邻苯二甲酸酯类和多环芳烃类代表物质联合雌性生殖毒性与健康风险评价研究
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摘要
环境污染物低剂量联合暴露对机体的健康危害是目前环境及毒理学界关注的重要问题。国内外多项研究显示,众多化学污染物中以POPs污染最为严重。三峡库区水环境POPs污染的主要特点是以邻苯二甲酸酯类(Phthalates acid esters, PAEs)和多环芳烃类(Polycyclic aromatic hydrocarbons, PAHs)物质为代表的复合性污染。PAEs和PAHs类物质大多具有明确雌性生殖毒性,但两者联合是否具有协同或拮抗生殖毒性尚不得而知,探究低剂量联合作用雌性生殖毒性及其机制势在必行。
基于PAEs和PAHs类物质潜在的女(雌性)性生殖危害,本研究选取PAEs和PAHs的代表性物质,具有明确雌性生殖毒性的邻苯二甲酸二(2-乙基己基)酯(di-(2-ethylhcxyl) phthalate, DEHP)与苯并[a]芘(benzo[a]pyrene, B[a]P)及其代谢产物单-(2-乙基己基)邻苯二甲酸酯(MEHP)与反式二氢二醇环氧苯并芘(BPDE),以整体动物和细胞模型为手段,探讨两种毒物低剂量联合作用下的雌性生殖危害的特点,并从颗粒细胞的结构、功能以及PPARs-Arom/17β-HSD信号通路调节雌激素合成等方面探讨其机制。此外,采用统计学析因分析对DEHP和B[a]P(MEHP和BPDE)的联合生殖毒性作用进行定性评价。最后,结合三峡库区水环境PAEs和PAHs类物质的现实污染状况,本研究构建了水环境POPs污染物健康风险评价模型,应用前期水环境污染监测资料,对三峡库区干流主要断面6种PAEs和PAHs代表物质进行健康风险评价。研究旨在探讨DEHP与B[a]P联合作用下雌性生殖毒性危害的特点及作用机制,并对环境PAEs和PAHs暴露对人群的健康危害进行评估,研究将为POPs的防治和人群健康的保护奠定基础,为决策机构提供决策依据和参考。
研究内容:
第一部分DEHP和B[a]P联合暴露对雌性大鼠的生殖毒性及其机制研究
1. 4～5周龄健康雌性SD大鼠随即分7组,每组12只。实验分组为:正常对照组(玉米油);B[a]P高、低剂量组(B[a]P10mg/kg和B[a]P5mg/kg);DEHP高、低剂量组( DEHP600mg/kg和DEHP300mg/kg ); B[a]P+DEHP高、低剂量组(B[a]P10mg/kg+DEHP600mg/kg和B[a]P5mg/kg+ DEHP300mg/kg)。
2.采用隔日灌胃方式连续染毒60 d,受试动物于染毒30 d和60 d结束后分两批次,清晨1%戊巴比妥钠腹腔麻醉后心脏取血,进行各项指标的取材、检测。观察动物的生长发育、脏器系数、动情周期、血清激素水平、卵巢卵泡发育及组织病理学改变、颗粒细胞凋亡等指标。运用RT-PCR、Western blot和免疫组化等方法检测雌激素合成通路PPARs、P450Arom和17β-HSD等基因和蛋白的表达差异。
第二部分MEHP和BPDE低剂量联合对体外卵巢颗粒细胞的毒性及其机制
1.采用机械分离结合胰蛋白酶消化与低速离心的方法分离培养卵巢颗粒细胞。
2.筛选MEHP和BPDE染毒剂量,建立MEHP和BPDE低剂量联合染毒颗粒细胞模型,进行联合染毒3×3析因分析。染毒分组:Control(1%DMSO); MEHP(25μM; 50μM); BPDE(10 nM; 100 nM); MEHP+BPDE(25μM+10 nM;25μM+100 nM;50μM+10 nM;50μM+100 nM)。通过MTT实验与细胞形态学观察评价MEHP和BPDE联合作用对颗粒细胞的毒性。
3.采用RT-PCR、细胞免疫化学染色法检测MEHP和BPDE联合染毒后颗粒细胞PPAR-γ和P450Arom基因和蛋白表达,在细胞水平探讨联合毒性机制。
第三部分三峡库区水环境邻苯二甲酸酯类和多环芳烃类代表物质健康风险评价
1.三峡库区干流设置7个水质监测点,采集2005年2月和8月枯、丰水期水样,采用瓦里安GC3800/MS200型气质联用仪进行水中POPs分析测定。
2.介绍健康风险评价方法,建立了水环境致癌物/非致癌物健康风险评价模型。选取水中邻苯二甲酸二丁酯(DBP)、邻苯二甲酸二乙基酯(DEP)和邻苯二甲酸二(2-乙基己基)酯(DEHP)、萘(NA)、芘(Pyr)、萤蒽(FLA)等6种主要PAEs和PAHs物质,分析污染数据并进行健康风险评价。
研究结果:
第一部分DEHP和B[a]P联合暴露雌性大鼠的生殖毒性及其机制
1. B[a]P、DEHP单独与B[a]P+DEHP染毒可造成动物体重增长减缓,但与正常对照相比未见显著差异(P>0.05)。B[a]P+DEHP染毒30 d即可导致卵巢重量减轻,卵体系数下降(P<0.05)。随着染毒时间延长、剂量增加,呈现剂量与时间反应趋势。B[a]P与B[a]P+DEHP相比,卵体系数具有显著差异(P<0.05)。析因分析表明,B[a]P+DEHP染毒30 d和60 d对卵体系数无显著交互作用。
2. B[a]P、DEHP单独与B[a]P+DEHP染毒导致EC延长、E逐渐缩短。染毒30 d, B[a]P10mg/kg+DEHP600mg/kg组EC和No E时间延长(P<0.05)。染毒60 d,各染毒组(B[a]P5mg/kg组除外)EC和No E时间显著延长(P<0.01),B[a]P+DEHP和DEHP染毒可导致E时间显著缩短(P<0.05);B[a]P10mg/kg+DEHP600mg/kg组分别与B[a]P5mg/kg+DEHP300mg/kg组和B[a]P10mg/kg组相比,EC和No E具有显著差异(P<0.01)。析因分析表明,B[a]P+DEHP染毒对EC和E无显著交互作用。
3. B[a]P+DEHP染毒导致血清E2、P和T含量显著降低。染毒30 d,DEHP600mg/kg和B[a]P10mg/kg+DEHP600mg/kg组大鼠血清E2和P含量明显降低(P<0.05)。染毒60 d,各染毒组(B[a]P5mg/kg组除外)血清E2和P含量继续下降(P<0.05);高、低剂量B[a]P+DEHP组分别与高、低剂量B[a]P组相比,血清E2水平显著降低(P<0.05)。染毒60 d,各组(DEHP300mg/kg组除外)血清T含量与对照组相比显著降低(P<0.05)。各染毒组血清FSH和LH含量未见显著改变。析因分析表明,B[a]P+DEHP联合染毒30 d和60 d对大鼠血清E2和P无显著交互作用(P>0.05)。
4. B[a]P+DEHP染毒导致卵巢原始卵泡和初/次级卵泡数目减少,闭锁卵泡数目增多。染毒30 d,B[a]P、DEHP(DEHP300mg/kg组除外)和B[a]P+DEHP染毒导致原始卵泡和初/次级卵泡数目减少(P<0.05),DEHP600mg/kg组和B[a]P10mg/kg+ DEHP600mg/kg组闭锁卵泡明显增加(P<0.05)。染毒60 d,B[a]P和B[a]P+DEHP染毒可导致原始卵泡、初/次级卵泡数目减少(P<0.05),DEHP和B[a]P+DEHP染毒可导致闭锁卵泡数目增多(P<0.05);B[a]P+DEHP组与DEHP组比较,原始卵泡数目具有显著差异(P<0.05),B[a]P10mg/kg+DEHP600mg/kg组与B[a]P10mg/kg组比较闭锁卵泡显著增加(P<0.05)。析因分析表明,B[a]P+DEHP染毒30 d和60 d对大鼠原始卵泡、初/次级卵泡、卵泡闭锁和黄体的数目无显著交互作用。
5.光镜下,B[a]P、DEHP单独与B[a]P+DEHP染毒均导致颗粒细胞结构疏松,变性、脱落,细胞数量减少;B[a]P和B[a]P+DEHP染毒出现颗粒细胞与膜细胞间隙增宽,细胞坏死。电镜下,B[a]P、DEHP单独与B[a]P+DEHP染毒均造成颗粒细胞线粒体肿胀与空泡化,内质网扩张甚至髓鞘样结构形成,可见凋亡小体。随着染毒时间延长、剂量增加,颗粒细胞损伤加重,局灶性坏死和髓鞘样结构形成增多。
6. B[a]P、DEHP单独与B[a]P+DEHP染毒30 d和60 d均造成颗粒细胞凋亡增加,呈现剂量-反应趋势(P<0.05),Caspase-3的激活参与了细胞凋亡启动,联合染毒卵泡闭锁增加与细胞凋亡发生高度关联。随着染毒时间延长和剂量增加,B[a]P和B[a]P+DEHP染毒组细胞凋亡反而出现下降趋势,B[a]P+DEHP组与B[a]P或DEHP组相比出现显著差异(P<0.05)。析因分析表明,B[a]P+DEHP染毒对卵巢颗粒细胞凋亡和Caspase-3表达均具有交互作用(P<0.01),提示两者联合主要表现为协同作用。
7.免疫组化和Western blot蛋白检测与基因表达结果一致。无论染毒30 d或60 d,B[a]P、DEHP和B[a]P+DEHP染毒导致P450Arom mRNA和蛋白表达下调,P450Arom mRNA下调与PPAR-α和PPAR-γmRNA蛋白表达上调高度关联(P<0.05);此外,DEHP和B[a]P+DEHP染毒组卵巢17β-HSDΠmRNA表达水平显著升高(P<0.05)。在就PPARs/P450Arom通路而言,DEHP在联合作用机制中发挥主导作用。析因分析表明,B[a]P+DEHP染毒对P450Arom、PPAR-α、PPAR-γ和17β-HSDΠmRNA表达无显著交互作用,而对P450Arom和PPAR-γ蛋白表达存在交互作用(P<0.05)。
