A review of the toxicity of ingredients in e-cigarettes, including those ingredients having the FDA's "Generally Recognized as Safe (GRAS)" regulatory status for use in food.

INTRODUCTION: Some firms and marketers of electronic cigarettes (e-cigarettes; a type of electronic nicotine delivery system (ENDS)) and refill liquids (e-liquids) have made claims about the safety of ingredients used in their products based on the term "GRAS or Generally Recognized As Safe" (GRAS). However, GRAS is a provision within the definition of a food additive under section 201(s) (21 U.S.C. 321(s)) of the U.S. Federal Food Drug and Cosmetic Act (FD&C Act). Food additives and GRAS substances are by the FD&C Act definition intended for use in food, thus safety is based on oral consumption; the term GRAS cannot serve as an indicator of the toxicity of e-cigarette ingredients when aerosolized and inhaled (i.e., vaped). There is no legal or scientific support for labeling e-cigarette product ingredients as "GRAS". This review discusses our concerns with the GRAS provision being applied to e-cigarette products and provides examples of chemical compounds that have been used as food ingredients but have been shown to lead to adverse health effects when inhaled. The review provides scientific insight into the toxicological evaluation of e-liquid ingredients and their aerosols to help determine the potential respiratory risks associated with their use in e-cigarettes. IMPLICATIONS: The rise in prevalence of e-cigarette use and emerging evidence of adverse effects, particularly on lung health, warrant assessing all aspects of e-cigarette toxicity. One development is manufacturers' stated or implied claims of the safety of using e-cigarette products containing ingredients determined to be "Generally Recognized As Safe" (GRAS) for use in food. Such claims, typically placed on e-cigarette product labels and used in marketing, are unfounded, as pointed out by the United States Food and Drug Administration (FDA)1 and the Flavor and Extract Manufacturers Association (FEMA)2. Assessment of inhalation health risks of all ingredients used in e-liquids, including those claimed to be GRAS, is warranted.

Acute Effects of Heated Tobacco Product (IQOS) Aerosol Inhalation on Lung Tissue Damage and Inflammatory Changes in the Lungs.

INTRODUCTION: Emerging heated tobacco products (HTPs) were designed to reduce exposure to toxicants from cigarette smoke (CS) by avoiding burning tobacco and instead heating tobacco. We studied the effects of short-term inhalation of aerosols emitted from HTP called IQOS, on lung damage and immune-cell recruitment to the lungs in mice. METHODS: Numerous markers of lung damage and inflammation including albumin and lung immune-cell infiltrates, proinflammatory cytokines, and chemokines were quantified in lungs and bronchoalveolar (BAL) fluid from IQOS, CS, or air-exposed (negative control) mice. RESULTS: Importantly, as a surrogate marker of lung epithelial-cell damage, we detected significantly increased levels of albumin in the BAL fluid of both HTP- and CS-exposed mice compared with negative controls. Total numbers of leukocytes infiltrating the lungs were equivalent following both IQOS aerosols and CS inhalation and significantly increased compared with air-exposed controls. We also observed significantly increased numbers of CD4+IL-17A+ T cells, a marker of a T-cell immune response, in both groups compared with air controls; however, numbers were the highest following CS exposure. Finally, the numbers of CD4+RORγt+ T cells, an inflammatory T-cell subtype expressing the transcription factor that is essential for promoting differentiation into proinflammatory Th17 cells, were significantly augmented in both groups compared with air-exposed controls. Levels of several cytokines in BAL were significantly elevated, reflecting a proinflammatory milieu. CONCLUSIONS: Our study demonstrates that short-term inhalation of aerosols from IQOS generates damage and proinflammatory changes in the lung that are substantially similar to that elicited by CS exposure. IMPLICATIONS: Exposure of mice to IQOS, one of the candidate modified-risk tobacco products, induces inflammatory immune-cell accumulation in the lungs and augments the levels of proinflammatory cytokines and chemokines in the BAL fluid. Such an exacerbated pulmonary proinflammatory microenvironment is associated with lung epithelial-cell damage in IQOS-exposed mice, suggesting a potential association with the impairment of lung function.

