Limited toxicity data on electronic cigarette (ECIG) impede evidence-based policy recommendations. We compared two popular mixed fruit flavored ECIG-liquids with and without nicotine aerosolized at 40 W (E-smoke) with respect to particle number concentrations, chemical composition, and response on physiologically relevant human bronchial and alveolar lung mucosa models cultured at air–liquid interface. E-smoke was characterized by significantly increased particle number concentrations with increased wattage (25, 40, and 55 W) and nicotine presence. The chemical composition of E-smoke differed across the two tested flavors in terms of cytotoxic compounds including p-benzoquinone, nicotyrine, and flavoring agents (for example vanillin, ethyl vanillin). Significant differences in the expression of markers for pro-inflammation, oxidative stress, tissue injury/repair, alarm anti-protease, anti-microbial defense, epithelial barrier function, and epigenetic modification were observed between the flavors, nicotine content, and/ or lung models (bronchial or alveolar). Our findings indicate that ECIG toxicity is influenced by combination of multiple factors including flavor, nicotine content, vaping regime, and the region of respiratory tree (bronchial or alveolar). Toxic chemicals and flavoring agents detected in high concentrations in the E-smoke of each flavor warrant independent evaluation for their specific role in imparting toxicity. Therefore, multi-disciplinary approaches are warranted for comprehensive safety profiling of ECIG.

Secreted phosphoprotein 1 (Spp1) is located within quantitative trait loci associated with lung function that was previously identified by contrasting C3H/HeJ and JF1/Msf mouse strains that have extremely divergent lung function. JF1/Msf mice with diminished lung function had reduced lung SPP1 transcript and protein during the peak stage of alveologenesis (postnatal day [P]14–P28) as compared with C3H/HeJ mice. In addition to a previously identified genetic variant that altered runt-related transcription factor 2 (RUNX2) binding in the Spp1 promoter, we identified another promoter variant in a putative RUNX2 binding site that increased the DNA protein binding. SPP1 induced dose-dependent mouse lung epithelial-15 cell proliferation. Spp1(−/−) mice have decreased specific total lung capacity/body weight, higher specific compliance, and increased mean airspace chord length (Lm) compared with Spp1(+/+) mice. Microarray analysis revealed enriched gene ontogeny categories, with numerous genes associated with lung development and/or respiratory disease. Insulin-like growth factor 1, Hedgehog-interacting protein, wingless-related mouse mammary tumor virus integration site 5A, and NOTCH1 transcripts decreased in the lung of P14 Spp1(−/−) mice as determined by quantitative RT-PCR analysis. SPP1 promotes pneumocyte growth, and mice lacking SPP1 have smaller, more compliant lungs with enlarged airspace (i.e., increased Lm). Microarray analysis suggests a dysregulation of key lung developmental transcripts in gene-targeted Spp1(−/−) mice, particularly during the peak phase of alveologenesis. In addition to its known roles in lung disease, this study supports SPP1 as a determinant of lung development in mice.

Diacetyl is an artificial flavouring agent, known to cause bronchiolitis obliterans. Diacetyl-induced pulmonary effects were assessed in human primary bronchial epithelial cells (PBEC) cultured at air-liquid interface (ALI). The PBEC-ALI models were exposed to clean air (sham) and diacetyl vapour (1, 3, 10 and 30 ppm) for 30 min. At 6 and 24 h post-exposure, cell medium was sampled for assessment of cytotoxicity measurement, and CXCL8, MMP9 secretion by ELISA. Pro-inflammatory, oxidative stress, tissue injury/repair, anti-protease and beta-defensin markers were assessed using qRT-PCR. Additionally, epidermal growth factor receptor ligands (amphiregulin) and anti-protease (SLPI) were analysed at 6 h, 8 h and 24 h post exposure to 1 and 10 ppm diacetyl. No significant cytotoxicity was observed at any exposure level. MMP9 was significantly increased in both apical and basal media at 24 h. Both SLPI and amphiregulin secretion were significantly increased following exposure to 10 ppm diacetyl. Exposure of PBEC-ALI model to diacetyl vapour resulted in significantly altered transcript expression of pro-inflammatory, oxidative stress, anti-protease, tissue injury/repair markers. Changes in transcript expression of significantly altered markers were more prominent 24 h post-exposure compared to 6 h. This study warrants further mechanistic investigations to elucidate the pulmonary effects of inhaled diacetyl vapour using physiologically relevant in vitro models.