第二部分MEHP和BPDE低剂量联合对卵巢颗粒细胞毒性及其机制
1.采用机械分离法结合胰蛋白酶消化及低速离心法分离培养的卵巢颗粒纯度高、活性好,48 h～96 h大量增殖,生长旺盛。HE和FSHR表达染色鉴定颗粒细胞是一种简便快速的方法。
2. MEHP和BPDE对体外培养卵巢颗粒细胞具有毒性,BPDE对颗粒细胞的毒性明显大于MEHP。MEHP 200μM染毒24 h颗粒细胞活性明显抑制;BPDE 10 nM染毒24 h颗粒细胞生长即出现显著抑制(P<0.05)。
3. BPDE和MEHP+BPDE染毒12 h和24 h均从一定程度上抑制颗粒细胞活性(P<0.05),析因分析表明,MEHP+BPDE染毒对颗粒细胞活性不存在交互作用(P>0.05),提示联合作用表现为相加作用。BPDE和MEHP+BPDE染毒可导致细胞萎缩、胞核缩小、出现空泡化与坏死,随着染毒剂量增加,毒性作用更为明显。
4. MEHP+BPDE染毒在一定剂量水平可导致颗粒细胞PPAR-γmRNA和蛋白表达上调,P450Arom mRNA和蛋白表达下调,PPARs/P450Arom途径可能是MEHP和BPDE联合作用颗粒细胞的毒性机制。析因分析表明,MEHP+BPDE染毒对颗粒细胞P450Arom和PPAR-γmRNA和蛋白表达均存在交互作用(P<0.05),提示联合毒性表现为协同作用。
第三部分三峡库区水环境PAEs和PAHs代表物质健康风险评价
1.三峡库区2005年枯、丰水期水源水分别检出有机物178种和144种,其中属于美国环保局优先控制污染物有18种,属于我国水环境优先控制污染物7种;检出率较高的是PAHs和PAEs两类物质。在7个监测点中,水体DBP、DEP、DEHP、NA、Pyr、FLA等6种物质检出范围为ND～3.60×10-3。
2.三峡库区水源水6种有机污染物由饮水途径所致健康危害的个人年风险介于6.34×10-14 a-1～3.99×10-11 a-1范围,按年风险大小排列为:DEHP>DBP>Pyr>NA>FLA>DEP;水中6种有机污染物对人体健康危害的年平均总风险仅为8.86×10-12 a-1,远低于国际辐射防护委员会(ICRP)推荐的最大可接受值5.0×10-5·a-1。在库区各断面中,处于长江和嘉陵江汇合的寸滩断面有机物健康危害风险最大,涪陵和开县断面次之。
结论
1. PAEs类和PAHs类代表性物质,DEHP和B[a]P及其代谢产物(MEHP和BPDE)联合具有雌性生殖毒性,卵巢颗粒细胞是两者共同作用的靶细胞。
2. DEHP和B[a]P联合染毒导致大鼠卵体系数降低,动情周期延长和动情期缩短,大鼠血清E2、P和T水平明显降低,卵巢原始卵泡、初/次级卵泡数目减少,闭锁卵泡数目增多,细胞结构出现病理损伤;DEHP和B[a]P联合对上述指标不具有交互作用,DEHP在毒性效应中发挥主导作用。MEHP和BPDE低剂量联合染毒导致体外培养颗粒细胞活性降低,细胞出现萎缩、空泡化与坏死;MEHP和BPDE低剂量联合染毒对卵巢颗粒细胞活性不具有交互作用,两者联合表现为相加作用。
3. DEHP和B[a]P联合染毒诱发颗粒细胞凋亡发生增加,细胞凋亡与卵泡闭锁增加高度关联;Caspase-3的激活在细胞凋亡发生过程中发挥调控作用。DEHP和B[a]P联合染毒对卵巢颗粒细胞凋亡和Caspase-3表达具有交互作用,两者联合主要表现为协同作用。
4.体内与体外实验相继证实,DEHP和B[a]P(MEHP和BPDE)联合毒性效应大于每种化学物质的单独作用,主要表现为毒性增强作用,联合类型以非交互作用为主。就毒性机制而言,DEHP和B[a]P(MEHP和BPDE)干扰PPARs/P450Arom信号通路导致雌激素合成障碍是联合毒性作用机制之一,DEHP和B[a]P(MEHP和BPDE)对P450Arom和PPARs蛋白表达具有交互作用,联合作用表现为协同作用。
5.三峡库区水环境PAEs和PAHs类有机物检出种类和含量较高,6种POPs污染物所致的健康危害年风险度目前还处于很低水平,但应引起管理部门的重视。
Background and objective
Nowadays, expanding urbanization and industrialization has led to ubiquitous chemical contaminants in environment that have clearly been defied as endocrine disrupting chemicals and these problems have been received much more concern by scientists. Persistent organic pollutants (POPs) are chemical substances that persist in the environment, bioaccumulate through the food web, and subsequently pose potential risks to human health. Many research studies showed that POPs pollution is becoming the most serious problem all over the world. The main pollution chemicals of POPs in the water body of the Three Georges reservoir was characterized by phthalates acid esters (PAEs) and polycyclic aromatic hydrocarbons (PAHs). Phthalates acid esters and polycyclic aromatic hydrocarbons have clearly been defied as endocrine disrupting chemicals, a great deal of literatures indicates that these environmental toxicants may accumulate in ovary and cause reproductive disorders. Traditionally, reproductive studies have focused on healthy effects associated with exposure to single environmental endocrine disrupting chemical, but exploration of interactions among these chemicals is an important and also key area of enquiry. The toxicity of chemical mixtures might be lower or higher than predicted from the known individual effects of each chemical, which could be a synergistic, antagonistic, potentiation or inhibitory interaction in any mixture-related studies. In addition, adult women, children, particularly neonates, can be biologically more sensitive to the mixture toxicants exposure on physiological functions and characteristics basis.
In our studies conducted in the Three Gorges reservoir, the higher average concentrations of PAEs and PAHs chemicals were found in aquatic environment, thus dietary exposure to these chemicals is possible which can occur simultaneously. Taking these facts into consideration, the present study was divided into three parts. In terms of part one and two, we chose the representative chemicals of PAEs and PAHs, namely DEHP and B[a]P separately, also their active metabolites MEHP and BPDE, as the model chemicals to evaluate the combined female reproductive toxicity as well as their possible mechanism in vivo and in vitro study. The third part introduced the health risk assessment method and established health risk assessment model for POPs contamination in water environmental so as to explore potential healthy hazards of POPs in the Three Gorges reservoir. The purpose of this study is to evaluate the combined reproductive toxicity and involved mechanism, provide experiment data and explore a method to evaluate the adverse effect of organic pollutants mixture on human health.
