基于流式细胞术的大鼠外周血网织红细胞微核试验方法的建立
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摘要
目的建立基于流式细胞术的大鼠外周血网织红细胞微核试验,检测阳性诱变剂甲基磺酸乙酯(ethyl methanesulphonate,EMS)和环磷酰胺(cyclophosphamide,CP)在所建立的试验方法中的表现。方法使用CD71-异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记大鼠外周血网织红细胞,核酸染料DRAQ5标记DNA,建立流式细胞术检测方案。采用该方案测定EMS和CP的遗传毒性时,将40只雄性SD大鼠随机分为8组,EMS染毒剂量为0、50、100、200mg/kg.bw,CP为0、5、10、15 mg/kg.bw,间隔24 h2次经口染毒。在给药前1 d和给药后24、48、72、96、120 h取尾静脉血,使用流式细胞术测定外周血网织红细胞和成熟红细胞微核率。结果本试验方法所得历史阴性对照成熟红细胞和网织红细胞背景微核率均数分别为0.04‰和0.42‰,标准差分别为0.02‰和0.18‰。在所建立的试验系统中观察到EMS及CP可诱导外周血网织红细胞微核显著升高。结论本实验室建立的基于CD71-FITC和DRAQ5双染的外周血流式微核技术操作简便快捷,背景微核率较低,检测结果稳定,在化学物的遗传毒性评价方面具有较大的推广价值和良好的应用前景。
Objective The aim of this work was to develop a study protocol for rat micronucleus test by flow cytometry, which was conducted on peripheral blood reticulocytes treated with known positive chemicals, ethyl methanesulphonate(EMS) and cyclophosphamide(CP). Methods CD71-fluorescein isothiocyanate(FITC) and DRAQ5 were adopted for the fluorescent staining of proteins and DNA, respectively, so as to detect micronuclei in rat peripheral blood erythrocytes. In this experiment, 40 male SD rats were randomized divided to 8 groups, i.e. 3 treatment groups of EMS(0, 50, 100, 200mg/kg·bw), 3 treatment groups of CP(0, 5, 10, 15 mg/kg·bw) and 2 solvent control groups(distilled water). Tail vein blood specimens for micronucleus test were collected on-1 d(one day before gavage) and 24, 48, 72, 96, and 120 h. The numbers of micronucleus, red blood cells and reticulocytes were acquired by flow cytometer. Results For negative historical control data(total: 125), the mean percentages of micronucleated red blood cells and micronucleated reticulocytes were0.04‰ and 0.42‰, respectively, and the standard deviations were 0.04‰ and 0.42‰ respectively. The flow cytometric method consistently demonstrated highly sensitive responses for MN production at all concentrations and all time points of EMS and CP. Conclusion These data suggested that the method reported here, combined with easier operation, lower background and better precision, could be an efficient system in chemical safety assessment of genotoxicity.
Objective The aim of this work was to develop a study protocol for rat micronucleus test by flow cytometry, which was conducted on peripheral blood reticulocytes treated with known positive chemicals, ethyl methanesulphonate(EMS) and cyclophosphamide(CP). Methods CD71-fluorescein isothiocyanate(FITC) and DRAQ5 were adopted for the fluorescent staining of proteins and DNA, respectively, so as to detect micronuclei in rat peripheral blood erythrocytes. In this experiment, 40 male SD rats were randomized divided to 8 groups, i.e. 3 treatment groups of EMS(0, 50, 100, 200mg/kg·bw), 3 treatment groups of CP(0, 5, 10, 15 mg/kg·bw) and 2 solvent control groups(distilled water). Tail vein blood specimens for micronucleus test were collected on-1 d(one day before gavage) and 24, 48, 72, 96, and 120 h. The numbers of micronucleus, red blood cells and reticulocytes were acquired by flow cytometer. Results For negative historical control data(total: 125), the mean percentages of micronucleated red blood cells and micronucleated reticulocytes were0.04‰ and 0.42‰, respectively, and the standard deviations were 0.04‰ and 0.42‰ respectively. The flow cytometric method consistently demonstrated highly sensitive responses for MN production at all concentrations and all time points of EMS and CP. Conclusion These data suggested that the method reported here, combined with easier operation, lower background and better precision, could be an efficient system in chemical safety assessment of genotoxicity.
