Tobacco Smoke Extract Research Articles

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.

Exposure to tobacco (Nicotiana tabacum) extract and cigarette smoke induces hematological and histopathological alterations in Swiss albino mice

Tobacco smoking is a widely practiced recreational activity and is associated with numerous health risks. Hence, the present study was conducted on Swiss albino mice to assess the impact of aqueous extract of tobacco and cigarette smoke on behavioral, hematological, and histopathological parameters of the lungs, liver, and kidney. The mice were divided into Control (Cont), Tobacco nicotiana (TN), and Cigarette smoke (CS) groups (n=6). TN group received aqueous tobacco orally at a dose of 5ml/kg BW for 21 days, whereas CS group was exposed to inhalational commercial cigarette smoke for the same duration. On day 22, blood samples and visceral organs were collected to evaluate hematological (TEC, TLC, Hb%) and histopathological parameters from euthanized mice. The TEC values were significantly elevated (P<0.05) in both groups. In contrast to the control group, the TLC value was lower in the CS group (P>0.05) than in the TN group, but the mean Hb% values showed no significant changes. Lower body weight, aggressive behavior, and weakness were observed in the treated groups. Histopathologically, the lung revealed emphysematous changes, alveolar septal thickening, inflammatory cells infiltration, congestion, and thrombus formation in pulmonary artery. Liver exhibited hepatocyte degeneration with karyomegaly and karyorrhexis, derangement of hepatic cords and sinusoids with central vein congestion. Derangement of renal tubules, edema, and glomerular degeneration observed in the kidney. These observations lead us to postulate that tobacco and cigarette smoke exert detrimental effects on the structure of some vital organs in the body after direct or indirect uptake.

Analysis of Influence of Cigarette Smoke on Signaling Pathways of Innate Immune System in Monocytes of Peripheral Blood

INTRODUCTION: Tobacco smoking is an important medical problem since it has a significant impact on the development and progression of chronic obstructive pulmonary disease (COPD). The components of tobacco smoke can initiate and support local and systemic inflammation with participation of monocytes and macrophages.
 AIM: To study molecular mechanisms associated with the impact of cigarette smoke on signaling pathways of the innate immune system in monocytes of peripheral blood.
 MATERIALS AND METHODS: The methods of in silico analysis was used to identify genes associated with the impact of tobacco smoke. On the basis of the data obtained, a cellular model of inflammation was created in vitro using tobacco smoke extract and monocytes of peripheral blood isolated by immunomagnetic separation. An enzyme-linked immunoassay (ELISA) kit was used to measure the concentration of tumor necrosis factor- (TNF-), interleukin-1 (IL-1) in cell supernatants, and of Toll-like receptor 4 (TLR4), ATP-binding cassette A1 (ABCA1) in homogenates of cell membranes of native monocytes and monocytes exposed to 4% tobacco smoke extract. These data were compared with the levels of TNF-, IL-1, TLR4 and ABCA1 in monocytes of peripheral blood of patients with COPD with frequent exacerbation phenotype and with obliterating atherosclerosis of lower limb arteries (OALLA). For statistical processing and visualization of the data, MedCalc 20.1.4 and R (version 4.2.2) software was used.
 RESULTS: Tobacco smoke influences TLR4, TNF- signaling pathways and lipid metabolism. Cigarette smoke extract enhanced the expression of proinflammatory cytokines TNF- and IL-1 in cell supernatants, increased the level of TLR4 and decreased that of ABCA1 in plasmolemma of monocytes of peripheral blood. In patients with COPD with frequent exacerbation phenotype and with OALLA, there were shown increase in the levels of proinflammatory TNF- and IL-1 cytokines in cell supernatants, increase in the level of TLR4 and reduction of the level of ABCA1 in plasmolemma of monocytes of peripheral blood compared to native monocytes of healthy individuals.
 CONCLUSION: Cigarette smoke enhances the production of proinflammatory TNF- and IL-1 cytokines, increases the levels of TLR4 protein and reduces the amount of ABCA1 transporter in membranes of monocytes of peripheral blood. This may partially explain the cause of the influence of cigarette smoke on development of the pulmonary and cardiovascular diseases. COPD with frequent exacerbation phenotype and OALLA are characterized by enhancement of inflammation with participation of monocytes.

