苯并[a]芘神经毒性与CYP1A1基因多态性的关系
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摘要
苯并[a]芘在环境中广泛存在,是有机物不完全燃烧的产物。苯并[a]芘的致癌性在人类和动物模型中已被确认。国内外关于苯并[a]芘的神经毒性研究鲜见报道。但有研究表明苯并[a]芘可引起染毒大鼠的行为学改变,人群调查表明焦化厂工人的认知功能损伤与接触苯并[a]芘的剂量有关,提示苯并[a]芘具有神经毒性。CYP1A1是参与苯并[a]芘和其他多环芳烃类化合物活化代谢的重要酶类,可直接影响苯并[a]芘和其它前致癌物的活化,CYP1A1遗传多态性与多种肿瘤的易感性之间存在着密切关系。脑组织中含有多种亚型CYP-450,有报道神经毒物林丹、锰等的神经毒性均与CYP-450有关,苯并[a]芘神经毒作用与血浆苯并[a]芘浓度和脑内苯并[a]芘代谢物浓度显著相关,提示苯并[a]芘的神经毒性可能通过CYP1A1介导,因而,苯并[a]芘的神经毒性应与CYP1A1的基因多态性有关。苯并[a]芘的职业和环境接触人群众多,以前可能由于关注其致癌性而忽略了其神经毒性,其神经毒性对作业工人的危害可能是一个重要的职业卫生问题,意义可能不亚于其致癌性。本研究从职业接触人群、实验动物、体外细胞培养三个方面研究了苯并[a]芘的神经毒性作用,并从CYP1A1的基因多态性的出发,初步探讨了苯并[a]芘神经毒性的易感因素和作用机制。本文共分三个部分。
第一部分职业接触苯并[a]芘的神经毒性研究
第一节焦炉作业工人的神经行为功能的研究
从某钢铁公司焦化厂选择健康成年男性工人200名。从该公司的原材料处和自动化处选择88名工人作为对照组。采用自行设计的、统一的调查表,询问调查对象的一般情况、文化程度、工种、工龄、吸烟饮酒情况、生活习惯、职业变动史、自觉症状、服药史、神经精神系统疾病史及家族史等,高效液相色谱法测定作业环境空气中苯并[a]芘,收集调查对象的班后尿,用高效液相色谱法检测尿中1-羟基芘的水平;用WHO推荐的神经行为核心测试组合(NCTB)测试焦炉工神经行为功能,包括情感状态和和6个行为操作项目。环境监测表明炉顶、炉侧的苯并[a]芘浓度分别为0.186μg/m~3和1.624μg/m~3,超过职业卫生标准0.15μg/m~3的0.24倍和9.83倍。炉底和对照区空气中苯并[a]芘浓度未超过标准。可见B[a]P浓度由炉底到炉顶大幅度升高。与对照组相比,焦炉工尿1-羟基芘的水平高于对照组,P＜0.05,接触组与对照组在年龄、工龄、吸烟、饮酒及吃烧烤食物情况间均衡,文化水平不均衡,以文化水平为协变量对进行协方差分析,焦炉工在总数字跨度、数字跨度顺序、二次打点总数、二次打点正确数得分低于对照组,差异有统计学意义(P＜0.05),按照尿1-羟基芘浓度30%和70%百分位数为分界点将所有研究对象分为三组,数字跨度总分、数字跨度顺序、数字跨度倒序、习惯用手、数字译码、视觉记忆得分随尿1-羟基芘浓度增高而降低;通过多元线性回归和偏相关分析表明,数字跨度、数字跨度顺序、数字译码和视觉记忆得分的回归方程可以引入尿1-羟基芘浓度,与尿1-羟基芘浓度存在负相关(P＜0.01)。结果表明,职业性苯并[a]芘接触引起神经行为中与记忆有关三项指标的相同改变,职业性苯并[a]芘可以引起接触人群感知和记忆功能降低。
第二节焦炉作业自主神经功能的研究
用Ewing DJ推荐的自主神经(ANS)功能测试项目测定288名研究对象的心率、血压变化,测试项目共包括Valaslva Manoeuvre心率反应(HR-V)、深呼吸时的心率变化(HR-DB)、即立心率反应(HR-IS,包括Re_(30:15)和RR_(max/min)两项)和即立血压反应(BP-IS)。以文化水平为协变量,进行协方差分析。结果显示,焦炉作业工人自主神经功能降低,表现反映副交感神经调节功能的HR-V值降低,接触组与对照组相比有显著性差异(P＜0.05),而反映交感神经调节功能的指标没有明显改变。按照尿1-羟基芘浓度30%和70%百分位数为分界点将所有研究对象分为三组,各组HR-V值比较有统计学差异,多元线性回归也表明尿1-羟基芘浓度,文化水平、炼焦工龄与心率反应有关。结果表明,焦炉工副交感神经调节功能下降,自主神经调节功能紊乱可能与焦炉生产中产生的苯并[a]芘有关。
第三节焦炉作业工人脑电图和脑电地形图研究
接触组按照炉顶、炉侧、炉底各工种按年龄随机选取15名作为研究对象,对照组按年龄随机选取25名,要求在年龄、文化水平、工龄、吸烟和饮酒习惯上尽量齐同,共70人作为研究对象。采用日产6518型脑电图机与北京太阳科技公司脑电处理系统对70名研究对象的脑电图和脑电地形图进行了采集,分析结果表明,经fisher检验,接触组与对照组均以α波为主(p=0.013),但是在接触组θ波出现的频率已高达13.33%。广泛性异常(p=0.2666)、弥散性异常(p=0.1718)和局限性异常(p=0.0089)高于对照组,脑电地形图则主要表现为各频带均为低能量值,α频带呈弥散性分布,有时出现不对称现象。提示焦炉作业工人大脑皮层神经功能低下。
第四节焦炉作业工人外周神经传导速度和诱发电位的研究
使用日本捷斯特公司生产Neuropack M1,检测第三节中选择研究对象的双侧正中神经和尺神经的感觉和运动神经(排除受伤神经)传导速度(SCV和MCV)、远端潜伏期(SL和ML)、体感诱发电位、听觉诱发电位和视觉诱发电位。结果表明,感觉和运动神经传导速度、远端潜伏期上接触组与对照组比较无显著性差异,两组体感诱发电位N9、N13、N20潜伏期之间无统计学差异,听觉诱发电位Ⅰ波峰潜伏期、Ⅲ波峰潜伏期、Ⅴ波峰潜伏期、Ⅰ～Ⅲ峰间潜伏期、Ⅲ～Ⅴ峰间潜伏期、Ⅰ～Ⅴ峰间潜伏期、和视觉诱发电位P100峰潜伏期各指标之间无统计学差异。可见多环芳烃未引起焦炉作业工人外周神经传导速度和诱发电位的改变。
第五节焦炉作业工人GYP1A1基因多态性分析
采用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)和等位基因特异性PCR(ASA-PCR)分别对CYP1A1基因Msp I位点多态性和Exon7 Ile—Val位点多态性进行了分析,结果显示,Msp I位点ml/ml、ml/m2、m2/m2基因型分布以及Ile-Val位点Ile/Ile、Ile/Val、Val/Val基因型分布在接触组与对照组都无显著性差异,两基因多态性位点等位基因频率经卡方检验,符合Hardy-weinberg平衡定律。焦炉工CYP1A1 MspⅠ各基因型个体中尿中1-羟基芘水平表现为野生型纯合子＜杂合子＜变异型纯合子的变化趋势,但差异无显著性,而GYP1A1第7外显子Val/Val个体尿中1-羟基芘水平与Ile/Ile比较有显著性差异,CYP1A1基因多态性影响了尿中1-羟基芘水平。接触组两多态性位点不同基因型个体神经行为功能和自主神经功能分析表明,MspⅠ位点ml/ml、ml/m2、m2/m2基因型间神经行为功能和自主神经功能各指标无统计学差异,Ile-Val位点Ile/Ile、Ile/Val、Val/Val基因型间上述指标比较,突变型(Val/Val)倒序数字跨度得分低于野生型(Ile/Ile),CYP1A1 Ile-Val位点多态性影响了倒序数字跨度,可见,CYP1A1基因多态性影响了苯并[a]芘的神经行为毒性。
第二部分苯并[a]芘神经毒性的体内实验研究
第一节单次侧脑室注射苯并[a]芘大鼠脑形态学改变
健康雄性SD大鼠40只,随机分为5组:空白对照组、二甲基亚砜(DMSO)组(溶剂对照组)、苯并[a]芘高浓度组(50mmol/L)、苯并[a]芘中浓度组(5mmol/L)、苯并[a]芘低浓度组(0.5mmol/L)。每组8只,用25%乌拉坦(0.4ml/100g)经腹腔注射麻醉后,将大鼠置于立体定向仪上,侧脑室插管,50μL微量进样器通过插管分别向高、中、低浓度组缓慢注入苯并[a]芘溶液10μL,溶剂对照组DMS010μl,空白对照组生理盐水10μl,动物插管给药后,各组都出现活动减少、反应迟钝、进食量减少、昏睡;三天后,中、高剂量组部分大鼠出现动作迟缓、步态蹒跚,少数表现烦燥易惊,未出现动物死亡,对照组和低剂量组仅表现为活动减少,无其他明显异常行为改变。病理结果显示,对照组小脑蒲肯野细胞数目形态正常,海马神经细胞排列整齐,数目形态正常;随着苯并[a]芘剂量的增高,神经细胞损伤越严重,特别高剂量组出现典型的神经细胞损伤现象出现,表现出小脑蒲氏细胞数目减少,部分细胞出现坏死表现,胞核聚集、浓缩、溶解;海马神经细胞出现排列紊乱,数目减少,胞浆透明,核皱缩,脑皮质未发现典型的坏死灶和胶质细胞增生,脑桥和延髓部分也未见明显改变。本次研究显示脑病理改变随苯并[a]芘剂量增加而加重,有一定的剂量一反应关系,为下一步的慢性染毒剂量选择提供了依据。
第二节亚慢性染毒大鼠学习记忆功能测试
健康雄性SD大鼠40只,经麻醉后定位插管,一周后称重后按体重编号,将动物随机分为4组:橄榄油组(溶剂对照组)、苯并[a]芘2.5mmol/L组、苯并[a]芘5mmol/L组、苯并[a]芘10mmol/L组。每组8只,麻醉后定位,应用微量注射器通过插管分别向高、中、低浓度组缓慢注入苯并[a]芘溶液10μL,溶剂对照组橄榄油10μl,缝皮。每周给药一次,连续给药三周。为探讨B[a]P对大鼠学习记忆功能的影响,本研究选择跳台试验和水迷宫实验对染毒大鼠进行学习记忆功能评价。大鼠染毒后跳台试验结果显示,随染毒剂量的增加,大鼠步下平台的潜伏期缩短,次数增多,有明显的剂量反应关系,结果表明苯并[a]芘可损害大鼠对被动逃避反应的学习和短期记忆能力。Morris水迷宫试验结果显示:随染毒剂量的增加,各组大鼠平均潜伏期延长,在原平台停留时间缩短,各剂量组与对照组比较有显著性差异,各剂量组间平均潜伏期比较无显著性差异,而在原平台象限停留时间高剂量组与低剂量组间有差异,P＜0.05。B[a]P可造成大鼠空间学习记忆功能的损害。这与人群调查发现的焦炉作业工人学习记忆能力降低相一致。
第三节亚慢性染毒大鼠海马的形态学改变和凋亡检测
大鼠染毒结束后,断头处死,取出海马。病理结果显示随着苯并[a]芘剂量的增高,有典型的神经细胞损伤现象出现,中、高剂量组海马神经细胞出现排列紊乱,数目减少,胞浆透明,核皱缩,细胞周围出现环状带,电镜结果显示,随着剂量的增高,出现细胞核皱缩,染色质边聚,核膜破裂等形态学的变化,线粒体形态逐渐出现肿胀,并可伴有线粒体嵴减少或消失,肿胀重者呈气球样变而且外膜破裂。海马神经细胞凋亡测定结果显示,随着B[a]P染毒剂量的增加,海马神经细胞凋亡指数增高,有剂量-反应关系。海马神经细胞凋亡可能是大鼠学习记忆功能降低的原因,线粒体形态改变提示,神经细胞凋亡可能与线粒体损害有关。
