全氟化合物PFOA、PFOS内分泌干扰效应的研究
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摘要
全氟化合物（PFCs）是一类具有重要应用价值的有机化合物，因其具有优良的稳定性、表面活性、疏水疏脂等特性，被广泛应用于工业和民用领域。PFCs广泛分布在各种环境介质和生物体内，且具有难降解性和生物蓄积性，是一种新型持久性有机污染物（POPs）。近年来，PFCs对生态环境和人体健康带来的不利影响受到全球的普遍关注，成为环境科学和毒理学领域的研究热点。
研究表明，PFCs能造成机体的内分泌系统发生紊乱，已成为环境内分泌干扰效应物（EDCs）筛检的重点。全氟辛酸（PFOA）和全氟辛烷磺酸（PFOS）是两种典型的PFCs，目前尚没有对其内分泌干扰活性的完整研究。国际上EDCs筛选程序中推荐采用体内（in vivo）和体外（in vitro）试验的组合测试法。本项目采用代表性PFCs（PFOA、PFOS）作为研究对象，通过体外成组方法筛选受试物的拟/抗雌激素受体（ER）、雄激素受体（AR）、甲状腺激素受体（TR）活性、过氧化物酶体增殖剂激活受体（PPAR）活性及对类固醇激素合成的影响，利用模式生物斑马鱼、SD大鼠，在体内水平研究PFOA、PFOS对机体内分泌系统的影响及作用机制，综合评价PFCs的内分泌干扰效应。
第一部分
体外试验筛选PFOA、PFOS的内分泌干扰活性
第一节PFOA、PFOS对激素受体的干扰活性
目的
环境化合物可以通过受体介导途径发挥内分泌干扰效应，采用受体介导的报告基因试验体系，检测和评价PFOA、PFOS对雌激素受体（ER）、雄激素受体（AR）、甲状腺激素受体（TR）、过氧化物酶体增殖剂激活受体（PPAR）激动或拮抗作用。
方法
通过瞬时转染，将激素受体（ER、TR、PPAR）表达质粒、含有相应激素反应元件的荧光素酶（Luc）报告基因质粒，转染到激素受体阴性的CV-1细胞中，建立ER、TR、PPAR介导的报告基因试验方法，用来检测PFOA、PFOS的拟/抗雌激素、甲状腺激素、过氧化物酶体增殖剂激活受体活性；利用稳定转染雄激素反应原件报告基因的MDA-Kb2细胞来检测PFOA、PFOS的拟/抗雄激素活性。
结果
在ER介导的报告基因试验中，FPOA、PFOS单独作用，不能显著诱导报告基因Luc的表达；与1×10-9M E2共同作用，在受试浓度3×10-9-3×10-7M内可协同E2增强报告基因Luc的表达。在AR介导的报告基因试验中，未发现PFOA、PFOS对Luc有明显的诱导和抑制作用。在TR介导的报告基因试验中，PFOA（1×10-8-3×10-7M）、PFOS（1×10-7M、3×10-7M）能抑制5×10-9M T3诱导的Luc表达量。在PPAR介导的报告基因试验中，PFOA、PFOS在受试范围内表现出PPAR激动作用。
结论
PFOA、PFOS具有雌激素协同活性、抗甲状腺激素活性、PPAR激动活性。
第二节PFOA、PFOS对类固醇激素合成的影响
目的
环境化合物可以通过非受体介导途径来发挥内分泌干扰效应，通过研究PFOA、PFOS对类固醇激素合成过程的影响，来评价它们的类固醇激素合成干扰效应。
方法
采用人肾上腺皮质瘤细胞H295R，检测PFOA、PFOS作用后对H295R细胞中类固醇激素（雌二醇和睾酮，即E2和T）水平、类固醇激素合成过程中关键酶基因及关键调控因子（SF-1）表达的影响。
结果
PFOA在1×10-8M及更高浓度时，增加细胞内E2的水平，在3×10-8M及以上，抑制T的水平。PFOS在3×10-8M及以上，增加E2的表达，在1×10-7M和3×10-7M浓度时，降低T的表达。PFOA、PFOS可以不同程度的改变类固醇合成过程中关键酶基因的表达。在10种与类固醇激素合成相关的基因中，PFOA作用后显著上调17βHSD1、3βHSD2、CYP11B2、CYP19的表达，下调CYP17、17βHSD4、CYP11A的表达。PFOS暴露后显著上调HMGR、StAR、CYP11A、CYP19、3βHSD2、CYP11B2的表达，下调CYP17、17βHSD1、CYP21的表达。PFOA暴露还可导致类固醇合成关键调控因子SF-1的基因和蛋白表达水平降低。
结论
PFOA、PFOS可以影响H295R细胞中类固醇激素的合成过程，具有类固醇激素合成干扰效应。
第二部分
体内试验研究PFOA、PFOS的内分泌干扰效应
第一节PFOA、PFOS对斑马鱼胚胎发育的影响
目的
利用模式生物斑马鱼，研究PFOA、PFOS短期暴露对斑马鱼胚胎发育、内分泌相关基因以及整体基因表达的影响。
方法
受精后4h（4hpf）的斑马鱼胚胎，用100、200、500μg/L的PFOA、PFOS染毒至120hpf。观察斑马鱼胚胎/幼鱼的生长状况，包括胚胎死亡、孵化、生长发育、有无胚胎发育畸形等情况。检测120hpf的斑马鱼幼鱼体内内分泌相关基因（包括雌激素受体表达基因esr1、esr2b，甲状腺早期分化关键基因hhex、pax8，类固醇激素合成基因cyp19a、cyp19b、cyp17）的表达情况。在此基础上，进行单一浓度多暴露终点的斑马鱼胚胎暴露试验。选择500μg/L的PFOA、PFOS在斑马鱼4hpf-120hpf期间染毒，染毒至12hpf、24hpf、48hpf、72hpf、96hpf、120hpf，观察各时间点的胚胎发育情况，选取暴露至120hpf的斑马鱼幼鱼，利用斑马鱼表达谱芯片检测整体基因的表达情况，分析筛选出关键差异表达基因。
