nano-TiO_2与PbAc联合诱导人胚肝细胞氧化应激和凋亡作用研究
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摘要
作为一种新型科技产物,nano-TiO2被广泛应用于涂料、化妆品、水污染处理等行业。但是大量研究证明nano-TiO2对人体健康和环境存在着危害作用。nano-TiO2可刺激机体组织产生炎症反应,降低细胞活性,促进细胞凋亡,以及导致DNA损伤等。nano-TiO2具有纳米尺度效应、表面效应、量子尺寸效应、宏观量子隧道效应、光催化效应。相对于大颗粒,nano-TiO2生物学效应的性质和强度也发生了质的变化。据目前研究发现,nano-TiO2对诱导损伤的发生机制主要跟炎症反应和氧化应激有关。
铅是一种被普遍应用于电池、颜料、汽油、油漆、仪器中的有毒重金属。在环境中广泛存在,能够在生物体内蓄积。铅污染是当前国际社会特别突出的环境问题。铅污染导致DNA损伤和基因毒性,损伤人体神经、造血、生殖等系统。近来研究表明铅对诱导损伤的发生机制主要跟氧化应激有关。
随着nano-TiO2的广泛使用,导致大量进入环境,特殊的表面效应使其极易与环境中的多种污染物结合。由于nano-TiO2的特殊理化性质,对环境中的金属离子或有机物具有很强的吸附能力。从而可能产生与其本身所致生物学效应的性质和强度不同的效应。pb2+广泛存在于环境当中,可吸附到nano-TiO2的表面,产生相互作用并共同作用于机体或环境。二者联合作用于机体可能导致的各种健康效应有何变化,至今尚无研究。
环境中的nano-TiO2和其他污染物含量很低,一般都是痕量存在。在本研究中以低浓度nano-TiO2与环境常见污染物PbAc在无紫外条件下联合作用于体外培养的人类胚胎肝细胞(L02细胞)24hr,探究nano-TiO2与PbAc联合作用对细胞活性、ROS生成、还原性物质、DNA损伤、DNA修复酶表达、细胞凋亡等情况的影响,系统了解nano-TiO2与PbAc联合作用所致氧化应激和凋亡效应。本文在当前单独PbAc与nano-TiO2的毒理学作用机理研究基础上,探讨了二者之间的相互作用及可能的联合毒作用机制,为完善nano-TiO2的毒理学评价和了解环境中nano-TiO2与PbAc之间的健康效应提供理论基础和实验依据。本研究分为四个部分：
第一部分nano-TiO2的团聚效应及其对PbAc的吸附作用研究
目的：研究纳米二氧化钛(nano-TiO2)在溶液中的团聚作用及TiO2对醋酸铅(PbAc)的吸附作用。方法：采用纳米粒度仪检测0.001、0.01、0.1、1、10μg/mL的nano-TiO2在水溶液中团聚后粒度大小；石墨炉原子吸收(Graphite furnace atomic absorption spectrometry, GFAAS)检测0.001、0.01、0.1、1、10μg/mL的nano-TiO2对1μg/mL的PbAc吸附量的大小。结果：各浓度纳米二氧化钛团聚后粒度平均大小为298.8 nm,之间并无显著性差异(P>0.05)；相比较于单一的纳米二氧化钛红外图谱,混合物显示有醋酸基团被吸附于纳米二氧化钛；原子吸收结果显示0.001、0.01μg/mL TiO2吸附的PbAc量显著低于0.1、1、10μg/mL的TiO2 (P< 0.05);并且吸附量随TiO2浓度呈剂量依赖关系。结论：单一nano-TiO2在没有分散剂的作用下,短时间内在水溶液中有团聚；对PbAc有明显吸附作用,并且随nano-TiO2浓度的升高,吸附量增大。
第二部分nano-TiO2与PbAc联合诱导人胚肝细胞氧化应激作用
目的：观察nano-TiO2与PbAc单独及联合作用对人胚肝细胞(L02)活性、活性氧(reactive oxygen species, ROS)含量及氧化应激的影响。方法：0.001、0.01、0.1、1、10μg/mL的nano-TiO2和1μg/mL的PbAc,以及0.001、0.01、0.1、1、10μg/mLnano-TiO2和1μg/mLPbAc的混合物(为了方便,在下面的描述中将TiO2和1μg/mL PbAc的混合物简写为10,1,0.1,0.01,0.001μg/mL混合物)联合作用于生长良好的L02细胞,二甲基亚砜(Dimethyl sulfoxide, DMSO,1μL/mL)为阴性对照,将L02细胞染毒24hr后,以MTT法检测nano-TiO2与PbAc单独及联合作用对L02细胞活性的影响；运用DCFH-DA (2',7'-dichlorofluorescin-diacetate)作为荧光探针,采用流式细胞检测技术检测nano-TiO2与PbAc联合作用下L02细胞内ROS含量,同时检测L02细胞内抗氧化酶超氧化物歧化酶(Superoxide dismutase, SOD)、谷胱甘肽(Glutathione, GSH)的含量。结果：相比阴性对照、PbAc和其他单一TiO2剂量组,10μg/mL TiO2单独作用L02细胞24hr可显著降低细胞活性(P<0.05);1μg/mL TiO2相比阴性对照,导致显著性的细胞活性降低。与阴性对照相比,0.1、1、10μg/mL的混合物联合作用L02细胞24hr可引起细胞活性显著性降低(P<0.05)；与1μg/mL的PbAc单独作用相比,1、10μg/mL的混合物联合作用L02细胞24hr可引起细胞活性显著性降低(P<0.05)；所有浓度的nano-TiO2和1μg/mL的PbAc单独作用不能引起ROS、SOD、GSH水平的改变(P>0.05); 0.001、0.01、0.1、1、10μg/mL的混合物联合作用L02细胞24hr可引起ROS显著性增加(P<0.05)；单一TiO2和混合物所诱导的细胞毒性、ROS生成显示出剂量依赖关系。0.01、0.1、1μg/mL的混合物联合作用使细胞内GSH水平较阴性对照组有显著升高(P<0.05),并且1μg/mL的混合物联合作用使细胞内GSH水平较1μg/mL的PbAc单独作用有显著升高(P<0.05)；与阴性对照相比,0.01、0.1、μg/mL的混合物联合作用使细胞中抗氧化酶SOD活力显著性升高(P<0.05)；单一TiO2所诱导的GSH和SOD水平显示出剂量依赖关系。析因分析结果显示两种受试物混和染毒对细胞内细胞活性、ROS、GSH、SOD水平不存在交互协同作用(P>0.05)。结论：证明在低浓度无紫外的条件下,nano-TiO2和PbAc联合作用可增强细胞毒性、促进活性氧的产生和抗氧化物质应激性升高；nano-TiO2和PbAc联合作用可诱导细胞氧化应激,低浓度混合物作用时,上升的氧化应激触发了细胞保护防御的正调节作用,使细胞还拥有一定的抗氧化能力从而阻碍进一步的氧化损伤；而高浓度时,则触发了负调节作用,从而导致抗氧化能力下降。
