CORESTA Congress, Quebec, 2014, Smoke Science/Product Technology Groups, STPOST 18

Development and validation of a high content screening in vitro micronucleus assay for the assessment of total particulate matter in cigarette smoke

GAO Qian(1); ZENG Wanli(1); ZHU Zhouhai(1); GUAN Ying(1); MI Qili(1); MIAO Mingming(1); LI Xuemei(1); YAO Jianhua(1); LI Ming(2)
(1) Yunnan Academy of Tobacco Science of CNTC, Kunming, P.R. China; (2) China Tobacco Society, Beijing, P.R. China

High Content Screening (HCS) is imaging based multi-parametric approaches to cell analysis at the single-cell level, which was originally developed as a complementary technology to traditional biochemical high-throughput screening in drug discovery. It has been applied in a far broader area of the life science as an unbiased method of imaging multiple cellular samples. To investigate the possibility of HCS for in vitro micronucleus assay of Total Particulate Matter (TPM) and improvements in the assay efficiency, Chinese Hamster Ovary (CHO) cells were treated with TPM produced from ten types of cigarettes at five concentrations (25-200 µg/ml). The two following methods were used to score the micronucleus (MN) frequency: (a) HCS with DAPI and FITC dyes, which differentially stained micronuclei and cytoplasm to enhance assay reliability; (b) Visual microcopy with Giemsa dye. The test results obtained using the two methods were compared using correlation analysis. The result showed that HCS method was effective for MN identification, the MN frequencies that were measured in the same samples by HCS and visual microscopy were highly correlated (R=0.950), and there were no significant differences (p=0.570). In conclusion, HCS can be used to evaluate the MN frequency induced by TPM.