邻苯二甲酸二丁酯和苯并[a]芘对大鼠精子的联合毒性研究
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摘要
持久性有机污染物(Persistent Organic Pollutants),简称POPs,指的是持久存在与环境中,具有很长半衰期,并且能通过食物链积聚,并对人类健康及环境造成不利影响的有机化学物质。POPs对人类和环境带来的危害已成为全球性环境问题。由于目前世界上POPs物质有几千种,在有限的财力、物力、人力条件下,很难全面进行研究。因此,选择本地区需要优先监测和关注的环境及人群体内的优先POPs(priority POPs)进行研究,对于高效率的环境监测、污染治理以及健康防护具有非常重要的意义。
本课题组前期研究发现三峡库区水环境中的污染物以多环芳烃类物质和邻苯二甲酸酯类的检出频率和检出浓度最高。本实验拟以含量较高的邻苯二甲酸二丁酯(DBP)做为邻苯二甲酸酯类物质代表,以毒性较强的苯并[a]芘(Bap)做为多环芳烃类物质代表,对这两类物质联合毒性进行研究。有文献报道,DBP、Bap均具有确切的雄性生殖毒性,但两者低剂量联合作用下雄性生殖毒性特别是针对生精细胞的毒性及机制研究尚未见报道。本研究注重突出联合亚慢性毒性,以DBP与Bap低剂量联合作用于雄性大鼠,从毒性效应和机制两个层次开展研究,观察其联合毒性作用及机制,可为客观评价及预测POPs污染对人群健康的损害、建立环境健康效应的预警体系打下很好的基础。
试验方法
1.实验动物及分组情况
4-5周龄健康雄性SPF级SD大鼠245只,由第三军医大学实验动物中心提供,按随机数字法随机分为7组(每组35只),分别为对照组(玉米油)、Bap低剂量组(Bap 1 mg/kg)、Bap高剂量组(Bap 5 mg/kg)、DBP低剂量组(DBP 50 mg/kg)、DBP高剂量组(DBP 250 mg/kg)、DBP+ Bap低剂量组(DBP 50 mg/kg +Bap 1 mg/kg)、DBP+ Bap高剂量组(DBP 250 mg/kg +Bap 5 mg/kg)。
2.染毒及取材
采取隔天灌胃的方式进行染毒,连续染毒90d。30d、60d、90d时每组各处死8只,处死前称体重,10%水合氯醛腹腔麻醉,采集心脏血后,立即处死剖取睾丸、附睾、心、肝、脾、肾,称重后将睾丸于冰上迅速切分为约50 mg- 100 mg的小块,分装于冻存管中液氮保存。血液离心后取血清于-70℃保存,用于测定激素。每组留4只90d后与健康雌性SPF级SD大鼠交配,观察生育能力。
每组随机取1只大鼠的左侧睾丸,冰上迅速取材后固定于2.5%戊二醛,然后梯度酒精脱水、环氧树脂渗透、包埋、切片,醋酸铀和柠檬酸铅染色,透射电镜下观察。
随机选取5只大鼠的右侧睾丸,4%多聚甲醛固定液中固定,常规石蜡包埋切片,HE染色,光镜下观察,拍照,通过计算机图像分析软件(DP72-BSW)测定生精小管和睾丸面积,计算生精小管平均横截面积和生精小管与睾丸面积比。
随机选取8只大鼠单侧附睾,置于0.09%生理盐水内洗去血水,将洗净的附睾置于生理盐水内用眼科剪剪碎,轻压组织碎块,静置2 min- 3 min释放出精子,显微镜下观察精子活性和畸形、计数精子数。
随机选取6只大鼠单侧睾丸(90d时相点对照组和高剂量染毒组(Bap 5组、DBP 250组、B5+ D250组)各多选1只用于蛋白表达分析)用组合酶法分离生精细胞。
生精细胞分化情况分析:将生精细胞悬液用PBS洗2次,加1 mg/ ml RNA酶37℃孵育30 min,加入碘化丙碇4℃染色30 min,通过流式细胞仪进行相关分析(分析软件:Cellquest)。
生精细胞蛋白表达分析:提取生精细胞悬液总蛋白进行等电聚焦和SDS-PAGE,使用软件ImageMaster 2D Platinum 5.0分析各组蛋白表达差异;切下具有表达差异的蛋白点使用MALDI-TOF/TOF质谱仪(Bruker Ultraflex)进行质谱分析(N2激光器(发射波长337 nm),加速电压25 KV),使用软件moverz和peakErazor分析质谱图,通过用mascot收索引擎检索鉴定蛋白。
3.统计方法
使用SPSS软件进行统计,交配实验结果使用卡方检验,其他结果使用单因素方差分析。
结果与讨论
1.邻苯二甲酸二丁酯和苯并[a]芘对大鼠生长发育的影响
各染毒组与对照组的大鼠体重变化各组之间无显著性差异,说明本实验两种化合物的染毒剂量及染毒时间对大鼠的消化吸收与能量代谢无明显影响,不会对大鼠生长发育没有造成过度的压力而影响实验结果。
各组大鼠的心脏系数、脾脏系数、肾脏系数和睾丸脏器系数各组间无显著性差异(P>0.05),可认为本实验两种化合物的染毒剂量及染毒时间不会对大鼠的心脏、脾脏、肾脏和睾丸产生明显毒性作用。
肝脏器系数在染毒30d时低剂量联合组出现明显降低(与其它各组比均有显著性,P<0.05),在染毒60d和90d时差异消失(P>0.05)。提示肝脏不仅是Bap和DBP的代谢器官,也是其靶器官,低剂量联合染毒可导致肝脏急性损伤,引起脏器系数的降低,这种肝脏毒性可能为短期效应,长期染毒时机体逐渐对其耐受。
染毒90d两者低剂量联合染毒组与对照组相比附睾脏器系数显著性下降(P<0.05),可能由于两者低剂量联合染毒对附睾的损伤逐渐超过机体的耐受能力,造成附睾萎缩。
2.邻苯二甲酸二丁酯和苯并[a]芘睾丸形态学影响
2.1电镜观察
与对照组相比,各染毒组电镜下均可见不同程度的细胞超微结构损伤,主要表现为生精细胞线粒体肿胀、固缩,内质网扩张。
Bap低剂量单独染毒组(Bap1组)前期损伤较小,染毒90d时出现染色质聚集和脂滴。DBP低剂量单独染毒组(DBP50组)在染毒初期和中期超微结构病理改变比较重,90d时开始恢复。Bap和DBP低剂量联合染毒组(B1+ D50组)从超微结构观察整个染毒过程中损伤较小,在60d时有内质网和线粒体肿胀形成髓鞘样结构以及染色质聚集。
Bap高剂量单独染毒组(Bap5组)在整个染毒过程中受影响较大,超微结构观察染毒30d时可见细胞严重损伤,结构破坏,层次不清;60d可见细胞水肿,间隙增大;90d时内质网扩张、线粒体水肿、出现大量脂滴。DBP高剂量单独染毒组(DBP250组)在30d和90d时未出现严重的超微结构病理改变,60d时可见细胞结构破坏、层次不清。Bap和DBP高剂量联合染毒组(B5+ D250组)与DBP类似,30d和90d时超微结构病理改变较小,存在部分线粒体固缩,60d时可见生精细胞从基膜脱落。
2.2光镜观察
光镜观察发现不论是Bap和DBP的单独染毒还是联合染毒,都存在一定层度的生精细胞层数减少、空泡化、生精细胞间隙增大以及生精细胞脱落,有的还能观察到间质细胞增生。与Bap相比,DBP对睾丸组织形态学影响出现的要早,在染毒30d时候就出现生精细胞间隙增大、层数减少;联合染毒组的表现主要在于高剂量联合染毒60d时可见间质细胞增生,而生精细胞本身变化在形态学上并不明显。
2.3计算机图像分析
我们运用计算机图像分析软件在光镜下进行分析,发现:
曲细精管横截面积比较可见各染毒组曲细精管均有不同程度的萎缩。Bap和DBP低剂量联合染毒组出现最早,30d就显著性差异(与对照组比,P<0.05),60d时差异不明显(与对照组比,P>0.05),90d时又出现显著性差异(与对照组比,P<0.05),体现了一个损伤→代偿→失代偿的过程;其次是DBP低剂量单独染毒组,在第60d开始出现曲细精管明显萎缩,与对照组比有显著性差异(P<0.05),90d是差异持续存在,是一个逐渐损伤的过程;其他4组(Bap低剂量单独染毒组、Bap高剂量单独染毒组、DBP高剂量单独染毒组、两者高剂量联合染毒组)均在30d、60d与对照比差异不明显(P>0.05),90d时与对照组比有显著性差异(P<0.05)。与其电镜、光镜观察结果相比较,可以看出一个由超微结构→细胞→组织的损伤或修复的时间顺序。
曲细精管占睾丸体积百分比在60d、90d时各染毒组与对照相比无显著差异(P>0.05),在30d时DBP单独染毒组(DBP50组、DBP250组)与其他各组相比均显著升高(P<0.05)。通过与曲细精管横截面积变化结果相比较,我们推测30d时DBP单独染毒组其曲细精管占睾丸体积百分比增加可能是由于其间质组织萎缩引起,而90d时各染毒组可能都出现了不同程度间质组织萎缩。
3.邻苯二甲酸二丁酯和苯并[a]芘对大鼠生殖能力的影响
3.1精子数量和质量分析
临床评价男性生育能力的三个常规指标精子计数、成活率以及活性,染毒90d时各组间无显著性差异(P>0.05),提示我们的染毒剂量外推到人群时,该剂量下临床生育能力检查可能不会发现明显改变,不易引起重视。
精子计数结果与90d时附睾脏器系数比较,我们认为两者低剂量联合染毒组90d时与对照组相比附睾脏器系数显著性下降(P<0.05),可能不是因为精子数下降而是由于附睾内附睾管上皮细胞减少,这些细胞参与构成附睾内精子成熟、精子运动能力和受精能力的获得和发育所必需的微环境,它们的损伤将会影响精子成熟以及精子运动能力和受精能力的获得和发育,此外作为附睾功能重要调控因素的睾酮在血清中浓度下降将会加剧这种影响。根据文献报道,进入附睾的精子在结构上已经趋于完整,因此这种影响在引起精子畸形方面主要表现为断头畸形的增加,这与我们的精子畸形观察结果一致,我们在试验中发现Bap和DBP低剂量联合暴露引起的精子畸形增加主要表现为断头畸形。
DBP高剂量染毒组在染毒90d时畸形精子的数量明显增加(与对照组比P<0.05),低剂量联合组在染毒90d精子畸形率增加更显著,高于对照组、单独剂量组以及联合高剂量组(P<0.05),结合附睾脏器系数的改变,我们认为在低剂量时,两者联合后在生殖毒性上显示出加强效应。
3.2生殖相关激素的改变
低剂量联合染毒组血清T水平与对照组、Bap低剂量组相比在30d、90d显著下降(P<0.05),而60d时无显著差异(P>0.05),呈现出一个典型的损伤→代偿→失代偿的过程。
血清LH水平变化与我们血清T水平的相反,低剂量联合染毒组90d时与对照组相比显著升高(P<0.05),推论本试验所用的染毒剂量长期单独或联合暴露染毒对青春期SD大鼠血清LH水平没有直接影响,其LH水平改变可能来源于血清T水平改变引起的负反馈调节,其对睾酮浓度的影响不是通过LH浓度改变,而是在LH之后的途径,例如抑制间质细胞功能、增强肝脏对睾酮代谢等。
3.3对大鼠繁殖能力影响的分析
Bap高剂量染毒和两者高剂量联合染毒会造成死胎和吸收胎出现率显著高于对照和DBP高剂量单独染毒(P<0.05)。因此我们认为虽然Bap高剂量单独或两者高剂量联合长期暴露后虽然在一般生长发育指标、生育能力常规指标(精子计数、精子存活率、精子活性、精子畸形)及激素水平上与对照相比均没有明显改变,但是其生育能力已经受到影响。
4.邻苯二甲酸二丁酯和苯并[a]芘对生精细胞分化和蛋白表达的影响
4.1生精细胞分化改变
我们发现Bap和DBP低剂量联合染毒在精子发生上表现出早期兴奋(30d、60d)而后期逐渐失代偿并出现抑制(90d)的效应,主要集中在减数分裂阶段;单独低剂量染毒也早期有类似效应,但出现兴奋的时间要晚于联合染毒(60d),晚期机体逐渐对其耐受,兴奋效应消失(90d),从而在联合效应上出现协同→拮抗→协同的变化。高剂量组Bap单独染毒对精子发生过程始终没有显著影响,DBP高剂量单独染毒和低剂量单独染毒类似,从中期(60d)开始出现兴奋效应;两者高剂量联合在中期开始表现出以DBP效应为主,Bap对其拮抗。
4.2生精细胞蛋白表达改变分析
通过比较各组二维电泳图,我们共得到35个表达差异蛋白点,我们将这35个点通过蛋白质谱鉴定,有5个点未得到结果(无峰),剩下30个点中有11个为阴性结果(分值小于60),最后得到19个有阳性意义的蛋白,对比这些蛋白在各组间的差异,我们发现:
与精子发生密切相关的核内不均一核糖核蛋白在高剂量联合染毒组表达与Bap高剂量单独染毒组相比增强;在精母细胞的分化、精子在附睾内的成熟、精子获能和顶体反应过程中起重要作用的肿瘤拒绝抗原gp96在高剂量联合染毒组中比Bap高剂量单独染毒组表达降低;参与精原细胞有丝分裂和减数分裂并在其中其重要作用的β肌动蛋白在高剂量联合染毒组中比Bap高剂量单独染毒组表达降低。这几种蛋白表达差异可能是造成染毒90d时高剂量联合染毒组中与Bap高剂量单独染毒组相比在光镜、电镜照片中形态学损伤小而在流式细胞计数中却出现明显的精子发生过程受影响、减数分裂受阻的原因。
参与染色质组装或分解、转录、调控转录以及DNA依赖、负调控转录、染色质修饰,与DNA结合、染色质结合、转录抑制因子相关的色素框同源物2在Bap高剂量单独染毒组表达消失(与高剂量联合染毒组、对照组比均有差异),这可能与Bap高剂量生精上皮结构破坏有关。
与减数分裂、精子尾部鞭毛装配、维持细胞核形态等密切相关β-微管蛋白在Bap高剂量单独染毒组中特异性表达(与对照组相比),推测是Bap高剂量单独染毒引起生精上皮结构破坏导致β-微管蛋白过度表达;白蛋白在Bap高剂量单独染毒时候在生精细胞中表达增加(与对照组、高剂量联合染毒组相比),可能是由于Bap高剂量单独染毒引起生精上皮结构破坏,血睾屏障被破坏而致使白蛋白渗漏进生精上皮内部引起。
与铁转运相关的血红素结合蛋白在各染毒组生精细胞中表达与对照组相比均上升,Bap高剂量单独染毒组与对照组相比还存在转铁蛋白表达上升,提示Bap和DBP长期染毒引起睾丸损伤作用与铁有关。
高剂量联合染毒与Bap高剂量单独染毒相比,与线粒体的能动性相关的多毛蛋白表达消失,提示高剂量联合染毒生精细胞内线粒体活动性比Bap高剂量单独染毒要强,这可能是我们在电镜照片下发现90d时高剂量联合染毒组线粒体损伤远低于Bap高剂量单独染毒组的原因之一。
广泛参与体内脂肪族及芳香族醛类代谢的线粒体乙醛脱氢酶在各染毒组生精细胞中均出现线粒体乙醛脱氢酶特异性表达(与对照组相比),推测其可能参与睾丸对DBP和Bap及其代谢产物的生物转化。
高剂量联合染毒组与DBP高剂量单独染毒组相比糖代谢途径限速酶之一的丙酮酸激酶3的2亚型表达消失,提示两者能量代谢系统存在差异。
结论
1. DBP和Bap染毒对大鼠生育能力影响
DBP和Bap长期低剂量联合染毒可引起血清T水平显著下降,同时还损伤附睾内微环境导致精子断头畸形增加。Bap高剂量单独或Bap和DBP高剂量联合长期暴露不会影响一般生长发育指标、生育能力常规指标及激素水平,但能够影响雄性大鼠的生殖能力。
睾丸形态学改变:①Bap低剂量单独染毒前期损伤较小,后期出现损伤;DBP低剂量单独染毒在初期损伤较大,后期开始恢复;②Bap和DBP低剂量联合染毒全程损伤较小,呈现拮抗效应,两者高剂量联合染毒在形态学方面表现以DBP表现为主;②各染毒组曲细精管均有不同程度的萎缩。
2. DBP和Bap染毒对大鼠生精细胞分化及蛋白表达的影响
DBP和Bap低剂量联合染毒主要影响精子减数分裂阶段,表现为早期兴奋而后期逐渐失代偿并抑制;单独低剂量染毒早期有类似效应,但出现兴奋的时间要晚于联合染毒,晚期兴奋效应消失,从而在联合效应上出现协同效应→拮抗效应→协同效应的变化。高剂量组Bap单独染毒对精子发生没有显著影响,DBP高剂量单独染毒和低剂量单独染毒类似,从中期开始出现兴奋效应;两者高剂量联合在中期开始表现出以DBP效应为主,Bap对其拮抗。
二维电泳和质谱分析表明各染毒组与对照组相比均出现血红素结合蛋白表达上升,线粒体乙醛脱氢酶特异性表达;Bap高剂量单独染毒组与对照组相比色素框同源物2表达消失,β-tubulin特异性表达,白蛋白、转铁蛋白表达上升;高剂量联合染毒组与DBP高剂量单独染毒组相比丙酮酸激酶3的2亚型表达消失,与Bap高剂量单独染毒组相比核内不均一核糖核蛋白增强,肿瘤拒绝抗原gp96、β-actin、白蛋白表达降低,多毛蛋白表达消失。