第三部分nano-TiO2与PbAc联合诱导人胚肝细胞DNA损伤及其对修复蛋白的影响研究
目的：研究nano-TiO2与PbAc单独及联合作用对L02细胞活性、DNA损伤以及对OGGl (8-oxoguanine DNA glycosylase homolog 1)修复蛋白表达的影响。方法：0.001、0.01、0.1、1、10μg/mL的nano-TiO2和1μg/mL的PbAc,以及0.001、0.01、0.1、1、10μg/mL的混合物联合作用于生长良好的L02细胞,DMSO (1mL/L)为阴性对照,L02细胞处理时间为24 hr；运用碱性彗星试验和DNA氧化损伤标志物8-OHdG (8-Hydroxydeoxyguanosine)的含量检测各组DNA断裂损伤程度；运用western blot检测单独及联合作用对细胞DNA修复蛋白OGG1的影响。结果：相比阴性对照1μg/mL PbAc,10μg/mL混合物作用L02细胞24 hr可导致显著性升高的Olive尾距(Olive tail moment, OTM) (P< 0.05);相比阴性对照,1μg/mL混合物作用导致显著性升高的OTM(P<0.05)。所有浓度的nano-TiO2和1μg/mL的PbAc单独作用不能引起8-OHdG的含量和细胞DNA修复蛋白OGG1表达相对于阴性对照的显著性改变(P>0.05)；1、10μg/mL的混合物联合作用L02细胞导致8-OHdG的含量显著高于阴性对照组(P<0.05); 10μg/mL混合物联合作用导致8-OHdG的含量显著高于PbAc单独作用(P<0.05)。0.001、0.01、0.1、1μg/mL的混合物联合作用使细胞内DNA修复蛋白OGG1表达水平较阴性对照组和PbAc单独作用有显著升高(P<0.05)。单一TiO2和混合物所导致的OTM、8-OHdG生成显示出剂量依赖关系。但只有单一TiO2导致的OGG1表达显示出剂量依赖关系。析因分析结果显示两种受试物混和染毒对DNA损伤修复蛋白OGG1的表达存在交互协同作用(P<0.05)。结论：nano-TiO2和PbAc联合作用可增强L02细胞DNA损伤,同时协同增强调节DNA修复蛋白OGG1修复损伤的DNA,细胞还拥有一定的修复损伤DNA的能力。
第四部分nano-TiO2与PbAc联合诱导人胚肝细胞凋亡效应研究
目的：研究nano-TiO2与PbAc单独及联合作用对L02凋亡效应及对凋亡关键蛋白Caspase 3 (Cysteine-asparate protease 3)表达的影响。方法：0.001、0.01、0.1、1、10μg/mL的nano-TiO2和1μg/mL的PbAc,以及0.001、0.01、0.1、1、10μg/mL的混合物联合作用于生长良好的L02细胞,DMSO (1 mL/L)为阴性对照,L02细胞处理时间为24 hr；运用流式细胞仪检测nano-TiO2与PbAc单独及联合作用对L02早期和晚期凋亡的效应的影响；运用western blot检测单独及联合作用对细胞凋亡调控蛋白Caspase 3的影响。结果：单一1μg/mL的PbAc单独作用不能引起细胞早期凋亡水平相对于阴性对照的显著性改变(P>0.05); 1、10μg/mL的nano-TiO2导致细胞早期凋亡水平显著高于DMSO、PbAc以及0.001、0.01和0.1μg/mL nano-TiO2单独作用(P<0.05); 0.1μg/mL的nano-TiO2导致细胞早期凋亡水平显著高于DMSO、PbAc、0.001、0.01μg/mL nano-TiO2单独作用(P<0.05)；而所有的混合物均导致细胞早期凋亡水平显著高于DMSO阴性对照和PbAc单独作用(P<0.05)。所有浓度的nano-TiO2和1μg/mL的PbAc单独作用不能引起细胞晚期凋亡水平相对于阴性对照的显著性改变(P>0.05)。但是,10μg/mL的混合物联合作用使细胞晚期凋亡水平较DMSO阴性对照、PbAc单独作用和其他混合物作用有显著升高(P<0.05)。所有浓度的nano-TiO2和1μg/mL的PbAc单独作用不能引起细胞凋亡关键蛋白Caspase 3表达相对于阴性对照的显著性改变(P>0.05)。除了0.001μg/mL,所有混合物作用均导致细胞Caspase 3表达水平较DMSO阴性对照显著升高(P<0.05); 10μg/mL的混合物联合作用使细胞Caspase 3表达水平较DMSO阴性对照、PbAc单独作用和其他混合物作用有显著升高(P<0.05)。单一nano-TiO2和混合物所导致的细胞早晚期凋亡显示出剂量依赖关系。析因分析结果显示两种受试物混和染毒对晚期凋亡水平存在交互协同作用(P<0.05)。结论：nano-TiO2和PbAc联合作用可增强诱导L02增加细胞的凋亡,并协同增强细胞的晚期凋亡水平,而Caspase 3在细胞凋亡作用中起着关键作用。
Titanium dioxide (TiO2) in nano-size is a new technology product which was widely used in cosmetics, paints and in decontamination water. Numerous studies demonstrated that nano-TiO2 in nano-size induced damage to human health and environment. Nano-TiO2 could stimulate inflammatory reaction of the organism, reduce the cell viability, and induce the apoptosis and DNA damage. Nano-TiO2 has some special properties, such as nano-scale effect, surface effect, quantum size effect, macroscopic quantum tunneling effect, photocatalytic effect. Compared with the large particles, nano-TiO2 on the nature and intensity of the biological effects of a qualitative change also occurred. According to the present study, mechanisms of damage induced by nano-TiO2 are mainly related to inflammation and oxidative stress.