Background. Chronic obstructive pulmonary disease (COPD) is a devastating condition with limited pharmacotherapeutic options and exceptionally high public-health burden globally as well as in India. Tobacco smoking is the primary cause for COPD among men in India. Systemic inflammation involving altered regulation of cytokines controlling the host defense mechanism is a hallmark of COPD pathogenesis. However, biomarker discovery studies are limited among Indian COPD patients.Methods. We assessed the serum concentrations [median (25th-75th percentile) pg/ml] of interleukin (IL)-2,4,6,8,10, granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) using a multiplexed immunoassay. Our study cohort consisted of 30 tobacco smokers with COPD (TS COPD) and 20 tobacco smokers without COPD (TS CONTROL) from South India. The study population was matched for age, sex (male), and tobacco consumption (pack-years). COPD was diagnosed according to the global initiative for chronic obstructive lung disease (GOLD) criteria of persistent airflow obstruction determined by the ratio of postbronchodilator forced expiratory volume in 1 second/forced vital capacity (FEV₁/FVC) of <0.7. A validated structured questionnaire-based survey [Burden of Obstructive Lung Disease (BOLD) study] and spirometry were performed during house to house visit of the field study. Statistical analysis included nonparametric (two-tailed) Mann–Whitney U and Spearman rank test, as appropriate (significance: p<0.05).Results. Serum GM-CSF [69.64 (46.67, 97.48); 36.78 (30.07, 53.88), p=0.014], IFN-γ[51.06 (17.00, 84.86); 11.70 (3.18, 32.81), p=0.017], IL-4 [9.09 (1.8, 19.9); 1.8 (1.8, 4.46); p=0.024], and TNF-α[20.68 (5.5, 29.26); 3.5 (3.5, 4.5); p<0.001] concentrations (pg/ml) were increased in TS COPD subjects compared to TS CONTROL. A weak correlation between lung function parameters and cytokine concentrations was detected.Conclusion. Our pilot study reveals GM-CSF, IFN-γ, IL-4, and TNF-αas plausible COPD susceptibility biomarkers within the investigated South Indian population that needs to be validated in a larger cohort.

Secretoglobin family 1A member 1 (SCGB1A1) alternatively known as club cell protein 16 is a protective pneumo-protein. Decreased serum levels of SCGB1A1 have been associated with tobacco smoke induced chronic obstructive pulmonary disease (TS-COPD). Exposure to biomass smoke (BMS) is an important COPD risk factor among women in low and lower-middle income countries. Therefore, in a cross-sectional study (n = 50/group; total 200 subjects) we assessed serum SCGB1A1 levels in BMS-COPD subjects (11 male, 39 female) compared to TS-COPD (all male) along with TS-CONTROL (asymptomatic smokers, all male) and healthy controls (29 male, 21 female) in an Indian population. Normal and chronic bronchitis like bronchial mucosa models developed at the air–liquid interface using human primary bronchial epithelial cells (3 donors, and three replicates per donor) were exposed to cigarette smoke condensate (CSC; 0.25, 0.5, and 1%) to assess SCGB1A1 transcript expression and protein secretion. Significantly (p < 0.0001) decreased serum SCGB1A1 concentrations (median, interquartile range, ng/mL) were detected in both BMS-COPD (1.6; 1.3–2.4) and TS-COPD (1.8; 1.4–2.5) subjects compared to TS-CONTROL (3.3; 2.9–3.5) and healthy controls (5.1; 4.5–7.2). The levels of SCGB1A1 were positively correlated (r = 0.7–0.8; p < 0.0001) with forced expiratory volume in 1 s, forced vital capacity, their ratios, and exercise capacity. The findings are also consistent within the BMS-COPD sub-group as well. Significantly (p < 0.03) decreased SCGB1A1 concentrations were detected with severity of COPD, dyspnea, quality of life, and mortality indicators. In vitro studies demonstrated significantly (p < 0.05) decreased SCGB1A1 transcript and/or protein levels following CSC exposure. Circulating SCGB1A1 levels may therefore also be considered as a potent marker of BMS-COPD and warrant studies in larger independent cohorts.

Failure to attain peak lung function by early adulthood is a risk factor for chronic lung diseases. Previously, we reported that C3H/HeJ mice have about twice total lung capacity (TLC) compared to JF1/MsJ mice. We identified seven lung function quantitative trait loci (QTL: Lfnq1-Lfnq7) in backcross/intercross mice derived from these inbred strains. We further demonstrated, superoxide dismutase 3, extracellular (Sod3), Kit oncogene (Kit) and secreted phosphoprotein 1 (Spp1) located on these Lfnqs as lung function determinants. Emanating from the concept of early origin of lung disease, we sought to identify novel candidate genes for pulmonary function by investigating lung transcriptome in C3H/HeJ and JF1/MsJ mice at the completion of embryonic development, bulk alveolar formation and maturity. Design-based stereological analysis was performed to study lung structure in C3H/HeJ and JF1/MsJ mice. Microarray was used for lung transcriptomic analysis [embryonic day 18, postnatal days 28, 70]. Quantitative real time polymerase chain reaction (qRT-PCR), western blot and immunohistochemical analysis were used to confirm selected differences. Stereological analysis revealed decreased alveolar number density, elastin to collagen ratio and increased mean alveolar volume in C3H/HeJ mice compared to JF1/MsJ. Gene ontology term “extracellular region” was enriched among the decreased JF1/MsJ transcripts. Candidate genes identified using the expression-QTL strategy include: ATP-binding cassette, sub-family G (WHITE), member 1 (Abcg1), formyl peptide receptor 1 (Fpr1), gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1); histocompatibility 2 genes: class II antigen E beta (H2-Eb1), D region locus 1 (H2-D1), and Q region locus 4 (H2-Q4); leucine rich repeat containing 6 (testis) (Lrrc6), radial spoke head 1 homolog (Rsph1), and surfactant associated 2 (Sfta2). Noteworthy genes selected as candidates for their consistent expression include: Wnt inhibitor factor 1 (Wif1), follistatin (Fst), chitinase-like 1 (Chil1), and Chil3. Comparison of late embryonic, adolescent and adult lung transcript profiles between mouse strains with extreme TLCs lead to the identification of candidate genes for pulmonary function that has not been reported earlier. Further mechanistic investigations are warranted to elucidate their mode of action in determining lung function.