E-cigarette-induced pulmonary inflammation and dysregulated repair are mediated by nAChR α7 receptor: role of nAChR α7 in SARS-CoV-2 Covid-19 ACE2 receptor regulation.

Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanisms that mediate toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChRα7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lung tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased SARS-Cov-2 Covid-19 ACE2 receptor, whereas nAChRα7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8 and MMP9, were altered both at the protein and mRNA transcript levels in female and male KO mice, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChRα7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin, significantly in a sex-dependent manner, but without the direct role of nAChRα7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChRα7 in a sex-dependent manner.

Cannabidiol differentially regulates basal and LPS-induced inflammatory responses in macrophages, lung epithelial cells, and fibroblasts.

INTRODUCTION: Cannabidiol (CBD) containing products are available in a plethora of flavors in oral, sublingual, and inhalable forms. Immunotoxicological effects of CBD containing liquids were assessed by hypothesizing that CBD regulates oxidative stress and lipopolysaccharide (LPS) induced inflammatory responses in macrophages, epithelial cells, and fibroblasts. METHODS: Epithelial cells (BEAS-2B and NHBE), macrophages (U937), and lung fibroblast cells (HFL-1) were treated with varying CBD concentrations or exposed to CBD aerosols. Generated reactive oxygen species (ROS) and inflammatory mediators were measured. Furthermore, monocytes and epithelial cells were stimulated with LPS in combination with CBD or dexamethasone to understand the anti-inflammatory effects of CBD. RESULTS: CBD showed differential effects on IL-8 and MCP-1, and acellular and cellular ROS levels. CBD significantly attenuated LPS-induced NF-κB activity, IL-8, and MCP-1 release from macrophages. Cytokine array data depicted a differential cytokine response due to CBD. Inflammatory mediators, IL-8, serpin E1, CXCL1, IL-6, MIF, IFN-γ, MCP-1, RANTES, and TNF-α were induced, whereas MCP-1/CCL2, CCL5, eotaxin, and IL-2 were reduced. CBD and dexamethasone treatments reduced the IL-8 level induced by LPS when the cells were treated individually, but showed antagonistic effects when used in combination via MCPIP (monocytic chemotactic protein-induced protein). CONCLUSION: CBD differentially regulated basal pro-inflammatory response and attenuated both LPS-induced cytokine release and NF-κB activity in monocytes, similar to dexamethasone. Thus, CBD has a differential inflammatory response and acts as an anti-inflammatory agent in pro-inflammatory conditions but acts as an antagonist with steroids, overriding the anti-inflammatory potential of steroids when used in combination.

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies.

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil-injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

Dietary intake of Deepwater Horizon oil-injected live food fish by double-crested cormorants resulted in oxidative stress.

The Deepwater Horizon oil spill released 134 million gallons of crude oil into the Gulf of Mexico making it the largest oil spill in US history and exposing fish, birds, and marine mammals throughout the Gulf of Mexico to its toxicity. Fish eating waterbirds such as the double-crested cormorant (Phalacrocorax auritus) were exposed to the oil both by direct contact with the oil and orally through preening and the ingestion of contaminated fish. This study investigated the effects of orally ingestedMC252 oil-contaminated live fish food by double-crested cormorants on oxidative stress. Total, reduced, and oxidized glutathione levels, superoxide dismutase and glutathione peroxidase activities, total antioxidant capacity and lipid peroxidation were assessed in the liver tissues of control and treated cormorants. The results suggest that ingestion of the oil-contaminated fish resulted in significant increase in oxidative stress in the liver tissues of these birds. The oil-induced increase in oxidative stress could have detrimental impacts on the bird's life-history.

Commonly used air filters fail to eliminate secondhand smoke induced oxidative stress and inflammatory responses.

