A new laboratory study found that cigarette smoke rapidly destroyed lung cell defenses, while e-cigarette vapor showed no significant damage in the same model, highlighting an important difference but not resolving the question of long-term safety in humans.
Study: Cigarette 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 reportresearchers from the University of Graz, Austria, compared the effects of cigarette smoke extract (CSE) and electronic cigarette vapor extract (EVE) on epithelial barrier integrity, inflammation, and deoxyribonucleic acid (DNA) damage in human lung cells.
Background of the lung epithelial barrier and smoke exposure
What happens in your lungs every time you inhale smoke or vapor? Every time you inhale smoke or vapor, your airway epithelium acts as a barrier, preventing harmful particles, chemicals, and other potentially pathogenic substances from entering your body.
Cigarette smoke damages this barrier and has been shown to be strongly associated with diseases such as chronic obstructive pulmonary disease (COPD). Although there is less evidence regarding the health effects of e-cigarette vapor, it is unclear whether e-cigarettes are less harmful than traditional cigarettes, and some studies have shown that e-cigarettes can increase inflammatory and oxidative stress.
Damage to the epithelial barrier can have serious respiratory effects, so it is critical to understand how exposure to e-cigarette vapor and exposure to tobacco smoke affects the respiratory system. However, further research is needed to determine the extent to which laboratory test results reflect human health outcomes.
Calu-3 lung cell research design
This study was conducted using human Calu-3 lung epithelial cells, which were cultured under controlled laboratory conditions and exposed to CSE or EVE. Three experimental approaches were adopted: cell layer development, formation of a fully formed epithelial barrier, and measurement of functional permeability using the Transwell system.
Aerosol extracts were generated by passing cigarette smoke or e-cigarette vapor through the 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 for defined time intervals. E-cigarette extract was produced from unflavored e-liquid containing nicotine.
Transepithelial electrical resistance (TEER) measurements and fluorescein isothiocyanate (FITC) dextran permeability assays were used to assess barrier integrity. Western blotting and real-time quantitative polymerase chain reaction (qPCR) were used to assess protein and gene expression of binding components such as claudin-1, occludin, and E-cadherin. The 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 a neutral comet assay that detects DNA double-strand breaks. Statistical analyzes included analysis of variance (ANOVA) and nonparametric tests.
Effects of CSE and EVE on barrier integrity and DNA damage
Exposure to CSE had a significant effect on epithelial barrier integrity. TEER measurements showed a significant decrease in barrier resistance, indicating impaired cell cohesion. This finding was supported by increased permeability in the FITC-dextran assay, indicating that harmful substances could more easily pass through the epithelial layer. We also observed that EVE did not damage the barrier and appeared to slightly improve stability.
CSE decreased gene and protein expression of key components of the apical junctional complex, such as claudin-1 and occludin, in maturation barrier experiments. Notably, CSE exposure reduced claudin-1 levels by 45%, indicating that claudin-1 is likely a vulnerable component of the epithelial barrier. Examination of the localization pattern of claudin-1 in smoke-exposed cells using confocal microscopy demonstrated that claudin-1 lost its normal localization and claudin-1 aggregated. Cells exposed to e-cigarette vapor showed a normal claudin-1 localization pattern.
CSE inhibited claudin-1 expression for long periods of time during barrier development, especially for 5–7 days. Expression of the E-cadherin gene was also reduced, but its protein levels remained largely unchanged, suggesting post-transcriptional regulation. EVE did not significantly alter these structural proteins.
The inflammatory response was significantly elevated in cells treated with cigarette smoke. IL-6 expression increased up to 10-fold during the early stages of barrier formation and remained significantly elevated in the mature cell layer. After long-term exposure, protein secretion of IL-6 increased approximately 3.5- to 4-fold. 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 further accumulation of DNA damage markers within the cell nucleus. Neutral comet assay showed a 2.7-fold increase in DNA strand breaks. In contrast, EVE produced much less obvious DNA damage with no significant increase in DNA strand breaks compared to untreated control cells in the neutral comet assay.
It has also been pointed out that cigarette smoke damages cells, but nicotine is not the only cause. E-cigarette vapor has higher nicotine concentrations than traditional cigarettes, indicating that other toxic components are likely present in cigarette smoke that are causing the observed cell destruction.
Respiratory health effects of CSE and EVE
CSE negatively affects the integrity of the lung epithelial barrier, increases inflammation, and causes DNA damage, all of which can contribute to the respiratory disease process. Conversely, EVE showed no significant negative effects on these parameters in this Calu-3 cell model under the conditions tested.
Although e-cigarettes appeared to have fewer harmful effects than traditional cigarettes at the cellular level 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 were obtained from in vitro models and should not be interpreted as direct evidence of improved health outcomes in vivo.
The authors also noted that using liquid extracts rather than direct exposure to aerosols and testing only unflavored e-liquids may limit the extent to which these findings can be generalized. Therefore, additional studies using more representative biological systems are needed to assess the long-term health effects of EVE use.
Reference magazines:
- Mayer, B., Kollau, A., Kappaun, W., Rauchegger, K., Wölkart, G., Toedtling, A., and Schrammel, A. (2026). Cigarette smoke, but not e-cigarette vapor, induces epithelial barrier disruption, inflammation, and DNA damage in human Calu-3 cells. science senator Doi: https://doi.org/10.1038/s41598-026-45438-9
