B[a]P致神经细胞凋亡的机制可能为AhR/ARNT-CYP1A1-p53-bcl-2/bax-caspase。
4.此外,B[a]P的代谢物也可能直接损伤线粒体以caspase依赖的方式导致细胞凋亡。
Metabolism and carcinogenicity of B[a]P depend on the activation of AhR/ARNT-CYP1A1 pathway.Specific binding of B[a]P and AhR was a prerequisite of the activation of AhR/ARNT-CYP1A1 pathway.So,AhR gene polymorphism may influence the sensitivity of neurotoxicity and apoptosis induced by B[a]P.Researches showed that B[a]P increased AhR expression.AhR,ARNT and CYP1A1 mRNA expression increased in a time and dose dependent manner in the process of B[a]P-induced hepatoma cell apoptosis.B[a]P caused hepatoma cell apoptosis until it metabolited into active forms after activating AhR and inducing CYP1A1.B[a]P-induced malignant cell apoptosis was related to CYP1A1 activation and mediated by CYP1A1.It is reported that CYP1A1 protein and enzyme activity in zebrafish signifiantly increased after B[a]P exposure.B[a]P-induced neurocyte apoptosis was related to CYP1A1 mRNA and protein.So in this part we would study the relationship of AhR gene polymorphism and neurotoxicity and cell apoptosis induced by B[a]P by analyzing populations' genotype,and the changes of AhR/ARNT-CYP1A1 pathway in the process of B[a]P-induced neural cell apoptosis in vivo and vitro.
ChapterⅠRelationship of AhR gene polymorphism and B[a]P's neurotoxieity and peripheral blood lymphocyte apoptosis
We recruited 214 male healthy coke-oven workers occupationally-exposed B[a]P as the exposed group and 81 warehouse workers in the raw material plant as the control group which were as possible as comparable with the exposed group except for never occupationally exposed B[a]P and other neurotoxicants.Neurobehavioral core test battery recommended by world health organization(NCTB/WHO) was used to test the neurobehavioral function of subjects;autonomic nerve system test battery recommended by Ewing DJ was adopted to measure the autonomic nervous function.And lymphocytes and genome DNA were isolated and extracted from the peripheral ulnar vein blood in the conditional of empty stomach.Apoptosis of lymphocytes were detected by the flow cytometry after stained with Annexin V-FITC and PI.Allele specific-polymerase chain reaction(AS-PCR) and restricted fragment length polymorphismpolymerase chain reaction(RFLP-PCR) methods were applied to determine the AhR gene variations in the 3 sites of C 1549T(Pro517Ser)、G1661A(Arg554Lys) and G 1708A(Va1570Ile) in the 10~(th) exon.Then we analyzed the relationship between AhR gene polymorphism and neurobehavioral function,autonomic nervous function(ANS) and lymphocyte apoptosis by SPSS FOR WINDOWS 10.0 SOFTWARE.The results were given in next.
1.Results of gene polymorphism analysis
Results of 295 subjects' AhR gene polymorphism indicated that the 2 sites variation including C1549T(Pro517Ser) and G1708A(Va1570Ile),were wild homozygotes and the mutant alleles were not detected in population of Taiyuan district.That is,the mutant allele frequencies of C1549T and G1708A were 0.There were 3 genotypes in G1661A sites including GG(wild type homozygote),GA(mutant heterozygote) and AA(mutant homozygote).The distributional frequencies of GG,GA and AA were 52.8%,27.6%and 19.6%in the exposed group and 67.9%, 19.8%and 12.3%in the control group,respectively.The allele frequencies of G and A were 77.8%and 22.2%in the exposed workers,66.6%and 33.4%in the controls,and no significant difference existed between the two groups(P＞0.05).The allele frequency distribution was consistent with the Hardy-Weinberg equilibrium law.Therefore,we analyzed the relationship between the gene polymorphism and neurotoxicity and lymphocyte apoptosis only in the exposed group.
2.Relationship between AhR G1661A gene polymorphism and neurobehavioral function
Compared to the controls,the scores of total digital span,serial digital span and digital decoding decreased significantly in the coke oven workers.The scores in other neurobehavioral function and profile of mood state had not significant difference.Analytical results show that the score of stumble-vast and hazy increased highly in the AA genotype than that in the GG or GA genotypes(P＜0.05).There were no significant difference among GG,GA and AA genotypes in anger-hostility,gloom-depression,tension-anxiety and power-activity(P＞0.05).The scores of total digital span,reversed digital span and vision retention presented the tendency of AA＜GA＜GG,however,the differences were not significant(P＞0.05).This result suggests that AhR genetic polymorphism influenced the profile of mood state in the coke oven workers,and the scores of stumble-vast and lazy significantly increased in the AA genotypes compared to the GG or GA genotype.
3.Relationship between AhR G1661A gene polymorphism and autonomic nervous function
The HR-V value reflecting the modulation of parasympathetic nerve significantly decreased in the exposed group compared to the control group(P＜0.05),other indices had no significant difference between the exposed group and the controls.Analytical results of gene polymorphism and autonomic nervous system show that no significant difference existed in HR-V,HR-DB, RR30:15,RRmax:min and BP-IS in the coke oven workers among GG,GA and AA genotypes (P＞0.05).All these results indicate that AhR G1661A gene polymorphism had no relation with the autonomic nevous function of the coke oven workers.
4.Relationship between AhR G1661A gene polymorphism and apoptosis rates of lymphocytes
The rates of early apoptosis,later apoptosis and total apoptosis of lymphocytes in the peripheral blood were higher in the coke-oven workers than that in the controls,but no significant differences existed(P＞0.05).Also the apoptosis rate of lymphocyte had no statistical difference(P＜0.05) among GG,GA and AA genotypes although there was a tendency of GG＜GA＜AA.Relationship between the AhR G1661A gene polymorphism and the lymphocyte apoptosis was not found in our study.
ChapterⅡRole of AhR/ARNT-CYP1A1 pathway in B[a]P -induced neurocyte apoptosis
1.Study on the neural cell apoptosis induced by B[a]P in vivo
Forty male adult and healthy Sprague-Dawley rats were divided randomly into 4 groups according to their weights after incubated in the cerebral ventrical:olive oil group(solvent control group),0.625 mmol/L B[a]P(low-dose) group,1.25 mmol/L B[a]P(mid-dose) group and 2.5 mmol/L B[a]P(high-dose) group.The low-dose,mid-dose and high-dose rats were injected 10μl B[a]P olive oil solution of different concentrations,respectively,and the control rats were injected the same volume solvent olive oil.The treatment was performed once a week for successive 4 times.After the treatment was accomplished,the rats were decapitated to death and isolated the brain tissues to be observed the morphologic and ultramicrostructure changes under the light and electric microscopes.Cell apoptosis was detected by Annexin V/PI staining in the flow cytometry.
(1) Morphology changes:under the light microscope,the hippocampus neurocytes arranged in order with normal shape.The higher of the B[a]P dose was,the more serious of the damage was. The impaired hippocampus cells was contracted,diminished and displayed disorderly and loosened,the karyon shrinked and even disappeared,and the chromatin condensed and margined. However,no obvious changes were observed in the cortex,cerebellum,bulb and pons.
(2) The ultrastructure changes in the electron microscope displayed:in the control group,the karyotin was round and distributed symmetrically with well-distributed chromatin.The mitochondria were regular and the bars were well-arranged.With the concentration of B[a]P increasing,the karyon became shrinken,assembled highly in the edge.The karyotheca was even broken in some severely-damaged cells.The mitochondria became swollen and sometimes like a balloon even with a broken outer-membrane.The bars were reduced,shortened,broken and even disappeared.
(3) The apoptotic rate of neurocytes increased with the concentration of B[a]P.Early apoptotic rate significantly increased in the 3 B[a]P groups compared to the controls(P＜0.01).The later apoptosis was notably higher in the high-dose group than that in other groups(P＜0.05),but which is not high in the middle group and the low group(P＞0.05).These results demonstrate that B[a]P can induce hippocampal cell apoptosis.
2.Research on the relationship of neurocyte apoptosis and AhR,ARNT and CYP1A1 mRNA and protein in vivo
(1) Results of real-time fluorescent quantitation show that no statistical significance were found in AhR and ARNT mRNA expression among groups but CYP1A1 mRNA significantly increased with the dose of B[a]P,which was higher in 3 treated groups than that in the solvent group (P＜0.05) and higher in the high-dose group than that in the low-dose group(P＜0.05).
(2) Immunohistochemistry results show that AhR and ARNT protein expressed in the cytoplasm and nucleus in the hippocampal cells,and increased with the increment of B[a]P dose.Compared to the controls,AhR protein had a significant increase in the 3 B[a]P-treated groups(P＜0.05), and ARNT protein statistically increased in the mid-dose and high-dose groups(P＜0.05),but not in the low-dose group(P＞0.05).
(3) Western-blot results indicate that AhR,ARNT and CYP1A1 protein increased in a B[a]P dose-dependent manner.AhR and ARNT protein were significantly higher in the mid-dose group and the high-dose group than those in the controls(P＜0.05).And CYP1A1 protein increased significantly in the the 3 B[a]P-treated groups compared to the solvent group(P＜0.05), furthermore which were higher in the mid-dose and high-dose groups than that in the low-dose group(P＜0.05).
(4) Correlated analysis manifested that a positive correlation existed between the cell apoptotic rates and CYP1A1 mRNA(r=0.804,P＜0.05).Positive correlations were also found in the cell apoptotic rates and AhR protein(r=0.845,P＜0.01),ARNT protein(r=0.781,P＜0.01) and CYP1A1 protein(r=0.807,P＜0.01),respectively.In addition,the positive correlations existed between AhR protein and CYP1A1 mRNA(r=0.826,P＜0.01),and between AhR.protein and CYP1A1 protein(r=0.774,P＜0.05).The disagreement in mRNA and protein of AhR and ARNT was possibly caused by the long duration of administration in which mRNA transcriptional expression had accomplished and translated into protein.All the results showed that AhR,ARNT and CYP1A1 possibly involved in the process of neurocyte apoptosis.