第四节亚慢性染毒大鼠海马神经生物化学的改变
大鼠染毒结束后,断头处死,取出海马。高效液相色谱法分别测定海马中谷氨酸(Glu)、天门冬氨酸(Asp)、甘氨酸(Cly)、γ-氨基丁酸(GABA)、多巴胺、5-羟色胺、5-羟吲哚乙酸含量,应用相应试剂盒测定海马SOD、MDA。结果显示,大鼠海马谷氨酸含量明显降低,各剂量组与对照组比较,p＜0.01,各剂量组间无显著性差异;而天门冬氨酸(Asp)、甘氨酸(Cly)、γ-氨基丁酸(GABA)含量各组未见明显变化。大鼠海马5-羟色胺(5-HT)含量明显增高,有剂量-反应关系,而多巴胺(DA)、5-羟吲哚乙酸(5-HIAA)含量各组未见明显变化。随苯并[a]芘剂量的增大,MDA含量增多,SOD活性下降。各剂量组与对照组比较,p＜0.05,各剂量组间比较,高剂量组与低剂量组比较有显著性差异,结果提示苯并[a]芘染毒可引起大鼠氨基酸递质、单胺类递质的改变,这可能大鼠学习记忆功能减低有关。大鼠海马随苯并[a]芘剂量的增大,MDA含量增多,SOD活性下降,显示海马发生脂质过氧化。
第五节亚慢性染毒大鼠海马CYP1A1基因和蛋白的表达
本次研究应用PT-PCR法研究CYP1A1mRNA表达,免疫组化SABC法和Western-blot检测了海马CYP1A1蛋白的表达。随苯并[a]芘剂量的增高,CYP1A1mRNA及蛋白表达增多,有剂量-反应关系,CYP1A1基因和蛋白表达与海马神经细胞凋亡指数的相关分析表明:海马神经细胞凋亡指数与CYP1A1mRNA表达呈正相关(r=0.871,P＜0.01);与Western-blot检测CYP1A1蛋白表达呈正相关(r=0.804,P＜0.01)。进而结合动物学习记忆功能、神经递质的改变,脂质过氧化水平的增高。我们推测CYP1A1表达增高可能是苯并[a]芘神经毒性作用的关键环节。这可部分解释人群CYP1A1基因多态性与焦炉作业工人学习记忆功能降低的关系。
第三部分苯并[a]芘神经毒性的体外研究
第一节苯并[a]芘致神经元形态改变和凋亡检测
选用新生1～3d的SD大鼠,取脑,分离大脑皮质进行神经元培养,加入0.5mg/ml阿糖胞苷(终浓度10μM)培养液以抑制胶质细胞增殖。经GFAP免疫组化证明,胶质细胞小于10%,在细胞培养第5天左右,选取生长良好的同批次神经细胞,以苯并[a]芘同时加或不加S9分别对神经元染毒,两种染毒方法都分为对照组、低剂量组、中剂量组、高剂量组,使苯并[a]芘终浓度分别为0μM、10μM、20μM,40μM。继续培养40h,观察细胞的生长状况和形态学改变。
结果表明,苯并[a]芘染毒后神经元树突数量减少,轴突减少变短,细胞与细胞间连接明显减少;加S9后,各剂量组比不加S9组细胞形态改变明显,两组相差一个剂量组,即低剂量加S9组形态改变基本相当于不加S9的中剂量组。高剂量加S9组形态改变最明显,部分神经元的大多数树突消失融解,细胞轴突也严重受损,胞体变圆、收缩。AO/EB染色结果表明苯并[a]芘可引起神经元轴突与树突的数量,细胞间和细胞连接减少。高剂量染毒组可见轴突与树突的数量进一步减少,细胞与突触间,胞体与胞体间连接明显减少甚至消失,胞体明显缩小,染色质聚集,细胞呈桔黄色;加S9后,各剂量组比不加S9组细胞形态改变明显,两组相差一个剂量组,即低剂量加S9组形态改变基本相当于不加S9的中剂量组。高剂量加S9组形态改变最明显,+S9高剂量组神经细胞胞核呈固缩状的桔红色荧光,轴突与树突的数量锐减,细胞与细胞间连接很少甚至消失,有核分裂边聚。本次应用Annexin V和PI双染法进行细胞凋亡的检测,结果发现,随着苯并[a]芘染毒剂量的加大,神经细胞的早期、晚期和总凋亡率逐渐增高,有剂量依赖性,未加S9的中、低剂量组早期和晚期凋亡率与对照组相比差异均无显著性意义(p＞0.05)。高剂量组与对照组相比差异有显著性意义,加入S9后,苯并[a]芘对神经细胞的损害作用明显加强,随着苯并[a]芘染毒剂量,神经细胞早期和晚期凋亡率明显升高,+S9的三个剂量组与对照组相比差异均有显著性意义,p＜0.05。细胞的凋亡率叠加示意图和直方图显示,神经细胞的早期、晚期和总凋亡率与染毒剂量呈明显的剂量依赖性。表明苯并[a]芘可引起神经元凋亡,而且加入肝S9后作用增强,代谢活化是苯并[a]芘引起凋亡的主要原因。
第二节苯并[a]芘染毒神经元的细胞活力改变和氧化损伤
应用Cell Counting Kit-8检测染毒后各组神经元的细胞活力,随着苯并[a]芘浓度的增加,神经细胞活力下降,未加S9组仅中、高剂量组与对照组比较有显著性差异,加S9组各剂量组与对照组比较均有显著性差异,而且各剂量组间有显著性差异,表现出较明显的剂量-反应关系。表明苯并[a]芘具有细胞毒性。染毒后的神经细胞内SOD和MDA测定表明,加S9组随着苯并[a]芘浓度的增加,神经细胞SOD活性下降,MDA含量升高,各剂量组与对照组比较有显著性差异,表明神经细胞发生脂质过氧化。加入罗丹明123(Rh123),应用流式细胞仪测定细胞线粒体膜电位,随着苯并[a]芘浓度的增加,细胞线粒体膜电位下降,未加S9组仅中、高剂量组与对照组比较有显著性差异,加S9组各剂量组与对照组比较均有显著性差异,而且各剂量组间有显著性差异,表现出较明显的剂量-反应关系,苯并[a]芘代谢活化引起脂质过氧化,进而线粒体膜电位的降低可能是凋亡的诱发途径。
第三节神经元CYP1A1蛋白及基因的表达与细胞损伤的关系
本次研究应用RT-PCR法对神经元CYP1A1mRNA表达,免疫组化SABC法和Western-blot检测了神经元CYP1A1蛋白的表达。结果随苯并[a]芘剂量的增高,CYP1A1mRNA及蛋白表达增多,有剂量-反应关系,CYP1A1基因和蛋白表达与神经元细胞凋亡率的相关分析表明:神经细胞凋亡率与CYP1A1mRNA表达呈正相关(r=0.831,P＜0.01);与CYP1A1蛋白表达呈正相关(r=0.780,P＜0.01)。结合所有+S9和—S9的检测结果,表明CYP1A1表达增高是细胞损伤的关键因素,这与整体动物试验的结果是一致的。
小结
1.职业性苯并[a]芘接触可引起明显的记忆功能降低和副交感神经调节功能降低。
2.苯并[a]芘引起的记忆功能降低与CYP1A1基因多态性有关。
3.体内和体外实验都证明苯并[a]芘诱导的CYP1A1表达增高是苯并[a]芘神经毒性的关键环节。
4.苯并[a]芘可引起大鼠学习记忆能力降低,可能与海马神经细胞凋亡,谷氨酸含量降低,5-羟色胺水平升高以及脂质过氧化有关。
5.苯并[a]芘可能通过线粒体途径诱发神经细胞凋亡。
Benzo(a)pyrene(B[a]P),one of polycyclic aromatic hydrocarbon(PAH)compounds,is product of incomplete combustion of organic matters and is widespread in the environment.The carcinogenicity of B[a]P is well established in human and animal model.Due to the widespread distribution of B[a]P in the environment and the potential exposure for human,the adverse effects of these compounds deserved to be investigated.Although the neurotoxic effect of B[a]P has not drawn much attention,.there were some related data implying that B[a]P showed the neurotoxic effects.when exposed to B[a]P by oral gavage,rats were induced behavioral impairments,and there was a significant correlation between neurotoxic effects and plasma B[a]P concentration,and its metabolite concentration in brain tissue.Coke-production plant workers in Poland developed neurotic syndromes with vegetative disregulation,and a loss of short-term memory;their prevalence depended on the level of exposure to B[a]P.B[a]P is metabolised to active metabolites that are tumourigenic.The bioactivation of B[a]P is catalyzed by some cytochrome P450(CYP)enzymes,particularly CYP 1A1.The brain tissues of various species contain almost all CYP-enzymes found in liver,including CYP1A1.CYP-enzymes in brain were associated with neurotoxic effects of some chemicals,such as lindane and manganes.The correlation between the neurotoxicity of B[a]P and B[a]P metabolite concentrations in brain implied that CYP1A1 may mediate the neurotoxicity of B[a]P.CYP1A1 gene polymorphisms have been studied in relation to various kinds of cancers.Our hypothesis is that CYP1A1 gene polymorphisms wound modulates the neurotoxicity of B[a]P.The neurotoxicity of B[a]P may be a important occupational risk like its carcinogenicity for occupational exposed populations.In this research,occupational exposed population,experimental animals and primarily cultured neuron exposed to B[a]P were investigated to preliminarily elucidate the mechanism of neurotoxicity of B[a]P and its association with CYP1A1 gene polymorphisms.The research was divided into three parts.