Materials and Methods
PartⅠFemale reproductive toxicity following DEHP and B[a]P combination.
1. This study was to determine the female reproductive toxicity and possible molecular mechanism of oral exposure to DEHP and B[a]P combination following 30 and 60days using Sprague-Dawley (SD) rat’s model. Females SD rats were randomly assigned to seven groups of 12 animals at each group. Female rats were given intragastrical administration of corn oil (control), B[a]P alone (B[a]P5mg/kg and B[a]P10mg/kg), DEHP alone (DEHP300mg/kg and DEHP600mg/kg) and B[a]P+DEHP (B[a]P5mg/kg+DEHP300mg/kg and B[a]P10mg/kg+DEHP600mg/kg) on alternate days for both 30 and 60days.
2. After the toxical exposure, rats were killed and general clinical observation, reproductive organs, serum hormone level, ovarian follicle population, cell apoptosis, ovary histopathology, mRNA levels of target genes and proteins expression including PPARs, P450Arom and 17β-HSD in granulosa cells were evaluated.
PartⅡCytotoxicity of MEHP+BPDE in cultured granulosa cells
1. Obtain and identify the cultured granulosa cells from the ovary of impuberism rats so as to establish a convenient and stable cell experiment model.
2. The doses of MEHP and BPDE intoxication was chosen and 3×3 factorial design model in vivo study was established, As detailed as below, Control (DMSO), MEHP(25μM; 50μM), BPDE(10nM; 100nM), MEHP+BPDE (25μM+10nM; 25μM+100nM; 50μM+10nM; 50μM+100nM). Cytotoxicity of MEHP or BPDE alone and concomitant treatments was evaluated by MTT and histopathology observation.
3. The target genes and proteins expression including P450Arom and PPARs in cultured granulosa cells were evaluated using RT-PCR and immunohistochemistry methods.
PartⅢWater environmental health risk assessment of PAEs and PAHs.
1. Seasonal samplings were carried out in Feb and Aug of 2005. Water samples were collected from seven different sampling sites of trunks and tributaries in the Three Gorges reservoir. The organic pollutants samples were analyzed by GC/MS.
2. U.S.EPA’S model recommended for water environmental health risk assessment was improved based on the water monitoring data in the year of 2005. The health risk assessment method was introduced for organic pollutants contamination in water environment and selected 6 kinds of organic pollutants (PAEs and PAHs), which is DBP, DEP, DEHP, NA, Pyr and FLA, to carry out data analysis and evaluate the total health risk.
Results
PartⅠIn vivo study
1. The growth and development of rats in DEHP and B[a]P alone and combination groups was not were obviously inhibited compared with control (P>0.05). The absolute ovary weight and ovary/body weight ratio of rats exposed to B[a]P+DEHP treatment for 30 and 60 days was significantly decreased compared to control (P<0.05), dose- and time-response tendency appeared. The joint effect of B[a]P+DEHP on ovary/body weight ratio appeared no interaction.
2. DEHP and B[a]P alone or combination resulted in prolonged estrous cycle as well as shorten estrus phase in animals following 30 and 60 days exposure compared to control (P<0.05). After 60 days exposure, B[a]P and DEHP alone or combination (besides B[a]P5mg/kg) resulted in prolonged EC and No E (P<0.01), the estrous phase of B[a]P10mg/kg+DEHP600mg/kg and DEHP600mg/kg treated groups was shortened (P<0.05), there was significant difference between B[a]P10mg/kg+DEHP600mg/kg and B[a]P10mg/kg or B[a]P5mg/kg+DEHP300mg/kg (P<0.01). The joint effect of B[a]P+DEHP on EC and E appeared no interaction.