3.Study on the neuronal cell apoptosis induced by B[a]P in vitro
Primary neuronal cells were dissociated and prepared from newborn 1-3 days neonate SD rats' cerebral cortex,and cultured in a 37℃and 5%CO_2 incubator after adjusted to 1×10~5 cells/ml.The fresh DMEM medium was displaced and added cytosine arabinoside(final concentration 10μM) to inhibit astrocytes growing after 24 hours.The astrocytes specific marker GFAP was identified by immunohistochemical method to ensure the neurons' purity. Astrocytes should be less than 10%of the total cells.In the 5th days,comparable well-growth neurons were divided randomly into 4 groups:the control group,the low-dose group,the mid-dose group and the high dose group,and then treated with 0.1%of the medium to the B[a]P final concentration of 0,10μM,20μM,40μM,respectively.S9 mixture was added to 0.3%of the medium at the same time.The control group was only added DMSO solvent.After a sequence maintenace for 40 h in the incubator,morphology changes were observed under the fluorescence inverted microscope and apoptotic rates were detected by the Annexin-V/PI double staining.
(1) Morphology changes of the neuronal cells demonstrated that the nucleus was big and clear with long and uniform neuraxons and many dendrites,and the cellular junctions were abundant and tight.With the dose of B[a]P increasing,the numbers of neuraxons and dendrites decreased and the conjunctions among cells were reduced evidently.The severity displayed that some neuraxons became shorter,even disappear,the cell body became shfinked and round.Dyeing with AO/EB fluorescent reagent,live neuron showed a bright green color,while the dead one was red,and the apoptosis was orange.According to the color of neuron,we can distinct living status of the neuron.With B[a]P dose increasing,the ratios of orange cells increased,and the numbers of neuraxons and dendrites and the conjunction among cells decreased,some neuraxons became shorter,even disappear,and the cell body became round,the karyon shrinked or aggregated in the edge.
(2) Annexin-V/PI double staining results showed that cell apoptosis rates in the mid-dose group and the high-dose group were significantly higher than that in the controls(P＜0.05),but no difference were found in apoptosis rates between the controls and the low-dose group(P＞0.05). The results indicated that B[a]P induced primary neuron cell apoptosis.
4.Research on the relationship of neurocyte apoptosis and AhR,ARNT and CYP1A1 mRNA and protein in vitro
Real-time fluorescent quantitation PCR results showed that AhR,ARNT and CYP1A1 mRNA expression significantly increased in the 3 B[a]P treated groups compared to the control group(P＜0.05),and CYP1A1 mRNA expression in the 40μM B[a]P group was statistically higher than that in the 10μM B[a]P group(P＜0.05).The data suggested that B[a]P can induce primary cultured neurons apoptosis and which related to the induction of AhR,ARNT and CYP1A1.Western-blot results indicated that AhR,ARNT and CYP1A1 protein increased with the dosage of B[a]P.Compared to the controls,AhR and ARNT protein in the mid-dose group and the high-dose group were statistically high(P＜0.05),but not in the low-dose group (P＞0.05).CYP1A1 protein were significantly higher in the 3 treated groups than that in the control group(P＜0.05),and which were higher in the mid-dose group and high-dose group than that in the control group(P＜0.05).This suggested that B[a]P-induced neurocyte apoptosis was related to the augment ofAhR,ARNT and CYP1A1 mRNA and protein expression.
PartⅡRole of p53-bcl-2/bax mitochondrial pathway in B[a]P-induced neurocyte apoptosis
B[a]P-induced cell apoptosis were related with the activation of p53 signals.P53 induces cell apoptosis via increasing bax protein expression and dicreasing the ratio of bcl-2/bax.Bcl-2 and bax is a couple of genes with opposite function regulating apoptosis.Bax homodimer urges mitochondrion releasing cytoshrome C into the cytoplasm which activates caspase family and causes cell apoptosis;however,bcl-2 inhibits cell apoptosis by heterodiming with bax and reducing bax contents.So the bcl-2/bax ratio determines the cell fate.This part would study the mechanism of p53-bcl-2/bax mitochondrial pathway in B[a]P-induced apoptosis.
1.Study on the relationship of nerve cell apoptosis and p53-bcl-2/bax mRNA and protein in vivo
(1) Results of real-time fluorescent quantitation PCR showed that p53 mRNA increased with the dose of B[a]P,which statistically increased in the 3 B[a]P-treated groups(P＜0.05),and was higher in the high-dose group than that in the low-dose group(P＜0.05).Bcl-2 mRNA expression decreased significantly in the 3 B[a]P-treated groups compared to the controls(P＜0.01),which was lower in the high-dose group than the control rats(P＜0.05).However,no significant difference was found in bax mRNA among groups although bax mRNA showed a tendency of decreasing post B[a]P treatment.
(2) Immunohistochemistry results showed p53 and bax protein rose and bcl-2 protein diminished with the B[a]P dose.Compared to the controls,p53 protein were significantly increased and bcl-2 protein were statistically decreased(P＜0.05),and bcl-2 protein was lower in the high-dose group than that in the control group(P＜0.05).Bax protein increased with the dosage of B[a]P and was higher in the mid-dose and high-dose group than the controls(P＜0.05),but not higher in the low-dose group(P＜0.05).Bcl-2/bax ratio had a significant decrease in the 3 B[a]P-treated groups compared to the control group.
(3) Western-blot results indicated a similar dose-dependent relationship between p53 protein and the dosage of B[a]P.Compared to the controls,p53 protein increased significantly in the mid-dose group and high-dose group(P＜0.05),and was higher in the high-dose group than that in the low-dose group(P＜0.05).Correlation analysis showed positive correlations existed between the apoptosis rate and p53 mRNA(r=0.864,P＜0.05),and p53 protein(r=0.757,P＜0.01). Negative relations existed between the cell apoptosis rate and bcl-2 mRNA(r=-0.924,P＜0.01), and bcl-2 protein(r=-0.893,P＜0.01),and bcl-2/bax ratio(r=-0.876,P＜0.01);and a positive correlation existed between the apoptosis rate and bax protein(r=0.872,P＜0.01).This results indicated that p53-bcl-2/bax was involved in the process of B[a]P induced neural cell apoptosis.
2.Study on the relationship of neural cell apoptosis and mitochondrial cytochrome C and caspase enzymes in vivo
B[a]P caused the mitochodria in neural cell swelling accompanied with bars shorten, segmentation and even deletion in the transmission electron microscope.The mitochondrial mean fluorescence density increased gradually with the dosage of B[a]P,and which was significantly higher in the high-dose group than that in the controls and other groups(P＜0.05). The membrane potential decreased significantly in the high-dose group compared with the low-dose group and the controls(P＜0.05).Western-blot results showed that cytochrome C in cytoplasm emitting from mitochondria increased with the dose of B[a]P and had significant diferrences in the 3 B[a]P-treated groups compared to the control group(P＜0.05).Compared to the controls,the specific activity of caspase-3 increased statistically in the mid-dose group and the high-dose group(P＜0.05),but not in the low-dose group.And the specific activity of caspase-9 were significanlty higher in the B[a]P treated groups than that in the control group (P＜0.05).However,there were no significant differences in caspase-8 specific activity among 4 groups.A negative correlation existed between the cell apoptosis rate and the mitochondrial membrane potential(r=-0.820,P＜0.01),and positive correlations between the apoptosis rate and cytochrome C(r=0.883,P＜0.01),and the specific activity of caspase-3(r=0.805,P＜0.01) and of caspase-9(r=-0.756,P＜0.01).This result indicated that cell apoptosis induced by B[a]P was dependent on the activation of caspase enzymes and related to the mitochondrion membrane potential decreasing and cytochrome C release.
3.Study on the relationship of neural cell apoptosis and p53-bcl-2/bax mRNA and protein in vitro
Results of real-time fluorescent quantitation PCR showed that p53 mRNA in neuron increased with the dose of B[a]P,and statistically increased in the mid-dose and high-dose groups(P＜0.05) compared to the controls but not in the low-dose group.Bcl-2 and bax mRNA expression had a significant decrease in the mid-dose and high-dose groups compared to the controls(P＜0.01),but which had not in the low-dose group(P＜0.05).Further analysis suggested that bcl-2 mRNA decreased much more than bax,and bcl-2/bax ratio was reduced significantly compared to the controls.P53 and bax protein rose and bcl-2 protein diminished with the dosage of B[a]P in immunohistochemistry results.In the western-blot results,Compared to the controls, p53 protein were significantly increased and bcl-2 protein were statistically decreased in the mid-dose and high-dose groups(P＜0.05),and bax protein was higher in the mid-dose and high-dose group than the controls(P＜0.05),but not higher in the low-dose group(P＜0.05).This results indicated that p53-bcl-2/bax was related to the process of B[a]P induced neural cell apoptosis.
4.Study on the relationship of neural cell apoptosis and mitochondrial cytochrome C and caspase enzymes in vitro
The mitochodria in neuron became swollen,and bars became shortened,broken and even deleted in the transmission electron microscope.The mitochondrial membrane potential decreased gradually with the dosage of B[a]P,and showed significant augment in the 3 B[a]P-treated groups compared to the controls(P＜0.05).Western-blot results showed that cytochrome C in cytoplasm emitting from mitochondria increased with the dose of B[a]P and were significantly higher in the 3 B[a]P-treated groups than that in the controls(P＜0.05).The specific activity of caspase-3 and caspase-9 increased statistically with the dose of B[a]P (P＜0.05),but no significant difference was found in caspase-8 activity among the 4 groups.
