Study on the Toxicity and Endocrine-disrupting Effects of Polyfluorinated iodine alkanes

全氟碘烷类化合物是一类合成有机氟化合物的重要中间体，例如合成有机氟表面活性剂、有机氟多聚物和有机氟橡胶等。在有机氟多聚物中已经检出全氟碘烷的残留，并且全氟碘烷已在大气中被检出。全氟碘烷的高产量以及全氟碘烷的环境释放已经引起广泛的关注，但是对于全氟碘烷的毒性研究还处于未知阶段。近年来有研究表明，某些全氟类化合物具有雌激素效应。我们通过细胞增殖实验、MVLN实验以及雌激素响应基因在MCF-7乳腺癌细胞中的表达实验研究了12种全氟碘烷的雌激素效应。在雌激素效应筛选之前，我们利用3种细胞评价了全氟碘烷的细胞毒性，以确定筛选暴露浓度。全氟碘烷的细胞毒性普遍不强，但是二碘全氟碘烷毒性较高。通过乳酸脱氢酶和CASPASE-7酶活的检测，发现二碘正己烷可能具有诱导细胞凋亡的能力。一碘全氟碘烷（FIAs）在全氟碳链末端上有一个碘取代基。在5种FIAs中，6碳和8碳的一碘全氟己烷和一碘全氟辛烷促进细胞的增殖，诱导MVLN细胞的虫荧光素酶活性，并且上调TFF1和EGR3的表达。全氟碘烷的雌激素效应与全氟碳链的长度和碘元素取代相关，但是全氟碘烷的雌激素构效关系和常规外源雌激素化合物不相似。我们推测全氟碘烷的雌激素效应是由其羟基化代谢产物所产生，具体的机理有待研究。全氟碘烷的环境行为和内分泌干扰效应需引起关注。其它的FIAs无此活性。二碘全氟碘烷(FDIAs)是在全氟碳链的两端各有一个碘原子取代的全氟碘烷类化合物。在碳链相同的情况下，FDIAs比FIAs在三项筛选实验中具有更强的雌激素效应。FIAs和FDIAs中都是六碳的全氟碘烷的雌激素效应最强。全氟调聚碘烷（FTIs）是在不饱和氟取代碳链末端有一个碘原子取代的全氟碘烷类化合物。但是四种FTIs在三项离体实验中都无雌激素效应。全氟辛烷羧酸（PFOA）、一氢全氟正辛烷（PFOC）、一溴全氟正辛烷（PFOB）和一碘正己烷和PFOI具有类似的化学结构，但是这些化合物在三项雌激素效应筛选实验中都无雌激素效应。<br />作为持久性有机污染物，全氟类化合物具有环境持久性、生物蓄积性、全球分布范围广和潜在的毒性效应等特点。关于全氟类化合物的内分泌干扰效应已经广泛研究，包括对甲状腺功能的影响，雌激素效应以及过氧化物酶体增殖体受体活性等。全氟碘烷广泛用于有机氟化工的合成。目前全氟碘烷的年产量超过4000吨。由于全氟碘烷的产量不断上升，以及它未知的环境行为，全氟碘烷类化合物的毒性有待系统评价。本研究中，我们利用H295R肾上腺皮质癌细胞在激素合成相关基因的表达水平和激素水平研究了四种全氟碘烷对离体细胞激素合成的影响。十种与激素合成相关的基因(StAR, HMGR, CYP21, CYP19, CYP17, CYP11B1, CYP11B2,CYP11A1, 17&beta;HSD1, 3&beta;HSD1, 3&beta;HSD2)通过定量PCR检测。培养基中分泌的雌激素17&beta;-雌二醇，雄激素二氢睾酮，糖皮质激素皮质醇，盐皮质激素醛固酮通过竞争性酶联放射性免疫法测定。细胞暴露于100 &mu;M PFHxI 和PFOI，10种基因都有相关上调趋势。CYP19, CYP11B1, CYP11B2, 3HSD1和3HSD2 显著上调 9.2, 9.4, 12.4, 10.3 和6.4倍。CYP19的上调导致雌激素水平上升，雄激素水平降低。醛固酮和皮质醇的水平有所上升。而PFOS和PFOA无此效应。结果表明全氟碘烷类化合物在离体条件下影响细胞激素的合成, 此效应与化合的碳链长度和碘取代基团有关。全氟碘烷可能会影响人类或野生动物的激素合成和内分泌系统。<br />由于全氟碘烷具有光敏感的性质，通过液相质谱联用仪检测了PFOI在不同物理条件下的转化规律，为研究全氟碘烷的环境转化和环境过程提供依据。将PFOI置于紫外线、太阳光、日光灯和加热条件下，比较不同物理过程条件下，PFOI的转化规律。研究表明，全氟碘烷对紫外线和太阳光照射敏感，4小时即有多种转化产物生成，产物为PFOI逐步脱去碳链的全氟类化合物。而PFOI对日光灯和加热不敏感，即使37度条件下加热24小时，PFOI只有少量发生转化，日光灯光照亦无明显变化。此研究表明，全氟碘烷对紫外光敏感，具有一定的热稳定性。Polyfluorinated iodine alkanes (PFIs) are important intermediates for the synthesis of organic fluoride products such as fluorosurfactants, fluoropolymers and fluororubber. PFIs have been detected in fluoropolymers as residual raw materials and atmosphere. The high production volume and environmental release of PFIs has been concerned, but the exposure risk and toxicity of PFIs is not known. Recent study suggests that some of the perfluorinated chemicals (PFCs) have estrogenic effects. In present work, we investigated the estrogenic effects of twelve PFIs by E-screen, MVLN assay and the evaluation of estrogen-responsive genes in MCF-7 cells. Before the estrogenic effects screening, the in vitro toxicity effects of PFIs was studied to identify the exposure concentrations. Most of PFIs are low toxic to cells, but FDIAs are high toxic. FDIAs could induce cell apoptosis by increasing the activity of CASPASE-7 and damage the cell membrance. Fluorinated iodine alkanes (FIAs) have an iodine atom at the ends of perfluorinated carbon chain. Two of the FIAs, 1-Iodoperfluorohexane (PFHxI) and 1-Iodoperfluorooctane (PFOI) promoted the proliferation of MCF-7 cells, induced the luciferase activity in MVLN cells and up-regulated the expression of TFF1 and EGR3 in MCF-7 cells. Other FIAs with shorter or longer chain length are not active in these assays. Fluorinated diiodine alkanes (FDIAs) have an iodine atom at both the ends of perfluorinated carbon chain. FDIAs exhibited stronger estrogenic effects than the corresponding FIAs with the same chain length in three estrogen-screening assays. The estogenic potencies of FIAs and FDIAs are in proportion to the carbon chain length. The optimum chain length for estrogenic potencies of FIAs and