PartⅠNeurotoxicity study on occupational population exposed to B[a]P
1.neurobehavioral function of coke oven wokers
200 healthy adult male coke oven workers were selected from coke plant of a state-owned enterprise in Taiyuan City.88 controls unexposed to polycyclic aromatic hydrocarbons(PAHs) occupationally were selected from the same enterprise.All the subjects participated this investigation freely according to their consent.Concentration of B[a]P in the working environment was monitored by High Performance Liquid Chromatography(HPLC).Urine samples were collected immediately after working shift.The level of urinary 1-hydroxypyrene was determined by High Performance Liquid Chromatography(HPLC).General informations of workers correlated to the investigation were collected by query and filled in the questionnaire according to the same criteria by well-trained investigators.WHO recommended neurobehavioral core test battery(NCTB)was performed on coke oven workers and controls to test the neurobehavioral changes and the mood state.The concentration of B[a]P at oven bottom,oven side and oven top were 0.0195μg/m3,,0.186μg/m3 and 1.624μg/m3 respectively,and Concentration of B[a]P at oven side and oven top were higher than the occupational hygiene criterion.Urinary 1-hydroxypyrene were significant difference between exposure group(3.42±0.98μmol/mol creatinine)and control group(2.75±1.09μmol/mol creatinine).No significant differences were found between exposure group and control group on age,length of service,smoking,drinking and unhealthy food consumption;however,compared to the controls,the scores of the coke oven workers were lower in the total digital span,the forward digital span,right dotting and total dotting,and the differences showed statistical significance(P＜0.05).Significant difference of the total digital span,the forward digital span, backward digital span,digit symbol and Benton visual retentions existed in different urinary 1-hydroxypyrene concentration groups and showed a dose-response tendency.Results of multiple stepwise regression analysis and correlation analysis showed that the level of urinary 1-hydroxypyrene affect memory and perception of coke oven workers and there wre negative correlations between the levels of urinary 1-hydroxypyrene and changes in neurobehavioral function.It was suggested that B[a]P wound mainly causes decrease of memory and perception in coke oven workers..
2.Function of autonomic nervous system(ANS)in coke oven wokers
Function of autonomic nervous system(ANS)of 288 subjiects were measured with the method recommended by Ewing DJ,which consists of 4 tests:Valaslva Manoeuvre heart rate variation(HR-V),variation of heart rate when deeply breathing(HR-DB),variation of heart rate when instantly standing up(HR-IS,including RR30:15 and RRmax:min)and variation of blood pressure when instantly standing up(BP-IS).Compared to controls,HR-V decreased significantly in coke oven workers(p＜0.01),which represents modulation of parasympathetic nervous function.But no statistical differences were found in HR-DB,RR30:15,RRmax:min and BP-IS between exposed groups and control group(p＞0.05).Significant difference of HR-V existed in different urinary 1-hydroxypyrene concentration groups.Results of multiple linear stepwise regression demonstrated that the level of urinary 1-hydroxypyrene and level of education affect HR-V.The results indicated that B[a]P affected autonomic nervous function of coke oven workers by mainly down- regulating parasympathetic nervous function.
3.Changes of electroencephalogram and brain electrical activity mapping in coke oven wokers
45 subjects,as a exposed group,were randomly selected from exposed groups according to their type of work in production and age,25 controls were chosen from unexposed group according to the same principal.All subjects were conducted electroencephalogram and brain electrical activity mapping.Compare with controls,the ratio of abnormal EEG increased,brain electrical activity mapping showed all frequency bands energy values were lowered,andαfrequency band distribution was diffuse,sometimes dissymmetrical.The results displayed B[a]P increased ratio of abnormal EEG.
