双苯氟嗪的致突变毒性研究
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摘要
双苯氟嗪（Dipfluzine，Dip）系由我校药学院研制并合成的桂利嗪类钙拮抗剂。先期的实验表明，Dip可以选择性地扩张椎动脉、基底动脉及冠状动脉，且作用强于桂利嗪；另外Dip也具有增加脑血流量，改善脑缺血、缺氧，及抗血小板凝集和血栓形成的作用，因此Dip很可能是治疗脑血管疾病极具临床开发价值的新型钙拮抗剂。目前，关于Dip的特殊毒性尚未见报道。本实验对Dip的体内外致突变毒性进行了研究，以便为开发此药提供遗传学资料。
目的：观察Dip的致突变毒性。
方法：应用鼠伤寒沙门菌回复突变实验（Ames实验）、哺乳动物培养细胞染色体畸变（Chromosome Aberration, CA）实验、小鼠微核（Micronucleus, MN）实验和哺乳动物骨髓细胞CA实验。
结果：1 Dip对Ames测试菌株的回复突变作用：对Ames测试菌株TA97、TA98、TA100、TA102， Dip浓度为2000、1000、100、10、1μg/皿时，无论加或不加S9混合物，其回复突变作用均未达到或超过自发回变菌落的2倍，也未见剂量反应关系，结果判为阴性。表明Dip在体外对原核细胞不具有移码型或碱基对置换型基因突变作用。
2 Dip对中国仓鼠肺成纤维 (Chinese hamster
lung,CHL)细胞的致突变作用：Dip浓度为125μg/ml、62.5μg/ml、31.3μg/ml与CHL细胞孵育培养，在培养24h和48h以及加S9混合物时培养24h收获细胞，染色体畸变率均小于4%，判定为阴性结果。表明Dip在体外对哺乳动物培养细胞染色体不具有致突变作用。
3小鼠微核实验：小鼠灌胃给Dip，剂量分别为2.8g/kg、1.4g/kg或0.7g/kg，于灌胃24h后取骨髓，计数各剂量组骨髓嗜多染红细胞微核（Micronucleus of polychromosome erythrocytes，MNPCE）率并与空白对照组比较，均无显著性差异，也无剂量反应关系，试验结果为阴性。表明Dip在小鼠体内不具有致突变作用。
4小鼠骨髓细胞CA实验：小鼠灌胃给 Dip，剂量分别为2.8g/kg、1.4g/kg或0.7g/kg，于灌胃12h、24h、48h后取骨髓进行染色体畸变分析，各剂量组与相应空白对照组相比无显著性差异，也无剂量反应关系，试验结果为阴性。表明Dip在小鼠体内不具有致突变作用。
结论：Dip在体外对原核细胞和哺乳动物真核细胞及在小鼠体内均不具有致突变毒性。
Dipfluzine(Dip), a novel calcium antagonist of diphenylpiperazines, was firstly sythensized by Institute of Pharmaceutic in Hebei Medical Univercity. Previous researches revealed that Dip selectively dilated vertebral artery，basilar artery and coronary artery. And the effects of Dip were more potent than those of flunarizine. In addition, Dip increased brain blood and improved brain ischemia as well as brain hypooxygen. It also inhibited thrombus formation and the platelet aggregation. Thus Dip is well likely to be a promising calcium antagonist to treat brain vascular diseases. At present, there is still no report about special toxicity of Dip. This experiment investigated the mutagenic toxicity of Dip in vivo and in vitro, which provided genetic data for the further study of Dip.
Aim: To investigate the mutagenic toxicity of Dip.
Method: salmonella typhimurium mutagenicity test (Ames test), chromosome aberration (CA) test of cultivated cells of mammalian, micronucleus test in mice and mammalian bone marrow CA test were used.
Result: 1 The revertant effect of Dip on Ames tester strains: TA97, TA98, TA100 and TA102 were used. At the concentrations of Dip 2000, 1000, 100, 10 and 1μg/plate, revertant number of four tester strains，were not up to or over the double number of spontaneous reversion in control group with or without mixture S9. Also，there was no dose-dependence in the number of revertants induced by Dip. So the effect of Dip on the number of revertants was qualified to be negative，indicating that Dip dose not genetic mutagenicity including base-pair substitutions and frameshift in vitro.