Secondhand smoke (SHS) causes approximately 50,000 deaths per year. Despite all the health warnings, smoking is still allowed indoors in many states exposing both workers and patrons to SHS on a daily basis. The opponents of smoking bans suggest that present day air filtration systems remove the health hazards of exposure to SHS. In this study, using an acute SHS exposure model, we looked at the impact of commonly used air filters (MERV-8 pleated and MERV-8 pleated activated charcoal) on SHS by assessing the inflammatory response and the oxidative stress response in C57BL/6 mice. In order to assess the inflammatory response, we looked at the tumor necrosis factor alpha (TNF-α) cytokine production by alveolar macrophages (AMs), and for the oxidative response, we quantified the products of lipid peroxidation and the total glutathione (tGSH) production in lung homogenates. Our results showed that SHS caused significant immune and oxidative stress responses. The tested filters resulted in only a modest alleviation of inflammatory and oxidative responses due to SHS exposure. Our data show that these air filters cannot eliminate the risk of SHS exposure and that a short-term exposure to SHS is sufficient to alter the inflammatory cytokine response and to initiate a complex oxidative stress response. Our results are consistent with the statement made by the Surgeon General's reports that there is no risk free level of exposure to SHS.

Club cell-specific telomere protection protein 1 (TPP1) protects against tobacco smoke-induced lung inflammation, xenobiotic metabolic dysregulation, and injurious responses.

Inhaling xenobiotics, such as tobacco smoke is a major risk factor for pulmonary diseases, e.g., COPD/emphysema, interstitial lung disease, and pre-invasive diseases. Shelterin complex or telosome provides telomeric end protection during replication. Telomere protection protein 1 (TPP1) is one of the main six subunits of the shelterin complex supporting the telomere stability and genomic integrity. Dysfunctional telomeres and shelterin complex are associated as a disease mechanism of tobacco smoke-induced pulmonary damage and disease processes. The airway epithelium is critical to maintaining respiratory homeostasis and is implicated in lung diseases. Club cells (also known as clara cells) play an essential role in the immune response, surfactant production, and metabolism. Disrupted shelterin complex may lead to dysregulated cellular function, DNA damage, and disease progression. However, it is unknown if the conditional removal of TPP1 from Club cells can induce lung disease pathogenesis caused by tobacco smoke exposure. In this study, conditional knockout of Club-cell specific TPP1 demonstrated the instability of other shelterin protein subunits, such as TRF1, dysregulation of cell cycle checkpoint proteins, p53 and downstream targets, and dysregulation of telomeric genes. This was associated with age-dependent senescence-associated genes, increased DNA damage, and upregulated RANTES/IL13/IL33 mediated lung inflammation and injury network by cigarette smoke (CS). These phenomena are also associated with alterations in cytochrome P450 and glutathione transferases, upregulated molecular pathways promoting lung lesions, bronchial neoplasms, and adenocarcinomas. These findings suggest a pivotal role of TPP1 in maintaining lung homeostasis and injurious responses in response to CS. Thus, these data TPP1 may have therapeutic value in alleviating telomere-related chronic lung diseases.

Challenges in current inhalable tobacco toxicity assessment models: A narrative review.

Emerging tobacco products such as electronic nicotine delivery systems (ENDS) and heated tobacco products (HTPs) have a dynamic landscape and are becoming widely popular as they claim to offer a low-risk alternative to conventional smoking. Most pre-clinical laboratories currently exploit in vitro, ex vivo, and in vivo experimental models to assess toxicological outcomes as well as to develop risk-estimation models. While most laboratories have produced a wide range of cell culture and mouse model data utilizing current smoke/aerosol generators and standardized puffing profiles, much variation still exists between research studies, hindering the generation of usable data appropriate for the standardization of these tobacco products. In this review, we discuss current state-of-the-art in vitro and in vivo models and their challenges, as well as insights into risk estimation of novel products and recommendations for toxicological parameters for reporting, allowing comparability of the research studies between laboratories, resulting in usable data for regulation of these products before approval by regulatory authorities.

Pulmonary immune response regulation, genotoxicity, and metabolic reprogramming by menthol- and tobacco-flavored e-cigarette exposures in mice.

