双酚A及其替代物双酚S对鱼类免疫和神经内分泌系统的毒性效应与作用机理
发布时间：2018-09-17 09:58
【摘要】：A(BPA)是公认的环境内分泌干扰物,广泛分布在河流、湖泊和其它水环境中,并能在水体、沉积物和水生生物中检出。双酚A主要用于生产聚碳酸酯、环氧树脂、增塑剂等物质的前体物质。由于其在日常生活中的广泛应用及环境中的普遍存在,BPA将对水生态系统产生很多潜在的不利影响。目前许多研究表明,双酚A具有潜在毒性,尤其对新生儿的发育毒性。因此,在部分国家包括中国,生产婴儿奶瓶及部分婴儿用品的原材料中已经禁用BPA。双酚S(BPS)是一类典型的BPA替代物,可以用来合成聚碳酸酯、环氧树脂、增塑剂等物质的前体物质。因为BPS具有和BPA类似的化学结构,且比BPA更为稳定的化学性质,近年已经被广泛应用在BPA free(不含双酚A)的产品上。然而,国内外目前对BPS的毒性研究却还是近乎空白。因此,迫切要求我们进行对BPS的毒性研究,评价标注BPA free的产品是否是真正的安全放心产品。本项研究以红鲤鱼原代巨噬细胞和模式生物斑马鱼为研究对象,开展环境相关水平BPA和BPS对鱼类的免疫细胞和神经内分泌系统的毒性效应和致毒机理研究,主要结果如下:1.环境相关浓度BPA(0.1,1,10,100和1000?g/L)的急性暴露(6小时)对红鲤鱼原代巨噬细胞具有免疫调节作用,结果如下:(1)BPA的所有浓度组暴露没有显著的影响巨噬细胞的成活率;(2)低浓度BPA暴露有促进细胞吞菌的作用,而随着浓度的增加,高浓度暴露造成溶菌酶活性降低,细胞吞菌能力受到抑制,表明BPA的暴露显著影响细胞的免疫功能;(3)高浓度的BPA暴露使得巨噬细胞胞内活性氧(ROS),羟自由基含量,一氧化氮,一氧化氮合酶以及多种抗氧化酶活性增加,显著干扰细胞的抗氧化系统;(4)高浓度的BPA使得细胞内典型的免疫相关基因Interleukin-10,interleukin-6 subfamily-like cytokine(M17)和(IL-1β)表达水平显著上调,细胞凋亡数量增多,明显干扰细胞的免疫调节作用。因此,我们推测低浓度BPA暴露可能激活了细胞的免疫功能,增加了溶菌酶的活性,促进巨噬细胞的吞菌活性,用以对抗外界环境的压力,而随着浓度的增加,高浓度的bpa对细胞造成氧化压迫,减弱细胞的免疫功能,干扰胞内免疫调节,可能对细胞造成一定程度的免疫损伤,威胁巨噬细胞的健康生长。同时,研究结果还表明,bpa短期急性暴露后,era和nfκb基因表达变化都呈现出浓度相关性增加,并且在加入雌激素受体抑制剂ici和nfκb通路拮抗剂pdtc与bpa共暴露后,使得原来由bpa单独暴露造成的基因诱导程度有所缓解。因此,我们进一步推测雌激素受体era和转录因子nf-κb共同参与bpa对红鲤鱼巨噬细胞的免疫调节作用。2.环境相关浓度bps的急性暴露(6小时)对红鲤鱼原代巨噬细胞具有与bpa相似的免疫调节作用,结果如下:(1)通过死亡率测试实验,本文首次提出bps对于巨噬细胞短期暴露的半致死浓度,5%致死浓度,无效应浓度,为后续实验提供了暴露浓度;(2)双酚s(0.1,1,10,100,1,000μg/l)的短期暴露显著增加了胞内ros和羟自由基的含量,增强胞内总抗氧化能力和诱导胞内产生脂质过氧化反应,表明双酚s的短期暴露对细胞造成了氧化压迫;(3)双酚s的短期暴露导致胞内溶菌酶活性降低,细胞吞菌能力减弱,表明双酚s的短期暴露削弱了原代巨噬细胞的免疫功能;(4)双酚s的短期暴露显著诱导大量免疫相关基因的表达,以及no和nos的含量,干扰巨噬细胞的免疫调节;(5)高浓度双酚s使得胞内caspase3活性升高,tunel检测凋亡细胞增多,出现明显的dnaladder条带,显著诱导细胞的凋亡。