Lead is widely applied heavy metal in batteries, paint, gasoline, paint, instruments. Lead exists in the environment widespread and accumulates in organism. Lead pollution is a particularly prominent environmental problem in the current situation. Lead pollution causes DNA damage and genetic toxicity, damages the nervous, hematopoietic, reproductive and other systems. Recent studies show that mechanisms of damage induced by lead major related to oxidative stress.
A large number of nano-Ti02 has been released into the environment with the widespread use. Nano-TiO2 could absorb a variety of pollutants in the environment because of the special surface effect. Nano-TiO2 has a strong adsorption capacity to metal ions or organic produce in the environment as the special physical and chemical properties. This may generate different effects compared with their own biological effects. Pb2+ exists in the environment can be adsorbed onto the surface of nano-TiO2, and interact with each other, and interact with the body or the environment. So far, the combined effects of Pb2+ and nano-TiO2 on the body were unknown.
The content of nano-TiO2 and other pollutants in the environment is very low, and exists in trace. In this study, to understand the combined effect of oxidative stress and apoptosis induced by nano-TiO2 mixed with PbAc, we assessed the cell activity, DNA damage, ROS generation, reduction enzyme changes, apoptosis, etc induced by TiO2 in trace mixed with on human embryo hepatocytes (L02) for a 24 hr treatment without photoactivation. Based on the researches on toxicity of nano-TiO2 and PbAc treated alone, the interaction of the mixtures was studied and potential toxicological mechanisms were also evaluated in this study, further consummated the toxicology evaluation and provided the theory basis of nano-TiO2, in addition to understand the healths effects and provide experimental evidence induced by nano-TiO2 and PbAc in trace.
The study is composed of the following four parts:
PartⅠ
The aggregation effect of nano-TiO2 and the adsorptive capacity of PbAc onto nano-Ti02
Objective:To observe the aggregation of nano-TiO2 and adsorption of nano-TiO2 on PbAc in the solution.
Methods:The aggregation size of 0.001,0.01,0.1,1,10μg/mL nano-TiO2 in aqueous solution was detected by a Nano-Particle Analyzer. The adsorption of Pb2+ onto nano-TiO2 in solution was detected by infrared scanning. The adsorptive capacity of 1μg/mL Pb2+ onto 0.001,0.01,0.1,1,10μg/mL nano-TiO2 were detected by graphite furnace atomic absorption spectrometry.
Results:The average size of aggregation is 298.8 nm and there is no significantly change in the given concentrations (P> 0.05). The absorptive capacity of PbAc by nano-TiO2 analyzed by GFAAS is showed that the mixture containing 0.001 and 0.01μg/mL nano-TiO2 absorbed significantly less PbAc than those of 0.1,1,10μg/mL nano-TiO2 (P< 0.05), and there was a dose-dependent absorption by nano-TiO2. Conclusion:nano-TiO2 treated alone in the absence of surfactants aggregated in the short time in water. In the mixtures, there was a significant adsorption on PbAc by nano-TiO2, and adsorption capacity increases with the concentration of nano-TiO2.
PartⅡ
Oxidative stress induced by a joint effect of titanium dioxide and lead acetate in human hepatocytes
Objective:To observe the cell viability, ROS generation and Oxidative stress induced by a joint effect of nano-TiO2 and PbAc in human hepatocytes.
Methods:L02 were exposed to nano-TiO2 in 10,1,0.1,0.01,0.001μg/mL and 1μg/mL PbAc treated alone and mixture for a period 24 hr (The mixtures of nano-TiO2 with 1μg/mL PbAc in the following descriptions were abbreviated as 10,1,0.1,0.01,0.001μg/mL mixture for convenience). In each experiment a negative control (1‰DMSO) was included. The cytotoxicity induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined using MTT assay. The production of ROS induced by nano-TiO2,1μg/mL PbAc and the mixtures were assessed by flow cytometry using DCFH-DA as fluorescent probe. The activities of GSH and SOD were determined meanwhile.
