Assessment of cigarette whole smoke biological activity
The aim of this work was to develop an in vitro model for direct exposure of human lung cells (A549) to native cigarette whole smoke, in a specially adapted exposure module based on the CULTEXTM cell cultivation system. Our system consists in three major elements: a smoking machine working with standard ISO parameters (35 mL puff volume, 2 s puff duration, one puff per minute), a dilution system for the fresh whole smoke, driven with synthetic clean air, and an exposure chamber for cells growing on cell culture inserts housed in a glass exposure device. We first checked that cellular viability was not affected by the synthetic clean air exposure: the cellular viability was maintained at more than 90% over a wide range of dilution (from 1.96 to 10.9). A reproducible dose-effect was observed in this range of dilution with the Neutral Red test. Classical in vitro tests assessing genotoxicity, cytotoxicity, mutagenicity, as well as new tests such as toxicogenomics have now to be developed with this whole smoke exposure system. We first developed a cytotoxicity test (Neutral Red) and a genotoxicity test (Comet assay), and compared the toxicity of whole smoke from various cigarettes. As an example, we tested the effect of the presence of charcoal in a cigarette filter. Cells were exposed to whole smoke from 4 cigarettes during 30 minutes at 2 dilution factors (4.81 and 2.92). With the charcoal filter, the whole smoke cytotoxicity decreased by around 50%, and the Tail Moment of the comet (parameter used to estimate DNA damage) decreased by around 3-fold in comparison with the control filter. In conclusion, our new approach to assess the whole smoke toxicity seems to be adapted to distinguish differences between two cigarettes in terms of genotoxicity and cytotoxicity.