【作者】 赵清；

【导师】 舒为群；

【作者基本信息】 第三军医大学 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 目前有机污染物在环境中广泛分布,尤其是持久性有机污染物( Persistent organic pollutions,POPs)对水环境的污染日益严重。我国水环境皆受到不同程度POPs的污染,污染种类以邻苯二甲酸酯类(Phthalatic acid esters, PAEs)和多环芳烃类(Polycyclic aromatic hydrocarbon, PAHs)尤为突出。人群可能同时长期低剂量暴露于这两类污染物,因此评价这两类化合物的联合作用对人类健康的影响很有必要。邻苯二甲酸二丁酯(Di-n-butyl phthalate,DBP)和苯并(a)芘(Benzo(a)pyrene, BaP)分别是这两类化合物中典型和具有代表性的污染物。虽然已有研究表明这两种化合物均具有雄性生殖发育毒性,但迄今为止未见有这两种物质联合作用下的生殖毒性研究报道,尤其是低剂量长期暴露的生殖毒性。既往研究表明,睾丸Leydig细胞是这两种化合物的主要靶细胞,可以影响睾酮的合成和分泌。本课题以DBP和BaP作为受试物分别代表PAEs和PAHs,以青春期雄性SD大鼠作为研究对象,采用亚慢性染毒方式,给予较低剂量的DBP和BaP,建立大鼠长期低剂量暴露模型;聚焦于观察DBP和BaP单独或联合染毒对青春期大鼠睾丸及Leydig细胞形态、结构和功能的影响。此外,Leydig细胞合成睾酮涉及多种调节因子和合成酶,因此,本研究采用Realtime-PCR、Western blot等手段检测与睾酮合成密切相关的胆固醇跨膜转运调节因子、睾酮合成关键酶、睾酮合成分泌调节激素受体、以及相关的调节因子在转录水平或翻译水平的变化以探讨相关机制,旨在增进对此类环境内分泌干扰物雄性生殖毒性及其机制的了解,为进一步深入研究奠定基础。实验方法1.实验动物及分组情况4-5周龄健康雄性SPF级SD大鼠168只,体重(89±17)g,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24只),即溶剂对照组(玉米油)、DBP低剂量组(50mg/kg)、DBP高剂量组(250mg/kg)、BaP低剂量组(1mg/kg)、BaP高剂量组(5mg/kg)、DBP+BaP低剂量联合组(50mg/kg DBP+ 1mg/kg BaP)、DBP+BaP高剂量联合组(250mg/kg DBP+ 5mg/kg BaP)。2.染毒方式及取材隔日染毒,等体积灌胃,灌胃体积2ml/kg,连续染毒90d。每天观察动物一般状况,每周称量体重1次,分别于染毒后30d、60d和90d,各组随机选取大鼠8只处死。分别取材用于检测各项指标。采集股动脉血,分离血清,采用放射免疫分析法测定血清睾酮(T)含量。采血后大鼠颈椎脱臼处死,立即剖取睾丸、附睾、心、肝、脾、肾,除去上述器官周围的脂肪结缔组织,吸尽脏器表面血液后,用电子天平称重,计算脏器系数,脏器系数以脏器重量(g)与体重(g)之比表示。睾丸称重后于冰上迅速切分为约50-100mg的小块,分装于冻存管中液氮保存。每组随机选取3个液氮冻存睾丸样本,用Realtime-PCR法检测睾丸胰岛素样因子3 (Insl3)、黄体生成素受体(LHR)、外周苯二氮卓受体(PBR)mRNA表达。每组随机选取3个液氮冻存睾丸样本,用Western blot法检测睾丸类固醇合成急性调节蛋白(StAR)、细胞色素胆固醇侧链裂解酶(P450scc)、3β-羟甾脱氢酶(3β-HSD)、17β-羟甾脱氢酶(17β-HSD)、转化生长因子α(TGF-α)和胰岛素样生长因子Ⅰ(IGF-Ⅰ)的蛋白表达。睾丸称重后每组选取3只大鼠的右侧睾丸,置于4%多聚甲醛固定液中固定,72h后常规石蜡包埋切片,HE染色,光镜下观察曲细精管和间质的组织病理学变化。每组随机选取3只大鼠的左侧睾丸,取材后迅速固定于2.5%的戊二醛,然后进行梯度酒精脱水、环氧树脂渗透、包埋、超薄切片,醋酸铀和柠檬酸铅染色,于透射电镜下观察超微结构的改变(TECNAI-10, Philip,荷兰)。3.统计学分析:数据经SPSS13.0统计软件处理,实验数据均以均数±标准差( x±s)的形式表示,组间均数差异的显著性比较采用单因素方差分析。结果与讨论一、邻苯二甲酸二丁酯和苯并(a)芘对大鼠生长发育及睾丸形态学的影响1.对体重和脏器系数的影响DBP单独染毒、BaP单独染毒以及DBP和BaP联合染毒在本研究的染毒剂量下未对大鼠一般生长发育情况和体重增长有明显影响。对大鼠睾丸、心脏、脾脏无明显毒性作用;1 mg/kg和5mg/kg BaP染毒60d可以使附睾系数比对照组下降(P<0.05);250mg/kg DBP染毒90d可升高肝脏系数(P<0.05),5mg/kg BaP染毒90d可降低肝脏系数(P<0.05);5mg/kg BaP染毒30d,以及50mg/kg DBP+ 1mg/kg BaP低剂量联合染毒30d都可使肾脏系数下降(P<0.05);250mg/kg DBP+ 5mg/kg BaP高剂量联合染毒60d使肾脏系数增高(P<0.05)。提示BaP对附睾的影响更明显,附睾可能是BaP的靶器官;肝脏、肾脏可能也是DBP和BaP的主要靶器官之一, DBP和BaP对肝、肾可能也有潜在毒性。见表1。2.对睾丸及Leydig细胞形态学的影响光镜下未观察到各染毒组睾丸形态有明显病理改变;电镜下各染毒组染毒90d均可引起大鼠睾丸Leydig细胞超微结构发生不同程度的变化,主要是线粒体、滑面内质网扩张,也可见核周隙增宽,髓样变,核固缩,染色质边集,细胞器固缩,密度增高,线粒体固缩。二、邻苯二甲酸二丁酯和苯并(a)芘对Leydig细胞激素合成的影响1.对血清睾酮的影响50mg/kg DBP染毒30d可升高血清睾酮含量(P<0.05),推测低剂量DBP染毒可能存在低剂量兴奋效应;1 mg/kg BaP染毒60d、5mg/kg BaP染毒30d-60d、50mg/kg DBP+ 1mg/kg BaP低剂量联合染毒30d可升高血清睾酮含量(P<0.05)。提示≤5mg/kg BaP染毒30d-60d可以干扰睾酮的合成,可能BaP在此染毒剂量也有低剂量兴奋效应。见表2。2 .对睾丸Insl3 mRNA表达的影响高、低剂量DBP和BaP单独染毒以及联合染毒30d、60d、90d都能使睾丸Insl3 mRNA的表达下降(P<0.01)。提示转录水平的Insl3比睾酮对低剂量DBP和BaP更敏感,青春期亚慢性暴露于低剂量DBP和BaP可影响Leydig细胞的功能。见表2。三、邻苯二甲酸二丁酯和苯并(a)芘对Leydig细胞睾酮合成的影响机制探讨DBP和BaP单独染毒以及联合染毒30d、60d、90d对胆固醇跨膜转运调节因子、睾酮合成关键酶、睾酮合成分泌调节激素受体、以及相关的调节因子在转录水平或翻译水平的影响结果总结于表3。1.对胆固醇跨膜转运调节因子的影响DBP和BaP单独染毒以及低剂量联合染毒60d对PBR基因表达的影响主要是下调(P<0.01;P<0.05);高剂量联合染毒60d都是增强PBR的基因表达(P<0.01)。提示DBP和BaP单独以及联合染毒都对PBR的基因表达有影响,但是60d睾酮升高可能与PBR的上调或下调无关。DBP和BaP染毒60d使StAR蛋白表达增强(P<0.01),与睾酮水平升高一致,提示StAR蛋白表达升高可能在睾酮升高中发挥作用,是致睾酮升高的可能机制之一。2.对睾酮合成关键酶的影响DBP和BaP单独染毒以及联合染毒60d对P450scc蛋白表达的影响无统计学意义。提示睾酮升高可能与P450scc蛋白无关。