A 6-month inhalation toxicology study in Apoe-/- mice demonstrates substantially lower effects of e-vapor aerosol compared with cigarette smoke in the respiratory tract

Abstract

Cigarette smoking is the major cause of chronic obstructive pulmonary disease. Considerable attention has been paid to the reduced harm potential of nicotine-containing inhalable products such as electronic cigarettes (e-cigarettes). We investigated the effects of mainstream cigarette smoke (CS) and e-vapor aerosols (containing nicotine and flavor) generated by a capillary aerosol generator on emphysematous changes, lung function, and molecular alterations in the respiratory system of female Apoe^-/- mice. Mice were exposed daily (3 h/day, 5 days/week) for 6 months to aerosols from three different e-vapor formulations-(1) carrier (propylene glycol and vegetable glycerol), (2) base (carrier and nicotine), or (3) test (base and flavor)-or to CS from 3R4F reference cigarettes. The CS and base/test aerosol concentrations were matched at 35 µg nicotine/L. CS exposure, but not e-vapor exposure, led to impairment of lung function (pressure-volume loop area, A and K parameters, quasi-static elastance and compliance) and caused marked lung inflammation and emphysematous changes, which were confirmed histopathologically and morphometrically. CS exposure caused lung transcriptome (activation of oxidative stress and inflammatory responses), lipidome, and proteome dysregulation and changes in DNA methylation; in contrast, these effects were substantially reduced in response to the e-vapor aerosol exposure. Compared with sham, aerosol exposure (carrier, base, and test) caused a slight impact on lung inflammation and epithelia irritation. Our results demonstrated that, in comparison with CS, e-vapor aerosols induced substantially lower biological and pathological changes in the respiratory tract associated with chronic inflammation and emphysema.

Keywords: COPD; Electronic cigarette; Emphysema; Inflammation; Smoking.

Fig. 1

Lung function following exposure to 3R4F CS or e-vapor aerosols. Pressure (Ptr) and volume (Vtr) were recorded at months 3 and 6 to generate pressure–volume (P–V) loops from the medians of replicate measurements. n = 12. CS, cigarette smoke

Fig. 2

Histopathological evaluation of structural and emphysematous changes in response to 3R4F CS or e-vapor aerosol exposure. Representative images of lung tissue sections stained with hematoxylin and eosin. CS, cigarette smoke

Fig. 3

Inflammatory mediators in BALF in response to 3R4F CS or e-vapor aerosol exposure. Inflammatory mediators in BALF. Cell-free BALF supernatants were analyzed using a multiplexed bead array. The ratio of inflammatory mediators is given as the median of levels in treated mice over the median of levels in sham-exposed mice at the same time point (see color scale). Analytes with statistically significant differences at least in one comparison with the sham group are shown. “ + ” denotes p < 0.05; “ +  +  + ” denotes p < 0.001; n = 10. Orange shades indicate significantly elevated analytes, and blue shades indicate significantly decreased analytes. The full list of analytes is provided on the INTERVALS website (10.26126/intervals.8lafdu.1). BALF bronchoalveolar lavage fluid

Fig. 4

Systems toxicology analysis of dysregulated genes in the lungs. a Number of differentially expressed genes. b RBIF for treatment versus sham. The percentages show the RBIF, which is derived from the cumulated network perturbations caused by the treatment relative to the reference, defined as the treatment comparison showing the highest perturbation (i.e., at the 6-month time point). c Heatmap of NPA scores summarizing subnetwork NPAs relative to the maximum NPA in each subnetwork. Stars indicate significant perturbations: A network is considered perturbed if, in addition to the significance of the NPA score with respect to the experimental variation, the two companion statistics (O and K), derived to inform on the specificity of the NPA score with respect to the biology described in the network, are significant. *O and K statistic p values < 0.05 and significant with respect to the experimental variation; n = 8–10. RBIF relative biological impact factor; NPA network perturbation amplitude

Fig. 5

Systems toxicology analysis of lipids and proteins in the lungs. a Differential abundance of lipids in the lungs; n = 10. Log₂ fold changes versus sham are color-coded, and statistical significance is indicated. *FDR-adjusted p < 0.01; ^XFDR-adjusted p < 0.05. Only lipids with significant differential abundance in any contrast are shown. b Differential expression of proteins involved in fatty acid metabolism and fatty acyl coenzyme A (CoA) synthesis (as defined by the KEGG and Reactome databases). Only proteins with significant differential abundance in any contrast are shown. c Differential abundance of surfactant proteins. PC phosphatidylcholine; PG phosphatidylglycerol. FDR false discovery rate; KEGG Kyoto Encyclopedia of Genes and Genomes