High-Field Asymmetric Waveform Ion Mobility Spectrometry Analysis of Carcinogenic Aromatic Amines in Tobacco Smoke with an Orbitrap Tribrid Mass Spectrometer.

Smoking is a risk factor for bladder cancer (BC), although the specific chemicals responsible for BC remain uncertain. Considerable research has focused on aromatic amines (AAs), including o-toluidine (o-tol), o-anisidine (o-anis), 2-naphthylamine (2-NA), and 4-aminobiphenyl (4-ABP), which are linked to human BC based on elevated BC incidence in occupationally exposed factory workers. These AAs arise at nanogram levels per combusted cigarette. The unambiguous identification of AAs, particularly low-molecular-weight monocyclic AAs in tobacco smoke extracts, by liquid chromatography-mass spectrometry (LC-MS) is challenging due to their poor performance on reversed-phase columns and co-elution with isobaric interferences from the complex tobacco smoke matrix. We employed a tandem liquid-liquid and solid-phase extraction method to isolate AAs from the basic fraction of tobacco smoke condensate (TSC) and utilized high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to high-resolution accurate mass (HRAM) Orbitrap LC-MS2 to assay AAs in TSC. The employment of FAIMS greatly reduced sample complexity by removing precursor co-isolation interfering species at the MS1 scan stage, resulting in dramatically improved signal-to-noise of the precursor ions and cleaner, high-quality MS2 spectra for unambiguous identification and quantification of AAs in TSC. We demonstrate the power of LC/FAIMS/MS2 by characterizing and quantifying two low-molecular-weight carcinogenic AAs, o-tol and o-anis, in TSC, using stable isotopically labeled internal standards. These results demonstrate the power of FAIMS in trace-level analyses of AA carcinogens in the complex tobacco smoke matrix.

Comparison of the effects of electronic cigarette vapours and tobacco smoke extracts on human neutrophilsin vitro

Electronic cigarettes (ECs) are electronic aerosol delivery systems composed of nicotine and various chemicals, which are widely used to facilitate smoking cessation. Although ECs are considered safer than cigarettes, they do, however, contain chemical toxicants, some of which may interact with cells of the host's innate immune system of which neutrophils constitute a key component. The current study was designed to compare the effects of aqueous EC aerosol extracts (ECEs; with or without nicotine) with those of cigarette smoke extract (CSE) on neutrophil and platelet reactivity in vitro. Neutrophil reactivity is characterised by the generation of reactive oxygen species (ROS), degranulation (elastase release) and the release of extracellular DNA (neutrophil extracellular trap (NET) formation: NETosis), which were measured using chemiluminescence, spectrophotometric and microscopic procedures, respectively. Platelet reactivity was measured according to the magnitude of upregulated expression of the adhesion molecule CD62P on activated cells using a flow cytometric procedure. Exposure of neutrophils to either ECEs or CSE caused a significant inhibition of ROS generation and elastase release by N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 µM)-activated neutrophils. Pre-treatment of neutrophils with CSE also resulted in a marked attenuation of phorbol 12-myristate 13-acetate (6.25 nM)-mediated release of extracellular DNA, which was unaffected by the ECEs. Similarly, CSE, but not the ECEs, inhibited the expression of CD62P by platelets activated with ADP (100 µM). These observations suggest that ECE aerosols may inhibit some of the immuno-protective activities of neutrophils such as ROS production and elastase release by activated cells, the effect of which was not enhanced by inclusion of nicotine. The inhibitory effects of CSE were significantly more pronounced than those of ECEs, especially so for suppression of NET formation and platelet activation. If operative in vivo, these harmful immunosuppressive effects of ECEs may compromise intrinsic pulmonary antimicrobial defence mechanisms, albeit less so than cigarette smoke.

