Exposure of human lung cells to inhalable substances: A novel test strategy involving clean air exposure periods using whole diluted cigarette mainstream smoke

An experimental approach was established for the validation of an in vitro test system for complex environmental test atmospheres consisting of both gaseous substances and particulates. Smoke from two different cigarette types (generated by an automatic cigarette-smoking machine) was employed to assess both the sensitivity and the specificity of the system. The smoke was diluted with synthetic air and used to expose human lung cells grown on microporous membranes. Cells were exposed alternately to diluted cigarette smoke and pure synthetic air. The effect of diluted smoke was assessed without humidification, addition of CO(2), or any other physical or chemical modification of the smoke. The experimental setup included online monitoring of the gas phase (by analysis of CO concentration) and particulate phase (by light-scattering photometry). Replicate experiments confirmed a reproducible generation and dilution of the smoke and a smoke age of about 7 s at the time it came into contact with the cells. Experiments using human lung cells revealed that smoke from the two different cigarette types induced different levels of dose-dependent toxicity. A cell exposure of 6 min using 6 alternating smoke and synthetic air periods was sufficient to cause different effects as measured by intracellular glutathione content. The fact that the system could differentiate between two different types of cigarette smoke demonstrated its high sensitivity and specificity. The system offers new ways to test native complex gaseous and aerosol mixtures in vitro using short exposure times and very small amounts of test substances