【作者】 张江华；

【导师】 周宜开； 吕斌；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2008， 博士

【摘要】 DDT是最早人工合成的有机氯杀虫剂,早先用于灭蚊防疟疾,战后开始广泛用于农业生产。由于DDT化学性质稳定,残留期长,到目前为止,土壤、水及动物脂肪组织中仍能检测到较高浓度的DDT及其代谢产物。大量研究表明DDT能够引起人类染色体断裂、白血病、肺癌等危害,国际癌症研究署(IARC)已将其列为人类可能致癌物。常规的物理、化学、生物方法难以满足净化处理在技术和经济上的要求,有机氯污染物的处理技术成为研究的热点。近些年发展了多项技术用于处理DDT污染的土壤和地表水,随着研究的深入,TiO2辅助的光催化技术应运而生并有了快速的发展。然而,关于光降解过程中的中间产物的毒性变化仍不清楚。本研究显示了在模拟太阳光光照12 h的过程中,中间产物对细胞的存活率和DNA损伤的影响逐渐降低。但是在光照条件不能满足时,DDT与nano-TiO2共存的情况并未考虑。当机体暴露在混合毒物时,共存的已知毒性的化合物在体内的相互作用,常常是通过改变机体的功能状态或代谢能力而实现的。它可发生在毒物摄入、吸收、分布、代谢转化、排泄等过程中,或是作用于同一靶器官而产生的相关作用效应。因此,研究纳米颗粒物与其它毒物之间的相互作用对评估人体健康效应具有非常重要的实用价值。通常情况下TiO2被认为是相对低毒的化合物。但是近来大量研究显示了TiO2对人体健康的不利影响。研究发现nano-TiO2较相同化学成分的大颗粒TiO2的毒作用更明显。纳米颗粒在理化性质发生巨变的同时,其生物学效应的性质和强度也可能发生质的变化。nano-TiO2刺激机体的免疫反应,引起炎症反应、DNA损伤、脂质过氧化。纳米颗粒诱导损伤的机制尚不明确,但是跟炎症反应和氧化应激有关。纳米材料具有大量的界面,具有很强的吸附能力,从而能对金属离子或有机物产生吸附作用。当纳米材料大量进入环境后,特殊的表面效应使得其极易与环境中的多种污染物结合。DDT广泛存在于环境当中,可吸附到nano-TiO2的表面,与nano-TiO2相互作用,纳米颗粒的理化性质利于污染物的吸收,促使其穿越各种生物屏障。二者对机体可能导致的各种健康效应有何变化,至今尚无研究。环境污染物一般都是痕量存在,因此本试验系统了解痕量nano-TiO2与环境常见污染物DDT的联合作用所致健康效应。本研究以体外培养的正常人类来源胚胎肝细胞(L-02细胞)为靶细胞,探究nano-TiO2与DDT联合作用对细胞ROS生成、DNA断裂损伤、染色体损伤及脂质过氧化等情况的影响,系统研究nano-TiO2与DDT联合作用所致氧化应激效应。本文在以往DDT与nano-TiO2的毒作用机理研究的基础上,用体外试验探讨了二者之间的相互作用及可能的毒作用机制,进一步完善光催化反应的毒理学评价,为nano-TiO2介导的光降解反应的可行性提供理论基础,为进一步深入了解环境中纳米金属氧化物的健康效应提供实验依据。本研究分为三个部分:第一部分纳米二氧化钛光催化降解DDT及中间产物的DNA损伤效应研究目的:观察纳米二氧化钛(nano-TiO2)光催化降解DDT不同时段的中间产物对人胚肝细胞株(L-02)毒性作用的变化。方法:采用nano-TiO2光催化降解法处理DDT的水溶液12 h,初始浓度为50μg/mL,取不同时段的中间产物染毒L-02细胞,染毒时间为24 h,检测其对L-02细胞的存活率、DNA损伤效应的影响。结果:50μg/mL的DDT能显著降低细胞的存活率(P < 0. 05),并引起明显的DNA损伤效应(P < 0. 05)。不同时段的DDT中间产物处理后,在0 min、2 min、5 min、10 min、30 min、40 min、50 min、1 h、1.5 h、2 h时间点L-02细胞的存活率与溶剂对照组比有显著性差异(P < 0. 05),且随着时间的增加存活率逐渐升高,在3 h以后的中间产物对细胞的存活率与溶剂对照组相比无显著性差异(P > 0. 05)。彗星试验显示40 min之前的DDT降解产物具有显著的DNA损伤作用(P < 0. 05),随着时间的增加DNA损伤效应逐渐减小,50 min以后的中间产物对细胞的DNA损伤效应与溶剂对照组相比无显著性差异(P >0. 05)。结论:DDT对L-02细胞具有细胞毒性和DNA损伤作用,但是随着光降解反应的进行,DDT光催化降解产物的毒性逐渐降低。第二部分DDT联合纳米二氧化钛协同诱导人胚肝细胞氧化应激作用目的:观察DDT、纳米二氧化钛(nano-TiO2)单独及联合作用对人胚肝细胞(L-02)内活性氧(reactive oxygen species,ROS)含量及氧化应激能力的影响。方法:生长良好的人胚肝细胞,分别加入0.001、0.01、0.1μmol/L的DDT,0.01、0.1、1μg/mL的nano-TiO2,以及上述各浓度的DDT和nano-TiO2联合作用,二甲基亚砜(DMSO,1 mL/L)为溶剂对照,将L-02细胞染毒24小时后,运用DCFH-DA作为荧光探针,采用流式细胞检测技术检测DDT与nano-TiO2联合作用下L-02细胞内ROS含量,同时检测L-02细胞内抗氧化酶超氧化物歧化酶(SOD)、脂质过氧化产物丙二醛(MDA)。结果:1μg/mL的nano-TiO2单独作用可引起ROS增加(P < 0.05);0.01μmol/L以上的DDT作用24h引起ROS增加(P < 0.05);nano-TiO2与DDT联合作用细胞内ROS水平较溶剂对照组有显著升高(P < 0.05)。与溶剂对照相比,0.1、1μg/mL的nano-TiO2单独作用使L-02细胞中抗氧化酶SOD活力显著性降低(P < 0.001,P < 0.001);0.1μmol/L的DDT作用24h显著降低SOD水平(P < 0.001);nano-TiO2与DDT联合作用细胞内SOD水平较溶剂对照组有显著降低(P < 0.05)。0.01、0.1、1μg/mL的nano-TiO2单独作用能明显增加L-02细胞中脂质过氧化产物MDA的含量(P<0.05,P<0.001,P<0.001);0.01、0.1μmol/L的DDT可诱导MDA含量显著增加(P < 0.001,P < 0.001);二者联合作用可促进MDA的生成(P < 0.05)。析因分析结果显示两种受试物混和染毒存在交互作用,反应曲面模型提示二者之间的相互作用为协同作用(P < 0.05)。在本试验浓度范围内,L-02细胞的SOD活力水平与ROS生成量呈负相关(r = -0.86901,P < 0.01),MDA含量与ROS生成量呈正相关(r = 0.91175,P < 0.01)。结论:痕量DDT和nano-TiO2联合作用可诱导L-02细胞氧化应激,使其脂质过氧化反应增强、抗氧化作用降低;二者引发的脂质过氧化反应和抗氧化力下降有可能是导致机体氧化损伤的影响因素。第三部分DDT与纳米二氧化钛对人胚肝细胞遗传毒性的协同效应研究目的:观察DDT、纳米二氧化钛(nano-TiO2)单独及联合作用对人胚肝细胞株(L-02)DNA、染色体损伤的影响。方法:生长良好的人胚肝细胞,分别加入0.001、0.01、0.1μmol/L的DDT,0.01、0.1、1μg/mL的nano-TiO2,以及上述各浓度的DDT和nano-TiO2联合作用,二甲基亚砜(DMSO,1 mL/L)为溶剂对照,L-02细胞处理时间为24h;运用碱性和中性两种彗星试验和DNA氧化损伤标志物8-OHdG的含量检测各组DNA断裂损伤程度,对各染毒组进行体外微核试验计算其微核率的改变。结果:各浓度的nano-TiO2均不能引起DNA单链断裂增加(P > 0.05);0.01μmol/L以上的DDT作用24h后DNA单链断裂增加(P<0.05);二者联合作用可促进DNA单链断裂损伤作用。而在中性彗星试验中,各实验组均无明显的DNA损伤(P > 0.05)。