Menthol and tobacco flavors are available for almost all tobacco products, including electronic cigarettes (e-cigs). These flavors are a mixture of chemicals with overlapping constituents. There are no comparative toxicity studies of these flavors produced by different manufacturers. We hypothesized that acute exposure to menthol and tobacco-flavored e-cig aerosols induces inflammatory, genotoxicity, and metabolic responses in mouse lungs. We compared two brands, A and B, of e-cig flavors (PG/VG, menthol, and tobacco) with and without nicotine for their inflammatory response, genotoxic markers, and altered genes and proteins in the context of metabolism by exposing mouse strains, C57BL/6J (Th1-mediated) and BALB/cJ (Th2-mediated). Brand A nicotine-free menthol exposure caused increased neutrophils and differential T-lymphocyte influx in bronchoalveolar lavage fluid and induced significant immunosuppression, while brand A tobacco with nicotine elicited an allergic inflammatory response with increased Eotaxin, IL-6, and RANTES levels. Brand B elicited a similar inflammatory response in menthol flavor exposure. Upon e-cig exposure, genotoxicity markers significantly increased in lung tissue. These inflammatory and genotoxicity responses were associated with altered NLRP3 inflammasome and TRPA1 induction by menthol flavor. Nicotine decreased surfactant protein D and increased PAI-1 by menthol and tobacco flavors, respectively. Integration of inflammatory and metabolic pathway gene expression analysis showed immunometabolic regulation in T cells via PI3K/Akt/p70S6k-mTOR axis associated with suppressed immunity/allergic immune response. Overall, this study showed the comparative toxicity of flavored e-cig aerosols, unraveling potential signaling pathways of nicotine and flavor-mediated pulmonary toxicological responses, and emphasized the need for standardized toxicity testing for appropriate premarket authorization of e-cigarette products.

Clearance of senescent cells reverts the cigarette smoke-induced lung senescence and airspace enlargement in p16-3MR mice.

Cigarette smoke (CS) leads to increased oxidative stress, inflammation, and exaggerated senescence, which are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). While the role of cellular senescence in COPD is known, it is not clear if the removal of senescent cells could alleviate the disease symptoms. To test this, we used the novel mouse model-p16-3MR, and studied the effect of ganciclovir (GCV)-mediated removal of senescent cells after chronic CS (3 months) and environmental tobacco smoke (ETS) (6 months) exposure to CS. Our results showed the reversal of CS-induced cellular senescence on the clearance of p16+ senesced cells by GCV treatment. Interestingly, the clearance of p16+ senescent cells via GCV led to a decrease in the neutrophil counts in the BALF of GCV-treated CS-exposed p16-3MR mice, as well as reversal of CS-mediated airspace enlargement in p16-3MR mice. Mice exposed to low dose ETS caused insignificant changes in the SA-ß-Gal+ senescent cells and airspace enlargement. Overall, our data provide evidence for the role of lung cellular senescence on smoke exposure and clearance of senescent cells in p16-3MR mice in the reversal of COPD/emphysema pathology with a possibility of senolytics as therapeutic interventions in COPD.

Correction: Lamb et al. Nose-Only Exposure to Cherry- and Tobacco-Flavored E-Cigarettes Induced Lung Inflammation in Mice in a Sex-Dependent Manner. Toxics 2022, 10, 471.

In the original publication [...].

Nose-Only Exposure to Cherry- and Tobacco-Flavored E-Cigarettes Induced Lung Inflammation in Mice in a Sex-Dependent Manner.

