【作者】 于文广；

【导师】 金一和；

【作者基本信息】 大连理工大学 ， 环境科学， 2010， 博士

【摘要】 全氟辛烷磺酸(Perfluorooctane sulfonate, PFOS)是全氟表面活性剂的代表。PFOS曾被广泛应用于纺织、造纸、皮革、石油化工表面活性剂、医药品、电子产品等生产和日常生活各个领域。PFOS具有难降解性、远距离传播性、生物富集性及多种生物毒性,2009年5月,PFOS被正式列入《关于持久性有机污染物的斯德哥尔摩公约》。甲状腺激素(TH)能够调节哺乳动物各项生理机能,对胚胎及幼龄动物的生长发育(尤其是神经智力发育)至关重要。PFOS的甲状腺毒作用研究是其内分泌研究的重要组成部分,也是PFOS人群健康风险评价的重要内容。本研究旨在阐明：①胚胎期接触PFOS与出生后通过乳汁接触PFOS对幼龄动物甲状腺功能的影响以及影响程度的差别；②PFOS干扰大鼠TH的作用机制。围绕以上两个实验目的,本论文开展了如下研究工作：(1)建立交叉哺育(cross-foster)实验动物模型,形成仔鼠胚胎期和出生后均不暴露(对照组)及胚胎期暴露、出生后暴露、胚胎期和出生后均暴露三种不同形式接触PFOS的组别,将胚胎期与出生后暴露PFOS分离开来,考察不同方式接触PFOS对不同发育时期仔鼠甲状腺功能的影响,阐明哪种暴露方式对仔鼠甲状腺功能影响程度较大。实验结果表明,PFOS既可以通过胎盘在胚胎体内积累,又可以通过乳汁在新生幼鼠体内积累；出生前和出生后均暴露PFOS仔鼠在出生后14、21和35天血清总甲状腺素(TT4)水平均显著降低,与同龄对照比较,分别降低了36.7、24.6和37.3%；随体内PFOS浓度逐步增加,出生后暴露PFOS组仔鼠在14、21和35天血清TT4水平比对照分别降低了11.9、28.6和35.9%；尽管仅胚胎期暴露组仔鼠在出生后21和35天血清中PFOS浓度水平显著降低,但二个时间点TT4水平仍然显著性下降,分别低于相应对照组20.3和19.4%。多重比较分析表明,胚胎期暴露PFOS或出生后通过乳汁积累PFOS对出生后14和21仔鼠血清TT4的抑制程度无统计学差异p>0.05)。(2)考察了不同暴露方式下,PFOS对仔鼠甲状腺滤泡形态以及肝脏组织中与TH代谢、转运、细胞作用等过程相关基因表达的影响。实验结果表明,甲状腺滤泡腔各形态学参数未发生显著性改变p>0.05),PFOS未对仔鼠甲状腺滤泡形态产生明显影响；各TH相关基因中,只有TH结合蛋白TTR,在出生前、后均暴露组的出生后21天仔鼠肝脏中表达显著增强,比对照组TTR表达提高了50%。(3)建立91天亚慢性PFOS染毒实验模型,形成1.7、5.0和15.0 mg／L三个染毒剂量组,从TH合成和肝脏代谢角度考察PFOS干扰TH的作用机制。实验结果表明,PFOS染毒显著降低了血清TT4水平,并与染毒剂量呈明显剂量效应关系。15.0 mg／L组,TT4浓度水平仅为对照组的20%。PFOS对TH合成的关键酶甲状腺过氧化物酶活性未产生影响。PFOS上调了肝脏尿苷二磷酸葡萄糖醛酸转移酶A1型酶(UGT1A1)mRNA的表达,在中、高剂量组,UGT1A1表达水平约为相应对照组的2.2和2.3倍。PFOS显著降低了肝脏中脱碘酶(DIO1)基因的表达,在中、高剂量组,DIO1表达为相应对照组的86%和44%。相关分析表明,血清TT4水平与肝脏中UGT1A1和DIO1表达相关性良好(r值分别为-0.754和0.565；p值分别为0.002和0.023),提示PFOS可能增强了T4与肝脏UGT结合水平,提高T4代谢,导致血清TT4水平下降。(4)建立成年大鼠亚急性暴露PFOS实验模型,经口连续染毒5天,染毒剂量分别为0.2、1.0和3.0 mg／kg,同时设置PTU染毒组(10 mg／kg)和10 mg／kg PTU+3.0 mg／kgPFOS的联合染毒组。从TH肝胆排泄和血清结合蛋白(TTR)角度考察PFOS降低血清TT4的机制。实验结果表明,血清中TT4浓度随PFOS染毒剂量增大而逐步降低,但胆汁中TT4浓度未受PFOS影响。PFOS对血清TTR水平及甲状腺球蛋白(TG)水平无显著影响。3.0 mg／kg PFOS上调了肝脏中负责摄入T4有机阴离子转运蛋白OAPT2的表达,约为对照组的1.48倍；PFOS对负责将肝脏T4排至胆汁的多药耐药蛋白MRP2表达影响显著,MRP2表达在1.0和3.0 mg／kg PFOS处理组为对照组的1.81和1.70倍。同时,血清TT4水平与肝脏MRP2相对表达水平具有良好相关性(r=-0.528；p=0.12)。PFOS和PTU对TH抑制不存在显著的联合效应,PTU对TH的抑制强于PFOS。综上研究结果得出,胚胎期暴露PFOS或出生后通过乳汁暴露PFOS对断乳仔鼠血清TT4水平的抑制程度无统计学差异；PFOS未对TH合成过程产生干扰；PFOS主要通过增强肝脏对T4摄入和排出,增强T4葡萄糖醛酸化,使得T4的肝脏代谢增强和肝胆排泄提高,进而抑制血清T4水平。PFOS与经典的抗甲状腺肿药物PTU的甲状腺毒作用机制存在本质区别。更多还原

