【作者】 崔永；

【导师】 陈子元；

【作者基本信息】 浙江大学 ， 生物物理学， 2006， 博士

【摘要】 毒死蜱(CAS编号:2921-88-2)是一种有机磷杀虫剂,其化学名称为:O,O-二乙基-O-(3,5,6-三氯-2-吡啶基)硫代磷酸酯,具杀虫活性是因为毒死蜱脱硫形成的毒死蜱氧磷使乙酰胆碱酯酶(AChE)磷酸化。磷酸化的AChE不能水解神经递质乙酰胆碱。过量的乙酰胆碱过度刺激乙酰胆碱受体,导致靶生物死亡。氯氰菊酯(CAS编号:52315-07-8)是一种拟除虫菊酯杀虫剂,其化学名称为:(RS)-a-氰基-3-苯氧苄基(1RS)-顺,反-3-(2,2-二氯乙烯基)-2,2-二甲基环丙烷羧酸酯,具杀虫活性是因为它使嵌于神经膜上的Na~+-通道持久开放,严重延迟Na~+-通道的激活、失活和去激活,引起神经细胞持续冲动,导致靶生物死亡。毒死蜱和氯氰菊酯是两种广泛使用的农药,但有关其遗传毒性的机制不很明确,本文围绕这一中心问题开展研究。 毒死蜱和氯氰菊酯均具有强疏水性。两种杀虫剂可能通过分配进入血脂和与血蛋白质结合而被运输。杀虫剂的运输直接影响其分布、代谢和排泄。白蛋白和血红蛋白是血液中两种重要的运输蛋白。杀虫剂与蛋白质结合将影响蛋白质的生理功能。为研究杀虫剂与血液蛋白质结合的特征,在1μmol/L牛血清白蛋白或牛血红蛋白溶液中,逐步注入微量的杀虫剂溶液,使杀虫剂与白蛋白或血红蛋白的摩尔比分别从0增至20或100。每次滴定后测量体系的荧光强度。采用改进的回归方法分析荧光强度的变化,计算结合常数和结合位点数。结果:两种杀虫剂均引起白蛋白荧光猝灭和血红蛋白荧光增强。毒死蜱和氯氰菊酯与白蛋白的结合常数和结合位点数分别是2.99×10~5和5.22×10~5 L/mol,1.25和0.78。毒死蜱和氯氰菊酯与血红蛋白的结合常数和结合位点数分别是2.94×10~4和2.48×10~4 L/mol,1.75和2.19。结论:毒死蜱和氯氰菊酯可与白蛋白和血红蛋白结合,两种杀虫剂与白蛋白的结合显著强于与血红蛋白的结合。 细胞色素P450(CYP)是细胞内重要的Ⅰ相代谢酶,参与物质的排泄。但是,某些P450酶如CYP1A1和3A可将前致癌物氧化为亲电性致癌物,后者可形成DNA加合物,启动癌症发生。物质增强前致癌物激活的能力称助癌性。CYP2B1和3A可产生活性氧(ROS)攻击DNA。为研究毒死蜱和氯氰菊酯对CYP活性更多还原

【Abstract】 Chlorpyrifos (CAS No: 2921-88-2) is an organophosphorus insecticide, and its chemical name is O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl)phosphorothionate. Chlorpyrifos has insecticidal activity because chlorpyrifos-oxon, which results from desulfuration of chlorpyrifos, can phosphorylate acetylcholine esterase (AChE). The phosphorylated AChE can not hydrolyze the neurotransmitter acetylcholine. Excess acetylcholine overstimulates acetylcholine receptors, leading to the death of target organisms. Cypermethrin (CAS No: 52315-07-8) is a pyrethroid insecticide, and its chemical name is (RS)-a-cyano-3-phenoxybenzyl (1RS)-cis ,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate. Cypermethrin has insecticidal activity because it causes the sustained opening of Na~+-channels embedded in nerve membranes and severely retards the activation, inactivation, and deactivation of Na+-channels, which causes the continued impulses in neurons, leading to the death of target organisms. Chlorpyrifos and cypermethrin are two widely used pesticides, but the mechanism of their genotoxicity is not well established. The focus of present study is on the mechanism.Both chlorpyrifos and cypermethrin possess strong hydrophobicity. It is probable that both insecticides are transported to target organs through partitioning into blood lipids and binding to blood proteins. The transportation of insecticides directly affects their distribution, metabolism and excretion. Albumin and hemoglobin are two improtant transport proteins in blood. Binding of an insecticide to the proteins will affect their physiological functions. The binding of insecticide to blood proteins is characterized. A minute volume of insecticide solution is progressively injected into 1 μmol/L bovine serum albumin or bovine hemoglobin. The molar ratio of insecticide to albumin and hemoglobin increases from 0 to 20 and 100, respectively. After every titration, the fluorescence of system is determined. A modified regression is used to analyze the alteration of fluorescence and calculate the association constant and the number of binding sites. It is found that both insecticides cause fluorescence quenching of thealbumin and fluorescence enhancement of the hemoglobin. The association constant and the number of binding sites of chlorpyrifos and cypermethrin binding to the albumin are 2.99xlO5 and 5.22xlO5 L/mol, 1.25 