4.Changes of conduction velocity of peripheral nerve and evoked potential in coke oven wokers
The conduction velocity of peripheral nerve and evoked potential were monitored by neuropack M1,(Nihon Kohden coporation),compared with controls,there is no significant difference in sensory conduction velocity and motorial conduction velocity of ulnar nerve and median nerve.The sensory evoked potencial,auditory evoked potencial and pattern-visual evoked potencial in two groups also showed no significant diffrence.It was suggested that B[a]P shoud have no effect on conduction velocity of peripheral nerve and evoked potential.
5.Analysis of CYP1A1 gene polymorphisms in coke oven wokers
CYP1A1 gene polymorphisms including MspⅠsite and Ile-Val site were monitored by PCR-RFLP and ASA-PCR,respectively.The results revealed that there were no statistic difference of the genotype frequencies of m1/m1、m1/m2、m2/m2 of MspⅠsite and that of Iie/Iie、Ile/Val、Val/Val of Ile-Val site between exposed group and contol group,In exposed group the level of urinary 1-hydroxypyrene of Val/Val was significantly higher than Ile/Ile wild homozygote.Although there was a tendency of m1/m1＜m1/m2＜m2/m2 in urinary 1-hydroxypyrene,there was no statistically significant difference.The scores of Dignity Span Backward with Val/Val gene type were higher than those with Iie/Iie gene type.Other index of NTCB and ANS showed no difference among three gene types of Ile-Val site.All index of NTCB and ANS showed no difference among three gene type of MspⅠsite.The present data revealed CYP1A1 Ile-Val site gene polymorphism modulats the neurobehavioral toxicity of B[a]P.
PARTⅡstudy ofneurotoxicity of B[a]P in vivo
1.single intracerebroventricular injection of benzo[a]pyrene induces morphological changes in rats
Forty male SD rats,adult and healthy,were divided into 5 groups randomly:normal control, DMSO control,B[a]P 0.5mmol/L,B[a]P 5mmol/L,B[a]P50mmol/L,all groups were administered by intracerebroventricular injection,and the injectant volume was 10μl.The morphology of neural cells in rats' hippocampus and cerebellum were observed by optical microscope.In cerebellum and hippocampus,arrangement and numbers of the neural cells were normal in control group,but with the increasing of the doses of B[a]P exposed,the arrangement of neural cells became disorderly,the quantity of cells became diminished,and the cells showed necrosis appearance.The results displayed that benzo[a]pyrene damaged brain tissues of rats,and the higher the dose of B[a]P is,the more serious the damage of brain is.
2.Subchronical intracerebroventricular injection of benzo[a]pyrene decreased the capacity of learning and memory in rats
Forty male SD rats,adult and healthy,were intubated,after one week,all incubated rats were divided into 4 groups randomly:normal control,olive oil control,B[a]P2.5mmol/L,B[a]P 5mmol/L,and B[a]P10mmol/L,all groups were administered by intracerebroventricular injection, and the injectant volume was 10μl.Step-down and Morris water maze tests were applied to assess the capacity of learning and memory in rats.With the dose of B[a]P increasing,the step down latency was shortern,and times of stepping down increased,there existed significant differences in three B[a]P -treated groups compared with controls.The results indicated that B[a]P can induce impairment of passive avoidance learning and short-term memory.In Morris water maze tests the mean escape latency was prolonged and the length of staying in the original quadrant was shorterned in treated groups while dose was increasing.There was an evident dose -response relationship between the dose of B[a]P and the data of Morris water maze tests,which suggested that B[a]P would affecte the spatial learning and memory functions.
3.Subchronicai intracerebroventricular injection of benzo[a]pyrene induces hippocampus morphological changes and apoptosis
The observation under light microcope showed:arrangement of the neural cells in rats' hippocampus was orderly in control group,but with the increasing of the doses of B[a]P exposed,the cells displayed disorderly arranged,the connections among the neural cells were loosened,the karyon shrinked,some rings around the cells were observed.Picture under electron microscope displayed:in control group,the karyotin distributed symmetrically,and its form was regular,the form of mitochondria was regular,the arrangement of bars was orderly,and with the increasing of the doses of B[a]P exposed,the karyon shrinked,the karyotin assembled in the edge,karyotheca was broken,mitochondria swelled,broken,the bars were reduced and disappeared.
With the increasing of the dose of B[a]P exposed,the apoptosis indices(AI)of neural cells in rats' hippocampus increased,and AI in 5mmol/L B[a]P group and 10mmol/L B[a]P group were significantly higher than that of controls(P＜0.05),and which in B[a]P 10mmol/L group were significantly higher than that of 2.5mmol/L B[a]P group.
4.Subchronicai intracerebroventricular injection of benzo[a]pyrene induces neurochemical changes of hippocampus in rats
To explore the mechanism of B[a]P induced neurotoxicity,amino acid transmitters, monoamine transmitters,SOD and MDA were determined.Compared with controls,GLU was decreased in three treated groups,and other amino acid transmitters showed no changes,while there was an increasing trend of 5-HT in treated groups,no differences of DA and 5-HIAA were found between treated groups and contol group.SOD activity decreased and content of MDA increased in three treated group compared with the conrols.All these data implied the potential mechanism of neurotoxicity of B[a]P.
5.Subchronical intracerebroventricular injection of benzo[a]pyrene induces CYP1A1 expression changes of hippocampus in rats
To explore the relationship between neurotoxicity of B[a]P and CYP1A1 expression, RT-PCR was used to determine CYP1A1 mPNA,and immunohistochemical staining and western bloting were performed to determine CYP1A1 protein.CYP1A1 mRNA and CYP1A1 protein increased significantly compared with the control group and in a dose dependent manner, a positive correlation between AI and the expression of CYP1A1mRNA,the expression of CYP1A1 protein were observed.Considering the data mentioned above and the capacity of learning and memory in rats and neurochemical changes prehensively,CYP1A1 expression may be thought to be the critical step in neurotoxicity of B[a]P.
PartⅢstudy of neurotoxicity of B[a]P in vitro
1.Changes of cell morphology and apoptosis of primary cultured neurons induced by B[a]P
primary neurons were dissociated from cerebral cortex of 1-3 days old SD rat and cultured with DMEM incubator at 37℃,cell density was adjusted at 1×10~5cells/ml,after 5 days' cultivation,the astrocyte GFAP was identified by immunohistochemical staining.If the astrocyte was less than 10%,the cultured cells was thought to be pure neurons.Then,they were divided into two groups,+S9 and -S9,and all the two groups of neurons were cultivated with B[a]P at 0,10,20,40μmol/L,respectively,for 40 hours.The morphology was observed,and apoptosis was measured.Under light microscope,with the increment of B[a]P dose,the numbers of neurites and dendrites of neurons and the conjunctions among cells were reduced evidently.The changes were more serious at 40μmol/L in +S9 group.The neurites of some neurons became shorter,even disappeared,and the cell body became round.Stained with AO/EB,under fluorescence light,live neuron showed a bright green color,while the dead one was red,as apoptotic cell was orange.Based on the color of neuron,we can distinguish living conditions of the neurons.With B[a]P dose increasing,the ratios of orange colored cells increased,and the numbers of neurites and dendrites of neurons and the conjunctions among cells decreased,+S9 group showed more severity,at 40μmol/L,the cell body became round and anumber of neurons were stained orange even red,and the neurite of some neuron became shorter,even disappear,the karyon shrinked or aggregated in the edge or cleaved.With AnnexinⅤand PI staining,the apoptosis ratios of neurons were measured,early-onset apoptosis ratio,late -onset apoptosis ratio and total apoptosis ratio increased in a dose dependent manner,and +S9 group showed more serious situation.The data suggested that B[a]P could induce apoptosis of neurons,and bioactivation of B[a]P was important to exert its adverse effects.
2.Changes of cell viability and the mitochondrial membrane potential(MMP)and lipid peroxidation induced by B[a]P
Neuron vilability decreased in—S9 group at dose of 20μmol/L compared with controls,in +S9 group there existed significant differences in three treated grous.The determination of MMP in neuron showed that MMP decreased in—S9 group at dose of 20μmol/L compared with controls,while in+S9 group,the MMP decresed in a dose dependent manner.The SOD activity decreased and MDA increased in treated group compared with controls.These results suggested that liquid peroxidation induced by BaP could lead to MMP changes,which may initiate neuron apoptosis and cell viability decline.
