Cytotoxic impact of nicotine products on periodontal ligament cells.

OBJECTIVES: The primary objective of this in vitro experiment was an assessment of proliferative capacity, metabolic activity, and potential cellular detriment of human periodontal ligament cells (hPDL) exposed to cigarette smoke (CS), electronic cigarette vapor (eCV), and heated tobacco product aerosol (HTP), or air (control). MATERIALS AND METHODS: Using a CAD/CAM-designed exposition chamber, hPDL were exposed to CS, eCV, HTP, or air (control) based on the Health Canada Intense Smoking Regime. Cell proliferation, metabolic activity, and cellular detriment were assessed at various time points. RESULTS: Compared to the control, hPDL exposed to CS exhibited significantly decreased cell numbers at all time points. HTP exposure led to reduced cell numbers 48 h and 72 h post-exposure, while eCV-exposed cells showed no significant decrease. The metabolic activity of eCV-treated hPDL was slightly reduced at 7 h but recovered at 24 h and 48 h. In contrast, CS-treated cells exhibited significantly decreased metabolic activity at 24 h and 48 h, and HTP-exposed cells showed a significant decrease after 48 h. Flow cytometry indicated both apoptotic and necrotic cell death following CS exposure, with necrotic cell death being more pronounced. CONCLUSIONS: eCV and HTP demonstrated comparatively reduced detrimental effects on hPDL compared to CS. CLINICAL RELEVANCE: The findings suggest that conventional cigarette smoke poses a substantial risk to periodontal health by significantly impairing cell proliferation and metabolic activity. However, alternatives such as eCV and HTP may offer a comparatively reduced risk.

Cytotoxic effects of the cigarette smoke extract of heated tobacco products on human oral squamous cell carcinoma: the role of reactive oxygen species and CaMKK2.

BACKGROUND: The increasing prevalence of heated tobacco products (HTPs) has heightened concerns regarding their potential health risks. Previous studies have demonstrated the toxicity of cigarette smoke extract (CSE) from traditional tobacco's mainstream smoke, even after the removal of nicotine and tar. Our study aimed to investigate the cytotoxicity of CSE derived from HTPs and traditional tobacco, with a particular focus on the role of reactive oxygen species (ROS) and intracellular Ca2+. METHODS: A human oral squamous cell carcinoma (OSCC) cell line, HSC-3 was utilized. To prepare CSE, aerosols from HTPs (IQOS) and traditional tobacco products (1R6F reference cigarette) were collected into cell culture media. A cell viability assay, apoptosis assay, western blotting, and Fluo-4 assay were conducted. Changes in ROS levels were measured using electron spin resonance spectroscopy and the high-sensitivity 2',7'-dichlorofluorescein diacetate assay. We performed a knockdown of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) by shRNA lentivirus in OSCC cells. RESULTS: CSE from both HTPs and traditional tobacco exhibited cytotoxic effects in OSCC cells. Exposure to CSE from both sources led to an increase in intracellular Ca2+ concentration and induced p38 phosphorylation. Additionally, these extracts prompted cell apoptosis and heightened ROS levels. N-acetylcysteine (NAC) mitigated the cytotoxic effects and p38 phosphorylation. Furthermore, the knockdown of CaMKK2 in HSC-3 cells reduced cytotoxicity, ROS production, and p38 phosphorylation in response to CSE. CONCLUSION: Our findings suggest that the CSE from both HTPs and traditional tobacco induce cytotoxicity. This toxicity is mediated by ROS, which are regulated through Ca2+ signaling and CaMKK2 pathways.

Secretome profiling of human epithelial cells exposed to cigarette smoke extract and their effect on human lung microvascular endothelial cells.