【Abstract】 Perfluorooctane sulfonate (PFOS) is an environmentally persistent organic pollutant (POPs), which was widely used in industrial and consumer applications. Related animal experiments suggest that PFOS is associated with hepatotoxicity, carcinogenicity, reproductive and developmental effects, immunotoxicity neuroendocrine and neurobehavioural effects as well as hormone disruption. In May 2009, PFOS was added to the Stockholm Convention on POPs.Thyroid hormone is essential for the normal physiological function of virtually all tissues in mammals, playing important roles in regulating metabolism, growth and development, especially for the development of central nervous system and brain function. The present paper investigated that whether prenatal or postnatal exposure to PFOS alone can disturb the thyroid function in rat pups, and if so, which kind of exposure is a major cause of thyroid disruption. Besides, mechanisms of action by which PFOS interferes with thyroid systems in rats were also investigated. The present study comprise of several parts as follows:(1) A cross-foster animal model was established, yield the following four groups: unexposed control (CC), prenatal exposure (TC), postnatal exposure (CT), and prenatal+ postnatal exposure (TT). Serum and liver PFOS concentrations as well as serum TH levels in rat pups at the age of various postnatal days 0,7,14,21 or 35 under different conditions of PFOS exposure were evaluated. The occurrence of PFOS transfer from dams to pups, both via placenta and milk, was observed. Neither total T3 (TT3) nor rT3 in pups was affected by PFOS exposure. Gestational exposure to PFOS alone (TC) significantly (p< 0.05) decreased total T4 (TT4) level in pups on PNDs 21 and 35,20.3 and 19.4% lower than the control on the same PND, respectively. Having postnatal accumulation of PFOS, TT4 levels were 11.9,28.6, and 35.9% lower than controls on PNDs 14,21 and 35, respectively. No significant difference in TT4 level (p> 0.05) was observed between TC and CT on PND 21.(2) Thyroid gland colloid histomorphology paremeters and hepatic expression of genes involved in TH transport, metabolism and receptors were also investigated in rat pups at the age of various postnatal days under different conditions of PFOS exposure. The results demonstrate that none of the thyroid gland colloid histomorphology paremeters, including colloid area, longest axis, shortest axis, roundness and aspect ratio, was affected by PFOS. None of the selected TH related transcripts was affected by PFOS in pups on PND 0. Only transcript level of transthyretin, TH binding protein, in group TT significantly increased to 150% of the control on PND 21.(3) A subchronic PFOS exposure animal model was established. Male rats were exposed to 1.7,5.0 and 15.0 mg/L of PFOS in drinking water for 91 consecutive days. Thyroid and liver were removed for the measurement of the endpoints closely related to TH biosynthesis and metabolism following PFOS exposure. The results showed that serum TT4 level decreased significantly at all applied dosages. Hepatic UGT1A1 mRNA but not UGT1A6 was up-regulated at 5.0 and 15.0 mg/L of PFOS. Correlation coefficients revealed that hepatic UGT1A1 mRNA expression correlated with TT4 level (r=-0.754; p= 0.002). Treatment with PFOS lowered hepatic DIO1 mRNA at 15.0 mg/L but increased thyroidal DIO1 mRNA dose-dependently. The activity of TPO was not affected by PFOS. NIS and TSHR mRNA in thyroid increased slightly but not significantly. These results indicate that increased hepatic T4 glucuronidation via UGT1A1 and increased thyroidal conversion of T4 to T3 via DIO1 were in part responsible for PFOS-induced hypothyroxinemia in rats.(4) A 5 day PFOS exposure was to further identify the major factors contributing to PFOS-induced TH decrease in rats.The consequence of exposure to PTU was also analyzed as a positive control of TH inhibition. The animals were given either (1) vehicle; (2) PFOS; (3) PTU; or (4) PTU+PFOS once a day by gavage for 5 consecutive days. Parameters including contents of TT4 and TT3 in both serum and bile, serum concentrations of transthyretin and thyroglobulin, as well as transcripts of transporters involved in hepatic uptake and efflux of T4 were determined in control and PFOS exposed groups. TT4 and TT3 were also analyzed in PTU and PTU+PFOS groups to reflect the different hormone effects between PFOS, PTU, and PFOS+PTU. Results showed that serum TT4 and TT3 decreased, while bile TT4 and TT3 remained stable following PFOS exposure. Exposure to 3.0 mg/kg of PFOS enhanced hepatic organic anions transporter OATP2 mRNA expression (1.43 times of control). Treatment with PFOS increased hepatic expression of multidrug resistance-associated protein MRP2, approximately 1.80 and 1.69 times of control in 1.0 and 3.0 mg/kg groups, respectively. Spearman’s correlation coefficients revealed that MRP2 mRNA expression correlated well with serum TT4 level (r=-0.528,p= 0.012). Serum thyroglobulin and transthyretin levels remained stable. In addition to serum TT4, PFOS and PTU groups demonstrated significantly difference with each other with respect to serum TT3, bile TT4, and bile TT3. No significant differences of TT4 and TT3 in both serum and bile were observed between PTU and PTU+PFOS (p> 0.05).In conclusion, gestational and lactational exposure to PFOS depresses TT4 in weaned rat to a similar extent. PFOS has little effects on the synthesis of TH. PFOS-induced TT4 deficiency is mainly due to increased hepatic uptake of T4 and enhanced hepatic metabolism and excretion of T4, which is probably different from the classic goitrogen, PTU.更多还原