POPs, short for Persistent Organic Pollutants, refers to the organic chemicals that could persistently exist in the environment, have a long half-life, accumulate through the food chain, and negatives affects human health and the environment. The harm POPs has on human beings and the environment has become a global environmental problem. As there are thousands of POPs substances worldwide, it is difficult to conduct a comprehensive study due to limited financial, material, and human resources. Therefore, it is important to choose to study the kind of POPs that needs concerns and monitoring, as well as people's priority POPs. This is vital for a high efficiency in environmental monitoring, pollution control and health protection.
Our previous studies show that polycyclic aromatic hydrocarbon (PAHs) and Phthalic Acid Esters (PAEs) are the most popular pollutants in the Three Gorges area. This experiment intended to study and use the DBP as a representative for PAEs, the Bap as a representative for PAHs. Previous studies have reported that both DBP and Bap have certain male reproductive toxicity, but the male reproductive toxicity under the exposure to Combination of Bap and DBP, especially for the toxicity of spermatogenic cell and its mechanism. In the present study, we test the single or combined reproductive toxicity of DBP and Bap on male rats, observe the toxic affection and discusses the effect of the combination of two toxic substances from the toxic effects level and mechanisms level. This will lay a solid foundation for objectively evaluating and predicting the possible damage POPs pollution will do to human beings and establishing an early-warning system for environmental health effects.
Methods
1. A total 245 male Sprague- Dawley rats (4-5 weeks old) were equally randomized into 7 groups (24 per group) and respectively received by gavage corn oil, 1 mg/ kg of Bap, 5 mg/ kg of Bap, 50mg/ kg of DBP, 250mg/ kg of DBP, 1 mg/ kg of Bap+ 50mg/ kg of DBP, 5 mg/ kg of Bap+ 250mg/ kg of DBP alt. dieb. For 90 days. At the 30, 60 and 90 days after gavage, 8 rats per group were sacrificed randomly. 4 rats per group were left for reproduction experiments.
2. treatment and methords
The testes, epididymis, liver and other visceral organs were harvested, weighed and the organ coefficients were counted. Blood samples were collected to assess the effect of Bap and DBP on plasma testosterone (T) and luteinizing hormone (LH) concentrations with Chemiluminescence immumo- assay. Cauda epididymides were isolated for the determination of progressive motility and density of stored spermatozoa.
Testes were prepared for histologic and morphometric analysis. Stained testis sections were evaluated using an OLYMPUS Photomicroscope equipped which coupled with a computerized morphometric planimetry system (DP72- BSW) to facilitate the measurement of tubule area and area percentages (volume percentages) occupied by seminiferous tubules.
The testes were surgically removed and germ cells were isolated. Flow cytometry (FCM) was used to detect changes of cell cycle and Two- dimensional electrophoresis (2-DE) was used to separate the proteins. The proteins which has different expression were analyzed using the Image master 5.0 and were identified by mass spectrometric analysis.
Results and Discussion
1. The effect of di-n-butyl phthalate and benzo(a)pyrene on rat growth
The exposure concentration of Bap and DBP used in this study did not affect weekly weight gains in exposed versus UNC rats (P>0.05). These data suggest that inhaled BaP, at the exposure concentration administered, the daily duration of exposures and the manner in which animals were restrained, did not impose undue stress on the rats, hence the identical weight gains between exposed and UNC rats.
The organ coefficients of heart, spleen, kidney and testis also had no changes between exposed versus UNC rats (P>0.05), which means the exposing dosage didn’t trigger a significant toxical effect in exposed rats.
Compared with other groups, the co- exposure to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 30 days could reduce liver coefficient (P<0.05), and the difference disappeared at 60 days and 90 days. It suggested that the liver is not only the organ where the Bap and DBP metabolize, but also the target organ of Bap and DBP. And the injuries to liver by Bap and DBP may be acute and provisional. The body may tolerated to them gradually.
The epididymis might be sensitive to the toxic effect of Bap and DBP. In our study, the epididymis coefficient were significant decreased at the co-exposure to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days compared with the UNC rats. Suggested that the long time co- exposure to Bap and DBP might have the potential toxic effext to epididymis and might cause the atrophy of it.
2. The effect of di-n-butyl phthalate and benzo(a)pyrene on rat fertility.
In our study, the stored sperm density, sperm viability and movement parameters, which often be used in semen analyses to appraise the male reproductive ability, were not changed both in mono- and co- exposure to B[a]p and DBP. Showed that these indicator may not sensitive to Bap and DBP exposure and routine clinical check to the male reproductive ability by semen analyses may not identify problems in the people exposed to Bap and DBP.
Spermatozoa with abnormal morphologies were disproportionately represented in samples of stored spermatozoa from exposed to DBP 250 mg/kg/d and co- exposed 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days to rats than their control counterparts (P<0.05).
Compared with the significant decreased of epididymis coefficient at co-exposure to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days. We thought that the decreased of epididymis coefficient may not arise from the decreased of sperm density, but from the regression in the epididymal epithelium, which construct the microenvironment in the epididymis for further sperm maturation and consequently. And the change in circulating testosterone concentrations may sharp the effect because the level of testosterone reaching the epididymides from general circulation and testicular fluid may not be sufficient to optimally regulate the epididymides. The increased percentage of decapitated spermatozoa in the cauda epididymides of co- exposed to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days compared with UNC could be likened to the degenerating conditions of stored spermatozoa due to damaged microenvironment in the epididymis and chronic deprivation of the epididymides of adequate testosterone.
