| Objective: The aim of study was to systematically study the genetic toxicity of triptolide by using the method of different genetic endpoints,which revealed the characteristics of its genetic toxicity and provided scientific basis for clinical rational use of triptolide.Methods: 1.Salmonella typhimurium reverse mutation test(Ames test)Histidine-deficient Salmonella typhimurium TA97,TA98,TA100,TA102 and TA1535 were used in this test.The test had eight groups: blank control,solvent control,positive control,1.6,8,40,200,and 1000 ?g per dish.Each dose group performed two parts parallel experiments with and without metabolic activation system(+/-s9).Each composed 3 parallel plates.After 48 h ~ 72 h inversion incubation in 37 oC,observation with the naked eye or low magnification microscope upbringing was used to determine whether there was bacteriostatic or bactericidal,and then count colonies.The test was repeated once.2.Chromosomal aberration test of CHO cell in vitroAccording to the half maximal inhibitory concentration(IC50)was 0.04 ?g/mL.The test had five groups: blank control,positive control,low dosage(0.01 ?g/mL)experiment group,moderate dosage(0.02 ?g/m L)experiment group,and high dosage(0.04 ?g/m L)experiment group.Each group set up two parallel samples simultaneously.CHO cells were treated with drugs for 4 hours in short term treatment group(with or without s9),while 24 hours in the length of treatment group(without s9).Colchicine was administered 24 hours after the start of the test.The cells,which stopped in the middle phase of mitosis,were collected.After low permeability,fixed,smear and dyeing,the chromosome number,the change of the structure,and the distortion rate were observed.3.ICR mouse bone marrow micronucleus testSexual maturity ICR mice were used in this test.According to the method of randomized,50 mice(half male and half female)were divided into five groups: the vehicle control group(10 m L/kg solvent volume),the positive control group(40 mg/kg cyclophosphamide),the low dose group(180 ?g/kg),the middle dose group(360 ?g/kg),and the high dose group(720 ?g/kg).The Mice leg femur marrow smears were made after 24 h and 48 h.After staining polychromatic erythrocytes(PCE),counted the number of PCE containing micronucleus,and then calculated micronucleus rate.Results: 1.Salmonella typhimurium reply mutation assay(Ames test)results showed that no pollution was found in each dose group and control group.Visible background lawn can be counted.Spontaneous revertant colonies of five strains were within the range of historical controls.A significant increase revertant colonies number in the positive reference strains was found compared to that in the control group.No inhibitory phenomenon was observed under the highest dose of 1000 ?g/dish.The back mutations induced by colony count in TA97,TA98,TA100,TA102 and TA1535 groups with or without metabolic activation system(+/-s9)were similar to that in the control group.No obvious dose-effect relationship was observed.2.Chromosomal aberration test of CHO cell in vitro results showed that chromosome aberrations of solvent control group in the +s9/4h and-s9/24 h were 1.0 % and 0.5 %,respectively,less than 5 %;chromosome aberrations of positive control group in the +s9/4h and-s9/24 h treatment were 14 % and 16 %,respectively,more than 10 %.The chromosome aberration rates of 0.01,0.02,and 0.04 ?g/mL under the condition of the-s9/24 h were 0,2.0 % and 0.5 %,respectively.The chromosome aberration rates of 0.01,0.02,and 0.04 ?g/mL under the condition of the-s9/4h were 0.5 %,0 and 1.0 %,respectively.The chromosome aberration rates of 0.01,0.02,and 0.04 ?g/m L under the condition of the +s9/24 h were 1.5 %,1.0 % and 0.5 %,respectively.Therefore,chromosome aberration rate in each dose group was less than 5.0 %.3.ICR mice bone marrow micronucleus test results showed that the inhibitory effect in mice bone marrow was not observed in each experimental dose.The bone marrow eosinophils polychromatic erythrocytes micronucleus rate of male and female mice in the solvent control group were 1.89 ‰ and 1.95 ‰,respectively,which in positive control group were 16.23 ‰ and 17.30 ‰,respectively.It had statistical significance between solvent control group and positive group(P<0.05).The bone marrow eosinophils polychromatic erythrocytes micronucleus rate of male and female mice in 180 ?g/kg dose group at 24 h were 2.15 ‰ and 2.78 ‰,respectively;which were 2.79 ‰ and 2.89 ‰ in 360 ?g/kg dose group(P>0.05 compared with solvent control group).The bone marrow eosinophils polychromatic erythrocytes micronucleus rate of male and female mice in 720 ?g/kg dose group at 24 h were 4.42 ‰ and 4.95 ‰,respectively;which were 3.80 ‰ and 3.65 ‰ at 48h(P<0.05 compared with solvent control group,and showed a dose-dependent manner).Conclusions: Triptolide did not show genetic toxicity based on the Ames test and chromosomal aberration.However,adversely effect in micronucleus formation was observed at the dosage of 720 ?g/kg Triptolide.The results suggest that triptolide may have potential genetic toxicity to human body.Attentions should be paid on the dose and the course of medication when using drugs containing triptolide in clinical,which prevent some adverse reactions including genetic toxicity. |
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