Results:Compared with the negative control, PbAc and the other nano-TiO2 treatments, cell viability decreased notably when treated with 10μg/mL nano-TiO2 (P< 0.05).1μg/mL nano-TiO2 induced significantly viability decreased compared with the negative control (P< 0.05). The mixtures from 0.1,1,10μg/mL induced significantly higher cytotoxicity than the negative control (P< 0.05). Mixtures of 1 and 10μg/mL induced significantly higher cytotoxicity than 1μg/mL PbAc treatment (P< 0.05). There were no significant increases in ROS, GSH, SOD levels caused by nano-TiO2 and PbAc-alone treatment (P> 0.05). In comparison with the negative control and 1μg/mL PbAc, the ROS generation significant increases in L02 cells after all mixtures treatment for 24 hr, respectively (P< 0.05). The 0.01,0.1 and 1μg/mL mixtures induced significant increases in GSH level, compared with the negative control (P< 0.05). The 1μg/mL mixture induced significant increases in GSH level compared with the 1μg/mL PbAc treated alone (P< 0.05). Furthermore, there were significant increases in SOD level induced by the 0.1 and 0.01μg/mL mixtures compared with the negative control (P< 0.05). There were dose-dependence patterns of cell viability and ROS generation induced by nano-TiO2 alone and the mixtures. However, there were only dose-dependence patterns of GSH and SOD levels induced by nano-TiO2 treated alone. There were no synergistic effect in cell viability, ROS, GSH and SOD levels caused by nano-TiO2 and PbAc (P> 0.05).
Conclusion:The joint effect of low (≤10μg/mL) concentrations of nano-TiO2 and PbAc enhances oxidative stress and cytotoxicity in the absence of UV in L02 over a 24 hr treatment period. The results suggest that the increased oxidative stress triggered the up regulation of defenses to protect cells at low concentrations but triggered the down regulation at higher concentrations of the mixtures.
PartⅢ
DNA damage and Repair protein expression induced by a joint effect of titanium dioxide and lead acetate in human hepatocytes
Objective:To observe the DNA damage, DNA adducts generation and Repair protein expression induced by a joint effect of nano-TiO2 and PbAc in human hepatocytes.
Methods:L02 was exposed to nano-TiO2 in 10,1,0.1,0.01,0.001μg/mL and 1μg/mL PbAc treated alone and mixture for a period 24 hr. In each experiment a negative control (1‰DMSO) was included. The DNA damage induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined using comet assay. The generation of DNA adducts induced by nano-TiO2,1μg/mL PbAc and the mixtures were assessed by HPLC. The expression of OGG1 induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined using western blotting assay.
Results:Compared with the negative control and 1μg/mL PbAc, OTM increased notably when treated with 10μg/mL mixture (P< 0.05).1μg/mL nano-TiO2 mixed with 1μg/mL PbAc induced significantly OTM increased compared with the negative control (P< 0.05). There were no significant increases in 8-OHdG formation and OGG1 expression caused by nano-TiO2 and PbAc-alone treatment (P> 0.05). The production of 8-OHdG induced by the 10 and 1μg/mL mixtures was significantly higher than the negative control (P< 0.05). In addition,10μg/mL mixtures caused higher 8-OHdG levels than 1μg/mL PbAc (P< 0.05). All mixtures exposures resulted in the increased OGG1 levels, except for the 10μg/mL mixtures treatment compared with the negative control and 1μg/mL PbAc, respectively (P< 0.05). Nano-TiO2 and the mixtures induced dose-dependence patterns of OTM,8-OHdG formation and OGG1 expression. Addition of nano-TiO2 with PbAC synergistically enhanced OGG1 expression in this study (P< 0.05).
Conclusion:The joint effect of low (≤10μg/mL) concentrations of nano-TiO2 and PbAc enhances oxidative stress and cytotoxicity in the absence of UV in L02 over a 24 hr treatment period. The results suggest that the increased oxidative stress triggered the up regulation of defenses to protect cells at low concentrations but triggered the down regulation at higher concentrations of the mixtures.
Part IV
Apoptosis induced by a joint effect of titanium dioxide and lead acetate in human hepatocytes
Objective:To observe the apoptosis effects induced by a joint effect of nano-TiO2 and PbAc in human hepatocytes.
Methods:L02 was exposed to nano-TiO2 in 10,1,0.1,0.01,0.001μg/mL and 1μg/mL PbAc treated alone and mixture for a period 24 hr. In each experiment a negative control (1‰DMSO) was included. The early and late apoptosis induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined by flow cytometry using FITC and PI as fluorescent probe. The expression of Caspase 3 induced by nano-TiO2, 1μg/mL PbAc and the mixtures were determined using western blotting assay.
Results:Compared with the negative control,1μg/mL PbAc induced no significantly early apoptosis increased (P> 0.05). There were significant increases in early apoptosis caused by 1 and 10μg/mL TiO2 compared with the negative control,1μg/mL PbAc and other concentrations TiO2 treated alone (P< 0.05). The 0.1μg/mL TiO2 induced notably early apoptosis increase compared with the negative control,1μg/mL PbAc,0.001,0.01μg/mL TiO2 treated alone (P< 0.05). All mixtures exposures resulted in the increased early apoptosis levels compared with the negative control and 1μg/mL PbAc, respectively (P< 0.05). All exposures of nano-TiO2 and 1μg/mL PbAc treated alone resulted in no notably late apoptosis increase compared with the negative control (P> 0.05). But late apoptosis increased notably caused by 10μg/mL mixtures treated alone compared with the negative control, PbAc and other mixtures treatments (P< 0.05). All exposures of nano-TiO2 and 1μg/mL PbAc treated alone resulted in no notably Caspase 3 expression increase compared with the negative control (P> 0.05). Compared with the negative control, the expression of Caspase 3 increased significantly when treated with all mixtures except 0.001μg/mL (P< 0.05). Caspase 3 expression increased significantly caused by 10μg/mL mixtures treated alone compared with the negative control, PbAc and other mixtures treatments (P< 0.05). Nano-TiO2 and the mixtures induced dose-dependence patterns of apoptosis. There was synergistic effect in late apoptosis caused by nano-TiO2 and PbAc (P< 0.05).