PartⅢchanges of apoptosis and apoptosis-related genes after AhR was antagonized or p53 was inhibited
Results in the former two parts suggested that AhR/ARNT-CYP1A1 pathway and p53-bcl-2/bax-caspase pathway may both involved in the process of B[a]P-induced neurocyte apoptosis.On the basis of that fact,the AhR/ARNT-CYP1A1 pathway mediates B[a]P's metabolism and the p53-bcl-2/bax-caspase pathway directly leads cell death,we guess the mechanism of B[a]P-induced neurocyte apoptosis should be AhR/ARNT-CYP1A1-p53-bcl-2/bax-caspase. We would confirm the mechanism in primary neuronal cell by adopting AhR antagonist ANF or p53 inhibitor pifithrin-α.Also considering that ANF and PFT-αmay exert potential induction or non-specific effects on CYP1A1 enzymes,we decide to clarify the possible mechanism by antagonizing AhR or inhibiting p53 after silenceing CYP1A1 gene by use of gene-interfering technique.Also it is hard to achieve high transfection efficiency in primary neuron cells due to its short life-span and non-division,we further reveal the mechanism of B[a]P-induced cell apoptosis in human neuroblastoma SH-SY5Y cell line by intefering CYP1A1 gene.
1.Changes of apoptosis-related genes after AhR was antagonized by ANF
Cells were added AhR antagonist ANF to the final concentration 50 nM 2 hours before being treated by B[a]P,other treatments were the same to the former.Early-stage,late-stage and total apoptosis rates of neurons presented no significant difference between the B[a]P treated groups and the control group.After AhR was antagonized by ANF,B[a]P still exerted some induction on the expression of AhR,ARNT and CYP1A1 mRNA of neuronal cells,but no significant difference were found.ANF could decrease B[a]P's induction to p53 mRNA expression in the low-dose group and the mid-dose group,but could not inhibit it in the high-dose group.ANF increased the bcl-2/bax ratio by increasing bcl-2 mRNA and decreasing bax mRNA.These results showed that ANF could prevent B[a]P-induced apoptosis mainly by blocking AhR transcriptional regulation and induction to CYP1A1 and was related to the reduction of B[a]P metabolism activation.
2.Changes of apoptosis-related genes expression after PFT-αinhibiting p53
P53 inhibitor PFT-αwas added into the medium to the final concentration 25μM 2 hours before B[a]P treatment,other treatments were the same to the forwards.Early-stage,later-stage and total apoptosis rates of neurons had not shown significant difference between the B[a]P treated groups and the control group.PFT-αcould not inhibit AhR,ARNT and CYP1A1 mRNA expression of neuronal cells induced by B[a]P,but could inhibit p53 mRNA expression and increase the bcl-2/bax ratio mainy by increasing bcl-2 mRNA.These results demonstrated that PFT-αinhibited B[a]P-induced neurons apoptosis mainly by reducing p53 mRNA level and inhibiting p53 transcriptional regulation for bcl-2 family,increasing the bcl-2/bax ratio by up-regulating anti-apoptosis gene bcl-2 and down-regulating pro-apoptosis gene bax.
5.B[a]P's toxicity on human SH-SY5Y cell line and effects of AhR and P53
Human neuroblastoma SH-SY5Y cell line growed in the RPMI1640 medium supplemented with 15%fetal bovine serum,2 mM L-glutamine,10 mM HEPES,80 units/ml penicillin,and 100μg/ml streptomycin.All cultured cells were kept at 37℃in 95%air/5%CO_2 environmant. Cell passage was performed every 3～4 days when cell density was ajusted to 1×10~5 cells/ml.24 hours later,the same generation of well-growth cells was randomly divided into 4 groups and treated with B[a]P:the control group,the low-dose group,the mid-dose group and the high-dose group.Cells were treated with B[a]P to the final concentration to 0,2.5 nM,5.0 nM and 10.0 nM and added 0.3%of the total medium $9 mixture after replacing the medium by RPMI1640 with 3%fetal bovine serum.The treated and control cells were incubated for 48 hours,and then harvested for the analysis.All compounds were dissolved in DMSO.DMSO was less than 0.3 %in the cultured medium.Inhibitors or antagonists were added to the culture 2 hours prior to the addition of B[a]P and s9 mixture.Morphologic changes were observed in the fluorescent reversed microscope.CCK-8 method was to measure the cell viability.Cell apoptosis was tested by Annexin V/PI staining(detecting phosphatidylserine) and PI staining method(detecting sub-diploid apoptotic peak).
Cells in the control group were in good condition with even size,well-adherence and abundant cell-cell junctions.Dendrites and neuraxises were more and fine.Few cells in the low-dose group was dead and floated,dendrites and neuraxises became less and shorter.The number of cells remarkably reduced,more cells were floated and some were shrinked and round. The dendrites and neuraxises became further less and shorter.And cell junctions became loose. Cells in the high-dose group were reduced notably in number,shrinked and round.Most cells were floated,and the dendrites and neuraxises shortened even disapear.The cell body shrinked severely and became round;the cell junctions bacame very loose even disapear.Hoechst 33258 was a dye specific binding to DNA and was used to observe the morphology changes.After dyeing by Hoechst 33258,normal karyon was round and even-size and uniform coloration. Apoptotic karyon was shrinked even broken,with deep and bad-distribution DNA coloration. The severity displayed karyon disintegrated and broken.But in this study no typical karyon changes was observed.AO/EB staining can dye live cells to light green,dead cells to red and apoptotic cells to orange.But in this study orange apoptotic cell was a few,however,the number of red dead cell increased.Cell viability decreased with the dose of B[a]P,and which was significantly lower in the high-dose group than that in the control group.AnnexinV/PI double-staining had not detected early-stage cell apoptosis,and PI staining detected few apoptosis according to the contents of sub-diploid DNA.But differences were not significant among groups.After CYP1A1 mRNA was silenced by specific SiRNA,ANF and PFT-αcould prevent cell viability decreasing after treated by B[a]P.Compared to the controls,cell viability increased significantly in the mid-dose group after ANF antagonizing AhR(P＜0.05),in the low-dose group after PFT-αinhibiting p53(P＜0.05).This study combined with the above results indicated that the AhR/ARNT-CYP1A1 pathway and 53-bcl-2/bax mitochondrial pathway participated in the process of B[a]P-induced neural cell apoptosis.