3. It showed that B[a]P+DEHP caused remarkable changes in serum hormone (E2 and P) levels following 30 day exposure. B[a]P (besides B[a]P5mg/kg) or DEHP alone and B[a]P+DEHP significantly reduced the serum E2 ,P and T levels following 60 days exposure compared to the control (P<0.05), serum E2 level was significantly decreased in B[a]P+DEHP compared to B[a]P intoxication (P<0.05). It seemed there was no significant changes in terms of serum FSH and LH levels in either following 30 or 60 days exposure (P>0.05). The joint effect of B[a]P+DEHP on serum E2 and P appeared no interaction.
4. B[a]P+DEHP resulted in decreased primordial and primary/secondary follicle populations, and increased atretic populations. After 30 days exposure, B[a]P or DEHP alone (besides DEHP300mg/kg) and B[a]P+DEHP had significantly reduced primordial follicle populations of ovaries compared to control (P<0.05). After 60 days exposure, it was shown that only the primordial populations significantly decreased in B[a]P+DEHP and B[a]P10mg/kg compared to control (P<0.05), The primordial follicle populations of B[a]P+DEHP significantly decreased compared to DEHP alone (P<0.05). The atretic numbers in the high dose of DEHP and B[a]P+DEHP significantly increased (P<0.05). The joint effect of B[a]P+DEHP on primordial follicles, primary/secondary follicles, atretic follicles and corpora lutea appeared no interaction.
5. B[a]P and DEHP alone or combination resulted in granulosa cell structure damage. cellular structure rarefaction, exfoliation and degeneration was observed in any treated groups under light microscope, additionally, the interspaces between granulosa cells and theca cells widened in B[a]P and B[a]P+DEHP treated groups. B[a]P+DEHP caused wide spread cellular swelling and lysis of plasma membranes. Mitochondrial cristae appeared swelling and collapse, cellular myelin figure and apoptotic body was observed with electron microscope. Accompanied by exposure extension, cellular damage was more serious, especially focal necrosis and myelin figure was observed frequently.
6. Whenever following 30 or 60 days exposure, B[a]P or DEHP alone (except DEHP300mg/kg) and B[a]P+DEHP dramatically increased the granulosa cells apoptosis in ovary compared to control (P<0.05), caspase-3 activation involved in the process of apoptosis occurrence (P<0.05). Remarkable increase of granulosa cell apoptosis was found in B[a]P+DEHP, however, cell apoptosis index decreased while following 60 days exposure. There was high correlation between apoptosis and atretic follicle occurrence. The joint effect of B[a]P+DEHP on granulosa cells apoptosis and caspase-3 expression appeared obvious interaction (P<0.05).
7. The target gene expression of P450Arom and PPARs was in coincidence with immunohistochemical and western blot results in granulosa cells. The P450Arom mRNA level and protein expression was obviously down-regulated in DEHP or B[a]P alone and combination groups compared to control (P<0.05), DEHP and B[a]P+DEHP treatment significantly up-regulated 17β-HSDΠmRNA level (P<0.05). In the two phase points of dose-dependent study, pronounced increases in PPAR-αand PPAR-γversus control were observed in any treatment groups (P<0.05). It was shown that the down-regulation of P450Arom was highly correlated with up-regulation of PPARs, DEHP played an important role in the combined mechanism of ovotoxic action. The joint effect of B[a]P+DEHP on P450Arom and PPAR-γprotein expression appeared obvious interaction (P<0.05).
PartⅡIn vitro study
1. More than 95%of highly purified granulosa cells could be obtained by mechanical method combined with trypsin digestion and brachytely centrifugation, the exponential phase of growth was between 48h and 96h incubation. Moreover, identifications of granulosa cells by HE and FSHR staining were volant and convenient approaches.
2. It showed that both MEHP and BPDE induced cytotoxicity in cultured granulosa cells, MEHP 200μM incubation for 24h obviously inhibited granulosa cells proliferation, and the viability of the cells were significantly inhibited after BPDE 10nM incubation for 24h (P<0.05).
3. MEHP+BPDE incubation for 12h and 24h, to a certain extent, inhibited granulosa cell viability compared to control (P<0.05), the joint effect of B[a]P+DEHP appeared no interaction, which suggesting addition joint action of the two compounds. Additionally, BPDE and MEHP+BPDE incubation for 24h resulted in significant cell damage as revealed by nuclear atrophy and cellular vacuolation and cellular structural damage, such as cell layer withdrawal and destruction.
4. MEHP or BPDE alone and combination, to a certain extent resulted in down regulation of P450Arom mRNA and protein expression, MEHP+BPDE significantly up regulated the PPAR-γmRNA and protein expression compared to control (P<0.05), the joint effect of B[a]P+DEHP on P450Arom and PPAR-γprotein expression appeared obvious interaction (P<0.05), which suggesting a synergistic action of the two compounds.
PartⅢWater environmental health risk assessment
1. Organic compounds were detected in water samples from the Three Gorges reservoir,which included 178 kinds of POPs in winter and 144 in summer. 18 kinds of POPs can be found on the EAP’s organic pollutants blacklist, 7 kinds of POPs can be found on the blacklist of Chinese government. The concentration of 6 kinds of pollutants (DBP, DEP, DEHP, NA, Pyr, FLA) in this study varied from ND～3.60×10-3.
2. The results showed that the greatest health risk to the individual person per year in terms of 6 kinds of organic pollutants through drinking is DEHP, followed by DBP, Pyr, NA, FLA and DEP, the health risk of the above organic pollutants varied from 6.34×10-14 a-1 to
3.99×10-11 a-1. The average total health risk caused by main organic pollutants (PAEs and PAHs) was 8.86×10-12 a-1 and even lower than the maximum tolerable value (5.0×10-5·a-1) recommended by ICRP. The most heavily polluted section located in Cuntan area among all the sampling sections and might have higher health risk.
Conclusion
1. DEHP and B[a]P, also their active metabolites MEHP and BPDE, possessed female reproductive toxicity, granulosa cell was the principal and common target cell of the cytotoxicity.
2. DEHP and B[a]P combination obviously exerted ovotoxicty in rats through decreasing ovary/body weight ratio, causing prolonged estrous cycle and shorten estrus, suppressing sex hormone levels, decreasing ovarian follicle populations, increasing atretic follicles and damaging the cell structure. The joint effect of DEHP and B[a]P combination on the above parameters appeared no interaction, DEHP played the principal role in the toxic actions. MEHP and BPDE combination inhibited granulosa cell viability and resulted in significant cell damage as revealed by nuclear atrophy, cellular vacuolation and cellular structural damage. The joint effect of DEHP and B[a]P combination appeared no interaction, which suggesting addition joint action of the two compounds.
3. DEHP and B[a]P combination obviously induced granulosa cells apoptosis, caspase-3 activation involved in the process of apoptosis occurrence, it was shown that atretic follicles were closely correlated with cell apoptosis. The joint effect of DEHP and B[a]P combination on granulosa cells apoptosis and caspase-3 expression appeared interaction.
4. According to in vivo and in vitro study, it is demonstrated that B[a]P+DEHP and MEHP+BPDE activated PPARs signaling pathway and then down-regulated P450Arom expression, subsequently, the balance of sex hormone synthesis, secretion and homeostasis was destroyed. As for interactive effect of the combination mechanism, we draw a conclusion that synergistic effects were observed in P450Arom and PPARs protein expression.