3.CYP1A1 expression induced by B[a]P in neuron
With the dose of B[a]P increasing,CYP1A1 mRNA and CYP1A1 protein increased,there existed significantly difference in treated groups compared with control group.A positive correlation between neuron apoptosis ratio and the expression of CYP1A1mRNA,as well as the expression of CYP1A1 protein were observed.The results indicated that CYP1A1 expression is related to cytotoxicity of B[a]P.
summary
1.B[a]P-related neurotoxicity in B[a]P exposed wokers are characterized by poor memory and down regulated parasympathetic nervous function.
2.The neurobehavioral effect of B[a]P is related to CYP1A1 gene polymorphism.
3.It had been proved that CYP1A1 expression induce by B[a]P may be the critical step in neurotoxicity of B[a]Rin vivo and in vitro.
4.The impairment of learning and memory by B[a]P was also found in rats,which may be related to neural cell apoptosis,variations of GLUand 5-HT and statue of lipid peroxidation in hippocampus.
5.B[a]P may induce neural cell apoptosis through mitochodria pathway.
第一部分职业接触苯并[a]芘的神经毒性研究
第一节焦炉作业工人的神经行为功能的研究
从某钢铁公司焦化厂选择健康成年男性工人200名。从该公司的原材料处和自动化处选择88名工人作为对照组。采用自行设计的、统一的调查表,询问调查对象的一般情况、文化程度、工种、工龄、吸烟饮酒情况、生活习惯、职业变动史、自觉症状、服药史、神经精神系统疾病史及家族史等,高效液相色谱法测定作业环境空气中苯并[a]芘,收集调查对象的班后尿,用高效液相色谱法检测尿中1-羟基芘的水平;用WHO推荐的神经行为核心测试组合(NCTB)测试焦炉工神经行为功能,包括情感状态和和6个行为操作项目。环境监测表明炉顶、炉侧的苯并[a]芘浓度分别为0.186μg/m~3和1.624μg/m~3,超过职业卫生标准0.15μg/m~3的0.24倍和9.83倍。炉底和对照区空气中苯并[a]芘浓度未超过标准。可见B[a]P浓度由炉底到炉顶大幅度升高。与对照组相比,焦炉工尿1-羟基芘的水平高于对照组,P＜0.05,接触组与对照组在年龄、工龄、吸烟、饮酒及吃烧烤食物情况间均衡,文化水平不均衡,以文化水平为协变量对进行协方差分析,焦炉工在总数字跨度、数字跨度顺序、二次打点总数、二次打点正确数得分低于对照组,差异有统计学意义(P＜0.05),按照尿1-羟基芘浓度30%和70%百分位数为分界点将所有研究对象分为三组,数字跨度总分、数字跨度顺序、数字跨度倒序、习惯用手、数字译码、视觉记忆得分随尿1-羟基芘浓度增高而降低;通过多元线性回归和偏相关分析表明,数字跨度、数字跨度顺序、数字译码和视觉记忆得分的回归方程可以引入尿1-羟基芘浓度,与尿1-羟基芘浓度存在负相关(P＜0.01)。结果表明,职业性苯并[a]芘接触引起神经行为中与记忆有关三项指标的相同改变,职业性苯并[a]芘可以引起接触人群感知和记忆功能降低。
第二节焦炉作业自主神经功能的研究
用Ewing DJ推荐的自主神经(ANS)功能测试项目测定288名研究对象的心率、血压变化,测试项目共包括Valaslva Manoeuvre心率反应(HR-V)、深呼吸时的心率变化(HR-DB)、即立心率反应(HR-IS,包括Re_(30:15)和RR_(max/min)两项)和即立血压反应(BP-IS)。以文化水平为协变量,进行协方差分析。结果显示,焦炉作业工人自主神经功能降低,表现反映副交感神经调节功能的HR-V值降低,接触组与对照组相比有显著性差异(P＜0.05),而反映交感神经调节功能的指标没有明显改变。按照尿1-羟基芘浓度30%和70%百分位数为分界点将所有研究对象分为三组,各组HR-V值比较有统计学差异,多元线性回归也表明尿1-羟基芘浓度,文化水平、炼焦工龄与心率反应有关。结果表明,焦炉工副交感神经调节功能下降,自主神经调节功能紊乱可能与焦炉生产中产生的苯并[a]芘有关。
第三节焦炉作业工人脑电图和脑电地形图研究
接触组按照炉顶、炉侧、炉底各工种按年龄随机选取15名作为研究对象,对照组按年龄随机选取25名,要求在年龄、文化水平、工龄、吸烟和饮酒习惯上尽量齐同,共70人作为研究对象。采用日产6518型脑电图机与北京太阳科技公司脑电处理系统对70名研究对象的脑电图和脑电地形图进行了采集,分析结果表明,经fisher检验,接触组与对照组均以α波为主(p=0.013),但是在接触组θ波出现的频率已高达13.33%。广泛性异常(p=0.2666)、弥散性异常(p=0.1718)和局限性异常(p=0.0089)高于对照组,脑电地形图则主要表现为各频带均为低能量值,α频带呈弥散性分布,有时出现不对称现象。提示焦炉作业工人大脑皮层神经功能低下。
第四节焦炉作业工人外周神经传导速度和诱发电位的研究
使用日本捷斯特公司生产Neuropack M1,检测第三节中选择研究对象的双侧正中神经和尺神经的感觉和运动神经(排除受伤神经)传导速度(SCV和MCV)、远端潜伏期(SL和ML)、体感诱发电位、听觉诱发电位和视觉诱发电位。结果表明,感觉和运动神经传导速度、远端潜伏期上接触组与对照组比较无显著性差异,两组体感诱发电位N9、N13、N20潜伏期之间无统计学差异,听觉诱发电位Ⅰ波峰潜伏期、Ⅲ波峰潜伏期、Ⅴ波峰潜伏期、Ⅰ～Ⅲ峰间潜伏期、Ⅲ～Ⅴ峰间潜伏期、Ⅰ～Ⅴ峰间潜伏期、和视觉诱发电位P100峰潜伏期各指标之间无统计学差异。可见多环芳烃未引起焦炉作业工人外周神经传导速度和诱发电位的改变。
第五节焦炉作业工人GYP1A1基因多态性分析
采用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)和等位基因特异性PCR(ASA-PCR)分别对CYP1A1基因Msp I位点多态性和Exon7 Ile—Val位点多态性进行了分析,结果显示,Msp I位点ml/ml、ml/m2、m2/m2基因型分布以及Ile-Val位点Ile/Ile、Ile/Val、Val/Val基因型分布在接触组与对照组都无显著性差异,两基因多态性位点等位基因频率经卡方检验,符合Hardy-weinberg平衡定律。焦炉工CYP1A1 MspⅠ各基因型个体中尿中1-羟基芘水平表现为野生型纯合子＜杂合子＜变异型纯合子的变化趋势,但差异无显著性,而GYP1A1第7外显子Val/Val个体尿中1-羟基芘水平与Ile/Ile比较有显著性差异,CYP1A1基因多态性影响了尿中1-羟基芘水平。接触组两多态性位点不同基因型个体神经行为功能和自主神经功能分析表明,MspⅠ位点ml/ml、ml/m2、m2/m2基因型间神经行为功能和自主神经功能各指标无统计学差异,Ile-Val位点Ile/Ile、Ile/Val、Val/Val基因型间上述指标比较,突变型(Val/Val)倒序数字跨度得分低于野生型(Ile/Ile),CYP1A1 Ile-Val位点多态性影响了倒序数字跨度,可见,CYP1A1基因多态性影响了苯并[a]芘的神经行为毒性。
第二部分苯并[a]芘神经毒性的体内实验研究
第一节单次侧脑室注射苯并[a]芘大鼠脑形态学改变
健康雄性SD大鼠40只,随机分为5组:空白对照组、二甲基亚砜(DMSO)组(溶剂对照组)、苯并[a]芘高浓度组(50mmol/L)、苯并[a]芘中浓度组(5mmol/L)、苯并[a]芘低浓度组(0.5mmol/L)。每组8只,用25%乌拉坦(0.4ml/100g)经腹腔注射麻醉后,将大鼠置于立体定向仪上,侧脑室插管,50μL微量进样器通过插管分别向高、中、低浓度组缓慢注入苯并[a]芘溶液10μL,溶剂对照组DMS010μl,空白对照组生理盐水10μl,动物插管给药后,各组都出现活动减少、反应迟钝、进食量减少、昏睡;三天后,中、高剂量组部分大鼠出现动作迟缓、步态蹒跚,少数表现烦燥易惊,未出现动物死亡,对照组和低剂量组仅表现为活动减少,无其他明显异常行为改变。病理结果显示,对照组小脑蒲肯野细胞数目形态正常,海马神经细胞排列整齐,数目形态正常;随着苯并[a]芘剂量的增高,神经细胞损伤越严重,特别高剂量组出现典型的神经细胞损伤现象出现,表现出小脑蒲氏细胞数目减少,部分细胞出现坏死表现,胞核聚集、浓缩、溶解;海马神经细胞出现排列紊乱,数目减少,胞浆透明,核皱缩,脑皮质未发现典型的坏死灶和胶质细胞增生,脑桥和延髓部分也未见明显改变。本次研究显示脑病理改变随苯并[a]芘剂量增加而加重,有一定的剂量一反应关系,为下一步的慢性染毒剂量选择提供了依据。
第二节亚慢性染毒大鼠学习记忆功能测试
健康雄性SD大鼠40只,经麻醉后定位插管,一周后称重后按体重编号,将动物随机分为4组:橄榄油组(溶剂对照组)、苯并[a]芘2.5mmol/L组、苯并[a]芘5mmol/L组、苯并[a]芘10mmol/L组。每组8只,麻醉后定位,应用微量注射器通过插管分别向高、中、低浓度组缓慢注入苯并[a]芘溶液10μL,溶剂对照组橄榄油10μl,缝皮。每周给药一次,连续给药三周。为探讨B[a]P对大鼠学习记忆功能的影响,本研究选择跳台试验和水迷宫实验对染毒大鼠进行学习记忆功能评价。大鼠染毒后跳台试验结果显示,随染毒剂量的增加,大鼠步下平台的潜伏期缩短,次数增多,有明显的剂量反应关系,结果表明苯并[a]芘可损害大鼠对被动逃避反应的学习和短期记忆能力。Morris水迷宫试验结果显示:随染毒剂量的增加,各组大鼠平均潜伏期延长,在原平台停留时间缩短,各剂量组与对照组比较有显著性差异,各剂量组间平均潜伏期比较无显著性差异,而在原平台象限停留时间高剂量组与低剂量组间有差异,P＜0.05。B[a]P可造成大鼠空间学习记忆功能的损害。这与人群调查发现的焦炉作业工人学习记忆能力降低相一致。
第三节亚慢性染毒大鼠海马的形态学改变和凋亡检测
大鼠染毒结束后,断头处死,取出海马。病理结果显示随着苯并[a]芘剂量的增高,有典型的神经细胞损伤现象出现,中、高剂量组海马神经细胞出现排列紊乱,数目减少,胞浆透明,核皱缩,细胞周围出现环状带,电镜结果显示,随着剂量的增高,出现细胞核皱缩,染色质边聚,核膜破裂等形态学的变化,线粒体形态逐渐出现肿胀,并可伴有线粒体嵴减少或消失,肿胀重者呈气球样变而且外膜破裂。海马神经细胞凋亡测定结果显示,随着B[a]P染毒剂量的增加,海马神经细胞凋亡指数增高,有剂量-反应关系。海马神经细胞凋亡可能是大鼠学习记忆功能降低的原因,线粒体形态改变提示,神经细胞凋亡可能与线粒体损害有关。