Conclusions:
1.AhR gene polymorphism is correlated with B[a]P neurotoxicity.
2.The AhR/ARNT-CYP1A1 pathway and p53-bcl-2/bax mitochodrion pathway may both involved in B[a]P-induced neuronal cell apoptosis process.
4.The mechanism of B[a]P-induced neural cell apoptosis may be AhR/ARNT-CYP1A1-p 53-bcl-2/bax-caspase pathway.
5.In addition,B[a]P's active metabolites may induced apoptosis in a caspase-dependent manner by damaging mitochondrion.
B[a]P在体内的代谢活化和致癌性是通过激活AhR/ARNT-CYP1A1途径实现的。而AhR与B[a]P的特异结合是AhR/ARNT-CYP1A1途径激活的前提条件。因此,AhR基因多态性变异可能影响个体对B[a]P神经毒性和细胞凋亡的易感性。研究表明,B[a]P可以引起AhR基因表达增高,B[a]P诱导的肝细胞凋亡中AhR、ARNT和CYP1A1 mRNA表达呈时间和剂量依赖性的上升。B[a]P必须经激活AhR,诱导CYP1A1代谢活化后才诱导肝细胞凋亡。B[a]P诱导的人宫颈癌细胞凋亡与CYP1A1激活有关,是由CYP1A1介导的。有报道鱼类暴露于B[a]P,CYP1A1蛋白水平和反应CYP1A1蛋白酶活力的EROD水平显著升高,二者高度相关。B[a]P诱导的神经细胞凋亡与CYP1A1 mRNA和蛋白的表达升高有关。由此可见,AhR/ARNT-CYP1A1途径可能介导B[a]P诱导的神经细胞凋亡。本部分内容通过人群研究分析AhR基因多态性与B[a]P神经毒性和致细胞凋亡的关系,通过动物体内外试验探讨AhR/ARNT-CYP1A1途径在B[a]P致神经细胞凋亡中的可能作用。
第一章AhR基因多态性与B[a]P神经毒性及外周血淋巴细胞凋亡的关系
我们选取了214名职业性接触B[a]P的男性健康焦炉工为接触组,81名原材料库的库工为对照组。对照组除了没有职业性接触B[a]P和其他神经毒物外,其他方面尽量与接触组相一致。采用世界卫生组织推荐的神经行为功能核心测试组合(NCTB/WHO)和Ewing DJ推荐的自主神经功能测试分别进行神经行为功能和自主神经功能测定;采集空腹肘静脉血,分离淋巴细胞和提取全血基因组DNA,用Annexin V-FITC/PI双染法测定淋巴细胞的凋亡率,用等位基因特异性PCR法和限制性片段长度多态性-PCR法检测了AhR基因第10号外显子C1549T(Pro517Ser)、G1661A(Arg554Lys)和G1708A(Val570Ile)3个位点的基因变异,分析这3种基因多态性与神经行为功能、自主神经功能和外周血淋巴细胞凋亡之间的关系。结果如下:
1.基因多态性分析结果:295名研究对象的基因多态性分析结果显示C1549T和G1708A这2个位点的基因在太原地区人群中为野生型纯合子,突变型基因的检测结果为0。G1661A位点基因可分为野生型纯合子GG、突变型杂合子GA和突变型纯合子AA三种基因型。GG、GA和AA三种基因型分布在接触组与对照组分别为52.8%、27.6%、19.6%和67.9%、19.8%、12.3%,差异没有统计学意义(P＞0.05);接触组和对照组G、A等位基因频率分别为77.8%、22.2%和66.6%、33.4%,差异没有统计学意义(P＞0.05);等位基因频率分布符合Hardy-Weinberg平衡定律。因此我们只分析接触组AhR基因多态性与神经毒性和淋巴细胞凋亡的关系。
2.AhR G1661A基因多态性与B[a]P神经行为毒性的关系:焦炉工总数字跨度得分、顺序数字跨度得分和数字译码得分明显低于对照组,差异有统计学意义(P＜0.05);其余神经行为功能和情感状态得分在接触组与对照组之间差异没有统计学意义(P＞0.05)。接触组按照G1661A3种不同的基因型分组后的分析结果表明,AA突变纯合子基因型携带者困惑-迷茫的得分显著高于GG野生型纯合子基因型携带者和GA杂合子基因型携带者,差异有统计学意义(P＜0.05);愤怒-敌意、忧郁-沮丧、疲惫-隋性、紧张-焦虑和有力-好动得分在三种基因型之间差异没有统计学意义(P＞0.05)。在神经行为功能方面,数字跨度得分、倒序和视觉记忆得分呈现AA＜GA＜GG基因型趋势,但是差异没有统计学意义(P＞0.05)。说明AhR基因多态性影响焦炉工的情感状态得分,AA突变纯合子基因型的携带者困惑-迷茫得分明显增加。
3.AhR G1661A基因多态性与B[a]P自主神经毒性的关系:与对照组相比,反应焦炉工副交感神经调节功能的HR-V值明显下降,差异有统计学意义(P＜0.05),其余指标的改变没有统计学意义。通过对焦炉工AhR G1661A位点三种基因型与自主神经功能的分析,结果显示GG、GA、AA三种基因型的副交感神经调节指标HR-V、HR-DB、RR30:15、RRmax:min和交感神经调节指标BP-IS方面,差异都没有统计学意义(P＞0.05)。说明AhR G1661A位点的基因多态性与焦炉工的自主神经功能无关。
4.AhR G1661A基因多态性与外周血淋巴细胞凋亡的关系:与对照组相比,焦炉工外周血淋巴细胞的早期凋亡率、晚期凋亡率和总凋亡率升高,但是差异没有统计学意义(P＞0.05)。接触组AhR基因多态性G1661A与外周血淋巴细胞凋亡的关系分析结果显示,焦炉工外周血淋巴细胞早期凋亡率、晚期凋亡率和总凋亡率呈现GG＜GA＜AA趋势,但是差异没有统计学意义(P＞0.05)。本研究未发现AhR G1661A基因多态性与外周血淋巴细胞凋亡有关。
第二章AhR/ARNT-CYP1A1途径在B[a]P致神经细胞凋亡中的作用研究
第一节B[a]P致神经细胞凋亡的体内研究
40只健康成年雄性SD大鼠,脑室插管一周后按体重随机分为4组:橄榄油组(溶剂对照组)、B[a]P低浓度组(0.625 mmol/L)、B[a]P中浓度组(1.25 mmol/L)、B[a]P低浓度组(2.5mmol/L)。高、中、低浓度组分别注入相应浓度的B[a]P溶液10μl,溶剂对照组注射10μl橄榄油。一周染毒一次,连续染毒4次。染毒结束后断头处死,分离脑组织,用光镜和电镜观察脑组织病理和超微结构的改变,用流式细胞仪检测神经细胞的凋亡率。
(1)病理形态改变光镜观察到,对照组大鼠海马神经细胞排列整齐,形态正常;随着B[a]P染毒剂量的增高,海马神经细胞出现体积缩小,细胞数目逐渐减少,排列紊乱,细胞核的皱缩、消失,染色质凝集、边聚,甚至有细胞核的溶解消失等现象。但是在皮质、小脑、延髓和脑桥部分未见明显改变。
(2)超微结构的改变透射电镜观察结果显示,对照组海马细胞核细胞核形态规则,对称分布,染色质分布均匀:线粒体形态规则,嵴排列整齐。随着B[a]P剂量的增高,细胞核皱缩,染色质边聚,核膜破裂,线粒体肿胀,甚至呈气球样变而且外膜破裂,并伴有线粒体嵴减少、变短、断裂甚至消失。
(3)神经细胞凋亡率的测定结果随着B[a]P染毒剂量的增加,大鼠神经细胞的早期凋亡率呈剂量反应性的上升。与对照组相比,各剂量组神经细胞的早期凋亡率显著上升,差异有统计学意义(P＜0.01);高剂量组的晚期凋亡率显著高于中、低剂量组和对照组,差异有统计学意义(P＜0.05),而中、低剂量组与对照组差异没有统计学意义(P＞0.05)。以上结果显示海马神经细胞凋亡可能是B[a]P神经毒性的主要机制之一。
第二节神经细胞凋亡与AhR、ARNT和CYP1A1基因和蛋白关系的体内研究
(1)实时荧光定量PCR结果显示:与对照组相比,各剂量组AhR和ARNT基因的表达差异没有统计学意义(P＞0.05);而CYP1A1的基因表达随着染毒剂量的增加而增加,与对照组相比,各剂量组的差异有统计学意义(P＜0.05);与低剂量组相比,高剂量组CYP1A1基因表达显著增加,差异有统计学意义(P＜0.05)。
(2)免疫组化结果显示:AhR、ARNT这2种蛋白质在海马汉神经细胞的胞浆和胞核都表达,而且表达量随着染毒剂量的增加而增加。与对照组相比,各染毒组AhR蛋白表达随着染毒剂量的增加都明显增加,差异有统计学意义(P＜0.05);ARNT蛋白的表达也随着染毒剂量的增加而增加,中、高剂量组的差异有统计学意义(P＜0.05),而低剂量组的差异没有统计学意义(P＞0.05)。
(3) Wesern-blot结果显示:AhR、ARNT和CYP1A1蛋白表达随着染毒剂量的增加而升高。与对照组相比,中、高剂量组AhR、ARNT蛋白的表达差异有统计学意义(P＜0.05);各剂量组CYP1A1的表达也显著高于对照组(P＜0.05),中、高剂量组CYP1A1蛋白表达显著增加高于低剂量组,差异有统计学意义(P＜0.05)。
(4)相关分析表明:神经细胞凋亡率与CYP1A1 mRNA表达呈正相关(r=0.804,P＜0.05),与AhR蛋白表达呈正相关(r=0.845,P＜0.01),与ARNT蛋白表达呈正相关(r=0.781,P＜0.05),与CYP1A1蛋白表达呈正相关(r=0.807,P＜0.01);AhR蛋白与CYP1A1 mRNA之间呈正相关(r=0.826,P＜0.05),与CYP1A1蛋白之间呈正相关(r=0.774,P＜0.05)。AhR和ARNT在mRNA和蛋白表达水平的差别可能与较长的染毒时间内,基因转录水平的变化已经完成并且已经翻译为蛋白质有关。以上结果表明AhR、ARNT和CYP1A1很可能参与了B[a]P致神经细胞的凋亡过程。
第三节B[a]P致神经细胞凋亡的体外研究
无菌条件下分离新生1～3d SD大乳鼠的皮质,将细胞浓度调至1×10~5个细胞/ml,于37℃、5%CO_2培养箱中培养,24 h后更换新鲜的DMEM培养液并加入0.5 mg/ml阿糖胞苷(终浓度10μM)培养液以抑制胶质细胞增殖。采用神经胶质细胞的特异标志物(GFAP)进行神经元的纯度检测。在细胞培养第5天左右,选取生长良好的同批次神经细胞,分为对照组、低、中和高剂量4个组,每组至少3孔,分别按照培养液总体积的0.%和0.3%加入B[a]P和S9,使B[a]P终浓度分别为0、10μM、20μM,40μM。继续培养40 h,观察细胞的生长状况和形态学改变,Annexin-V/PI双染法检测神经细胞的凋亡。
(1)神经细胞的形态学改变光学显微镜下可见,对照组神经细胞细胞核大而清晰,轴突长且比较均一,树突较多,细胞连接丰富而紧密。随着染毒剂量的增加,神经细胞的轴突逐渐变短,树突数量减少,细胞连接减少,严重者出现细胞体积收缩、变圆,部分树突融解、消失,轴突也严重受损,细胞连接明显减少。AO/EB荧光染色能将活细胞为亮绿色,死细胞染为红色,凋亡细胞染为桔色。根据细胞的颜色就可以区别细胞的生存状况。随着B[a]P染毒剂量的增加,被染成桔色的细胞比例逐渐增多,细胞轴突和树突数量减少,细胞间的连接也减少,有些细胞轴突变短、甚至消失,胞体变圆,胞核皱缩、边聚。
(2) Annexin-V/PI双染法检测到中、高剂量组细胞凋亡率显著高于对照组,差异有统计学意义(P＜0.05),但是低剂量组差异没有统计学意义(P＞0.05)。说明B[a]P能诱导原代培养神经元的凋亡。
第四节神经细胞凋亡与AhR、ARNT和CYP1A1基因和蛋白关系的体外研究
原代培养神经细胞的实时荧光定量PCR结果显示各剂量组AhR、ARNT和CYP1A1基因表达明显高于对照组,差异有统计学意义(P＜0.05);高剂量组CYP1A1基因表达显著高于低剂量组,差异有统计学意义(P＜0.05)。Western-blot检测结果也显示AhR、ARNT和CYP1A1蛋白表达随着染毒剂量的增加逐渐增高。与对照组相比,中、高剂量组AhR、ARNT蛋白的表达差异有统计学意义(P＜0.05);各剂量组CYP1A1的表达也显著高于对照组(P＜0.05),中、高剂量组CYP1A1蛋白表达显著高于低剂量组,差异有统计学意义(P＜0.05)。说明B[a]P诱导的神经细胞凋亡与AhR、ARNT和CYP1A1 mRNA和蛋白表达增加有关。
第二部分p53-bcl-2/bax线粒体途径在B[a]P致神经细胞凋亡中的作用