Cigarette smoke (CS) is one of the leading causes of pulmonary diseases and can induce lung secretome alteration. CS exposure-induced damages to human pulmonary epithelial cells and microvascular endothelial cells have been extensively demonstrated; however, the effects of the secretome of lung epithelial cells exposed to CS extracts (CSE) on lung microvascular endothelial cells are not fully understood. In this study, we aimed to determine the effects of the secretome of lung epithelial cells exposed to CSE on lung microvascular endothelial cells. Human lung epithelial cells, A549, were exposed to CSE, and the secretome was collected. Human lung microvascular endothelial cells, HULEC-5a, were used to evaluate the effect of the secretome of A549 exposed to CSE. Secretome profile, endothelial cell death, inflammation, and permeability markers were determined. CSE altered the secretome expression of A549 cells, and secretome derived from CSE-exposed A549 cells caused respiratory endothelial cell death, inflammation, and moderately enhanced endothelial permeability. This study demonstrates the potential role of cellular interaction between endothelial and epithelial cells during exposure to CSE and provides novel therapeutic targets or beneficial biomarkers using secretome analysis for CSE-related respiratory diseases.

Protective Effect of the Total Alkaloid Extract from Bulbus Fritillariae pallidiflorae in a Mouse Model of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

Purpose: In recent years, the incidence of chronic obstructive pulmonary disease (COPD) has been increasing year by year, but therapeutic drugs has no breakthrough. The total alkaloid extract from Bulbus Fritillariae pallidiflorae (BFP-TA) is widely used in treating lung diseases. Therefore, this study aimed to investigate the protective effect and mechanism of BFP-TA in COPD mice. Methods: BFP-TA was prepared by macroporous adsorbent resin, and the material basis of BFP-TA was analyzed by HPLC-ELSD and UHPLC-MS/MS. Then, the COPD mouse model was induced by cigarette smoke (CS) for 12 weeks, administered at weeks 9-12. Subsequently, the body weight, lung-body ratio, pulmonary function, histopathology, and the levels of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and oxidative stress markers in the serum of mice were determined. The expressions of related protein of EMT and MAPK signaling pathways in the lung tissues of mice were detected by Western blot. Results: The alkaloid relative content of BFP-TA is 64.28%, and nine alkaloids in BFP-TA were identified and quantified by UHPLC-MS/MS. Subsequently, the animal experiment showed that BFP-TA could improve pulmonary function, and alleviate inflammatory cell infiltration, pulmonary emphysema, and collagen fiber deposition in the lung of COPD mice. Furthermore, BFP-TA could decrease the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), MMPs (MMP-9 and MMP-12) and MDA, while increase the levels of TIMP-1 and SOD. Moreover, BFP-TA could decrease the protein expressions of collagen I, vimentin, α-SMA, MMP-9, MMP-9/TIMP-1, Bax, p-JNK/JNK, p-P38/P38, and p-ERK/ERK, while increase the level of E-cadherin. Conclusion: This study is the first to demonstrate the protective effect of BFP-TA in CS-induced COPD mouse model. Furthermore, BFP-TA may improve airway remodeling by inhibiting the EMT process and potentially exert anti-inflammatory effect by inhibiting the MAPK signaling pathway.

Dual Exposure to E-Cigarette Vapour and Cigarette Smoke Results in Poorer Airway Cell, Monocyte, and Macrophage Function Than Single Exposure.

E-cigarette users predominantly also continue to smoke cigarettes. These Dual Users either consume e-cigarettes in locations where smoking is not allowed, but vaping is, or to reduce their consumption of cigarettes, believing it will lead to harm reduction. Whilst it is known that e-cigarette vapour is chemically less complex than cigarette smoke, it has a distinct chemical profile, and very little is known about the health impacts of exposure to both chemical profiles vs. either alone. We simultaneously exposed cells in vitro to non-toxic levels of e-cigarette vapour extract (EVE) and cigarette smoke extract (CSE) to determine their effects on 16HBE14o- airway epithelial cell metabolism and inflammatory response, as well as immune cell (THP-1 cells and monocyte-derived macrophages (MDM) from healthy volunteers) migration, phagocytosis, and inflammatory response. We observed increased toxicity, reduced metabolism (a marker of proliferation) in airway epithelial cells, and reduced monocyte migration, macrophage phagocytosis, and altered chemokine production after exposure to either CSE or EVE. These cellular responses were greater after dual exposure to CSE and EVE. The airway epithelial cells from smokers showed reduced metabolism after EVE (the Switcher model) and dual CSE and EVE exposure. When EVE and CSE were allowed to interact, the chemicals were found to be altered, and new chemicals were also found compared to the CSE and EVE profiles. Dual exposure to e-cigarette vapour and cigarette smoke led to worse functional outcomes in cells compared to either single exposure alone, adding to limited data that dual use may be more dangerous than smoking only.