Flavoring chemicals in electronic nicotine delivery systems have been shown to cause cellular inflammation; meanwhile, the effects of fruit and tobacco flavors on lung inflammation by nose-only exposures to mice are relatively unknown. We hypothesized that exposure to flavored e-cigarettes would cause lung inflammation in C57BL/6 J mice. The mice were exposed to air, propylene glycol/vegetable glycerin, and flavored e-liquids: Apple, Cherry, Strawberry, Wintergreen, and Smooth & Mild Tobacco, one hour per day for three days. Quantification of flavoring chemicals by proton nuclear magnetic resonance spectroscopy (1H NMR), differential cell counts by flow cytometry, pro-inflammatory cytokines/chemokines by ELISA, and matrix metalloproteinase levels by western blot were performed. Exposure to PG/VG increased neutrophil cell count in lung bronchoalveolar lavage fluid (BALF). KC and IL6 levels were increased by PG/VG exposure and female mice exposed to Cherry flavored e-cigarettes, in lung homogenate. Mice exposed to PG/VG, Apple, Cherry, and Wintergreen increased MMP2 levels. Our results revealed flavor- and sex-based e-cigarette effects in female mice exposed to cherry-flavored e-liquids and male mice exposed to tobacco-flavored e-liquids, namely, increased lung inflammation.

Noninvasive systemic biomarkers of e-cigarette or vaping use-associated lung injury: a pilot study.

Background: Electronic cigarette (e-cigarette) vaping, containing nicotine and/or Δ8, Δ9 or Δ10 or Δo tetrahydrocannabinol (Δn-THC), is associated with an outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). Despite thousands being hospitalised with EVALI, much remains unknown about diagnosis, treatment and disease pathogenesis. Biomarkers of inflammation, oxidative stress and lipid mediators may help identify e-cigarette users with EVALI. Methods: We collected plasma and urine along with demographic and vaping-related data of EVALI subjects (age 18-35 years) and non-users matched for sex and age in a pilot study. Biomarkers were assessed by ELISA/EIA and Luminex-based assays. Results: Elevated levels of THC metabolite (11-nor-9-carboxy-Δ9-THC) were found in plasma from EVALI subjects compared to non-users. Levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidative DNA damage biomarker, and 8-isoprostane, an oxidative stress marker, were slightly increased in urine samples from EVALI subjects compared to non-users. Conversely, plasma levels of lipid mediators, including resolvin D1 (RvD1) and prostaglandin E2 (PGE2), were significantly lower in EVALI subjects compared to non-users. Both pro-inflammatory biomarkers, such as tumour necrosis factor-α, macrophage inflammatory protein-1ß, RANTES (regulated on activation, normal T-cell expressed and secreted) and granulocyte-macrophage colony-stimulating factor, as well as anti-inflammatory biomarkers, such as interleukin-9 and CC10/16, were decreased in plasma from EVALI subjects compared to non-users, supportive of a possible dysregulated inflammatory response in EVALI subjects. Conclusions: Significant elevations in urine and plasma biomarkers of oxidative stress, as well as reductions in lipid mediators, were shown in EVALI subjects. These noninvasive biomarkers (8-OHdG, 8-isoprostane, RvD1 and CC10/16), either individually or collectively, may serve as tools in diagnosing future EVALI subjects.

Comparative Reactive Oxygen Species (ROS) Content among Various Flavored Disposable Vape Bars, including Cool (Iced) Flavored Bars.

Studies have shown that aerosols generated from flavored e-cigarettes contain Reactive Oxygen Species (ROS), promoting oxidative stress-induced damage within pulmonary cells. Our lab investigated the ROS content of e-cigarette vapor generated from disposable flavored e-cigarettes (vape bars) with and without nicotine. Specifically, we analyzed vape bars belonging to multiple flavor categories (Tobacco, Minty Fruit, Fruity, Minty/Cool (Iced), Desserts, and Drinks/Beverages) manufactured by various vendors and of different nicotine concentrations (0-6.8%). Aerosols from these vape bars were generated via a single puff aerosol generator; these aerosols were then individually bubbled through a fluorogenic solution to semi-quantify ROS generated by these bars in H2O2 equivalents. We compared the ROS levels generated by each vape bar as an indirect determinant of their potential to induce oxidative stress. Our results showed that ROS concentration (µM) within aerosols produced from these vape bars varied significantly among different flavored vape bars and identically flavored vape bars with varying nicotine concentrations. Furthermore, our results suggest that flavoring chemicals and nicotine play a differential role in generating ROS production in vape bar aerosols. Our study provides insight into the differential health effects of flavored vape bars, in particular cool (iced) flavors, and the need for their regulation.

