HBCD神经发育毒性的分子机制和HBCD异构体代谢及毒性研究

发布时间：2018-05-02 11:05

本文选题：六溴环十二烷 + SH-SY5Y细胞　；参考：《上海大学》2016年博士论文

【摘要】：以急性毒性指标可以将六溴环十二烷(HBCD)界定为低毒化合物,但研究表明HBCD具有内分泌干扰毒性、免疫抑制毒性、生殖发育毒性、神经毒性等效应。其中,神经发育毒性是HBCD一个较为敏感的毒性终点,氧化应激会引起线粒体功能障碍,从而导致的能量代谢失常和线粒体依赖性凋亡是HBCD诱发神经发育毒性的机制之一。本研究使用β-NGF诱导神经母细胞瘤SH-SY5Y分化模型,研究HBCD对分化细胞的毒性。应用免疫荧光、流式细胞术、蛋白免疫印迹、高效液相色谱等技术检测了β-NGF和HBCD单独及联合作用于SH-SY5Y细胞后突起的生长状况、细胞凋亡、ROS的产生、线粒体膜电位的变化、细胞内钙离子的改变、细胞ATP含量,并用N-乙酰-L-半胱氨酸(NAC)消除ROS后再检测相关指标变化,结果表明:1.分化发育过程中的SH-SY5Y细胞比不分化的细胞更易受到损伤。在无凋亡诱导浓度下,分化细胞在HBCD作用下的凋亡率显著升高,且具有时间效应关系。同时,线粒体依赖性凋亡相关蛋白Cyt-c、Apaf-1和Caspase-9表达明显上升,表明HBCD启动了细胞的线粒体依赖性凋亡途径。2.β-NGF诱导SH-SY5Y细胞分化时线粒体膜电位上升,ROS和胞内钙离子略升高,与多数研究发现神经细胞分化过程中ROS水平和线粒体膜电位上升的结果相符。而HBCD作用于分化中的SH-SY5Y细胞时,线粒体膜电位显著下降,伴随ROS和细胞内钙浓度明显升高;抗氧化剂NAC预处理可明显抑制HBCD诱导的氧化应激效应,细胞ROS水平与对照组相近,线粒体膜电位回升,胞内钙离子浓度回落,同时凋亡率有所下降,可见氧化应激引起线粒体损伤诱导凋亡是HBCD神经发育毒性的原因之一。3.HBCD可抑制β-NGF诱导的SH-SY5Y细胞突起的生长,神经树突标志性蛋白MAP2表达下降,同时伴随胞内ATP水平降低,细胞能势下降;NAC能部分减轻HBCD对神经突起生长的抑制作用,提高ATP合成的水平和细胞能势,但对MAP2表达没有作用。以上结果表明HBCD对突起生长的抑制作用很可能通过HBCD刺激ROS累积,引起线粒体功能障碍继而影响ATP合成,无法提供足够供细胞分化的能量而引起,但ROS蓄积并不能解释MAP2蛋白的表达被抑制的原因。4.继续对能量敏感的蛋白AMPK和神经分化密切相关的PI3K/AKT/m TOR通路进行分析后发现,在β-NGF作用下,AMPK、PI3K、AKT磷酸化水平均显著上升,m TOR磷酸化低于对照组,并具有时间效应关系。另外,AMPK的表达与细胞内AMP/ATP比值水平呈正相关,并促进MAP2表达。HBCD作用后细胞内m TOR磷酸化水平随时间延长而显著上升,AMPK磷酸化受抑制,用PI3K和AKT抑制剂(LY294002,MK-2006)处理细胞后,AMPK磷酸化显著增加,MAP2表达水平回升。以上结果表明,在β-NGF诱导SH-SY5Y分化时m TOR受到AMPK的负调控,HBCD作用时m TOR磷酸化水平升高并下调了AMPK的磷酸化,阻碍了MAP2的表达,HBCD的发育毒性作用与PI3K/AKT/m TOR通路对AMPK的负调控有关。HBCD的立体异构体选择性和对映异构体选择性代谢在不同物种,不同组织器官都有差异,没有普适的规律可以进行模式生物的研究并进行异构体单体的风险评价。肝脏是HBCD的主要代谢器官,为了解HBCD异构体在人体内的代谢选择性和毒性差异,本研究利用正常人肝细胞L-02和人肝癌细胞Hep G2进行HBCD异构体的代谢和毒性研究,分别以10-7,10-6,10-5 mol/L的α-、β-和γ-HBCD染毒L-02和Hep G2细胞1 d、2 d、4 d和6 d,采用LC-MS/MS对细胞内三种异构体的含量变化、生物异构化情况和对映异构体选择性进行分析,应用CCK8、荧光探针法及彗星实验检测HBCD对L-02和Hep G2细胞的生物学效应;Real-time PCR检测代谢酶的表达变化;结果发现:1.α-HBCD、β-HBCD和γ-HBCD在两种肝细胞内的半衰期为α-HBCDγ-HBCDβ-HBCD,α-HBCD和γ-HBCD的代谢速率到第6天时基本一致。同时,Hep G2对HBCD的代谢能力比L-02强,同时间段Hep G2细胞内的异构体比L-02细胞内同一种异构体含量更低。2.在Hep G2细胞中,α-HBCD和β-HBCD能发生异构化转化成γ-HBCD,而γ-HBCD染毒组不能检出α-HBCD或β-HBCD;在L-02细胞中,β-HBCD染毒组能检出少量γ-HBCD,其它两个异构体没有检测到生物转化产物。3.在L-02细胞中,α-,β-,γ-HBCD的手性富集值EF分别为0.54±0.02,0.80±0.02,0.31±0.01;在Hep G2细胞中α-,β-,γ-HBCD的EF分别为0.54±0.01,0.79±0.02,0.32±0.02,两种细胞均表现为(+)-α-HBCD略高于(-)-α-HBCD,而(+)-β-HBCD和(-)-γ-HBCD为优势异构体。4.在L-02细胞中,β-HBCD和γ-HBCD对细胞增殖的抑制率高于α-HBCD,而β-HBCD对Hep G2细胞的抑制率高于α-HBCD和γ-HBCD;α-HBCD、β-HBCD和γ-HBCD均可诱导细胞ROS升高和线粒体膜电位降低,效应强度为β-HBCDγ-HBCDα-HBCD,且Hep G2细胞比L-02细胞更加敏感,同一种异构体在Hep G2细胞内刺激ROS产生的效应浓度要低于L-02细胞的效应浓度;β-HBCD诱导DNA损伤的效应最强,在L-02细胞中,α-HBCD和γ-HBCD之间没有差别,在Hep G2细胞中,α-HBCD较之γ-HBCD更易引起DNA损伤,两种细胞L-02细胞比Hep G2细胞更易产生DNA损伤。5.β-HBCD可下调L-02细胞中P450酶CYP1A1的表达,在Hep G2细胞中产生该效应的是α-HBCD和γ-HBCD。β-HBCD和γ-HBCD在L-02细胞中诱导CYP2B6的表达,且γ-HBCD还诱导了L-02细胞GST基因的表达,在两种细胞中,三种异构体对代谢酶的表达有明显差异,且L-02细胞比Hep G2细胞更易受到诱导。综上所述,HBCD能抑制SH-SY5Y细胞分化,线粒体依赖性凋亡和能量代谢障碍引起的突起生长障碍是重要的原因之一,PI3K/AKT/m TOR通路在调控AMPK的磷酸化和MAP2的表达中有重要作用,该条通路的激活是HBCD神经发育毒性的重要分子机制。HBCD在L-02和Hep G2两种细胞的代谢有立体异构体选择性和对映异构体选择性;α-、β-和γ-HBCD三种异构体的细胞毒性效应在L-02中比HepG2弱,但在DNA损伤效应强度上L-02细胞反应比Hep G2强。三种异构体在两种细胞中对CYP和GST的诱导效应有差异,推测这是L-02细胞对HBCD毒性反应程度比HepG2较弱的原因之一,也可能是β-HBCD毒性较强的原因之一。