FBXL19 Targeted STK11 Degradation Enhances Cigarette Smoke-Induced Airway Epithelial Cell Cytotoxicity.

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11's role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs-human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

[Aloperine suppresses TLR4/NF-κB/NLRP3 signaling to ameliorate cigarette smoke-induced injury to human bronchial epithelial cells].

Objective To explore the effects of aloperine (Alo) on cigarette smoke-induced injury in human bronchial epithelial cells and its potential mechanism. Methods After human bronchial epithelial 16HBE cells were co-treated by 100 mL/L cigarette smoke extract (CSE) and various concentrations (50,100 and 200 µmol/L) of Alo, cell viability was assessed using CCK-8 assay. Lactate dehydrogenase (LDH) activity was measured with a related kit. Cell apoptosis was evaluated using the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) and Western blot analysis. The levels of inflammatory factors were detected by ELISA. Oxidative stress levels were assessed using 2'7'-dichlorofluorescin diacetate (DCFH-DA) staining. The expression of Toll-like receptor 4 (TLR4)/nuclear factor-kappaB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3) signaling-associated proteins was measured by Western blot analysis. After cells were co-treated with 100 mL/L CSE and 200 µmol/L Alo, the aforementioned assays were applied to evaluate the effects of TLR4 overexpression on the TLR4/NF-κB/NLRP3 signaling, LDH activity, apoptosis, inflammatory response and oxidative stress in cells. Results CSE exposure might inhibit 16HBE cell viability, increase LDH activity, apoptosis, inflammatory response and oxidative stress levels and activate TLR4/NF-κB/NLRP3 signaling. Treatment with Alo promoted cell viability, decreased LDH activity, cell apoptosis, inflammation and oxidative stress levels, and inactivated TLR4/NF-κB/NLRP3 signaling. Furthermore, TLR4 overexpression might reverse the protective role of Alo treatment in CSE-induced injury in 16HBE cells. Conclusion Alo may ameliorate CSE-induced injury in human bronchial epithelial cells via inhibiting TLR4/NF-κB/NLRP3 signaling.

Exposure to fine particulate matter 2.5 from wood combustion smoke causes vascular changes in placenta and reduce fetal size.

During gestation, maternal blood flow to the umbilical cord and placenta increases, facilitating efficient nutrient absorption, waste elimination, and effective gas exchange for the developing fetus. However, the effects of exposure to wood smoke during this period on these processes are unknown. We hypothesize that exposure to PM2.5, primarily sourced from wood combustion for home heating, affects placental vascular morphophysiology and fetal size. We used exposure chambers that received either filtered or unfiltered air. Female rats were exposed to PM2.5 during pre-gestational and/or gestational stages. Twenty-one days post-fertilization, placentas were collected via cesarean section. In these placentas, oxygen diffusion capacity was measured, and the expression of angiogenic factors was analyzed using qPCR and immunohistochemistry. In groups exposed to PM2.5 during pre-gestational and/or gestational stages, a decrease in fetal weight, crown-rump length, theoretical and specific diffusion capacity, and an increase in HIF-1α expression were observed. In groups exposed exclusively to PM2.5 during the pre-gestational stage, there was an increase in the expression of placental genes Flt-1, Kdr, and PIGF. Additionally, in the placental labyrinth region, the expression of angiogenic factors was elevated. Changes in angiogenesis and angiogenic factors reflect adaptations to hypoxia, impacting fetal growth and oxygen supply. In conclusion, this study demonstrates that exposure to PM2.5, emitted from wood smoke, in both pre-gestational and gestational stages, affects fetal development and placental health. This underscores the importance of addressing air pollution in areas with high levels of wood smoke, which poses a significant health risk to pregnant women and their fetuses.