1μg/mL的nano-TiO2使L-02细胞中DNA氧化损伤标志物8-OHdG生成量显著性增加(P < 0.05);0.001μmol/L以上的DDT作用L-02细胞8-OHdG生成量显著增加(P<0.05, P<0.001, P<0.001);二者联合作用可促进8-OHdG的生成。各浓度的nano-TiO2均不能引起微核率增加(P > 0.05);0.01μmol/L以上的DDT作用L-02细胞微核率明显增加(P<0.001);二者联合作用可促进微核率的增加。析因分析结果显示两种受试物混和染毒存在交互作用(P < 0.05),反应曲面模型提示二者的相互作用为协同作用(P < 0.05)。在本试验浓度范围内,L-02细胞的DNA单链断裂损伤程度与ROS生成量呈正相关(r = 0.85838,P < 0.01);8-OHdG水平与ROS生成量呈正相关(r = 0.86691,P < 0.01);微核率水平与ROS生成量亦呈正相关(r = 0.80569,P < 0.01)。结论:痕量DDT和nano-TiO2联合作用可协同增加各自对L-02细胞内DNA损伤及染色体损伤的影响,对细胞的损伤作用增大。诱导产生自由基并导致DNA损伤以及非整倍体诱变作用导致染色体损伤很可能是DDT和nano-TiO2引起机体损伤的主要机制之一。更多还原

【Abstract】 DDT is an organochlorine insecticide which could cause chromosomal damage, leukemia and lung cancer in humans. International Agency for Research on Cancer (IARC) has placed DDT on the list of possibly human carcinogen. It has been widely used in agriculture in the past and now still exists at high concentrations along with its metabolites throughout the world for the reason of its long half-lives in soil, water and adipose tissue of animals. Conventional water treatment plants are typically not effective in remediating organochlorine contaminants. Many technologies have been developed to deal with DDT-polluted earth and surface waters in recent years. TiO2-assisted photocatalysis stands out from various environmental technologies. However, little was known about toxicity changes of intermediates with time during the process of degradation. In the experiment, the toxic effects of DDT degradation intermediates on human derived fetal hepatocytes were investigated. The results showed that the survival rate and DNA damage of cells gradually decreased in the time period 0– 12 h after the start of illumination using a simulated sun. But coexistence of DDT and nano-TiO2 in the absence of light was not considered yet. When organisms exposed to a mixture of different toxicants, their simultaneous presence that biologically modify cellular conformation and metabolic activity might induce non-overlapping toxic effects although the toxicity of the single compounds might be well known. The interaction may happen during the process of absorption, distribution, metabolic transformation and excretion or depend among other things on the affinity to target sites at cellular level. Therefore, studies of interactions among nanoparticles and other toxicants are of fundamental interest and practical importance to evaluate the exact health impact.In general, TiO2 was thought as a relatively low toxic compound. But recently numerous studies showed adverse effect of TiO2 on health. Several studies found that nano-TiO2 were more toxic than equivalent larger fine particles of the same chemical composition. Nano-TiO2 could stimulate immune response and cause inflammation, DNA damage and lipid peroxidation. The mechanism of nanoparticles induced DNA damage is not understood, but appears to be related to inflammation and oxidative stress. Nanomaterials have massive interface and strong adsorption ability. Thus nanomaterials can adsorb mental ions or organic compounds efficiently. Because of nanomaterials’special surface effect, they will combine with existed pollutants after they enter the environment. DDT in environment can be easily absorbed on the surface of TiO2 and interact with DDT. In addition to facilitating the absorption of pollutants, the physicochemical characteristics of nanoparticles enable them to cross various biological barriers