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.

Figure 1

Schematic presentation of the overall experimental design outlining the exposure regimen and endpoints. ALI: air–liquid interface; alv-ALI: alveolar mucosa model at ALI; bro-ALI: bronchial mucosa model at ALI; h: hours; H441: NCI-H441 (ATCC HTB-174) cell line; HTP: heated tobacco product; IFNγ: interferon gamma; IL: interleukin; IQOS: I quit ordinary smoking; LDH: lactate dehydrogenase; MDA: malondialdehyde; NFkB: nuclear factor kappa-light-chain-enhancer of activated B cells; PARP: cleaved poly [ADP-Ribose] polymerase; PBEC: human primary bronchial epithelial cells; TNFα: tumor necrosis factor alpha; γH2AX: phosphorylated histone H2AX.

Figure 2

Assessment of oxidative stress response by measurement of (a, b) total cellular reactive oxygen species (ROS) and (c, d) expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) p65 subunit by flow cytometry in sham exposed and HTP-smoke exposed bro-ALI and alv-ALI models. Data are shown as medians and interquartile ranges. n = 6 per exposure condition; non-parametric statistical analysis (Wilcoxon signed rank test), *p < 0.05. ALI: air–liquid interface; alv-ALI: alveolar mucosa model at ALI; bro-ALI: bronchial mucosa model at ALI; HTP: heated tobacco product, MFI: mean fluorescent intensity.

Figure 3

Assessment of the levels of DNA damage markers (a, b) 8-hydroxy-2′-deoxyguanosine (8-OHdG; pg/mL), (c, d) cellular phosphorylated histone H2AX (γH2AX) and (e, f) cleaved poly [ADP-Ribose] polymerase (PARP) levels in sham exposed and HTP-smoke exposed bro-ALI and alv-ALI models. Data are shown as medians and interquartile ranges. n = 6 per exposure condition; non-parametric statistical analysis (Wilcoxon signed rank test), *p < 0.05. ALI: air–liquid interface; alv-ALI: alveolar mucosa model at ALI; bro-ALI: bronchial mucosa model at ALI; HTP: heated tobacco product; MFI: mean fluorescent intensity.

Figure 4

Heatmap of the top 25 up regulated and 25 down regulated genes in the bro-ALI model following exposure to HTP-smoke. n = 7 per exposure condition; significantly (raw p < 0.01) regulated genes with the highest fold changes are shown. Genes were ordered by fold-change (HTP-smoke vs Sham) and relative gene expression values are shown across samples (z-scales to mean expression per row). A complete list of the 724 differentially regulated genes is provided in Supplementary Table S1. bro-ALI: bronchial mucosa model at air–liquid interface; HTP: heated tobacco product.

Figure 5

Concentration of secreted proinflammatory cytokines in the basal media of sham exposed and HTP-smoke exposed bro-ALI model. IFNγ: interferon gamma; IL: interleukin; TNFα: tumor necrosis factor alpha. Data are shown as medians and interquartile ranges. n = 6 per exposure condition; non-parametric statistical analysis (Wilcoxon signed rank test); *Significance: p < 0.05. bro-ALI: bronchial mucosa model at air–liquid interface; HTP: heated tobacco product.

Figure 6

Heatmap of the top 25 up regulated and 25 down regulated genes in the alv-ALI model following exposure to HTP-smoke. n = 6 per exposure condition; significantly (raw p < 0.01) regulated genes with the highest fold changes are shown Genes were ordered by fold-change (HTP-smoke vs Sham) and relative gene expression values are shown across samples (z-scales to mean expression per row). A complete list of the 121 differentially regulated genes is provided in Supplementary Table S3. alv-ALI: alveolar mucosa model at air–liquid interface; HTP: heated tobacco product.

Figure 7

Concentration of secreted proinflammatory cytokines in the basal media of sham exposed and HTP-smoke exposed alv-ALI model. IFNγ: interferon gamma; IL: interleukin; TNFα: tumor necrosis factor alpha. Data are shown as medians and interquartile ranges. n = 6 per exposure condition; non-parametric statistical analysis (Wilcoxon signed rank test); *Significance: p < 0.05. alv-ALI: alveolar mucosa model at air–liquid interface; HTP: heated tobacco product.

Figure 8

Assessment of lipid peroxidation levels in the bro-ALI and alv-ALI models due to sham exposure and HTP-smoke exposure. (a, b) Colorimetric malondialdehyde (MDA) assay and (c, d) BODIPY 581/591 C11 (Lipid Peroxidation Sensor) assay using ferrostatin-1 by flow cytometry. Data are shown as medians and interquartile ranges. n = 6 per exposure condition; non-parametric statistical analysis (Wilcoxon signed rank test or Friedman test followed by Wilcoxon signed rank test, as appropriate); ns: not significant; ^#,¤,$,*p < 0.05. MDA levels were below the limit of detection (LOD) in both bro-ALI and alv-ALI sham samples. Therefore, a value just below the LOD (0.85 nM) of MDA is assigned to all the sham samples for statistical analysis. ALI: air–liquid interface; alv-ALI: alveolar mucosa model at ALI; bro-ALI: bronchial mucosa model at ALI; Fer-1: ferrostatin-1; HTP: heated tobacco product; MFI: mean fluorescent intensity.