Systemic immunological responses are dependent on sex and ovarian hormone presence following acute inhaled woodsmoke exposure.

BACKGROUND: Rural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility. METHODS: This exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode. RESULTS: Metals analysis of WS particulate matter (PM) revealed significantly increased levels of 63Cu, 182W, 208Pb, and 238U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1ß, TNF-α, CXCL-1, CCL-5, TGF-ß, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1ß, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1ß, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche. CONCLUSIONS: In summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.

Cigarette smoke-induced dysbiosis: comparative analysis of lung and intestinal microbiomes in COPD mice and patients.

BACKGROUND: The impact of cigarette smoke (CS) on lung diseases and the role of microbiome dysbiosis in chronic obstructive pulmonary disease (COPD) have been previously reported; however, the relationships remain unclear. METHODS: Our research examined the effects of 20-week cigarette smoke (CS) exposure on the lung and intestinal microbiomes in C57BL/6JNarl mice, alongside a comparison with COPD patients' intestinal microbiome data from a public dataset. RESULTS: The study found that CS exposure significantly decreased forced vital capacity (FVC), thickened airway walls, and induced emphysema. Increased lung damage was observed along with higher lung keratinocyte chemoattractant (KC) levels by CS exposure. Lung microbiome analysis revealed a rise in Actinobacteriota, while intestinal microbiome showed significant diversity changes, indicating dysbiosis. Principal coordinate analysis highlighted distinct intestinal microbiome compositions between control and CS-exposed groups. In the intestinal microbiome, notable decreases in Patescibacteria, Campilobacterota, Defferibacterota, Actinobacteriota, and Desulfobacterota were observed. We also identified correlations between lung function and dysbiosis in both lung and intestinal microbiomes. Lung interleukins, interferon-É£, KC, and 8-isoprostane levels were linked to lung microbiome dysbiosis. Notably, dysbiosis patterns in CS-exposed mice were similar to those in COPD patients, particularly of Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 4 patients. This suggests a systemic impact of CS exposure. CONCLUSION: In summary, CS exposure induces significant dysbiosis in lung and intestinal microbiomes, correlating with lung function decline and injury. These results align with changes in COPD patients, underscoring the important role of microbiome in smoke-related lung diseases.

NADH Intraperitoneal Injection Prevents Lung Inflammation in a BALB/C Mice Model of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

Cigarette smoke is one of the main factors in Chronic Obstructive Pulmonary Disease (COPD), a respiratory syndrome marked by persistent respiratory symptoms and increasing airway obstruction. Perturbed NAD+/NADH levels may play a role in various diseases, including lung disorders like COPD. In our study, we investigated the preventive effect of NADH supplementation in an experimental model of COPD induced by cigarette smoke extract (CSE). N = 64 mice randomly distributed in eight groups were injected with NADH (two doses of 100 mg/kg or 200 mg/kg) or dexamethasone (2 mg/kg) before being exposed to CSE for up to 9 weeks. Additionally, NADH supplementation preserved lung antioxidant defenses by preventing the functional loss of key enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase, and the expression levels of glutathione (GSH) (n = 4, p < 0.001). It also reduced oxidative damage markers, such as malondialdehyde (MDA) and nitrites (n = 4, p < 0.001). A marked increase in tissue myeloperoxidase activity was assessed (MPO), confirming neutrophils implication in the inflammatory process. The latter was significantly ameliorated in the NADH-treated groups (p < 0.001). Finally, NADH prevented the CSE-induced secretion of cytokines such as Tumor Necrosis Factor alpha (TNF-α), IL-17, and IFN-y (n = 4, p < 0.001). Our study shows, for the first time, the clinical potential of NADH supplementation in preventing key features of COPD via its unique anti-inflammatory and antioxidant properties.