In Vitro effects of alternative smoking devices on oral cells: Electronic cigarette and heated tobacco product versus tobacco smoke

ObjectiveTo investigate the biological effects of electronic cigarette (e-cigarette) and heated tobacco product extracts respect to tobacco smoke extract on human gingival fibroblasts and human oral keratinocytes analysing cell viability, morphology, migration, apoptosis, cell cycle and epithelial-mesenchymal transition (EMT). DesignHuman gingival fibroblasts and human oral keratinocytes viability was analysed by MTT assay, cell morphology using scanning electron microscope (SEM) and cell migration by Scratch assay, a method that mimics the cell migration during wound healing in vivo. Apoptosis and cell cycle were analysed with flow cytometry and the related-gene expression of TP53, BCL2, CDKN2A and CDKN1A was indagated using real-time polymerase chain reaction. EMT process was analysed through expression of specific markers: CDH1, SNAI2, TWIST1, MMP2, FN1 and VIM. All investigations were evaluated after 24 h an in vitro exposure. ResultsUndiluted tobacco smoke extract induced significant inhibition of cell viability and cell migration, caused morphological alterations and induced an increase in cell death. No alterations or damage were observed after treatment with e-cigarette extracts. Heated tobacco product extract induced proliferation as highlighted by an increase of cell viability, cell migration and alterations of cycle analysis. ConclusionsComparing the different cigarette extracts, tobacco smoke turns out to be the most harmful, e-cigarette did not determine morphological and functional alterations and heated tobacco product must be carefully investigated for its possible clinical effects on oral cell populations.

HIV-1 gp120 and tobacco smoke synergistically disrupt the integrity of the blood-brain barrier

In the United States, the Centers for Disease Control and Prevention (CDC) terms HIV and tobacco use among the ten most important public health challenges we face today. In the last decade, there has been a remarkable decrease in the number of deaths due to HIV/AIDS, especially after the widespread availability and use of combination antiretroviral therapy (cART). However, people living with HIV/AIDS have a heightened risk of chronic complications and comorbidities, including neurological disorders. Around 40–60 % of HIV-infected individuals progress to NeuroAIDS, a group of disorders caused primarily by HIV-mediated damage to the central and peripheral nervous systems, despite receiving cART. The detrimental effects of chronic smoking on the cerebrovascular system are also well studied and reported. Addictive behavior, such as smoking, is more common in HIV patients compared to the general population. In this context, given the existing immune suppression, smoking can pose a significant risk for the progression of the disease to NeuroAIDS by disrupting the integrity of the blood-brain barrier (BBB). Here we show that co-treatment with Tobacco Smoke Extract (TSE) and HIV-1 gp120 (HIV envelope glycoprotein) in primary cultures of human brain microvascular endothelial cells promoted heightened cellular stress responses compared to control and individual treatments. Our findings suggest that a potential synergistic effect between smoke exposure and gp120 can worsen the loss of BBB viability, possibly exacerbating NeuroAIDS progression.

Role of cytoglobin in cigarette smoke constituent-induced loss of nitric oxide bioavailability in vascular smooth muscle cells

Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease.

Effects of environmental air pollutants on CFTR expression and function in human airway epithelial cells

The airway epithelium is exposed to a variety of air pollutants, which have been associated with the onset and worsening of respiratory diseases. These air pollutants can vary depending on their composition and associated chemicals, leading to different molecular interactions and biological effects. Mucociliary clearance is an important host defense mechanism against environmental air pollutants and this process is regulated by various ion transporters including the cystic fibrosis transmembrane conductance regulator (CFTR). With evidence suggesting that environmental air pollutants can lead to acquired CFTR dysfunction, it may be possible to leverage therapeutic approaches used in cystic fibrosis (CF) management. The aim of our study was to test whether environmental air pollutants tobacco smoke extract, urban particulate matter, and diesel exhaust particles lead to acquired CFTR dysfunction and whether it could be rescued with pharmacological interventions. Human airway epithelial cells (Calu-3) were exposed to air pollutant extracts for 24 h, with and without pharmacological interventions, with readouts of CFTR expression and function. We demonstrate that both tobacco smoke extract and diesel exhaust particles led to acquired CFTR dysfunction and that rescue of acquired CFTR dysfunction is possible with pharmacological interventions in diesel exhaust particle models. Our study emphasizes that CFTR function is not only important in the context of CF but may also play a role in other respiratory diseases impacted by environmental air pollutants. In addition, the pharmacological interventions approved for CF management may be more broadly leveraged for chronic respiratory disease management.