FDIAs are all six carbons. Fluorinated telomer iodides (FTIs) have an iodine atom at the end of partially fluorinated carbon chain. However, four FTIs did not show estrogenic effects in any of the in vitro assays. Perfluorooctanoic acid (PFOA), 1H-Perfluorooctane (PFOC), 1-Bromoperfluorooctane (PFOB) and 1-iodohexane are structure similar to PFOI. These chemicals showed negative results in all of estrogen-screening assays. The estrogenic potencies of the PFIs correlated well within three in vitro assays. The estrogenic effects of PFIs are dependent on the structure features of iodine substitution and perfluorinated alkyl chain. However, the structure features of PFIs are not similar to the general xnoestrogens. We hypothesized that the estrogenic activities of PFIs were caused by their hydroxylated metabolites in situ, but the underlying mechanisms need further studies. The environmental behavior and endocrine-disrupting effects of PFIs should be concerned.<br />Perfluorinated chemicals (PFCs) as a kind of persistent organic pollutants, are globally distributed, environmentally persistent, bioaccumulative, and potentially harmful. The endocrine-disrupting effects of PFCs were studied in diverse aspect, including thyroid function, estrogenic effects and PPAR activity. PFIs have been widely used in the synthesis of organic fluoride chemicals. The production volume of PFIs exceeded 1000 tons per years. It is necessary to study the toxicity effects of PFIs due to their high production volume and the unknown environmental behaviors. In this study, the steroidogenic effects of four PFIs were assessed at both the gene and enzyme/hormone levels in the H295R human adrenocortical carcinoma cell line. The expression levels of 10 steroidogenic genes (StAR, HMGR, CYP21, CYP19, CYP17, CYP11B1, CYP11B2, CYP11A1, 17HSD1, 3HSD1, 3HSD2) were measured using quantitative real-time PCR (Q-RT-PCR). Sex steroid (testosterone and 17&beta;-estradiol), cortisol and aldolestone concentrations in the culture medium were measured by competitive enzyme-linked immuno sorbent assay. Exposure to 100 &mu;M of PFHxI and PFOI, ten steroidogenic genes were up-regulated to some extent. The expression of CYP19, CYP11B1, CYP11B2, 3HSD1 and 3HSD2 were significantly up-regulated by 9.2, 9.4, 12.4, 10.3 and 6.4-folds. The up-regulation of CYP19 resulted in the increasing production of 17&beta;-estradiol and decreasing production of testosterone.The production of cortisol and aldolestone also increased. However, PFOS and PFOA showed no effect on steroidgenesis. Our results indicate that PFOI affect steroidogenesis in vitro, and the effect was related to the perfluorinated carbon chain length and iodine substitution. PFIs may have similar potential to affect steroidogenesis and endocrine function in human and wildlife.<br />Due to the PFIs is light sensitive, we studied the physical transforming regulation of PFOI by LC-MS-MS. It is helpful for the study of the environmental transformation and environmental processes of PFOI. We compared the effects of sunshine, ultraviolet light, lamp light and heat on PFOI. It was suggested that PFOI was sensitive to sunshine and ultraviolet light, 4 hour&rsquo; radiation resulted in the generation of transformation products. The products are the shorter perfluorinated carbon chain chemicals. However, PFOI was not sensitive to the heat and lamp light. 24 hour&rsquo; radiations lead to no obvious changes. Less transforming products was formed after 24 hour&rsquo;s heating at 37 ℃. PFOI was sensitive to ultraviolet light and thermo-stable to some extent.