Persistently Increased Systemic ACE2 Activity Is Associated With an Increased Inflammatory Response in Smokers With COVID-19.

Background: Tobacco smoking is known to be involved in the pathogenesis of several cardiopulmonary diseases. Additionally, smokers are highly susceptible to infectious agents due to weakened immunity. However, the progression of lung injury based on SARS-CoV-2-mediated COVID-19 pathogenesis amongst smokers and those with pre-existing pulmonary diseases is not known. We determined the systemic levels and activity of COVID-19 associated proteins, cytokine/chemokines, and lipid mediators (lipidomics) amongst COVID-19 patients with and without a history of smoking to understand the underlying susceptible factor in the pathogenesis of COVID-19. Methods: We obtained serum from healthy (CoV-), COVID-19 positive (CoV+), and COVID-19 recovered (CoV Rec) subjects with and without a history of smoking. We conducted a Luminex multiplex assay (cytokine levels), LC/MS (eicosanoids or oxylipin panel), and ACE2 enzymatic activity assays on the serum samples to determine the systemic changes in COVID-19 patients. Results: On comparing the levels of serum ACE2 amongst COVID-19 (positive and recovered) patients and healthy controls, we found a pronounced increase in serum ACE2 levels in patients with COVID-19 infection. Furthermore, ACE2 enzyme activity was significantly increased amongst COVID-19 patients with a smoking history. Also, we analyzed the levels of Angiotensin 1-7 (Ang1-7) peptide, the product of enzymatic action of ACE2, in the serum samples. We found significantly high levels of Ang1-7 in the serum of both CoV+ and CoV Rec patients. Our data further demonstrated a smoking-induced increase in serum furin and inflammatory cytokine [IFNγ(p = 0.0836), Eotaxin (p < 0.05), MCP-1 (p < 0.05), and IL-9 (p = 0.0991)] levels in COVID-19 patients as compared to non-smoking controls. Overall, our results show that smoking adversely affects the levels of systemic inflammatory markers and COVID-19 associated proteins, thus suggesting that COVID-19 infection may have severe outcomes amongst smokers.

Epithelial Ablation of Miro1/Rhot1 GTPase Augments Lung Inflammation by Cigarette Smoke.

Mitochondrial quality control is sustained by Miro1 (Rhot1), a calcium-binding membrane-anchored GTPase during mitophagy. The exact mechanism that operates the interaction of Miro1 with mitophagy machinery and their role in cigarette smoke (CS)-induced mitochondrial dysfunction that often results in lung inflammation is unclear. We hypothesized that Miro1 plays an important role in regulating mitophagy machinery and the resulting lung inflammation by CS exposure to mice. The lung epithelial Rhot1fl/fl (WT) and Rhot1CreCC10 mice were exposed to mainstream CS for 3 days (acute) and 4 months (chronic). Acute CS exposure showed a notable increase in the total inflammatory cells, macrophages, and neutrophils that are associated with inflammatory mediators. Chronic exposure showed increased infiltration of neutrophils versus air controls. The effects of acute and chronic CS exposure were augmented in the Rhot1CreCC10 group, indicating that epithelial Miro1 ablation led to the augmentation of inflammatory cell infiltration with alteration in the inflammatory mediators. Thus, Rhot1/Miro1 plays an important role in regulating CS-induced lung inflammatory responses with implications in mitochondrial quality control.

Multi-Walled Carbon Nanotubes (MWCNTs) Cause Cellular Senescence in TGF-ß Stimulated Lung Epithelial Cells.

