体内Pig-a基因突变试验组合评估丁香酚的遗传毒性
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摘要
目的采用大鼠3天染毒与体内Pig-a基因突变试验、彗星试验和微核试验组成的试验组合,评价丁香酚的体内遗传毒性。方法雄性SD大鼠连续3天灌胃给予丁香酚,剂量为241.25 mg/kg·bw(低)、482.5 mg/kg·bw(中)、965.0 mg/kg·bw(高),另设阳性对照组(N-乙基-N-亚硝基脲)和空白对照组。在第0、7、14、29天收集尾部静脉血进行体内Pig-a基因突变试验,检测样本中红细胞及网织红细胞突变率;在末次灌胃6 h后收集尾部静脉血进行彗星试验;在末次灌胃36 h后收集尾部静脉血进行外周血微核试验。结果体内Pig-a基因突变试验中,各组间各时间点外周血突变红细胞和突变网织红细胞率差异均无统计学意义;彗星试验中,各组间彗星尾长和Tail-DNA%差异无统计学意义,但中、高剂量组Olive尾矩分别为6.42μm和8.35μm,与空白对照组比较差异有统计学意义,且呈剂量反应关系;微核试验中,高剂量组外周血网织红细胞微核率为0.45%,与空白对照组比较差异有统计学意义。结论在本实验条件下,丁香酚在较高剂量下可能具有一定的遗传毒性。
Objective To verify the mutagenicity of eugenol by the Pig-a gene mutation assay,micronucleus test and comet test in vivo.Methods The SPF-grade male SD rats were randomly assigned to five groups which included three treatment groups(241.25,482.5,965.0 mg/kg·bw),a control group(olive oil) and a positive control group(N-ethyl-N-nitrosourea).All lab animals were administrated by gavage for continuous 3 days.Tail vein blood was collected on days 0,7,14,29 for vivo Pig-a gene mutation assay.The rate of mutant erythrocyte and reticulocyte was detected.Tail vein blood was collected for comet assay after 6 hours of the last gavage.After the final gavage for 36 hours,the tail vein blood was collected for the micronucleus test.Results In Pig-a gene mutation assay,there was no significant difference among the groups in mutant cell frequency of erythrocyte and reticulocyte at all 4 timepoints.In the comet assay,there was no significant difference among the groups in tail length and tail-DNA%,while there was a statistical difference in the middle(6.42μm) and high(8.35μm) eugenol groups with a good dose-response relationship in Olive tail moment.In the micronucleus test,there was a statistical difference between the solvent control group and high eugenol group in the MNRET%,which was high in the high-dose group.Conclusion Eugenol may have certain vivo genotoxicity at certain doses.
Objective To verify the mutagenicity of eugenol by the Pig-a gene mutation assay,micronucleus test and comet test in vivo.Methods The SPF-grade male SD rats were randomly assigned to five groups which included three treatment groups(241.25,482.5,965.0 mg/kg·bw),a control group(olive oil) and a positive control group(N-ethyl-N-nitrosourea).All lab animals were administrated by gavage for continuous 3 days.Tail vein blood was collected on days 0,7,14,29 for vivo Pig-a gene mutation assay.The rate of mutant erythrocyte and reticulocyte was detected.Tail vein blood was collected for comet assay after 6 hours of the last gavage.After the final gavage for 36 hours,the tail vein blood was collected for the micronucleus test.Results In Pig-a gene mutation assay,there was no significant difference among the groups in mutant cell frequency of erythrocyte and reticulocyte at all 4 timepoints.In the comet assay,there was no significant difference among the groups in tail length and tail-DNA%,while there was a statistical difference in the middle(6.42μm) and high(8.35μm) eugenol groups with a good dose-response relationship in Olive tail moment.In the micronucleus test,there was a statistical difference between the solvent control group and high eugenol group in the MNRET%,which was high in the high-dose group.Conclusion Eugenol may have certain vivo genotoxicity at certain doses.
引文
[1] 孔晓军,刘希望,李剑勇,等.丁香酚的药理学作用研究进展[J].湖北农业科学,2013,52(3):508-511.
[2] 俞懿强,邹德荣.丁香酚的药理作用及在口腔医学应用的研究进展[J].临床口腔医学杂志,2009,25(3):187-189.