第四节亚慢性染毒大鼠海马神经生物化学的改变
大鼠染毒结束后,断头处死,取出海马。高效液相色谱法分别测定海马中谷氨酸(Glu)、天门冬氨酸(Asp)、甘氨酸(Cly)、γ-氨基丁酸(GABA)、多巴胺、5-羟色胺、5-羟吲哚乙酸含量,应用相应试剂盒测定海马SOD、MDA。结果显示,大鼠海马谷氨酸含量明显降低,各剂量组与对照组比较,p＜0.01,各剂量组间无显著性差异;而天门冬氨酸(Asp)、甘氨酸(Cly)、γ-氨基丁酸(GABA)含量各组未见明显变化。大鼠海马5-羟色胺(5-HT)含量明显增高,有剂量-反应关系,而多巴胺(DA)、5-羟吲哚乙酸(5-HIAA)含量各组未见明显变化。随苯并[a]芘剂量的增大,MDA含量增多,SOD活性下降。各剂量组与对照组比较,p＜0.05,各剂量组间比较,高剂量组与低剂量组比较有显著性差异,结果提示苯并[a]芘染毒可引起大鼠氨基酸递质、单胺类递质的改变,这可能大鼠学习记忆功能减低有关。大鼠海马随苯并[a]芘剂量的增大,MDA含量增多,SOD活性下降,显示海马发生脂质过氧化。
第五节亚慢性染毒大鼠海马CYP1A1基因和蛋白的表达
本次研究应用PT-PCR法研究CYP1A1mRNA表达,免疫组化SABC法和Western-blot检测了海马CYP1A1蛋白的表达。随苯并[a]芘剂量的增高,CYP1A1mRNA及蛋白表达增多,有剂量-反应关系,CYP1A1基因和蛋白表达与海马神经细胞凋亡指数的相关分析表明:海马神经细胞凋亡指数与CYP1A1mRNA表达呈正相关(r=0.871,P＜0.01);与Western-blot检测CYP1A1蛋白表达呈正相关(r=0.804,P＜0.01)。进而结合动物学习记忆功能、神经递质的改变,脂质过氧化水平的增高。我们推测CYP1A1表达增高可能是苯并[a]芘神经毒性作用的关键环节。这可部分解释人群CYP1A1基因多态性与焦炉作业工人学习记忆功能降低的关系。
第三部分苯并[a]芘神经毒性的体外研究
第一节苯并[a]芘致神经元形态改变和凋亡检测
选用新生1～3d的SD大鼠,取脑,分离大脑皮质进行神经元培养,加入0.5mg/ml阿糖胞苷(终浓度10μM)培养液以抑制胶质细胞增殖。经GFAP免疫组化证明,胶质细胞小于10%,在细胞培养第5天左右,选取生长良好的同批次神经细胞,以苯并[a]芘同时加或不加S9分别对神经元染毒,两种染毒方法都分为对照组、低剂量组、中剂量组、高剂量组,使苯并[a]芘终浓度分别为0μM、10μM、20μM,40μM。继续培养40h,观察细胞的生长状况和形态学改变。
结果表明,苯并[a]芘染毒后神经元树突数量减少,轴突减少变短,细胞与细胞间连接明显减少;加S9后,各剂量组比不加S9组细胞形态改变明显,两组相差一个剂量组,即低剂量加S9组形态改变基本相当于不加S9的中剂量组。高剂量加S9组形态改变最明显,部分神经元的大多数树突消失融解,细胞轴突也严重受损,胞体变圆、收缩。AO/EB染色结果表明苯并[a]芘可引起神经元轴突与树突的数量,细胞间和细胞连接减少。高剂量染毒组可见轴突与树突的数量进一步减少,细胞与突触间,胞体与胞体间连接明显减少甚至消失,胞体明显缩小,染色质聚集,细胞呈桔黄色;加S9后,各剂量组比不加S9组细胞形态改变明显,两组相差一个剂量组,即低剂量加S9组形态改变基本相当于不加S9的中剂量组。高剂量加S9组形态改变最明显,+S9高剂量组神经细胞胞核呈固缩状的桔红色荧光,轴突与树突的数量锐减,细胞与细胞间连接很少甚至消失,有核分裂边聚。本次应用Annexin V和PI双染法进行细胞凋亡的检测,结果发现,随着苯并[a]芘染毒剂量的加大,神经细胞的早期、晚期和总凋亡率逐渐增高,有剂量依赖性,未加S9的中、低剂量组早期和晚期凋亡率与对照组相比差异均无显著性意义(p＞0.05)。高剂量组与对照组相比差异有显著性意义,加入S9后,苯并[a]芘对神经细胞的损害作用明显加强,随着苯并[a]芘染毒剂量,神经细胞早期和晚期凋亡率明显升高,+S9的三个剂量组与对照组相比差异均有显著性意义,p＜0.05。细胞的凋亡率叠加示意图和直方图显示,神经细胞的早期、晚期和总凋亡率与染毒剂量呈明显的剂量依赖性。表明苯并[a]芘可引起神经元凋亡,而且加入肝S9后作用增强,代谢活化是苯并[a]芘引起凋亡的主要原因。
第二节苯并[a]芘染毒神经元的细胞活力改变和氧化损伤
应用Cell Counting Kit-8检测染毒后各组神经元的细胞活力,随着苯并[a]芘浓度的增加,神经细胞活力下降,未加S9组仅中、高剂量组与对照组比较有显著性差异,加S9组各剂量组与对照组比较均有显著性差异,而且各剂量组间有显著性差异,表现出较明显的剂量-反应关系。表明苯并[a]芘具有细胞毒性。染毒后的神经细胞内SOD和MDA测定表明,加S9组随着苯并[a]芘浓度的增加,神经细胞SOD活性下降,MDA含量升高,各剂量组与对照组比较有显著性差异,表明神经细胞发生脂质过氧化。加入罗丹明123(Rh123),应用流式细胞仪测定细胞线粒体膜电位,随着苯并[a]芘浓度的增加,细胞线粒体膜电位下降,未加S9组仅中、高剂量组与对照组比较有显著性差异,加S9组各剂量组与对照组比较均有显著性差异,而且各剂量组间有显著性差异,表现出较明显的剂量-反应关系,苯并[a]芘代谢活化引起脂质过氧化,进而线粒体膜电位的降低可能是凋亡的诱发途径。
第三节神经元CYP1A1蛋白及基因的表达与细胞损伤的关系
本次研究应用RT-PCR法对神经元CYP1A1mRNA表达,免疫组化SABC法和Western-blot检测了神经元CYP1A1蛋白的表达。结果随苯并[a]芘剂量的增高,CYP1A1mRNA及蛋白表达增多,有剂量-反应关系,CYP1A1基因和蛋白表达与神经元细胞凋亡率的相关分析表明:神经细胞凋亡率与CYP1A1mRNA表达呈正相关(r=0.831,P＜0.01);与CYP1A1蛋白表达呈正相关(r=0.780,P＜0.01)。结合所有+S9和—S9的检测结果,表明CYP1A1表达增高是细胞损伤的关键因素,这与整体动物试验的结果是一致的。
小结
1.职业性苯并[a]芘接触可引起明显的记忆功能降低和副交感神经调节功能降低。
2.苯并[a]芘引起的记忆功能降低与CYP1A1基因多态性有关。
3.体内和体外实验都证明苯并[a]芘诱导的CYP1A1表达增高是苯并[a]芘神经毒性的关键环节。
4.苯并[a]芘可引起大鼠学习记忆能力降低,可能与海马神经细胞凋亡,谷氨酸含量降低,5-羟色胺水平升高以及脂质过氧化有关。
5.苯并[a]芘可能通过线粒体途径诱发神经细胞凋亡。
Benzo(a)pyrene(B[a]P),one of polycyclic aromatic hydrocarbon(PAH)compounds,is product of incomplete combustion of organic matters and is widespread in the environment.The carcinogenicity of B[a]P is well established in human and animal model.Due to the widespread distribution of B[a]P in the environment and the potential exposure for human,the adverse effects of these compounds deserved to be investigated.Although the neurotoxic effect of B[a]P has not drawn much attention,.there were some related data implying that B[a]P showed the neurotoxic effects.when exposed to B[a]P by oral gavage,rats were induced behavioral impairments,and there was a significant correlation between neurotoxic effects and plasma B[a]P concentration,and its metabolite concentration in brain tissue.Coke-production plant workers in Poland developed neurotic syndromes with vegetative disregulation,and a loss of short-term memory;their prevalence depended on the level of exposure to B[a]P.B[a]P is metabolised to active metabolites that are tumourigenic.The bioactivation of B[a]P is catalyzed by some cytochrome P450(CYP)enzymes,particularly CYP 1A1.The brain tissues of various species contain almost all CYP-enzymes found in liver,including CYP1A1.CYP-enzymes in brain were associated with neurotoxic effects of some chemicals,such as lindane and manganes.The correlation between the neurotoxicity of B[a]P and B[a]P metabolite concentrations in brain implied that CYP1A1 may mediate the neurotoxicity of B[a]P.CYP1A1 gene polymorphisms have been studied in relation to various kinds of cancers.Our hypothesis is that CYP1A1 gene polymorphisms wound modulates the neurotoxicity of B[a]P.The neurotoxicity of B[a]P may be a important occupational risk like its carcinogenicity for occupational exposed populations.In this research,occupational exposed population,experimental animals and primarily cultured neuron exposed to B[a]P were investigated to preliminarily elucidate the mechanism of neurotoxicity of B[a]P and its association with CYP1A1 gene polymorphisms.The research was divided into three parts.