2 Mutagenetic effect of Dip on CHL cells: Dip，at the concentration of 125μg/ml, 62.5μg/ml and 31.3μg/ml, was incubated with CHL cells for 24h and 48h with or without mixture S9. CHL cells were respectively harvested at the time 24h and 48h without S9 or 24h with S9 , and the CA was observed . It was found that CA rates in CHL cells incubated with Dip were less than 4%, which was qualified to be negative. The result indicates that Dip dose not induce chromosome aberration in vitro.
3 Micronucleus test in mice: The mice were treated with Dip p.o. at the doses of 2.8g/kg, 1.4g/kg and 0.7g/kg, the bone marrow of mice was taken out at 24h after Dip treatment. MNPCE were counted and MNPCE rates were calculated. MNPCE rates in Dip groups had no significant difference from those in control group, and Dip has no
effect on MNPCE rates in a dose-dependent way. Thus the effect of Dip on MNPCE was qualified to be negative, indicating that Dip has no mutagenicity in vivo.
4 CA test of mouse marrow cells: The mice were treated with Dip p.o. at the doses of 2.8g/kg, 1.4g/kg and 0.7g/kg, the bone marrow of mice was taken out at 12h, 24h and 48h respectively after Dip treatment. There was no significant difference in CA rate between Dip and control groups. Also, there was no dose-dependence in CA rate induced by Dip. Thus the effect of Dip on CA rate was qualified to be negative, indicating that Dip has no mutagenicity in vivo.
Conclusion: Dip has no mutagenic toxicity on prokaryocyte and eukaryocyte in vitro or in vivo.
目的：观察Dip的致突变毒性。
方法：应用鼠伤寒沙门菌回复突变实验（Ames实验）、哺乳动物培养细胞染色体畸变（Chromosome Aberration, CA）实验、小鼠微核（Micronucleus, MN）实验和哺乳动物骨髓细胞CA实验。
结果：1 Dip对Ames测试菌株的回复突变作用：对Ames测试菌株TA97、TA98、TA100、TA102， Dip浓度为2000、1000、100、10、1μg/皿时，无论加或不加S9混合物，其回复突变作用均未达到或超过自发回变菌落的2倍，也未见剂量反应关系，结果判为阴性。表明Dip在体外对原核细胞不具有移码型或碱基对置换型基因突变作用。
2 Dip对中国仓鼠肺成纤维 (Chinese hamster
lung,CHL)细胞的致突变作用：Dip浓度为125μg/ml、62.5μg/ml、31.3μg/ml与CHL细胞孵育培养，在培养24h和48h以及加S9混合物时培养24h收获细胞，染色体畸变率均小于4%，判定为阴性结果。表明Dip在体外对哺乳动物培养细胞染色体不具有致突变作用。
3小鼠微核实验：小鼠灌胃给Dip，剂量分别为2.8g/kg、1.4g/kg或0.7g/kg，于灌胃24h后取骨髓，计数各剂量组骨髓嗜多染红细胞微核（Micronucleus of polychromosome erythrocytes，MNPCE）率并与空白对照组比较，均无显著性差异，也无剂量反应关系，试验结果为阴性。表明Dip在小鼠体内不具有致突变作用。
4小鼠骨髓细胞CA实验：小鼠灌胃给 Dip，剂量分别为2.8g/kg、1.4g/kg或0.7g/kg，于灌胃12h、24h、48h后取骨髓进行染色体畸变分析，各剂量组与相应空白对照组相比无显著性差异，也无剂量反应关系，试验结果为阴性。表明Dip在小鼠体内不具有致突变作用。
结论：Dip在体外对原核细胞和哺乳动物真核细胞及在小鼠体内均不具有致突变毒性。
Dipfluzine(Dip), a novel calcium antagonist of diphenylpiperazines, was firstly sythensized by Institute of Pharmaceutic in Hebei Medical Univercity. Previous researches revealed that Dip selectively dilated vertebral artery，basilar artery and coronary artery. And the effects of Dip were more potent than those of flunarizine. In addition, Dip increased brain blood and improved brain ischemia as well as brain hypooxygen. It also inhibited thrombus formation and the platelet aggregation. Thus Dip is well likely to be a promising calcium antagonist to treat brain vascular diseases. At present, there is still no report about special toxicity of Dip. This experiment investigated the mutagenic toxicity of Dip in vivo and in vitro, which provided genetic data for the further study of Dip.