B[a]P诱导的多种细胞凋亡都与p53相关信号途径的激活有关。P53通过诱导bax蛋白表达增加,降低bcl-2/bax的比例诱导细胞凋亡。bcl-2和bax是参与调节细胞凋亡的的一对功能相反的基因。bax通过形成二聚体,促使线粒体释放细胞色素C,激活caspase家族诱导细胞凋亡;bcl-2通过与bax形成异二聚体,减少bax二聚体的形成抑制细胞凋亡。因此bcl-2/bax的比例决定着细胞的存亡。本研究将对B[a]P诱导神经细胞凋亡的p53-bcl-2/bax线粒体机制进行研究。
第一节神经细胞凋亡与p53-bcl-2/bax基因和蛋白关系的体内研究
(1)实时荧光定量PCR结果显示:p53 mRNA表达随着染毒剂量的增加而上升,与对照组相比,各剂量组的差异有统计学意义(P＜0.05),高剂量组p53 mRNA水平明显高于低剂量组,差异有统计学意义(P＜0.05);各剂量组bcl-2 mRNA表达显著低于对照组,差异有统计学意义(P＜0.01),高剂量组bcl-2 mRNA水平明显低于低剂量组,差异有统计学意义(P＜0.05);而bax mRNA表达在各组间差异没有统计学意义(P＞0.05)。
(2)免疫组化结果显示随着B[a]P染毒剂量的增加,p53和bax蛋白的表达逐渐增加,bcl-2蛋白逐渐下降。与对照组相比,各剂量组p53蛋白显著增加,bcl-2蛋白显著下降,差异有统计学意义(P＜0.01),高剂量组bcl-2蛋白显著低于低剂量组,差异有统计学意义(P＜0.05);与对照组相比,中、高剂量组bax蛋白显著升高,差异有统计学意义(P＜0.05),但是低剂量组差异没有统计学意义(P＞0.05)。与对照组相比,各剂量组bcl-2/bax比值显著下降,差异有统计学意义(P＜0.05)。
(3) Wesern-blot检测p53蛋白随着染毒剂量的增加而增加,与对照组相比,中、高剂量组p53蛋白水平显著增高,差异有统计学意义(P＜0.05)。相关分析结果显示,细胞凋亡率与p53 mRNA表达量之间呈明显的正相关(r=0.864,P＜0.05),与p53蛋白水平之间也呈明显的正相关(r=0.757,P＜0.01);海马神经细胞凋亡率与bcl-2 mRNA表达呈负相关(r=-0.924,P＜0.01),与Bcl-2蛋白呈负相关(r=-0.893,P＜0.01),与Bax蛋白呈正相关(r=0.872,P＜0.01),与bcl-2/bax比值呈负相关(r=-0.876,P＜0.01)。以上说明p53-Bcl-2/bax途径也参与了B[a]P诱导的神经细胞凋亡。
第二节神经细胞凋亡与线粒体途径细胞色素C和caspase酶关系的体内研究
透射电镜下观察到B[a]P主要使神经细胞线粒体肿胀变形,伴有嵴缩短、断裂甚至缺失等现象。线粒体平均荧光强度随着染毒剂量的增加逐渐升高,膜电位逐渐下降。高剂量组平均荧光强度明显高于对照组、低剂量组和中剂量,差异有统计学意义(P＜0.05);高剂量组膜电位下降百分率较对照组和低剂量组显著下降,差异有统计学意义(P＜0.05)。Western-blot结果显示线粒体释放到胞浆中的细胞色素C含量随着染毒剂量的增加逐渐增加,与对照组相比,各剂量组差异有统计学意义(P＜0.05)。caspase-3,8,9蛋白酶活力测定结果显示中、高剂量组的caspase-3比活力显著高于对照组,差异有统计学意义(P＜0.05),而低剂量组差异没有统计学意义(P＞0.05);各剂量组caspase-9的比活性都显著高于对照组,差异有统计学意义(P＜0.05);但是caspase-8的活力在对照组与各剂量组之间差异没有统计学意义(P＞0.05)。海马神经细胞凋亡率与线粒体膜电位呈负相关(r=-0.820,P＜0.01),与胞浆细胞色素C水平和caspase以及caspase-9活力呈正相关,相关系数分别为0.883(P＜0.01),0.805(P＜0.01)和0.756(P＜0.01),表明B[a]P诱导的神经细胞凋亡依赖于caspase酶的激活,与线粒体膜电位的下降和细胞色素C释放有关。
第三节神经细胞凋亡与p53-bcl-2/bax基因和蛋白关系的体外研究
实时荧光定量PCR结果显示神经元p53 mRNA的表达随着染毒剂量的增加而上升。与对照组相比,中、高剂量组的差异有统计学意义(P＜0.05),而低剂量组的差异没有统计学意义(P＞0.05);bcl-2和bax mRNA的表达随着染毒剂量的增加呈下降趋势,与对照组相比,中、高剂量组bcl-2和bax mRNA的差异有统计学意义(P＜0.01),而低剂量组bcl-2和bax的差异没有统计学意义(P＞0.05);进一步分析发现bcl-2 mRNA下降较baxmRNA明显,bcl-2/bax mRNA比值随着染毒剂量的增加而下降。免疫组化结果显示bcl-2随着染毒剂量的增加而下降,bax随着染毒剂量的增加而升高。Western-blot结果也显示与对照组相比,中、高剂量组p53蛋白表达显著增加,bcl-2明显下降,差异有统计学意义(P＜0.05),但是低剂量组差异没有统计学意义。中、高剂量组bax蛋白表达明显高于对照组,差异有统计学意义(P＜0.05)。说明B[a]P诱导的神经细胞凋亡与p53-bcl-2/bax途径有关。
第四节神经细胞凋亡与线粒体、细胞色素C和caspase酶关系的体外研究
透射电镜观察到体外原代培养神经元线粒体肿胀,嵴变短、断裂甚至消失。线粒体膜电位随着染毒剂量的增加逐渐下降,与对照组相比,各剂量组差异有统计学意义(P＜0.05);胞浆细胞色素C含量逐渐增加,与对照组相比,各剂量组差异有统计学意义(P＜0.05);caspase-3,9的酶活力随着染毒剂量的增加而逐渐上升,差异有统计学意义(P＜0.05),但是caspase-8的活力变化在组间没有统计学意义(P＞0.05)。
第三部分拮抗AhR或抑制p53后细胞凋亡及凋亡相关基因的改变
前两部分的研究结果提示AhR/ARNT-CYP1A1和p53-bcl-2/bax-caspase途径共同参与B[a]P诱导的神经细胞凋亡过程。由于AhR/ARNT-CYP1A1途径主要介导B[a]P的代谢活化,p53-bcl-2/bax-caspase途径直接参与凋亡的发生。那么这两条信号途径一定存在着某种内在的联系。我们的猜想是:B[a]P通过激活AhR/ARNT-CYP1A1途径,诱导p53-bcl-2/bax-caspase机制活化诱导神经细胞的凋亡。为此我们采用原代培养的神经细胞,用AhR拮抗剂α-萘黄酮(ANF)或p53抑制剂pifithrin-α(PFT-α)来证实这一猜想。
由于ANF和PFT-α可能对CYP1A1有潜在的诱导性或非特异干扰效应,我们又采用特异性基因干扰技术沉默CYP1A1后,研究拮抗AhR或抑制p53后B[a]P毒性的改变,阐明B[a]P致神经细胞凋亡的可能机制。由于原代培养神经元生命周期短暂,不能自我分裂,很难达到较高的转染效率,我们干扰人神经母细胞瘤细胞株SH-SY5Y细胞的CYP1A1基因后进一步研究B[a]P诱导神经细胞凋亡的机制。
第一节ANF拮抗AhR后神经元凋亡与凋亡相关基因的改变
B[a]P染毒前2小时加入AhR拮抗剂ANF,使终浓度为50 nM,其余同前。凋亡测定结果表明B[a]P诱导的神经细胞早期凋亡率、晚期凋亡率和总凋亡率在对照组与各染毒组间差异没有统计学意义(P＞0.05)。ANF拮抗AhR后,B[a]P对神经细胞AhR、ARNT以及CYP1A1mRNA的表达仍有一定的诱导作用,但是组间差异没有统计学意义(P＞0.05)。ANF能使神经细胞p53 mRNA的表达在低、中剂量组迅速降低,但是ANF不能降低高剂量B[a]P对p53的诱导;ANF能使神经细胞bcl-2 mRNA的表达增高,bax mRNA的表达降低,bcl-2/bax比值升高。说明ANF阻止B[a]P诱导的神经细胞凋亡,其主要机制可能与ANF通过阻断B[a]P对CYP1A1的转录调控和诱导,与降低B[a]P的代谢活化有关。
第二节PFT-α抑制后神经元凋亡与凋亡相关基因的改变
B[a]P染毒前2小时加入p53抑制剂PFT-α,使终浓度为25μM,其余同前。凋亡测定结果显示B[a]P诱导的神经细胞早期凋亡率、晚期凋亡率和总凋亡率在对照组与各染毒组间差异没有统计学意义(P＞0.05)。实时荧光定量PCR结果显示AhR、ARNT以及CYP1A1 mRNA表达量随着S[a]P染毒剂量增加而升高,差异有统计学意义(P＜0.05)。各组间p53 mRNA水平差异没有统计学意义(P＞0.05),说明PFT-α不能抑制B[a]P对神经细胞AhR、ARNT以及CYP1A1 mRNA的诱导,但是能抑制B[a]P对p53 mRNA诱导,使bcl-2 mRNA表达增高,使bcl-2/bax比值升高。说明PFT-α抑制B[a]P诱导的神经细胞凋亡的主要机制可能是:降低p53 mRNA水平和对bcl-2家族的转录调控能力,使抑凋亡基因bcl-2表达上升,促凋亡基因bax表达下调,提高bcl-2/bax比值。
第三节B[a]P对人SH-SY5Y细胞的毒性以及AhR和p53的作用
人SH-SY5Y细胞用RPMI1640培养液培养(含15%胎牛血清,2 mM L-谷氨酰胺,10mM HEPES,80单位/ml青霉素和100μg/ml链霉素),于37℃、5%CO2培养箱中培养。每3～4天传代一次。传代24h后选择处于对数生长期的同一代细胞,分为对照组、低剂量组、中剂量组、高剂量组进行染毒。染毒时换用含3%胎牛血清的RPMI1640培养液,分别按照培养液总体积的0.3%和0.1%加入S9混合液和B[a]P,混匀,使B[a]P终浓度分别为0、2.5 nM、5.0 nM和10.0 nM。所有的化合物都用DMSO溶解,保证DMSO不超过培养液总体积的0.3%。抑制物或者拮抗剂在染毒前2小时加入。染毒后继续培养48 h后进行检测用荧光倒置显微镜观察细胞的形态学改变,用CCK-8法测定细胞活力,通过检测磷脂酰丝氨酸的Annexin V/PI方法和检测亚二倍体凋亡峰的PI染色法测定细胞凋亡。
对照组细胞状态良好,大小均匀,贴壁好,细胞间连接较多,细胞树突和轴突长而多;低剂量组有少部分死亡细胞,呈漂浮状,树突和轴突减少变短;中剂量组细胞数量明显减少,漂浮的细胞较多,部分细胞皱缩,突起明显缩短;高剂量组细胞数量进一步减少,突起进一步缩短甚至消失,细胞皱缩呈圆形,细胞间连接异常松散甚至消失。hoechst 33258是一种特异性与DNA结合的染料,用于观察细胞核形态的变化。正常细胞核呈圆形,着色均匀,凋亡细胞核皱缩,DNA高度凝聚使着色深,有的出现核的崩解、破裂。但是hoechst33258染色没有观察到典型的凋亡细胞核的变化。AO/EB染色将活细胞染成亮绿色,死细胞染成红色,而凋亡细胞染成桔色。结果显示随着剂量的增加,染成红色的坏死细胞有所增加,但是被染成橘黄色的凋亡细胞数量很少。细胞活力测定结果显示随着B[a]P染毒剂量的增加,细胞活力逐渐降低,与对照组相比,高剂量细胞活力下降差异有统计学意义(P＜0.05);Annexin V/PI法没有检测到细胞早期凋亡率,PI法检测到亚二倍体的DNA,有少量细胞凋亡,但是差异没有统计学意义(P＞0.05)。沉默CYP1A1基因后,ANF和PFT-α能够阻止B[a]P诱导的细胞活力下降。与对照组相比,ANT拮抗后中剂量组细胞活力明显增高,差异有统计学意义(P＜0.05);PFT-α抑制后低剂量组细胞活力明显增高,差异有统计学意义(P＜0.05)。结合前面的研究结果,我们认为AhR/ARNT-CYP1A1途径和p53-bcl-2/bax线粒体途径共同参与了B[a]P诱导的神经细胞凋亡。
结论:
1.AhR基因多态性与B[a]P神经毒性有关。
2.AhR/ARNT-CYP1A1途径和p53-bcl-2/bax-caspase途径共同介导和参与了B[a]P诱导的神经细胞凋亡。
3.B[a]P致神经细胞凋亡的机制可能为AhR/ARNT-CYP1A1-p53-bcl-2/bax-caspase。
4.此外,B[a]P的代谢物也可能直接损伤线粒体以caspase依赖的方式导致细胞凋亡。
Metabolism and carcinogenicity of B[a]P depend on the activation of AhR/ARNT-CYP1A1 pathway.Specific binding of B[a]P and AhR was a prerequisite of the activation of AhR/ARNT-CYP1A1 pathway.So,AhR gene polymorphism may influence the sensitivity of neurotoxicity and apoptosis induced by B[a]P.Researches showed that B[a]P increased AhR expression.AhR,ARNT and CYP1A1 mRNA expression increased in a time and dose dependent manner in the process of B[a]P-induced hepatoma cell apoptosis.B[a]P caused hepatoma cell apoptosis until it metabolited into active forms after activating AhR and inducing CYP1A1.B[a]P-induced malignant cell apoptosis was related to CYP1A1 activation and mediated by CYP1A1.It is reported that CYP1A1 protein and enzyme activity in zebrafish signifiantly increased after B[a]P exposure.B[a]P-induced neurocyte apoptosis was related to CYP1A1 mRNA and protein.So in this part we would study the relationship of AhR gene polymorphism and neurotoxicity and cell apoptosis induced by B[a]P by analyzing populations' genotype,and the changes of AhR/ARNT-CYP1A1 pathway in the process of B[a]P-induced neural cell apoptosis in vivo and vitro.
