【作者】 涂宏刚；

【导师】 袁宗辉；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2007， 硕士

【摘要】 喹喔啉类药物为人工合成的、具有喹喔啉-1，4-二氧基本结构的动物专用药，具有提高饲料效率、促进动物生长和广谱的抗菌作用，曾作为抗菌促生长剂在世界范围内广泛应用。本类早期品种喹乙醇和卡巴氧由于对人和动物有不同程度的毒副作用已经被禁止或严格限制使用。中国农业科学院兰州研究所八十年代研制的乙酰甲喹由于毒性大，主要作为抗菌剂使用。喹烯酮和喹赛多是新一代喹喔啉类抗菌促生长剂，毒性低，毒副作用小，对食品动物具有良好的促生长效果，具有广阔的应用前景。目前的研究表明，喹喔啉类药物对多种细菌有致突变性，能引起细菌基因点突变或移码突变，但能否引起哺乳动物细胞突变还有待进一步的研究。本试验选用结构类似的五种喹喔啉类药物乙酰甲喹、卡巴氧、喹乙醇、喹烯酮和喹赛多，在体外试验条件下探讨其对中国仓鼠肺成纤维细胞（V79）hgprt基因位点和人外周血淋巴细胞DNA的影响，为进一步探讨喹喔啉类药物的遗传毒性机理奠定基础。体外哺乳细胞致突变试验以五种喹喔啉类药物为受试物，在加与不加S9代谢活化系统条件下观察五种喹喔啉类药物对V79细胞的致突变作用。每种受试药物设四个剂量组、阴性对照组（DMEM，-S9与+S9）、溶剂对照组（DMSO，-S9与+S9）和阳性对照组EMS（-S9）与B（a）P（+S9）。乙酰甲喹、卡巴氧、喹乙醇和喹烯酮给药剂量相同，终浓度分别为10μg／mL、5μg／mL、2.5μg／mL和1.25μg／mL，而喹赛多的终浓度分别为5μg／mL、2.5μg／mL、1.25μg／mL和0.625μg／mL，阴性对照（DMEM）与溶剂对照（DMSO）为总体积的0.5％，阳性对照终浓度EMS为1mg／mL，B（a）P为2μg／mL。经不同的处理4h后，采用克隆形成法观察五种喹喔啉类药物对V79细胞的慢性毒性作用，并在此基础上观察其对V79细胞hgprt基因位点突变频率的影响。结果发现不加S9时，阳性对照EMS的突变率为溶剂对照组的67倍以上；加S9时，阳性对照B（a）P的突变率为溶剂对照组的32倍以上，两者都为典型阳性反应。不论加与不加S9代谢活化系统，乙酰甲喹、卡巴氧和喹乙醇都能引起V79细胞hgprt基因突变率显著增加，并且突变率随着浓度的增加而增大，呈现剂量反应关系；喹烯酮仅在10μg／mL的剂量下与溶剂对照组差异显著，而喹赛多在0.625～5μg／mL的剂量范围内，对V79细胞hgprt基因的突变率与溶剂对照组相比差异不显著。试验结果表明，在本试验条件下，乙酰甲喹、卡巴氧和喹乙醇能引起V79细胞hgprt基因位点突变，而喹烯酮和喹赛多对V79细胞无诱变作用。在程序外DNA合成试验中，以五种喹喔啉类药物为受试物，在加与不加S9代谢活化系统条件下观察不同剂量喹喔啉类药物对人外周血淋巴细胞DNA损伤修复的影响。每种受试药物各设四个剂量组，同时设阴性对照组（RPMI1640，-S9与+S9）、溶剂对照组（DMSO，-S9与+S9）和阳性对照组HN₂·HCl（-S9）与B（a）P（+S9）。乙酰甲喹、卡巴氧、喹乙醇和喹烯酮的终浓度分别为20μg／mL、10μg／mL、5μg／mL和2.5μg／mL，喹赛多终浓度分别为10μg／mL、5μg／mL、2.5μg／mL和1.25μg／mL；阴性对照组（RPMI1640）和溶剂对照组（DMSO）为总体积的1％，阳性对照组HN₂·HCl终浓度为20μg／mL，B（a）P终浓度为25μg／mL。经不同浓度的药物、羟基脲和³H-TdR处理细胞4h后，收集细胞至玻璃纤维纸上，干燥后用液体闪烁仪测定DNA修复合成时³H-TdR的掺入量（CPM）。结果发现，阳性对照HN₂·HCl（-S9）和B（a）P（+S9）的CPM值与溶剂对照组差异极显著，为典型的阳性反应。不论加与不加S9代谢活化系统，在1.25～10μg／mL的浓度范围内，喹赛多不会引起人外周血淋巴细胞程序外DNA合成显著增加，对细胞DNA无损伤作用。而喹烯酮、喹乙醇、卡巴氧和乙酰甲喹的CPM值与溶剂对照组相比差异显著。喹烯酮10～20μg／mL的浓度，喹乙醇、卡巴氧和乙酰甲喹5～20μg／mL的浓度能引起人外周血淋巴细胞程序外DNA合成显著增加，呈现剂量反应关系，这表明喹烯酮、喹乙醇、卡巴氧和乙酰甲喹能引起人外周血淋巴细胞DNA损伤。以上结果显示喹赛多对哺乳动物细胞无遗传毒性，喹烯酮在高剂量时对哺乳动物细胞有一定的遗传毒性，而喹乙醇，卡巴氧和乙酰甲喹对哺乳动物细胞有比较强的遗传毒性。更多还原