without a specific transporter. But to the best of our knowledge, no study focused on health effect resulted from DDT and nano-TiO2 was reported yet.Environmental pollutants normally exist in trace concentration, therefore the co-effects of trace nano-TiO2 and DDT in human fetal hepatocytes (L-02) cells were studied in this study. This paper mainly discuss the effect of nano-TiO2 and DDT on ROS generation, DNA strand breaks, chromosome damage and lipid peroxidation in L-02 cells by a series of in vitro experiments, which were used to explore the oxidative stress level. Based on departed researches on toxicity of DDT and nano-TiO2, the interaction of both compounds was studied and potential toxicological mechanisms were also evaluated in vitro studies, further consummated the toxicology evaluation of photocatalyzed reactions, then provided the theory basis for photodegradation mediated by nano-TiO2 and provide experiment evidences to the health impact of nanoparticles.The whole study was composed of the following three parts: PartⅠ: Degradation of DDT in water using nano-TiO2 photocatalyst and DNA damage effect caused by intermediate productsObjective: To observe the toxic effects of DDT degradation intermediates on human derived fetal hepatocytes.Methods: An initial DDT concentration of 50μg/mL in water was degraded by nano-TiO2 photocatalyzed reactions under simulated sun for 12 h. Human derived fetal hepatocytes were exposed to DDT degradation intermediates. Survival rate and DNA damage were measured.Results: 50μg/mL DDT could decrease the cell viability (P < 0.05) and induce obvious DNA damage effect (P < 0.05). After being exposed to DDT degradation intermediates in the time period 0-2 h after the start of illumination, the survival rate of L-02 cells significantly decreased in comparison with the control (P < 0.05). Cell viability increased with time and the intermediates was similar to the control after 3 h. The comet assay indicated that significant DNA damage was observed exposed intermediates before 40 min (P < 0.05) and the damage effect decreased with time.Conclusion: DDT could cause cytotoxic effect and DNA damage to L-02 cells. With the development of photocatalyzed reaction, the toxicity of DDT degradation intermediates decreased.PartⅡ: Oxidative stress induced by DDT and nano-TiO2 in human derived fetal hepatocytesObjective: To investigate the levels of oxidative stress induced by DDT, nano-TiO2, DDT+nano-TiO2 in human derived fetal hepatocytes.Methods: L-02 cells were exposed to single DDT (0.001, 0.01, 0.1μmol/L), single nano-TiO2 (0.01, 0.1, 1μg/mL), combined different concentration of DDT with nano-TiO2 respectively. DMSO (1 mL/L) was used as solvent control. Reactive oxygen species (ROS) level was determined by flow cytometry. SOD, MDA, which were the product of the representative of antioxidative enzyme, lipid peroxidation respectively, were detected in L-02 cells after exposure.Results: It can be observed that cells treated with 0.01μmol/L or above concentration of DDT or 1μg/mL nano-TiO2 alone respectively showed a significantly increased level of ROS production compared with control cells (P < 0.05). ROS level generated by a mixture of nano-TiO2 and DDT was markedly elevated (P < 0.05). 