Component Of Cigarette Smoke Research Articles

Abstract 532: Dioxin Modifies The Transcriptomic And Epigenetic Landscape Of Smooth Muscle Cells Leading To Adverse Remodeling Of Atherosclerosis

Background: Environmental exposure to dioxin, an active component of cigarette smoke and air pollution has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity. We have previously found strong dioxin response element activity in modulated SMC of atherosclerotic lesion. In this study, we aimed to understand the effect of dioxin on vascular SMC and atherosclerosis phenotype. Methods: Primary human coronary artery SMC (HCASMC) treated in culture with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or vehicle control were used to perform RNA-Seq and ATAC-Seq. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21. Tamoxifen inducible SMC-lineage tracing reporter mice were used to assess the in-vivo effect of TCDD on aortic SMC. After 8 weeks of high fat diet (HFD), followed by 8 weeks of TCDD injection and HFD, the aortic sinus was collected for histology and single-cell sequencing (10X Genomics). Results: Analysis of the RNA-Seq from TCDD treated HCASMC showed differential enrichment of pathways related to cell migration and vascular development. Further, ATAC-Seq data showed a significant enrichment for pathways regulating vascular development, cell migration, and apoptosis. There was an overall increase in chromatin accessibility suggesting transcriptional activation with TCDD. We observed enrichment for AHR ChIP-Seq peaks in the open chromatin regions along with a significant reciprocal reduction in TCF21 occupancy. The scRNA-Seq of mice treated with TCDD exposure showed differential gene expression of SMC-lineage cells enriching for extracellular matrix (ECM) organization, inflammation, unfolded protein response, and apoptotic process, and histological analysis of the aortic sinus showed increased lesion area with increased presence of SMC-lineage cells and Cd68 + cells compared to control. Conclusion: Dioxin adversely remodels atherosclerotic plaque affecting pathways of ECM organization, inflammation and unfolded protein response in SMC, partly by modifying chromatin accessibility and occupancy of key transcription factors of phenotypic modulation.

P233 Environmental and occupational exposures in the development of systemic lupus erythematosus in adults: a narrative review

Abstract Background/Aims Systemic lupus erythematosus (SLE) is a chronic inflammatory disease, of an autoimmune nature, of unknown origin, characterized by loss of tolerance to nuclear antigens and the deposition of immunecomplexes in the tissues leading to inflammation that can affect various organs and systems of the body. This narrative review aims to demonstrate that occupational factors and environmental exposure contribute to the development of SLE in adults. Methods This is a narrative review of literature that seeks to analyse the scientific production on environmental and occupational exposure in the development of systemic lupus erythematosus in adults, published in health journals. A literature search was conducted in the period between November 2020 and July 2021, considering the object of study, initially the articles related to the theme addressed were searched in the PubMed database, in the US National Institutes of Health (NIH) and Scientific Electronic Library Online (SCIELO) portals. Results Exposure to respirable silica dust (i.e. the crystalline form of quartz) is an established risk factor for SLE and other systemic autoimmune diseases. Exposure to toxic components of cigarette smoke (e.g., tar, nicotine, carbon monoxide, polycyclic aromatic hydrocarbons, and free radicals) can induce oxidative stress and directly damage endogenous proteins and DNA, leading to genetic mutations and gene activation, which may be involved in the development of SLE. Smoking stimulates the expression of CD95 on B and CD4 on T-cell surfaces, potentially inducing autoimmunity, and also increases the production of pro- inflammatory cytokines. Exposure to ultraviolet (UV) radiation may exacerbate pre-existing SLE; it is not yet known whether UV exposure plays a role in the pathogenesis of SLE. Thus, UV may result in a redistribution of nuclear antigens to the cell surface or in the production of new forms of autoantigens, effects that may be relevant given the mechanisms believed to be involved in SLE etiology. Several hypotheses have been postulated that air pollution may contribute to autoimmune diseases via oxidative stress, nitrosative stress, and systemic inflammation. Traffic-related air pollutants may cause oxidative stress and and systemic inflammation. The combination of CO with hemoglobin (Hb) to form carboxyhemoglobin (COHb) reduces the oxygen-carrying capacity of the blood and causes tissue hypoxia. Conclusion The articles analysed identified risk factors for exposure to current smoking, silica, ultraviolet radiation, and environmental pollution from CO, NO2, and minute particles. These are caused by oxidative stress, apoptosis inducers, systemic inflammation, genetic mutations, and activation of genes that will lead to immunological changes with increased pro-inflammatory cytokines, activation of genes, and increased autoantigens, leading to predisposition of these exposures in the development of systemic lupus erythematosus. In contrast, alcohol has been shown to be a neutralizing factor in systemic inflammation, causing a decrease in cellular responses and suppressing the synthesis of pro-inflammatory cytokines, showing the association of alcohol with a lower risk of developing SLE. Disclosure B.G. Monteiro: Grants/research support; FUNADESP. C.B. Pizarro: Grants/research support; FUNADESP.