引文
[1] Hamada S, Sutou S, Morita T, et al.Evaluation of the rodent micronucleus assay by a 28-day treatment protocol:Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test(CSGMT)/Environmental Mutagen Society of Japan(JEMS)-Mammalian Mutagenicity Study Group(MMS)[J]. Environmental and Molecular Mutagenesis, 2001, 37(2):93-110.
[2] Organization for Economic Cooperation and Development(OECD).OECD Guidelines for the testing of chemicals:mammalian erythrocyte micronucleus test, Test No. 474[M/OL]. Paris:OECD, 2016:474[2018-12-05]. http://www.oecd-ilibrary.org/environment/oecdguidelines-for-the-testing-of-chemicals-section-4-health-effects_20745788.
[3] Dertinger SD, Torous DK, Hayashi MA. Flow cytometric scoring of micronucleated erythrocytes:an efficient platform for assessing in vivo cytogenetic damage[J]. Mutagenesis, 2011, 26(1, SI):139-145.
[4] Kirkland D, Kasper P, Martus HJ, et al. Updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests[J]. Mutation Research. Genetic Toxicology and Environmental Mutagenesis, 2016,795:7-30.
[5] GrawéJ, Zetterberg G, Amnéus H. Flow-cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood[J]. Cytometry, 1992, 13(7):750-758.
[6] Criswell KA, Krishna G, Zielinski D, et al. Validation of a flow cytometric acridine orange micronuclei methodology in rats[J]. Mutation Research, 2003, 528(1-2):1-18.
[7] Dertinger SD, Camphausen K, Macgregor JT, et al. Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood[J]. Environmental and Molecular Mutagenesis, 2004, 44(5):427-435.
[8] Wakata A, Miyamae Y, Sato SI, et al. Evaluation of the rat micronucleus test with bone marrow and peripheral blood:summary of the9th collaborative study by CSGMT/JEMS. MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenicity Study Group[J]. Environmental and Molecular Mutagenesis, 1998:32(1):84-100.
[9] Cammerer Z, Elhajouji A, Suter W. In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals[J]. Mutation Research, 2007, 626(1-2):26-33.
[10] Macgregor JT, Wehr CM, Henika PR, et al. The in vivo erythrocyte micronucleus test:measurement at steady state increases assay efficiency and permits integration with toxicity studies[J]. Fundamental and Applied Toxicology:Official Journal of the Society of Toxicology,1990, 14(3):513-522.
[2] Organization for Economic Cooperation and Development(OECD).OECD Guidelines for the testing of chemicals:mammalian erythrocyte micronucleus test, Test No. 474[M/OL]. Paris:OECD, 2016:474[2018-12-05]. http://www.oecd-ilibrary.org/environment/oecdguidelines-for-the-testing-of-chemicals-section-4-health-effects_20745788.
[3] Dertinger SD, Torous DK, Hayashi MA. Flow cytometric scoring of micronucleated erythrocytes:an efficient platform for assessing in vivo cytogenetic damage[J]. Mutagenesis, 2011, 26(1, SI):139-145.
[4] Kirkland D, Kasper P, Martus HJ, et al. Updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests[J]. Mutation Research. Genetic Toxicology and Environmental Mutagenesis, 2016,795:7-30.
[5] GrawéJ, Zetterberg G, Amnéus H. Flow-cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood[J]. Cytometry, 1992, 13(7):750-758.
[6] Criswell KA, Krishna G, Zielinski D, et al. Validation of a flow cytometric acridine orange micronuclei methodology in rats[J]. Mutation Research, 2003, 528(1-2):1-18.
[7] Dertinger SD, Camphausen K, Macgregor JT, et al. Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood[J]. Environmental and Molecular Mutagenesis, 2004, 44(5):427-435.
[8] Wakata A, Miyamae Y, Sato SI, et al. Evaluation of the rat micronucleus test with bone marrow and peripheral blood:summary of the9th collaborative study by CSGMT/JEMS. MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenicity Study Group[J]. Environmental and Molecular Mutagenesis, 1998:32(1):84-100.
[9] Cammerer Z, Elhajouji A, Suter W. In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals[J]. Mutation Research, 2007, 626(1-2):26-33.
[10] Macgregor JT, Wehr CM, Henika PR, et al. The in vivo erythrocyte micronucleus test:measurement at steady state increases assay efficiency and permits integration with toxicity studies[J]. Fundamental and Applied Toxicology:Official Journal of the Society of Toxicology,1990, 14(3):513-522.