5. The results showed that PAEs and PAHs pollution was widespread in aquatic environment of the Three Gorges reservoir. The total health risk caused by the six kinds of organic pollutants (PAEs and PAHs) was in low level. However, it also should be paid more attention by our environment protection management.
基于PAEs和PAHs类物质潜在的女(雌性)性生殖危害,本研究选取PAEs和PAHs的代表性物质,具有明确雌性生殖毒性的邻苯二甲酸二(2-乙基己基)酯(di-(2-ethylhcxyl) phthalate, DEHP)与苯并[a]芘(benzo[a]pyrene, B[a]P)及其代谢产物单-(2-乙基己基)邻苯二甲酸酯(MEHP)与反式二氢二醇环氧苯并芘(BPDE),以整体动物和细胞模型为手段,探讨两种毒物低剂量联合作用下的雌性生殖危害的特点,并从颗粒细胞的结构、功能以及PPARs-Arom/17β-HSD信号通路调节雌激素合成等方面探讨其机制。此外,采用统计学析因分析对DEHP和B[a]P(MEHP和BPDE)的联合生殖毒性作用进行定性评价。最后,结合三峡库区水环境PAEs和PAHs类物质的现实污染状况,本研究构建了水环境POPs污染物健康风险评价模型,应用前期水环境污染监测资料,对三峡库区干流主要断面6种PAEs和PAHs代表物质进行健康风险评价。研究旨在探讨DEHP与B[a]P联合作用下雌性生殖毒性危害的特点及作用机制,并对环境PAEs和PAHs暴露对人群的健康危害进行评估,研究将为POPs的防治和人群健康的保护奠定基础,为决策机构提供决策依据和参考。
研究内容:
第一部分DEHP和B[a]P联合暴露对雌性大鼠的生殖毒性及其机制研究
1. 4～5周龄健康雌性SD大鼠随即分7组,每组12只。实验分组为:正常对照组(玉米油);B[a]P高、低剂量组(B[a]P10mg/kg和B[a]P5mg/kg);DEHP高、低剂量组( DEHP600mg/kg和DEHP300mg/kg ); B[a]P+DEHP高、低剂量组(B[a]P10mg/kg+DEHP600mg/kg和B[a]P5mg/kg+ DEHP300mg/kg)。
2.采用隔日灌胃方式连续染毒60 d,受试动物于染毒30 d和60 d结束后分两批次,清晨1%戊巴比妥钠腹腔麻醉后心脏取血,进行各项指标的取材、检测。观察动物的生长发育、脏器系数、动情周期、血清激素水平、卵巢卵泡发育及组织病理学改变、颗粒细胞凋亡等指标。运用RT-PCR、Western blot和免疫组化等方法检测雌激素合成通路PPARs、P450Arom和17β-HSD等基因和蛋白的表达差异。
第二部分MEHP和BPDE低剂量联合对体外卵巢颗粒细胞的毒性及其机制
1.采用机械分离结合胰蛋白酶消化与低速离心的方法分离培养卵巢颗粒细胞。
2.筛选MEHP和BPDE染毒剂量,建立MEHP和BPDE低剂量联合染毒颗粒细胞模型,进行联合染毒3×3析因分析。染毒分组:Control(1%DMSO); MEHP(25μM; 50μM); BPDE(10 nM; 100 nM); MEHP+BPDE(25μM+10 nM;25μM+100 nM;50μM+10 nM;50μM+100 nM)。通过MTT实验与细胞形态学观察评价MEHP和BPDE联合作用对颗粒细胞的毒性。
3.采用RT-PCR、细胞免疫化学染色法检测MEHP和BPDE联合染毒后颗粒细胞PPAR-γ和P450Arom基因和蛋白表达,在细胞水平探讨联合毒性机制。
第三部分三峡库区水环境邻苯二甲酸酯类和多环芳烃类代表物质健康风险评价
1.三峡库区干流设置7个水质监测点,采集2005年2月和8月枯、丰水期水样,采用瓦里安GC3800/MS200型气质联用仪进行水中POPs分析测定。
2.介绍健康风险评价方法,建立了水环境致癌物/非致癌物健康风险评价模型。选取水中邻苯二甲酸二丁酯(DBP)、邻苯二甲酸二乙基酯(DEP)和邻苯二甲酸二(2-乙基己基)酯(DEHP)、萘(NA)、芘(Pyr)、萤蒽(FLA)等6种主要PAEs和PAHs物质,分析污染数据并进行健康风险评价。
研究结果:
第一部分DEHP和B[a]P联合暴露雌性大鼠的生殖毒性及其机制
1. B[a]P、DEHP单独与B[a]P+DEHP染毒可造成动物体重增长减缓,但与正常对照相比未见显著差异(P>0.05)。B[a]P+DEHP染毒30 d即可导致卵巢重量减轻,卵体系数下降(P<0.05)。随着染毒时间延长、剂量增加,呈现剂量与时间反应趋势。B[a]P与B[a]P+DEHP相比,卵体系数具有显著差异(P<0.05)。析因分析表明,B[a]P+DEHP染毒30 d和60 d对卵体系数无显著交互作用。
2. B[a]P、DEHP单独与B[a]P+DEHP染毒导致EC延长、E逐渐缩短。染毒30 d, B[a]P10mg/kg+DEHP600mg/kg组EC和No E时间延长(P<0.05)。染毒60 d,各染毒组(B[a]P5mg/kg组除外)EC和No E时间显著延长(P<0.01),B[a]P+DEHP和DEHP染毒可导致E时间显著缩短(P<0.05);B[a]P10mg/kg+DEHP600mg/kg组分别与B[a]P5mg/kg+DEHP300mg/kg组和B[a]P10mg/kg组相比,EC和No E具有显著差异(P<0.01)。析因分析表明,B[a]P+DEHP染毒对EC和E无显著交互作用。
3. B[a]P+DEHP染毒导致血清E2、P和T含量显著降低。染毒30 d,DEHP600mg/kg和B[a]P10mg/kg+DEHP600mg/kg组大鼠血清E2和P含量明显降低(P<0.05)。染毒60 d,各染毒组(B[a]P5mg/kg组除外)血清E2和P含量继续下降(P<0.05);高、低剂量B[a]P+DEHP组分别与高、低剂量B[a]P组相比,血清E2水平显著降低(P<0.05)。染毒60 d,各组(DEHP300mg/kg组除外)血清T含量与对照组相比显著降低(P<0.05)。各染毒组血清FSH和LH含量未见显著改变。析因分析表明,B[a]P+DEHP联合染毒30 d和60 d对大鼠血清E2和P无显著交互作用(P>0.05)。
4. B[a]P+DEHP染毒导致卵巢原始卵泡和初/次级卵泡数目减少,闭锁卵泡数目增多。染毒30 d,B[a]P、DEHP(DEHP300mg/kg组除外)和B[a]P+DEHP染毒导致原始卵泡和初/次级卵泡数目减少(P<0.05),DEHP600mg/kg组和B[a]P10mg/kg+ DEHP600mg/kg组闭锁卵泡明显增加(P<0.05)。染毒60 d,B[a]P和B[a]P+DEHP染毒可导致原始卵泡、初/次级卵泡数目减少(P<0.05),DEHP和B[a]P+DEHP染毒可导致闭锁卵泡数目增多(P<0.05);B[a]P+DEHP组与DEHP组比较,原始卵泡数目具有显著差异(P<0.05),B[a]P10mg/kg+DEHP600mg/kg组与B[a]P10mg/kg组比较闭锁卵泡显著增加(P<0.05)。析因分析表明,B[a]P+DEHP染毒30 d和60 d对大鼠原始卵泡、初/次级卵泡、卵泡闭锁和黄体的数目无显著交互作用。
5.光镜下,B[a]P、DEHP单独与B[a]P+DEHP染毒均导致颗粒细胞结构疏松,变性、脱落,细胞数量减少;B[a]P和B[a]P+DEHP染毒出现颗粒细胞与膜细胞间隙增宽,细胞坏死。电镜下,B[a]P、DEHP单独与B[a]P+DEHP染毒均造成颗粒细胞线粒体肿胀与空泡化,内质网扩张甚至髓鞘样结构形成,可见凋亡小体。随着染毒时间延长、剂量增加,颗粒细胞损伤加重,局灶性坏死和髓鞘样结构形成增多。
6. B[a]P、DEHP单独与B[a]P+DEHP染毒30 d和60 d均造成颗粒细胞凋亡增加,呈现剂量-反应趋势(P<0.05),Caspase-3的激活参与了细胞凋亡启动,联合染毒卵泡闭锁增加与细胞凋亡发生高度关联。随着染毒时间延长和剂量增加,B[a]P和B[a]P+DEHP染毒组细胞凋亡反而出现下降趋势,B[a]P+DEHP组与B[a]P或DEHP组相比出现显著差异(P<0.05)。析因分析表明,B[a]P+DEHP染毒对卵巢颗粒细胞凋亡和Caspase-3表达均具有交互作用(P<0.01),提示两者联合主要表现为协同作用。