结果
在4hpf-120hpf染毒期间，受试浓度下的PFOA、PFOS对斑马鱼胚胎/幼鱼的生长发育没有产生明显影响，但可引起斑马鱼幼鱼内分泌相关基因的表达发生改变。与对照组相比，500μg/L的PFOA引起esr1基因水平的上调，对esr2b的表达无影响；200μg/L、500μg/L的PFOA能显著增加hhex、pax8的表达量；PFOA对类固醇合成酶相关基因的影响不大。在最高浓度下，PFOS暴露使esr1的表达量增加、esr2b的表达量降低；PFOS浓度依赖性的增加hhex和pax8的表达；200μg/L、500μg/L的PFOS暴露能显著抑制cyp19a、cyp19b、cyp17的表达。120hpf的斑马鱼表达谱芯片结果提示，多个通路上的多种基因在PFOS暴露后发生改变，差异表达基因主要参与到细胞过程、代谢过程、生物调控、发育等生物学过程。
结论
PFOA、PFOS暴露可对斑马鱼的基因表达产生影响，发挥潜在的干扰效应。
第二节PFOA、PFOS对大鼠青春期发育的影响
目的
环境化合物暴露与青春期启动时间的异常密切相关。流行病学研究发现PFOA、PFOS的暴露和青春期启动的改变有关，但对其干扰青春期发育的相关机制一直未得到阐明。我们采用雌性大鼠模型，研究PFOA、PFOS在关键时间段暴露对雌性青春期启动和发育的影响，并探究其相关机制。
方法
采用SD雌性大鼠，在新生期（出生后1-5天，PND1-5）和青春前期（出生后26-30天，PND26-30）两个暴露阶段分别进行不同剂量的PFOA、PFOS（0.1mg/kg、1mg/kg、10mg/kg）染毒。测定大鼠出生时、PND15、PND25、PND35、阴道开口日的体重和肛门-生殖器距离（AGD），记录雌鼠进入青春期的年龄（阴道开口时间）、首次发情期时间、雌性周期，检测血清雌激素和促黄体生成素（LH）的水平，计算卵巢系数，进行卵巢组织病理分析和卵泡计数。采用Q-RT-PCR分析下丘脑Kisspeptin/GPR54系统中Kiss1、Kiss1r、ERα、ERβ基因表达的变化，采用免疫组织化学方法检测kisspeptin蛋白表达水平，以探讨该系统在PFOA、PFOS所致的青春期启动时间改变中发挥的作用。选择染毒雌鼠的肝脏组织，测定肝脏系数，进行肝脏功能相关血生化指标的检测，采用GC-MS进行血清脂肪酸组、肝脏代谢组测定，以探讨PFOA、PFOS在发育关键期短期暴露对肝脏功能的影响。
结果
新生期、青春前期10mg/kg PFOA、1mg/kg和10mg/kg PFOS短期暴露，可引起雌鼠阴道开口时间比对照组早4-5天，提前进入青春期。新生期PFOA、PFOS暴露后，以非剂量依赖性的方式增加雌鼠体重和AGD；青春前期暴露也可增加雌鼠PND35的体重。在两个暴露阶段处理后，低、中剂量的PFOA、PFOS染毒组大鼠都能出现不规律的雌性周期，表现为延长的静止期或发情后期。PND1-5和PND26-30暴露于PFOA、PFOS的雌鼠血清雌激素和LH水平上升，新生期对激素的影响强于青春前期。化合物染毒未引起卵巢组织结构发生明显改变，但可影响卵泡数量，在新生期低剂量的PFOA、PFOS组大鼠可见生长卵泡数量减少。下丘脑前腹侧室周核（AVPV）和弓状核（ARC）区域Kiss1、Kiss1r、ERα的基因水平下降，免疫组化结果显示kisspeptin纤维密度在PFOA、PFOS的高剂量暴露组受到抑制，与基因表达的结果趋势大体相同。对于大鼠肝脏，高剂量组的PFOA、PFOS可以增加肝脏重量。GC-MS分析结果显示，血清中多种脂肪酸的含量发生改变，肝脏代谢小分子分析显示，PFOA、PFOS导致的差异表达物质主要集中在代谢通路、半乳糖代谢通路、脂肪酸合成通路等。
结论
新生期、青春前期短期暴露于环境剂量下的PFOA、PFOS能够影响下丘脑-垂体-性腺（HPG）轴的调控，导致雌性青春期启动的提前，其中kisspeptin/GPR54系统在其中起了关键作用。新生期暴露于PFOA、PFOS也可影响体内脂肪酸代
Perfluorinated compounds (PFCs) are synthetic fully fluorinated organiccompounds that have been widely used as surfactants in industrial and commercialapplications. They are stable chemicals that is both lipid-and water-repellent.Moreover, PFCs have been listed as persistent organic pollutants (POPs). Thewidespread distribution and persistence of PFCs in humans and the environment havegenerated great concern about potential health impacts.
