【摘要】：A(BPA)是公認的環(huán)境內分泌干擾物,廣泛分布在河流、湖泊和其它水環(huán)境中,并能在水體、沉積物和水生生物中檢出。雙酚A主要用于生產聚碳酸酯、環(huán)氧樹脂、增塑劑等物質的前體物質。由于其在日常生活中的廣泛應用及環(huán)境中的普遍存在,BPA將對水生態(tài)系統(tǒng)產生很多潛在的不利影響。目前許多研究表明,雙酚A具有潛在毒性,尤其對新生兒的發(fā)育毒性。因此,在部分國家包括中國,生產嬰兒奶瓶及部分嬰兒用品的原材料中已經禁用BPA。雙酚S(BPS)是一類典型的BPA替代物,可以用來合成聚碳酸酯、環(huán)氧樹脂、增塑劑等物質的前體物質。因為BPS具有和BPA類似的化學結構,且比BPA更為穩(wěn)定的化學性質,近年已經被廣泛應用在BPA free(不含雙酚A)的產品上。然而,國內外目前對BPS的毒性研究卻還是近乎空白。因此,迫切要求我們進行對BPS的毒性研究,評價標注BPA free的產品是否是真正的安全放心產品。本項研究以紅鯉魚原代巨噬細胞和模式生物斑馬魚為研究對象,開展環(huán)境相關水平BPA和BPS對魚類的免疫細胞和神經內分泌系統(tǒng)的毒性效應和致毒機理研究,主要結果如下:1.環(huán)境相關濃度BPA(0.1,1,10,100和1000?g/L)的急性暴露(6小時)對紅鯉魚原代巨噬細胞具有免疫調節(jié)作用,結果如下:(1)BPA的所有濃度組暴露沒有顯著的影響巨噬細胞的成活率;(2)低濃度BPA暴露有促進細胞吞菌的作用,而隨著濃度的增加,高濃度暴露造成溶菌酶活性降低,細胞吞菌能力受到抑制,表明BPA的暴露顯著影響細胞的免疫功能;(3)高濃度的BPA暴露使得巨噬細胞胞內活性氧(ROS),羥自由基含量,一氧化氮,一氧化氮合酶以及多種抗氧化酶活性增加,顯著干擾細胞的抗氧化系統(tǒng);(4)高濃度的BPA使得細胞內典型的免疫相關基因Interleukin-10,interleukin-6 subfamily-like cytokine(M17)和(IL-1β)表達水平顯著上調,細胞凋亡數(shù)量增多,明顯干擾細胞的免疫調節(jié)作用。因此,我們推測低濃度BPA暴露可能激活了細胞的免疫功能,增加了溶菌酶的活性,促進巨噬細胞的吞菌活性,用以對抗外界環(huán)境的壓力,而隨著濃度的增加,高濃度的bpa對細胞造成氧化壓迫,減弱細胞的免疫功能,干擾胞內免疫調節(jié),可能對細胞造成一定程度的免疫損傷,威脅巨噬細胞的健康生長。同時,研究結果還表明,bpa短期急性暴露后,era和nfκb基因表達變化都呈現(xiàn)出濃度相關性增加,并且在加入雌激素受體抑制劑ici和nfκb通路拮抗劑pdtc與bpa共暴露后,使得原來由bpa單獨暴露造成的基因誘導程度有所緩解。因此,我們進一步推測雌激素受體era和轉錄因子nf-κb共同參與bpa對紅鯉魚巨噬細胞的免疫調節(jié)作用。2.環(huán)境相關濃度bps的急性暴露(6小時)對紅鯉魚原代巨噬細胞具有與bpa相似的免疫調節(jié)作用,結果如下:(1)通過死亡率測試實驗,本文首次提出bps對于巨噬細胞短期暴露的半致死濃度,5%致死濃度,無效應濃度,為后續(xù)實驗提供了暴露濃度;(2)雙酚s(0.1,1,10,100,1,000μg/l)的短期暴露顯著增加了胞內ros和羥自由基的含量,增強胞內總抗氧化能力和誘導胞內產生脂質過氧化反應,表明雙酚s的短期暴露對細胞造成了氧化壓迫;(3)雙酚s的短期暴露導致胞內溶菌酶活性降低,細胞吞菌能力減弱,表明雙酚s的短期暴露削弱了原代巨噬細胞的免疫功能;(4)雙酚s的短期暴露顯著誘導大量免疫相關基因的表達,以及no和nos的含量,干擾巨噬細胞的免疫調節(jié);(5)高濃度雙酚s使得胞內caspase3活性升高,tunel檢測凋亡細胞增多,出現(xiàn)明顯的dnaladder條帶,顯著誘導細胞的凋亡。(6)對比相同濃度bps和bpa對原代巨噬細胞的免疫毒性,bps和bpa都能夠顯著影響免疫基因,免疫相關蛋白,以及胞內活性氧的水平,并且在誘導程度上,bps和bpa有著非常類似的效應。因此,雙酚s的短期6h暴露,有類似bpa的作用,會顯著影響紅鯉魚原代巨噬細胞的免疫系統(tǒng),干擾其免疫調節(jié)作用�；趯ps作用機理的探討還發(fā)現(xiàn),bps短期急性暴露使得erα和erβ2基因表達出現(xiàn)顯著誘導,而在加入抑制劑ici后,都使得原來由bps單獨暴露造成的基因誘導程度有所緩解。因此,我們推測雌激素受體erα和erβ2可能參與bps對紅鯉魚巨噬細胞的免疫調節(jié)作用的。3.研究雙酚a和雙酚s的短期暴露對斑馬魚胚胎發(fā)育階段的神經內分泌系統(tǒng)具有相似的毒性效應,結果如下:(1)gnrh3轉基因斑馬魚胚胎經bpa暴露,在環(huán)境相關濃度1和10μg/L時,顯著誘導斑馬魚胚胎孵化率;(2)在環(huán)境濃度10和100μg/L BPA暴露下,斑馬魚三叉神經(TN)和下丘腦(HYPO)中GnRH3神經元數(shù)量出現(xiàn)顯著性增多,暗示BPA會對斑馬魚胚胎發(fā)育階段神經內分泌系統(tǒng)產生影響;(3)BPA的暴露進一步顯著影響了神經內分泌系統(tǒng)相關基因(Kiss 1、Kiss 1r、GnRH3、LHβ、FSHβ、SV2c)的表達,更加證實BPA會對斑馬魚胚胎發(fā)育階段神經內分泌系統(tǒng)產生影響;(4)相同濃度的BPS能夠顯著誘導GnRH3神經元的發(fā)育和神經內分泌系統(tǒng)相關基因的表達水平,表明BPS有類似BPA的效應,能夠顯著干擾魚類神經內分泌系統(tǒng);(5)研究結果還表明,雌激素受體,甲狀腺受體和芳香化酶通的抑制劑能夠顯著抑制BPA或BPS對內分泌系統(tǒng)相關基因包括Kiss 1、Kiss 1r、GnRH3、LHβ、FSHβ和ERa誘導水平,表明BPA和BPS對魚類的神經內分泌系統(tǒng)干擾作用與雌激素受體,甲狀腺受體和芳香化酶三條通路之間存在密切的關系。綜上,本論文研究結果揭示了環(huán)境雌激素雙酚A和其替代物雙酚S能夠顯著干擾魚類的免疫和神經內分泌系統(tǒng),揭示了魚類的先天性免疫系統(tǒng)和神經內分泌系統(tǒng)對環(huán)境雌激素類物質有一定的敏感性。因而,水環(huán)境中魚類在低劑量環(huán)境雌激素中的暴露風險可能被低估。免疫相關基因,活性氧,抗氧化酶活性和神經內分泌系統(tǒng)相關基因也可以作為生物標志物來進一步研究BPA和BPS對水環(huán)境的生態(tài)風險。同時本文首次報道BPS會對魚類的免疫系統(tǒng)和神經內分泌系統(tǒng)產生影響,暗示我們BPA free的產品不一定安全,為制定更高雙酚類物質在塑料等日用品上使用的標準提供依據(jù)。
[Abstract]:BPA is widely distributed in rivers, lakes and other aquatic environments and can be detected in water, sediments and aquatic organisms. BPA is mainly used to produce precursors of polycarbonates, epoxy resins, plasticizers and other substances. PA will have many potential adverse effects on aquatic ecosystems. Many studies have shown that BPA is potentially toxic, especially for neonatal development. Therefore, BPA. BPS has been banned in some countries, including China, as a typical BPA substitute for baby bottles and some baby products. In recent years, BPS has been widely used in BPA free (BPA free) products because of its similar chemical structure and more stable chemical