Conclusion:The joint effect of low (≤10μg/mL) concentrations of nano-TiO2 and PbAc enhances apoptosis and relate key protein expression in L02. The results suggest that the Caspase 3 play a very important role in the apoptosis of the cell.
铅是一种被普遍应用于电池、颜料、汽油、油漆、仪器中的有毒重金属。在环境中广泛存在,能够在生物体内蓄积。铅污染是当前国际社会特别突出的环境问题。铅污染导致DNA损伤和基因毒性,损伤人体神经、造血、生殖等系统。近来研究表明铅对诱导损伤的发生机制主要跟氧化应激有关。
随着nano-TiO2的广泛使用,导致大量进入环境,特殊的表面效应使其极易与环境中的多种污染物结合。由于nano-TiO2的特殊理化性质,对环境中的金属离子或有机物具有很强的吸附能力。从而可能产生与其本身所致生物学效应的性质和强度不同的效应。pb2+广泛存在于环境当中,可吸附到nano-TiO2的表面,产生相互作用并共同作用于机体或环境。二者联合作用于机体可能导致的各种健康效应有何变化,至今尚无研究。
环境中的nano-TiO2和其他污染物含量很低,一般都是痕量存在。在本研究中以低浓度nano-TiO2与环境常见污染物PbAc在无紫外条件下联合作用于体外培养的人类胚胎肝细胞(L02细胞)24hr,探究nano-TiO2与PbAc联合作用对细胞活性、ROS生成、还原性物质、DNA损伤、DNA修复酶表达、细胞凋亡等情况的影响,系统了解nano-TiO2与PbAc联合作用所致氧化应激和凋亡效应。本文在当前单独PbAc与nano-TiO2的毒理学作用机理研究基础上,探讨了二者之间的相互作用及可能的联合毒作用机制,为完善nano-TiO2的毒理学评价和了解环境中nano-TiO2与PbAc之间的健康效应提供理论基础和实验依据。本研究分为四个部分：
第一部分nano-TiO2的团聚效应及其对PbAc的吸附作用研究
目的：研究纳米二氧化钛(nano-TiO2)在溶液中的团聚作用及TiO2对醋酸铅(PbAc)的吸附作用。方法：采用纳米粒度仪检测0.001、0.01、0.1、1、10μg/mL的nano-TiO2在水溶液中团聚后粒度大小；石墨炉原子吸收(Graphite furnace atomic absorption spectrometry, GFAAS)检测0.001、0.01、0.1、1、10μg/mL的nano-TiO2对1μg/mL的PbAc吸附量的大小。结果：各浓度纳米二氧化钛团聚后粒度平均大小为298.8 nm,之间并无显著性差异(P>0.05)；相比较于单一的纳米二氧化钛红外图谱,混合物显示有醋酸基团被吸附于纳米二氧化钛；原子吸收结果显示0.001、0.01μg/mL TiO2吸附的PbAc量显著低于0.1、1、10μg/mL的TiO2 (P< 0.05);并且吸附量随TiO2浓度呈剂量依赖关系。结论：单一nano-TiO2在没有分散剂的作用下,短时间内在水溶液中有团聚；对PbAc有明显吸附作用,并且随nano-TiO2浓度的升高,吸附量增大。
第二部分nano-TiO2与PbAc联合诱导人胚肝细胞氧化应激作用
目的：观察nano-TiO2与PbAc单独及联合作用对人胚肝细胞(L02)活性、活性氧(reactive oxygen species, ROS)含量及氧化应激的影响。方法：0.001、0.01、0.1、1、10μg/mL的nano-TiO2和1μg/mL的PbAc,以及0.001、0.01、0.1、1、10μg/mLnano-TiO2和1μg/mLPbAc的混合物(为了方便,在下面的描述中将TiO2和1μg/mL PbAc的混合物简写为10,1,0.1,0.01,0.001μg/mL混合物)联合作用于生长良好的L02细胞,二甲基亚砜(Dimethyl sulfoxide, DMSO,1μL/mL)为阴性对照,将L02细胞染毒24hr后,以MTT法检测nano-TiO2与PbAc单独及联合作用对L02细胞活性的影响；运用DCFH-DA (2',7'-dichlorofluorescin-diacetate)作为荧光探针,采用流式细胞检测技术检测nano-TiO2与PbAc联合作用下L02细胞内ROS含量,同时检测L02细胞内抗氧化酶超氧化物歧化酶(Superoxide dismutase, SOD)、谷胱甘肽(Glutathione, GSH)的含量。结果：相比阴性对照、PbAc和其他单一TiO2剂量组,10μg/mL TiO2单独作用L02细胞24hr可显著降低细胞活性(P<0.05);1μg/mL TiO2相比阴性对照,导致显著性的细胞活性降低。与阴性对照相比,0.1、1、10μg/mL的混合物联合作用L02细胞24hr可引起细胞活性显著性降低(P<0.05)；与1μg/mL的PbAc单独作用相比,1、10μg/mL的混合物联合作用L02细胞24hr可引起细胞活性显著性降低(P<0.05)；所有浓度的nano-TiO2和1μg/mL的PbAc单独作用不能引起ROS、SOD、GSH水平的改变(P>0.05); 0.001、0.01、0.1、1、10μg/mL的混合物联合作用L02细胞24hr可引起ROS显著性增加(P<0.05)；单一TiO2和混合物所诱导的细胞毒性、ROS生成显示出剂量依赖关系。0.01、0.1、1μg/mL的混合物联合作用使细胞内GSH水平较阴性对照组有显著升高(P<0.05),并且1μg/mL的混合物联合作用使细胞内GSH水平较1μg/mL的PbAc单独作用有显著升高(P<0.05)；与阴性对照相比,0.01、0.1、μg/mL的混合物联合作用使细胞中抗氧化酶SOD活力显著性升高(P<0.05)；单一TiO2所诱导的GSH和SOD水平显示出剂量依赖关系。析因分析结果显示两种受试物混和染毒对细胞内细胞活性、ROS、GSH、SOD水平不存在交互协同作用(P>0.05)。结论：证明在低浓度无紫外的条件下,nano-TiO2和PbAc联合作用可增强细胞毒性、促进活性氧的产生和抗氧化物质应激性升高；nano-TiO2和PbAc联合作用可诱导细胞氧化应激,低浓度混合物作用时,上升的氧化应激触发了细胞保护防御的正调节作用,使细胞还拥有一定的抗氧化能力从而阻碍进一步的氧化损伤；而高浓度时,则触发了负调节作用,从而导致抗氧化能力下降。
第三部分nano-TiO2与PbAc联合诱导人胚肝细胞DNA损伤及其对修复蛋白的影响研究
