Transcriptomic changes in oxidative stress, immunity, and cancer pathways caused by cannabis vapor on alveolar epithelial cells

Abstract

As legalization of cannabis increases worldwide, vaping cannabis is gaining popularity due to the belief that it is less harmful than smoking cannabis. However, the safety of cannabis vaping remains untested. To address this, we developed a physiologically relevant method for in vitro assessment of cannabis vapor on alveolar epithelial cell cultures. We compared the transcriptional response in three in vitro models of cannabis vapor exposure using A549 epithelial cells in submerged culture, pseudo-air liquid interface (ALI) culture, and ALI culture coupled with the expoCube™ advanced exposure system. Baseline gene expression in ALI-maintained A549 cells showed higher expression of type 2 alveolar epithelial (AEC2) genes related to surfactant production, ion movement, and barrier integrity. Acute exposure to cannabis vapor significantly affected gene expression in AEC2 cells belonging to pathways related to cancer, oxidative stress, and the immune response without being associated with a DNA damage response. This study identifies potential risks of cannabis vaping and underscores the need for further exploration into its respiratory health implications.

Keywords: Air–liquid interface; Cannabis vapor; Lung cancer; New approach methods.

Fig. 1

CaVE causes inflammation in A549 cells cultured under submerged conditions. (a) Cytotoxicity of CaVE or volume-equivalent vehicle control across a dose range of 0–4.5 µg ${\mathrm{\Delta }}^9$-THC/ml. (b) PCA of cell cultures treated with CaVE, ethanol vehicle control, or negative control (c) Volcano plot showing the transcriptional response of CaVE versus vehicle control in submerged A549 cells. (d) GSEA of pathways significantly enriched in response to CaVE. (e) Cnetplot illustrating gene similarities and overlaps among the top enriched pathways. (f–k) Heatmaps with z-scores of genes involved in the top enriched pathways in response to CaVE, including TNF Signaling (f), Inflammatory Response (g), Hypoxia (h), E2F Targets (i), G2M Checkpoint (j), and Cholesterol Homeostasis (k)

Fig. 2

Cells cultured at ALI show decreased markers of NSCLC and increased markers of alveolar epithelial cells. (a) Heatmap displaying z-scores of NSCLC markers and cell cycle regulators across submerged, pseudo-ALI, and ALI culture conditions. (b) Expression of the AEC2 marker SFTPC is elevated in pseudo-ALI and ALI cultures. (c-d) Culturing cells at ALI enhances the expression of genes associated with alveolar functions, including (c) barrier integrity markers CDH1, TJP3, OCLN, and CLDN4 and (d) ion transport-related genes AQP3, SLC4A4, and CFTR. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 3

CaVE alters metabolism and cell growth pathways in pseudo-ALI cultures. (a) Cytotoxicity of CaVE, volume-equivalent ethanol control and negative control in pseudo-ALI cultures. (b) PCA of transcriptional data from pseudo-ALI A549 cultures treated with CaVE, vehicle control, or negative control. (c) Volcano plot of the transcriptional response of CaVE versus vehicle control in pseudo-ALI A549 cells. (d) GSEA of significantly enriched pathways in response to CaVE. (e) Cnetplot showing gene similarities and overlaps among the top enriched pathways. (f–i) Heatmaps with z-scores of genes involved in the top enriched pathways, including Mitotic Spindle (f), G2M Checkpoint (g), E2F Targets (h), and Oxidative Phosphorylation (i)

Fig. 4

Cannabis vapor alters metabolism and cell growth pathways in ALI cultures. (a) Cannabis vapor deposition in cell culture. (b) Cytotoxicity of cannabis vapor or air vehicle control compared to negative control. (c) PCA of transcriptional data from A549 cells treated with cannabis vapor, vehicle control, or negative control. (d) Volcano plot of the transcriptional response of cannabis vapor versus vehicle control in ALI A549 cells. (e) GSEA of significantly enriched pathways in response to cannabis vapor exposure. (f) Cnetplot showing gene similarities and overlaps among the top enriched pathways. (g–l) Heatmaps with z-scores of genes involved in the top enriched pathways, including Unfolded Protein Response (g), MTORC1 Signaling (h), Epithelial-Mesenchymal Transition (i), Complement (j), KRAS Signaling (k), and Estrogen Response (l)

Fig. 5

Cannabis vapor does not elicit genotoxic effects in alveolar epithelial cells. (a) Heatmap, (b) PCA, and (c) hierarchical clustering using Euclidean distances with average linkage of transcriptome profiling data illustrating co-expressed sets of genes associated with genotoxic and non-genotoxic compounds

Fig. 6

Transcriptional changes in A549 cells in response to cannabis vapor exposure across submerged, pseudo-ALI, and ALI models. (a) Venn diagram showing DEGs upregulated by cannabis vapor across models. (b) Venn diagram showing DEGs downregulated by cannabis across models. (c) GSEA of DEGs common to all models. (d) Cnetplot of enriched pathways. (e–h) Heatmaps displaying log2 fold change values of DEGs of cannabis vapor versus vehicle control in each model related to (e) Mitotic Spindle, (f) UV Response, (g) Epithelial-Mesenchymal Transition, and (h) Oxidative Phosphorylation pathways