(6)对比相同浓度bps和bpa对原代巨噬细胞的免疫毒性,bps和bpa都能够显著影响免疫基因,免疫相关蛋白,以及胞内活性氧的水平,并且在诱导程度上,bps和bpa有着非常类似的效应。因此,双酚s的短期6h暴露,有类似bpa的作用,会显著影响红鲤鱼原代巨噬细胞的免疫系统,干扰其免疫调节作用。基于对bps作用机理的探讨还发现,bps短期急性暴露使得erα和erβ2基因表达出现显著诱导,而在加入抑制剂ici后,都使得原来由bps单独暴露造成的基因诱导程度有所缓解。因此,我们推测雌激素受体erα和erβ2可能参与bps对红鲤鱼巨噬细胞的免疫调节作用的。3.研究双酚a和双酚s的短期暴露对斑马鱼胚胎发育阶段的神经内分泌系统具有相似的毒性效应,结果如下:(1)gnrh3转基因斑马鱼胚胎经bpa暴露,在环境相关浓度1和10μg/L时,显著诱导斑马鱼胚胎孵化率;(2)在环境浓度10和100μg/L BPA暴露下,斑马鱼三叉神经(TN)和下丘脑(HYPO)中GnRH3神经元数量出现显著性增多,暗示BPA会对斑马鱼胚胎发育阶段神经内分泌系统产生影响;(3)BPA的暴露进一步显著影响了神经内分泌系统相关基因(Kiss 1、Kiss 1r、GnRH3、LHβ、FSHβ、SV2c)的表达,更加证实BPA会对斑马鱼胚胎发育阶段神经内分泌系统产生影响;(4)相同浓度的BPS能够显著诱导GnRH3神经元的发育和神经内分泌系统相关基因的表达水平,表明BPS有类似BPA的效应,能够显著干扰鱼类神经内分泌系统;(5)研究结果还表明,雌激素受体,甲状腺受体和芳香化酶通的抑制剂能够显著抑制BPA或BPS对内分泌系统相关基因包括Kiss 1、Kiss 1r、GnRH3、LHβ、FSHβ和ERa诱导水平,表明BPA和BPS对鱼类的神经内分泌系统干扰作用与雌激素受体,甲状腺受体和芳香化酶三条通路之间存在密切的关系。综上,本论文研究结果揭示了环境雌激素双酚A和其替代物双酚S能够显著干扰鱼类的免疫和神经内分泌系统,揭示了鱼类的先天性免疫系统和神经内分泌系统对环境雌激素类物质有一定的敏感性。因而,水环境中鱼类在低剂量环境雌激素中的暴露风险可能被低估。免疫相关基因,活性氧,抗氧化酶活性和神经内分泌系统相关基因也可以作为生物标志物来进一步研究BPA和BPS对水环境的生态风险。同时本文首次报道BPS会对鱼类的免疫系统和神经内分泌系统产生影响,暗示我们BPA free的产品不一定安全,为制定更高双酚类物质在塑料等日用品上使用的标准提供依据。
[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.
【学位授予单位】：上海大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X171.5
本文编号：2245510
[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.
【学位授予单位】：上海大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X171.5
【参考文献】
相关期刊论文 前2条
1 汤娇雯;张富;陈兆波;;我国鱼类生物多样性保护策略[J];淡水渔业;2009年04期
2 宋宏宇,王捷;环境内分泌干扰物与农药[J];农药科学与管理;2001年02期
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