Coeoposure to low dose of Bap and DBP for 30, 90 days could reduce the circulating testosterone concentrations (P<0.05). Decreased plasma concentrations of testosterone in coexposed rats to low dose of Bap and DBP were accompanied by concomitant increases in plasma LH concentrations throughout the time periods studied, indicating that coeoposure to low dose of Bap and DBP did not have a direct effect on this pituitary gonadotropin, but rather a reflection of Leydig cell failure to respond to LH. It is also very likely that the reduction in the circulating testosterone concentrations by coexposed to low dose of Bap and DBP is secondarily contributed to by the heightened induction of the liver cytochrome P450s that are necessary for detoxification of Bap and DBP. The observed elevated plasma concentrations of LH among could result from the negative feedback on GnRH- induced LH synthesis and release.
Compared with the control group, the stillbirth rate and absorption rate are increased at the groups exposed to high dosage of Bap and co- exposed to high dose of Bap and DBP, which had not significant changes in the determination of progressive motility and density of stored spermatozoa and plasma testosterone and luteinizing hormone concentrations.
3. The effect of di-n-butyl phthalate and benzo(a)pyrene on histologic and morphometric analysis.
The testicular ultra structure observed by electronic microscope showed that both of DBP and Bap caused mitochondrion dropsy and endoplasmic reticulum expanding in testicular cells. The adverse effect of combined groups were similar with Bap groups. The toxicity in combined groups were not stronger than that of DBP or Bap groups alone.
The changes of histomorphology in the groups co- exposed to Bap and DBP were insignificant, but the reductions in the number of spermatogenic cell, sloughing of immature sperm atogenic cels and vacuoles degeneration of seminiferous epithelium were observed in the groups treated with Bap or DBP at both dosages.
We analysis the morphometric characteristics of rat testis exposed to Bap and DBP versus UNC. The atrophy seminiferous tubules were observed in the groups treated with Bap and DBP at both dosages. It occurred earliest at the testes from co- exposed to Bap and DBP at low dosage at 30 days. Secondary was the testes from expososed to DBP at low dosage at 60 days. Other group exposed to Bap and DBP occurred at 90 days.
Volume percentages occupied by seminiferous tubules in testis was increased in the groups exposed to DBP at 30 days. Compared with the results of tubule area, we ascribed the increasing of Volume percentages to the shrinkage of interstitium.
4. The change in cell cycle and total proteic expression.
On the basis of DNA content, three main germ cell peaks could be identified flow cytometrically: 2C (spermatogonia), 4C (primary spermatocytes), 1C (round and elongated spermatids). The region between the 2C and 4C peaks is comprised of cells actively synthesizing DNA and is termed as S-phase (synthetic phase). We compared the mean relative percentage of the different germ cell populations and germ cell ratios of 1C:2C, 1C:4C, 2C:4C. The effect of Bap and DBP in spermatogenesis focus on meiosis. The coexposure to Bap and DBP at low dosage had the early hormesis at 30d and 60d and became decompensation and inhibition at 90d. the mono- exposure to Bap and DBP at low dosage had the same effect, but the hormesis came later than co- exposure (60d ). No significant difference in the population of germ cell types was detected in the group treated with Bap at high dosage. The exposure to DBP at high dosage had the same effect with the exposure to it at low dosage and the co-exposure at high dosage could have interaction effects which demonstrated as antagonism.
Two- dimensional electrophoresis (2-DE) was used to separate the proteins and ImageMaster 2D Platinum 5.0 analysis revealed 35 different proteins in UNC and treated group. The 35 proteins expressed were by mass spectrometric analysis. The expression of hemopexin and mitochondrial aldehyde dehydrogenase precursor had enhanced at treated group versus UNC. Compared with UNC, the expression of chromobox homolog 2 disappeared and the expression of albumin, tubulin beta and transferring enhanced at Bap treated group. Compared with the co- exposed group, the expression of pyruvate kinase 3 isoform 2 disappeared at the group exposed to DBP, and the expression of Heterogeneous nuclear ribonucleoprotein D-like enhanced and the expression of beta actin, tumor rejection antigen gp96, albumin and trichoplein decreased.
Conclusion
Both DBP and Bap can cause certain adverse effect on the male reproductive system at the dosages of our experiment, including a reduction in mean tubular area and the epididymis coefficients, affect in spermatogenesis and histopathology observation, the increasing of the sperm deformation, and the different at proteins expression. But the semen analyses including stored sperm density, sperm viability and movement parameters had not changed. Suggesting that when we evaluate the reproductive risk associated with exposure to organic pollution in water, we should considered the combined effects of the toxicant and the impact in sub-clinical conditions, and can not only rely on the toxicological results of the single toxicant and the epidemiological investigations.
本课题组前期研究发现三峡库区水环境中的污染物以多环芳烃类物质和邻苯二甲酸酯类的检出频率和检出浓度最高。本实验拟以含量较高的邻苯二甲酸二丁酯(DBP)做为邻苯二甲酸酯类物质代表,以毒性较强的苯并[a]芘(Bap)做为多环芳烃类物质代表,对这两类物质联合毒性进行研究。有文献报道,DBP、Bap均具有确切的雄性生殖毒性,但两者低剂量联合作用下雄性生殖毒性特别是针对生精细胞的毒性及机制研究尚未见报道。本研究注重突出联合亚慢性毒性,以DBP与Bap低剂量联合作用于雄性大鼠,从毒性效应和机制两个层次开展研究,观察其联合毒性作用及机制,可为客观评价及预测POPs污染对人群健康的损害、建立环境健康效应的预警体系打下很好的基础。
试验方法
1.实验动物及分组情况
4-5周龄健康雄性SPF级SD大鼠245只,由第三军医大学实验动物中心提供,按随机数字法随机分为7组(每组35只),分别为对照组(玉米油)、Bap低剂量组(Bap 1 mg/kg)、Bap高剂量组(Bap 5 mg/kg)、DBP低剂量组(DBP 50 mg/kg)、DBP高剂量组(DBP 250 mg/kg)、DBP+ Bap低剂量组(DBP 50 mg/kg +Bap 1 mg/kg)、DBP+ Bap高剂量组(DBP 250 mg/kg +Bap 5 mg/kg)。
2.染毒及取材
采取隔天灌胃的方式进行染毒,连续染毒90d。30d、60d、90d时每组各处死8只,处死前称体重,10%水合氯醛腹腔麻醉,采集心脏血后,立即处死剖取睾丸、附睾、心、肝、脾、肾,称重后将睾丸于冰上迅速切分为约50 mg- 100 mg的小块,分装于冻存管中液氮保存。血液离心后取血清于-70℃保存,用于测定激素。每组留4只90d后与健康雌性SPF级SD大鼠交配,观察生育能力。
每组随机取1只大鼠的左侧睾丸,冰上迅速取材后固定于2.5%戊二醛,然后梯度酒精脱水、环氧树脂渗透、包埋、切片,醋酸铀和柠檬酸铅染色,透射电镜下观察。
随机选取5只大鼠的右侧睾丸,4%多聚甲醛固定液中固定,常规石蜡包埋切片,HE染色,光镜下观察,拍照,通过计算机图像分析软件(DP72-BSW)测定生精小管和睾丸面积,计算生精小管平均横截面积和生精小管与睾丸面积比。
随机选取8只大鼠单侧附睾,置于0.09%生理盐水内洗去血水,将洗净的附睾置于生理盐水内用眼科剪剪碎,轻压组织碎块,静置2 min- 3 min释放出精子,显微镜下观察精子活性和畸形、计数精子数。
随机选取6只大鼠单侧睾丸(90d时相点对照组和高剂量染毒组(Bap 5组、DBP 250组、B5+ D250组)各多选1只用于蛋白表达分析)用组合酶法分离生精细胞。
生精细胞分化情况分析:将生精细胞悬液用PBS洗2次,加1 mg/ ml RNA酶37℃孵育30 min,加入碘化丙碇4℃染色30 min,通过流式细胞仪进行相关分析(分析软件:Cellquest)。
生精细胞蛋白表达分析:提取生精细胞悬液总蛋白进行等电聚焦和SDS-PAGE,使用软件ImageMaster 2D Platinum 5.0分析各组蛋白表达差异;切下具有表达差异的蛋白点使用MALDI-TOF/TOF质谱仪(Bruker Ultraflex)进行质谱分析(N2激光器(发射波长337 nm),加速电压25 KV),使用软件moverz和peakErazor分析质谱图,通过用mascot收索引擎检索鉴定蛋白。