Unburned Tobacco Smoke Affects Neuroinflammation-Related Pathways in the Rat Mesolimbic System.

Tobacco use disorder represents a significant public health challenge due to its association with various diseases. Despite awareness efforts, smoking rates remain high, partly due to ineffective cessation methods and the spread of new electronic devices. This study investigated the impact of prolonged nicotine exposure via a heat-not-burn (HnB) device on selected genes and signaling proteins involved in inflammatory processes in the rat ventral tegmental area (VTA) and nucleus accumbens (NAc), two brain regions associated with addiction to different drugs, including nicotine. The results showed a reduction in mRNA levels for PPARα and PPARγ, two nuclear receptors and anti-inflammatory transcription factors, along with the dysregulation of gene expression of the epigenetic modulator KDM6s, in both investigated brain areas. Moreover, decreased PTEN mRNA levels and higher AKT phosphorylation were detected in the VTA of HnB-exposed rats with respect to their control counterparts. Finally, significant alterations in ERK 1/2 phosphorylation were observed in both mesolimbic areas, with VTA decrease and NAc increase, respectively. Overall, the results suggest that HnB aerosol exposure disrupts intracellular pathways potentially involved in the development and maintenance of the neuroinflammatory state. Moreover, these data highlight that, similar to conventional cigarettes, HnB devices use affects specific signaling pathways shaping neuroinflammatory process in the VTA and NAc, thus triggering mechanisms that are currently considered as potentially relevant for the development of addictive behavior.

Therapeutic effect of ginkgetin on smoke-induced airway inflammation by down-regulating the c/EBPß signaling pathway and CCL2 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba leaf and seed have been traditionally used in ancient China for the treatment of cough and asthma. However, there is limited literature available on the anti-COPD effects and mechanisms of Ginkgo biloba. AIMS OF THE STUDY: The aim of this study was to comprehensively investigate the therapeutic potential of ginkgo extracts in COPD through a combination of in vivo and in vitro functional experiments. Transcriptomic analyses were also employed to uncover novel molecular mechanisms underlying the therapeutic effects of ginkgetin in COPD. MATERIALS AND METHODS: The therapeutic efficacy of ginkgo extracts was assessed in a COPD model. The anti-inflammatory effects of ginkgetin and its underlying molecular mechanisms were examined in A549 cells treated with cigarette smoke extract (CSE). Additionally, transcriptomic analyses were conducted to identify novel molecular pathways influenced by ginkgetin. These findings were further validated using quantitative real-time polymerase chain reaction (qPCR) and Western blot techniques. RESULTS: The ethyl acetate extract of Ginkgo biloba L. seeds and ginkgetin treatment significantly reduced cytokine production in COPD mice. Following drug administration, lung function improved in different groups. The transcriptome data strongly supports the inhibitory effect of ginkgetin on CSE-induced inflammation through the downregulation of the c/EBPß signaling pathway and subsequent inhibition of CCL2 expression. CONCLUSION: Our results demonstrate that ginkgetin, one of the biflavones found in Ginkgo biloba, exhibits inhibitory effects on smoke-induced airway inflammation. This effect is achieved through the downregulation of the c/EBPß signaling pathway and the reduction of CCL2 expression.

Cigarette smoke sustains immunosuppressive microenvironment inducing M2 macrophage polarization and viability in lung cancer settings.