Multi-walled carbon nanotubes are engineered nanomaterials (ENMs) that have a fiber-like structure which may be a concern for the development of cellular senescence. Premature senescence, a state of irreversible cell cycle arrest, is implicated in the pathogenesis of chronic lung diseases such as pulmonary fibrosis (PF). However, the crosstalk between downstream pathways mediating fibrotic and senescent responses of MWCNTs is not well-defined. Here, we exposed human bronchial epithelial cells (BEAS-2B) to MWCNTs for up to 72 h and demonstrate that MWCNTs increase reactive oxygen species (ROS) production accompanied by inhibition of cell proliferation. In addition, MWCNT exposure resulted in the increase of p21 protein abundance and senescence associated ß-galactosidase (SA ß-gal) activity. We also determined that co-exposure with the cytokine, transforming growth factor-ß (TGF-ß) exacerbated cellular senescence indicated by increased protein levels of p21, p16, and γH2A.X. Furthermore, the production of fibronectin and plasminogen activator inhibitor (PAI-1) was significantly elevated with the co-exposure compared to MWCNT or TGF-ß alone. Together, our study suggests that the cellular senescence potential of MWCNTs may be enhanced by pro-fibrotic mediators, such as TGF-ß in the surrounding microenvironment.

Molecular clock REV-ERBα regulates cigarette smoke-induced pulmonary inflammation and epithelial-mesenchymal transition.

Cigarette smoke (CS) is the main etiological factor in the pathogenesis of emphysema/chronic obstructive pulmonary disease (COPD), which is associated with abnormal epithelial-mesenchymal transition (EMT). Previously, we have shown an association among circadian rhythms, CS-induced lung inflammation, and nuclear heme receptor α (REV-ERBα), acting as an antiinflammatory target in both pulmonary epithelial cells and fibroblasts. We hypothesized that molecular clock REV-ERBα plays an important role in CS-induced circadian dysfunction and EMT alteration. C57BL/6J WT and REV-ERBα heterozygous (Het) and -KO mice were exposed to CS for 30 days (subchronic) and 4 months (chronic), and WT mice were exposed to CS for 10 days with or without REV-ERBα agonist (SR9009) administration. Subchronic/chronic CS exposure caused circadian disruption and dysregulated EMT in the lungs of WT and REV-ERBα-KO mice; both circadian and EMT dysregulation were exaggerated in the REV-ERBα-KO condition. REV-ERBα agonist, SR9009 treatment reduced acute CS-induced inflammatory response and abnormal EMT in the lungs. Moreover, REV-ERBα agonist (GSK4112) inhibited TGF-ß/CS-induced fibroblast differentiation in human fetal lung fibroblast 1 (HFL-1). Thus, CS-induced circadian gene alterations and EMT activation are mediated through a Rev-erbα-dependent mechanism, which suggests activation of REV-ERBα as a novel therapeutic approach for smoking-induced chronic inflammatory lung diseases.

Pod-based menthol and tobacco flavored e-cigarettes cause mitochondrial dysfunction in lung epithelial cells.

Current FDA regulations have resulted in a ban of flavored e-cigarette pods, with only menthol and tobacco flavored pods being exempted. Previous work using menthol and tobacco-flavored e-cigarettes have been shown to induce mitochondrial reactive oxygen species. We hypothesized that exposure to pod-based JUUL Menthol and Virginia Tobacco aerosols will alter mitochondrial respiration and electron transport chain protein levels. We determined mitochondrial respiration by using a Seahorse technique and electron transport chain complexes by total OXPHOS antibodies after exposing lung epithelial cells, Beas-2b, to pod-based Menthol and Virginia Tobacco flavored aerosols. Menthol pod exposure resulted in an immediate increase in proton leak and decrease in coupling efficiency, as well as a decrease in complex I, II, and IV. Menthol pod exposure twenty-four hour post-exposure resulted in a decrease in basal respiration, maximal respiration, and spare capacity, as well as a decrease in complex I. Tobacco pod exposure resulted in no significant alterations to mitochondrial respiration, but immediately post final exposure resulted in a significant increase in complex I, IV, and V. Our results indicate that exposure to Menthol flavored e-cigarette pods cause mitochondrial dysfunction in lung epithelial cells.