3.统计方法
使用SPSS软件进行统计,交配实验结果使用卡方检验,其他结果使用单因素方差分析。
结果与讨论
1.邻苯二甲酸二丁酯和苯并[a]芘对大鼠生长发育的影响
各染毒组与对照组的大鼠体重变化各组之间无显著性差异,说明本实验两种化合物的染毒剂量及染毒时间对大鼠的消化吸收与能量代谢无明显影响,不会对大鼠生长发育没有造成过度的压力而影响实验结果。
各组大鼠的心脏系数、脾脏系数、肾脏系数和睾丸脏器系数各组间无显著性差异(P>0.05),可认为本实验两种化合物的染毒剂量及染毒时间不会对大鼠的心脏、脾脏、肾脏和睾丸产生明显毒性作用。
肝脏器系数在染毒30d时低剂量联合组出现明显降低(与其它各组比均有显著性,P<0.05),在染毒60d和90d时差异消失(P>0.05)。提示肝脏不仅是Bap和DBP的代谢器官,也是其靶器官,低剂量联合染毒可导致肝脏急性损伤,引起脏器系数的降低,这种肝脏毒性可能为短期效应,长期染毒时机体逐渐对其耐受。
染毒90d两者低剂量联合染毒组与对照组相比附睾脏器系数显著性下降(P<0.05),可能由于两者低剂量联合染毒对附睾的损伤逐渐超过机体的耐受能力,造成附睾萎缩。
2.邻苯二甲酸二丁酯和苯并[a]芘睾丸形态学影响
2.1电镜观察
与对照组相比,各染毒组电镜下均可见不同程度的细胞超微结构损伤,主要表现为生精细胞线粒体肿胀、固缩,内质网扩张。
Bap低剂量单独染毒组(Bap1组)前期损伤较小,染毒90d时出现染色质聚集和脂滴。DBP低剂量单独染毒组(DBP50组)在染毒初期和中期超微结构病理改变比较重,90d时开始恢复。Bap和DBP低剂量联合染毒组(B1+ D50组)从超微结构观察整个染毒过程中损伤较小,在60d时有内质网和线粒体肿胀形成髓鞘样结构以及染色质聚集。
Bap高剂量单独染毒组(Bap5组)在整个染毒过程中受影响较大,超微结构观察染毒30d时可见细胞严重损伤,结构破坏,层次不清;60d可见细胞水肿,间隙增大;90d时内质网扩张、线粒体水肿、出现大量脂滴。DBP高剂量单独染毒组(DBP250组)在30d和90d时未出现严重的超微结构病理改变,60d时可见细胞结构破坏、层次不清。Bap和DBP高剂量联合染毒组(B5+ D250组)与DBP类似,30d和90d时超微结构病理改变较小,存在部分线粒体固缩,60d时可见生精细胞从基膜脱落。
2.2光镜观察
光镜观察发现不论是Bap和DBP的单独染毒还是联合染毒,都存在一定层度的生精细胞层数减少、空泡化、生精细胞间隙增大以及生精细胞脱落,有的还能观察到间质细胞增生。与Bap相比,DBP对睾丸组织形态学影响出现的要早,在染毒30d时候就出现生精细胞间隙增大、层数减少;联合染毒组的表现主要在于高剂量联合染毒60d时可见间质细胞增生,而生精细胞本身变化在形态学上并不明显。
2.3计算机图像分析
我们运用计算机图像分析软件在光镜下进行分析,发现:
曲细精管横截面积比较可见各染毒组曲细精管均有不同程度的萎缩。Bap和DBP低剂量联合染毒组出现最早,30d就显著性差异(与对照组比,P<0.05),60d时差异不明显(与对照组比,P>0.05),90d时又出现显著性差异(与对照组比,P<0.05),体现了一个损伤→代偿→失代偿的过程;其次是DBP低剂量单独染毒组,在第60d开始出现曲细精管明显萎缩,与对照组比有显著性差异(P<0.05),90d是差异持续存在,是一个逐渐损伤的过程;其他4组(Bap低剂量单独染毒组、Bap高剂量单独染毒组、DBP高剂量单独染毒组、两者高剂量联合染毒组)均在30d、60d与对照比差异不明显(P>0.05),90d时与对照组比有显著性差异(P<0.05)。与其电镜、光镜观察结果相比较,可以看出一个由超微结构→细胞→组织的损伤或修复的时间顺序。
曲细精管占睾丸体积百分比在60d、90d时各染毒组与对照相比无显著差异(P>0.05),在30d时DBP单独染毒组(DBP50组、DBP250组)与其他各组相比均显著升高(P<0.05)。通过与曲细精管横截面积变化结果相比较,我们推测30d时DBP单独染毒组其曲细精管占睾丸体积百分比增加可能是由于其间质组织萎缩引起,而90d时各染毒组可能都出现了不同程度间质组织萎缩。
3.邻苯二甲酸二丁酯和苯并[a]芘对大鼠生殖能力的影响
3.1精子数量和质量分析
临床评价男性生育能力的三个常规指标精子计数、成活率以及活性,染毒90d时各组间无显著性差异(P>0.05),提示我们的染毒剂量外推到人群时,该剂量下临床生育能力检查可能不会发现明显改变,不易引起重视。
精子计数结果与90d时附睾脏器系数比较,我们认为两者低剂量联合染毒组90d时与对照组相比附睾脏器系数显著性下降(P<0.05),可能不是因为精子数下降而是由于附睾内附睾管上皮细胞减少,这些细胞参与构成附睾内精子成熟、精子运动能力和受精能力的获得和发育所必需的微环境,它们的损伤将会影响精子成熟以及精子运动能力和受精能力的获得和发育,此外作为附睾功能重要调控因素的睾酮在血清中浓度下降将会加剧这种影响。根据文献报道,进入附睾的精子在结构上已经趋于完整,因此这种影响在引起精子畸形方面主要表现为断头畸形的增加,这与我们的精子畸形观察结果一致,我们在试验中发现Bap和DBP低剂量联合暴露引起的精子畸形增加主要表现为断头畸形。
DBP高剂量染毒组在染毒90d时畸形精子的数量明显增加(与对照组比P<0.05),低剂量联合组在染毒90d精子畸形率增加更显著,高于对照组、单独剂量组以及联合高剂量组(P<0.05),结合附睾脏器系数的改变,我们认为在低剂量时,两者联合后在生殖毒性上显示出加强效应。
3.2生殖相关激素的改变
低剂量联合染毒组血清T水平与对照组、Bap低剂量组相比在30d、90d显著下降(P<0.05),而60d时无显著差异(P>0.05),呈现出一个典型的损伤→代偿→失代偿的过程。
血清LH水平变化与我们血清T水平的相反,低剂量联合染毒组90d时与对照组相比显著升高(P<0.05),推论本试验所用的染毒剂量长期单独或联合暴露染毒对青春期SD大鼠血清LH水平没有直接影响,其LH水平改变可能来源于血清T水平改变引起的负反馈调节,其对睾酮浓度的影响不是通过LH浓度改变,而是在LH之后的途径,例如抑制间质细胞功能、增强肝脏对睾酮代谢等。
3.3对大鼠繁殖能力影响的分析
Bap高剂量染毒和两者高剂量联合染毒会造成死胎和吸收胎出现率显著高于对照和DBP高剂量单独染毒(P<0.05)。因此我们认为虽然Bap高剂量单独或两者高剂量联合长期暴露后虽然在一般生长发育指标、生育能力常规指标(精子计数、精子存活率、精子活性、精子畸形)及激素水平上与对照相比均没有明显改变,但是其生育能力已经受到影响。
4.邻苯二甲酸二丁酯和苯并[a]芘对生精细胞分化和蛋白表达的影响
4.1生精细胞分化改变
我们发现Bap和DBP低剂量联合染毒在精子发生上表现出早期兴奋(30d、60d)而后期逐渐失代偿并出现抑制(90d)的效应,主要集中在减数分裂阶段;单独低剂量染毒也早期有类似效应,但出现兴奋的时间要晚于联合染毒(60d),晚期机体逐渐对其耐受,兴奋效应消失(90d),从而在联合效应上出现协同→拮抗→协同的变化。高剂量组Bap单独染毒对精子发生过程始终没有显著影响,DBP高剂量单独染毒和低剂量单独染毒类似,从中期(60d)开始出现兴奋效应;两者高剂量联合在中期开始表现出以DBP效应为主,Bap对其拮抗。
4.2生精细胞蛋白表达改变分析
通过比较各组二维电泳图,我们共得到35个表达差异蛋白点,我们将这35个点通过蛋白质谱鉴定,有5个点未得到结果(无峰),剩下30个点中有11个为阴性结果(分值小于60),最后得到19个有阳性意义的蛋白,对比这些蛋白在各组间的差异,我们发现:
与精子发生密切相关的核内不均一核糖核蛋白在高剂量联合染毒组表达与Bap高剂量单独染毒组相比增强;在精母细胞的分化、精子在附睾内的成熟、精子获能和顶体反应过程中起重要作用的肿瘤拒绝抗原gp96在高剂量联合染毒组中比Bap高剂量单独染毒组表达降低;参与精原细胞有丝分裂和减数分裂并在其中其重要作用的β肌动蛋白在高剂量联合染毒组中比Bap高剂量单独染毒组表达降低。这几种蛋白表达差异可能是造成染毒90d时高剂量联合染毒组中与Bap高剂量单独染毒组相比在光镜、电镜照片中形态学损伤小而在流式细胞计数中却出现明显的精子发生过程受影响、减数分裂受阻的原因。
参与染色质组装或分解、转录、调控转录以及DNA依赖、负调控转录、染色质修饰,与DNA结合、染色质结合、转录抑制因子相关的色素框同源物2在Bap高剂量单独染毒组表达消失(与高剂量联合染毒组、对照组比均有差异),这可能与Bap高剂量生精上皮结构破坏有关。
与减数分裂、精子尾部鞭毛装配、维持细胞核形态等密切相关β-微管蛋白在Bap高剂量单独染毒组中特异性表达(与对照组相比),推测是Bap高剂量单独染毒引起生精上皮结构破坏导致β-微管蛋白过度表达;白蛋白在Bap高剂量单独染毒时候在生精细胞中表达增加(与对照组、高剂量联合染毒组相比),可能是由于Bap高剂量单独染毒引起生精上皮结构破坏,血睾屏障被破坏而致使白蛋白渗漏进生精上皮内部引起。
与铁转运相关的血红素结合蛋白在各染毒组生精细胞中表达与对照组相比均上升,Bap高剂量单独染毒组与对照组相比还存在转铁蛋白表达上升,提示Bap和DBP长期染毒引起睾丸损伤作用与铁有关。
高剂量联合染毒与Bap高剂量单独染毒相比,与线粒体的能动性相关的多毛蛋白表达消失,提示高剂量联合染毒生精细胞内线粒体活动性比Bap高剂量单独染毒要强,这可能是我们在电镜照片下发现90d时高剂量联合染毒组线粒体损伤远低于Bap高剂量单独染毒组的原因之一。
广泛参与体内脂肪族及芳香族醛类代谢的线粒体乙醛脱氢酶在各染毒组生精细胞中均出现线粒体乙醛脱氢酶特异性表达(与对照组相比),推测其可能参与睾丸对DBP和Bap及其代谢产物的生物转化。
高剂量联合染毒组与DBP高剂量单独染毒组相比糖代谢途径限速酶之一的丙酮酸激酶3的2亚型表达消失,提示两者能量代谢系统存在差异。
结论
1. DBP和Bap染毒对大鼠生育能力影响
DBP和Bap长期低剂量联合染毒可引起血清T水平显著下降,同时还损伤附睾内微环境导致精子断头畸形增加。Bap高剂量单独或Bap和DBP高剂量联合长期暴露不会影响一般生长发育指标、生育能力常规指标及激素水平,但能够影响雄性大鼠的生殖能力。
睾丸形态学改变:①Bap低剂量单独染毒前期损伤较小,后期出现损伤;DBP低剂量单独染毒在初期损伤较大,后期开始恢复;②Bap和DBP低剂量联合染毒全程损伤较小,呈现拮抗效应,两者高剂量联合染毒在形态学方面表现以DBP表现为主;②各染毒组曲细精管均有不同程度的萎缩。
2. DBP和Bap染毒对大鼠生精细胞分化及蛋白表达的影响
DBP和Bap低剂量联合染毒主要影响精子减数分裂阶段,表现为早期兴奋而后期逐渐失代偿并抑制;单独低剂量染毒早期有类似效应,但出现兴奋的时间要晚于联合染毒,晚期兴奋效应消失,从而在联合效应上出现协同效应→拮抗效应→协同效应的变化。高剂量组Bap单独染毒对精子发生没有显著影响,DBP高剂量单独染毒和低剂量单独染毒类似,从中期开始出现兴奋效应;两者高剂量联合在中期开始表现出以DBP效应为主,Bap对其拮抗。
二维电泳和质谱分析表明各染毒组与对照组相比均出现血红素结合蛋白表达上升,线粒体乙醛脱氢酶特异性表达;Bap高剂量单独染毒组与对照组相比色素框同源物2表达消失,β-tubulin特异性表达,白蛋白、转铁蛋白表达上升;高剂量联合染毒组与DBP高剂量单独染毒组相比丙酮酸激酶3的2亚型表达消失,与Bap高剂量单独染毒组相比核内不均一核糖核蛋白增强,肿瘤拒绝抗原gp96、β-actin、白蛋白表达降低,多毛蛋白表达消失。
POPs, short for Persistent Organic Pollutants, refers to the organic chemicals that could persistently exist in the environment, have a long half-life, accumulate through the food chain, and negatives affects human health and the environment. The harm POPs has on human beings and the environment has become a global environmental problem. As there are thousands of POPs substances worldwide, it is difficult to conduct a comprehensive study due to limited financial, material, and human resources. Therefore, it is important to choose to study the kind of POPs that needs concerns and monitoring, as well as people's priority POPs. This is vital for a high efficiency in environmental monitoring, pollution control and health protection.
Our previous studies show that polycyclic aromatic hydrocarbon (PAHs) and Phthalic Acid Esters (PAEs) are the most popular pollutants in the Three Gorges area. This experiment intended to study and use the DBP as a representative for PAEs, the Bap as a representative for PAHs. Previous studies have reported that both DBP and Bap have certain male reproductive toxicity, but the male reproductive toxicity under the exposure to Combination of Bap and DBP, especially for the toxicity of spermatogenic cell and its mechanism. In the present study, we test the single or combined reproductive toxicity of DBP and Bap on male rats, observe the toxic affection and discusses the effect of the combination of two toxic substances from the toxic effects level and mechanisms level. This will lay a solid foundation for objectively evaluating and predicting the possible damage POPs pollution will do to human beings and establishing an early-warning system for environmental health effects.