7.免疫组化和Western blot蛋白检测与基因表达结果一致。无论染毒30 d或60 d,B[a]P、DEHP和B[a]P+DEHP染毒导致P450Arom mRNA和蛋白表达下调,P450Arom mRNA下调与PPAR-α和PPAR-γmRNA蛋白表达上调高度关联(P<0.05);此外,DEHP和B[a]P+DEHP染毒组卵巢17β-HSDΠmRNA表达水平显著升高(P<0.05)。在就PPARs/P450Arom通路而言,DEHP在联合作用机制中发挥主导作用。析因分析表明,B[a]P+DEHP染毒对P450Arom、PPAR-α、PPAR-γ和17β-HSDΠmRNA表达无显著交互作用,而对P450Arom和PPAR-γ蛋白表达存在交互作用(P<0.05)。
第二部分MEHP和BPDE低剂量联合对卵巢颗粒细胞毒性及其机制
1.采用机械分离法结合胰蛋白酶消化及低速离心法分离培养的卵巢颗粒纯度高、活性好,48 h～96 h大量增殖,生长旺盛。HE和FSHR表达染色鉴定颗粒细胞是一种简便快速的方法。
2. MEHP和BPDE对体外培养卵巢颗粒细胞具有毒性,BPDE对颗粒细胞的毒性明显大于MEHP。MEHP 200μM染毒24 h颗粒细胞活性明显抑制;BPDE 10 nM染毒24 h颗粒细胞生长即出现显著抑制(P<0.05)。
3. BPDE和MEHP+BPDE染毒12 h和24 h均从一定程度上抑制颗粒细胞活性(P<0.05),析因分析表明,MEHP+BPDE染毒对颗粒细胞活性不存在交互作用(P>0.05),提示联合作用表现为相加作用。BPDE和MEHP+BPDE染毒可导致细胞萎缩、胞核缩小、出现空泡化与坏死,随着染毒剂量增加,毒性作用更为明显。
4. MEHP+BPDE染毒在一定剂量水平可导致颗粒细胞PPAR-γmRNA和蛋白表达上调,P450Arom mRNA和蛋白表达下调,PPARs/P450Arom途径可能是MEHP和BPDE联合作用颗粒细胞的毒性机制。析因分析表明,MEHP+BPDE染毒对颗粒细胞P450Arom和PPAR-γmRNA和蛋白表达均存在交互作用(P<0.05),提示联合毒性表现为协同作用。
第三部分三峡库区水环境PAEs和PAHs代表物质健康风险评价
1.三峡库区2005年枯、丰水期水源水分别检出有机物178种和144种,其中属于美国环保局优先控制污染物有18种,属于我国水环境优先控制污染物7种;检出率较高的是PAHs和PAEs两类物质。在7个监测点中,水体DBP、DEP、DEHP、NA、Pyr、FLA等6种物质检出范围为ND～3.60×10-3。
2.三峡库区水源水6种有机污染物由饮水途径所致健康危害的个人年风险介于6.34×10-14 a-1～3.99×10-11 a-1范围,按年风险大小排列为:DEHP>DBP>Pyr>NA>FLA>DEP;水中6种有机污染物对人体健康危害的年平均总风险仅为8.86×10-12 a-1,远低于国际辐射防护委员会(ICRP)推荐的最大可接受值5.0×10-5·a-1。在库区各断面中,处于长江和嘉陵江汇合的寸滩断面有机物健康危害风险最大,涪陵和开县断面次之。
结论
1. PAEs类和PAHs类代表性物质,DEHP和B[a]P及其代谢产物(MEHP和BPDE)联合具有雌性生殖毒性,卵巢颗粒细胞是两者共同作用的靶细胞。
2. DEHP和B[a]P联合染毒导致大鼠卵体系数降低,动情周期延长和动情期缩短,大鼠血清E2、P和T水平明显降低,卵巢原始卵泡、初/次级卵泡数目减少,闭锁卵泡数目增多,细胞结构出现病理损伤;DEHP和B[a]P联合对上述指标不具有交互作用,DEHP在毒性效应中发挥主导作用。MEHP和BPDE低剂量联合染毒导致体外培养颗粒细胞活性降低,细胞出现萎缩、空泡化与坏死;MEHP和BPDE低剂量联合染毒对卵巢颗粒细胞活性不具有交互作用,两者联合表现为相加作用。
3. DEHP和B[a]P联合染毒诱发颗粒细胞凋亡发生增加,细胞凋亡与卵泡闭锁增加高度关联;Caspase-3的激活在细胞凋亡发生过程中发挥调控作用。DEHP和B[a]P联合染毒对卵巢颗粒细胞凋亡和Caspase-3表达具有交互作用,两者联合主要表现为协同作用。
4.体内与体外实验相继证实,DEHP和B[a]P(MEHP和BPDE)联合毒性效应大于每种化学物质的单独作用,主要表现为毒性增强作用,联合类型以非交互作用为主。就毒性机制而言,DEHP和B[a]P(MEHP和BPDE)干扰PPARs/P450Arom信号通路导致雌激素合成障碍是联合毒性作用机制之一,DEHP和B[a]P(MEHP和BPDE)对P450Arom和PPARs蛋白表达具有交互作用,联合作用表现为协同作用。
5.三峡库区水环境PAEs和PAHs类有机物检出种类和含量较高,6种POPs污染物所致的健康危害年风险度目前还处于很低水平,但应引起管理部门的重视。
Background and objective
Nowadays, expanding urbanization and industrialization has led to ubiquitous chemical contaminants in environment that have clearly been defied as endocrine disrupting chemicals and these problems have been received much more concern by scientists. Persistent organic pollutants (POPs) are chemical substances that persist in the environment, bioaccumulate through the food web, and subsequently pose potential risks to human health. Many research studies showed that POPs pollution is becoming the most serious problem all over the world. The main pollution chemicals of POPs in the water body of the Three Georges reservoir was characterized by phthalates acid esters (PAEs) and polycyclic aromatic hydrocarbons (PAHs). Phthalates acid esters and polycyclic aromatic hydrocarbons have clearly been defied as endocrine disrupting chemicals, a great deal of literatures indicates that these environmental toxicants may accumulate in ovary and cause reproductive disorders. Traditionally, reproductive studies have focused on healthy effects associated with exposure to single environmental endocrine disrupting chemical, but exploration of interactions among these chemicals is an important and also key area of enquiry. The toxicity of chemical mixtures might be lower or higher than predicted from the known individual effects of each chemical, which could be a synergistic, antagonistic, potentiation or inhibitory interaction in any mixture-related studies. In addition, adult women, children, particularly neonates, can be biologically more sensitive to the mixture toxicants exposure on physiological functions and characteristics basis.
