Detection of cytotoxicity in the water-insoluble fraction of cigarette smoke gas vapor phase using a whole smoke exposure system
When biological evaluations of cigarette smoke are conducted, the particulate phase (PP) and gas vapor phase (GVP) of mainstream smoke are collected separately and exposed to cultured cells. GVP is generally collected in phosphate buffer saline (PBS) by bubbling method. However, using this methodology for collecting GVP means that water-insoluble fraction of the GVP will not be collected. Therefore, the toxic potential of the water-insoluble fraction of the GVP has hardly been investigated. The objective of this study is to research toxic effects of water-insoluble fraction of GVP using a whole smoke exposure system such as CULTEX→ system that enables cultured cells to be exposed to PP and GVP of native cigarette smoke including water-insoluble fraction. For the research on the water-insoluble fraction of the GVP from mainstream smoke of research cigarette K2R4F, we placed a piece of Cambridge filter and 6 impingers connected in tandem with each other between the cigarette ports and the cylinder in the smoking machine VC10. The PP of K2R4F was removed by the Cambridge filter and the GVP was forwarded to the 6 tandem impingers containing PBS to remove the water-soluble fraction. The water-insoluble fraction passing through the impingers was directly exposed to Chinese hamster ovary cells (CHO-K1). After the exposure, we performed neutral red uptake assay (NR uptake assay) to research the cytotoxicity of the water-insoluble fraction of the GVP. To confirm the removal of the water-soluble components, we analyzed cytotoxic aldehydes as markers using High Performance Liquid Chromatography (HPLC) and found that no aldehydes were detected after the 6th impinger. As a result, the cytotoxicity was detected even from the water-insoluble fraction of the GVP.