Methods
1. A total 245 male Sprague- Dawley rats (4-5 weeks old) were equally randomized into 7 groups (24 per group) and respectively received by gavage corn oil, 1 mg/ kg of Bap, 5 mg/ kg of Bap, 50mg/ kg of DBP, 250mg/ kg of DBP, 1 mg/ kg of Bap+ 50mg/ kg of DBP, 5 mg/ kg of Bap+ 250mg/ kg of DBP alt. dieb. For 90 days. At the 30, 60 and 90 days after gavage, 8 rats per group were sacrificed randomly. 4 rats per group were left for reproduction experiments.
2. treatment and methords
The testes, epididymis, liver and other visceral organs were harvested, weighed and the organ coefficients were counted. Blood samples were collected to assess the effect of Bap and DBP on plasma testosterone (T) and luteinizing hormone (LH) concentrations with Chemiluminescence immumo- assay. Cauda epididymides were isolated for the determination of progressive motility and density of stored spermatozoa.
Testes were prepared for histologic and morphometric analysis. Stained testis sections were evaluated using an OLYMPUS Photomicroscope equipped which coupled with a computerized morphometric planimetry system (DP72- BSW) to facilitate the measurement of tubule area and area percentages (volume percentages) occupied by seminiferous tubules.
The testes were surgically removed and germ cells were isolated. Flow cytometry (FCM) was used to detect changes of cell cycle and Two- dimensional electrophoresis (2-DE) was used to separate the proteins. The proteins which has different expression were analyzed using the Image master 5.0 and were identified by mass spectrometric analysis.
Results and Discussion
1. The effect of di-n-butyl phthalate and benzo(a)pyrene on rat growth
The exposure concentration of Bap and DBP used in this study did not affect weekly weight gains in exposed versus UNC rats (P>0.05). These data suggest that inhaled BaP, at the exposure concentration administered, the daily duration of exposures and the manner in which animals were restrained, did not impose undue stress on the rats, hence the identical weight gains between exposed and UNC rats.
The organ coefficients of heart, spleen, kidney and testis also had no changes between exposed versus UNC rats (P>0.05), which means the exposing dosage didn’t trigger a significant toxical effect in exposed rats.
Compared with other groups, the co- exposure to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 30 days could reduce liver coefficient (P<0.05), and the difference disappeared at 60 days and 90 days. It suggested that the liver is not only the organ where the Bap and DBP metabolize, but also the target organ of Bap and DBP. And the injuries to liver by Bap and DBP may be acute and provisional. The body may tolerated to them gradually.
The epididymis might be sensitive to the toxic effect of Bap and DBP. In our study, the epididymis coefficient were significant decreased at the co-exposure to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days compared with the UNC rats. Suggested that the long time co- exposure to Bap and DBP might have the potential toxic effext to epididymis and might cause the atrophy of it.
2. The effect of di-n-butyl phthalate and benzo(a)pyrene on rat fertility.
In our study, the stored sperm density, sperm viability and movement parameters, which often be used in semen analyses to appraise the male reproductive ability, were not changed both in mono- and co- exposure to B[a]p and DBP. Showed that these indicator may not sensitive to Bap and DBP exposure and routine clinical check to the male reproductive ability by semen analyses may not identify problems in the people exposed to Bap and DBP.
Spermatozoa with abnormal morphologies were disproportionately represented in samples of stored spermatozoa from exposed to DBP 250 mg/kg/d and co- exposed 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days to rats than their control counterparts (P<0.05).
Compared with the significant decreased of epididymis coefficient at co-exposure to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days. We thought that the decreased of epididymis coefficient may not arise from the decreased of sperm density, but from the regression in the epididymal epithelium, which construct the microenvironment in the epididymis for further sperm maturation and consequently. And the change in circulating testosterone concentrations may sharp the effect because the level of testosterone reaching the epididymides from general circulation and testicular fluid may not be sufficient to optimally regulate the epididymides. The increased percentage of decapitated spermatozoa in the cauda epididymides of co- exposed to 1 mg/kg/d Bap and 50 mg/kg/d DBP for 90 days compared with UNC could be likened to the degenerating conditions of stored spermatozoa due to damaged microenvironment in the epididymis and chronic deprivation of the epididymides of adequate testosterone.
Coeoposure to low dose of Bap and DBP for 30, 90 days could reduce the circulating testosterone concentrations (P<0.05). Decreased plasma concentrations of testosterone in coexposed rats to low dose of Bap and DBP were accompanied by concomitant increases in plasma LH concentrations throughout the time periods studied, indicating that coeoposure to low dose of Bap and DBP did not have a direct effect on this pituitary gonadotropin, but rather a reflection of Leydig cell failure to respond to LH. It is also very likely that the reduction in the circulating testosterone concentrations by coexposed to low dose of Bap and DBP is secondarily contributed to by the heightened induction of the liver cytochrome P450s that are necessary for detoxification of Bap and DBP. The observed elevated plasma concentrations of LH among could result from the negative feedback on GnRH- induced LH synthesis and release.
Compared with the control group, the stillbirth rate and absorption rate are increased at the groups exposed to high dosage of Bap and co- exposed to high dose of Bap and DBP, which had not significant changes in the determination of progressive motility and density of stored spermatozoa and plasma testosterone and luteinizing hormone concentrations.
3. The effect of di-n-butyl phthalate and benzo(a)pyrene on histologic and morphometric analysis.
The testicular ultra structure observed by electronic microscope showed that both of DBP and Bap caused mitochondrion dropsy and endoplasmic reticulum expanding in testicular cells. The adverse effect of combined groups were similar with Bap groups. The toxicity in combined groups were not stronger than that of DBP or Bap groups alone.
The changes of histomorphology in the groups co- exposed to Bap and DBP were insignificant, but the reductions in the number of spermatogenic cell, sloughing of immature sperm atogenic cels and vacuoles degeneration of seminiferous epithelium were observed in the groups treated with Bap or DBP at both dosages.
We analysis the morphometric characteristics of rat testis exposed to Bap and DBP versus UNC. The atrophy seminiferous tubules were observed in the groups treated with Bap and DBP at both dosages. It occurred earliest at the testes from co- exposed to Bap and DBP at low dosage at 30 days. Secondary was the testes from expososed to DBP at low dosage at 60 days. Other group exposed to Bap and DBP occurred at 90 days.
Volume percentages occupied by seminiferous tubules in testis was increased in the groups exposed to DBP at 30 days. Compared with the results of tubule area, we ascribed the increasing of Volume percentages to the shrinkage of interstitium.
4. The change in cell cycle and total proteic expression.
On the basis of DNA content, three main germ cell peaks could be identified flow cytometrically: 2C (spermatogonia), 4C (primary spermatocytes), 1C (round and elongated spermatids). The region between the 2C and 4C peaks is comprised of cells actively synthesizing DNA and is termed as S-phase (synthetic phase). We compared the mean relative percentage of the different germ cell populations and germ cell ratios of 1C:2C, 1C:4C, 2C:4C. The effect of Bap and DBP in spermatogenesis focus on meiosis. The coexposure to Bap and DBP at low dosage had the early hormesis at 30d and 60d and became decompensation and inhibition at 90d. the mono- exposure to Bap and DBP at low dosage had the same effect, but the hormesis came later than co- exposure (60d ). No significant difference in the population of germ cell types was detected in the group treated with Bap at high dosage. The exposure to DBP at high dosage had the same effect with the exposure to it at low dosage and the co-exposure at high dosage could have interaction effects which demonstrated as antagonism.
Two- dimensional electrophoresis (2-DE) was used to separate the proteins and ImageMaster 2D Platinum 5.0 analysis revealed 35 different proteins in UNC and treated group. The 35 proteins expressed were by mass spectrometric analysis. The expression of hemopexin and mitochondrial aldehyde dehydrogenase precursor had enhanced at treated group versus UNC. Compared with UNC, the expression of chromobox homolog 2 disappeared and the expression of albumin, tubulin beta and transferring enhanced at Bap treated group. Compared with the co- exposed group, the expression of pyruvate kinase 3 isoform 2 disappeared at the group exposed to DBP, and the expression of Heterogeneous nuclear ribonucleoprotein D-like enhanced and the expression of beta actin, tumor rejection antigen gp96, albumin and trichoplein decreased.
Conclusion
Both DBP and Bap can cause certain adverse effect on the male reproductive system at the dosages of our experiment, including a reduction in mean tubular area and the epididymis coefficients, affect in spermatogenesis and histopathology observation, the increasing of the sperm deformation, and the different at proteins expression. But the semen analyses including stored sperm density, sperm viability and movement parameters had not changed. Suggesting that when we evaluate the reproductive risk associated with exposure to organic pollution in water, we should considered the combined effects of the toxicant and the impact in sub-clinical conditions, and can not only rely on the toxicological results of the single toxicant and the epidemiological investigations.
引文
1.成毅明,石心泉,于和鸣.β-肌动蛋白在大鼠精子发生过程中的特殊表达[J].中华男科学杂志, 2005, 11(10): 755-760.
2.陈敏,谢吉民,闫永胜.火焰原子吸收光谱法研究五种螯合剂对染镉小鼠睾丸中镉钙铁锌浓度的影响[J].理化检验:化学分册, 2007, (9): 709-712.
3.曹波,任谦,崔志鸿,等.嘉陵江C市段源水中有机污染物定性研究[J].四川环境, 2006, 25(06):78-80.
4.常兵,刘德瑜,梁玉香,等.邻苯二甲酸二丁酯对青春期雄性大鼠的生殖毒性研究[J].中国自然医学杂志, 2007, 9(3): 164-168.
5.黄宇烽.主编精液细胞学与超微结构图谱.第二军医大学出版社, 2002. 11-13.
6.金名华,石龙,刘小梅,等.苯并[a]芘对小鼠睾丸细胞周期的影响[J].中国公共卫生, 2005,20(2):153-154.
7.廖晓岗, Nobuo TERADA,李子龙.体内冷冻的正常及镉处理小鼠睾丸内血清蛋白质的免疫组织化学研究[J].生殖与避孕, 2006, 26(3): 135-141.
8.刘晓梅,金明华,王雯等.苯并[a]芘对雄性小鼠生殖细胞的影响[J].毒理学杂志, 2005, 19(S1): 190.
9.邱志群,舒为群,陈济安.邻苯二甲酸二丁酯和苯并[a]芘对大鼠睾丸支持细胞波形蛋白和微管蛋白表达的影响[J].癌变·畸变·突变, 2009, 21(03): 165-168.
10.王玉邦,宋玲,朱正平,等.邻苯二甲酸二丁酯对睾酮合成的影响[J].中国公共卫生, 2005, 21(10): 1168-1170.
11.赵清,舒为群,曹佳,等.邻苯二甲酸二丁酯亚慢性染毒对大鼠睾丸Insl3mRNA表达的影响[J].癌变·畸变·突变, 2009, 21(3): 161-164.
12.赵清,舒为群,曹佳,等.苯并[a]芘对大鼠睾丸Insl3mRNA基因表达的影响[J].环境与健康杂志, 2009, 26(5): 377-379.
13. Alnouti Y, Klaassen CD. Tissue distribution, ontogeny, and regulation of aldehyde dehydrogenase (Aldh) enzymes mRNA by prototypical microsomal enzyme induce in mice[J]. Toxicology Science, 2008, 101(1): 51—64.