Self-administration by female rats of low doses of nicotine alone vs. nicotine in tobacco smoke extract

BackgroundNicotine has reinforcing effects, but there are thousands of other compounds in tobacco, some of which might interact with nicotine reinforcement. AimsThis rat study was conducted to determine if nicotine self-administration is altered by co-administration of the complex mixture of compounds in tobacco smoke extract (TSE). MethodsFemale Sprague-Dawley rats were tested for self-administration of low doses of nicotine (3 or 10 µg/kg/infusion) at three different rates of reinforcement (FR1, FR3 and FR5) over three weeks either alone or together with the complex mixture of tobacco smoke extract (TSE). ResultsRats self-administering 3 µg/kg/infusion of nicotine alone showed a rapid initiation on an FR1 schedule, but declined with FR5. Rats self-administering nicotine in TSE acquired self-administration more slowly, but increased responding over the course of the study. With 10 µg/kg/infusion rats self-administered significantly more nicotine alone than rats self-administering the same nicotine dose in TSE. Rats self-administering nicotine alone took significantly more infusions with the 10 than the 3 µg/kg/infusion dose, whereas rats self-administering nicotine in TSE did not. Nicotine in TSE led to a significantly greater locomotor hyperactivity at a dose of 0.1 mg/kg compared to rats that received nicotine alone. Rats self-administering nicotine alone had significantly more responding on the active vs. inactive lever, but rats self-administering the same nicotine doses in TSE did not. ConclusionsSelf-administration of nicotine in a purer form appears to be more clearly discriminated and dose-related than nicotine self-administered in the complex mixture of TSE.

Abstract PO033: Exposure to E-cigarette aerosol reduces the expression of Toll-like receptor 3 in lung epithelial cells

Abstract Background and Aim: The use of conventional cigarettes is a major and preventable health problem and the leading cause of preventable morbidity and mortality. Electronic cigarettes have been shown to yield plasma nicotine levels similar to that seen in traditional cigarette users. E-cigarettes have been touted as a healthier alternative to traditional cigarettes and as a smoking cessation tool with mixed results. The increasing use of e-cigarettes by youth is concerning with 27.5% of high school students and 10.5% of middle school students reporting using e-cigarettes in 2019. Use among young adults (18-24) is increasing with 7.6% reporting use in 2018. Toll-like receptors (TLRs) are key sensors in the activation of innate immunity. In addition to activating antiviral and pro-inflammatory responses, TLR3 in particular has been shown to behave as a death receptor, causing apoptosis in cancer cells. TLR3 expression is reduced in the airway epithelial cells of smokers, leading to a reduction in the downstream expression of type I interferons following viral infection. Methods: E-cigarette aerosol extracts were prepared from two brands of e-cigarettes, as previously described. Mainstream tobacco smoke extract was used as a positive control. Normal lung epithelial cells were exposed for 2 weeks to e-cigarette aerosol extracts at nicotine doses comparable to those observed in e-cigarette users. Whole-cell RNA was isolated. Gene expression was assessed by RNA-sequencing and validated by western blot. Results: Exposure of lung epithelial cells to e-cigarette aerosol extracts significantly decreased the mRNA expression levels of TLR3 (p<0.0001). A significant reduction (p<0.0001) in the mRNA expression of downstream innate immune system molecules including RIG1, IRF7 and interferons was also observed. The reduction in gene expression was similar to that observed from traditional cigarette smoke extracts. Conclusions: Our study suggests that exposure to e-cigarette aerosol can decrease the expression of key genes involved in innate immune response. TLR3 expression has been associated with good prognosis in early stage lung cancer. A decrease in TLR3 expression could allow tumors to evade immune surveillance and apoptosis Citation Format: Daniel Brobst, Jimmy Manyanga, Constantin Georgescu, Jonathan Wren, Lurdes Queimado. Exposure to E-cigarette aerosol reduces the expression of Toll-like receptor 3 in lung epithelial cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO033.