[Abstract]:Six bromo ring twelve alkanes (HBCD) can be defined as low toxic compounds with acute toxicity, but the study shows that HBCD has endocrine disrupting toxicity, immunosuppressive toxicity, reproductive development toxicity, neurotoxicity and other effects. Among them, neurodevelopmental toxicity is a more sensitive toxicity end of HBCD, and oxidative stress causes mitochondrial dysfunction, The resulting energy metabolic disorder and mitochondrial dependent apoptosis are one of the mechanisms of HBCD induced neurodevelopmental toxicity. This study used beta -NGF to induce the SH-SY5Y differentiation model of neuroblastoma and study the toxicity of HBCD to differentiated cells. Immunofluorescence, flow cytometry, protein immunization, high performance liquid chromatography and other techniques were used to detect beta -NG. The growth of F and HBCD alone and in combination with SH-SY5Y cells, apoptosis, ROS production, changes in mitochondrial membrane potential, intracellular calcium ion change, cell ATP content, and N- acetyl -L- cysteine (NAC) elimination of ROS after ROS and then change the correlation index, the results show that the ratio of SH-SY5Y cells in 1. differentiation and development process The undifferentiated cells are more susceptible to damage. The apoptosis rate of the differentiated cells under the action of HBCD is significantly increased and has a time effect relationship. At the same time, the expression of mitochondrial dependent apoptosis related protein Cyt-c, Apaf-1 and Caspase-9 increased obviously, indicating that HBCD activated the mitochondria dependent apoptosis pathway.2. beta -NGF of the cells. The mitochondrial membrane potential increased and the ROS and intracellular calcium ions increased slightly in the induction of SH-SY5Y cells differentiation. The results were consistent with the results of most studies, which were found to be consistent with the results of ROS and mitochondrial membrane potential in the process of neural differentiation. While HBCD acted on the differentiated SH-SY5Y cells, the mitochondrial membrane potential decreased significantly, with ROS and intracellular calcium concentration rising obviously. The antioxidant NAC pretreatment could obviously inhibit the oxidative stress induced by HBCD, the ROS level of the cells was similar to the control group, the mitochondrial membrane potential recovered, the intracellular calcium ion concentration fell, and the apoptosis rate decreased. The oxidative stress induced apoptosis induced by mitochondrial damage was one of the causes of HBCD neurotoxicity,.3.HBCD could inhibit beta. The growth of -NGF induced SH-SY5Y cell protuberance, the decrease of the