14. Anesti V, Scorrano L. The relationship between mitochondrial shape and function and the cytoskeleton. Biochimica et Biophysica Acta (BBA), 2006, 1757(5-6): 692-699.
15. Archibong AE, England DC, Stormshak F. Factors contributing to embryonic mortality in gilts bred at pubertal estrus[J]. Animal Science, 1987, (64):474-478.
16. Asquith KL, Harman AJ., McLaughlin EA, et a1.Localization and significance of molecular chaperones, heat shock protein 1, and tumor rejection antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction[J].Biology of Reproduction, 2005, (72): 328-337.
17. Barthelemy C, Khalfoun B, Guillaumin JM, et a1. Seminal fluid transferrin as an index of gonadal function in men[J]. Journal of reproduction and fertility, 1988, (82): ll3-ll8.
18. Carlsen E, Giwercman A, Keidiing N, et al. Evidence for decreasing quality of semen during past 50 years[J]. British Medical Journal, 1992, (305): 609-613.
19. Chen JA, Luo JH, Qiu ZQ, et al. PCDDs/PCDFs and PCBs in water samples from the Three Gorge Reservoir[J]. Chemosphere, 2008, (70): 1545-1551.
20. Clubb BH, Locke M.β-actin forms transient perinuclear shells at the mitosis -interphase transition[J]. Cell motility and the cytoskeleton, 1996, 33(2): 151-162.
21. Davis MTB, Miller SG. Expression of a testisβ-tubulin gene in Heliothis virescens during spermatogenesis[J]. Insect Biochemistry, 1988, 18(4): 389-394.
22. Elena Kleymenova, Cynthia Swanson, Kim Boekelheide, et al. Exposure in utero to di(n-butyl)phthalate alters the vimentin cytoskeleton of fetal ratSertoli cells and disrupts Sertoli cell-gonocyte contact[J]. Biology of Reproduction. 2005, 73(3): 482-490.
23. Foresta C, Manoni F, Businaro V, et a1. PossiNe significance of transferrin levels in seminal plasma of fertile and infertile men[J]. Journal of andrology, 1986, 7(2): 77-82.
24. Foster PMD, Mylchreest E, Gaido KW, et al. Effects of phthalate esters on the developing reproductive tract of male rats[J]. Human Reproduction Update, 2001, 7(3): 231-235.
25. Inyang F., Ramesh A., Kopsombut P., et al. Disruption of testicular steroidogenesis and epididymal function by inhaled benzo[a]pyrene[J]. Reproductive Toxicology, 2003, (17): 527-537.
26. Harris CA, Henttu P, Parker MG, et al. The estrogenic activity of phthalate esters in vitro[J]. Environ Health Perspect, 1997, 105(8): 802-811.
27. Hisashi Tsutsumi, Kenzaburo Tani, Hisaichi Fujii. Expression of L- and M-typepyruvate kinase in human tissues. Genomics, 1988, 2(1): 86-89.
28. Inyang F, Ramesh A, Kopsombut P, et al. Disruption of testicular steroidogenesis and epididymal function by inhaled benzo[a]pyrene[J]. Reproductive Toxicology, 2003,(17): 527-537.
29. Jeyaraj DA, Grossman G, Petrusz P. Altered bioavailability of testosterone in androgen-binding protein-transgenic mice[J]. Steroids, 2005, (70): 704-714.
30. Ju Young Ryu, Byung Mu Lee, Sam Kacew, et al. Identification of differentially expressed genes in the testis of Sprague- Dawley rats treated with di(n-butyl) phthalate [J]. Toxicology, 2007, (234): 103-112.
31. Karabelyos CS, Csaba G. Benzpyrene treatment decreases the sexual activity of adult rats, what is reversed in neonatally allylestrenol treated animals[J]. Acta Physiologica Hungarica, 1996, 84:131-137.
32. Kavlock R, Boekelheide K, Chapin R, et al. NTP center for the evaluation of risks to human reproduction: phthalates expert panel report on the reproductive and developmental toxicity of di-n-butyl phthalate[J]. Reproductive Toxicology, 2002, 16(5): 489-527.
33. Knuckles ME, Inyang F, Ramesh A. Acute and subchronic oral toxicities of benzo(a)pyrene in F-344 rats[J]. Toxicological Sciences, 2001, (61): 382-388.
34. Marsman D. NTP technical report on the toxicity studies of Dibutyl Phthalate (CAS No. 84-74-2) Administered in Feed to F344/N Rats and B6C3F1[J]. Toxicity report series, 1995, (30): G1-G5.
35. Payne A. The pathological effects of the intraperitoneal injection of 3, 4-benzopyrene in rats and mice[J]. British Journal of Cancer, 1958, (12):65-74.
36. Ramesh A, Inyang F, Lunstra DD, et al. Alteration of fertility endpoints in adult male F-344 rats by subchronic exposure to inhaled benzo(a)pyrene[J]. Experimental and Toxicologiy Pathology, 2008, (60): 269-280.
37. Raychoudhury SS, Kubinski D. Polycyclic aromatic hydrocarbon induced cytotoxicity in cultured rat sertoli cells involves differential apoptotic response[J]. Environmental Health Perspectives, 2003, (111): 33-38.
38. Revel A, Roanani H, Younglai E, et al. Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects sperm from DNA damage and apoptosis caused bybenzo(a)pyrene[J]. Reproductive Toxicology, 2001, (15): 479-86.
39. Rochira V, Zirilli L, Genazzani AD, et al. Hypothalamic-pituitary-gonadal axis in two men with aromatase deficiency: evidence that circulating estrogens are required at the hypothalamic level for the integrity of gonadotropin negative feedback[J]. Endocrinology, 2006, (155): 513-522.
40. Shultz VD, Phillips S, Sar M, Foster P.M., et al. Altered gene profiles in fetal rat testes after in utero exposure to di (n-butyl) phthalate[J]. Toxicology, 2001,(64): 233-242.
41. Van PN, Peter F, Soling HD. Four intracisternal calcium -binding glycoproteins from rat liver microsomes with high af2 finity for calcium: No indication for calsequestrin -like proteins in inositol 1,4,5 -trisphosphate -sensitive calcium sequestering rat liver vesicles. The Journal of biological chemistry, 1989, (264): 17494-17501.
42. Weber KL, Sokac AM, Berg JS, et al. A microtubule-binding myosin required for nuclear anchoring and spindle assembly[J]. Nautre, 2004, (431): 325-329.
43. Wu AT, Sutovsky P, Xu W. The postacrosomal assembly of sperm head protein, PAWP, is independent of acrosome formation and dependent on microtubular manchette transport[J]. Developmental Biology, 2007, 312(2): 471-483.
44. Yilei Liu, Cyril F Bourgeois, Shaochen Pang. The Germ Cell Nuclear Proteins hnRNP G-T and RBMY Activate a Testis-Specific Exon. PLoS Genetics, 2009, 5(11): e1000707.
1. Moline, J. M.; Golden, A. L.; Bar-Chama, N.; Smith, E.; Rauch, M. E.; Chapin, R. E.; Perreault, S. D.; Schrader, S. M.; Suk, W. A.; Landrigan, P.J.: Exposure to hazardous substances and male reproductive health: a research framework. Environ Health Perspect. 2000 108, 803-813.
2. Solomon, G.M.; Schettler, T.: Environment and health: 6. Endocrine disruption and potential human health implications. CMAJ 2000 163, 1471-1476.
3. Arcand-hoy, L. D.; Nimrod, A. C.; Benson, W. H.: Endocrine-modulating substances in the environment:estrogenic effects of pharmaceutical products. Int. J. Toxicol. 1998 17, 139– 158.
4. Jarow, J. P.; Zirkin, B.: The androgen microenvironment of the human testis and hormonal control of spermatogenesis. Ann NY Sci 2005, 1061, 208-220.
5. Paoletti P, Marchesi E, Velona F,et al. Air pollution and health[J]. Nature.1992,355(6358):290.
6. Yoo EJ, Lee BM. Chemopreventive effects of aloe against genotoxicity induced by benzo[a]pyrene[J]. J Toxicol Environ Health A. 2005,68(21):1841-1860.
7. Lehr RE, Jerina DM. Metabolic activations of polycyclic hydrocarbons. Structure-activity relationships. Arch Toxicol. 1977,39(1-2):1-6.
8. Kim JH, Stansbury KH, Walker NJ, Trush MA, Strickland PT, Sutter TR. Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1. Carcinogenesis. 1998 Oct;19(10):1847–1853.
9. Payne, S., 1958. The pathological effects of the intraperitoneal injection of 3:4-benzopyrene into rats and mice. Br. J. Cancer 12, pp. 65–74.
10. Inyang, F., Ramesh, A., Kopsombut, P., Niaz, M.S., Hood, D.B., Nyanda, A.M. and Archibong, A.E., 2003. Disruption of testicular steroidogenesis and epididymal function by inhaled benzo[a]pyrene. Reprod. Toxicol. 17, pp. 527–537.
11. Ciolino HP, Daschner PJ, Yeh GC. Resveratrol inhibits transcription of CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor[J]. Cancer Res.1998,58:5707-5712.
12. Mandal, P. K.; McDaniel, L. R.; Prough, R. A.; Clark, B. J.: 7, 12-Dimethlbenz(a)anthracene inhibition of steroid production in MA-10 mouse Leydig tumor cells is not directly linked to induction of CYP1B1. Toxicol Appl Pharmacol., 2001, 175, 200-208.
13. Ramesh, A.; Inyang, F.; Hood, D. B.; Knuckles, M. E.: Aryl hydrocarbon hydroxylase activity in F-344 rats subchronically exposed to benzo(a)pyrene and fluoranthene through diet. J Biochem Mol Toxicol., 2000, 14, 155-161.
14. Ramesh, A.; Hood, D. B.; Inyang, F.; Greenwood, M.; Archibong, A.E.; Knuckles, M. E et al.: Comparative metabolism, bioavailability and toxicokinetics of benzo(a)pyrene in rats after acute oral, inhalation, and intravenous administration. Polycyclic Aromatic Compounds, 2002, 22: 969-980.
15. Lee, I. P.; Nagayama, J.: Metabolism of benzo(a)pyrene by the isolated perfused rat testis. Cancer Res., 1980, 40, 3297-3303.
16. Polyakov, L. M.; Chasovskikh, M. I.; Panin, L. E.: Binding and transport of benzo(a)pyrene by blood plasma lipoproteins: the possible role of apolipoprotein B in this process. Bioconjugate Chem 1996, 7, 396-400.
17. Williams, J. A.; Martin, F. C.; Muir, G. H.; Hewer, A.; Grover, P. L.; Phillips, D. H.: Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue. Carcinogenesis, 2000, 21, 1683-1689.
18. Senft, A. P.; Dalton, T. P.; Nebert, D. W.; Genter, M. B.; Puga, A.; Hutchinson, R. J, et al.: Mitochondrial reactive oxygen production is dependent on the aromatic hydrocarbon receptor. Free Radic Biol Med, 2002, 33, 1268-1278.
19. Baird WM, Ralston SL. Carcinogenic polycyclic aromatic hydrocarbons. In: Bowden GT, Fischer S, editors. Comprehensive toxicology. Chemical carcinogens and anticarcinogens, vol. 12; 2004. p. 171–200.
20. Diemer, T.; Allen, J. A.; Hales, K. H.; Hales, D. B.: Reactive oxygen disrupts mitochondriain MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis. Endocrinology, 2003, 144, 2882-2891.