In vitro cytoxicity profile of e-cigarette liquid samples on primary human bronchial epithelial cells.

Cigarette smoke is associated to severe chronic diseases. The most harmful components of cigarette smoke derive from the combustion process, which are significantly reduced in the electronic cigarette aerosol, thus providing a valid option in harm reduction strategies. To develop safer products, it is therefore necessary to screen electronic cigarette liquids (e-liquids) to meet high safety standards defined by government regulations. The aim of the present study was to evaluate the presence of metal- and plastic-derived contaminants in four different commercial e-liquids with high concentration of nicotine and their cytotoxic effect in normal human bronchial epithelial cells by a number of in vitro assays, in comparison with the 1R6F reference cigarette, using an air-liquid interface (ALI) exposure system. Moreover, we evaluated the effect of aerosol exposure on oxidative stress by measuring the production of reactive oxygen species and mitochondrial potential. Our results showed no contaminants in all e-liquids and a significantly reduced cytotoxic effect of e-liquid aerosol compared to cigarette smoke as well as a maintained mitochondria integrity. Moreover, no production of reactive oxygen species was detected with e-cigarette aerosol. In conclusion, these results support the reduced toxicity potential of e-cigs compared to tobacco cigarettes in an in vitro model resembling real life smoke exposure.

HYDROQUINONE,A CIGARETTE SMOKE COMPOUND, AFFECTS CARTILAGE HOMEOSTASISTHROUGHACTIVATION OF THE ARYL HYDROCARBON RECEPTOR PATHWAY

Purpose: Exposure to cigarette smoke has a proven detrimental impact on different aspects of human health. Increasing evidence linked smoking to degeneration of joint tissues. However, the toxic mechanisms elicited by the different components of cigarette smoke have not been fully understood. We have previously shown that exposure to hydroquinone (HQ), a pro-oxidative chemical present in cigarette smoke, can promote joint tissue degradation in murine models of rheumatoid arthritis through the activation of the aryl hydrocarbon receptor (AhR) pathway.

Existence of multiple organ aging in animal model of emphysema induced by cigarette smoke extract.

INTRODUCTIONIt is commonly considered that COPD or at least emphysema represents accelerated lung aging induced in part by oxidative damage from cigarette smoke components. However, the issue if there are any aging signs in other organs in patients with COPD or emphysema remains unclear. The aim of this study is to explore whether there is multiple organ aging in the animal model of emphysema induced by cigarette smoke extract (CSE), and to ascertain the possible mechanisms, if any.METHODSThe animal model of emphysema was induced by CSE. Histomorphological changes in lung, heart, liver, kidney and spleen tissues were measured after staining with hematoxylin and eosin (H&E). The concentrations of stem cell factor (SCF), CyclinD1 and superoxide dismutase (SOD) in serum were determined by ELISA kit. The expressions of p16 (INK4a), Sca-1, eNOS proteins and mRNA in lung, heart, liver, kidney and spleen tissues were detected by Western blotting and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), respectively. Decitabine (Dec) was applied to examine whether it could alter the changes caused by CSE.RESULTSThe histomorphology of lung tissue was significantly changed, while other organs exhibited normal structure and histomorphology. The concentrations of SCF, CyclinD1 and SOD in serum were lower in the CSE group than in the control group. The expression levels of p16(INK4a) protein and mRNA in lung, heart, liver, kidney and spleen tissues were higher in the CSE group than in the control group, while the expression levels of Sca-1 and eNOS proteins and mRNA were lower in the CSE group than in the control group, in the tissues described above. Dec could partly alleviate the damages caused by CSE and the degree of alleviation resulted by Dec varied from organ to organ.CONCLUSIONSIn addition to the aging of the lung tissue in the emphysema animal model induced by CSE, the tissues of the heart, liver, kidney and spleen were also in the progress of aging, but the sensibility and affinity of lung to CSE were higher than those of the other organs. Multiple organ aging may also exist in the animal model of emphysema induced by CSE. DEC can partly alleviate the multiple organ aging caused by CSE.

