| 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.
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