PartⅠNeurotoxicity study on occupational population exposed to B[a]P
1.neurobehavioral function of coke oven wokers
200 healthy adult male coke oven workers were selected from coke plant of a state-owned enterprise in Taiyuan City.88 controls unexposed to polycyclic aromatic hydrocarbons(PAHs) occupationally were selected from the same enterprise.All the subjects participated this investigation freely according to their consent.Concentration of B[a]P in the working environment was monitored by High Performance Liquid Chromatography(HPLC).Urine samples were collected immediately after working shift.The level of urinary 1-hydroxypyrene was determined by High Performance Liquid Chromatography(HPLC).General informations of workers correlated to the investigation were collected by query and filled in the questionnaire according to the same criteria by well-trained investigators.WHO recommended neurobehavioral core test battery(NCTB)was performed on coke oven workers and controls to test the neurobehavioral changes and the mood state.The concentration of B[a]P at oven bottom,oven side and oven top were 0.0195μg/m3,,0.186μg/m3 and 1.624μg/m3 respectively,and Concentration of B[a]P at oven side and oven top were higher than the occupational hygiene criterion.Urinary 1-hydroxypyrene were significant difference between exposure group(3.42±0.98μmol/mol creatinine)and control group(2.75±1.09μmol/mol creatinine).No significant differences were found between exposure group and control group on age,length of service,smoking,drinking and unhealthy food consumption;however,compared to the controls,the scores of the coke oven workers were lower in the total digital span,the forward digital span,right dotting and total dotting,and the differences showed statistical significance(P＜0.05).Significant difference of the total digital span,the forward digital span, backward digital span,digit symbol and Benton visual retentions existed in different urinary 1-hydroxypyrene concentration groups and showed a dose-response tendency.Results of multiple stepwise regression analysis and correlation analysis showed that the level of urinary 1-hydroxypyrene affect memory and perception of coke oven workers and there wre negative correlations between the levels of urinary 1-hydroxypyrene and changes in neurobehavioral function.It was suggested that B[a]P wound mainly causes decrease of memory and perception in coke oven workers..
2.Function of autonomic nervous system(ANS)in coke oven wokers
Function of autonomic nervous system(ANS)of 288 subjiects were measured with the method recommended by Ewing DJ,which consists of 4 tests:Valaslva Manoeuvre heart rate variation(HR-V),variation of heart rate when deeply breathing(HR-DB),variation of heart rate when instantly standing up(HR-IS,including RR30:15 and RRmax:min)and variation of blood pressure when instantly standing up(BP-IS).Compared to controls,HR-V decreased significantly in coke oven workers(p＜0.01),which represents modulation of parasympathetic nervous function.But no statistical differences were found in HR-DB,RR30:15,RRmax:min and BP-IS between exposed groups and control group(p＞0.05).Significant difference of HR-V existed in different urinary 1-hydroxypyrene concentration groups.Results of multiple linear stepwise regression demonstrated that the level of urinary 1-hydroxypyrene and level of education affect HR-V.The results indicated that B[a]P affected autonomic nervous function of coke oven workers by mainly down- regulating parasympathetic nervous function.
3.Changes of electroencephalogram and brain electrical activity mapping in coke oven wokers
45 subjects,as a exposed group,were randomly selected from exposed groups according to their type of work in production and age,25 controls were chosen from unexposed group according to the same principal.All subjects were conducted electroencephalogram and brain electrical activity mapping.Compare with controls,the ratio of abnormal EEG increased,brain electrical activity mapping showed all frequency bands energy values were lowered,andαfrequency band distribution was diffuse,sometimes dissymmetrical.The results displayed B[a]P increased ratio of abnormal EEG.
4.Changes of conduction velocity of peripheral nerve and evoked potential in coke oven wokers
The conduction velocity of peripheral nerve and evoked potential were monitored by neuropack M1,(Nihon Kohden coporation),compared with controls,there is no significant difference in sensory conduction velocity and motorial conduction velocity of ulnar nerve and median nerve.The sensory evoked potencial,auditory evoked potencial and pattern-visual evoked potencial in two groups also showed no significant diffrence.It was suggested that B[a]P shoud have no effect on conduction velocity of peripheral nerve and evoked potential.