There is growing evidence that PFCs may disrupt the endocrine system.Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) are therepresentative and commonly used PFCs. Further characterization of theendocrine-disrupting effects of PFOA and PFOS is required for assessing potentialhealth risks. Methods developed by U.S.EPA involve a battery of in vitro and in vivoscreening assays to identify substances that interact with endocrine systems. Toinvestigate the endocrine disruption potencies of PFOA and PFOS, a combination of in vitro and in vivo assays was employed in the present study. Reporter gene assayswere used to detect receptor mediated (anti-)estrogenic,(anti-)androgenic,(anti-)thyroid hormone activities and PPAR activation of PFOA/PFOS. Thesteroidogenic effects of PFOA/PFOS were measured at both the hormone and genelevels in the human adrenocortical carcinoma cell line H295R. Additionally, azebrafish-based short-term screening method and female pubertal rat assay weredeveloped to detect the potential effect of PFOA/PFOS on endocrine function in vivo.Our research focused on a detailed characterization of PFOA/PFOS in an attempt todetermine the endocrine-disrupting effects of these chemicals.
Part I Assessing the endocrine-disrupting effects of PFOAand PFOS by in vitro assays
Chapter I Assessing hormone receptor activities of PFOAand PFOS in reporter gene assays
Objective
The major way by which endocrine disrupting chemicals (EDCs) disrupt thephysiological process is through interaction with nuclear hormone receptors. Reportergene assay has been established as a powerful tool for testing receptor agonists andantagonists among chemicals. Hormone receptor mediated reporter gene assays wereused to detect (anti-)estrogenic (anti-)androgenic,(anti-)thyroid hormone activitiesand PPAR activation of PFOA and PFOS.
Methods
In ER mediated reporter gene assay, CV-1cell line was transfected with ER expression vector rER/pCI and the reporter plasmid pERE-TATA-Luc+. In TR mediated reporter gene assay, CV-1cell line was transfected with TR expressionvector pGal4-L-TR and the reporter plasmid pUAS-tk-luc. In PPAR mediatedreporter gene assay, CV-1cell line was transfected with PPAR expression vectorPG-PPAR and the reporter plasmid PBR-PPRE. Cells were treated with PFOA orPFOS alone to investigate the agonistic activities of these chemicals. For antagonisticactivity tests, cells were treated with PFOA/PFOS with E2/T3. MDA-kb2cell linehad been stably transfected with the pMMTV.neo.luc reporter gene plasmid. Theywere treated with various concentrations of tested chemicals with or without DHT toinvestigate the agonistic and antagonistic activities.
Results
PFOA and PFOS acted alone had no effect on reporter gene expression ascompared with vehicle control. PFOA and PFOS co-treatment with1×10-9M E2could increase the luciferase expression at the tested concentrations (3×10-9-3×10-7M). None of the tested groups showed androgenic and antiandrogenic activities in ARreporter gene assay. PFOA and PFOS did not have TR agonistic activity in TRreporter gene assay. PFOA (1×10-8-3×10-7M) displayed anti-thyroid hormone effectswith5×10-9M T3in the medium. While PFOS co-treated with T3could also suppressthe expression of luciferase at concentrations of1×10-7M and3×10-7M. In PPAR reporter gene assay, PFOA and PFOS activated PPAR at all concentrations.
Conclusion
Results here demonstrated that PFOA and PFOS has additive estrogenic activity,anti-thyroid hormone activity and PPAR activation in vitro. They could have effectson the endocrine system through interfering with nuclear receptor.
Chapter II The steroidogenic effects of PFOA and PFOSin H295R steroidgenesis Assay
Objective
Chemicals can exert endocrine-disrupting effects via non-receptor mediatedpathways. The human adrenocortical carcinoma cell line H295R has been shown tobe a potential in vitro model for screening adverse effects of chemicals onsteroidogenesis. In this study, H295R cells were used to determine the effects ofPFOA and PFOS on steroidogenesis.
Methods
In this study, the steroidogenic effects of PFOA and PFOS were measured inH295R cell. The hormone levels of estradiol (E2) and testosterone (T), the expressionof major steroidogenic genes and the key steroidogenic gene regulator steroidogenicfactors1(SF-1) were measured after PFOA and PFOS exposure in H295Rsteroidogenesis assay.