21. Ramesh A, Inyang F, Hood DB, Archibong AE, Knuckles ME, Nyanda AM. Metabolism, bioavailability, and toxicokinetics of benzo(a)pyrene in F-344 rats following oral administration. Exp Toxicol Pathol 2001;53:275–90.
22. Ramesh A, Hood DB, Inyang F, Greenwood M, Archibong AE, Knuckles ME, et al.Comparative metabolism, bioavailability and toxicokinetics of benzo(a)pyrene in rats after acute oral, inhalation, and intravenous administration. Polycyclic Aromat Compd 2002;22:969–80.
23. Lee IP, Nagayama J. Metabolism of benzo(a)pyrene by the isolated perfused rat testis. Cancer Res 1980;40:3297–303.
24. Williams JA, Martin FC, Muir GH, Hewer A, Grover PL, Phillips DH. Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue. Carcinogenesis 2000;21:1683–9.
25. Yardimci S, Atan A, Delibasi T, Sunguroglu K, Guven MC. Long-term effects of cigarette-smoke exposure on plasma testosterone, luteinizing hormone and follicle-stimulating hormone levels in male rats. Br J Urol 1997;79:66–9.
26. Archibong AE, Ramesh A, Niaz MS, Brook CM, Roberson SI, Lunstra DD. Effects of Benzo(a)pyrene on intra-testicular function in F-344 rats. Int. J. Environ. Res. Public Health. 2008;5:32–40.
27. Archibong AE, Inyang F, Ramesh A, Greenwood M, Nayyar T, Kopsombut P, et al. Alteration of pregnancy related hormones and fetal survival in F-344 rats by inhaled benzo(a)pyrene. Reprod Toxicol 2002;16:801–8.
28. Raj AS, Katz M. Inhibitory effects of alpha- and betanaphthoflavones on DMBA-induced anomalies in germ cells assessed by sperm normality assay. Mutat Res 1984; 136:81–4.
29.金明华,石龙,刘晓梅,孙志伟,郝民,刘亚辉.苯并[a]芘对雄性小鼠睾丸细胞周期的影响.中国公共卫生,2004, 20(2):153-154
30.郝民.苯并[a]芘的生殖毒性及其作用机制研究[M]. 1999-2002硕士学位论文.
31. A. Ramesh, F. Inyang, D.D. Lunstra, M.S. Niaz, P. Kopsombut, K.M. Jones, D.B. Hoode, E.R. Hills and A.E. Archibong, Alteration of fertility endpoints in adult male F-344 rats by subchronic exposure to inhaled benzo(a)pyrene, Exp. Toxicol. Pathol. 60 (2008), pp. 269–280.
32.陈晓东,吴玲,罗绥兰.香烟烟雾提取物致雄性小鼠生殖细胞染色体畸变与睾丸组织中SOD和MDA含量的变化[J].预防医学文献信息,20028(1):46-47.
33. Vine MF. Smoking and male reproduction:a review. Int Jandrol.1996,19:323-37.
34. Choi H, Jedrychowski W, Spengler J, et al. International studies of prenatal exposure topolycyclic aromatic hydrocarbons and fetal growth[J]. Environ Health Perspect. 2006,114(11):1744-1750.
35. Pierantoni R, Cobellis G, Meccariello R, et al. The amphibian testis as model to study germ cell apoptosis during spermatogenesis[J]. Comp Biochem Physiol Biochem Mol Biol, 2002, 132(1): 131-139.
36. Raychoudhury SS, Kubinski D. Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response[J]. Environ Health Perspect. 2003,111(1):33-38.
37. Vine MF, Margolin BH, Morrison HI, et al. Cigarette smoking and sperm density: a meta-analysis[J]. Fertil Steril. 1994 ,61(1):35-43.
38. Maeda Y, Shiratsuchi A, Namiki M, et al. Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility[J]. Cell Death Differ, 2002,9 (7): 742-749.
39. Taylor CT. Antioxidants and reactive oxygen species in human fertility. Environ Toxicol Pharmacol 2001;10:189–98.
40. Aitken RJ, Harkiss D, Buckingham D. Relationship between iron-catalyzed lipid peroxidation and human sperm function. J Reprod Fertil 1993;98:257–65.
41. Selevan SG, Borkovec L, Zudova Z, Slott V, Hanjova R, Rubes J, et al. Semen quality in young men and air pollution in two Czech communities. Epidemiology 1995;6(Suppl):S85.
42. Sram RJ, Binkova B, R?ssner P, Rube? J, Topinka J, Dejmek J. Adverse reproductive outcomes from exposure to environmental mutagens. Mutat Res 1999;428:203–15.
43.徐晨,周作民.生殖生物学理论与实践[M].上海:上海科学技术文献出版社,2005.
44. Vinggaard AM, Hnida C, Larsen JC. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro. Toxicology 2000;145:173–83.
2.陈敏,谢吉民,闫永胜.火焰原子吸收光谱法研究五种螯合剂对染镉小鼠睾丸中镉钙铁锌浓度的影响[J].理化检验:化学分册, 2007, (9): 709-712.
3.曹波,任谦,崔志鸿,等.嘉陵江C市段源水中有机污染物定性研究[J].四川环境, 2006, 25(06):78-80.
4.常兵,刘德瑜,梁玉香,等.邻苯二甲酸二丁酯对青春期雄性大鼠的生殖毒性研究[J].中国自然医学杂志, 2007, 9(3): 164-168.
5.黄宇烽.主编精液细胞学与超微结构图谱.第二军医大学出版社, 2002. 11-13.
6.金名华,石龙,刘小梅,等.苯并[a]芘对小鼠睾丸细胞周期的影响[J].中国公共卫生, 2005,20(2):153-154.
7.廖晓岗, Nobuo TERADA,李子龙.体内冷冻的正常及镉处理小鼠睾丸内血清蛋白质的免疫组织化学研究[J].生殖与避孕, 2006, 26(3): 135-141.
8.刘晓梅,金明华,王雯等.苯并[a]芘对雄性小鼠生殖细胞的影响[J].毒理学杂志, 2005, 19(S1): 190.
9.邱志群,舒为群,陈济安.邻苯二甲酸二丁酯和苯并[a]芘对大鼠睾丸支持细胞波形蛋白和微管蛋白表达的影响[J].癌变·畸变·突变, 2009, 21(03): 165-168.
10.王玉邦,宋玲,朱正平,等.邻苯二甲酸二丁酯对睾酮合成的影响[J].中国公共卫生, 2005, 21(10): 1168-1170.
11.赵清,舒为群,曹佳,等.邻苯二甲酸二丁酯亚慢性染毒对大鼠睾丸Insl3mRNA表达的影响[J].癌变·畸变·突变, 2009, 21(3): 161-164.
12.赵清,舒为群,曹佳,等.苯并[a]芘对大鼠睾丸Insl3mRNA基因表达的影响[J].环境与健康杂志, 2009, 26(5): 377-379.
13. Alnouti Y, Klaassen CD. Tissue distribution, ontogeny, and regulation of aldehyde dehydrogenase (Aldh) enzymes mRNA by prototypical microsomal enzyme induce in mice[J]. Toxicology Science, 2008, 101(1): 51—64.
14. Anesti V, Scorrano L. The relationship between mitochondrial shape and function and the cytoskeleton. Biochimica et Biophysica Acta (BBA), 2006, 1757(5-6): 692-699.
15. Archibong AE, England DC, Stormshak F. Factors contributing to embryonic mortality in gilts bred at pubertal estrus[J]. Animal Science, 1987, (64):474-478.
16. Asquith KL, Harman AJ., McLaughlin EA, et a1.Localization and significance of molecular chaperones, heat shock protein 1, and tumor rejection antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction[J].Biology of Reproduction, 2005, (72): 328-337.
17. Barthelemy C, Khalfoun B, Guillaumin JM, et a1. Seminal fluid transferrin as an index of gonadal function in men[J]. Journal of reproduction and fertility, 1988, (82): ll3-ll8.
18. Carlsen E, Giwercman A, Keidiing N, et al. Evidence for decreasing quality of semen during past 50 years[J]. British Medical Journal, 1992, (305): 609-613.
19. Chen JA, Luo JH, Qiu ZQ, et al. PCDDs/PCDFs and PCBs in water samples from the Three Gorge Reservoir[J]. Chemosphere, 2008, (70): 1545-1551.
20. Clubb BH, Locke M.β-actin forms transient perinuclear shells at the mitosis -interphase transition[J]. Cell motility and the cytoskeleton, 1996, 33(2): 151-162.
21. Davis MTB, Miller SG. Expression of a testisβ-tubulin gene in Heliothis virescens during spermatogenesis[J]. Insect Biochemistry, 1988, 18(4): 389-394.
22. Elena Kleymenova, Cynthia Swanson, Kim Boekelheide, et al. Exposure in utero to di(n-butyl)phthalate alters the vimentin cytoskeleton of fetal ratSertoli cells and disrupts Sertoli cell-gonocyte contact[J]. Biology of Reproduction. 2005, 73(3): 482-490.
23. Foresta C, Manoni F, Businaro V, et a1. PossiNe significance of transferrin levels in seminal plasma of fertile and infertile men[J]. Journal of andrology, 1986, 7(2): 77-82.
24. Foster PMD, Mylchreest E, Gaido KW, et al. Effects of phthalate esters on the developing reproductive tract of male rats[J]. Human Reproduction Update, 2001, 7(3): 231-235.
25. Inyang F., Ramesh A., Kopsombut P., et al. Disruption of testicular steroidogenesis and epididymal function by inhaled benzo[a]pyrene[J]. Reproductive Toxicology, 2003, (17): 527-537.
26. Harris CA, Henttu P, Parker MG, et al. The estrogenic activity of phthalate esters in vitro[J]. Environ Health Perspect, 1997, 105(8): 802-811.
27. Hisashi Tsutsumi, Kenzaburo Tani, Hisaichi Fujii. Expression of L- and M-typepyruvate kinase in human tissues. Genomics, 1988, 2(1): 86-89.
28. Inyang F, Ramesh A, Kopsombut P, et al. Disruption of testicular steroidogenesis and epididymal function by inhaled benzo[a]pyrene[J]. Reproductive Toxicology, 2003,(17): 527-537.
29. Jeyaraj DA, Grossman G, Petrusz P. Altered bioavailability of testosterone in androgen-binding protein-transgenic mice[J]. Steroids, 2005, (70): 704-714.
30. Ju Young Ryu, Byung Mu Lee, Sam Kacew, et al. Identification of differentially expressed genes in the testis of Sprague- Dawley rats treated with di(n-butyl) phthalate [J]. Toxicology, 2007, (234): 103-112.
31. Karabelyos CS, Csaba G. Benzpyrene treatment decreases the sexual activity of adult rats, what is reversed in neonatally allylestrenol treated animals[J]. Acta Physiologica Hungarica, 1996, 84:131-137.
32. Kavlock R, Boekelheide K, Chapin R, et al. NTP center for the evaluation of risks to human reproduction: phthalates expert panel report on the reproductive and developmental toxicity of di-n-butyl phthalate[J]. Reproductive Toxicology, 2002, 16(5): 489-527.
33. Knuckles ME, Inyang F, Ramesh A. Acute and subchronic oral toxicities of benzo(a)pyrene in F-344 rats[J]. Toxicological Sciences, 2001, (61): 382-388.
34. Marsman D. NTP technical report on the toxicity studies of Dibutyl Phthalate (CAS No. 84-74-2) Administered in Feed to F344/N Rats and B6C3F1[J]. Toxicity report series, 1995, (30): G1-G5.
35. Payne A. The pathological effects of the intraperitoneal injection of 3, 4-benzopyrene in rats and mice[J]. British Journal of Cancer, 1958, (12):65-74.