Hydroquinone increases risk for retinal pigment epithelial (RPE) cell degeneration

AbstractPurposeCigarette smoke increases the risk for age‐related macular degeneration (AMD) e.g. through oxidative stress and mitochondrial damage in human retinal pigment epithelial (RPE) cells. Vascular endothelial growth factor (VEGF) promotes choroidal neovascularization in wet AMD, for which anti‐VEGF injections are used as therapy. Since VEGF is important for the RPE cell functionality and integrity, too low VEGF levels increase the risk for RPE cell degeneration. Pigment epithelium‐derived factor (PEDF) improves mitochondrial activity and reduces neovascularization. The aim of the present study was to examine the effects of the cigarette smoke component hydroquinone on the VEGF and PEDF levels in RPE cells.MethodsIL‐1α‐primed ARPE‐19 cells were exposed to hydroquinone (10 μM, 50 μM, 200 μM; 18 h, 1 h or 15 min). Enzyme‐linked immunosorbent assay (ELISA) was used to detect the levels of intracellular VEGF as well as secreted VEGF and PEDF. The 2′,7′‐dichlorofluorescin diacetate (DCFDA) probe was used to detect ROS production. Lactate dehydrogenase (LDH) and intracellular ATP were measured using commercial kits, and total protein concentrations using a protocol based on the Bradford method.ResultsHydroquinone reduced the levels of both intra‐ and extracellular VEGF. In addition, the release of PEDF as well as the ratio between VEGF and PEDF were reduced at all used concentrations. ROS production increased at 50 μM hydroquinone, and the cell membrane was compromised at 200 μM hydroquinone after 15 min exposure. ATP levels were reduced from the 50 μM hydroquinone concentration upwards and total protein levels at all concentrations, already without cytotoxicity.ConclusionsAccording to our present data, hydroquinone favors anti‐angiogenic conditions but predisposes to the RPE cell degeneration, impaired mitochondrial function, and reduced protein synthesis in human RPE cells.

Genome-wide methylation and expression analyses reveal the epigenetic landscape of immune-related diseases for tobacco smoking

BackgroundSmoking is a major causal risk factor for lung cancer, chronic obstructive pulmonary disease (COPD), cardiovascular disease (CVD), and is the main preventable cause of deaths in the world. The components of cigarette smoke are involved in immune and inflammatory processes, which may increase the prevalence of cigarette smoke-related diseases. However, the underlying molecular mechanisms linking smoking and diseases have not been well explored. This study was aimed to depict a global map of DNA methylation and gene expression changes induced by tobacco smoking and to explore the molecular mechanisms between smoking and human diseases through whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq).ResultsWe performed WGBS on 72 samples (36 smokers and 36 nonsmokers) and RNA-seq on 75 samples (38 smokers and 37 nonsmokers), and cytokine immunoassay on plasma from 22 males (9 smokers and 13 nonsmokers) who were recruited from the city of Jincheng in China. By comparing the data of the two groups, we discovered a genome-wide methylation landscape of differentially methylated regions (DMRs) associated with smoking. Functional enrichment analyses revealed that both smoking-related hyper-DMR genes (DMGs) and hypo-DMGs were related to synapse-related pathways, whereas the hypo-DMGs were specifically related to cancer and addiction. The differentially expressed genes (DEGs) revealed by RNA-seq analysis were significantly enriched in the “immunosuppression” pathway. Correlation analysis of DMRs with their corresponding gene expression showed that genes affected by tobacco smoking were mostly related to immune system diseases. Finally, by comparing cytokine concentrations between smokers and nonsmokers, we found that vascular endothelial growth factor (VEGF) was significantly upregulated in smokers.ConclusionsIn sum, we found that smoking-induced DMRs have different distribution patterns in hypermethylated and hypomethylated areas between smokers and nonsmokers. We further identified and verified smoking-related DMGs and DEGs through multi-omics integration analysis of DNA methylome and transcriptome data. These findings provide us a comprehensive genomic map of the molecular changes induced by smoking which would enhance our understanding of the harms of smoking and its relationship with diseases.