BACKGROUND: It is amply demonstrated that cigarette smoke (CS) has a high impact on lung tumor progression worsening lung cancer patient prognosis and response to therapies. Alteration of immune cell types and functions in smokers' lungs have been strictly related with smoke detrimental effects. However, the role of CS in dictating an inflammatory or immunosuppressive lung microenvironment still needs to be elucidated. Here, we investigated the effect of in vitro exposure to cigarette smoke extract (CSE) focusing on macrophages. METHODS: Immortalized murine macrophages RAW 264.7 cells were cultured in the presence of CS extract and their polarization has been assessed by Real-time PCR and cytofluorimetric analysis, viability has been assessed by SRB assay and 3D-cultures and activation by exposure to Poly(I:C). Moreover, interaction with Lewis lung carcinoma (LLC1) murine cell models in the presence of CS extract were analyzed by confocal microscopy. RESULTS: Obtained results indicate that CS induces macrophages polarization towards the M2 phenotype and M2-phenotype macrophages are resistant to the CS toxic activity. Moreover, CS impairs TLR3-mediated M2-M1 phenotype shift thus contributing to the M2 enrichment in lung smokers. CONCLUSIONS: These findings indicate that, in lung cancer microenvironment of smokers, CS can contribute to the M2-phenotype macrophages prevalence by different mechanisms, ultimately, driving an anti-inflammatory, likely immunosuppressive, microenvironment in lung cancer smokers.

Tobacco Smoke Condensate Induces Morphologic Changes in Human Papillomavirus-Positive Cervical Epithelial Cells Consistent with Epithelial to Mesenchymal Transition (EMT) with Activation of Receptor Tyrosine Kinases and Regulation of TGFB.

High-risk human papillomavirus (HR-HPV; HPV-16) and cigarette smoking are associated with cervical cancer (CC); however, the underlying mechanism(s) remain unclear. Additionally, the carcinogenic components of tobacco have been found in the cervical mucus of women smokers. Here, we determined the effects of cigarette smoke condensate (CSC; 3R4F) on human ectocervical cells (HPV-16 Ect/E6E7) exposed to CSC at various concentrations (10-6-100 µg/mL). We found CSC (10-3 or 10 µg/mL)-induced proliferation, enhanced migration, and histologic and electron microscopic changes consistent with EMT in ectocervical cells with a significant reduction in E-cadherin and an increase in the vimentin expression compared to controls at 72 h. There was increased phosphorylation of receptor tyrosine kinases (RTKs), including Eph receptors, FGFR, PDGFRA/B, and DDR2, with downstream Ras/MAPK/ERK1/2 activation and upregulation of common EMT-related genes, TGFB SNAI2, PDGFRB, and SMAD2. Our study demonstrated that CSC induces EMT in ectocervical cells with the upregulation of EMT-related genes, expression of protein biomarkers, and activation of RTKs that regulate TGFB expression, and other EMT-related genes. Understanding the molecular pathways and environmental factors that initiate EMT in ectocervical cells will help delineate molecular targets for intervention and define the role of EMT in the initiation and progression of cervical intraepithelial neoplasia and CC.

Involvement of GPR43 Receptor in Effect of Lacticaseibacillus rhamnosus on Murine Steroid Resistant Chronic Obstructive Pulmonary Disease: Relevance to Pro-Inflammatory Mediators and Oxidative Stress in Human Macrophages.