36. Ramesh A, Inyang F, Lunstra DD, et al. Alteration of fertility endpoints in adult male F-344 rats by subchronic exposure to inhaled benzo(a)pyrene[J]. Experimental and Toxicologiy Pathology, 2008, (60): 269-280.
37. Raychoudhury SS, Kubinski D. Polycyclic aromatic hydrocarbon induced cytotoxicity in cultured rat sertoli cells involves differential apoptotic response[J]. Environmental Health Perspectives, 2003, (111): 33-38.
38. Revel A, Roanani H, Younglai E, et al. Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects sperm from DNA damage and apoptosis caused bybenzo(a)pyrene[J]. Reproductive Toxicology, 2001, (15): 479-86.
39. Rochira V, Zirilli L, Genazzani AD, et al. Hypothalamic-pituitary-gonadal axis in two men with aromatase deficiency: evidence that circulating estrogens are required at the hypothalamic level for the integrity of gonadotropin negative feedback[J]. Endocrinology, 2006, (155): 513-522.
40. Shultz VD, Phillips S, Sar M, Foster P.M., et al. Altered gene profiles in fetal rat testes after in utero exposure to di (n-butyl) phthalate[J]. Toxicology, 2001,(64): 233-242.
41. Van PN, Peter F, Soling HD. Four intracisternal calcium -binding glycoproteins from rat liver microsomes with high af2 finity for calcium: No indication for calsequestrin -like proteins in inositol 1,4,5 -trisphosphate -sensitive calcium sequestering rat liver vesicles. The Journal of biological chemistry, 1989, (264): 17494-17501.
42. Weber KL, Sokac AM, Berg JS, et al. A microtubule-binding myosin required for nuclear anchoring and spindle assembly[J]. Nautre, 2004, (431): 325-329.
43. Wu AT, Sutovsky P, Xu W. The postacrosomal assembly of sperm head protein, PAWP, is independent of acrosome formation and dependent on microtubular manchette transport[J]. Developmental Biology, 2007, 312(2): 471-483.
44. Yilei Liu, Cyril F Bourgeois, Shaochen Pang. The Germ Cell Nuclear Proteins hnRNP G-T and RBMY Activate a Testis-Specific Exon. PLoS Genetics, 2009, 5(11): e1000707.
1. Moline, J. M.; Golden, A. L.; Bar-Chama, N.; Smith, E.; Rauch, M. E.; Chapin, R. E.; Perreault, S. D.; Schrader, S. M.; Suk, W. A.; Landrigan, P.J.: Exposure to hazardous substances and male reproductive health: a research framework. Environ Health Perspect. 2000 108, 803-813.
2. Solomon, G.M.; Schettler, T.: Environment and health: 6. Endocrine disruption and potential human health implications. CMAJ 2000 163, 1471-1476.
3. Arcand-hoy, L. D.; Nimrod, A. C.; Benson, W. H.: Endocrine-modulating substances in the environment:estrogenic effects of pharmaceutical products. Int. J. Toxicol. 1998 17, 139– 158.
4. Jarow, J. P.; Zirkin, B.: The androgen microenvironment of the human testis and hormonal control of spermatogenesis. Ann NY Sci 2005, 1061, 208-220.
5. Paoletti P, Marchesi E, Velona F,et al. Air pollution and health[J]. Nature.1992,355(6358):290.
6. Yoo EJ, Lee BM. Chemopreventive effects of aloe against genotoxicity induced by benzo[a]pyrene[J]. J Toxicol Environ Health A. 2005,68(21):1841-1860.
7. Lehr RE, Jerina DM. Metabolic activations of polycyclic hydrocarbons. Structure-activity relationships. Arch Toxicol. 1977,39(1-2):1-6.
8. Kim JH, Stansbury KH, Walker NJ, Trush MA, Strickland PT, Sutter TR. Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1. Carcinogenesis. 1998 Oct;19(10):1847–1853.
9. Payne, S., 1958. The pathological effects of the intraperitoneal injection of 3:4-benzopyrene into rats and mice. Br. J. Cancer 12, pp. 65–74.
10. Inyang, F., Ramesh, A., Kopsombut, P., Niaz, M.S., Hood, D.B., Nyanda, A.M. and Archibong, A.E., 2003. Disruption of testicular steroidogenesis and epididymal function by inhaled benzo[a]pyrene. Reprod. Toxicol. 17, pp. 527–537.
11. Ciolino HP, Daschner PJ, Yeh GC. Resveratrol inhibits transcription of CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor[J]. Cancer Res.1998,58:5707-5712.
12. Mandal, P. K.; McDaniel, L. R.; Prough, R. A.; Clark, B. J.: 7, 12-Dimethlbenz(a)anthracene inhibition of steroid production in MA-10 mouse Leydig tumor cells is not directly linked to induction of CYP1B1. Toxicol Appl Pharmacol., 2001, 175, 200-208.
13. Ramesh, A.; Inyang, F.; Hood, D. B.; Knuckles, M. E.: Aryl hydrocarbon hydroxylase activity in F-344 rats subchronically exposed to benzo(a)pyrene and fluoranthene through diet. J Biochem Mol Toxicol., 2000, 14, 155-161.
14. Ramesh, A.; Hood, D. B.; Inyang, F.; Greenwood, M.; Archibong, A.E.; Knuckles, M. E et al.: Comparative metabolism, bioavailability and toxicokinetics of benzo(a)pyrene in rats after acute oral, inhalation, and intravenous administration. Polycyclic Aromatic Compounds, 2002, 22: 969-980.
15. Lee, I. P.; Nagayama, J.: Metabolism of benzo(a)pyrene by the isolated perfused rat testis. Cancer Res., 1980, 40, 3297-3303.
16. Polyakov, L. M.; Chasovskikh, M. I.; Panin, L. E.: Binding and transport of benzo(a)pyrene by blood plasma lipoproteins: the possible role of apolipoprotein B in this process. Bioconjugate Chem 1996, 7, 396-400.
17. Williams, J. A.; Martin, F. C.; Muir, G. H.; Hewer, A.; Grover, P. L.; Phillips, D. H.: Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue. Carcinogenesis, 2000, 21, 1683-1689.
18. Senft, A. P.; Dalton, T. P.; Nebert, D. W.; Genter, M. B.; Puga, A.; Hutchinson, R. J, et al.: Mitochondrial reactive oxygen production is dependent on the aromatic hydrocarbon receptor. Free Radic Biol Med, 2002, 33, 1268-1278.
19. Baird WM, Ralston SL. Carcinogenic polycyclic aromatic hydrocarbons. In: Bowden GT, Fischer S, editors. Comprehensive toxicology. Chemical carcinogens and anticarcinogens, vol. 12; 2004. p. 171–200.
20. Diemer, T.; Allen, J. A.; Hales, K. H.; Hales, D. B.: Reactive oxygen disrupts mitochondriain MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis. Endocrinology, 2003, 144, 2882-2891.
21. Ramesh A, Inyang F, Hood DB, Archibong AE, Knuckles ME, Nyanda AM. Metabolism, bioavailability, and toxicokinetics of benzo(a)pyrene in F-344 rats following oral administration. Exp Toxicol Pathol 2001;53:275–90.
22. Ramesh A, Hood DB, Inyang F, Greenwood M, Archibong AE, Knuckles ME, et al.Comparative metabolism, bioavailability and toxicokinetics of benzo(a)pyrene in rats after acute oral, inhalation, and intravenous administration. Polycyclic Aromat Compd 2002;22:969–80.
23. Lee IP, Nagayama J. Metabolism of benzo(a)pyrene by the isolated perfused rat testis. Cancer Res 1980;40:3297–303.
24. Williams JA, Martin FC, Muir GH, Hewer A, Grover PL, Phillips DH. Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue. Carcinogenesis 2000;21:1683–9.
25. Yardimci S, Atan A, Delibasi T, Sunguroglu K, Guven MC. Long-term effects of cigarette-smoke exposure on plasma testosterone, luteinizing hormone and follicle-stimulating hormone levels in male rats. Br J Urol 1997;79:66–9.
26. Archibong AE, Ramesh A, Niaz MS, Brook CM, Roberson SI, Lunstra DD. Effects of Benzo(a)pyrene on intra-testicular function in F-344 rats. Int. J. Environ. Res. Public Health. 2008;5:32–40.
27. Archibong AE, Inyang F, Ramesh A, Greenwood M, Nayyar T, Kopsombut P, et al. Alteration of pregnancy related hormones and fetal survival in F-344 rats by inhaled benzo(a)pyrene. Reprod Toxicol 2002;16:801–8.
28. Raj AS, Katz M. Inhibitory effects of alpha- and betanaphthoflavones on DMBA-induced anomalies in germ cells assessed by sperm normality assay. Mutat Res 1984; 136:81–4.
29.金明华,石龙,刘晓梅,孙志伟,郝民,刘亚辉.苯并[a]芘对雄性小鼠睾丸细胞周期的影响.中国公共卫生,2004, 20(2):153-154
30.郝民.苯并[a]芘的生殖毒性及其作用机制研究[M]. 1999-2002硕士学位论文.
31. A. Ramesh, F. Inyang, D.D. Lunstra, M.S. Niaz, P. Kopsombut, K.M. Jones, D.B. Hoode, E.R. Hills and A.E. Archibong, Alteration of fertility endpoints in adult male F-344 rats by subchronic exposure to inhaled benzo(a)pyrene, Exp. Toxicol. Pathol. 60 (2008), pp. 269–280.
32.陈晓东,吴玲,罗绥兰.香烟烟雾提取物致雄性小鼠生殖细胞染色体畸变与睾丸组织中SOD和MDA含量的变化[J].预防医学文献信息,20028(1):46-47.
33. Vine MF. Smoking and male reproduction:a review. Int Jandrol.1996,19:323-37.
34. Choi H, Jedrychowski W, Spengler J, et al. International studies of prenatal exposure topolycyclic aromatic hydrocarbons and fetal growth[J]. Environ Health Perspect. 2006,114(11):1744-1750.
35. Pierantoni R, Cobellis G, Meccariello R, et al. The amphibian testis as model to study germ cell apoptosis during spermatogenesis[J]. Comp Biochem Physiol Biochem Mol Biol, 2002, 132(1): 131-139.
36. Raychoudhury SS, Kubinski D. Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response[J]. Environ Health Perspect. 2003,111(1):33-38.
37. Vine MF, Margolin BH, Morrison HI, et al. Cigarette smoking and sperm density: a meta-analysis[J]. Fertil Steril. 1994 ,61(1):35-43.
38. Maeda Y, Shiratsuchi A, Namiki M, et al. Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility[J]. Cell Death Differ, 2002,9 (7): 742-749.
39. Taylor CT. Antioxidants and reactive oxygen species in human fertility. Environ Toxicol Pharmacol 2001;10:189–98.
40. Aitken RJ, Harkiss D, Buckingham D. Relationship between iron-catalyzed lipid peroxidation and human sperm function. J Reprod Fertil 1993;98:257–65.
41. Selevan SG, Borkovec L, Zudova Z, Slott V, Hanjova R, Rubes J, et al. Semen quality in young men and air pollution in two Czech communities. Epidemiology 1995;6(Suppl):S85.
42. Sram RJ, Binkova B, R?ssner P, Rube? J, Topinka J, Dejmek J. Adverse reproductive outcomes from exposure to environmental mutagens. Mutat Res 1999;428:203–15.
43.徐晨,周作民.生殖生物学理论与实践[M].上海:上海科学技术文献出版社,2005.
44. Vinggaard AM, Hnida C, Larsen JC. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro. Toxicology 2000;145:173–83.