Effect of 2,6-xylidine (DMA) on secretion of biomarkers for inflammation and neurodevelopment by the placenta

Cigarette smoke enhances placental inflammation and interferes with steroidogenesis. However, the chemicals in the smoke responsible for these biological activities are unclear. 2,6 xylidine (also called 2,6 Dimethylaniline, DMA) is a component of cigarette smoke that has carcinogenic properties but its effects on the placenta are unknown. Therefore, we hypothesized that DMA may interfere with placental steroidogenesis or enhance placental inflammation. Placental explant cultures were treated with 0–50,000 nM DMA and concentrations of progesterone (P4), estradiol (E2), testosterone (T), IL-1β, TNF-α, IL-6, sgp130, HO-1, IL-10, 8-Isoprostane (8-IsoP), and BDNF in the conditioned medium were quantified. Since many environmental toxins enhance the proinflammatory host response to infection, we also performed experiments on placental cultures co-stimulated with 107 heat-killed E. coli. DMA alone significantly reduced P4 and T secretion but enhanced E2 secretion. The toxin also reduced placental secretion of IL-6, sgp130, and BDNF. For bacteria-stimulated cultures, DMA increased secretion of P4 and T, and proinflammatory cytokines (IL-1β, TNF-α) but had mixed effects on anti-inflammatory markers, increasing some (sgp130, IL-10) and reducing others (HO-1). However, DMA enhanced 8-IsoP levels by bacteria-stimulated placental cultures, suggesting that it increases oxidative stress by the tissues. These studies suggest that DMA affects secretion of biomarkers by the placenta and may promote inflammation. Further studies are needed to determine if these observed changes occur in vivo and the extent to which DMA exposure increases the risk of adverse pregnancy outcomes associated with smoking in pregnancy.

Do Genetic Variants Modify the Effect of Smoking on Risk of Preeclampsia in Pregnancy?

Maternal smoking is associated with as much as a 50% reduced risk of preeclampsia, despite increasing risk of other poor pregnancy outcomes that often co-occur with preeclampsia, such as preterm birth and fetal growth restriction. Researchers have long sought to understand whether this perplexing association is biologically based, or a result of noncausal mechanisms. We examined whether smoking-response genes modify the smoking-preeclampsia association to investigate potential biological explanations. We conducted a nested case-control study within the Norwegian Mother, Father and Child Birth Cohort (1999-2008) of 2,596 mother-child dyads. We used family-based log-linear Poisson regression to examine modification of the maternal smoking-preeclampsia relationship by maternal and fetal single nucleotide polymorphisms involved in cellular processes related to components of cigarette smoke (n = 1,915 with minor allele frequency ≥10%). We further investigated the influence of smoking cessation during pregnancy. Three polymorphisms showed overall (p < 0.001) multiplicative interaction between smoking and maternal genotype. For rs3765692 (TP73) and rs10770343 (PIK3C2G), protection associated with smoking was reduced with two maternal copies of the risk allele and was stronger in continuers than quitters (interaction p = 0.02 for both loci, based on testing 3-level smoking by 3-level genotype). For rs2278361 (APAF1) the inverse smoking-preeclampsia association was eliminated by the presence of a single risk allele, and again the trend was stronger in continuers than in quitters (interaction p = 0.01). Evidence for gene-smoking interaction was limited, but differences by smoking cessation warrant further investigation. We demonstrate the potential utility of expanded dyad methods and gene-environment interaction analyses for outcomes with complex relationships between maternal and fetal genotypes and exposures. · Maternal and fetal genotype may differentially influence preeclampsia.. · Smoking-related genes did not strongly modify smoking-preeclampsia association.. · Smoking cessation reduced strength of gene by smoking interactions..

Evidence for Alpha7 Nicotinic Receptor Activation During the Cough Suppressing Effects Induced by Nicotine and Identification of ATA-101 as a Potential Novel Therapy for the Treatment of Chronic Cough.