5mg/kg BaP单独染毒30 d使3β-HSD蛋白表达增高(P>0.05),提示3β-HSD可能与5mg/kg BaP染毒30 d睾酮升高有关。250mg/kg DBP染毒60d可使17β-HSD蛋白表达升高(P<0.01);1mg/kg BaP染毒90d使其下调(P<0.01)。提示BaP染毒30 d引起睾酮升高可能没有17β-HSD的参与。3.对激素受体和相关调节因子的影响DBP和BaP单独染毒以及联合染毒都可使LHR mRNA表达下降(P<0.01);提示LHR在转录水平对DBP和BaP的毒性敏感,但是LHR基因表达的下降可能并非染毒60d血清睾酮升高的原因,DBP和BaP对睾酮的影响与受体调节途径无关,可能有非依赖LH通路的作用。50mg/kg DBP和1mg/kg BaP染毒30d、60d都使TGFα蛋白表达增高(P<0.01),5mg/kg BaP以及高、低剂量联合染毒30d都上调TGFα蛋白表达(P<0.01)。提示睾酮升高与TGFα蛋白表达上调一致,推测可能BaP通过增加TGFα的表达来调节睾酮。染毒组对IGF-Ⅰ蛋白表达的影响,30d、60d主要是上调(P<0.01;P<0.05),90d时下调(P<0.01;P<0.05)。BaP组染毒60d IGF-Ⅰ蛋白表达上调与睾酮升高一致,说明IGF-Ⅰ蛋白表达上调可能是本研究中DBP或BaP导致睾酮升高的机制之一。4. DBP和BaP高低剂量联合染毒对各观察指标的联合作用总结于表4。DBP和BaP联合染毒可发生交互作用,主要表现为拮抗,其次是协同,少数呈现非交互作用-相加。结论亚慢性DBP和BaP单独或联合暴露可对青春期SD雄鼠产生不利影响,不同的染毒时间产生不同程度的有害效应。BaP染毒可引起附睾、肝脏、肾脏系数改变,对这几个器官有潜在毒性。BaP染毒60d可使睾酮升高,推测StAR、3β-HSD、TGFα、IGF-Ⅰ蛋白的上调与其作用机制有关。50mg/kg DBP染毒60d升高血清睾酮,可能是DBP的低剂量兴奋效应,TGFα在其中可能发挥重要作用。由于联合作用主要表现为拮抗,因此本研究中染毒剂量下DBP和BaP联合染毒未见比单独染毒明显的毒性效应。本研究观察到血清睾酮主要是升高而并非下降,分析原因可能主要与染毒剂量和暴露年龄有关。一是本研究染毒剂量相对较低,可能出现低剂量兴奋效应;另外大鼠并非宫内暴露,而是青春期才开始暴露,因此没有宫内暴露敏感。DBP和BaP的联合毒性作用及机制还有待进一步验证和深入探讨。更多还原

【Abstract】 At present, organic contaminants are extensively distributed in the environment. Water surroundings were especially polluted by persistent organic pollutions(POPs) with increasing severity. Aquatic environment in China were contaminated by POPs at different degrees. Phthalatic acid esters (PAEs) and Polycyclic aromatic hydrocarbon (PAHs) are the most popular pollutants. As human beings could be exposed to PAEs and PAHs at a relatively low level for a long time, it’s necessary to evaluate the toxic effects of PAEs and PAHs on human health. Di-n-butyl phthalate(DBP) and Benzo(a)pyrene (BaP) were typical pollutants of PAEs and PAHs, respectively. Although many researches have showed that PAEs and PAHs have male reproductive developmental toxicity, data of the combined toxicity and potential threats to male reproduction posed by DBP and BaP remain limited. Many studies have revealed that Leydig cell of testes is one of the main target cells of DBP or BaP, and testosterone synthesis and secretion could be impacted by DBP or BaP. In the present study we chose DBP and BaP as the model chemicals of organic pollutants, and tested the single or combined effect of them on morphology and function of testicle leydig cell in puberty SD male rats. In addition, some regulatory factors and synthetase were involved in testosterone synthesis pathway in Leydig cells, therefore, we observed the effect of DBP and BaP on the mRNA and protein expression of these factors, and further studied the relationship among these factors and steroidogenesis induced by DBP and BaP, which would provide some reference indicators for deep understanding of its mechanism.Methods1. AnimalsA total of 168 male Sprague-Dawley rats (4-5 weeks old) were equally randomized into 7 groups (24 per group), alt. dieb., and respectively received by gavage corn oil , 50 mg/ kg of DBP , 250 mg/ kg of DBP ,1 mg/ kg of BaP,5mg/ kg of BaP, 50 mg/ kg of DBP +1 mg/ kg of BaP , 250 mg/ kg of DBP +5 mg/ kg of BaP for 90days. At the end of experimental period, i.e. 30,60 and 90 day after gavage of DBP or BaP, 8 rats per group were sacrificed randomly.2. treatment and methodsThe rats were anesthetized with pentobarbital and sacrificed