内分泌干扰物双酚A对秀丽隐杆线虫的生态毒性效应及其作用机制研究

发布时间：2018-11-08 15:57

【摘要】：双酚A(BPA)是世界公认的环境内分泌干扰物,一直是毒理学研究中的明星化合物。BPA的世界年需求量超过400万吨/年,环境中的污染也日趋严重,在土壤、水、大气和沉积物中都发现了BPA的踪迹,甚至在人体组织和体液中也检测到了BPA的存在。体内和体外实验已经证明BPA暴露能够引起内分泌干扰效应、神经毒性、生殖毒性、免疫毒性和致癌致畸性等。人群流行病学研究也发现,糖尿病、肥胖、神经行为障碍、肝功能异常、复发性流产、发育性疾病都与BPA暴露有关。目前关于BPA暴露对无脊椎动物的毒性报道较少。作为一种从土壤中分离出来的代表性的线虫种类,秀丽隐杆线虫是环境暴露评价以及生态毒理学研究中最常用的模式生物之一。本研究选取秀丽隐杆线虫作为受试生物,主要从生理、生化、分子水平探究环境相关浓度下BPA急性与亚急性暴露、慢性暴露和多代连续暴露对秀丽隐杆线虫的生态毒性效应；此外,本研究还考察了BPA的替代品双酚S(BPS)对秀丽隐杆线虫的毒性影响,并与BPA的毒性进行对比,分析BPA替代品的生态风险性。本研究主要的研究结果如下：(1)BPA急性与亚急性暴露能够显著抑制秀丽隐杆线虫的体长、运动行为水平以及子代数目,显著提高细胞凋亡水平以及胁迫相关基因的表达量。急性暴露后,秀丽隐杆线虫的头部摆动频率是最敏感的生理指标,暴露浓度在0.01μM即可对秀丽隐杆线虫产生显著的负面效应,而亚急性暴露后,体长和头部摆动频率相比其他生理指标更加敏感,同样在暴露浓度为0.01μM时能够对秀丽隐杆线虫产生显著的抑制作用。BPA急性和亚急性暴露对线虫体内脂褐素累积和ROS水平没有产生显著影响,但当暴露浓度达到0.1μM时能够显著提高线虫体内细胞凋亡水平,表明实验浓度下BPA暴露对秀丽隐杆线虫产生的毒性效应并不是通过氧化损伤途径,而很可能与细胞凋亡水平升高有关。BPA亚急性暴露能够导致秀丽隐杆线虫体内绝大部分胁迫相关基因表达量的升高,即使在最低暴露浓度0.001 μM, BPA暴露也能够引起绝大多数胁迫相关基因表达量的显著提升。(2)BPA慢性暴露能够显著抑制秀丽隐杆线虫的体长、运动行为水平、寿命以及种群数目,显著提高线虫体内细胞凋亡水平、胁迫相关基因表达以及转基因品系TJ375的荧光蛋白表达量。慢性暴露后,头部摆动频率是最敏感的生理指标,暴露浓度在0.001μM即可对秀丽隐杆线虫产生显著的负面效应。BPA慢性暴露对线虫体内脂褐素累积同样没有显著影响,但能够在暴露浓度达到0.01μM以上时,显著地提高秀丽隐杆线虫体内的细胞凋亡水平。在实验浓度范围内,BPA慢性暴露能够明显地提高线虫体内绝大部分胁迫相关基因的表达量。与亚急性暴露的结果相比,同一暴毒浓度下,BPA慢性暴露能够引起胁迫相关基因的mRNA水平更为明显的提高。当BPA暴露浓度达到0.1μM时,秀丽隐杆线虫的种群数目显著减小。转基因品系TJ375只能够检测环境浓度达到1μM时的BPA毒性,与其它测试指标相比不够灵敏。(3)BPA多代连续暴露能够在暴露浓度达到0.01μM以上时对秀丽隐杆线虫的生理指标产生显著的毒性影响。在较低暴露浓度时,子代线虫会出现毒性逐代修复或者恶化的情况,造成秀丽隐杆线虫生理水平上的世代毒性差异,而在最高暴露浓度10μM时,秀丽隐杆线虫G1代和G4代在毒性效应上没有显著性的差异。在秀丽隐杆线虫G1代和G4代中,与高浓度暴露组相比,低浓度(≤0.01 μM)暴露组能够引起体内胁迫相关基因表达量更为明显的提高；与G4代相比,秀丽隐杆线虫的G1代体内胁迫相关基因的表达量增加更为显著。(4)BPS亚急性暴露后,秀丽隐杆线虫的头部摆动频率是最敏感的生理指标,暴露浓度在1 μM以上才能够对秀丽隐杆线虫产生显著的负面效应,而其他生理指标只有在最高浓度暴露组才会受到显著的负面影响。亚急性暴露后,秀丽隐杆线虫体内脂褐素的累积没有受到显著影响,细胞凋亡水平也只有在暴露浓度为1μM时显著提高。BPS亚急性暴露能够引起秀丽隐杆线虫体内绝大部分基因表达量的小幅度上升。综上所述,低浓度的BPA暴露能够对秀丽隐杆线虫产生发育毒性、神经行为毒性以及生殖毒性。BPA暴露对秀丽隐杆线虫生理水平的毒性影响很可能与体内细胞凋亡水平提高有关；BPA暴露能够显著提高秀丽隐杆线虫体内胁迫相关基因的表达,破坏机体的稳态；转基因品系TJ375对于BPA暴露不敏感,不能有效监测BPA的毒性；与BPA毒性相比,BPS的毒性明显较弱。
[Abstract]:Bisphenol A (BPA) is a world-recognized environmental endocrine disruptor and has been a star compound in toxicology studies. The world's annual demand for BPA is more than 4 million tons/ year, and the pollution in the environment is becoming more and more serious. The trace of BPA is found in soil, water, atmosphere and sediment, and BPA is also detected in human tissue and body fluids. In vivo and in vitro experiments have shown that the exposure of BPA can cause endocrine disruption, neurotoxicity, reproductive toxicity, immunotoxicity, and carcinogenicity. The epidemiological study of the population also found that diabetes, obesity, neurobehavioral disorders, liver function abnormalities, recurrent spontaneous abortion, and developmental disorders were associated with exposure to BPA. At present, the toxicity of BPA exposure to invertebrates is less. As a representative nematode species isolated from the soil, the Caenorhabditis elegans is one of the most commonly used modes in environmental exposure assessment and ecological toxicology studies. The study was conducted to study the ecological toxicity of Caenorhabditis elegans as the tested organism, mainly from the physiological, biochemical and molecular level to the acute and subacute exposure of BPA, the chronic exposure and the continuous exposure of multiple generations to the enorhabditis elegans; in addition, The toxicity of biphenol S (BPS) to the caenorhabditis elegans was also investigated in this study, and the toxicity of BPA was compared with the toxicity of BPA, and the ecological risk of the substitute of BPA was analyzed. The main results of this study are as follows: (1) The acute and subacute exposure of BPA can significantly inhibit the body length, the level of activity and the number of offspring of the caenorhabditis elegans, and significantly increase the level of cell apoptosis and the expression of stress-related genes. after acute exposure, the head swing frequency of the caenorhabditis elegans is the most sensitive physiological index, and the exposure concentration is 0. 