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Insight into the pulmonary molecular toxicity of heated tobacco products using human bronchial and alveolar mucosa models at air-liquid interface

ABSTRACT: Heated tobacco products (HTP) are novel nicotine delivery products with limited toxicological data. HTP uses heating instead of combustion to generate aerosol (HTP-smoke). Physiologically relevant human bronchial and alveolar lung mucosa models developed at air-liquid interface were exposed to HTP-smoke to assess broad toxicological response (n=6-7; ISO puffing regimen; compared to sham; non-parametric statistical analysis; significance: p<0.05). Elevated levels of total cellular reactive oxygen species, stress responsive nuclear factor kappa-B, and DNA damage markers [8-hydroxy-2΄-deoxyguanosine, phosphorylated histone H2AX, cleaved poly-(ADP-Ribose) polymerase] were detected in HTP-smoke exposed bronchial and/ or alveolar models. RNA sequencing detected differential regulation of 724 genes in the bronchial- and 121 genes in the alveolar model following HTP-smoke exposure (cut off: p≤0.01; fold change: ≥2). Common enriched pathways included estrogen biosynthesis, ferroptosis, superoxide radical degradation, xenobiotics, and α-tocopherol degradation. Secreted levels of interleukin (IL)1ꞵ and IL8 increased in the bronchial model whereas in the alveolar model, interferon-γ and IL4 increased and IL13 decreased following HTP-smoke exposure. Increased lipid peroxidation was detected in HTP-smoke exposed bronchial and alveolar models which was inhibited by ferrostatin-1. The findings form a basis to perform independent risk assessment studies on different flavours of HTP using different puffing topography and corresponding chemical characterization.

ORGANISM(S): Homo sapiens

PROVIDER: GSE198082 | GEO | 2022/09/16

REPOSITORIES: GEO

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Project description:Chronic obstructive pulmonary disease (COPD) has the highest increased risk due to household air pollution arising from biomass fuel burning. However, knowledge on COPD patho-mechanisms is mainly limited to tobacco smoke exposure. In this study, a repeated direct wood smoke (WS) exposure was performed using normal- (bro-ALI) and chronic bronchitis-like bronchial (bro-ALI-CB), and alveolar (alv-ALI) lung mucosa models at air-liquid interface (ALI) to assess broad toxicological end points. The bro-ALI and bro-ALI-CB models were developed using human primary bronchial epithelial cells and the alv-ALI model was developed using a representative type-II pneumocyte cell line. The lung models were exposed to WS (10 minutes/exposure; 5-exposures over 3-days; n=6-7 independent experiments). Sham exposed samples served as control. WS composition was analyzed following passive sampling. Cytotoxicity, total cellular reactive oxygen species (ROS) and stress responsive NFkB were assessed by flow cytometry. WS exposure induced changes in gene expression were evaluated by RNA-seq (p≤0.01) followed by pathway enrichment analysis. Secreted levels of proinflammatory cytokines were assessed in the basal media. Non-parametric statistical analysis was performed. 147 unique compounds were annotated in WS of which 42 compounds have inhalation toxicity (9 very high). WS exposure resulted in significantly increased ROS in bro-ALI (11.2%) and bro-ALI-CB (25.7%) along with correspondingly increased NFkB levels (bro-ALI: 35.6%; bro-ALI-CB: 18.1%). A total of 1262 (817-up and 445-down), 329 (141-up and 188-down), and 102 (33-up and 69-down) genes were differentially regulated in the WS-exposed bro-ALI, bro-ALI-CB, and alv-ALI models respectively. The enriched pathways included the terms acute phase response, mitochondrial dysfunction, inflammation, oxidative stress, NFkB, ROS, xenobiotic metabolism of AHR, and chronic respiratory disorder. The enrichment of the ‘cilium’ related genes was predominant in the WS-exposed bro-ALI (180-up and 7-down). The pathways primary ciliary dyskinesia, ciliopathy, and ciliary movement were enriched in both WS-exposed bro-ALI and bro-ALI-CB. Interleukin-6 and tumor necrosis factor-α were reduced (p<0.05) in WS-exposed bro-ALI and bro-ALI-CB.

Project description:Exposure to cigarette smoke is a leading cause of lung diseases including chronic obstructive pulmonary disease and cancer. Cigarette smoke is a complex aerosol containing over 6000 chemicals, and thus it is difficult to determine individual contributions to overall toxicity, and the molecular mechanisms by which smoke constituents exert their effects. We selected three well-known harmful and potentially harmful constituents (HPHCs) in tobacco smoke: acrolein, formaldehyde and catechol and established a High Content Screening (HCS) method using normal human bronchial epithelial cells, which are the first bronchial cells in contact with cigarette smoke. The impact of each HPHC was investigated using 13 multi-parametric indicators of cellular toxicity and complemented with a microarray-based whole transcriptome analysis followed by a computational approach leveraging mechanistic network models to identify and quantify perturbed molecular pathways. HPHCs were evaluated over a wide range of concentrations and at different exposure time points (4 h, 8 h, and 24 h). By High Content Screening, the toxic effects of the three HPHCs could only be observed at the highest doses. Whole genome transcriptomics unraveled toxicity mechanisms at lower doses and earlier time points. The most prevalent toxicity mechanisms observed were: DNA damage/growth arrest, oxidative stress, mitochondrial stress and apoptosis/necrosis. In summary, combination of multiple toxicological endpoints with a systems-based impact assessment allows for a more robust scientific basis for the toxicological assessment of HPHCs that allows insight into time- and dose-dependent molecular perturbations of specific biological pathways. This approach allowed us to establish an in vitro Systems Toxicology platform that can be applied to a broader selection of HPHCs and their mixtures and can serve more generally as the basis for testing the impact of other environmental toxicants on normal bronchial epithelial cells.

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Insight into the pulmonary molecular toxicity of heated tobacco products using human bronchial and alveolar mucosa models at air-liquid interface

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