ChapterⅠRelationship of AhR gene polymorphism and B[a]P's neurotoxieity and peripheral blood lymphocyte apoptosis
We recruited 214 male healthy coke-oven workers occupationally-exposed B[a]P as the exposed group and 81 warehouse workers in the raw material plant as the control group which were as possible as comparable with the exposed group except for never occupationally exposed B[a]P and other neurotoxicants.Neurobehavioral core test battery recommended by world health organization(NCTB/WHO) was used to test the neurobehavioral function of subjects;autonomic nerve system test battery recommended by Ewing DJ was adopted to measure the autonomic nervous function.And lymphocytes and genome DNA were isolated and extracted from the peripheral ulnar vein blood in the conditional of empty stomach.Apoptosis of lymphocytes were detected by the flow cytometry after stained with Annexin V-FITC and PI.Allele specific-polymerase chain reaction(AS-PCR) and restricted fragment length polymorphismpolymerase chain reaction(RFLP-PCR) methods were applied to determine the AhR gene variations in the 3 sites of C 1549T(Pro517Ser)、G1661A(Arg554Lys) and G 1708A(Va1570Ile) in the 10~(th) exon.Then we analyzed the relationship between AhR gene polymorphism and neurobehavioral function,autonomic nervous function(ANS) and lymphocyte apoptosis by SPSS FOR WINDOWS 10.0 SOFTWARE.The results were given in next.
1.Results of gene polymorphism analysis
Results of 295 subjects' AhR gene polymorphism indicated that the 2 sites variation including C1549T(Pro517Ser) and G1708A(Va1570Ile),were wild homozygotes and the mutant alleles were not detected in population of Taiyuan district.That is,the mutant allele frequencies of C1549T and G1708A were 0.There were 3 genotypes in G1661A sites including GG(wild type homozygote),GA(mutant heterozygote) and AA(mutant homozygote).The distributional frequencies of GG,GA and AA were 52.8%,27.6%and 19.6%in the exposed group and 67.9%, 19.8%and 12.3%in the control group,respectively.The allele frequencies of G and A were 77.8%and 22.2%in the exposed workers,66.6%and 33.4%in the controls,and no significant difference existed between the two groups(P＞0.05).The allele frequency distribution was consistent with the Hardy-Weinberg equilibrium law.Therefore,we analyzed the relationship between the gene polymorphism and neurotoxicity and lymphocyte apoptosis only in the exposed group.
2.Relationship between AhR G1661A gene polymorphism and neurobehavioral function
Compared to the controls,the scores of total digital span,serial digital span and digital decoding decreased significantly in the coke oven workers.The scores in other neurobehavioral function and profile of mood state had not significant difference.Analytical results show that the score of stumble-vast and hazy increased highly in the AA genotype than that in the GG or GA genotypes(P＜0.05).There were no significant difference among GG,GA and AA genotypes in anger-hostility,gloom-depression,tension-anxiety and power-activity(P＞0.05).The scores of total digital span,reversed digital span and vision retention presented the tendency of AA＜GA＜GG,however,the differences were not significant(P＞0.05).This result suggests that AhR genetic polymorphism influenced the profile of mood state in the coke oven workers,and the scores of stumble-vast and lazy significantly increased in the AA genotypes compared to the GG or GA genotype.
3.Relationship between AhR G1661A gene polymorphism and autonomic nervous function
The HR-V value reflecting the modulation of parasympathetic nerve significantly decreased in the exposed group compared to the control group(P＜0.05),other indices had no significant difference between the exposed group and the controls.Analytical results of gene polymorphism and autonomic nervous system show that no significant difference existed in HR-V,HR-DB, RR30:15,RRmax:min and BP-IS in the coke oven workers among GG,GA and AA genotypes (P＞0.05).All these results indicate that AhR G1661A gene polymorphism had no relation with the autonomic nevous function of the coke oven workers.
4.Relationship between AhR G1661A gene polymorphism and apoptosis rates of lymphocytes
The rates of early apoptosis,later apoptosis and total apoptosis of lymphocytes in the peripheral blood were higher in the coke-oven workers than that in the controls,but no significant differences existed(P＞0.05).Also the apoptosis rate of lymphocyte had no statistical difference(P＜0.05) among GG,GA and AA genotypes although there was a tendency of GG＜GA＜AA.Relationship between the AhR G1661A gene polymorphism and the lymphocyte apoptosis was not found in our study.
ChapterⅡRole of AhR/ARNT-CYP1A1 pathway in B[a]P -induced neurocyte apoptosis
1.Study on the neural cell apoptosis induced by B[a]P in vivo
Forty male adult and healthy Sprague-Dawley rats were divided randomly into 4 groups according to their weights after incubated in the cerebral ventrical:olive oil group(solvent control group),0.625 mmol/L B[a]P(low-dose) group,1.25 mmol/L B[a]P(mid-dose) group and 2.5 mmol/L B[a]P(high-dose) group.The low-dose,mid-dose and high-dose rats were injected 10μl B[a]P olive oil solution of different concentrations,respectively,and the control rats were injected the same volume solvent olive oil.The treatment was performed once a week for successive 4 times.After the treatment was accomplished,the rats were decapitated to death and isolated the brain tissues to be observed the morphologic and ultramicrostructure changes under the light and electric microscopes.Cell apoptosis was detected by Annexin V/PI staining in the flow cytometry.