Studies performed in healthy smokers have documented a diminished responsiveness to tussive challenges, and several lines of experimental evidence implicate nicotine as an antitussive component in both cigarette smoke and the vapors generated by electronic cigarettes (eCigs). We set out to identify the nicotinic receptor subtype involved in the antitussive actions of nicotine and to further evaluate the potential of nicotinic receptor-selective agonists as cough-suppressing therapeutics. We confirmed an antitussive effect of nicotine in guinea pigs. We additionally observed that the alpha-4 beta-2 (α 4 β 2)-selective agonist Tc-6683 was without effect on evoked cough responses in guinea pigs, while the α 7-selective agonist PHA 543613 dose-dependently inhibited evoked coughing. We subsequently describe the preclinical evidence in support of ATA-101, a potent and highly selective (α 7) selective nicotinic receptor agonist, as a potential candidate for antitussive therapy in humans. ATA-101, formerly known as Tc-5619, was orally bioavailable and moderately central nervous system (CNS) penetrant and dose-dependently inhibited coughing in guinea pigs evoked by citric acid and bradykinin. Comparing the effects of airway targeted administration versus systemic dosing and the effects of repeated dosing at various times prior to tussive challenge, our data suggest that the antitussive actions of ATA-101 require continued engagement of α 7 nicotinic receptors, likely in the CNS. Collectively, the data provide the preclinical rationale for α 7 nicotinic receptor engagement as a novel therapeutic strategy for cough suppression. The data also suggest that α 7 nicotinic acetylcholine receptor (nAChR) activation by nicotine may be permissive to nicotine delivery in a way that may promote addiction. SIGNIFICANCE STATEMENT: This study documents the antitussive actions of nicotine and identifies the α7 nicotinic receptor subtype as the target for nicotine during cough suppression described in humans. We additionally present evidence suggesting that ATA-101 and other α7 nicotinic receptor-selective agonists may be promising candidates for the treatment of chronic refractory cough.

Nicotine exacerbates tacrolimus-induced renal injury by programmed cell death.

Background/AimsCigarette smoking is an important modifiable risk factor in kidney disease progression. However, the underlying mechanisms for this are lacking. This study aimed to assess whether nicotine (NIC), a major toxic component of cigarette smoking, would exacerbates tacrolimus (TAC)-induced renal injury.MethodsSprague-Dawley rats were treated daily with NIC, TAC, or both drugs for 4 weeks. The influence of NIC on TAC-caused renal injury was examined via renal function, histopathology, oxidative stress, mitochondria, endoplasmic reticulum (ER) stress, and programmed cell death (apoptosis and autophagy).ResultsBoth NIC and TAC significantly impaired renal function and histopathology, while combined NIC and TAC treatment aggravated these parameters beyond the effects of either alone. Increased oxidative stress, ER stress, mitochondrial dysfunction, proinflammatory and profibrotic cytokine expressions, and programmed cell death from either NIC or TAC were also aggravated by the two combined.ConclusionsOur observations suggest that NIC exacerbates chronic TAC nephrotoxicity, implying that smoking cessation may be beneficial for transplant smokers taking TAC.

Transcriptional Response of Mycobacterium tuberculosis to Cigarette Smoke Condensate.

Smoking is known to be an added risk factor for tuberculosis (TB), with nearly a quarter of the TB cases attributed to cigarette smokers in the 22 countries with the highest TB burden. Many studies have indicated a link between risk of active TB and cigarette smoke. Smoking is also known to significantly decrease TB cure and treatment completion rate and increase mortality rates. Cigarette smoke contains thousands of volatile compounds including carcinogens, toxins, reactive solids, and oxidants in both particulate and gaseous phase. Yet, to date, limited studies have analyzed the impact of cigarette smoke components on Mycobacterium tuberculosis (Mtb), the causative agent of TB. Here we report the impact of cigarette smoke condensate (CSC) on survival, mutation frequency, and gene expression of Mtb in vitro. We show that exposure of virulent Mtb to cigarette smoke increases the mutation frequency of the pathogen and strongly induces the expression of the regulon controlled by SigH—a global transcriptional regulator of oxidative stress. SigH has previously been shown to be required for Mtb to respond to oxidative stress, survival, and granuloma formation in vivo. A high-SigH expression phenotype is known to be associated with greater virulence of Mtb. In patients with pulmonary TB who smoke, these changes may therefore play an important, yet unexplored, role in the treatment efficacy by potentially enhancing the virulence of tubercle bacilli.

Cigarette smoke and nicotine effects on behavior in HIV transgenic rats

HIV-related neurocognitive impairment can be worsened by cigarette smoking and be more severe in women. Therefore, we analyzed the effects of sex on behavioral function in HIV transgenic (Tg) rats that were exposed to either nicotine alone, to smoke from either nicotine-containing or nicotine-free cigarettes, or non-exposed. The animals were then assessed on the open field test for the total distance traveled and for the fraction of the total distance traveled and the total time spent in the center of the field, and the results then compared to WT rats subjected to the same exposures and testing. Higher total distances indicate greater locomotor activity and a higher center field measures imply a lower anxiety state. Total distances were overall higher for female and for Tg rats exposed to nicotine-free CS. Also, the total distance and both center field measures were overall higher for female rats in the control and nicotine-free CS-exposed groups. This was observed specifically for WT females as compared to WT males and, for the center field measures, for WT females as compared to Tg males. No genotype or sex-related differences were found for rats in the nicotine-free cigarette smoke (CS) and nicotine-containing CS exposed groups. Therefore, nicotine exposure did not impact genotype- and sex-related differences in motor responses and anxiety levels that were found in the control state. However, exposure to the non-nicotine components of CS resulted in locomotor activation in the presence of the HIV genes and was anxiogenic in WT and Tg male animals.