5.Analysis of CYP1A1 gene polymorphisms in coke oven wokers
CYP1A1 gene polymorphisms including MspⅠsite and Ile-Val site were monitored by PCR-RFLP and ASA-PCR,respectively.The results revealed that there were no statistic difference of the genotype frequencies of m1/m1、m1/m2、m2/m2 of MspⅠsite and that of Iie/Iie、Ile/Val、Val/Val of Ile-Val site between exposed group and contol group,In exposed group the level of urinary 1-hydroxypyrene of Val/Val was significantly higher than Ile/Ile wild homozygote.Although there was a tendency of m1/m1＜m1/m2＜m2/m2 in urinary 1-hydroxypyrene,there was no statistically significant difference.The scores of Dignity Span Backward with Val/Val gene type were higher than those with Iie/Iie gene type.Other index of NTCB and ANS showed no difference among three gene types of Ile-Val site.All index of NTCB and ANS showed no difference among three gene type of MspⅠsite.The present data revealed CYP1A1 Ile-Val site gene polymorphism modulats the neurobehavioral toxicity of B[a]P.
PARTⅡstudy ofneurotoxicity of B[a]P in vivo
1.single intracerebroventricular injection of benzo[a]pyrene induces morphological changes in rats
Forty male SD rats,adult and healthy,were divided into 5 groups randomly:normal control, DMSO control,B[a]P 0.5mmol/L,B[a]P 5mmol/L,B[a]P50mmol/L,all groups were administered by intracerebroventricular injection,and the injectant volume was 10μl.The morphology of neural cells in rats' hippocampus and cerebellum were observed by optical microscope.In cerebellum and hippocampus,arrangement and numbers of the neural cells were normal in control group,but with the increasing of the doses of B[a]P exposed,the arrangement of neural cells became disorderly,the quantity of cells became diminished,and the cells showed necrosis appearance.The results displayed that benzo[a]pyrene damaged brain tissues of rats,and the higher the dose of B[a]P is,the more serious the damage of brain is.
2.Subchronical intracerebroventricular injection of benzo[a]pyrene decreased the capacity of learning and memory in rats
Forty male SD rats,adult and healthy,were intubated,after one week,all incubated rats were divided into 4 groups randomly:normal control,olive oil control,B[a]P2.5mmol/L,B[a]P 5mmol/L,and B[a]P10mmol/L,all groups were administered by intracerebroventricular injection, and the injectant volume was 10μl.Step-down and Morris water maze tests were applied to assess the capacity of learning and memory in rats.With the dose of B[a]P increasing,the step down latency was shortern,and times of stepping down increased,there existed significant differences in three B[a]P -treated groups compared with controls.The results indicated that B[a]P can induce impairment of passive avoidance learning and short-term memory.In Morris water maze tests the mean escape latency was prolonged and the length of staying in the original quadrant was shorterned in treated groups while dose was increasing.There was an evident dose -response relationship between the dose of B[a]P and the data of Morris water maze tests,which suggested that B[a]P would affecte the spatial learning and memory functions.
3.Subchronicai intracerebroventricular injection of benzo[a]pyrene induces hippocampus morphological changes and apoptosis
The observation under light microcope showed:arrangement of the neural cells in rats' hippocampus was orderly in control group,but with the increasing of the doses of B[a]P exposed,the cells displayed disorderly arranged,the connections among the neural cells were loosened,the karyon shrinked,some rings around the cells were observed.Picture under electron microscope displayed:in control group,the karyotin distributed symmetrically,and its form was regular,the form of mitochondria was regular,the arrangement of bars was orderly,and with the increasing of the doses of B[a]P exposed,the karyon shrinked,the karyotin assembled in the edge,karyotheca was broken,mitochondria swelled,broken,the bars were reduced and disappeared.
With the increasing of the dose of B[a]P exposed,the apoptosis indices(AI)of neural cells in rats' hippocampus increased,and AI in 5mmol/L B[a]P group and 10mmol/L B[a]P group were significantly higher than that of controls(P＜0.05),and which in B[a]P 10mmol/L group were significantly higher than that of 2.5mmol/L B[a]P group.
4.Subchronicai intracerebroventricular injection of benzo[a]pyrene induces neurochemical changes of hippocampus in rats
To explore the mechanism of B[a]P induced neurotoxicity,amino acid transmitters, monoamine transmitters,SOD and MDA were determined.Compared with controls,GLU was decreased in three treated groups,and other amino acid transmitters showed no changes,while there was an increasing trend of 5-HT in treated groups,no differences of DA and 5-HIAA were found between treated groups and contol group.SOD activity decreased and content of MDA increased in three treated group compared with the conrols.All these data implied the potential mechanism of neurotoxicity of B[a]P.
5.Subchronical intracerebroventricular injection of benzo[a]pyrene induces CYP1A1 expression changes of hippocampus in rats
To explore the relationship between neurotoxicity of B[a]P and CYP1A1 expression, RT-PCR was used to determine CYP1A1 mPNA,and immunohistochemical staining and western bloting were performed to determine CYP1A1 protein.CYP1A1 mRNA and CYP1A1 protein increased significantly compared with the control group and in a dose dependent manner, a positive correlation between AI and the expression of CYP1A1mRNA,the expression of CYP1A1 protein were observed.Considering the data mentioned above and the capacity of learning and memory in rats and neurochemical changes prehensively,CYP1A1 expression may be thought to be the critical step in neurotoxicity of B[a]P.
PartⅢstudy of neurotoxicity of B[a]P in vitro
1.Changes of cell morphology and apoptosis of primary cultured neurons induced by B[a]P
primary neurons were dissociated from cerebral cortex of 1-3 days old SD rat and cultured with DMEM incubator at 37℃,cell density was adjusted at 1×10~5cells/ml,after 5 days' cultivation,the astrocyte GFAP was identified by immunohistochemical staining.If the astrocyte was less than 10%,the cultured cells was thought to be pure neurons.Then,they were divided into two groups,+S9 and -S9,and all the two groups of neurons were cultivated with B[a]P at 0,10,20,40μmol/L,respectively,for 40 hours.The morphology was observed,and apoptosis was measured.Under light microscope,with the increment of B[a]P dose,the numbers of neurites and dendrites of neurons and the conjunctions among cells were reduced evidently.The changes were more serious at 40μmol/L in +S9 group.The neurites of some neurons became shorter,even disappeared,and the cell body became round.Stained with AO/EB,under fluorescence light,live neuron showed a bright green color,while the dead one was red,as apoptotic cell was orange.Based on the color of neuron,we can distinguish living conditions of the neurons.With B[a]P dose increasing,the ratios of orange colored cells increased,and the numbers of neurites and dendrites of neurons and the conjunctions among cells decreased,+S9 group showed more severity,at 40μmol/L,the cell body became round and anumber of neurons were stained orange even red,and the neurite of some neuron became shorter,even disappear,the karyon shrinked or aggregated in the edge or cleaved.With AnnexinⅤand PI staining,the apoptosis ratios of neurons were measured,early-onset apoptosis ratio,late -onset apoptosis ratio and total apoptosis ratio increased in a dose dependent manner,and +S9 group showed more serious situation.The data suggested that B[a]P could induce apoptosis of neurons,and bioactivation of B[a]P was important to exert its adverse effects.
2.Changes of cell viability and the mitochondrial membrane potential(MMP)and lipid peroxidation induced by B[a]P
Neuron vilability decreased in—S9 group at dose of 20μmol/L compared with controls,in +S9 group there existed significant differences in three treated grous.The determination of MMP in neuron showed that MMP decreased in—S9 group at dose of 20μmol/L compared with controls,while in+S9 group,the MMP decresed in a dose dependent manner.The SOD activity decreased and MDA increased in treated group compared with controls.These results suggested that liquid peroxidation induced by BaP could lead to MMP changes,which may initiate neuron apoptosis and cell viability decline.
3.CYP1A1 expression induced by B[a]P in neuron
With the dose of B[a]P increasing,CYP1A1 mRNA and CYP1A1 protein increased,there existed significantly difference in treated groups compared with control group.A positive correlation between neuron apoptosis ratio and the expression of CYP1A1mRNA,as well as the expression of CYP1A1 protein were observed.The results indicated that CYP1A1 expression is related to cytotoxicity of B[a]P.
summary
1.B[a]P-related neurotoxicity in B[a]P exposed wokers are characterized by poor memory and down regulated parasympathetic nervous function.
2.The neurobehavioral effect of B[a]P is related to CYP1A1 gene polymorphism.
3.It had been proved that CYP1A1 expression induce by B[a]P may be the critical step in neurotoxicity of B[a]Rin vivo and in vitro.
4.The impairment of learning and memory by B[a]P was also found in rats,which may be related to neural cell apoptosis,variations of GLUand 5-HT and statue of lipid peroxidation in hippocampus.
5.B[a]P may induce neural cell apoptosis through mitochodria pathway.
引文
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