by decapitation. Arteria femoralis blood was immediately collected before decapitation. The serum testosterone was separated and was determined with commercially available radioimmunoassay kits, according to the manufacturer’s instruction. Samples and standards were measured in duplicates in the same assay and the CV% were always <15%.Rats were immediately subjected to necropsy and the testes, epididymides and main organ samples were promptly removed, and weighed. Testes was used for gene expression of insulin-like factor 3(Insl3), luteinizing hormone receptor (LHR), peripheral benzodiazepine receptor (PBR) and protein expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 Side Chain Cleavage Enzyme (P450scc), 3 beta hydroxysteroid dehydrogenase (3β-HSD), 17 beta hydroxysteroid dehydrogenase (17β-HSD), transforming growth factor alpha (TGFα) and insulin-like growth factor 1 (IGF-Ⅰ)assay, and histopathological examination.3 rats randomly selected from each group were subjected to necropsy and the right testis was fixed in 4% Polyoxymethylene solution. One piece of tissues from each side of the testis were embedded in paraffin, sectioned at 4μm, stained with hematoxylin and eosin (H&E), and examined histologically. For electron microscope observation, testis of each group were selected randomly and fixed in 2.5% glutaraldehyde immediately after removal. After fixation, the specimens were processed through standard dehydration in graded ethanol before infiltration in Epon and embedded. The ultrathin sections were stained with uranyl acetate and lead citrate and viewed under transmission electron microscope (TECNAI-10, Philip, Netherlands).3. Statistical analysisStatistical significance was established using one-way analysis of variance (ANOVA) followed by the least significant difference test when multiple treatment groups were compared. The data were expressed as mean±standard deviation (SD) of 8 animals per group. The software package SPSS 13.0 was used for statistical analysis. For all statistical tests, significance was determined at a level of P<0.05. Results and discussion1. The effect of di-n-butyl phthalate and benzo(a)pyrene on rat growth and testis morphology.1.1 On the weight and organ coefficientAfter DBP individual exposure, BaP individual exposure and DBP/BaP coexposure at the dosage used in this study, no significant effect was found on rat growth or its weight increase, and no significant toxical effect was found in rat testis, heart and spleen. Compared with control, exposure to 1 mg/kg and 5 mg/kg BaP for 60 days could decrease liver coefficient (P<0.05); exposure to 250 mg/kg DBP for 90 days could increase liver coefficient (P <0.05) and exposure to 50 mg/kg BaP for 30 days could reduce liver coefficient (P <0.05); exposure to 5 mg/kg BaP for 30 days and coexposure to 50 mg/kg DBP and 1 mg/kg BaP for 30 days at low doses could reduce kidney coefficient (P <0.05). Coexposure to 250 mg/kg DBP and 5 mg/kg BaP for 60 days at hige doses could increase kidney coefficient (P <0.05). These results revealed that BaP had a greater effect on epididymis which might be the target organ of BaP. Liver and kidney might be two of the main target organs for DBP and BaP, which showed that they might have potential