Lab study shows cigarette smoke damaged lung cells more than e-cigarette vapor

A new laboratory study found that cigarette smoke sharply disrupted lung cell defenses, while e-cigarette vapor showed no significant damage in the same model, highlighting important differences but not settling the question of long-term safety in humans.

Study: Tobacco smoke but not e-cigarette vapor induces epithelial barrier disruption, inflammation, and DNA damage in human Calu-3 cells. Image Credit: Antonio Marca / Shutterstock

In a recent study published in the journal Scientific Reports, researchers at the University of Graz, Austria, compared the effects of cigarette smoke extract (CSE) and e-cigarette vapor extract (EVE) on epithelial barrier integrity, inflammation, and deoxyribonucleic acid (DNA) damage in human lung cells.

Lung Epithelial Barrier and Smoke Exposure Background

What happens inside your lungs every time you inhale smoke or vapor? Each time you breathe in smoke or vapor, your airway epithelium acts as a barrier to protect you against harmful particles, chemicals, and other potentially pathogenic substances from entering the body.

Cigarette smoke has been shown to damage this barrier and is strongly linked to diseases like chronic obstructive pulmonary disease (COPD). While there is less evidence on the health effects of e-cigarette vapor, it is unclear whether e-cigarettes are less harmful than traditional cigarettes, and some studies show that they may increase inflammatory and oxidative stress.

Understanding how e-cigarette vapor exposure or cigarette smoke exposure affects the respiratory system is very important, as damage to the epithelial barrier may have significant respiratory consequences; however, more research is needed to determine how well laboratory findings reflect human health outcomes.

Calu-3 Lung Cell Study Design

The study was conducted using human Calu-3 lung epithelial cells, and cells were cultured under controlled laboratory conditions and exposed to CSE or EVE. Three experimental approaches were employed: developing cell layers, forming fully formed epithelial barriers, and measuring functional permeability using Transwell systems.

Aerosol extracts were generated by passing cigarette smoke or e-cigarette vapor through culture medium using a standardized puffing system to ensure consistent exposure conditions. Nicotine concentrations were quantified to allow accurate comparisons between treatments. Cells were exposed to diluted CSE and undiluted EVE over defined time intervals. The e-cigarette extract was generated from nicotine-containing, unflavored e-liquid.

Transepithelial electrical resistance (TEER) measurements and a fluorescein isothiocyanate (FITC)-dextran permeability assay were used to evaluate barrier integrity. Western blotting and real-time quantitative polymerase chain reaction (qPCR) were used to assess protein and gene expression of junctional components, including claudin-1, occludin, and E-cadherin. An inflammatory response was assessed by interleukin-6 (IL-6) expression and secretion using enzyme-linked immunosorbent assay (ELISA).

DNA damage was measured using phosphorylated histone H2AX (γH2AX) immunostaining and neutral comet assays, which detect DNA double-strand breaks. Statistical analyses included analysis of variance (ANOVA) and non-parametric tests.

CSE and EVE Effects on Barrier Integrity and DNA Damage

Exposure to CSE had profound effects on epithelial barrier integrity. TEER measurements showed a significant reduction in barrier resistance, indicating compromised cell cohesion. This finding was supported by increased permeability in FITC-dextran assays, showing that harmful substances could more easily pass through the epithelial layer. It was also observed that EVE did not damage the barrier and appeared to slightly improve stability.

CSE decreased gene and protein expression for key components of the apical junctional complex, including claudin-1 and occludin, in mature barrier experiments. Notably, the decline in claudin-1 levels upon exposure to CSE was 45%, indicating that claudin-1 is likely a vulnerable component of the epithelial barrier. Inspection of claudin-1 localization patterns in smoke-exposed cells using confocal microscopy demonstrated that claudin-1 had lost its normal localization and that claudin-1 was aggregating. Cells exposed to e-cigarette vapor displayed normal claudin-1 localization patterns.

CSE inhibited claudin-1 expression for a longer period during the development of barriers, particularly between five and seven days. While E-cadherin gene expression was also reduced, its protein levels remained largely unchanged, suggesting post-transcriptional regulation. EVE did not significantly alter these structural proteins.

Inflammatory responses were markedly elevated in cigarette smoke-treated cells. IL-6 expression increased up to tenfold during early stages of barrier formation and remained significantly elevated in mature cell layers. Protein secretion of IL-6 increased by approximately 3.5- to 4-fold after prolonged exposure. In contrast, EVE showed no significant effect on IL-6 levels under the experimental conditions tested.

CSE also caused significant damage to DNA, and γH2AX staining revealed more accumulation of DNA damage markers within cell nuclei. Neutral comet assay showed a 2.7-fold increase in DNA strand breaks. In contrast, EVE led to much less evident DNA damage, with no significant increase in DNA strand breaks compared with untreated control cells in the neutral comet assay.

It was also noted that cigarette smoke damages cells, but it is not only due to nicotine. E-cigarette vapor has higher concentrations of nicotine than traditional cigarettes, which shows that it is likely that there are other toxic components in cigarette smoke that are causing the observed cellular disruption.

Respiratory Health Implications of CSE Versus EVE

CSE negatively impacts the integrity of the lung epithelial barrier, increases inflammation, and leads to DNA damage, all of which may contribute to respiratory disease processes. Conversely, EVE showed no significant adverse effects on these parameters in this Calu-3 cell model under the conditions tested.

Although at the cellular level, e-cigarettes appeared to exert less harmful effects than traditional cigarettes in this experimental system, there is still uncertainty about their long-term effects in humans.

This is an important distinction for individuals and the public health community because these findings come from an in vitro model and should not be interpreted as direct evidence of improved health outcomes in vivo.

The authors also noted that the use of liquid extracts rather than direct aerosol exposure, and the testing of unflavored e-liquid only, may limit how broadly these findings can be generalized. Therefore, additional studies using more representative biological systems are necessary to assess the long-term health effects of EVE use.