The Synergistic Effect of Nicotine and Staphylococcus aureus on Peri-Implant Infections.

Smoking is considered a key risk factor for implant survival; however, how it interacts with the pathogens in peri-implant infections is not clear. Here, we identified that nicotine, the key component of cigarette smoking, can interact with Staphylococcus aureus and synergistically induce peri-implant infections in a rat osteolysis model. The nicotine–S. aureus combination group increased the gross bone pathology, osteolysis, periosteal reactions, and bone resorption compared to the nicotine or S. aureus single treated group (p < 0.05). Nicotine did not promote the proliferation of S. aureus both in vitro and in vivo, but it can significantly upregulate the expression of staphylococcal protein A (SpA), a key virulence factor of S. aureus. The nicotine–S. aureus combination also synergistically activated the expression of RANKL (receptor activator of nuclear factor-kappa B ligand, p < 0.05) to promote the development of peri-implant infections. The synergistic effects between nicotine and S. aureus infection can be a new target to reduce the peri-implant infections.

Nicotine facilitates pancreatic fibrosis by promoting activation of pancreatic stellate cells via α7nAChR-mediated JAK2/STAT3 signaling pathway in rats

AimSmoking has been considered as a risk factor of chronic pancreatitis (CP), but the potential mechanism is still unknown. The major pathological feature of CP is pancreatic fibrosis, whose major functional cells are pancreatic stellate cells (PSCs). Nicotine is the major component of cigarette smoke, our recent study suggested that nicotine has the potential to facilitate pancreatic fibrosis in CP. This study was aimed to analyze the function and mechanism of nicotine on PSCs and pancreatic fibrosis in rats. Materials and methodsIn vivo, a rat CP model was induced by intraperitoneal injection of 20 % L-arginine hydrochloride (200 mg/100 g) at 1 h intervals twice per week, nicotine was injected subcutaneously at a dose of 1 mg/kg body weight per day. After four weeks, the pancreatic tissue was collected for H&E, Masson and immunohistochemical staining. In vitro, primary rPSCs were isolated from rats and treated with nicotine (0.1 μM and 1 μM). The proliferation、apoptosis、α-SMA expression、extracellular matrix (ECM) metabolism and α7nAChR-mediated JAK2/STAT3 signaling pathway of rPSCs were detected by CCK-8 assay、flow cytometry、real-time Q-PCR and western blotting analysis. The α7nAChR antagonist α-bungarotoxin (α-BTX) was used to perform inhibition experiments. Key findingsNicotine increased pancreatic damage, collagen deposition and activation of PSCs in the CP rat model. In rPSCs, the proliferation, α-SMA expression and ECM formation were significantly promoted by nicotine in a dose-dependent manner. Meanwhile, the apoptosis of rPSCs was significantly reduced after nicotine treatment. Moreover, nicotine also activated the α7nAChR-mediated JAK2/STAT3 signaling pathway in rPSCs. These effects of nicotine on rPSCs were blocked by α-BTX. SignificanceOur finding in this research suggests that nicotine facilitates pancreatic fibrosis by promoting activation of pancreatic stellate cells via α7nAChR-mediated JAK2/STAT3 signaling pathway in rats, partly revealing the mechanism of smoking on chronic pancreatitis.

