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目的检测连翘酯苷冻干粉的遗传毒性,为临床前安全性评价提供依据。方法分别应用Ames试验、小鼠骨髓微核试验、体外培养CHO细胞染色体畸变试验、CHO细胞和正常人肝细胞Chang liver两个细胞株单细胞凝胶电泳法。结果Ames试验选用组氨酸营养缺陷型鼠伤寒沙门氏菌(S.typhimurium)TA97、TA98、TA100、TA102及TA1535为指示菌株,加和不加代谢活化系统(S9)时对鼠伤寒沙门氏菌均无致突变性。小鼠骨髓微核试验采用ICR小鼠,尾静脉注射给药,剂量分别为0.2、0.4和0.8g/kg,结果显示:雌性小鼠微核诱发率分别为1.50、2.67和6.75%,雄性小鼠微核诱发率分别为1.87、5.79和6.57%;其中雌性小鼠在0.8g/kg剂量下、雄性小鼠在0.4、0.8g/kg剂量下的微核诱发率与阴性对照组比较差异有统计学意义(P<0.05)。表明受试物连翘酯苷冻干粉在0.4、0.8g/kg剂量下对ICR小鼠有诱发骨髓嗜多染红细胞微核的效应。CHO细胞染色体畸变试验结果显示:受试物连翘酯苷冻干粉在受试剂量下作用24h,-S9的染色体畸变率分别为3%、7%和9%,与阴性对照组比较164.0和328.0μg/ml2个剂量组均显示差异有统计学意义(P<0.05);-S9作用48h的染色体畸变率分别为3%、5%和8%,与阴性对照组比较164.0和328.0μg/ml剂量组差异有统计学意义(P<0.05);+S9的染色体畸变率分别为1%、1%和6%,各剂量组与阴性对照组比较差异均无统计学意义(P>0.05)。重复试验结果一致。以上结果说明连翘酯苷冻干粉在无代谢活化系统时,作用24h328μg/ml以上剂量对CHO细胞有致染色体畸变作用,作用48h164μg/ml以上剂量对CHO细胞具有致染色体畸变作用,在有S9代谢活化系统时,在受试剂量下对CHO细胞均无致染色体畸变作用。细胞彗星试验结果显示,在各试剂量的拖尾率和尾长与溶剂对照组相比较差异无统计学上意义,表明酯苷冻干粉在受试剂量下无损伤CHO细胞和人肝细胞Chang liverDNA的能力。结论连翘酯苷在体外培养CHO细胞染色体畸变试验和微核试验中,在较高剂量时呈阳性,无损伤CHO细胞和人肝细胞Chang liver DNA的能力。连翘酯苷冻干粉有一定的遗传毒性,分析连翘酯苷所致染色体畸变和微核的机制,可能不是通过损伤DNA机制诱导的,而是通过抑制DNA合成的、抑制拓扑异构酶、细胞毒性等非DNA损伤所致。
Objective To detect the genotoxicity of forsythiaside lyophilized powder and provide evidence for preclinical safety evaluation. Methods Ames test, mouse bone marrow micronucleus test, chromosome aberration test of CHO cells in vitro, single cell gel electrophoresis of CHO cells and normal human hepatocytes Chang liver were used. Results The Ames test used histidine auxotrophs S.typhimurium TA97, TA98, TA100, TA102 and TA1535 as indicator strains, and there was no mutagenicity to Salmonella typhimurium when the additive and non-metabolic activation system (S9) was used. Sex. In the mouse bone marrow micronucleus test, ICR mice were administered by tail vein injection at doses of 0.2, 0.4, and 0.8 g/kg. The results showed that the micronucleus induction rate in female mice was 1.50, 2.67, and 6.75%, respectively. The micronucleus induction rate was 1.87, 5.79, and 6.57%, respectively. The difference in the frequency of micronucleus induction in female mice at doses of 0.8 and 0.4 g/kg and the doses of 0.4 and 0.8 g/kg in male mice was significantly different from that in the negative control group. Statistical significance (P<0.05). The results showed that the test compound Forsythiaside lyophilized powder at 0.4,0.8g/kg dose of ICR mice induced bone marrow polychromatic erythrocyte micronucleus effect. Chromosome aberration test of CHO cells showed that the forsythiasid lyophilized powder was treated for 24 h under test dose, the chromosome aberration rate of -S9 was 3%, 7% and 9%, respectively, compared with negative control group 164.0 and 328.0μg/ml 2 dose groups showed a significant difference (P <0.05); -S9 effect 48h chromosomal aberration rate was 3%, 5% and 8%, compared with negative control group 164.0 and 328.0μg/ml The difference between the dose group was statistically significant (P<0.05); the chromosomal aberration rates of +S9 were 1%, 1%, and 6%, respectively, and there was no significant difference between each dose group and the negative control group (P>0.05). Repeated test results are consistent. The above results showed that the forsythiaside lyophilized powder in the absence of metabolic activation system, the role of 24h328μg/ml more than the dose of CHO cell chromosomal aberrations, the role of more than 48h164μg/ml CHO cells have a chromosomal aberration effect, in the S9 metabolism When the system was activated, there was no chromosomal aberration effect on CHO cells at the dose tested. The result of cell comet assay showed that there was no statistically significant difference in the tailing rate and tail length of each reagent compared with the solvent control group, indicating that the esterosterone lyophilized powder had no damage to CHO cells and human hepatocytes at the doses tested. The power of liverDNA. Conclusion Forsythiaside was positive in high doses in the chromosomal aberration test and micronucleus test of CHO cells cultured in vitro, without the ability to damage CHO cells and human liver cells Chang liver DNA. Forsythiaside lyophilized powder has a certain degree of genotoxicity. Analysis of the mechanism of chromosome aberration and micronucleus caused by forsythiaside may not be induced by damage to the DNA mechanism, but by inhibition of DNA synthesis, inhibition of topoisomerase , cytotoxicity and other non-DNA damage.