目的：研究nano-TiO2与PbAc单独及联合作用对L02细胞活性、DNA损伤以及对OGGl (8-oxoguanine DNA glycosylase homolog 1)修复蛋白表达的影响。方法：0.001、0.01、0.1、1、10μg/mL的nano-TiO2和1μg/mL的PbAc,以及0.001、0.01、0.1、1、10μg/mL的混合物联合作用于生长良好的L02细胞,DMSO (1mL/L)为阴性对照,L02细胞处理时间为24 hr；运用碱性彗星试验和DNA氧化损伤标志物8-OHdG (8-Hydroxydeoxyguanosine)的含量检测各组DNA断裂损伤程度；运用western blot检测单独及联合作用对细胞DNA修复蛋白OGG1的影响。结果：相比阴性对照1μg/mL PbAc,10μg/mL混合物作用L02细胞24 hr可导致显著性升高的Olive尾距(Olive tail moment, OTM) (P< 0.05);相比阴性对照,1μg/mL混合物作用导致显著性升高的OTM(P<0.05)。所有浓度的nano-TiO2和1μg/mL的PbAc单独作用不能引起8-OHdG的含量和细胞DNA修复蛋白OGG1表达相对于阴性对照的显著性改变(P>0.05)；1、10μg/mL的混合物联合作用L02细胞导致8-OHdG的含量显著高于阴性对照组(P<0.05); 10μg/mL混合物联合作用导致8-OHdG的含量显著高于PbAc单独作用(P<0.05)。0.001、0.01、0.1、1μg/mL的混合物联合作用使细胞内DNA修复蛋白OGG1表达水平较阴性对照组和PbAc单独作用有显著升高(P<0.05)。单一TiO2和混合物所导致的OTM、8-OHdG生成显示出剂量依赖关系。但只有单一TiO2导致的OGG1表达显示出剂量依赖关系。析因分析结果显示两种受试物混和染毒对DNA损伤修复蛋白OGG1的表达存在交互协同作用(P<0.05)。结论：nano-TiO2和PbAc联合作用可增强L02细胞DNA损伤,同时协同增强调节DNA修复蛋白OGG1修复损伤的DNA,细胞还拥有一定的修复损伤DNA的能力。
第四部分nano-TiO2与PbAc联合诱导人胚肝细胞凋亡效应研究
目的：研究nano-TiO2与PbAc单独及联合作用对L02凋亡效应及对凋亡关键蛋白Caspase 3 (Cysteine-asparate protease 3)表达的影响。方法：0.001、0.01、0.1、1、10μg/mL的nano-TiO2和1μg/mL的PbAc,以及0.001、0.01、0.1、1、10μg/mL的混合物联合作用于生长良好的L02细胞,DMSO (1 mL/L)为阴性对照,L02细胞处理时间为24 hr；运用流式细胞仪检测nano-TiO2与PbAc单独及联合作用对L02早期和晚期凋亡的效应的影响；运用western blot检测单独及联合作用对细胞凋亡调控蛋白Caspase 3的影响。结果：单一1μg/mL的PbAc单独作用不能引起细胞早期凋亡水平相对于阴性对照的显著性改变(P>0.05); 1、10μg/mL的nano-TiO2导致细胞早期凋亡水平显著高于DMSO、PbAc以及0.001、0.01和0.1μg/mL nano-TiO2单独作用(P<0.05); 0.1μg/mL的nano-TiO2导致细胞早期凋亡水平显著高于DMSO、PbAc、0.001、0.01μg/mL nano-TiO2单独作用(P<0.05)；而所有的混合物均导致细胞早期凋亡水平显著高于DMSO阴性对照和PbAc单独作用(P<0.05)。所有浓度的nano-TiO2和1μg/mL的PbAc单独作用不能引起细胞晚期凋亡水平相对于阴性对照的显著性改变(P>0.05)。但是,10μg/mL的混合物联合作用使细胞晚期凋亡水平较DMSO阴性对照、PbAc单独作用和其他混合物作用有显著升高(P<0.05)。所有浓度的nano-TiO2和1μg/mL的PbAc单独作用不能引起细胞凋亡关键蛋白Caspase 3表达相对于阴性对照的显著性改变(P>0.05)。除了0.001μg/mL,所有混合物作用均导致细胞Caspase 3表达水平较DMSO阴性对照显著升高(P<0.05); 10μg/mL的混合物联合作用使细胞Caspase 3表达水平较DMSO阴性对照、PbAc单独作用和其他混合物作用有显著升高(P<0.05)。单一nano-TiO2和混合物所导致的细胞早晚期凋亡显示出剂量依赖关系。析因分析结果显示两种受试物混和染毒对晚期凋亡水平存在交互协同作用(P<0.05)。结论：nano-TiO2和PbAc联合作用可增强诱导L02增加细胞的凋亡,并协同增强细胞的晚期凋亡水平,而Caspase 3在细胞凋亡作用中起着关键作用。
Titanium dioxide (TiO2) in nano-size is a new technology product which was widely used in cosmetics, paints and in decontamination water. Numerous studies demonstrated that nano-TiO2 in nano-size induced damage to human health and environment. Nano-TiO2 could stimulate inflammatory reaction of the organism, reduce the cell viability, and induce the apoptosis and DNA damage. Nano-TiO2 has some special properties, such as nano-scale effect, surface effect, quantum size effect, macroscopic quantum tunneling effect, photocatalytic effect. Compared with the large particles, nano-TiO2 on the nature and intensity of the biological effects of a qualitative change also occurred. According to the present study, mechanisms of damage induced by nano-TiO2 are mainly related to inflammation and oxidative stress.
