Comparative Assessment of Acute Pulmonary Effects Induced by Heat-Not-Burn Tobacco Aerosol Inhalation in a Murine Model

Abstract

Tobacco smoking remains a major global health concern, causing preventable deaths and economic strain. Although new tobacco products such as heat-not-burn (HnB) are safer alternatives to traditional cigarettes, research on their associated risks remains limited. This study aimed to investigate the effects of HnB smoke exposure on the lungs compared to those of traditional cigarettes and the combined use of HnB and cigarettes using experiments with a mouse model. We quantitatively analyzed changes in the levels of 92 blood plasma proteins using the proximity extension assay method and observed significant changes in their levels in mice exposed to different smoke conditions; specifically, the levels of certain proteins, including Ccl20, Cxcl1, and Pdgfb, increased in the HnB smoke-exposed group, suggesting activation of nicotine pathways. Comparative analysis with traditional cigarette smoke-exposed mice further highlighted similarities and differences in their protein expression profiles. This study contributes to an improved understanding of the biological mechanisms underlying the harmful effects of alternative nicotine delivery systems and identifies potential biomarkers associated with the harmful effects of HnB smoke exposure. However, the precise impact of nicotine on the immune system may be influenced by various factors, necessitating further research.

Keywords: electronic nicotine delivery systems; lung injury; model animal; smoking; toxicity test.

Figure 1

Heatmap illustrating unique proteins. Unique proteins were identified using the proximity extension assay, which was logarithmically scaled, and their normalized protein expression (NPX) values were standardized to zero. Positive values within the heatmap indicate NPX levels that are above the detection threshold and higher than the average scaled NPX value.

Figure 2

Detection of biomarker candidates using proximity extension assay (PEA) to compare control mice with mice exposed to cigarette smoke. Volcano plot of the 92 proteins analyzed using the PEA. The estimated difference is presented on the x-axis and −log10(p-value) is presented on the y-axis. The horizontal dotted line indicates a raw p-value of 0.05, and the vertical dotted line indicates the threshold in the log2 ratio of fold change. Dots are colored based on the criteria for significant results.

Figure 3

Detection of biomarker candidates using proximity extension assay (PEA) to compare control mice with mice exposed to both cigarette and heat-not-burn smoke. Volcano plot of the 92 proteins analyzed using the PEA. The estimated difference is presented on the x-axis and −log10(p-value) is presented on the y-axis. The horizontal dotted line indicates a raw p-value of 0.05 and the vertical dotted line indicates the threshold in the log2 ratio of fold change. Dots are colored based on the criteria for significant results.

Figure 4

Detection of biomarker candidates using proximity extension assay (PEA) to compare control mice with mice exposed to heat-not-burn smoke. Volcano plot of the 92 proteins analyzed using the proximity extension assay. The estimated difference is presented on the x-axis and −log10(p-value) is presented on the y-axis. The horizontal dotted line indicates a raw p-value of 0.05 and the vertical dotted line indicates the threshold in the log2 ratio of fold change. Dots are colored based on the criteria for significant results.

Figure 5

Schematic diagram illustrating smoking exposure to mice. Experimental animals were exposed to tobacco and heat-not-burn smoke five days a week for four weeks using the Smoking Tester Line System of Three Shine Inc. Each group was exposed to the smoke of 20 cigarettes or heat-not-burn sticks, five at a time, over the course of approximately 30–40 min. The mixed-use group was alternately exposed to the smoke of 10 cigarettes and 10 IQOS sticks.