N‐acetylcysteine is more effective than ellagic acid in preventing acrolein induced dysfunction in mitochondria isolated from rat liver

Acrolein, a common environmental, food, and water pollutant, has been linked to the pathology of several diseases. This toxic substance is an unsaturated aldehyde and a major component of cigarette smoke and also produced during the processing of fat-containing foods. This study aimed to evaluate the protective effect of ellagic acid and N-acetylcysteine (NAC) in acrolein-induced toxicity in mitochondria isolated from the rat liver. The mitochondria were exposed to different concentrations of acrolein for 40min, then functionality was assessed. Contact with acrolein rapidly and remarkably depleted the intracellular glutathione and antioxidant capacity, because of increased ROS production and lipid peroxidation which may lead to the cell death. Mitochondria were then pre-exposed to different concentrations of ellagic acid, NAC, and IC50 concentration of acrolein. Consistent with the results, acrolein decreased GSH content and increased ROS level and lipid peroxidation, which led to ATP depletion and mitochondrial dysfunction. While ellagic acid has been able to reduce ROS and therefore the permeability of the mitochondrial membrane potential (MMP), presumably via its antioxidant properties, we've not detected its favorable effect on GSH and ATP restoration and also on mitochondrial complex II function. However, NAC strongly decreased ROS, lipid peroxidation and MMP and improved GSH content and complex II activity. These results showed that ellagic acid while reported to possess some cellular protective properties, did not prevent mitochondria from being affected by acrolein during this in vitro study. PRACTICAL APPLICATIONS: Ellagic acid is found in fruits, vegetables, and nuts which are revealed to possess strong antioxidant and protective properties. Mitochondrial dysfunction has been implicated in the pathogenesis of some chronic diseases including cancer, diabetes, liver disease, and neurodegenerative disorders, and presumably, ellagic acid by its mitochondrial protective effects can be helpful in these chronic conditions. Acrolein is an α,β-unsaturated aldehyde that can be produced during cooking at high temperature. By increasing the ROS level and lipid peroxidation and depleting the glutathione content, acrolein induces cellular damage and mitochondrial toxicity. This toxicant is taken into account as a carcinogen and mutagen. In this study, the protective effect of ellagic acid in comparison with N-acetylcysteine has been investigated during the toxicity of acrolein in the rat liver mitochondria to look for evidence of whether it is useful or not through this insult.

Long‐term exposure to cigarette smoke influences characteristics in human gingival fibroblasts

Periodontal disease is a chronic inflammatory disease caused by periodontopathic bacteria accumulated in the gingival sulcus and periodontal pocket. Cigarette smoking is a well-established risk factor for periodontal disease, and periodontal tissues in smokers are chronically exposed to cigarette smoke on a long-term basis. In this study, we investigated the effects of long-term exposure to nicotine or cigarette smoke condensate (CSC) on cellular functions of human gingival fibroblasts (HGFs). In vitro-maintained HGFs were divided into two groups. The HGFs of the short-term and the long-term culture groups were cultured for 4 and 25days, respectively, in the presence or absence of nicotine, which is one of the main components of cigarette smoke, or CSC. The cellular proliferation and migration capacities of HGFs exposed to nicotine or CSC were evaluated by WST-1 and wound healing assays. The effects of exposure to nicotine or CSC on the expression of various extracellular matrix (ECM) components, inflammatory cytokines, and senescence-related genes were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The cellular senescence of HGFs exposed to nicotine or CSC was detected by the senescence-associated β-galactosidase (SA-β-gal) assay. To explore the senescence-associated microRNA (miRNA), we extracted miRNA from the HGFs and the expression profiles were examined by miRNA array. In short-term culture, no significant changes were observed. Long-term exposure of HGFs to nicotine or CSC significantly suppressed their cellular proliferation and migration and upregulated type Ⅰ collagen, type Ⅲ collagen, interleukin (IL)-6, IL-8, p16, p21, and p53mRNA expression, and IL-6 and IL-8 protein expression. Furthermore, long-term nicotine or CSC exposure significantly increased the percentage of SA-β-gal-positive HGFs. In addition, long-term nicotine or CSC exposure reduced miR-29b and miR-199a expression to less than 50% of that in the unstimulated HGFs. These data suggest that long-term smoking habits may reduce wound healing ability, modulate ECM protein homeostasis, stimulate the inflammatory response, and accelerate cellular senescence in HGFs, and consequently accelerate the progression of periodontal diseases.

