title: Heme oxygenase-1 gene expression in human alveolar epithelial cells (A549) following exposure to whole cigarette smoke on a direct in vitro exposure system.
abstract: Many in vitro studies have employed cigarette smoke condensates or soluble smoke components to investigate the biological effects of cigarette smoke. However, neither of these methods evaluates the biological effects of fresh whole cigarette smoke. It is most desirable to conduct in vitro biological studies under conditions which accommodate the dynamic physicochemical character of fresh cigarette smoke. Previously we reported the development of a whole smoke exposure system to assess the biological effects of mainstream cigarette smoke. The exposure system design was based on a combination of the sedimentation procedure and the CULTEX cultivation technique, which includes a systemized air/liquid interface methodology and exposes the cells to fresh smoke at every puff. The aim of this study was to adopt the other biological endpoint to our whole smoke exposure system. We focused on heme oxygenase (HO)-1 mRNA gene expression, an enzyme which has recently been shown to be highly responsible for oxidative stress. In the present study, a dose-response relationship between the HO-1 mRNA expression based on the reverse transcription real-time PCR method and total exposure to cigarette smoke was observed. When a Cambridge filter pad was placed between the cigarette and exposure module, to ensure the cells were only exposed to the gas/vapor phase, the latter, as well as the whole smoke, induced HO-1 mRNA dose dependently. For the next step, acetate plain and charcoal filters with the same pressure drop were prepared to assess the potential ability of charcoal filters with regard to the vapor phase performance. The results revealed reduced HO-1 mRNA gene expression when a charcoal filter was used. Direct whole smoke exposure is a significant approach and may reflect the conditions of exposure essentially resulting from direct contact between cells and a dynamic mixture of gaseous and particulate constituents. We were able to adopt a gene expression assay for oxidative stress to the whole smoke exposure system, following the adaptation of cytotoxicity assays. This system, which includes several advantages involving the post-exposure washing of cells, by adding the exchanging medium and assuring the exposure of the particulate phase through the sedimentation method, may have potential for further investigations into the molecular basis of smoking-related lung disease.
authors.affiliation: Tobacco Science Research Center, Japan Tobacco Inc., 6-2, Umegaoka, Aoba Ward, Yokohama, Kanagawa 227-8512, Japan. yasuo.fukano@ims.jti.co.jp
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Fukano
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yoshimura
authors.forename: Takemi
authors.initials: T
authors.lastname: Yoshida
mesh_terms: D000393:Air Pollutants / Q000009:adverse effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects*; D051547:Heme Oxygenase-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D011650:Pulmonary Alveoli / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012906:Smoke / Q000009:adverse effects*; D014026:Nicotiana
chemical_list: D000393:Air Pollutants; D012333:RNA, Messenger; D012906:Smoke; D051547:Heme Oxygenase-1