| The micronucleus assay is a kind of genotoxicity evaluation method by observing the formation of micronucleus in cells,which can detect genetic damage induced by various physical and chemical factors more economically, conveniently and accurately. It is playing important role in assessing genotoxicity of compounds. The traditional micronuclues assay by microscope has demerits of time-consuming and tedious for operator, and the results is susceptible to subjective factors. It cann’t meet the needs of quickly detecting for a large amount of compounds. While the automatic methods of detecting micronucleus on basis of instrument aims to assay genotoxicity of compounds rapidly and high-throughput screen.In this paper,the hamster ovary cells (CHO cells) which were general cell line for domestic and overseas toxicology evaluation were chosed as material, and research on automated micronucleus detecting techniques was conducted using flow cytomety (FCM) a-nd laser scanning cytomety(LSC) in purpose of detectiong genotoxicity of compounds rapidly and accurately.In flow cytometry method, Micronucleus in CHO cells induced by cyclophosphamide (CP) and Colchicine which caused breakage of chromosome and formation of aneuploidy respectively were detected by FCM conbined with cytokinesis block micronucleus assay using In Vitro MicroFlow kit. Furthermore,this results of micronucleus frequencies were compared with the results from method of manual scoring of slides. According to Method Comparison and Bias Estimation recommended by National Committee for Clinical Laboratory Standards (NCCLS)document EP9-A2,Outlier Tests on Within-Method Duplicates, Test for Adequate Range of X,Linear Regression were analyzed.The results showed that micronucleus frequencies scored by FCM have a good correlation with that by the traditional microscopy. an automatic detection of micronucleus in CHO cells by flow cytometry was established. In order to futher understand the reliability and applicability of this method,4kinds of food additives,4kinds of drugs,3kinds of pesticides,4kinds of hair colorings,4kinds of polluted water, heavy metal ion and2kinds of the Total Particulate Matter(TPM) from2Cigarette samples were respectively treated with CHO and detected micronucleus in cells by FCM, comparing with method of manual scoring of slides.The results showed that there was no outliers and the two methods had a good correlation.So it is suitable for evaluating genetic damage induced by food additives, drugs, pesticides, hair colorings, polluted water, and TPM from cigrates.The test result is reliable and accurate.In laser scanning cytometry(LSC) method, combined with cytokinesis block micronucleus assay,micronucleus of CHO cells induced by cyclophosphamide (CP) were detected by LSC using two kinds of fluorescent dyes and this results were compared with that scoring by a microscope.The results showed that there was no outliers and the two methods had a good correlation. An assessment method of in vitro micronucleus scored by LSC was established. Furthermore, this method was applied in detection of13kinds of compounds, comparing with method of manual scoring of slides.By comparsion of micronucleus frequencies scored by LSC and Microscope, The datas were analyzed according to analysis method of document EP9-A2,It turned out that there was no Outliers, and the two methods have a good correlation.The results showed that the method by LSC is reliable and, is an high throughput screen for detecting micronucleus induced by different kinds of compounds.With strong advantage,The flow cytometric automatic method and laser scanning cytometric automatic method which established in this research could detect in vitro micronucleus in CHO cells rapidly and accurately, the methods was simple and applicability was good. This study provides reference data for research on automated methods of micronucleus assays and provide the fast method for screening of mutants,food toxicology assay and monitoring marine environment pollution. |
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