and 0.78, respectively. The association constant and the number of binding sites of chlorpyrifos and cypermethrin binding to the hemoglobin are 2.94xlO4 and 2.48xlO4 L/mol, 1.75 and 2.19, respectively. The results indicate that chlorpyrifos and cypermethrin can- bind to albumin and hemoglobin, and the binding of insecticides to albumin is significantly stronger than that to hemoglobin.Cytochrome P450 (CYP) are the phase I enzymes of great importance in cells and involved in the excretion of substance. However, some CYP such as CYP1 Al and 3A can oxidize precarcinogen into electrophilic carcinogen, which may form DNA adducts, initiating carcinogenesls. The ability of substance to substantiate activation of precarcinogen is called cocarcinogenicity. CYP2B1 and 3A can produce reactive oxygen species (ROS) which may insult DNA. The effect of chlorpyrifos and cypermethrin on activities of CYP is studied. ICR (Institute of Cancer Research) mice are intraperitoneally administrated with chlorpyrifos (25.0, 50.0 mg/kg bw/d) or cypermethrin (100.0, 200.0 mg/kg bw/d) for 3 days. On the 4th day, the activities of CYP in liver, kidney and lung are determined. It is found that both insecticides increase activities of CYP in most cases. For example, the treatment of 50.0 mg/kg chlorpyrifos increases the activities of lung CYP1A1 of male and female mice by 86% and 62%, respectively. The treatment of 200.0 mg/kg cypermethrin increases the activities of lung CYP1A2 of female mice and kidney CYP2B1 of male mice by 109% and 69%, respectively. Both insecticides exert sex-specific influence on activities of some CYP, such as CYP1A1 in liver and kidney, CYP1A2 in kidney, CYP2E1 in liver, CYP3A in liver and lung for chlorpyrifos, and CYP1A2 in liver and lung, CYP2B1 in kidney, CYP2E1 in liver, kidney and lung for cypermethrin. Both insecticides also exert organ-specific influence on activities of some CYP, such as CYP1A1, 1A2, 2B1, 2E1 of female and male mice, CYP3A of male mice for chlorpyrifos, and CYP1A2, 2E1, 3A of female and male mice, CYP2B1 of female mice for cypermethrin. Chlorpyrifos significantly increases activities of CYP1A1 in lung, and CYP2B1 in kidney and lung. Cypermethrin significantly increases activities of CYP1A1 in liver, kidney and lung, CYP2B1 in liver and lung, and CYP3A in liver. Theresults suggest that chlorpyrifos and cypermethrin have cocarcinogenicity and indirectly damage DNA through increase of reactive oxygen species (ROS) that result from the increase of CYP activities.The types of DNA damage by chlorpyrifos and cypermethrin are studied through four assays, which are performed as follows.1) Single cell gel electrophoresis (comet) assay. ICR mouse primary lymphocytes are treated with 3.1-50.0 ^g/mL chlorpyrifos or 6.3-100.0 ^wg/mL cypermethrin for 2 h. Frequency of comet cells is determined. It is found that under the treatment of PBS buffer or 1% dimethyl sulfoxide (DMSO), the frequency of comet cells is 6%. Chlorpyrifos and cypermethrin significantly increase the frequency of comet cells to 13-45% and 17-37% (P < 0.001), respectively. It is concluded that both chlorpyrifos and cypermethrin can lead to DNA strand breakage.2) Bioluminescence assay. ICR mouse primary hepatocytes are treated with 0.39-100.00 jug/mL chlorpyrifos or 0.78-200.00 /(g/mL cypermethrin for 4 h. DNA adduct coefficients are determined and DNA adduct coefficient by 1% DMSO is defined as 0. It is found that