Flavour-Specific Pro-Oxidative and Pro-Inflammatory Activation of Adherent and Non-Adherent THP-1 Monocytic Cells Induced by E-Cigarette Aerosols

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Abstract
Electronic cigarettes are widely promoted as a less harmful alternative to conventional cigarettes, but their cellular effects remain incompletely defined and may vary by flavouring composition. This study examined the effects of four flavoured e-cigarette aerosols, cigarette smoke, and nicotine on human THP-1 monocytic cells, focusing on cytotoxicity, oxidative stress, inflammatory activation, adhesion, and phagocytic function. Cells were exposed for 24 hours to conditioned media generated using a standardised puffing protocol and then analysed by microscopy, trypan blue exclusion, flow cytometry, and dual-fluorescence scatterplot analysis. Cigarette smoke-conditioned medium produced the greatest acute toxicity, significantly increasing cell death, membrane damage, and DNA fragmentation. In contrast, the tested e-cigarette aerosol-conditioned media and nicotine alone produced little overt cytotoxicity under the same experimental conditions. However, e-cigarette exposures increased cell adhesion and were associated with formulation- and cell subpopulation–dependent changes in CD11a, CD11b, and CD54 expression. E-cigarettes increased intracellular DCF-associated oxidative fluorescence and TNF expression, with the watermelon and strawberry formulations producing the strongest responses. Nicotine alone did not reproduce these pro inflammatory/pro oxidant effects to the same extent as the full aerosol. Instead, in non adherent cells, nicotine behaved more like an antioxidant/anti inflammatory agent, while in adherent cells, it showed a tendency toward pro oxidant/pro inflammatory behaviour, though generally weaker than cigarette smoke and e cigarette aerosols. These effects indicate that other aerosol components substantially contribute to biological activity, and the effect may be bimodal. Phagocytosis was reduced in selected groups, suggesting that some exposures may impair this cellular function. Bivariate fluorescence revealed exposure-dependent differences in the relationship between oxidative stress and TNF expression, with cigarette smoke producing the most pronounced disruption. Overall, these findings support the potential utility of bivariate fluorescence analysis as a complementary in vitro approach for detecting and comparing oxidative and inflammatory cellular responses to different e-cigarette aerosols.

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