BACKGROUND: Cytokine storm and oxidative stress are present in chronic obstructive pulmonary disease (COPD). Individuals with COPD present high levels of NF-κB-associated cytokines and pro-oxidant agents as well as low levels of Nrf2-associated antioxidants. This condition creates a steroid-resistant inflammatory microenvironment. Lacticaseibacillus rhamnosus (Lr) is a known anti-cytokine in lung diseases; however, the effect of Lr on lung inflammation and oxidative stress in steroid-resistant COPD mice remains unknown. OBJECTIVE: Thus, we investigated the Lr effect on lung inflammation and oxidative stress in mice and macrophages exposed to cigarette smoke extract (CSE) and unresponsive to steroids. METHODS: Mice and macrophages received dexamethasone or GLPG-094 (a GPR43 inhibitor), and only the macrophages received butyrate (but), all treatments being given before CSE. Lung inflammation was evaluated from the leukocyte population, airway remodeling, cytokines, and NF-κB. Oxidative stress disturbance was measured from ROS, 8-isoprostane, NADPH oxidase, TBARS, SOD, catalase, HO-1, and Nrf2. RESULTS: Lr attenuated cellularity, mucus, collagen, cytokines, ROS, 8-isoprostane, NADPH oxidase, and TBARS. Otherwise, SOD, catalase, HO-1, and Nrf2 were upregulated in Lr-treated COPD mice. Anti-cytokine and antioxidant effects of butyrate also occurred in CSE-exposed macrophages. GLPG-094 rendered Lr and butyrate less effective. CONCLUSIONS: Lr attenuates lung inflammation and oxidative stress in COPD mice, suggesting the presence of a GPR43 receptor-dependent mechanism also found in macrophages.

Effects of tobacco smoke on the expression of virulence genes in Escherichia coli.

It is widely acknowledged that smoking exacerbates the severity of infectious diseases. A presumed mechanism involves the damage inflicted by tobacco smoke on the organs of host organisms. In this study, an alternative hypothesis was explored: smoking enhances the virulence of bacteria. This possibility was investigated using Escherichia coli as the model bacteria and Drosophila as the host organism. Our inquiry focused on the potential gene expression changes in E. coli subsequent to exposure to tobacco smoke extracts. Analysis of the transcription promoter activity of genes encoding proteins within the E. coli two-component system, a regulatory machinery governing gene expression, revealed the suppression of thirteen out of 23 promoters in response to tobacco smoke extracts. Subsequently, Drosophila was infected with E. coli exposed to tobacco smoke extracts or left untreated. Interestingly, there were no significant differences observed in the survival periods of Drosophila following infection with E. coli, whether treated or untreated with tobacco smoke extracts. Contrary to the initial hypothesis, the findings suggest that while tobacco smoke extracts alter gene expression in E. coli, these changes do not appear to impact bacterial virulence. Although this study has illuminated the influence of tobacco smoke extracts on the gene expression of E. coli, further analyses are necessary to elucidate the implications of these changes. Nevertheless, the results imply that smoking affects not only host organisms but may also exert influence on invading bacteria.

Exploring the adverse effect of fine particulate matter (PM2.5) on wildland firefighters' pulmonary function and DNA damage.

Chiang Mai encounters severe pollution during the wildfire season. Wildland firefighters encounter various hazards while engaged in fire suppression operations, which encompass significant exposure to elevated concentrations of air pollutants resulting from combustion, especially particulate matter. The adverse effects of wildfire smoke on respiratory health are a significant concern. The objective of this study was to examine the potential adverse effects of PM2.5 exposure on the respiratory function and DNA damage of wildland firefighters. This prospective cohort study conducted in Chiang Mai from January to May 2022 planned to evaluate the health status of wildland firefighters during the pre-peak, peak, and post-peak ambient air pollution seasons. The measurement of PM2.5 was done at every forest fire station, as well as utilizing data from the Pollution Control Department. Participants received general health examinations, spirometry evaluations, and blood tests for DNA damage analysis. Pair t-tests and multiple regression models were used to examine the connection between pulmonary function parameters (FVC, FEV1) and PM2.5 concentration, with a significance level of P < 0.05. Thirty-three peak-season and twenty-one post-peak-season participants were enrolled. Four pre-peak-season wildland firefighters had FVC and FEV1 declines of > 15%. Multiple regression analysis showed a negative association between PM2.5 exposure and FVC% predicted (- 2.81%, 95% CI - 5.27 to - 0.34%, P = 0.027) and a marginally significant negative correlation with FVC (- 114.38 ml, 95% CI - 230.36 to 1.59 ml, P = 0.053). The remaining pulmonary measures showed a statistically insignificant decline. There were no significant changes in DNA damage detected. Wildland firefighters suffered a significant decline in pulmonary function associated with PM2.5 exposure. Spirometry is crucial for monitoring and promptly identifying respiratory issues that occur during wildfire seasons. Further research is recommended to explore DNA damage alterations and their potential association with PM2.5.