Results
PFOA and PFOS could induce E2production and reduce T production in aconcentration-dependent manner. E2level were significantly increased by PFOA andPFOS at1×10-8M and3×10-8M, respectively. While T level were decreased byPFOA and PFOS at3×10-8M and1×10-7M, respectively. The expression patterns ofthe10steroidogenic genes were altered by PFOA and PFOS exposure. PFOAsignificantly induced the expression of17βHSD1,3βHSD2, CYP11B2, CYP19.Expressions of CYP17,17βHSD4, CYP11A were decreased by PFOA exposure. Genelevels of HMGR, StAR, CYP11A, CYP19,3βHSD2, CYP11B2were elevated by PFOS.For CYP17,17βHSD1and CYP21, the relative responses were suppressed after PFOStreatment. For the key steroidogenic gene regulator SF-1, gene expression wasdown-regulated within the tested concentration range. SF-1protein level gradually decreased in H295R cells as PFOA exposure concentration increased.
Conclusion
PFOA and PFOS could alter steroid hormone synthesis and the majorsteroidogenic genes expression in H295R steroidogenesis assay. Results heredemonstrated that PFOA and PFOS have the ability to alter steroidogenesis.
Part II Assessing the endocrine-disrupting effects of PFOAand PFOS by in vivo assays
Chapter I Effects of PFOA and PFOS on development andendocrine function in zebrafish
Objective
A short-term zebrafish embryo exposure assay was developed to detect thepotential effects of PFOA and PFOS on endocrine system development and functionin vivo.
Methods
Zebrafish embryos from4h post fertilization (hpf) to120hpf were exposed to100μg/L,200μg/L, and500μg/L PFOA or PFOS. The embryos’ survivals and thestage of embryonic development were recorded daily. Genes related to estrogenreceptor production (esr1and esr2b), early thyroid development (hhex and pax8), andsteroidogenic enzyme synthesis (cyp17, cyp19a, and cyp19b) were quantified byQ-RT-PCR. To identify genes and pathways involved in the endocrine-disruptingeffects, mRNA microarray analysis were conducted.
Results
Exposure to PFOA and PFOS did not affect hatching and larval survival rates inzebrafish embryos. No significant lethal and developmental abnormality wasobserved during the whole exposure time. PFOA cause a significantly increase ofesr1at500μg/L and no changes for esr2b. Exposure to PFOA up-regulated hhex andpax8expression levels at200and500μg/L. No significant changes of cyp17, cyp19a,and cyp19b were observed. Exposure to PFOS (500μg/L) led to an increase in theexpression level of esr1but a decrease in the expression of esr2b. An apparentconcentration-dependent increase of hhex and pax8expression was observed afterPFOS treatment. Suppression of cyp17, cyp19a, and cyp19b was found at200and500μg/L PFOS exposure. Microarray analysis revealed several classes ofsignificantly regulated genes which could be related to cellular, metabolic,development and biological regulation processes in zebrafish.
Conclusion
In short-term zebrafish embryo exposure assay, PFOA and PFOS could alter theexpressions of estrogen receptor, early thyroid development and steroid synthesisrelated genes. Further large-scale genetic study using microarrays provided moreinformation on exploring the mechanisms of PFCs-mediated endocrine-related effectsin zebrafish.
Chapter II Effects of PFOA and PFOS on pubertydevelopment
Objective
Limited information about effects of PFOA and PFOS on puberty developmentis available. We evaluated the effects and explored the neuroendocrine mechanism of neonatal and juvenile PFOA/PFOS exposure on female puberty maturation. We alsoevaluated the effects of PFOA/PFOS on liver.
Methods
Female rats were injected with PFOA or PFOS at0.1,1and10mg/kg/dayduring postnatal day (PND)1-5or26-30. Body weight, anogenital distance (AGD),vaginal opening (VO), estrous cyclicity and other development indexes were recorded.Serum levels, ovaries histology and follicles numbers were evaluated. Geneexpressions of Kiss1, Kiss1r, ERα, ERβ and immunohistochemistry of kisspeptin inthe hypothalamic anteroventral periventricular (AVPV) and arcuate (ARC) nucleiwere quantified. Liver weight were measured. Fatty acids in serum were determinedby GC/MS. Analysis of biomarkers for hepatic metabolism were also assessed.
Results
VO and first estrus were significantly advanced in10mg/kg PFOA,1and10mg/kg PFOS groups after neonatal and juvenile exposure. Neonatal PFOA/PFOSexposure increased body weight and AGD in a non-dose-dependent manner. Estradioland luteinizing hormone (LH) levels were increased after neonatal and juveniletreatments. Irregular estrous cycles occurred more frequently in0.1and1mg/kgPFOA/PFOS groups. Although no altered ovarian morphology was observed, folliclesnumbers were reduced in neonatal groups. Kiss1, Kiss1r, ERα mRNA expressions inAVPV and ARC were decreased after two periods’ exposure. Suppression ofkisspeptin fiber intensities were also evoked by PFOA/PFOS exposure especially atthe high dose in AVPV and ARC. Results also showed that liver weight weresignificantly increased in the highest PFOA/PFOS treated groups.Changes in serumfatty acids composition and metabolic disturbance in liver by both PFOA/PFOS wereobserved.