Electronic cigarette aerosols alter the expression of cisplatin transporters and increase drug resistance in oral cancer cells

Tobacco smoking is the leading preventable cause of cancer. Moreover, continued smoking during cancer therapy reduces overall survival. Aware of the negative consequences of tobacco smoking and the challenges of smoking cessation, cancer patients are inquiring whether they should switch to electronic cigarettes (e-cigarettes). To obtain evidence-based data to inform this decision, we examined the effects of e-cigarette aerosol exposure on cisplatin resistance in head and neck cancer cells. Our results show that cancer cells exposed to e-cigarette aerosol extracts and treated with cisplatin have a significant decrease in cell death, increase in viability, and increase in clonogenic survival when compared to non-exposed cells. Moreover, exposure to e-cigarette aerosol extracts increased the concentration of cisplatin needed to induce a 50% reduction in cell growth (IC50) in a nicotine-independent manner. Tobacco smoke extracts induced similar increases in cisplatin resistance. Changes in the expression of drug influx and efflux transporters, rather than activation of cell growth-promoting pathways or DNA damage repair, contribute to e-cigarette induced cisplatin resistance. These results suggest that like combustible tobacco, e-cigarette use might increase chemotherapy resistance, and emphasize the urgent need for rigorous evaluation of e-cigarettes health effects to ensure evidence-based public health policies.

Myo‐Inositol Improves Ischemic Stroke Outcome After both Nicotine Containing Electronic Cigarette and Tobacco Smoke Exposure

Ischemic stroke occurs in 87% of all strokes and is the leading cause of disability in the United States. Smoking has been shown to increase brain edema after ischemic stroke and worsen long‐term clinical outcomes. Electronic nicotine delivery systems or e‐cigarettes (e‐Cigs) are gaining popularity over traditional tobacco smoke cigarettes, and have been reported influence stroke outcome. Previous work from our lab reported nicotine and e‐Cig exposure decreased glucose utilization in the brain in ischemic stroke models, further exacerbating the effects of ischemic injury. Myo‐inositol(MI), a major organic osmolyte that promotes cell membrane stability and various cellular functions, has been reported to reduce infarcted area and edema ratio in a diabetic ischemic mouse model. We also sought to understand the role of MI protection with regard to additional stroke comorbidities, such as e‐Cig or tobacco smoke exposure. In this study, we investigated the neuroprotective role of MI transporters during stroke and post‐stroke outcomes, especially related to the role of glial cells and post‐stroke reorganization. We hypothesize MI plays a role in increased recovery of motor function, after stroke, in animal groups exposed to tobacco smoke or e‐Cig vape. Primary astrocytes and primary neurons were used for in vitro studies and a middle cerebral artery occlusion (MCAO) mouse model for in vivo stroke studies. Immunocytochemistry (ICC) studies showed astrocytes express sodium dependent myo‐inositol transporter 1 (SMIT1) and sodium‐dependent glucose transporter 6 (SGLT6). Our data illustrated expression of both transporters increased in a time‐dependent manner during oxygen‐glucose deprivation (OGD), in tobacco smoke extract (TSE), and e‐Cig extract (ECE) treated cells. Further, this was confirmed by an increase of MI uptake in TSE treated astrocytes. Altered cell mitochondrial respiration levels were observed in astrocytes and neurons incubated for 24hr with TSE and ECE followed by OGD exposure using Seahorse XF Cell Mito Stress. Animal studies demonstrated post‐stroke administration of MI, reduced infarction size and edema ratios in tobacco smoke compared to non‐tobacco smoke animals. An improvement of motor function was also seen in selected behavioral tests. E‐cig exposed animals had a slight increase in infarction size compared to non‐e‐Cig animals. This preliminary data suggest myo‐inositol may have a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome in both tobacco smokers and e‐Cig users. Further investigation will include assessment of sex differences with regard to the neuroprotective mechanisms of MI in e‐Cig and tobacco smoke exposed animals.Support or Funding Information1R01DA0497375 and R01DA029121 to TJA and LC

Tobacco Smoke Extract Causes Mitochondrial Dysfunction Associated with Myotube Atrophy