【Abstract】 Quinoxalines, which have the structure of quinoxaline-1, 4-dioxides are syntheticantimicrobial agents, which have the characteristic for improving feed efficiency,promoting growth and increasing the rate of weight gain. For these reasons, they havebeen widely used as growth promoters in the most countries of the world. However,carbadox and olaquindox, which the first were developed and used, have been banned orstrictly limited to use due to their toxicities. Mequindox was synthesized by LanzhouInstitute of Animal and Veterinary Pharmaceutics sciences of CAAS in China, and usedas antibacterial agent. Quinocetone and Cyadox, which were new growth promoters ofthe quinoxalines family, had the characteristic of low toxicity, less side effect and obviousgrowth promotion in food-producing animals. Some researches showed that quinoxalineshad the mutagenic activity in germ and could induce base pair substitutions andframeshift mutations in germ. But, the mutagenicity of quinoxalines in mammalian cellsin vitro systems have few reported. In order to investigate the genotoxicity ofquinoxaline-1,4-dioxides in mammalian in vitro, five similar structural compounds:mequindox, carbadox, olaquindox, quinocetone and cyadox were chosen and tested theeffects of hgprt gene mutation in V79 cells and unscheduled DNA synthesis in Humanperipheral blood lymphocytes.Five quinoxalines were investigated for their capabilities of inducing HGPRT genemutation in V79 cells in the absence and presence of rats S9, which were PCB-induced asmetabolism activation systems. Every drug was divided four groups: differentconcentrations groups, negative control （DMEM, -S9与+S9）, solvent control （DMSO,-S9与+S9）, EMS（-S9） and B（a）P（+S9） control. Mequindox, carbadox, olaquindox andquinocetone had the same final concentration: 10μg/mL, 5μg/mL, 2.5μg/mL and1.25μg/mL, the final concentration of cyadox were 5μg/mL, 2.5μg/mL, 1.25μg/mL and0.625μg/mL, the negative control（DMEM） and the solvent control（DMSO） were 0.5%（V/V）, the final concentration of positive control EMS was 1mg/mL and B（a）P was 2μg/mL. After V79 cells were treated for 4h, the cytotoxicity of quinoxalines wereevaluated in V79 cells line by using clone formation assay. Based on the results, wefurther observed the effects of quinoxalinees on hgprt locus mutation frequency. Theresults showed that the mutation frequency of the positive control EMS was more 67times than that of the solvent control in the absence of S9. B（a）P was more 32 times thanthat of the solvent control in the presence of S9. Both of them are typical positivecompounds. Mequindox, carbadox and olaquindox all induced gene mutation at the hgprtlocus in the absence and presence of S9-mix, and there were significant increase of hgprtgene mutation frequency with an approximately dose-effect relationship. However,quinocetone only produced a slight increasing of the mutant frequency at a concentrationof 10μg/mL, and cyadox failed to produce significant increase in the frequency of thehgprt locus mutation at concentration from 0.625 to 5μg/mL whether S9-mix was addedto the plates or not. The results suggest that mequindox, carbadox and olaquindox canlead to mutation of the hgprt locus in V79 cells, while quinocetone and cyadox can notinduce V79 gene mutation.To study the effects of quinoxalines on DNA damage and repair in Humanperipheral blood lymphocytes, unscheduled DNA synthesis test was used in the absenceand presence of S9-mix in vitro. Every drug was divided four groups: four differentconcentration groups, negative control （RPMI1640, -S9与+S9）, solvent control （DMSO,-S9与+S9）, positive control HN₂·HCl（-S9） and B（a）P （+S9）. Mequindox, carbadox,olaquindox and quinocetone have the same final concentration:20μg/mL, 10μg/mL,5μg/mL and 2.5μg/mL, the final concentration of cyadox were 10μg/mL, 5μg/mL,2.5μg/mL and 1.25μg/mL, the negative control（RPMI1640） and the solventcontrol（DMSO） were 1%（V/V）, the final concentration of positive control HN₂·HCl was20μg/mL and B（a）P was 25μg/mL. Human peripheral blood lymphocytes were treatedwith quinoxalines, hydroxyurea and ³H-TdR for 4h, then were collected on glass fiberfilters. The filters were dried and counted CPM in the Bckman liquid scintillator. Theresults showed that the CPM of HN₂·HCl and B（a）P groups had significant increase ofthat of the solvent control, which suggests that they are both typical positive compounds.Cyadox could not make the significance increase of unscheduled DNA synthesis inhuman peripheral blood lymphocytes at a concentration from 1.25 to 10μg/mL in theabsence and presence of S9-mix, suggest that it will not induce DNA damage. However,the CPM of mequindox, carbadox, olaquindox and quinocetone had significant difference from that of the solvent control. Quinocetone could produce the significance increase ofunscheduled DNA synthesis at a concentration from 10 to 20μg/mL, carbadox,olaquindox and mequindox make the similar effect at concentrations from 5 to 20μg/mL,and they were an approximately dose-effect relationship, the results suggested quincetone,olaquindox, carbadox and mequindox could cause the lymphocytes DNA damage inHuman peripheral blood.Based on the results, we found cyadox had no genotoxicity in mammalian cells;quinocetone at high concentration had slight genotoxicity; olaquindox, carbadox andmequindox had strong genotoxicity in mammalian cells.更多还原