Heated Tobacco Aerosol Triggers Weaker Oxidative and Inflammatory Responses Than Cigarette Smoke in Lung Cells

Heated tobacco products have been marketed as a cleaner way to consume nicotine, but the cellular consequences of their aerosols remain only partly mapped. A new laboratory study published in Frontiers in Physiology compared the effects of cigarette smoke extract and heated tobacco aerosol extract on human lung fibroblasts, and found that while both exposures triggered oxidative stress and inflammatory signaling, the cigarette smoke extract produced measurably stronger responses at the concentrations compared. The findings add granular detail to an ongoing debate about how far reduced-toxicant nicotine products actually differ from conventional cigarettes at the level of individual cells.

The research team, drawn from Universitas Padjadjaran in Indonesia and the University of Catania in Italy, including investigators affiliated with the Center of Excellence for the Acceleration of Harm Reduction, worked with MRC-5 cells, a widely used line of human fetal lung fibroblasts. Fibroblasts are central players in the lung’s response to injury: they drive tissue remodeling, participate in inflammatory signaling and sit at the center of the oxidative stress responses implicated in chronic diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis. Cigarette smoke is a major external source of reactive oxygen species that disrupt cellular redox balance, and elevated oxidant levels are known to feed inflammatory pathways, including the expression of pro-inflammatory cytokines.

To generate the exposures, the researchers bubbled mainstream smoke from Marlboro Red cigarettes through ice-cold phosphate-buffered saline, producing a cigarette smoke extract stock of 2 puffs per milliliter. For the heated tobacco product, they used an IQOS 3 Duo device with HEETS Sienna heatsticks, capturing mainstream aerosol from 30 heatsticks under identical conditions to yield a stock of 9 puffs per milliliter. Crucially, the two products were generated under different standardized puffing regimens: cigarette smoke followed the ISO 3308:2012 regimen with 35-milliliter puffs taken every 60 seconds, while the heated tobacco aerosol was generated under the Health Canada Intense regimen with larger 55-milliliter puffs at 30-second intervals. Because puff volume, duration and interval differ, one puff of one product is not chemically equivalent to one puff of the other, a caveat that shapes every comparison in the study.

The first endpoint was cytotoxicity. Cells were exposed for 24 hours to graded concentrations of each extract, and viability was measured with a resazurin reduction assay. Cigarette smoke extract reduced viability significantly from 0.5 puffs per milliliter onward, dropping to 57.3 percent of the vehicle control at 0.7 puffs per milliliter and just 20.4 percent at 1.0 puffs per milliliter. The heated tobacco aerosol extract only began to significantly reduce viability at 3.5 puffs per milliliter, reaching 38.2 percent at the highest concentration tested, 5.5 puffs per milliliter. Nonlinear regression yielded a 24-hour half-maximal inhibitory concentration of 0.70 puffs per milliliter for cigarette smoke extract and 4.76 puffs per milliliter for the heated tobacco aerosol, meaning the aerosol required far higher nominal puff concentrations to inflict comparable loss of viability under these specific conditions.

Morphology told a parallel story. Under phase-contrast microscopy, control fibroblasts displayed their characteristic spindle shape, intact junctions and a confluent monolayer. Cigarette smoke extract drove progressive shrinkage, rounding and detachment, with more rounded, condensed cells and debris appearing at higher concentrations. The heated tobacco aerosol produced a more gradual response: at lower concentrations cells largely kept their normal shape and attachment, and even at higher exposures a substantial proportion remained adherent, although cytoplasmic condensation, rounding and debris increased with dose. The authors note that these qualitative observations serve only as supporting evidence for the quantitative endpoints.

Oxidative stress was assessed with dihydroethidium, a fluorescent probe whose oxidation-sensitive signal serves here as an index of intracellular oxidant activity rather than a specific measurement of superoxide. After a 90-minute exposure, both extracts raised the fluorescence signal in a concentration-dependent manner. Cigarette smoke extract lifted the signal from 1.47-fold over vehicle at the lowest concentration to 3.52-fold at the highest, while the heated tobacco aerosol raised it from 1.77-fold to 2.73-fold. Every concentration of both products differed significantly from the vehicle control, and both series showed strong concentration trends. Antimycin A, a positive control that disrupts mitochondrial respiration, produced the largest signals on both plates, confirming that the assay was responsive throughout.