INTRODUCTIONWhereas the link between chronic tobacco smoke (TS) exposure and development of disease (e.g., chronic obstructive pulmonary disease; COPD) is well established, how chronic TS exposure relates to the mitochondrial dysfunction and muscle atrophy seen in COPD remains unclear.METHODSWe exposed C2C12 myotubes to TS extract (TSE) on day 5 post‐differentiation to determine the impact of TS on mitochondrial function (respiration, reactive oxygen species [ROS] emission and sensitivity to mitochondrial permeability transition [MPT]), and myotube atrophy. Briefly, for respiration experiments myotubes were treated with 0.02% TSE or vehicle (DMSO) for 24 h, and subsequently washed and assessed for maximal uncoupled respiration in an oxygraph using carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP; 250 nM – 1.5 μM). For ROS experiments, following 24 h TSE or DMSO treatment myotubes were incubated with the superoxide indicator MitoSox (500 nM) for 15 min and imaged for MitoSox positive area. For MPT experiments, after 24 h TSE or DMSO treatment myotubes were fixed in 4% PFA and incubated with anti‐Apoptosis Inducing Factor (AIF; a protein released from mitochondria during MPT) and NucBlue (to stain nuclei). Images (n=5 per well, 8 wells per treatment) were analysed using Image J to quantify the fraction of AIF co‐localizing with nuclei using the Manders coefficient. For myotube atrophy experiments, 24 h following TSE or DMSO treatment myotubes were fixed, labelled with anti‐sarcomeric myosin, and subsequently imaged to assess myotube area using automated batch processing to avoid human bias. Two‐tailed Students t‐tests were used to evaluate differences between DMSO vs. CSE with an alpha level of 0.05. Data are presented as mean±SD with exact p‐values.RESULTSTreatment of C2C12’s with TSE reduced maximal uncoupled respiration (TSE: 312.0±24.0 vs. DMSO: 415.1±45.5 pmoles/sec/μg, p=0.002) and increased MitoSox positive area (TSE: 8,607.0±2,963.1 vs. DMSO: 2,254.7±1,034.7 μm2, p=0.0001); the latter suggesting TSE increases ROS. AIF: nuclear co‐localization was greater in C2C12’s treated with TSE compared with vehicle (Manders coefficient in TSE: 0.66±0.08 vs. DMSO: 0.46±0.05, p=0.0001), suggesting TSE causes MPT. We also observed significantly smaller myotube area in TSE‐treated cells compared with vehicle (TSE: 62,972.9±43,527.5 vs. DMSO: 85,284.9±28,136.1 μm2, p=0.048), consistent with TS inducing myotube atrophy.DISCUSSIONOur experiments demonstrate that TSE exposure causes significant mitochondrial impairment, as evidenced by reduced respiratory capacity, increased ROS generation, and nuclear co‐localization with a mitochondrial protein released during MPT. Furthermore, TSE treatment caused significant myotube atrophy. These observations are consistent with chronic TS exposure playing an important role in the mitochondrial dysfunction and muscle atrophy seen with the TS‐related disease COPD.

Gestational exposure to nicotine and/or benzo[a]pyrene causes long-lasting neurobehavioral consequences.

Tobacco smoke is a complex mixture that includes thousands of compounds. Previously, we have found that gestational exposure to the complex mixture of tobacco smoke extract caused long-term neurobehavioral impairments. In this study, we examined the interaction of two of the most biologically active, nicotine and benzo[a]pyrene (BaP). Developmental effects were determined in Sprague-Dawley rats prenatally exposed to low doses of BaP and nicotine (0.03 mg/kg/day of BaP and 2 mg/kg/day of nicotine) via maternal osmotic minipumps throughout gestation. Behavioral function was assessed in the offspring via a battery of tests through adolescence into adulthood. There were sex-selective effects in four of the behavioral tests. In the elevated plus maze, there was a significant interaction of BaP and sex, where BaP-treated males showed a trend for increased activity. In the novelty suppressed feeding test, there were significant sex selective effects in males such that the normal sex difference in the behavior in this test was eliminated. Male offspring with prenatal exposure to either nicotine or BaP showed significant reductions in fear response. In the Figure-8 locomotor activity test, BAP-exposed male offspring were significantly hyperactive. This also eliminated the sex difference typically seen in this test. This effect persisted into adulthood. In the attention task, males exposed to nicotine during gestation showed a significant percent hit impairment. BaP reversed this effect. No significant effects were seen with percent correct rejection. These data show that both nicotine and BaP cause persisting sex-selective behavioral effects that persist into adulthood.