Conclusion
Neonatal and juvenile exposure to environmental levels of PFOA/PFOS couldaffect the regulation of hypothalamic-pituitary-gonadal (HPG) axis leading toadvancing puberty onset. Kisspeptin/GPR54system played a key role in the effects.Neonatal exposure to low-dose of PFOA/PFOS could affect serum fatty acidmetabolism and other metabolic processes in liver.
研究表明，PFCs能造成机体的内分泌系统发生紊乱，已成为环境内分泌干扰效应物（EDCs）筛检的重点。全氟辛酸（PFOA）和全氟辛烷磺酸（PFOS）是两种典型的PFCs，目前尚没有对其内分泌干扰活性的完整研究。国际上EDCs筛选程序中推荐采用体内（in vivo）和体外（in vitro）试验的组合测试法。本项目采用代表性PFCs（PFOA、PFOS）作为研究对象，通过体外成组方法筛选受试物的拟/抗雌激素受体（ER）、雄激素受体（AR）、甲状腺激素受体（TR）活性、过氧化物酶体增殖剂激活受体（PPAR）活性及对类固醇激素合成的影响，利用模式生物斑马鱼、SD大鼠，在体内水平研究PFOA、PFOS对机体内分泌系统的影响及作用机制，综合评价PFCs的内分泌干扰效应。
第一部分
体外试验筛选PFOA、PFOS的内分泌干扰活性
第一节PFOA、PFOS对激素受体的干扰活性
目的
环境化合物可以通过受体介导途径发挥内分泌干扰效应，采用受体介导的报告基因试验体系，检测和评价PFOA、PFOS对雌激素受体（ER）、雄激素受体（AR）、甲状腺激素受体（TR）、过氧化物酶体增殖剂激活受体（PPAR）激动或拮抗作用。
方法
通过瞬时转染，将激素受体（ER、TR、PPAR）表达质粒、含有相应激素反应元件的荧光素酶（Luc）报告基因质粒，转染到激素受体阴性的CV-1细胞中，建立ER、TR、PPAR介导的报告基因试验方法，用来检测PFOA、PFOS的拟/抗雌激素、甲状腺激素、过氧化物酶体增殖剂激活受体活性；利用稳定转染雄激素反应原件报告基因的MDA-Kb2细胞来检测PFOA、PFOS的拟/抗雄激素活性。
结果
在ER介导的报告基因试验中，FPOA、PFOS单独作用，不能显著诱导报告基因Luc的表达；与1×10-9M E2共同作用，在受试浓度3×10-9-3×10-7M内可协同E2增强报告基因Luc的表达。在AR介导的报告基因试验中，未发现PFOA、PFOS对Luc有明显的诱导和抑制作用。在TR介导的报告基因试验中，PFOA（1×10-8-3×10-7M）、PFOS（1×10-7M、3×10-7M）能抑制5×10-9M T3诱导的Luc表达量。在PPAR介导的报告基因试验中，PFOA、PFOS在受试范围内表现出PPAR激动作用。
结论
PFOA、PFOS具有雌激素协同活性、抗甲状腺激素活性、PPAR激动活性。
第二节PFOA、PFOS对类固醇激素合成的影响
目的
环境化合物可以通过非受体介导途径来发挥内分泌干扰效应，通过研究PFOA、PFOS对类固醇激素合成过程的影响，来评价它们的类固醇激素合成干扰效应。
方法
采用人肾上腺皮质瘤细胞H295R，检测PFOA、PFOS作用后对H295R细胞中类固醇激素（雌二醇和睾酮，即E2和T）水平、类固醇激素合成过程中关键酶基因及关键调控因子（SF-1）表达的影响。
结果
PFOA在1×10-8M及更高浓度时，增加细胞内E2的水平，在3×10-8M及以上，抑制T的水平。PFOS在3×10-8M及以上，增加E2的表达，在1×10-7M和3×10-7M浓度时，降低T的表达。PFOA、PFOS可以不同程度的改变类固醇合成过程中关键酶基因的表达。在10种与类固醇激素合成相关的基因中，PFOA作用后显著上调17βHSD1、3βHSD2、CYP11B2、CYP19的表达，下调CYP17、17βHSD4、CYP11A的表达。PFOS暴露后显著上调HMGR、StAR、CYP11A、CYP19、3βHSD2、CYP11B2的表达，下调CYP17、17βHSD1、CYP21的表达。PFOA暴露还可导致类固醇合成关键调控因子SF-1的基因和蛋白表达水平降低。
结论
PFOA、PFOS可以影响H295R细胞中类固醇激素的合成过程，具有类固醇激素合成干扰效应。
第二部分
体内试验研究PFOA、PFOS的内分泌干扰效应
第一节PFOA、PFOS对斑马鱼胚胎发育的影响
目的
利用模式生物斑马鱼，研究PFOA、PFOS短期暴露对斑马鱼胚胎发育、内分泌相关基因以及整体基因表达的影响。
方法
受精后4h（4hpf）的斑马鱼胚胎，用100、200、500μg/L的PFOA、PFOS染毒至120hpf。观察斑马鱼胚胎/幼鱼的生长状况，包括胚胎死亡、孵化、生长发育、有无胚胎发育畸形等情况。检测120hpf的斑马鱼幼鱼体内内分泌相关基因（包括雌激素受体表达基因esr1、esr2b，甲状腺早期分化关键基因hhex、pax8，类固醇激素合成基因cyp19a、cyp19b、cyp17）的表达情况。在此基础上，进行单一浓度多暴露终点的斑马鱼胚胎暴露试验。选择500μg/L的PFOA、PFOS在斑马鱼4hpf-120hpf期间染毒，染毒至12hpf、24hpf、48hpf、72hpf、96hpf、120hpf，观察各时间点的胚胎发育情况，选取暴露至120hpf的斑马鱼幼鱼，利用斑马鱼表达谱芯片检测整体基因的表达情况，分析筛选出关键差异表达基因。
