Investigative analysis of blood-brain barrier penetrating potential of electronic nicotine delivery systems (e-cigarettes) chemicals using predictive computational models.

INTRODUCTION: Seizures are known potential side effects of nicotine toxicity and have been reported in electronic nicotine delivery systems (ENDS, e-cigarettes) users, with the majority involving youth or young adults. AREAS COVERED: Using chemoinformatic computational models, chemicals (including flavors) documented to be present in ENDS were compared to known neuroactive compounds to predict the blood-brain barrier (BBB) penetration potential, central nervous system (CNS) activity, and their structural similarities. The literature search used PubMed/Google Scholar, through September 2023, to identify individual chemicals in ENDS and neuroactive compounds.The results show that ENDS chemicals in this study contain >60% structural similarity to neuroactive compounds based on chemical fingerprint similarity analyses. The majority of ENDS chemicals we studied were predicted to cross the BBB, with approximately 60% confidence, and were also predicted to have CNS activity; those not predicted to passively diffuse through the BBB may be actively transported through the BBB to elicit CNS impacts, although it is currently unknown. EXPERT OPINION: In lieu of in vitro and in vivo testing, this study screens ENDS chemicals for potential CNS activity and predicts BBB penetration potential using computer-based models, allowing for prioritization for further study and potential early identification of CNS toxicity.

Transcriptome analysis reveals lung-specific miRNAs associated with impaired mucociliary clearance induced by cigarette smoke in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is strongly associated with impaired mucociliary clearance (MCC), which has been implicated in the pathogenesis of CS-induced respiratory diseases, such as chronic obstructive pulmonary diseases (COPD). In this study, we aimed to identify microRNAs (miRNAs) that are associated with impaired MCC caused by CS in an in vitro human air-liquid-interface (ALI) airway tissue model. ALI cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min of clean air) from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 1 week (a total of 3 days, 40 min/day). Transcriptome analyses of ALI cultures exposed to the high concentration of CS identified 5090 differentially expressed genes and 551 differentially expressed miRNAs after the third exposure. Genes involved in ciliary function and ciliogenesis were significantly perturbed by repeated CS exposures, leading to changes in cilia beating frequency and ciliary protein expression. In particular, a time-dependent decrease in the expression of miR-449a, a conserved miRNA highly enriched in ciliated airway epithelia and implicated in motile ciliogenesis, was observed in CS-exposed cultures. Similar alterations in miR-449a have been reported in smokers with COPD. Network analysis further indicates that downregulation of miR-449a by CS may derepress cell-cycle proteins, which, in turn, interferes with ciliogenesis. Investigating the effects of CS on transcriptome profile in human ALI cultures may provide not only mechanistic insights, but potential early biomarkers for CS exposure and harm.

Integration of transcriptome analysis with pathophysiological endpoints to evaluate cigarette smoke toxicity in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is a known risk factor in the pathogenesis of smoking-caused diseases, such as chronic obstructive pulmonary diseases (COPD) and lung cancer. To assess the effects of CS on the function and phenotype of airway epithelial cells, we developed a novel repeated treatment protocol and comprehensively evaluated the progression of key molecular, functional, and structural abnormalities induced by CS in a human in vitro air-liquid-interface (ALI) airway tissue model. Cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min clean air) generated from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 4 weeks (3 days per week, 40 min/day). By integrating the transcriptomics-based approach with the in vitro pathophysiological measurements, we demonstrated CS-mediated effects on oxidative stress, pro-inflammatory cytokines and matrix metalloproteinases (MMPs), ciliary function, expression and secretion of mucins, and squamous cell differentiation that are highly consistent with abnormalities observed in airways of smokers. Enrichment analysis on the transcriptomic profiles of the ALI cultures revealed key molecular pathways, such as xenobiotic metabolism, oxidative stress, and inflammatory responses that were perturbed in response to CS exposure. These responses, in turn, may trigger aberrant tissue remodeling, eventually leading to the onset of respiratory diseases. Furthermore, changes of a panel of genes known to be disturbed in smokers with COPD were successfully reproduced in the ALI cultures exposed to CS. In summary, findings from this study suggest that such an integrative approach may be a useful tool for identifying genes and adverse cellular events caused by inhaled toxicants, like CS.

In vitro and in silico genetic toxicity screening of flavor compounds and other ingredients in tobacco products with emphasis on ENDS.

Electronic nicotine delivery systems (ENDS) are regulated tobacco products and often contain flavor compounds. Given the concern of increased use and the appeal of ENDS by young people, evaluating the potential of flavors to induce DNA damage is important for health hazard identification. In this study, alternative methods were used as prioritization tools to study the genotoxic mode of action (MoA) of 150 flavor compounds. In particular, clastogen-sensitive (γH2AX and p53) and aneugen-sensitive (p-H3 and polyploidy) biomarkers of DNA damage in human TK6 cells were aggregated through a supervised three-pronged ensemble machine learning prediction model to prioritize chemicals based on genotoxicity. In addition, in silico quantitative structure-activity relationship (QSAR) models were used to predict genotoxicity and carcinogenic potential. The in vitro assay identified 25 flavors as positive for genotoxicity: 15 clastogenic, eight aneugenic and two with a mixed MoA (clastogenic and aneugenic). Twenty-three of these 25 flavors predicted to induce DNA damage in vitro are documented in public literature to be in e-liquid or in the aerosols produced by ENDS products with youth-appealing flavors and names. QSAR models predicted 46 (31%) of 150 compounds having at least one positive call for mutagenicity, clastogenicity or rodent carcinogenicity, 49 (33%) compounds were predicted negative for all three endpoints, and remaining compounds had no prediction call. The parallel use of these predictive technologies to elucidate MoAs for potential genetic damage, hold utility as a screening strategy. This study is the first high-content and high-throughput genotoxicity screening study with an emphasis on flavors in ENDS products.

Evaluating Mode of Action of Acrolein Toxicity in an In Vitro Human Airway Tissue Model.

Acrolein is a reactive unsaturated aldehyde and is found at high concentrations in both mainstream and side-stream tobacco smoke. Exposure to acrolein via cigarette smoking has been associated with acute lung injury, chronic obstructive pulmonary diseases (COPDs), and asthma. In this study, we developed an in vitro treatment strategy that resembles the inhalation exposure to acrolein experienced by smokers and systematically examined the adverse respiratory effects induced by the noncytotoxic doses of acrolein in a human airway epithelial tissue model. A single 10-min exposure to buffered saline containing acrolein significantly induced oxidative stress and inflammatory responses, with changes in protein oxidation and GSH depletion occurring immediately after the treatment whereas responses in inflammation requiring a manifestation time of at least 24 h. Repeated exposure to acrolein for 10 consecutive days resulted in structural and functional changes that recapitulate the pathological lesions of COPD, including alterations in the beating frequency and structures of ciliated cells, inhibition of mucin expression and secretion apparatus, and development of squamous differentiation. Although some of the early responses caused by acrolein exposure were reversible after a 10-day recovery, perturbations in the functions and structures of the air-liquid-interface (ALI) cultures, such as mucin production, cilia structures, and morphological changes, failed to fully recover over the observation period. Taken together, these findings are consistent with its mode of action that oxidative stress and inflammation have fundamental roles in acrolein-induced tissue remodeling. Furthermore, these data demonstrate the usefulness of analytical methods and testing strategy for recapitulating the key events in acrolein toxicity using an in vitro model.