toxic effect on liver and kidney. (Table 1) 1.2 On the testis and Leydig cell morphologyNo significant pathological changes were observed by light microscope; After all groups exposed for 90 days, different levels of changes in ultramicrostructure of Leydig cells was found by electron microscope, which mainly demonstraed as the extension of mitochondria and smooth endoplasmic reticulum.2. The effect of di-n-butyl phthalate and benzo(a)pyrene on hormone synthesis in Leydig cells2.1 On serum testosteroneExposure to 50 mg/kg DBP could increase serum testosterone levels (P <0.05), which indicated that exposure to low doses of DBP might led to hormesis effect of low doses; exposure to 1 mg/kg BaP for 60 days, exposure to 5mg/kg BaP for 30-60 days, and coexposure to 50 mg/kg DBP and 1 mg/kg BaP for 30 days could increase the level of surum testosterone (P <0.05). This indicated that exposure to≤5mg/kg BaP for 30-60 days could interrupt the synthesis of serum testosterone and could have hormesis effect of low doses (Table 2).2.2 On the expression of Insl3 RNA in the testisExposure and coexposure to high/low doses of DBP and BaP for 30, 60 and 90 days could reduce the expression of Insl3 mRNA in the testis (P < 0.01). This indicated that Insl3 at the transcriptional level was more sensitive than the low doses of DBP and BaP. In adolecent age, subchronical exposure to low doses of DBP and BaP might influence the function of Leydig cells (Table 2). 3. Discussion on the influence mechanisms of di-n-butyl phthalate and benzo(a)pyrene on testosterone synthesis in Leydig cells.Table 3 demonstrated the effect of individual exposure and coexposure to DBP and BaP (30 days, 60 days and 90 days) on cholesterol transmembrane transport regulatory factor, key enzymes in testosterone synthesis, hormone receptor and hormone-related adjusting factors and the related factors at the transcriptional level or translational level.3.1 The influence on cholesterol transmembrane transport regulatory factorIndividual exposure to DBP and BaP, and coexposure to low doses of DBP and BaP for 60 days mainly decreased PBR gene expression (P <0.01; P <0.05); coexposure to high doses of DBP and BaP for 60 days could increase PBR gene expression (P <0.01). This indicated individual exposure or coexposure to DBP and BaP had an effect on PBR gene expression, however, the increase of testosterone level on the 60th day might have little to do with the upregulation or downregulation of PBR.Exposure to DBP and BaP for 60 days could increase the expression of StAR protein (P >0.05), which is in accordance with the increase of testosterone level. This revealed that the increase of StAR protein expression might play a role in increasing the testosterone level, which might be one of the mechanisms for increasing the testosterone level.3.2 On key enzymes in testosterone synthesisThere is no statistical significance between individual exposure or coexposure to DBP and BaP for 60 days and P450scc protein expression,which demonstrated that the increase of testosterone level might be irrelevant to P450scc protein expression.Individual exposure to 5 mg/kg BaP for 30 days raised the expression of 3β-HSD protein (P >0.05). This suggested that 3β-HSD protein might be related to