In our studies conducted in the Three Gorges reservoir, the higher average concentrations of PAEs and PAHs chemicals were found in aquatic environment, thus dietary exposure to these chemicals is possible which can occur simultaneously. Taking these facts into consideration, the present study was divided into three parts. In terms of part one and two, we chose the representative chemicals of PAEs and PAHs, namely DEHP and B[a]P separately, also their active metabolites MEHP and BPDE, as the model chemicals to evaluate the combined female reproductive toxicity as well as their possible mechanism in vivo and in vitro study. The third part introduced the health risk assessment method and established health risk assessment model for POPs contamination in water environmental so as to explore potential healthy hazards of POPs in the Three Gorges reservoir. The purpose of this study is to evaluate the combined reproductive toxicity and involved mechanism, provide experiment data and explore a method to evaluate the adverse effect of organic pollutants mixture on human health.
Materials and Methods
PartⅠFemale reproductive toxicity following DEHP and B[a]P combination.
1. This study was to determine the female reproductive toxicity and possible molecular mechanism of oral exposure to DEHP and B[a]P combination following 30 and 60days using Sprague-Dawley (SD) rat’s model. Females SD rats were randomly assigned to seven groups of 12 animals at each group. Female rats were given intragastrical administration of corn oil (control), B[a]P alone (B[a]P5mg/kg and B[a]P10mg/kg), DEHP alone (DEHP300mg/kg and DEHP600mg/kg) and B[a]P+DEHP (B[a]P5mg/kg+DEHP300mg/kg and B[a]P10mg/kg+DEHP600mg/kg) on alternate days for both 30 and 60days.
2. After the toxical exposure, rats were killed and general clinical observation, reproductive organs, serum hormone level, ovarian follicle population, cell apoptosis, ovary histopathology, mRNA levels of target genes and proteins expression including PPARs, P450Arom and 17β-HSD in granulosa cells were evaluated.
PartⅡCytotoxicity of MEHP+BPDE in cultured granulosa cells
1. Obtain and identify the cultured granulosa cells from the ovary of impuberism rats so as to establish a convenient and stable cell experiment model.
2. The doses of MEHP and BPDE intoxication was chosen and 3×3 factorial design model in vivo study was established, As detailed as below, Control (DMSO), MEHP(25μM; 50μM), BPDE(10nM; 100nM), MEHP+BPDE (25μM+10nM; 25μM+100nM; 50μM+10nM; 50μM+100nM). Cytotoxicity of MEHP or BPDE alone and concomitant treatments was evaluated by MTT and histopathology observation.
3. The target genes and proteins expression including P450Arom and PPARs in cultured granulosa cells were evaluated using RT-PCR and immunohistochemistry methods.
PartⅢWater environmental health risk assessment of PAEs and PAHs.
1. Seasonal samplings were carried out in Feb and Aug of 2005. Water samples were collected from seven different sampling sites of trunks and tributaries in the Three Gorges reservoir. The organic pollutants samples were analyzed by GC/MS.
2. U.S.EPA’S model recommended for water environmental health risk assessment was improved based on the water monitoring data in the year of 2005. The health risk assessment method was introduced for organic pollutants contamination in water environment and selected 6 kinds of organic pollutants (PAEs and PAHs), which is DBP, DEP, DEHP, NA, Pyr and FLA, to carry out data analysis and evaluate the total health risk.
Results
PartⅠIn vivo study
1. The growth and development of rats in DEHP and B[a]P alone and combination groups was not were obviously inhibited compared with control (P>0.05). The absolute ovary weight and ovary/body weight ratio of rats exposed to B[a]P+DEHP treatment for 30 and 60 days was significantly decreased compared to control (P<0.05), dose- and time-response tendency appeared. The joint effect of B[a]P+DEHP on ovary/body weight ratio appeared no interaction.
2. DEHP and B[a]P alone or combination resulted in prolonged estrous cycle as well as shorten estrus phase in animals following 30 and 60 days exposure compared to control (P<0.05). After 60 days exposure, B[a]P and DEHP alone or combination (besides B[a]P5mg/kg) resulted in prolonged EC and No E (P<0.01), the estrous phase of B[a]P10mg/kg+DEHP600mg/kg and DEHP600mg/kg treated groups was shortened (P<0.05), there was significant difference between B[a]P10mg/kg+DEHP600mg/kg and B[a]P10mg/kg or B[a]P5mg/kg+DEHP300mg/kg (P<0.01). The joint effect of B[a]P+DEHP on EC and E appeared no interaction.
3. It showed that B[a]P+DEHP caused remarkable changes in serum hormone (E2 and P) levels following 30 day exposure. B[a]P (besides B[a]P5mg/kg) or DEHP alone and B[a]P+DEHP significantly reduced the serum E2 ,P and T levels following 60 days exposure compared to the control (P<0.05), serum E2 level was significantly decreased in B[a]P+DEHP compared to B[a]P intoxication (P<0.05). It seemed there was no significant changes in terms of serum FSH and LH levels in either following 30 or 60 days exposure (P>0.05). The joint effect of B[a]P+DEHP on serum E2 and P appeared no interaction.
