title: Automation of the in vitro micronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas-vapor phase of cigarette smoke.
abstract: Total particulate matter (TPM) and the gas-vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24 h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.
authors.affiliation: a Philip Morris Products S.A., Philip Morris International R&D , Neuchâtel , Switzerland .
authors.forename: Ewald
authors.initials: E
authors.lastname: Roemer
authors.forename: Volker
authors.initials: V
authors.lastname: Zenzen
authors.forename: Lynda L
authors.initials: LL
authors.lastname: Conroy
authors.forename: Kathrin
authors.initials: K
authors.lastname: Luedemann
authors.forename: Ruth
authors.initials: R
authors.lastname: Dempsey
authors.forename: Christian
authors.initials: C
authors.lastname: Schunck
authors.forename: Edgar Trelles
authors.initials: ET
authors.lastname: Sticken
mesh_terms: D000393:Air Pollutants / Q000633:toxicity; D000818:Animals; D001331:Automation; D002470:Cell Survival / Q000187:drug effects; D002869:Chromosome Aberrations / Q000187:drug effects*; D006224:Cricetinae; D003412:Cricetulus; D048749:Cytokinesis; D005347:Fibroblasts / Q000187:drug effects; D005740:Gases / Q000633:toxicity; D008677:Metaphase / Q000187:drug effects; D015162:Micronucleus Tests / Q000191:economics / Q000295:instrumentation* / Q000379:methods*; D009152:Mutagenicity Tests / Q000191:economics / Q000295:instrumentation* / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D052638:Particulate Matter / Q000633:toxicity; D012906:Smoke / Q000009:adverse effects*; D014026:Nicotiana
chemical_list: D000393:Air Pollutants; D005740:Gases; D009153:Mutagens; D052638:Particulate Matter; D012906:Smoke
keywords: Automation; chromosome aberration assay; cigarette smoke; cytokinesis-block proliferation index; cytotoxicity; genotoxicity; micronucleus assay