结果
在4hpf-120hpf染毒期间，受试浓度下的PFOA、PFOS对斑马鱼胚胎/幼鱼的生长发育没有产生明显影响，但可引起斑马鱼幼鱼内分泌相关基因的表达发生改变。与对照组相比，500μg/L的PFOA引起esr1基因水平的上调，对esr2b的表达无影响；200μg/L、500μg/L的PFOA能显著增加hhex、pax8的表达量；PFOA对类固醇合成酶相关基因的影响不大。在最高浓度下，PFOS暴露使esr1的表达量增加、esr2b的表达量降低；PFOS浓度依赖性的增加hhex和pax8的表达；200μg/L、500μg/L的PFOS暴露能显著抑制cyp19a、cyp19b、cyp17的表达。120hpf的斑马鱼表达谱芯片结果提示，多个通路上的多种基因在PFOS暴露后发生改变，差异表达基因主要参与到细胞过程、代谢过程、生物调控、发育等生物学过程。
结论
PFOA、PFOS暴露可对斑马鱼的基因表达产生影响，发挥潜在的干扰效应。
第二节PFOA、PFOS对大鼠青春期发育的影响
目的
环境化合物暴露与青春期启动时间的异常密切相关。流行病学研究发现PFOA、PFOS的暴露和青春期启动的改变有关，但对其干扰青春期发育的相关机制一直未得到阐明。我们采用雌性大鼠模型，研究PFOA、PFOS在关键时间段暴露对雌性青春期启动和发育的影响，并探究其相关机制。
方法
采用SD雌性大鼠，在新生期（出生后1-5天，PND1-5）和青春前期（出生后26-30天，PND26-30）两个暴露阶段分别进行不同剂量的PFOA、PFOS（0.1mg/kg、1mg/kg、10mg/kg）染毒。测定大鼠出生时、PND15、PND25、PND35、阴道开口日的体重和肛门-生殖器距离（AGD），记录雌鼠进入青春期的年龄（阴道开口时间）、首次发情期时间、雌性周期，检测血清雌激素和促黄体生成素（LH）的水平，计算卵巢系数，进行卵巢组织病理分析和卵泡计数。采用Q-RT-PCR分析下丘脑Kisspeptin/GPR54系统中Kiss1、Kiss1r、ERα、ERβ基因表达的变化，采用免疫组织化学方法检测kisspeptin蛋白表达水平，以探讨该系统在PFOA、PFOS所致的青春期启动时间改变中发挥的作用。选择染毒雌鼠的肝脏组织，测定肝脏系数，进行肝脏功能相关血生化指标的检测，采用GC-MS进行血清脂肪酸组、肝脏代谢组测定，以探讨PFOA、PFOS在发育关键期短期暴露对肝脏功能的影响。
结果
新生期、青春前期10mg/kg PFOA、1mg/kg和10mg/kg PFOS短期暴露，可引起雌鼠阴道开口时间比对照组早4-5天，提前进入青春期。新生期PFOA、PFOS暴露后，以非剂量依赖性的方式增加雌鼠体重和AGD；青春前期暴露也可增加雌鼠PND35的体重。在两个暴露阶段处理后，低、中剂量的PFOA、PFOS染毒组大鼠都能出现不规律的雌性周期，表现为延长的静止期或发情后期。PND1-5和PND26-30暴露于PFOA、PFOS的雌鼠血清雌激素和LH水平上升，新生期对激素的影响强于青春前期。化合物染毒未引起卵巢组织结构发生明显改变，但可影响卵泡数量，在新生期低剂量的PFOA、PFOS组大鼠可见生长卵泡数量减少。下丘脑前腹侧室周核（AVPV）和弓状核（ARC）区域Kiss1、Kiss1r、ERα的基因水平下降，免疫组化结果显示kisspeptin纤维密度在PFOA、PFOS的高剂量暴露组受到抑制，与基因表达的结果趋势大体相同。对于大鼠肝脏，高剂量组的PFOA、PFOS可以增加肝脏重量。GC-MS分析结果显示，血清中多种脂肪酸的含量发生改变，肝脏代谢小分子分析显示，PFOA、PFOS导致的差异表达物质主要集中在代谢通路、半乳糖代谢通路、脂肪酸合成通路等。
结论
新生期、青春前期短期暴露于环境剂量下的PFOA、PFOS能够影响下丘脑-垂体-性腺（HPG）轴的调控，导致雌性青春期启动的提前，其中kisspeptin/GPR54系统在其中起了关键作用。新生期暴露于PFOA、PFOS也可影响体内脂肪酸代
Perfluorinated compounds (PFCs) are synthetic fully fluorinated organiccompounds that have been widely used as surfactants in industrial and commercialapplications. They are stable chemicals that is both lipid-and water-repellent.Moreover, PFCs have been listed as persistent organic pollutants (POPs). Thewidespread distribution and persistence of PFCs in humans and the environment havegenerated great concern about potential health impacts.