(1) Morphology changes:under the light microscope,the hippocampus neurocytes arranged in order with normal shape.The higher of the B[a]P dose was,the more serious of the damage was. The impaired hippocampus cells was contracted,diminished and displayed disorderly and loosened,the karyon shrinked and even disappeared,and the chromatin condensed and margined. However,no obvious changes were observed in the cortex,cerebellum,bulb and pons.
(2) The ultrastructure changes in the electron microscope displayed:in the control group,the karyotin was round and distributed symmetrically with well-distributed chromatin.The mitochondria were regular and the bars were well-arranged.With the concentration of B[a]P increasing,the karyon became shrinken,assembled highly in the edge.The karyotheca was even broken in some severely-damaged cells.The mitochondria became swollen and sometimes like a balloon even with a broken outer-membrane.The bars were reduced,shortened,broken and even disappeared.
(3) The apoptotic rate of neurocytes increased with the concentration of B[a]P.Early apoptotic rate significantly increased in the 3 B[a]P groups compared to the controls(P＜0.01).The later apoptosis was notably higher in the high-dose group than that in other groups(P＜0.05),but which is not high in the middle group and the low group(P＞0.05).These results demonstrate that B[a]P can induce hippocampal cell apoptosis.
2.Research on the relationship of neurocyte apoptosis and AhR,ARNT and CYP1A1 mRNA and protein in vivo
(1) Results of real-time fluorescent quantitation show that no statistical significance were found in AhR and ARNT mRNA expression among groups but CYP1A1 mRNA significantly increased with the dose of B[a]P,which was higher in 3 treated groups than that in the solvent group (P＜0.05) and higher in the high-dose group than that in the low-dose group(P＜0.05).
(2) Immunohistochemistry results show that AhR and ARNT protein expressed in the cytoplasm and nucleus in the hippocampal cells,and increased with the increment of B[a]P dose.Compared to the controls,AhR protein had a significant increase in the 3 B[a]P-treated groups(P＜0.05), and ARNT protein statistically increased in the mid-dose and high-dose groups(P＜0.05),but not in the low-dose group(P＞0.05).
(3) Western-blot results indicate that AhR,ARNT and CYP1A1 protein increased in a B[a]P dose-dependent manner.AhR and ARNT protein were significantly higher in the mid-dose group and the high-dose group than those in the controls(P＜0.05).And CYP1A1 protein increased significantly in the the 3 B[a]P-treated groups compared to the solvent group(P＜0.05), furthermore which were higher in the mid-dose and high-dose groups than that in the low-dose group(P＜0.05).
(4) Correlated analysis manifested that a positive correlation existed between the cell apoptotic rates and CYP1A1 mRNA(r=0.804,P＜0.05).Positive correlations were also found in the cell apoptotic rates and AhR protein(r=0.845,P＜0.01),ARNT protein(r=0.781,P＜0.01) and CYP1A1 protein(r=0.807,P＜0.01),respectively.In addition,the positive correlations existed between AhR protein and CYP1A1 mRNA(r=0.826,P＜0.01),and between AhR.protein and CYP1A1 protein(r=0.774,P＜0.05).The disagreement in mRNA and protein of AhR and ARNT was possibly caused by the long duration of administration in which mRNA transcriptional expression had accomplished and translated into protein.All the results showed that AhR,ARNT and CYP1A1 possibly involved in the process of neurocyte apoptosis.
3.Study on the neuronal cell apoptosis induced by B[a]P in vitro
Primary neuronal cells were dissociated and prepared from newborn 1-3 days neonate SD rats' cerebral cortex,and cultured in a 37℃and 5%CO_2 incubator after adjusted to 1×10~5 cells/ml.The fresh DMEM medium was displaced and added cytosine arabinoside(final concentration 10μM) to inhibit astrocytes growing after 24 hours.The astrocytes specific marker GFAP was identified by immunohistochemical method to ensure the neurons' purity. Astrocytes should be less than 10%of the total cells.In the 5th days,comparable well-growth neurons were divided randomly into 4 groups:the control group,the low-dose group,the mid-dose group and the high dose group,and then treated with 0.1%of the medium to the B[a]P final concentration of 0,10μM,20μM,40μM,respectively.S9 mixture was added to 0.3%of the medium at the same time.The control group was only added DMSO solvent.After a sequence maintenace for 40 h in the incubator,morphology changes were observed under the fluorescence inverted microscope and apoptotic rates were detected by the Annexin-V/PI double staining.
(1) Morphology changes of the neuronal cells demonstrated that the nucleus was big and clear with long and uniform neuraxons and many dendrites,and the cellular junctions were abundant and tight.With the dose of B[a]P increasing,the numbers of neuraxons and dendrites decreased and the conjunctions among cells were reduced evidently.The severity displayed that some neuraxons became shorter,even disappear,the cell body became shfinked and round.Dyeing with AO/EB fluorescent reagent,live neuron showed a bright green color,while the dead one was red,and the apoptosis was orange.According to the color of neuron,we can distinct living status of the neuron.With B[a]P dose increasing,the ratios of orange cells increased,and the numbers of neuraxons and dendrites and the conjunction among cells decreased,some neuraxons became shorter,even disappear,and the cell body became round,the karyon shrinked or aggregated in the edge.
(2) Annexin-V/PI double staining results showed that cell apoptosis rates in the mid-dose group and the high-dose group were significantly higher than that in the controls(P＜0.05),but no difference were found in apoptosis rates between the controls and the low-dose group(P＞0.05). The results indicated that B[a]P induced primary neuron cell apoptosis.
4.Research on the relationship of neurocyte apoptosis and AhR,ARNT and CYP1A1 mRNA and protein in vitro
Real-time fluorescent quantitation PCR results showed that AhR,ARNT and CYP1A1 mRNA expression significantly increased in the 3 B[a]P treated groups compared to the control group(P＜0.05),and CYP1A1 mRNA expression in the 40μM B[a]P group was statistically higher than that in the 10μM B[a]P group(P＜0.05).The data suggested that B[a]P can induce primary cultured neurons apoptosis and which related to the induction of AhR,ARNT and CYP1A1.Western-blot results indicated that AhR,ARNT and CYP1A1 protein increased with the dosage of B[a]P.Compared to the controls,AhR and ARNT protein in the mid-dose group and the high-dose group were statistically high(P＜0.05),but not in the low-dose group (P＞0.05).CYP1A1 protein were significantly higher in the 3 treated groups than that in the control group(P＜0.05),and which were higher in the mid-dose group and high-dose group than that in the control group(P＜0.05).This suggested that B[a]P-induced neurocyte apoptosis was related to the augment ofAhR,ARNT and CYP1A1 mRNA and protein expression.
PartⅡRole of p53-bcl-2/bax mitochondrial pathway in B[a]P-induced neurocyte apoptosis
B[a]P-induced cell apoptosis were related with the activation of p53 signals.P53 induces cell apoptosis via increasing bax protein expression and dicreasing the ratio of bcl-2/bax.Bcl-2 and bax is a couple of genes with opposite function regulating apoptosis.Bax homodimer urges mitochondrion releasing cytoshrome C into the cytoplasm which activates caspase family and causes cell apoptosis;however,bcl-2 inhibits cell apoptosis by heterodiming with bax and reducing bax contents.So the bcl-2/bax ratio determines the cell fate.This part would study the mechanism of p53-bcl-2/bax mitochondrial pathway in B[a]P-induced apoptosis.
1.Study on the relationship of nerve cell apoptosis and p53-bcl-2/bax mRNA and protein in vivo
(1) Results of real-time fluorescent quantitation PCR showed that p53 mRNA increased with the dose of B[a]P,which statistically increased in the 3 B[a]P-treated groups(P＜0.05),and was higher in the high-dose group than that in the low-dose group(P＜0.05).Bcl-2 mRNA expression decreased significantly in the 3 B[a]P-treated groups compared to the controls(P＜0.01),which was lower in the high-dose group than the control rats(P＜0.05).However,no significant difference was found in bax mRNA among groups although bax mRNA showed a tendency of decreasing post B[a]P treatment.
(2) Immunohistochemistry results showed p53 and bax protein rose and bcl-2 protein diminished with the B[a]P dose.Compared to the controls,p53 protein were significantly increased and bcl-2 protein were statistically decreased(P＜0.05),and bcl-2 protein was lower in the high-dose group than that in the control group(P＜0.05).Bax protein increased with the dosage of B[a]P and was higher in the mid-dose and high-dose group than the controls(P＜0.05),but not higher in the low-dose group(P＜0.05).Bcl-2/bax ratio had a significant decrease in the 3 B[a]P-treated groups compared to the control group.
(3) Western-blot results indicated a similar dose-dependent relationship between p53 protein and the dosage of B[a]P.Compared to the controls,p53 protein increased significantly in the mid-dose group and high-dose group(P＜0.05),and was higher in the high-dose group than that in the low-dose group(P＜0.05).Correlation analysis showed positive correlations existed between the apoptosis rate and p53 mRNA(r=0.864,P＜0.05),and p53 protein(r=0.757,P＜0.01). Negative relations existed between the cell apoptosis rate and bcl-2 mRNA(r=-0.924,P＜0.01), and bcl-2 protein(r=-0.893,P＜0.01),and bcl-2/bax ratio(r=-0.876,P＜0.01);and a positive correlation existed between the apoptosis rate and bax protein(r=0.872,P＜0.01).This results indicated that p53-bcl-2/bax was involved in the process of B[a]P induced neural cell apoptosis.