01 & mu; m, which can produce a significant negative effect on the caenorhabditis elegans, and after the subacute exposure, the body length and the head swing frequency are more sensitive than other physiological indexes, It is also possible to have a significant inhibitory effect on the caenorhabditis elegans at a concentration of 0.01. m u.M. The acute and subacute exposure of BPA did not have a significant effect on the accumulation of lipofuscin and the level of ROS in the nematode, but when the exposure concentration reached 0.1. m The results show that the toxic effect of BPA exposure to the caenorhabditis elegans is not the way of oxidative damage, and it is likely to be related to the increase of the level of apoptosis. The subacute exposure of BPA can lead to a significant increase in the expression of most stress-related genes in the Caenorhabditis elegans, even when the minimum exposure concentration is 0. 001. m u.M, and the exposure of BPA can also cause a significant increase in the expression of most stress-related genes. (2) The chronic exposure of BPA can significantly inhibit the body length, the activity level, the service life and the population number of the caenorhabditis elegans, and obviously improve the cell apoptosis level, the stress-related gene expression and the fluorescent protein expression amount of the transgenic line TJ375. After chronic exposure, the swing frequency of the head is the most sensitive physiological index, and the exposure concentration is 0. 001. m The chronic exposure of BPA did not have a significant effect on the accumulation of lipofuscin in Nematode, but it was able to significantly increase the level of apoptosis in the Caenorhabditis elegans when the exposure concentration was above 0.01. m In the experimental concentration range, the chronic exposure of BPA can significantly improve the expression of most stress-related genes in the nematode. Compared with the results of subacute exposure, the chronic exposure of BPA could lead to a more significant increase in the mRNA level of stress-related genes. When the exposure concentration of BPA reached 0.1. m u.M, the number of Caenorhabditis elegans was significantly reduced. The transgenic strain TJ375 can only detect the toxicity of BPA when the environmental concentration reaches 1. m u.M, and is not sensitive to other test indicators. (3) The continuous exposure of BPA can have a significant effect on the physiological index of the Caenorhabditis elegans when the exposure concentration is above 0.01. m In the case of lower exposure, the generation toxicity of Nematode in the offspring was different from generation to generation, while at the highest exposure concentration of 10. m u.M, there was no significant difference in the toxicity effect of the G1 and G4 of the Caenorhabditis elegans. in that G1 and G4 generation of the Caenorhabditis elegans, the exposure group of low concentration (0. 01. mu.M) can induce a more significant increase in the expression of the related gene in the in vivo compared with the high-concentration exposure group; compared with the G4 generation, The expression of the stress-related genes in the G1-generation of the Caenorhabditis elegans was more significant. (4) After the subacute exposure of BPS, the head swing frequency of the caenorhabditis elegans is the most sensitive physiological index, and the exposure concentration is more than 1. m while other physiological indicators will be significantly negatively affected only in the highest concentration exposure group. After sub-acute exposure, the accumulation of lipofuscin in the caenorhabditis elegans was not significantly affected, and the level of apoptosis was significantly increased only when the exposure concentration was 1. m The sub-acute exposure of BPS can cause a small increase in the expression of most of the gene in the Caenorhabditis elegans. In conclusion, exposure of low concentrations of BPA can produce developmental toxicity, neurobehavioral toxicity, and reproductive toxicity to the Caenorhabditis elegans. The effect of BPA exposure on the physiological level of Caenorhabditis elegans is likely to be related to the improvement of the level of cell apoptosis in vivo; the exposure of BPA can significantly improve the expression of stress-related genes in the Caenorhabditis elegans and destroy the steady state of the body; the transgenic line TJ375 is not sensitive to the exposure of BPA, The toxicity of BPA could not be effectively monitored; the toxicity of BPS was significantly less than the toxicity of BPA.
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X171.5

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