Abstract 80: Electronic cigarette aerosols increase clonogenic survival in oral cancer cells after cisplatin treatment

Abstract Background and Aim: Are electronic cigarettes (ECs) safer than combustible tobacco? No one knows the answer yet. ECs are devices that deliver an aerosol from a heated mixture of propylene glycol, vegetable glycerin, and flavors, with or without nicotine. Short-term EC studies are mostly limited and inconclusive. Despite unknown long-term health effects, the use of ECs has increased alarmingly over the past decade. EC aerosols contain unique chemicals as well as other chemicals similar to those found in tobacco smoke. Tobacco smoking during cancer treatment has been associated with increased drug resistance and reduced overall survival rate. The awareness of the dangers of cigarette smoking and the paucity of studies evaluating the safety of ECs pose a dilemma for cancer patients who might be considering switching to ECs during chemotherapy. Here, we sought to examine the effects of EC aerosol extracts exposure on cell survival in oral cancer cells during cisplatin treatment. Methods: EC aerosol extracts were collected from two brands of ECs as previously described. Mainstream tobacco smoke (MS) extract was used as a positive control. To evaluate clonogenic survival after cisplatin treatment, oral cancer cells were exposed for 48 h to EC aerosol extracts at nicotine doses comparable to those observed in EC users. Next, cells were treated with both EC aerosol extracts and cisplatin (or vehicle-control) for another 48 h. After treatment, cells were counted and seeded in 6-well plates and colony formation was assessed two weeks later. Colonies were fixed with methanol followed by staining with 0.5% crystal violet in 25% methanol and counted manually. Only colonies with at least 50 cells were counted. Data were analyzed by Student's t-tests and one-way analysis of variance (ANOVA). Results: After cisplatin treatment, cells exposed to EC aerosol extracts showed a significant increase in clonogenic survival fraction compared to vehicle-control treated cells. Furthermore, cells exposed to EC aerosol extracts formed visibly larger colonies. An increase in clonogenic survival was also observed in MS extract-treated cells. Conclusions: Collectively, our data suggest that short-term exposure to EC aerosol can induce cisplatin resistance in oral cancer cells, thus allowing the cells to survive and form colonies. These findings are particularly important in paving the way to understanding whether ECs are a safer alternative for oral cancer patients undergoing chemotherapy. Citation Format: Jimmy Manyanga, Toral R. Mehta, Lurdes Queimado. Electronic cigarette aerosols increase clonogenic survival in oral cancer cells after cisplatin treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 80.

Reactive Oxygen Species-Dependent Calpain Activation Contributes to Airway and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease.

Aims: Airway and pulmonary vascular remodeling is an important pathological feature in the pathogenesis of chronic obstructive pulmonary disease (COPD). Tobacco smoke (TS) induces the production of large amounts of reactive oxygen species (ROS) in COPD lungs. We investigated how ROS lead to airway and pulmonary vascular remodeling in COPD. Results: We used in vitro bronchial and pulmonary artery smooth muscle cells (BSMCs and PASMCs), in vivo TS-induced COPD rodent models, and lung tissues of COPD patients. We found that H2O2 and TS extract (TSE) induced calpain activation in BSMCs and PASMCs. Calpain activation was elevated in smooth muscle of bronchi and pulmonary arterioles in COPD patients and TS-induced COPD rodent models. Calpain inhibition attenuated H2O2- and TSE-induced collagen synthesis and proliferation of BSMCs and PASMCs. Exposure to TS causes increases in airway resistance, right ventricular systolic pressure (RVSP), and thickening of bronchi and pulmonary arteries. Calpain inhibition by smooth muscle-specific knockout of calpain and the calpain inhibitor MDL28170 attenuated increases in airway resistance, RVSP, and thickening of bronchi and pulmonary arteries. Moreover, smooth muscle-specific knockout of calpain did not reduce TS-induced emphysema in the mouse model, but MDL28170 did reduce TS-induced emphysema in the rat model. Innovation: This study provides the first evidence that ROS-induced calpain activation contributes to airway and pulmonary vascular remodeling in TS-induced COPD. Calpain might be a novel therapeutic target for the treatment of COPD. Conclusion: These results indicate that ROS-induced calpain activation contributes to airway and pulmonary vascular remodeling and pulmonary hypertension in COPD.