Lead is widely applied heavy metal in batteries, paint, gasoline, paint, instruments. Lead exists in the environment widespread and accumulates in organism. Lead pollution is a particularly prominent environmental problem in the current situation. Lead pollution causes DNA damage and genetic toxicity, damages the nervous, hematopoietic, reproductive and other systems. Recent studies show that mechanisms of damage induced by lead major related to oxidative stress.
A large number of nano-Ti02 has been released into the environment with the widespread use. Nano-TiO2 could absorb a variety of pollutants in the environment because of the special surface effect. Nano-TiO2 has a strong adsorption capacity to metal ions or organic produce in the environment as the special physical and chemical properties. This may generate different effects compared with their own biological effects. Pb2+ exists in the environment can be adsorbed onto the surface of nano-TiO2, and interact with each other, and interact with the body or the environment. So far, the combined effects of Pb2+ and nano-TiO2 on the body were unknown.
The content of nano-TiO2 and other pollutants in the environment is very low, and exists in trace. In this study, to understand the combined effect of oxidative stress and apoptosis induced by nano-TiO2 mixed with PbAc, we assessed the cell activity, DNA damage, ROS generation, reduction enzyme changes, apoptosis, etc induced by TiO2 in trace mixed with on human embryo hepatocytes (L02) for a 24 hr treatment without photoactivation. Based on the researches on toxicity of nano-TiO2 and PbAc treated alone, the interaction of the mixtures was studied and potential toxicological mechanisms were also evaluated in this study, further consummated the toxicology evaluation and provided the theory basis of nano-TiO2, in addition to understand the healths effects and provide experimental evidence induced by nano-TiO2 and PbAc in trace.
The study is composed of the following four parts:
PartⅠ
The aggregation effect of nano-TiO2 and the adsorptive capacity of PbAc onto nano-Ti02
Objective:To observe the aggregation of nano-TiO2 and adsorption of nano-TiO2 on PbAc in the solution.
Methods:The aggregation size of 0.001,0.01,0.1,1,10μg/mL nano-TiO2 in aqueous solution was detected by a Nano-Particle Analyzer. The adsorption of Pb2+ onto nano-TiO2 in solution was detected by infrared scanning. The adsorptive capacity of 1μg/mL Pb2+ onto 0.001,0.01,0.1,1,10μg/mL nano-TiO2 were detected by graphite furnace atomic absorption spectrometry.
Results:The average size of aggregation is 298.8 nm and there is no significantly change in the given concentrations (P> 0.05). The absorptive capacity of PbAc by nano-TiO2 analyzed by GFAAS is showed that the mixture containing 0.001 and 0.01μg/mL nano-TiO2 absorbed significantly less PbAc than those of 0.1,1,10μg/mL nano-TiO2 (P< 0.05), and there was a dose-dependent absorption by nano-TiO2. Conclusion:nano-TiO2 treated alone in the absence of surfactants aggregated in the short time in water. In the mixtures, there was a significant adsorption on PbAc by nano-TiO2, and adsorption capacity increases with the concentration of nano-TiO2.
PartⅡ
Oxidative stress induced by a joint effect of titanium dioxide and lead acetate in human hepatocytes
Objective:To observe the cell viability, ROS generation and Oxidative stress induced by a joint effect of nano-TiO2 and PbAc in human hepatocytes.
Methods:L02 were exposed to nano-TiO2 in 10,1,0.1,0.01,0.001μg/mL and 1μg/mL PbAc treated alone and mixture for a period 24 hr (The mixtures of nano-TiO2 with 1μg/mL PbAc in the following descriptions were abbreviated as 10,1,0.1,0.01,0.001μg/mL mixture for convenience). In each experiment a negative control (1‰DMSO) was included. The cytotoxicity induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined using MTT assay. The production of ROS induced by nano-TiO2,1μg/mL PbAc and the mixtures were assessed by flow cytometry using DCFH-DA as fluorescent probe. The activities of GSH and SOD were determined meanwhile.