Inflammation was quantified with an in-cell Western assay measuring intracellular TNF-α and IL-6 protein after four hours of exposure, with signals normalized to a cell-content dye to correct for differences in cell number. Both cytokines rose concentration-dependently with both products. Normalized TNF-α climbed from 1.30-fold over vehicle at the lowest cigarette smoke concentration to 2.63-fold at the highest, and from 1.16-fold to 2.34-fold across the heated tobacco range. IL-6 rose from 1.32-fold to 3.25-fold with cigarette smoke extract, and from 1.04-fold, which was not statistically significant, to 2.95-fold with the aerosol, becoming significant from 4.0 puffs per milliliter onward. Importantly, the increases were also present in the absolute, non-normalized signals, indicating they were not artifacts of the normalization procedure, although cell content fell at the highest concentrations, which inflates the normalized ratios relative to the absolute fold changes.

The study also included ATG5, an autophagy-related protein, as an exploratory endpoint, and here the results were inconclusive. No concentration of either extract differed from the untreated control. The complication is that the vehicle wells on that particular plate carried an anomalously low ATG5 signal, only about 36 percent of the untreated control despite equivalent cell content, while behaving normally for the cytokine measurements. A technical problem is the more likely explanation, the authors write, but it could not be confirmed retrospectively. Because the apparent rises in normalized ATG5 tracked the falling cell-content denominator rather than any absolute increase, the team concluded that the data provide no evidence of an increase in ATG5 protein, and drew no conclusions about autophagy. They emphasize that a single marker cannot establish autophagic flux, which would require flux-based measurements such as LC3B turnover with and without lysosomal inhibition.

Because the two products’ concentration-response lines were not parallel on a logarithmic scale, no single relative-potency factor could describe their difference. Instead, the researchers ran an exploratory comparison at the tested concentrations closest to each product’s own 24-hour IC50: 0.7 puffs per milliliter for cigarette smoke extract and 4.75 puffs per milliliter for the aerosol. At these points, cigarette smoke extract produced a larger oxidative signal, 3.17-fold versus 2.31-fold over vehicle, a larger IL-6 signal, 3.06-fold versus 1.94-fold, and a larger normalized TNF-α signal, 2.30-fold versus 1.77-fold, although the difference in absolute TNF-α did not reach statistical significance. The authors caution that equivalent viability at the earlier 90-minute and 4-hour endpoints was not established, and that the aerosol wells contained a larger fraction of extract and less culture medium than the smoke wells, an unmatched dilution that limits the comparison.

The broader significance of the work lies in what it does and does not show. Heated tobacco products heat rather than burn tobacco, operating below 350 degrees Celsius compared with the more than 800 degrees Celsius of combustion, which sharply reduces carbonyl compounds, volatile organic compounds and particulate matter in the aerosol. Yet biologically active constituents, including nicotine and toxic carbonyl species, remain present, and the present findings confirm that reduced emissions do not mean biological inertness: the aerosol extract still raised oxidant activity and intracellular cytokine levels in these cells. The authors are explicit about the limitations. The extracts were not chemically characterized, pH and osmolality were not standardized, a single fetal fibroblast line cannot recapitulate adult lung physiology or disease microenvironments, and acute submerged exposure to aqueous extracts differs from repeated whole-aerosol exposure at an air-liquid interface. Three biological replicates limit precision, and the findings do not establish relative product safety or population-level risk. What the study delivers is a carefully bounded observation: under product-specific laboratory conditions, cigarette smoke extract elicited stronger oxidative and inflammatory responses than heated tobacco aerosol extract, and confirming the real-world meaning of that gap will require chemically anchored dosing, secreted-cytokine measurements and more physiologically faithful exposure models.

Subject of Research: Comparative oxidative stress and inflammatory responses to cigarette smoke extract versus heated tobacco aerosol extract in human lung fibroblasts

Article Title: Comparative oxidative and inflammatory responses to heated tobacco aerosol and cigarette smoke extracts in human lung fibroblasts

Article References: Abelia, X. A., Lesmana, R., Barliana, M. I., Caruso, M., & Polosa, R. (2026). Comparative oxidative and inflammatory responses to heated tobacco aerosol and cigarette smoke extracts in human lung fibroblasts. Frontiers in Physiology, 17, 1902675. https://doi.org/10.3389/fphys.2026.1902675

Image Credits: AI Generated

DOI: 10.3389/fphys.2026.1902675

Keywords: heated tobacco products, cigarette smoke extract, lung fibroblasts, oxidative stress, inflammation, reactive oxygen species, TNF-alpha, IL-6, ATG5, MRC-5 cells, harm reduction, in vitro toxicology