properties than BPA. However, the toxicity of BPS is still almost blank at home and abroad. In this study, the toxicity of BPA and BPS to immune cells and neuroendocrine system of fish and the toxic mechanism of BPA and BPS were studied in the primary macrophages and zebrafish. The main results were as follows: 1. Acute exposure of BPA (0.1, 1, 10, 100 and 1000?G/L) at environmental related concentrations (6 hours) had immunomodulatory effects on primary macrophages of red carp. The results were as follows: (1) Exposure to all concentrations of BPA had no significant effect on the survival rate of macrophages; (2) Exposure to BPA at low concentrations could promote cell phagocytosis, but with the increase of BPA concentration, the survival rate of macrophages was increased. With the increase of concentration, the lysozyme activity was decreased and the phagocytosis ability was inhibited, indicating that BPA exposure significantly affected the cellular immune function; (3) High concentration of BPA exposure increased the intracellular reactive oxygen species (ROS), hydroxyl free radical content, nitric oxide, nitric oxide synthase and a variety of antioxidant enzymes activities in macrophages. (4) High concentration of BPA significantly increased the expression levels of typical immune-related genes Interleukin-10, interleukin-6 subfamily-like cytokine (M17) and (IL-1 beta), increased the number of apoptosis and interfered with the immune regulation of cells. It can increase the activity of lysozyme and promote the phagocytosis of macrophages to resist the pressure of external environment. With the increase of concentration, high concentration of BPA will cause oxidative stress on cells, weaken the immune function of cells, interfere with the regulation of intracellular immunity, and may cause certain degree of immune damage to cells. At the same time, the results also showed that the expression of era and NF - kappa B genes increased in a concentration-dependent manner after short-term acute exposure to bpa, and the gene induction caused by BPA exposure alone was slowed down after co-exposure with estrogen receptor inhibitor ICI and NF - kappa B pathway antagonist PDTC and bpa. Therefore, we further speculate that estrogen receptor era and transcription factor NF-kappa B may participate in the immunomodulatory effect of BPA on red carp macrophages. 2. acute exposure to environmental-related concentrations of BPS (6 hours) has a similar immunomodulatory effect to that of BPA on red carp primary macrophages. the results are as follows: (1) through the mortality test, this paper reports the results of this study. For the first time, the semi-lethal concentration, 5% lethal concentration and ineffective stress concentration of BPS for short-term exposure of macrophages were proposed, which provided the exposure concentration for subsequent experiments. (2) Short-term exposure of bisphenols (0.1,1,10,100,1,000 ug/l) significantly increased the content of intracellular ROS and hydroxyl radicals, enhanced the total intracellular antioxidant capacity and induced intracellular lipid peroxidation. The results showed that short-term exposure to bisphenols resulted in oxidative stress on cells; (3) short-term exposure to bisphenols resulted in decreased intracellular lysozyme activity and decreased phagocytosis, indicating that short-term exposure to bisphenols weakened the immune function of primary macrophages; (4) short-term exposure to bisphenols significantly induced the expression of a large number of immune-related genes, as well as n. The contents of O and NOS