[3] Hikiba H,Watanabe E,Barrett JC,et al.Ability of fourteen chemical agents used in dental practice to induce chromosome aberrations in Syrian hamster embryo cells[J].Journal of Pharmacological Sciences,2005,97(1):146-152.
[4] Ishidate MJ,Miura KF,Sofuni T.Chromosome aberration assays in genetic toxicology testing in vitro[J].Mutation Research,1998,404(1/2):167-172.
[5] Mcnamee JP,Bellier PV.Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds;ampicillin trihydrate,1,2-dimethylhydrazine dihydrochloride,and N-nitrosodimethylamine[J].Mutation Research-Genetic Toxicology and Environmental Mutagenesis,2015,786(SI):158-164.
[6] The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1)[EB/OL].[2019-04-22].http://ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S2_R1/Step4/S2R1_Step4.pdf.
[7] 马思佳,霍娇,陈锦瑶,等.大鼠体内Pig-a基因突变试验的优化及ENU时-效关系和量-效关系评估[J].四川大学学报(医学版),2017,48(1):127-131,139.
[8] 陈奇言,马思佳,陈锦瑶,等.体内Pig-a基因突变试验检测亚硒酸钠的致突变作用[J].现代预防医学,2017,44(15):2797-2801.
[9] Schuler M,Gollapudi BB,Thybaud V,et al.Need and potential value of the Pig-ain vivo mutation assay-a HESI perspective[J].Environmental and Molecular Mutagenesis,2011,52(9):685-689.
[10] Maura A,Pino A,Ricci R,et al.Negative evidence in vivo of DNA-damaging,mutagenic and chromosomal effects of eugenol[J].Mutation Research Letters,1989,227(2):125-129.
[11] Woolverton CJ,Fotos PG,Mokas M,et al.Evaluation of eugenol for mutagenicity by the mouse micronucleus test[J].Journal of Oral Pathology,1986,15(8):450-453.
[12] Sancar A,Lindsey-Boltz LA,Unsal-Ka?maz K,et al.Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints[J].Annual Review of Biochemistry,2004,73(73):39-85.
[2] 俞懿强,邹德荣.丁香酚的药理作用及在口腔医学应用的研究进展[J].临床口腔医学杂志,2009,25(3):187-189.
[3] Hikiba H,Watanabe E,Barrett JC,et al.Ability of fourteen chemical agents used in dental practice to induce chromosome aberrations in Syrian hamster embryo cells[J].Journal of Pharmacological Sciences,2005,97(1):146-152.
[4] Ishidate MJ,Miura KF,Sofuni T.Chromosome aberration assays in genetic toxicology testing in vitro[J].Mutation Research,1998,404(1/2):167-172.
[5] Mcnamee JP,Bellier PV.Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds;ampicillin trihydrate,1,2-dimethylhydrazine dihydrochloride,and N-nitrosodimethylamine[J].Mutation Research-Genetic Toxicology and Environmental Mutagenesis,2015,786(SI):158-164.
[6] The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1)[EB/OL].[2019-04-22].http://ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S2_R1/Step4/S2R1_Step4.pdf.
[7] 马思佳,霍娇,陈锦瑶,等.大鼠体内Pig-a基因突变试验的优化及ENU时-效关系和量-效关系评估[J].四川大学学报(医学版),2017,48(1):127-131,139.
[8] 陈奇言,马思佳,陈锦瑶,等.体内Pig-a基因突变试验检测亚硒酸钠的致突变作用[J].现代预防医学,2017,44(15):2797-2801.
[9] Schuler M,Gollapudi BB,Thybaud V,et al.Need and potential value of the Pig-ain vivo mutation assay-a HESI perspective[J].Environmental and Molecular Mutagenesis,2011,52(9):685-689.
[10] Maura A,Pino A,Ricci R,et al.Negative evidence in vivo of DNA-damaging,mutagenic and chromosomal effects of eugenol[J].Mutation Research Letters,1989,227(2):125-129.
[11] Woolverton CJ,Fotos PG,Mokas M,et al.Evaluation of eugenol for mutagenicity by the mouse micronucleus test[J].Journal of Oral Pathology,1986,15(8):450-453.
[12] Sancar A,Lindsey-Boltz LA,Unsal-Ka?maz K,et al.Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints[J].Annual Review of Biochemistry,2004,73(73):39-85.
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