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Comparison of the Biological Effects of Long-Term Exposure of Human Bronchial Epithelial Cells to Total Particulate Matter from 3R4F Cigarette Smoke or Aerosol from the Candidate Modified Risk Tobacco Product (cMRTP) THS2.2

Chronic cigarette smoke exposure leads to airway epithelial changes underlying lung tumorigenesis, although the molecular events are not fully elucidated. We mimicked chronic exposure in smokers’ airways by continuously exposing BEAS-2B cells to TPM from 3R4F cigarette smoke or aerosol of the Tobacco Heating System 2.2 (THS2.2), a cMRTP, for 12 weeks.

Several endpoints including proliferation, DNA damage, oxidative stress, epithelial-mesenchymal transition (EMT) and wound repair were assessed monthly. Soft-agar assays were performed after 12 weeks, and resulting clones were tested for invasion. Cells were also collected regularly for systems toxicological analysis. Increased oxidative stress and DNA damage were noticeable within the first 2 weeks of 3R4F TPM treatment, but subsided thereafter, indicating adaptation.

Four-week 3R4F TPM treatment resulted in crisis and EMT. By week 8, cells regained E-cadherin expression, suggesting EMT was reversible. Increased MMP levels and decreased wound repair were noted in 3R4F TPM-treated cells at week 12.

However, these changes were not observed following prolonged treatment of BEAS-2B cells with the same or a 5-fold higher concentration of THS2.2 TPM, while a 20-fold higher concentration also increased oxidative burden, DNA damage and caused reversible EMT. In addition, anchorage-independent growth was observed in 3R4F and THS2.2 TPM-treated BEAS-2B cells. 3R4F TPM-derived clones were invasive, while THS2.2 TPM clones were not. Systems toxicological analysis indicated an overall smaller biological impact of THS2.2 compared with 3R4F TPM, confirming a differential effect of the two TPMs. Further analysis by DNA sequencing and in vitro xenograft assay is currently underway.