the increase of testosterone level after 30 days of 5 mg/kg BaP exposure.Exposure to 250 mg/kg DBP for 60 days could raise the expression of 17β-HSD protein (P <0.01) while exposure to 1 mg/kg BaP for 90 days could decrease its expression (P <0.01). This indicated that after exposed to BaP for 30 days 17β-HSD might not participate in the increase of testosterone level.3.3 On hormone receptor and hormone-related adjusting factorsBoth individual exposure and coexposure to DBP and BaP could reduce LHR mRNA expression (P <0.01). This revealed that LHR was sensitive to DBP and BaP at the transcriptional level, however the decrease of LHR mRNA expression might not be induced by the increase of serum testosterone after 60 days exposure, and the influence of DBP and BaP on the testosterone level was unrelated to the receptor adjusting pathway . This might relate to LH-independent pathway.Exposure to 50 mg/kg DBP and 1mg/kg BaP for 30 days and 60 days could raise the TGFαprotein expression, exposure to 5 mg/kg BaP and coexposure to high/low doses of DBP and BaP for 30 days could increase TGFαprotein expression (P <0.01). This demonstrated that the increase of testosterone level was in accord with the increase of TGFαprotein expression, which might prove BaP adjusting testosterone level by increasing TGFαprotein expression.The effect of these exposure group on IGF-I protein expression: exposure for 30 days and 60 days upregulated IGF-I protein expression (P <0.01; P <0.05) and 90 days exposure downregulated IGF-I protein expression (P <0.01; P <0.05). In this study, after exposure to BaP for 60 days, the upregulation of IGF-I protein expression is the same as the increase of testosterone level, which revealed that the upregulation of IGF-I protein expression was one of the mechanisms for DBP or BaP induced testosterone level increase.3.4 The combined effect of exposure to high/low doses of DBP and BaP showed in Table 4. ConclusionSubchoronic individual exposure or coexposure to DBP and BaP had negative effects on the adolecent SD male rat and different exposure time could induce different degrees of damage. Exposure to BaP could change epididymis coefficient,liver coefficient and kidney coefficient. Exposure to BaP for 60 days could increase testosterone level, which indicated that the upregulation of StAR protein, 3β-HSD protein, TGFαprotein and IGF-I protein was related to their functional mechanisms. After 60-day exposure to 50 ma/kg DBP, serum testosterone level was raised, which might be hormesis effect induced by low dose of DBP exposure. And TGFαmight play an important role in this process. Coexposure to DBP and BaP could have interaction effects, which demonstrated as antagonism and synergism with few showed non-interaction effect (addition). In this study, serum testosterone level was increased but not decreased, which might relate to the exposure doses and exposure age: the DBP exposure dosage adopted in this study was camparetively low, which might induce hormesis effect; and adolecent exposure were used instead of utero exposure which might not cause the rat be sensible to intrauterine exposure.Further studies is required to provide more evidence for the toxical effect of DBP and BaP coexposure and its mechanisms.更多还原