Role of reactive carbonyls and superoxide radicals in protein damage by cigarette smoke extracts: Comparison of Heat-not-Burn e-cigarettes to conventional cigarettes.

Oxidative protein damage involving carbonylation of respiratory tract proteins typically accompanies exposure to tobacco smoke. Such damage can arise via multiple mechanisms, including direct amino acid oxidation by reactive oxygen species or protein adduction by electrophilic aldehydes. This study investigated the relative importance of these pathways during exposure of a model protein to fresh cigarette emission extracts. Briefly, protein carbonyl adducts were estimated in bovine serum albumin following incubation in buffered solutions with whole cigarette emissions extracts prepared from either a single 1R6F research cigarette or a single "Heat-not-Burn" e-cigarette. Although both extracts caused concentration-dependent protein carbonylation, conventional cigarette extracts produced higher adduct yields than e-cigarette extracts. Superoxide radical generation by conventional and e-cigarette emissions was assessed by monitoring nitro blue tetrazolium reduction and was considerably lower in extracts made from "Heat-Not-Burn" e-cigarettes. The superoxide dismutase/catalase mimic EUK-134 strongly suppressed radical production by whole smoke extracts from conventional cigarettes, however, it did not diminish protein carbonyl adduction when incubating smoke extracts with the model protein. In contrast, edaravone, a neuroprotective drug with strong carbonyl-trapping properties, strongly suppressed protein damage without inhibiting superoxide formation. Although these findings require extension to appropriate cell-based and in vivo systems, they suggest reactive aldehydes in tobacco smoke make greater contributions to oxidative protein damage than smoke phase radicals.

Burn Pit Smoke Condensate-Mediated Toxicity in Human Airway Epithelial Cells.

Burn pits are a method of open-air waste management that was common during military operations in Iraq, Afghanistan, and other regions in Southwest Asia. Veterans returning from deployment have reported respiratory symptoms, potentially from exposure to burn pit smoke, yet comprehensive assessment of such exposure on pulmonary health is lacking. We have previously shown that exposure to condensates from burn pit smoke emissions causes inflammation and cytotoxicity in mice. In this study, we explored the effects of burn pit smoke condensates on human airway epithelial cells (HAECs) to understand their impact on cellular targets in the human lung. HAECs were cultured at the air-liquid interface (ALI) and exposed to burn pit waste smoke condensates (plywood, cardboard, plastic, mixed, and mixed with diesel) generated under smoldering and flaming conditions. Cytotoxicity was evaluated by measuring transepithelial electrical resistance (TEER) and lactate dehydrogenase (LDH) release; toxicity scores (TSs) were quantified for each exposure. Pro-inflammatory cytokine release and modulation of gene expression were examined for cardboard and plastic condensate exposures. Burn pit smoke condensates generated under flaming conditions affected cell viability, with flaming mixed waste and plywood exhibiting the highest toxicity scores. Cardboard and plastic smoke condensates modulated cytokine secretion, with GM-CSF and IL-1ß altered in more than one exposure group. Gene expression of detoxifying enzymes (ALDH1A3, ALDH3A1, CYP1A1, CYP1B1, NQO1, etc.), mucins (MUC5AC, MUC5B), and cytokines was affected by several smoke condensates. Particularly, expression of IL6 was elevated following exposure to all burn pit smoke condensates, and polycyclic aromatic hydrocarbon acenaphthene was positively associated with the IL-6 level in the basolateral media of HAECs. These observations demonstrate that exposure to smoke condensates of materials present in burn pits adversely affects HAECs and that aberrant cytokine secretion and altered gene expression profiles following burn pit material smoke exposure could contribute to the development of airway disease.