DNA adduct coefficients by chlorpyrifos are 0.01-0.53, which are not significantly different from that by 1% DMSO (P > 0.05). DNA adduct coefficients by cypermethrin are 0.71-0.90, which are significantly higher than that by 1% DMSO (P < 0.05, P < 0.01). The results illustrate that cypermethrin can lead to the formation of DNA adducts but chlorpyrifos cannot.3) K+-SDS precipitation assay. ICR mouse primary hepatocytes are treated with 0.39-100.00 jug/mL chlorpyrifos or 0.78-200.00 //g/mL cypermethrin for 4 h. DNA-protein crosslink coefficients are determined and DNA-protein crosslink coefficient by 1% DMSO is defined as 1. It is found that DNA-protein crosslink coefficients by chlorpyrifos and cypermethrin are 0.80-0.94 and 0.68-1.07, respectively, which are not significantly different from that by 1% DMSO (P > 0.05). It is concluded that neither chlorpyrifos nor cypermethrin cause DNA-protein crosslinks.4) Ethidium bromide fluorescence assay. Calf thymus DNA and ICR mouse primary hepatocytes are solely treated with 0.39-100.00 ^g/mL chlorpyrifos or 0.78-200.00 jug/mL cypermethrin for 4 h. A parallel group of hepatocytes is co-treated with insecticideand SKF-525A, a cytochrome P450 inhibitor. The ratio (R) of DNA fluorescence intensity after denaturing to that before denaturing is calculated. It is found that under the treatment of 1% DMSO, the R of calf thymus DNA, hepatocytes, and hepatocytes co-treated with SKF-525A are 0.600, 0.608 and 0.596, respectively. Under the treatment of chlorpyrifos, the corresponding R are 0.591-0.609, 0.612-0.626, and 0.587-0.610, respectively, which are not significantly different from the corresponding R by 1% DMSO (P > 0.05). Under the treatment of cypermethrin, the R of calf thymus DNA and hepatocytes co-treated with SKF-525A are 0.586-0.592 and 0.573-0.592, respectively, which are not significantly different from the corresponding R by 1% DMSO (P > 0.05). It is worth notice that the R of hepatocytes treated with 0.78-12.50 ,Mg/mL cypermethrin increase to 0.634-0.654, which are significantly different from the corresponding R by 1% DMSO (P < 0.05, P < 0.001). However, the R of hepatocytes treated with 50.00 and 200.00 fig/mL cypermethrin decrease to the level of corresponding control. The results indicate that chlorpyrifos can not cause formation of DNA interstrand crosslinks, but cypermethrin can cause it. Active metabolites of cypermethrin instead of cypermethrin itself cause DNA interstrand crosslinks. Cytochrome P450 may be involved in the activation of cypermethrin. Cypermethrin of high concentrations may inhibit the activation of cypermethrin by cytochrome P450.DNA repair under the treatment of chlorpyrifos or cypermethrin is studied. ICR mouse primary lymphocytes and hepatocytes are treated with 3.1-50.0 //g/mL chlorpyrifos or 6.3-100.0 jug/mh cypermethrin for 3 h. The unscheduled DNA synthesis (UDS) assay is performed. It is discovered that under the treatments of PBS buffer, 1% DMSO, chlorpyrifos, and cypermethrin, the incorporations of 3H-thymidine (3H-TdR) into DNA, which is extracted from 1.6xlO6 lymphocytes, are 632, 676, 568-922, and 482-630 disintegrations/min (dpm), respectively. The corresponding incorporations of 3H-TdR into 40 n% DNA of hepatocytes are 615, 680, 344-391, and 335-553 dpm, respectively. It is displayed that the unscheduled DNA synthesis in lymphocytes or hepatocytes treated with chlorpyrifos or cypermethrin is not