Aim: To investigate the mutagenic toxicity of Dip.
Method: salmonella typhimurium mutagenicity test (Ames test), chromosome aberration (CA) test of cultivated cells of mammalian, micronucleus test in mice and mammalian bone marrow CA test were used.
Result: 1 The revertant effect of Dip on Ames tester strains: TA97, TA98, TA100 and TA102 were used. At the concentrations of Dip 2000, 1000, 100, 10 and 1μg/plate, revertant number of four tester strains，were not up to or over the double number of spontaneous reversion in control group with or without mixture S9. Also，there was no dose-dependence in the number of revertants induced by Dip. So the effect of Dip on the number of revertants was qualified to be negative，indicating that Dip dose not genetic mutagenicity including base-pair substitutions and frameshift in vitro.
2 Mutagenetic effect of Dip on CHL cells: Dip，at the concentration of 125μg/ml, 62.5μg/ml and 31.3μg/ml, was incubated with CHL cells for 24h and 48h with or without mixture S9. CHL cells were respectively harvested at the time 24h and 48h without S9 or 24h with S9 , and the CA was observed . It was found that CA rates in CHL cells incubated with Dip were less than 4%, which was qualified to be negative. The result indicates that Dip dose not induce chromosome aberration in vitro.
3 Micronucleus test in mice: The mice were treated with Dip p.o. at the doses of 2.8g/kg, 1.4g/kg and 0.7g/kg, the bone marrow of mice was taken out at 24h after Dip treatment. MNPCE were counted and MNPCE rates were calculated. MNPCE rates in Dip groups had no significant difference from those in control group, and Dip has no
effect on MNPCE rates in a dose-dependent way. Thus the effect of Dip on MNPCE was qualified to be negative, indicating that Dip has no mutagenicity in vivo.
4 CA test of mouse marrow cells: The mice were treated with Dip p.o. at the doses of 2.8g/kg, 1.4g/kg and 0.7g/kg, the bone marrow of mice was taken out at 12h, 24h and 48h respectively after Dip treatment. There was no significant difference in CA rate between Dip and control groups. Also, there was no dose-dependence in CA rate induced by Dip. Thus the effect of Dip on CA rate was qualified to be negative, indicating that Dip has no mutagenicity in vivo.
Conclusion: Dip has no mutagenic toxicity on prokaryocyte and eukaryocyte in vitro or in vivo.
引文
1新药(西药)临床前研究指导原则汇编（药学 药理学 毒理学）.中华人民共和国卫生部药政局,1992,211~216
2 Wang YL, He RR. Selective vasodilatory effect of dipfluzine on vertebral artery in anesthetized dogs. Acta Pharmacol Sin , 1993; 14(2): 124~127.
3 Wang YL, He RR. Protective effect of dipfluzine on
experimental brain edema in rats. Acta Pharmacol Sin, 1994; 15(3): 201~205.
4 Wang YL, He RR. Effect of dipfluzine on platelet aggregation and thrombus formation. Acta Pharmacol Sin, 1994, 15(5): 443~446.