0.1μg/mL or above concentration of nano-TiO2 alone and 0.1μmol/L DDT alone significantly decreased SOD activity (P < 0.001) in comparison with solvent control. The content of MDA was significantly increased in L-02 cells exposed to nano-TiO2 alone at the concentration of 0.01μg/mL or above concentration (P<0.05, P<0.001, P<0.001). Cells treated with greater than 0.01μmol/L DDT showed a significantly increased level of MDA (P < 0.001,P < 0.001). Mixture of trace amount of DDT and nano-TiO2 was synergistic to decrease SOD level (P < 0.05), increase MDA generation (P < 0.05) and ROS formation (P < 0.05). The results of factorial analysis showed an interaction between both compounds (P < 0.05). Response surface model indicated that the interaction was synergism (P < 0.05). During the concentration tested, there was a negative association between SOD level and ROS generation (r = -0.86901,P < 0.01) and a striking positive association between MDA content and 8-OHdG level (r = 0.91175,P < 0.01).Conclusion: Our study indicated that coexistence of trace amount of DDT and nano-TiO2 could induce oxidative stress in L-02 cells including increase of lipid peroxidation and decrease of antioxidative enzyme. Oxidative damage in L-02 cells might be ascribed to DDT and nano-TiO2 induced lipid peroxidation and the weakening of antioxidation ability.PartⅢ: Synergistic genotoxicity caused by DDT and nano-TiO2 in human derived fetal hepatocytesObjective: To investigate the effects of DDT, nano-TiO2, DDT+nano-TiO2 on DNA and chromosome damage level in human derived fetal hepatocytes.Methods: L-02 cells were exposed to single DDT (0.001, 0.01, 0.1μmol/L), single nano-TiO2 (0.01, 0.1, 1μg/mL), combined different concentration of DDT with nano-TiO2 respectively. DMSO (1 mL/L) was used as solvent control. DNA damage was measured by comet assay and 8-OHdG which is the marker of DNA oxidative damage. And chromosome change was investigated using micronucleus test.Results: No significant effects on DNA damage were observed in single nano-TiO2 groups (P > 0.05) and 0.01μmol/L or above concentration of DDT could induce DNA damage (P < 0.05). There was no statistically significant increase of OTM in all groups compared with control group in neutral comet assay (P > 0.05). 1μg/mL nano-TiO2 could increase 8-OHdG formation significantly (P < 0.05). And 0.001μmol/L or above concentration of DDT alone significantly increase 8-OHdG level (P<0.05, P<0.001, P<0.001). Nano-TiO2 could not induce MN frequency in concentrations ranging from 0 to 1μg/mL (P > 0.05). MN frequency significantly increased in the cells treated with greater than 0.01μmol/L DDT (P<0.001). Coexistence of both compounds could enhance the MN frequency. Factorial design showed that there was an interaction between both tested substances (P < 0.05) and response surface model indicated that the interaction was synergism (P < 0.05). There was a striking positive association between DNA single strand breaks and ROS generation (r = 0.85838, P < 0.01), between 8-OHdG level and ROS generation (r = 0.86691, P < 0.01) and between MN frequency and ROS generation (r = 0.80569, P < 0.01) in L-02 cells exposed to DDT and nano-TiO2 with concentrations tested.Conclusion: Exposure with trace amount of DDT and nano-TiO2 synergistically enhanced their capability in DNA damage and chromosome change on L-02 cells. The primary mechanism of toxicity may be that DDT and nano-TiO2 can induce the free radical generation, followed by DNA damage and result in chromosome damage via aneugenic activity.更多还原