Lung cells are frequently used in in vitro studies to assess the impact of cigarette smoke on defense and repair mechanisms of the lung. Previously, we identified a panel of fifteen genes representing inflammation, oxidative stress, metabolism and cellular growth regulation that were common in vitro to human ex-vivo lung comparisons.

The objective of this study was to evaluate the acute response of the panel of genes following exposure to tobacco smoke and tobacco extracts in two in vitro lung cell models – normal human bronchial epithelial (NHBE) and nuclear related factor 2 (Nrf2) luciferase/oxidative stress reporter cells. NHBE cells were initially evaluated following exposure to whole smoke from Kentucky Reference 3R4F (K3R4F) cigarettes via quantitative reverse transcriptase/polymerase chain reaction (QRT/PCR). Inflammatory responses were further assessed by multiplex cytokine analyses of the conditioned media. Subsequently, both models were exposed to total particulate matter (TPM) from K3R4F cigarettes and three smokeless tobacco extracts (STEs) from moist snuff, dry snuff and a smokeless tobacco blend. Gene expression and Nrf2 promoter regulation were then assessed via QRT/PCR and luciferase reporter assays.

Compared to air controls, whole smoke exposure induced gene expression levels that reached 5-fold (metabolism), 9-fold (inflammatory) and 20- to 100-fold (stress response). Elevated cytokine release confirmed the inflammatory indicators suggested by the mRNA changes. TPM exposure elicited gene expression responses ≥ 2-fold in the panel of genes in both NHBE (9 genes) and Nrf2 parental (BEAS2b) cells (7 genes). However, the targets were minimally impacted by the STEs as shown by ≤ 2-fold modifications of 14 of 15 of the targets. K3R4F TPM induced ~ 20-fold increase in Nrf2-regulated luciferase activity compared to the vehicle control while the STEs modulated the Nrf2 promoter ≤ 2-fold.

Collectively, the data indicate that the gene panel represents putative biomarkers of effect. The genes were responsive to tobacco exposures from multiple formats, were differentially regulated by combustible and non-combustible tobacco exposures and may further our understanding of the biological relevance of responses in in vitro models.