5 徐淑云，卞如濂，陈修主编.药理实验方法学,人民卫生出版社,1991, 第二版, 209~216
6 张均田.现代药理实验方法,北京医科大学中国协和医科大学联合出版社，1998,第一版:1828~1840
7 Ames BN. Methods for detecting carcinogens and mutagens with salmonlla /mamalianimi-chrosome mutagenicity test ．Mutat Res, 1975, 31: 347
8 Maron DM, Ames BN, Revised methods for Salmonella mutagenicity test . Mutat Res, 1983, 113: 173~215
9 Dayan J, Deguingand S, Truzman C, Study of the mutagenic activity of 6 hepatotoxic pharmaceutical drugs in the Salmonella typhimmurium microsome test, and the HGPRT and Na+/K+ ATPase system in cultured mammalian cells, Mutat Res, 1985, 157: 1~12
10 黄幸纾,陈星若.环境化学物致突变致畸致癌实验方法.第一版,浙江科学技术出版社,1985, 202~217
11 陆竞恒，汪岱迪，史清水.尼索地平的鼠伤寒沙门氏菌诱变性实验.首都医药,2000，7（11）: 36
12 Ferguson LR, Baguley BC, Verapamil as a co-mutagen in the Salmonella/mammlian microsome mutagenicity test,Mutat Res, 1988, 209: 57~ 62
13 Rottgers U. Vntersuchungen an menschlichen lymphozytenuber die beeinflussung der durch das zytostatikum bleomycin induzier ten Chromosomenaberrationen durch Calciumantagonisten. Thesis, 1991
14 黄念君.染色体畸变实验中CHL细胞系/剂量法则的应用遗传.HEREDITAS（Beijing）1984, 6：26~28
15 Nesterova EV, Durnev AD, Seredenin SB. Verapamil contributes to the clastogenic effects of acrylamide, cyclophosphamide, and dioxidine on somatic cells of BALB/C and C57BL/6mice, Mutat Res , 1999, 440: 171~179
2 Wang YL, He RR. Selective vasodilatory effect of dipfluzine on vertebral artery in anesthetized dogs. Acta Pharmacol Sin , 1993; 14(2): 124~127.
3 Wang YL, He RR. Protective effect of dipfluzine on
experimental brain edema in rats. Acta Pharmacol Sin, 1994; 15(3): 201~205.
4 Wang YL, He RR. Effect of dipfluzine on platelet aggregation and thrombus formation. Acta Pharmacol Sin, 1994, 15(5): 443~446.
5 徐淑云，卞如濂，陈修主编.药理实验方法学,人民卫生出版社,1991, 第二版, 209~216
6 张均田.现代药理实验方法,北京医科大学中国协和医科大学联合出版社，1998,第一版:1828~1840
7 Ames BN. Methods for detecting carcinogens and mutagens with salmonlla /mamalianimi-chrosome mutagenicity test ．Mutat Res, 1975, 31: 347
8 Maron DM, Ames BN, Revised methods for Salmonella mutagenicity test . Mutat Res, 1983, 113: 173~215
9 Dayan J, Deguingand S, Truzman C, Study of the mutagenic activity of 6 hepatotoxic pharmaceutical drugs in the Salmonella typhimmurium microsome test, and the HGPRT and Na+/K+ ATPase system in cultured mammalian cells, Mutat Res, 1985, 157: 1~12
10 黄幸纾,陈星若.环境化学物致突变致畸致癌实验方法.第一版,浙江科学技术出版社,1985, 202~217
11 陆竞恒，汪岱迪，史清水.尼索地平的鼠伤寒沙门氏菌诱变性实验.首都医药,2000，7（11）: 36
12 Ferguson LR, Baguley BC, Verapamil as a co-mutagen in the Salmonella/mammlian microsome mutagenicity test,Mutat Res, 1988, 209: 57~ 62
13 Rottgers U. Vntersuchungen an menschlichen lymphozytenuber die beeinflussung der durch das zytostatikum bleomycin induzier ten Chromosomenaberrationen durch Calciumantagonisten. Thesis, 1991
14 黄念君.染色体畸变实验中CHL细胞系/剂量法则的应用遗传.HEREDITAS（Beijing）1984, 6：26~28
15 Nesterova EV, Durnev AD, Seredenin SB. Verapamil contributes to the clastogenic effects of acrylamide, cyclophosphamide, and dioxidine on somatic cells of BALB/C and C57BL/6mice, Mutat Res , 1999, 440: 171~179