in-vitro-cannabis-exposures-lung-epithelial-cells-at-air-liquid

<em>In vitro</em> Cannabis Exposures of Lung Epithelial Cells at the Air-Liquid Interface

Cannabis is used by an estimated 192 million people around the world. Most people use cannabis through the inhalation of cannabis smoke, which contains ...

McGill University

Research Institute of the McGill University Heath Centre

Cannabis (marijuana) is the most commonly used illicit product in the world and is the second most smoked plant after tobacco. There has been a rapid increase in the number of countries legalizing cannabis for both recreational and medicinal purposes. Smoking cannabis in the form of a joint is the most common mode of cannabis consumption. Combustion of cannabis smoke generates many of the same chemicals as tobacco smoke. Although the impact of tobacco smoke on respiratory health is well-known, the consequence of cannabis smoke on the respiratory system and, in particular, the inflammatory response is unclear. Besides the combustion products present in cannabis smoke, cannabis also contains cannabinoids including Δ-tetrahydrocannabinol (Δ-THC) and cannabidiol (CBD). These compounds are hydrophobic and not present in aqueous solutions. In order to understand the impact of cannabis smoke on pathological mechanisms associated with adverse respiratory outcomes, the development of  surrogates of cannabis smoke exposure is needed. Therefore, we developed a standardized protocol for the generation of cannabis smoke extract (CaSE) to investigate its effect on cellular mechanisms . First, we determined the concentration of Δ-THC, one of the major cannabinoids, by ELISA and found that addition of methanol to the cell culture media during generation of the aqueous smoke extract significantly increased the amount of Δ-THC. We also observed by LC-MS/MS that CaSE preparation with methanol contains CBD. Using a functional assay in cells for CB1 receptors, the major target of cannabinoids, we found that this CaSE contains Δ-THC which activates CB1 receptors. Finally, this standardized preparation of CaSE induces an inflammatory response in human lung fibroblasts. This study provides an optimized protocol for aqueous CaSE preparation containing biologically active cannabinoids that can be used for  experimentation of cannabis smoke and its potential impact on various indices of pulmonary health.

Standardized Cannabis Smoke Extract Induces Inflammation in Human Lung Fibroblasts.

The lungs, in addition to participating in gas exchange, represent the first line of defense against inhaled pathogens and respiratory toxicants. Cells lining the airways and alveoli include epithelial cells and alveolar macrophages, the latter being resident innate immune cells important in surfactant recycling, protection against bacterial invasion and modulation of lung immune homeostasis. Environmental exposure to toxicants found in cigarette smoke, air pollution and cannabis can alter the number and function of immune cells in the lungs. Cannabis (marijuana) is a plant-derived product that is typically inhaled in the form of smoke from a joint. However, alternative delivery methods such as vaping, which heats the plant without combustion, are becoming more common. Cannabis use has increased in recent years, coinciding with more countries legalizing cannabis for both recreational and medicinal purposes. Cannabis may have numerous health benefits owing to the presence of cannabinoids that dampen immune function and therefore tame inflammation that is associated with chronic diseases such as arthritis. The health effects that could come with cannabis use remain poorly understood, particularly inhaled cannabis products that may directly impact the pulmonary immune system. Herein, we first describe the bioactive phytochemicals present in cannabis, with an emphasis on cannabinoids and their ability to interact with the endocannabinoid system. We also review the current state-of-knowledge as to how inhaled cannabis/cannabinoids can shape immune response in the lungs and discuss the potential consequences of altered pulmonary immunity. Overall, more research is needed to understand how cannabis inhalation shapes the pulmonary immune response to balance physiological and beneficial responses with potential deleterious consequences on the lungs.

Modulation of pulmonary immune function by inhaled cannabis products and consequences for lung disease.

The increasing shift from cannabis smoking to cannabis vaping is largely driven by the perception that vaping to form an aerosol represents a safer alternative to smoking and is a form of consumption appealing to youth. Herein, we compared the chemical composition and receptor-mediated activity of cannabis smoke extract (CaSE) to cannabis vaping extract (CaVE) along with the biological response in human bronchial epithelial cells. Chemical analysis using HPLC and GC/MS revealed that cannabis vaping aerosol contained fewer toxicants than smoke; CaSE and CaVE contained teratogens, carcinogens, and respiratory toxicants. A bioluminescence resonance energy transfer (BRET)-based biosensor detected the receptor-mediated activity of the extracts, primarily driven by Δ-tetrahydrocannabinol (Δ-THC) concentration. RNA-sequencing showed both CaSE and CaVE induced similar transcriptional responses, significantly upregulating genes within pathways related to inflammation, cancer, and cellular stress. This was paralleled by downregulation of pathways related to lipid synthesis and metabolism from both CaSE and CaVE. Targeted metabolomics revealed significant changes in metabolites involved in lipid and membrane metabolism, energy production, nucleotide/DNA/RNA pathways, and oxidative stress response, suggesting potential impairment of lung epithelial cell repair and function. In addition, the upregulation of 5-hydroxymethylcytosine (5hmC) indicates epigenetic changes potentially contributing to inflammation, oxidative stress, and an increased risk of cancer. These findings challenge the notion that cannabis vaping is risk-free, highlighting an urgent need for comprehensive research into its respiratory health effects. This comparison of cannabis consumption methods offers insights that could inform public health policies and raise consumer awareness regarding the potential risks of inhaling cannabis aerosol. Cannabis use is increasing worldwide amid broad acceptance and legalization. The prevalence of traditional smoking is diminishing in favor of vaping dry flower. This is the first study to provide initial evidence that cannabis aerosol contains carcinogenic, teratogenic, and respiratory toxicants that induce transcriptional responses in epithelial cells analogous to those from cannabis smoke, suggesting potential adverse pulmonary effects.

Cannabis vaping elicits transcriptomic and metabolomic changes involved in inflammatory, oxidative stress, and cancer pathways in human bronchial epithelial cells.

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.

Article	Year
Standardized Cannabis Smoke Extract Induces Inflammation in Human Lung Fibroblasts. Frontiers in pharmacology\| PubMed ID: 35418857	2022
Modulation of pulmonary immune function by inhaled cannabis products and consequences for lung disease. Respiratory research\| PubMed ID: 36978106	2023
Cannabis vaping elicits transcriptomic and metabolomic changes involved in inflammatory, oxidative stress, and cancer pathways in human bronchial epithelial cells. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 39823205	2025
Transcriptomic changes in oxidative stress, immunity, and cancer pathways caused by cannabis vapor on alveolar epithelial cells. Cell biology and toxicology\| PubMed ID: 40056285	2025

Emily T. Wilson

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