There is growing evidence that PFCs may disrupt the endocrine system.Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) are therepresentative and commonly used PFCs. Further characterization of theendocrine-disrupting effects of PFOA and PFOS is required for assessing potentialhealth risks. Methods developed by U.S.EPA involve a battery of in vitro and in vivoscreening assays to identify substances that interact with endocrine systems. Toinvestigate the endocrine disruption potencies of PFOA and PFOS, a combination of in vitro and in vivo assays was employed in the present study. Reporter gene assayswere used to detect receptor mediated (anti-)estrogenic,(anti-)androgenic,(anti-)thyroid hormone activities and PPAR activation of PFOA/PFOS. Thesteroidogenic effects of PFOA/PFOS were measured at both the hormone and genelevels in the human adrenocortical carcinoma cell line H295R. Additionally, azebrafish-based short-term screening method and female pubertal rat assay weredeveloped to detect the potential effect of PFOA/PFOS on endocrine function in vivo.Our research focused on a detailed characterization of PFOA/PFOS in an attempt todetermine the endocrine-disrupting effects of these chemicals.
Part I Assessing the endocrine-disrupting effects of PFOAand PFOS by in vitro assays
Chapter I Assessing hormone receptor activities of PFOAand PFOS in reporter gene assays
Objective
The major way by which endocrine disrupting chemicals (EDCs) disrupt thephysiological process is through interaction with nuclear hormone receptors. Reportergene assay has been established as a powerful tool for testing receptor agonists andantagonists among chemicals. Hormone receptor mediated reporter gene assays wereused to detect (anti-)estrogenic (anti-)androgenic,(anti-)thyroid hormone activitiesand PPAR activation of PFOA and PFOS.
Methods
In ER mediated reporter gene assay, CV-1cell line was transfected with ER expression vector rER/pCI and the reporter plasmid pERE-TATA-Luc+. In TR mediated reporter gene assay, CV-1cell line was transfected with TR expressionvector pGal4-L-TR and the reporter plasmid pUAS-tk-luc. In PPAR mediatedreporter gene assay, CV-1cell line was transfected with PPAR expression vectorPG-PPAR and the reporter plasmid PBR-PPRE. Cells were treated with PFOA orPFOS alone to investigate the agonistic activities of these chemicals. For antagonisticactivity tests, cells were treated with PFOA/PFOS with E2/T3. MDA-kb2cell linehad been stably transfected with the pMMTV.neo.luc reporter gene plasmid. Theywere treated with various concentrations of tested chemicals with or without DHT toinvestigate the agonistic and antagonistic activities.
Results
PFOA and PFOS acted alone had no effect on reporter gene expression ascompared with vehicle control. PFOA and PFOS co-treatment with1×10-9M E2could increase the luciferase expression at the tested concentrations (3×10-9-3×10-7M). None of the tested groups showed androgenic and antiandrogenic activities in ARreporter gene assay. PFOA and PFOS did not have TR agonistic activity in TRreporter gene assay. PFOA (1×10-8-3×10-7M) displayed anti-thyroid hormone effectswith5×10-9M T3in the medium. While PFOS co-treated with T3could also suppressthe expression of luciferase at concentrations of1×10-7M and3×10-7M. In PPAR reporter gene assay, PFOA and PFOS activated PPAR at all concentrations.
Conclusion
Results here demonstrated that PFOA and PFOS has additive estrogenic activity,anti-thyroid hormone activity and PPAR activation in vitro. They could have effectson the endocrine system through interfering with nuclear receptor.
Chapter II The steroidogenic effects of PFOA and PFOSin H295R steroidgenesis Assay
Objective
Chemicals can exert endocrine-disrupting effects via non-receptor mediatedpathways. The human adrenocortical carcinoma cell line H295R has been shown tobe a potential in vitro model for screening adverse effects of chemicals onsteroidogenesis. In this study, H295R cells were used to determine the effects ofPFOA and PFOS on steroidogenesis.
Methods
In this study, the steroidogenic effects of PFOA and PFOS were measured inH295R cell. The hormone levels of estradiol (E2) and testosterone (T), the expressionof major steroidogenic genes and the key steroidogenic gene regulator steroidogenicfactors1(SF-1) were measured after PFOA and PFOS exposure in H295Rsteroidogenesis assay.