expression of the nerve dendrite marker protein MAP2 and the decrease of the intracellular ATP level and the decrease of the cell energy potential; NAC can partially alleviate the inhibitory effect of HBCD on the growth of the neurite protuberance, improve the level of the ATP synthesis and the cell energy potential, but have no effect on the expression of MAP2. The above results indicate that HBCD is protruding. The inhibitory effect of growth is likely to stimulate the accumulation of ROS through HBCD, causing mitochondrial dysfunction and then affecting the synthesis of ATP, which can not provide enough energy for cell differentiation, but the accumulation of ROS does not explain the reason for the inhibition of the expression of MAP2 protein,.4. continues to be closely related to the energy sensitive protein AMPK and PI3K/AKT/m. After the analysis of TOR pathway, the phosphorylation level of AMPK, PI3K and AKT increased significantly under the action of beta -NGF, and the phosphorylation of M TOR was lower than that of the control group and had a time effect relationship. In addition, the expression of AMPK was positively correlated with the level of AMP/ATP ratio in the cells, and the phosphorylation level of the M cells in the cells increased with time after the MAP2 expression was.HBCD. The phosphorylation of AMPK was inhibited, and the phosphorylation of AMPK increased significantly with the treatment of PI3K and AKT inhibitor (LY294002, MK-2006), and the expression level of MAP2 increased. The above results showed that m TOR was regulated by AMPK in the induction of SH-SY5Y differentiation by beta -NGF. The developmental toxicity of HBCD is related to the negative regulation of the PI3K/AKT/m TOR pathway to the negative regulation of AMPK, the selectivity of the isomer of.HBCD and the selective metabolism of enantiomers in different species, different tissues and organs, and there is no universal law for the study of model organisms and the risk assessment of isomer monomers. The liver is H The main metabolic organs of BCD are to understand the metabolic selectivity and toxicity difference of HBCD isomers in the human body. The metabolism and toxicity of HBCD isomers in normal human hepatocyte L-02 and human hepatoma cell Hep G2 were studied in this study, respectively, with the alpha, beta and gamma -HBCD L-02 and Hep G2 cells of 10-7,10-6,10-5 mol/L, respectively, 2, 4 and 6. The changes in the content of three isomers, bioisomerization and enantiomer selectivity were analyzed by MS/MS. The biological effects of HBCD on L-02 and Hep G2 cells were detected by CCK8, fluorescence probe and comet assay, and Real-time PCR detected the changes in the expression of metabolic enzymes. The results showed that 1. alpha -HBCD, beta -HBCD and gamma -HBCD were in two kinds of liver. The half-life of the cell is alpha -HBCD gamma -HBCD beta -HBCD, the metabolic rate of alpha -HBCD and gamma -HBCD is basically the same as the sixth day. At the same time, the metabolic ability of Hep G2 to HBCD is stronger than that of L-02. The isomers in the Hep G2 cells of the same time period are lower than the same isomers in the L-02 cells. In gamma -HBCD, alpha -HBCD or beta -HBCD can not be detected by gamma -HBCD; in L-02 cells, a small amount of gamma -HBCD can be detected in the beta -HBCD group, and the other two isomers are not detected in the biotransformation product.3. in L-02 cells. The chiral enrichment value of alpha, beta, and gamma -HBCD is 0.54 + + + 0.01, respectively. The EF of D was 0.54 + 0.01,0.79 + 0.02,0.32 + 0.02 respectively. The two cells were (+) - alpha -HBCD was slightly higher than (-) - alpha -HBCD, while (+) - beta -HBCD and (-) - gamma -HBCD were dominant isomers in L-02 cells, and the inhibition rate of beta -HBCD and gamma -HBCD on cell proliferation was higher than that of alpha. Both -HBCD and gamma -HBCD can induce the increase of cell ROS and the decrease of mitochondrial membrane potential, the effect intensity is beta -HBCD gamma -HBCD alpha -HBCD, and Hep G2 cells are more sensitive than L-02 cells. The effect concentration of the same isomer in Hep G2 cells is lower than the effect concentration of the cell. In the cell, there is no difference between alpha -HBCD and gamma -HBCD. In Hep G2 cells, the alpha -HBCD is more likely to cause DNA damage than that of gamma -HBCD. The two cell L-02 cells are more likely to produce DNA damage.5. beta than Hep G2 cells. The expression of CYP2B6 was induced in the cells, and the expression of GST gene in L-02 cells was induced by gamma -HBCD. In two cells, the expression of metabolic enzymes was significantly different from three isomers, and L-02 cells were more easily induced than Hep G2 cells. To sum up, HBCD could inhibit the differentiation of SH-SY5Y cells, mitochondrial dependent apoptosis and energy metabolism disorder. The PI3K/AKT/m TOR pathway plays an important role in the regulation of the phosphorylation of AMPK and the expression of MAP2. The activation of this pathway is an important molecular mechanism of HBCD neurodevelopmental toxicity,.HBCD, in the metabolism of two cells of L-02 and Hep G2, which have stereoselective selectivity and enantiomer selectivity; alpha, beta - - The cytotoxic effects of three isomers of -HBCD and gamma are weaker than HepG2 in L-02, but L-02 cell reaction is stronger than Hep G2 in the intensity of DNA damage effect. The induction effect of CYP and GST in the three isomers is different in two cells. It is presumed that this is one of the reasons that L-02 cells are less toxic to HBCD than HepG2, and may also be beta toxicity. One of the stronger reasons.

【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

【相似文献】