Anti‐tumor and Anti‐Angiogenic activity of the Choline Acetyltransferase Inhibitor BW813U, in Human Lung Adenocarcinoma

Lung adenocarcinoma (LACs) accounts for about 60% of all lung cancer cases in the United States. Epidemiological studies show that the development of lung adenocarcinoma is strongly correlated to smoking habits. Although, cigarette smoke is a complex mixture of over 4000 chemicals, nicotine is the active and addictive component of cigarette smoke. Our previous data show that nicotine accelerates the growth of human lung cancers by promoting angiogenesis. The endogenous ligand for nicotine in lung cancer cells is the neurotransmitter ACh. The enzyme choline acetyltransferase (ChAT) synthesizes ACh in mammalian cells. The present study aims to determine the anti-tumor and anti-angiogenic activity of BW813U in human LACs. Human microvascular endothelial cells from the lung (HMEC-L) robustly express enzymatically active ChAT. BW813U robustly blocked the proliferation and angiogenic tubule formation of HMEC-Ls. These experiments were repeated in lung adenocarcinoma-associated endothelial cells (LAAECs) and similar results were obtained. The anti-angiogenic activity of BW813 was confirmed in rat retinal explants assay and chicken chorioallantoic membrane (CAM) assays. Finally, the administration of BW813U potently suppressed the growth of human LAC tumors xenotransplanted in athymic mice. Immunoshistochemical experiments revealed that BW813U-treated human LAC tumors (isolated from athymic mice) contained fewer number of blood vessels than vehicle-treated mice. The anti-angiogenic activity of BW813U was mediated by nicotinic acetylcholine receptors on HMEC-Ls and required the Akt pathway. The administration of BW813U did not cause any discomfort or toxicity in mice. Therefore ChAT blockers (like BW813U) may be useful in suppressing angiogenesis and growth of human LACs.

TRPA1 function in human pulmonary fibroblasts

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective Ca2+ permeable cation channel. While TRPA1 was mostly studied in neuronal cells, it was originally cloned from cultured human lung fibroblasts. TRPA1-mediated Ca2+ entry is evoked by exposure to several chemicals including allyl isothiocyanate (AITC) and inhaled components of cigarette smoke, suggesting a possible role of TRPA1 in progression of lung disease. However, a protective effect of TRPA1 activation in the development of cardiac fibrosis was proposed. Yet, the function of TRPA1 in transforming growth factor β1 (TGF-β1)-driven fibroblast to myofibroblast differentiation as well as the development of pulmonary fibrosis remains elusive. TRPA1 expression and function was analyzed in cultured primary human pulmonary fibroblasts (HPF) obtained from three different donors before and after incubation with TGF-β1. Transcriptome sequencing, qRT-PCR and immunoblotting were used to identify target genes on mRNA and protein levels. TRPA1 specific siRNAs were established to assess the role of the channel in cell function during fibroblast myofibroblasts differentiation. TRPA1 mRNA is highly expressed in HPF and levels were significantly reduced after adding TGF-β1. Expression of fibrosis markers, e.g. alpha smooth muscle actin (αSMA), plasminogen activator inhibitor (PAI), fibronectin (FN1) and collagen (Col1a1), was increased after down-regulation of TRPA1-mRNA by siRNA targeting in HPF. Moreover, TGF-β1 treatment of HPFs reduced Ca2+ entry evoked by the TRPA1 activator AITC, while increasing barrier function and contractile characteristics of the cells. Effects of siRNA-mediated knock-down of TRPA1 on cell contraction, migration and resistance of HPF will be quantified and compared to TGF-β1-induced fibroblast myofibroblast differentiation. Our results suggest an inhibitory function of TRPA1 channels in TGF-β1-driven fibroblast to myofibroblast differentiation. Therefore, activation of TRPA1 channels might protect from the development of pulmonary fibrosis in patients

Identification of Isobars and Isomers in Cigarette Sidestream Smoke in Real Time by Synchrotron Radiation Photoionization Mass Spectrometry and Multiple Linear Regression

Direct analysis of chemical components in fresh cigarette smoke in real time is a challenging task. In this work, by using a novel continuous cigarette-pushing and smoke-introducing setup combined with synchrotron radiation photoionization mass spectrometry (SR-PIMS), the photoionization mass spectra of fresh gaseous cigarette sidestream smoke (SSS) from the combustion of solid tobacco could be recorded in real time, and the photoionization efficiency (PIE) curves of each mass peak could be obtained for the first time. Hence, lots of well-known chemical components and even isomers could be identified by their discriminated onsets or PIE curve simulation. Moreover, diimine, 2H-azirine, and sulfur monoxide, which have never been reported in cigarette smoke, were observed in cigarette SSS, and even two intermediates, ethenol and propen-2-ol, anticipated to exist were actually observed and distinguished. To increase the qualification accuracy, a new simulation method based on multiple linear regression (MLR) was developed and applied for the PIE curve simulation, where qualification mistakes caused by subjective judgements could be eliminated as far as possible.