Results
PFOA and PFOS could induce E2production and reduce T production in aconcentration-dependent manner. E2level were significantly increased by PFOA andPFOS at1×10-8M and3×10-8M, respectively. While T level were decreased byPFOA and PFOS at3×10-8M and1×10-7M, respectively. The expression patterns ofthe10steroidogenic genes were altered by PFOA and PFOS exposure. PFOAsignificantly induced the expression of17βHSD1,3βHSD2, CYP11B2, CYP19.Expressions of CYP17,17βHSD4, CYP11A were decreased by PFOA exposure. Genelevels of HMGR, StAR, CYP11A, CYP19,3βHSD2, CYP11B2were elevated by PFOS.For CYP17,17βHSD1and CYP21, the relative responses were suppressed after PFOStreatment. For the key steroidogenic gene regulator SF-1, gene expression wasdown-regulated within the tested concentration range. SF-1protein level gradually decreased in H295R cells as PFOA exposure concentration increased.
Conclusion
PFOA and PFOS could alter steroid hormone synthesis and the majorsteroidogenic genes expression in H295R steroidogenesis assay. Results heredemonstrated that PFOA and PFOS have the ability to alter steroidogenesis.
Part II Assessing the endocrine-disrupting effects of PFOAand PFOS by in vivo assays
Chapter I Effects of PFOA and PFOS on development andendocrine function in zebrafish
Objective
A short-term zebrafish embryo exposure assay was developed to detect thepotential effects of PFOA and PFOS on endocrine system development and functionin vivo.
Methods
Zebrafish embryos from4h post fertilization (hpf) to120hpf were exposed to100μg/L,200μg/L, and500μg/L PFOA or PFOS. The embryos’ survivals and thestage of embryonic development were recorded daily. Genes related to estrogenreceptor production (esr1and esr2b), early thyroid development (hhex and pax8), andsteroidogenic enzyme synthesis (cyp17, cyp19a, and cyp19b) were quantified byQ-RT-PCR. To identify genes and pathways involved in the endocrine-disruptingeffects, mRNA microarray analysis were conducted.
Results
Exposure to PFOA and PFOS did not affect hatching and larval survival rates inzebrafish embryos. No significant lethal and developmental abnormality wasobserved during the whole exposure time. PFOA cause a significantly increase ofesr1at500μg/L and no changes for esr2b. Exposure to PFOA up-regulated hhex andpax8expression levels at200and500μg/L. No significant changes of cyp17, cyp19a,and cyp19b were observed. Exposure to PFOS (500μg/L) led to an increase in theexpression level of esr1but a decrease in the expression of esr2b. An apparentconcentration-dependent increase of hhex and pax8expression was observed afterPFOS treatment. Suppression of cyp17, cyp19a, and cyp19b was found at200and500μg/L PFOS exposure. Microarray analysis revealed several classes ofsignificantly regulated genes which could be related to cellular, metabolic,development and biological regulation processes in zebrafish.
Conclusion
In short-term zebrafish embryo exposure assay, PFOA and PFOS could alter theexpressions of estrogen receptor, early thyroid development and steroid synthesisrelated genes. Further large-scale genetic study using microarrays provided moreinformation on exploring the mechanisms of PFCs-mediated endocrine-related effectsin zebrafish.
Chapter II Effects of PFOA and PFOS on pubertydevelopment
Objective
Limited information about effects of PFOA and PFOS on puberty developmentis available. We evaluated the effects and explored the neuroendocrine mechanism of neonatal and juvenile PFOA/PFOS exposure on female puberty maturation. We alsoevaluated the effects of PFOA/PFOS on liver.
Methods
Female rats were injected with PFOA or PFOS at0.1,1and10mg/kg/dayduring postnatal day (PND)1-5or26-30. Body weight, anogenital distance (AGD),vaginal opening (VO), estrous cyclicity and other development indexes were recorded.Serum levels, ovaries histology and follicles numbers were evaluated. Geneexpressions of Kiss1, Kiss1r, ERα, ERβ and immunohistochemistry of kisspeptin inthe hypothalamic anteroventral periventricular (AVPV) and arcuate (ARC) nucleiwere quantified. Liver weight were measured. Fatty acids in serum were determinedby GC/MS. Analysis of biomarkers for hepatic metabolism were also assessed.
Results
VO and first estrus were significantly advanced in10mg/kg PFOA,1and10mg/kg PFOS groups after neonatal and juvenile exposure. Neonatal PFOA/PFOSexposure increased body weight and AGD in a non-dose-dependent manner. Estradioland luteinizing hormone (LH) levels were increased after neonatal and juveniletreatments. Irregular estrous cycles occurred more frequently in0.1and1mg/kgPFOA/PFOS groups. Although no altered ovarian morphology was observed, folliclesnumbers were reduced in neonatal groups. Kiss1, Kiss1r, ERα mRNA expressions inAVPV and ARC were decreased after two periods’ exposure. Suppression ofkisspeptin fiber intensities were also evoked by PFOA/PFOS exposure especially atthe high dose in AVPV and ARC. Results also showed that liver weight weresignificantly increased in the highest PFOA/PFOS treated groups.Changes in serumfatty acids composition and metabolic disturbance in liver by both PFOA/PFOS wereobserved.
Conclusion
Neonatal and juvenile exposure to environmental levels of PFOA/PFOS couldaffect the regulation of hypothalamic-pituitary-gonadal (HPG) axis leading toadvancing puberty onset. Kisspeptin/GPR54system played a key role in the effects.Neonatal exposure to low-dose of PFOA/PFOS could affect serum fatty acidmetabolism and other metabolic processes in liver.
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