4. B[a]P+DEHP resulted in decreased primordial and primary/secondary follicle populations, and increased atretic populations. After 30 days exposure, B[a]P or DEHP alone (besides DEHP300mg/kg) and B[a]P+DEHP had significantly reduced primordial follicle populations of ovaries compared to control (P<0.05). After 60 days exposure, it was shown that only the primordial populations significantly decreased in B[a]P+DEHP and B[a]P10mg/kg compared to control (P<0.05), The primordial follicle populations of B[a]P+DEHP significantly decreased compared to DEHP alone (P<0.05). The atretic numbers in the high dose of DEHP and B[a]P+DEHP significantly increased (P<0.05). The joint effect of B[a]P+DEHP on primordial follicles, primary/secondary follicles, atretic follicles and corpora lutea appeared no interaction.
5. B[a]P and DEHP alone or combination resulted in granulosa cell structure damage. cellular structure rarefaction, exfoliation and degeneration was observed in any treated groups under light microscope, additionally, the interspaces between granulosa cells and theca cells widened in B[a]P and B[a]P+DEHP treated groups. B[a]P+DEHP caused wide spread cellular swelling and lysis of plasma membranes. Mitochondrial cristae appeared swelling and collapse, cellular myelin figure and apoptotic body was observed with electron microscope. Accompanied by exposure extension, cellular damage was more serious, especially focal necrosis and myelin figure was observed frequently.
6. Whenever following 30 or 60 days exposure, B[a]P or DEHP alone (except DEHP300mg/kg) and B[a]P+DEHP dramatically increased the granulosa cells apoptosis in ovary compared to control (P<0.05), caspase-3 activation involved in the process of apoptosis occurrence (P<0.05). Remarkable increase of granulosa cell apoptosis was found in B[a]P+DEHP, however, cell apoptosis index decreased while following 60 days exposure. There was high correlation between apoptosis and atretic follicle occurrence. The joint effect of B[a]P+DEHP on granulosa cells apoptosis and caspase-3 expression appeared obvious interaction (P<0.05).
7. The target gene expression of P450Arom and PPARs was in coincidence with immunohistochemical and western blot results in granulosa cells. The P450Arom mRNA level and protein expression was obviously down-regulated in DEHP or B[a]P alone and combination groups compared to control (P<0.05), DEHP and B[a]P+DEHP treatment significantly up-regulated 17β-HSDΠmRNA level (P<0.05). In the two phase points of dose-dependent study, pronounced increases in PPAR-αand PPAR-γversus control were observed in any treatment groups (P<0.05). It was shown that the down-regulation of P450Arom was highly correlated with up-regulation of PPARs, DEHP played an important role in the combined mechanism of ovotoxic action. The joint effect of B[a]P+DEHP on P450Arom and PPAR-γprotein expression appeared obvious interaction (P<0.05).
PartⅡIn vitro study
1. More than 95%of highly purified granulosa cells could be obtained by mechanical method combined with trypsin digestion and brachytely centrifugation, the exponential phase of growth was between 48h and 96h incubation. Moreover, identifications of granulosa cells by HE and FSHR staining were volant and convenient approaches.
2. It showed that both MEHP and BPDE induced cytotoxicity in cultured granulosa cells, MEHP 200μM incubation for 24h obviously inhibited granulosa cells proliferation, and the viability of the cells were significantly inhibited after BPDE 10nM incubation for 24h (P<0.05).
3. MEHP+BPDE incubation for 12h and 24h, to a certain extent, inhibited granulosa cell viability compared to control (P<0.05), the joint effect of B[a]P+DEHP appeared no interaction, which suggesting addition joint action of the two compounds. Additionally, BPDE and MEHP+BPDE incubation for 24h resulted in significant cell damage as revealed by nuclear atrophy and cellular vacuolation and cellular structural damage, such as cell layer withdrawal and destruction.
4. MEHP or BPDE alone and combination, to a certain extent resulted in down regulation of P450Arom mRNA and protein expression, MEHP+BPDE significantly up regulated the PPAR-γmRNA and protein expression compared to control (P<0.05), the joint effect of B[a]P+DEHP on P450Arom and PPAR-γprotein expression appeared obvious interaction (P<0.05), which suggesting a synergistic action of the two compounds.
PartⅢWater environmental health risk assessment
1. Organic compounds were detected in water samples from the Three Gorges reservoir,which included 178 kinds of POPs in winter and 144 in summer. 18 kinds of POPs can be found on the EAP’s organic pollutants blacklist, 7 kinds of POPs can be found on the blacklist of Chinese government. The concentration of 6 kinds of pollutants (DBP, DEP, DEHP, NA, Pyr, FLA) in this study varied from ND～3.60×10-3.
2. The results showed that the greatest health risk to the individual person per year in terms of 6 kinds of organic pollutants through drinking is DEHP, followed by DBP, Pyr, NA, FLA and DEP, the health risk of the above organic pollutants varied from 6.34×10-14 a-1 to
3.99×10-11 a-1. The average total health risk caused by main organic pollutants (PAEs and PAHs) was 8.86×10-12 a-1 and even lower than the maximum tolerable value (5.0×10-5·a-1) recommended by ICRP. The most heavily polluted section located in Cuntan area among all the sampling sections and might have higher health risk.
Conclusion
1. DEHP and B[a]P, also their active metabolites MEHP and BPDE, possessed female reproductive toxicity, granulosa cell was the principal and common target cell of the cytotoxicity.
2. DEHP and B[a]P combination obviously exerted ovotoxicty in rats through decreasing ovary/body weight ratio, causing prolonged estrous cycle and shorten estrus, suppressing sex hormone levels, decreasing ovarian follicle populations, increasing atretic follicles and damaging the cell structure. The joint effect of DEHP and B[a]P combination on the above parameters appeared no interaction, DEHP played the principal role in the toxic actions. MEHP and BPDE combination inhibited granulosa cell viability and resulted in significant cell damage as revealed by nuclear atrophy, cellular vacuolation and cellular structural damage. The joint effect of DEHP and B[a]P combination appeared no interaction, which suggesting addition joint action of the two compounds.
3. DEHP and B[a]P combination obviously induced granulosa cells apoptosis, caspase-3 activation involved in the process of apoptosis occurrence, it was shown that atretic follicles were closely correlated with cell apoptosis. The joint effect of DEHP and B[a]P combination on granulosa cells apoptosis and caspase-3 expression appeared interaction.
4. According to in vivo and in vitro study, it is demonstrated that B[a]P+DEHP and MEHP+BPDE activated PPARs signaling pathway and then down-regulated P450Arom expression, subsequently, the balance of sex hormone synthesis, secretion and homeostasis was destroyed. As for interactive effect of the combination mechanism, we draw a conclusion that synergistic effects were observed in P450Arom and PPARs protein expression.
5. The results showed that PAEs and PAHs pollution was widespread in aquatic environment of the Three Gorges reservoir. The total health risk caused by the six kinds of organic pollutants (PAEs and PAHs) was in low level. However, it also should be paid more attention by our environment protection management.
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