Role of Myo‐inositol in Ischemic Stroke Outcome in a Preclinical Tobacco Smoke Exposed Mouse Model

Tobacco smoking is a preventable risk factor for ischemic stroke in both males and females and contributes up to 14% of all stroke deaths. It has been reported that an increase in brain edema after ischemic stroke worsens long‐term clinical outcome, such as seen in tobacco smokers. Myo‐inositol (MI) is a major organic “non‐perturbing” osmolyte that promotes cell membrane stability and also a precursor for phosphoinositide signaling pathway. Our previous work reported that MI attenuated brain infarct and edema ratio in an ischemic stroke model coupled to preclinical diabetes and suggests that MI and MI transporters provide neuroprotection during/following stroke both in non‐diabetic and diabetic conditions through protection from brain edema. Since our lab has shown that tobacco smoke exposure worsens stroke edema similarly to diabetic conditions, the aim of the present study was to assess the neuroprotective effects of MI associated with ischemic mice exposed to tobacco smoke. In this study, we also investigated the role of MI transporters during stroke and post‐stroke outcomes especially related to the role of astrocytes cells, which are the most abundant glial cells in the brain and play an important role in post‐stroke reorganization, and indirectly protecting neuronal metabolic support. Primary mouse astrocytes were incubated with tobacco smoke extract (TSE) overnight prior for in vitro studies. Toxicity of TSE was measured using MTT assay after incubating astrocytes with TSE 6hr and 24hr. Therapeutic efficacy of MI was evaluated in a transient‐middle cerebral artery occlusion (tMCAO) mouse model after smoking 3R4F research cigarettes for 10 days. Uptake studies showed an increase of MI uptake increased with longer incubation of TSE in normoxic conditions. Additionally, it was also observed that uptake of MI increased during Oxygen Glucose Deprivation(OGD) conditions with prior exposure of TSE. These results are in support with our previous reports, where we found an increased expression of MI transports, Sodium Myo‐inositol Transporter 1 (SMIT1) and Sodium Dependent Glucose Transporter 6 (SGLT6) during OGD exposure. Preliminary animal studies using middle cerebral artery occlusion mouse model for in vivo stroke studies, demonstrated that post‐stroke administration of MI attenuated infarction size and edema ratios in tobacco smoke exposed animals compared to non‐tobacco smoked ischemic animals and also improved motor function in behavioral tests. Taken together with previous work, this study provides an insight into how MI may play a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome, especially in tobacco smoke populations. Further studies are required to understand the neuroprotective mechanisms of MI in ischemic stroke injury in reducing brain edema.Support or Funding InformationR01NS076012 and R01DA029121This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Comparative Study on the Effect of Tobacco Smoke and Tobbaco Salivary Extract on Serum Lipid Profile

A Comparative Study on the Effect of Tobacco Smoke and Tobbaco Salivary Extract on Serum Lipid Profile

Critical developmental periods for effects of low-level tobacco smoke exposure on behavioral performance

Tobacco exposure during development leads to neurobehavioral dysfunction in children, even when exposure is limited to secondhand smoke. We have previously shown in rats that developmental exposure to tobacco smoke extract (TSE), at levels mimicking secondhand smoke, starting preconception and extending throughout gestation, evoked subsequent locomotor hyperactivity and cognitive impairment. These effects were greater than those caused by equivalent exposures to nicotine alone, implying that other agents in tobacco smoke contributed to the adverse behavioral effects. In the present study, we examined the critical developmental windows of vulnerability for these effects, restricting TSE administration (0.2 mg/kg/day nicotine equivalent, or DMSO vehicle, delivered by subcutaneously-implanted pumps) to three distinct 10 day periods: the 10 days preceding mating, the first 10 days of gestation (early gestation), or the second 10 days of gestation (late gestation). The principal behavioral effects revealed a critical developmental window of vulnerability, as well as sex selectivity. Late gestational TSE exposure significantly increased errors in the initial training on the radial-arm maze in female offspring, whereas no effects were seen in males exposed during late gestation, or with either sex in the other exposure windows. In attentional testing with the visual signal detection test, male offspring exposed to TSE during early or late gestation showed hypervigilance during low-motivating conditions. These results demonstrate that gestational TSE exposure causes persistent behavioral effects that are dependent on the developmental window in which exposure occurs. The fact that effects were seen at TSE levels modeling secondhand smoke, emphasizes the need for decreasing involuntary tobacco smoke exposure, particularly during pregnancy.