significantly different from that by PBS buffer or 1% DMSO (P > 0.05). The results illustrate that there is not significant DNA repair when chlorpyrifos or cypermethrin damages DNA.DNA methylation plays an important role in the regulation of gene expression in eukaryotes. Effect of chlorpyrifos and cypermethrin on DNA methylation level is studied. ICR mouse primary hepatocytes are treated with 3.1-50.0 /ig/mL chlorpyrifos or 6.3-100.0 jug/mL cypermethrin for 4 h. High performance liquid chromatography (HPLC) is used to determine the content of DNA bases. It is found that DNA methylation levels in hepatocytes treated with PBS buffer and 1% DMSO are 62.8% and 52.2%, repectively. The levels in hepatocytes treated with chlorpyrifos and cypermethrin are 16.9-35.1% and 18.8-26.4%, repectively, which are significantly lower than that by PBS buffer or 1% DMSO (P < 0.001). The results indicate that both chlorpyrifos and cypermethrin can decrease DNA methylation levels.Since chlorpyrifos and cypermethrin can damage DNA of somatic cells, they may damage DNA of germ cells through similar mechanisms, impacting on mammalian reproduction and development. A preliminary study on reproductive toxicity of chlorpyrifos and cypermethrin is performed. ICR male mice are intraperitoneally administrated with chlorpyrifos (25.0, 50.0 mg/kg bw/d) or cypermethrin (100.0, 200.0 mg/kg bw/d) for 4 days. On 7th day, ratio of bilaterally testicular weight to body weight (i?t/b) and frequency of abnonnal sperm are determined. It is shown that the i?t/b of mice treated with corn oil is 8.49x10"3. Under the treatments of low and high doses of chlorpyrifos, and low and high doses of cypermethrin, the /?t/b are 7.93x10"3, 8.53x10"3, and 8.98x10"3, 7.88x10"’, respectively, which are not significantly different from that by corn oil (P > 0.05). Frequency of abnormal sperm of mice treated with corn oil is l%o. Under the treatments of low and high doses of chlorpyrifos, or low and high doses of cypermethrin, the frequency of abnormal sperm are 40%o and 71%o, or 99%o and 125%o, respectively, which are significantly higher than that by corn oil (P < 0.001). It is illustrated that both chlorpyrifos and cypermethrin have reproductive toxicity.From the present study, two new ideas are brought forth as follows.(A) To obtain the association constant and the number of binding sites between chemical and protein, a new parameter, i.e., the fluorescence intensity of system when adequate chemical is added, is introduced in linear regression analysis. The modified method well describes both quenching and enhancement of fluorescence.(B) The probable pathways that cypermethrin is activated are put forward. The product from hydrolysis of cypermethrin, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, can be hydroxylated in two ways: 1) the methyl group on cyclopropane group is hydroxylated and the product is oxidized into an epoxide, 3-(2,2-dichloro-l,2-epoxyethyl)-2-hydroxymethyl- 2-methylcyclopropanecarboxylic acid, which can form DNA monoadducts. 2) 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid is hydroxylated via rearrangement of radical and formation of carbocation, and the products are oxidized into two diepoxides: 5,5-dichloro-3-hydroxy-2-(l,2-epoxyisopropyl)-4,5-epoxypentanoic acid and5,5-dichloro-2-hydroxy-3-(l,2-epoxyisopropyl)-4,5-epoxypentanoic acid, which can cause DNA monoadducts and DNA interstrand crosslinks.更多还原