Results:Compared with the negative control, PbAc and the other nano-TiO2 treatments, cell viability decreased notably when treated with 10μg/mL nano-TiO2 (P< 0.05).1μg/mL nano-TiO2 induced significantly viability decreased compared with the negative control (P< 0.05). The mixtures from 0.1,1,10μg/mL induced significantly higher cytotoxicity than the negative control (P< 0.05). Mixtures of 1 and 10μg/mL induced significantly higher cytotoxicity than 1μg/mL PbAc treatment (P< 0.05). There were no significant increases in ROS, GSH, SOD levels caused by nano-TiO2 and PbAc-alone treatment (P> 0.05). In comparison with the negative control and 1μg/mL PbAc, the ROS generation significant increases in L02 cells after all mixtures treatment for 24 hr, respectively (P< 0.05). The 0.01,0.1 and 1μg/mL mixtures induced significant increases in GSH level, compared with the negative control (P< 0.05). The 1μg/mL mixture induced significant increases in GSH level compared with the 1μg/mL PbAc treated alone (P< 0.05). Furthermore, there were significant increases in SOD level induced by the 0.1 and 0.01μg/mL mixtures compared with the negative control (P< 0.05). There were dose-dependence patterns of cell viability and ROS generation induced by nano-TiO2 alone and the mixtures. However, there were only dose-dependence patterns of GSH and SOD levels induced by nano-TiO2 treated alone. There were no synergistic effect in cell viability, ROS, GSH and SOD levels caused by nano-TiO2 and PbAc (P> 0.05).
Conclusion:The joint effect of low (≤10μg/mL) concentrations of nano-TiO2 and PbAc enhances oxidative stress and cytotoxicity in the absence of UV in L02 over a 24 hr treatment period. The results suggest that the increased oxidative stress triggered the up regulation of defenses to protect cells at low concentrations but triggered the down regulation at higher concentrations of the mixtures.
PartⅢ
DNA damage and Repair protein expression induced by a joint effect of titanium dioxide and lead acetate in human hepatocytes
Objective:To observe the DNA damage, DNA adducts generation and Repair protein expression induced by a joint effect of nano-TiO2 and PbAc in human hepatocytes.
Methods:L02 was exposed to nano-TiO2 in 10,1,0.1,0.01,0.001μg/mL and 1μg/mL PbAc treated alone and mixture for a period 24 hr. In each experiment a negative control (1‰DMSO) was included. The DNA damage induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined using comet assay. The generation of DNA adducts induced by nano-TiO2,1μg/mL PbAc and the mixtures were assessed by HPLC. The expression of OGG1 induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined using western blotting assay.
Results:Compared with the negative control and 1μg/mL PbAc, OTM increased notably when treated with 10μg/mL mixture (P< 0.05).1μg/mL nano-TiO2 mixed with 1μg/mL PbAc induced significantly OTM increased compared with the negative control (P< 0.05). There were no significant increases in 8-OHdG formation and OGG1 expression caused by nano-TiO2 and PbAc-alone treatment (P> 0.05). The production of 8-OHdG induced by the 10 and 1μg/mL mixtures was significantly higher than the negative control (P< 0.05). In addition,10μg/mL mixtures caused higher 8-OHdG levels than 1μg/mL PbAc (P< 0.05). All mixtures exposures resulted in the increased OGG1 levels, except for the 10μg/mL mixtures treatment compared with the negative control and 1μg/mL PbAc, respectively (P< 0.05). Nano-TiO2 and the mixtures induced dose-dependence patterns of OTM,8-OHdG formation and OGG1 expression. Addition of nano-TiO2 with PbAC synergistically enhanced OGG1 expression in this study (P< 0.05).
Conclusion:The joint effect of low (≤10μg/mL) concentrations of nano-TiO2 and PbAc enhances oxidative stress and cytotoxicity in the absence of UV in L02 over a 24 hr treatment period. The results suggest that the increased oxidative stress triggered the up regulation of defenses to protect cells at low concentrations but triggered the down regulation at higher concentrations of the mixtures.
Part IV
Apoptosis induced by a joint effect of titanium dioxide and lead acetate in human hepatocytes
Objective:To observe the apoptosis effects induced by a joint effect of nano-TiO2 and PbAc in human hepatocytes.
Methods:L02 was exposed to nano-TiO2 in 10,1,0.1,0.01,0.001μg/mL and 1μg/mL PbAc treated alone and mixture for a period 24 hr. In each experiment a negative control (1‰DMSO) was included. The early and late apoptosis induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined by flow cytometry using FITC and PI as fluorescent probe. The expression of Caspase 3 induced by nano-TiO2, 1μg/mL PbAc and the mixtures were determined using western blotting assay.
Results:Compared with the negative control,1μg/mL PbAc induced no significantly early apoptosis increased (P> 0.05). There were significant increases in early apoptosis caused by 1 and 10μg/mL TiO2 compared with the negative control,1μg/mL PbAc and other concentrations TiO2 treated alone (P< 0.05). The 0.1μg/mL TiO2 induced notably early apoptosis increase compared with the negative control,1μg/mL PbAc,0.001,0.01μg/mL TiO2 treated alone (P< 0.05). All mixtures exposures resulted in the increased early apoptosis levels compared with the negative control and 1μg/mL PbAc, respectively (P< 0.05). All exposures of nano-TiO2 and 1μg/mL PbAc treated alone resulted in no notably late apoptosis increase compared with the negative control (P> 0.05). But late apoptosis increased notably caused by 10μg/mL mixtures treated alone compared with the negative control, PbAc and other mixtures treatments (P< 0.05). All exposures of nano-TiO2 and 1μg/mL PbAc treated alone resulted in no notably Caspase 3 expression increase compared with the negative control (P> 0.05). Compared with the negative control, the expression of Caspase 3 increased significantly when treated with all mixtures except 0.001μg/mL (P< 0.05). Caspase 3 expression increased significantly caused by 10μg/mL mixtures treated alone compared with the negative control, PbAc and other mixtures treatments (P< 0.05). Nano-TiO2 and the mixtures induced dose-dependence patterns of apoptosis. There was synergistic effect in late apoptosis caused by nano-TiO2 and PbAc (P< 0.05).
Conclusion:The joint effect of low (≤10μg/mL) concentrations of nano-TiO2 and PbAc enhances apoptosis and relate key protein expression in L02. The results suggest that the Caspase 3 play a very important role in the apoptosis of the cell.
引文
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