2.Study on the relationship of neural cell apoptosis and mitochondrial cytochrome C and caspase enzymes in vivo
B[a]P caused the mitochodria in neural cell swelling accompanied with bars shorten, segmentation and even deletion in the transmission electron microscope.The mitochondrial mean fluorescence density increased gradually with the dosage of B[a]P,and which was significantly higher in the high-dose group than that in the controls and other groups(P＜0.05). The membrane potential decreased significantly in the high-dose group compared with the low-dose group and the controls(P＜0.05).Western-blot results showed that cytochrome C in cytoplasm emitting from mitochondria increased with the dose of B[a]P and had significant diferrences in the 3 B[a]P-treated groups compared to the control group(P＜0.05).Compared to the controls,the specific activity of caspase-3 increased statistically in the mid-dose group and the high-dose group(P＜0.05),but not in the low-dose group.And the specific activity of caspase-9 were significanlty higher in the B[a]P treated groups than that in the control group (P＜0.05).However,there were no significant differences in caspase-8 specific activity among 4 groups.A negative correlation existed between the cell apoptosis rate and the mitochondrial membrane potential(r=-0.820,P＜0.01),and positive correlations between the apoptosis rate and cytochrome C(r=0.883,P＜0.01),and the specific activity of caspase-3(r=0.805,P＜0.01) and of caspase-9(r=-0.756,P＜0.01).This result indicated that cell apoptosis induced by B[a]P was dependent on the activation of caspase enzymes and related to the mitochondrion membrane potential decreasing and cytochrome C release.
3.Study on the relationship of neural cell apoptosis and p53-bcl-2/bax mRNA and protein in vitro
Results of real-time fluorescent quantitation PCR showed that p53 mRNA in neuron increased with the dose of B[a]P,and statistically increased in the mid-dose and high-dose groups(P＜0.05) compared to the controls but not in the low-dose group.Bcl-2 and bax mRNA expression had a significant decrease in the mid-dose and high-dose groups compared to the controls(P＜0.01),but which had not in the low-dose group(P＜0.05).Further analysis suggested that bcl-2 mRNA decreased much more than bax,and bcl-2/bax ratio was reduced significantly compared to the controls.P53 and bax protein rose and bcl-2 protein diminished with the dosage of B[a]P in immunohistochemistry results.In the western-blot results,Compared to the controls, p53 protein were significantly increased and bcl-2 protein were statistically decreased in the mid-dose and high-dose groups(P＜0.05),and bax protein was higher in the mid-dose and high-dose group than the controls(P＜0.05),but not higher in the low-dose group(P＜0.05).This results indicated that p53-bcl-2/bax was related to the process of B[a]P induced neural cell apoptosis.
4.Study on the relationship of neural cell apoptosis and mitochondrial cytochrome C and caspase enzymes in vitro
The mitochodria in neuron became swollen,and bars became shortened,broken and even deleted in the transmission electron microscope.The mitochondrial membrane potential decreased gradually with the dosage of B[a]P,and showed significant augment in the 3 B[a]P-treated groups compared to the controls(P＜0.05).Western-blot results showed that cytochrome C in cytoplasm emitting from mitochondria increased with the dose of B[a]P and were significantly higher in the 3 B[a]P-treated groups than that in the controls(P＜0.05).The specific activity of caspase-3 and caspase-9 increased statistically with the dose of B[a]P (P＜0.05),but no significant difference was found in caspase-8 activity among the 4 groups.
PartⅢchanges of apoptosis and apoptosis-related genes after AhR was antagonized or p53 was inhibited
Results in the former two parts suggested that AhR/ARNT-CYP1A1 pathway and p53-bcl-2/bax-caspase pathway may both involved in the process of B[a]P-induced neurocyte apoptosis.On the basis of that fact,the AhR/ARNT-CYP1A1 pathway mediates B[a]P's metabolism and the p53-bcl-2/bax-caspase pathway directly leads cell death,we guess the mechanism of B[a]P-induced neurocyte apoptosis should be AhR/ARNT-CYP1A1-p53-bcl-2/bax-caspase. We would confirm the mechanism in primary neuronal cell by adopting AhR antagonist ANF or p53 inhibitor pifithrin-α.Also considering that ANF and PFT-αmay exert potential induction or non-specific effects on CYP1A1 enzymes,we decide to clarify the possible mechanism by antagonizing AhR or inhibiting p53 after silenceing CYP1A1 gene by use of gene-interfering technique.Also it is hard to achieve high transfection efficiency in primary neuron cells due to its short life-span and non-division,we further reveal the mechanism of B[a]P-induced cell apoptosis in human neuroblastoma SH-SY5Y cell line by intefering CYP1A1 gene.
1.Changes of apoptosis-related genes after AhR was antagonized by ANF
Cells were added AhR antagonist ANF to the final concentration 50 nM 2 hours before being treated by B[a]P,other treatments were the same to the former.Early-stage,late-stage and total apoptosis rates of neurons presented no significant difference between the B[a]P treated groups and the control group.After AhR was antagonized by ANF,B[a]P still exerted some induction on the expression of AhR,ARNT and CYP1A1 mRNA of neuronal cells,but no significant difference were found.ANF could decrease B[a]P's induction to p53 mRNA expression in the low-dose group and the mid-dose group,but could not inhibit it in the high-dose group.ANF increased the bcl-2/bax ratio by increasing bcl-2 mRNA and decreasing bax mRNA.These results showed that ANF could prevent B[a]P-induced apoptosis mainly by blocking AhR transcriptional regulation and induction to CYP1A1 and was related to the reduction of B[a]P metabolism activation.
2.Changes of apoptosis-related genes expression after PFT-αinhibiting p53
P53 inhibitor PFT-αwas added into the medium to the final concentration 25μM 2 hours before B[a]P treatment,other treatments were the same to the forwards.Early-stage,later-stage and total apoptosis rates of neurons had not shown significant difference between the B[a]P treated groups and the control group.PFT-αcould not inhibit AhR,ARNT and CYP1A1 mRNA expression of neuronal cells induced by B[a]P,but could inhibit p53 mRNA expression and increase the bcl-2/bax ratio mainy by increasing bcl-2 mRNA.These results demonstrated that PFT-αinhibited B[a]P-induced neurons apoptosis mainly by reducing p53 mRNA level and inhibiting p53 transcriptional regulation for bcl-2 family,increasing the bcl-2/bax ratio by up-regulating anti-apoptosis gene bcl-2 and down-regulating pro-apoptosis gene bax.
5.B[a]P's toxicity on human SH-SY5Y cell line and effects of AhR and P53
Human neuroblastoma SH-SY5Y cell line growed in the RPMI1640 medium supplemented with 15%fetal bovine serum,2 mM L-glutamine,10 mM HEPES,80 units/ml penicillin,and 100μg/ml streptomycin.All cultured cells were kept at 37℃in 95%air/5%CO_2 environmant. Cell passage was performed every 3～4 days when cell density was ajusted to 1×10~5 cells/ml.24 hours later,the same generation of well-growth cells was randomly divided into 4 groups and treated with B[a]P:the control group,the low-dose group,the mid-dose group and the high-dose group.Cells were treated with B[a]P to the final concentration to 0,2.5 nM,5.0 nM and 10.0 nM and added 0.3%of the total medium $9 mixture after replacing the medium by RPMI1640 with 3%fetal bovine serum.The treated and control cells were incubated for 48 hours,and then harvested for the analysis.All compounds were dissolved in DMSO.DMSO was less than 0.3 %in the cultured medium.Inhibitors or antagonists were added to the culture 2 hours prior to the addition of B[a]P and s9 mixture.Morphologic changes were observed in the fluorescent reversed microscope.CCK-8 method was to measure the cell viability.Cell apoptosis was tested by Annexin V/PI staining(detecting phosphatidylserine) and PI staining method(detecting sub-diploid apoptotic peak).
Cells in the control group were in good condition with even size,well-adherence and abundant cell-cell junctions.Dendrites and neuraxises were more and fine.Few cells in the low-dose group was dead and floated,dendrites and neuraxises became less and shorter.The number of cells remarkably reduced,more cells were floated and some were shrinked and round. The dendrites and neuraxises became further less and shorter.And cell junctions became loose. Cells in the high-dose group were reduced notably in number,shrinked and round.Most cells were floated,and the dendrites and neuraxises shortened even disapear.The cell body shrinked severely and became round;the cell junctions bacame very loose even disapear.Hoechst 33258 was a dye specific binding to DNA and was used to observe the morphology changes.After dyeing by Hoechst 33258,normal karyon was round and even-size and uniform coloration. Apoptotic karyon was shrinked even broken,with deep and bad-distribution DNA coloration. The severity displayed karyon disintegrated and broken.But in this study no typical karyon changes was observed.AO/EB staining can dye live cells to light green,dead cells to red and apoptotic cells to orange.But in this study orange apoptotic cell was a few,however,the number of red dead cell increased.Cell viability decreased with the dose of B[a]P,and which was significantly lower in the high-dose group than that in the control group.AnnexinV/PI double-staining had not detected early-stage cell apoptosis,and PI staining detected few apoptosis according to the contents of sub-diploid DNA.But differences were not significant among groups.After CYP1A1 mRNA was silenced by specific SiRNA,ANF and PFT-αcould prevent cell viability decreasing after treated by B[a]P.Compared to the controls,cell viability increased significantly in the mid-dose group after ANF antagonizing AhR(P＜0.05),in the low-dose group after PFT-αinhibiting p53(P＜0.05).This study combined with the above results indicated that the AhR/ARNT-CYP1A1 pathway and 53-bcl-2/bax mitochondrial pathway participated in the process of B[a]P-induced neural cell apoptosis.
Conclusions:
1.AhR gene polymorphism is correlated with B[a]P neurotoxicity.
2.The AhR/ARNT-CYP1A1 pathway and p53-bcl-2/bax mitochodrion pathway may both involved in B[a]P-induced neuronal cell apoptosis process.
4.The mechanism of B[a]P-induced neural cell apoptosis may be AhR/ARNT-CYP1A1-p 53-bcl-2/bax-caspase pathway.
5.In addition,B[a]P's active metabolites may induced apoptosis in a caspase-dependent manner by damaging mitochondrion.
引文
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