interfered with the immune regulation of macrophages; (5) high concentration of bisphenols increased the activity of caspase 3 in the cells, and TUNEL detected the apoptotic cells increased, there were obvious DNA ladder bands, significantly induced cell apoptosis. (6) compared with the same concentration of BP s and BPA on the immunotoxicity of primary macrophages, BP s and BPA can significantly affect the immune basis. BP s and BPA have very similar effects on the level of immune-related proteins and intracellular reactive oxygen species. Therefore, short-term exposure to bisphenols for 6 hours, similar to bpa, can significantly affect the immune system of primary macrophages of red carp and interfere with their immune regulation. Short-term acute exposure to BPS resulted in significant induction of ER-alpha and er-beta-2 gene expression, which was alleviated by adding inhibitor ici. Therefore, we speculated that estrogen receptor ER-alpha and er-beta-2 might be involved in the immunomodulatory effect of BPS on red carp macrophages. 3. Bisphenol-a and bisphenol-A were studied. Short-term exposure to bisphenols has similar toxic effects on the neuroendocrine system of zebrafish embryonic development stage. The results are as follows: (1) Gnrh3 transgenic zebrafish embryos exposed to BPA significantly induced the hatching rate of zebrafish embryos at environmental related concentrations of 1 and 10 ug/L; (2) Zebrafish trigemina at environmental concentrations of 10 and 100 ug/L BPA The number of GnRH3 neurons in (TN) and hypothalamus (HYPO) increased significantly, suggesting that BPA could affect the neuroendocrine system in zebrafish embryonic development; (3) BPA exposure further significantly affected the expression of neuroendocrine system-related genes (Kiss 1, Kiss 1r, GnRH3, LH beta, FSH beta, SV2c), further confirming that BPA could affect the embryonic development of zebrafish. (4) BPS at the same concentration can significantly induce the development of GnRH3 neurons and the expression of genes related to the neuroendocrine system, suggesting that BPS has a BPA-like effect and can significantly interfere with the neuroendocrine system of fish; (5) The results also show that estrogen receptor, thyroid receptor and thyroid receptor can significantly interfere with the neuroendocrine system of fish. Inhibitors of aromatase can significantly inhibit the induction of genes related to endocrine system by BPA or BPS, including Kiss 1, Kiss 1r, GnRH3, LH beta, FSH beta and ERa, suggesting that there is a close relationship between the interference of BPA and BPS on neuroendocrine system of fish and estrogen receptor, thyroid receptor and aromatase pathways. The results revealed that environmental estrogens bisphenol A and its substitutes bisphenol S could significantly interfere with the immune and neuroendocrine systems of fish, and the innate immune and neuroendocrine systems of fish were sensitive to environmental estrogens. The risk of exposure may be underestimated. Immune-related genes, reactive oxygen species, antioxidant enzymes and neuroendocrine system-related genes can also be used as biomarkers to further study the ecological risk of BPA and BPS to the aquatic environment. BPA free products are not necessarily safe, providing a basis for the development of higher standards for the use of bisphenols in plastics and other daily necessities.
【學位授予單位】：上海大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：X171.5

【參考文獻】

相關期刊論文 前2條

1 湯嬌雯;張富;陳兆波;;我國魚類生物多樣性保護策略[J];淡水漁業(yè);2009年04期

2 宋宏宇,王捷;環(huán)境內分泌干擾物與農藥[J];農藥科學與管理;2001年02期

，

本文編號：2245510