title: In vitro micronucleus assay for the analysis of total particulate matter in cigarette smoke: comparison of flow cytometry and laser scanning cytometry with microscopy.
abstract: The possible genotoxicity of the total particulate matter (TPM) in cigarette smoke has typically been evaluated using the in vitro micronucleus assay. In recent years, automated scoring techniques have been developed to replace the manual counting process in this assay. However, these automated scoring techniques have not been applied in routine genotoxicity assays for the analysis of TPM to improve the assay efficiency. Chinese hamster ovary (CHO) cells were treated with TPM produced from 14 types of cigarettes at five concentrations (25-200μg/ml) without exogenous metabolic activation. The three following methods were used to score the micronucleus (MN) frequency: (a) flow cytometry with SYTOX and EMA dyes, which differentially stain micronuclei and apoptotic/necrotic chromatin to enhance assay reliability; (b) laser scanning cytometry with FITC and PI dyes, which is a system that combines the analytical capabilities of flow and image cytometry; and (c) visual microcopy with Giemsa dye. The test results obtained using the three methods were compared using correlation analysis. The key findings for this set of compounds include the following: (a) both flow cytometry- and laser scanning cytometry-based methods were effective for MN identification, (b) the three scoring methods could detect dose-dependent micronucleus formation for the 14 types of TPM, and (c) the MN frequencies that were measured in the same samples by flow cytometry, laser scanning cytometry, and visual microscopy were highly correlated, and there were no significant differences (p>0.05). In conclusion, both flow cytometry and laser scanning cytometry can be used to evaluate the MN frequency induced by TPM without exogenous metabolic activation. The simpler and faster processing and the high correlation of the results make these two automatic methods appropriate tools for use in in vitro micronucleus assays for the analysis of TPM using CHO cells.
authors.affiliation: Faculty of Environmental Science and Engineering, Kunming University of Science & Technology, Kunming 650224, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Yao
authors.forename: Qian
authors.initials: Q
authors.lastname: Gao
authors.forename: Qili
authors.initials: Q
authors.lastname: Mi
authors.forename: Xuemei
authors.initials: X
authors.lastname: Li
authors.forename: Mingming
authors.initials: M
authors.lastname: Miao
authors.forename: Peng
authors.initials: P
authors.lastname: Cheng
authors.forename: Ying
authors.initials: Y
authors.lastname: Luo
mesh_terms: D000818:Animals; D001386:Azides; D001399:Azure Stains; D016466:CHO Cells / Q000187:drug effects; D004396:Coloring Agents; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D066298:In Vitro Techniques; D015162:Micronucleus Tests / Q000379:methods*; D008853:Microscopy / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods*; D009930:Organic Chemicals; D052638:Particulate Matter / Q000032:analysis* / Q000633:toxicity; D012906:Smoke / Q000009:adverse effects / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods; D062789:Tobacco Products
chemical_list: D001386:Azides; D001399:Azure Stains; D004396:Coloring Agents; D009930:Organic Chemicals; D052638:Particulate Matter; C402795:SYTOX Green; D012906:Smoke; C014535:8-azidoethidium
keywords: CHO; Chinese hamster ovary; Cigarette smoke; EMA; Ethidium monoazide; FCM; FITC; Flow cytometry; Fluorescein 5(6)-isothiocyanate; Green nucleic acid stain; LSC; Laser scanning cytometry; MN; Micronucleus; Micronucleus frequencies; OECD; Organisation for Economic Co-operation and Development; PI; Propidium iodide; SYTOX; TPM; Total particulate matter; VM; Visual microscopy