Component Of Cigarette Smoke Research Articles

Simultaneous Determination of Four Aldehydes in Gas Phase of Mainstream Smoke by Headspace Gas Chromatography-Mass Spectrometry.

A method for simultaneous determination of acetaldehyde, propionaldehyde, acrolein, and crotonaldehyde in gas phase of cigarette mainstream smoke by headspace gas chromatography-mass spectrometry was developed and validated. Gas phase components of mainstream cigarette smoke were extracted with methanol, and then the samples were separated on a DB 624 (60 m, 0.32 mm x 1.8 mm) column, analyzed with headspace gas chromatography-mass spectrometry, and quantified by isotope internal standard. The linearities of acetaldehyde, propionaldehyde, acrolein, and crotonaldehyde were good (R2>0.992). The recoveries of acetaldehyde, propionaldehyde, acrolein, and crotonaldehyde were between 78.5% and 115%. The relative standard deviations were less than 10%. The limits of detection and limits of quantitation were 0.014 μg/cigarette ~0.12 μg/cigarette and 0.045 μg/cigarette ~0.38 μg/cigarette, respectively. The method had advantage of high sensitivity, it did not require derivatization of 2,4-dinitrophenylhydrazine and avoided a large number of adverse reactions during the process of derivation to improve the accuracy of result, and it was suitable for quantitative analysis of four aldehydes in gas phase of cigarette mainstream smoke.

Polycyclic Aromatic Hydrocarbons and Endocrine Disruption: Role of Testicular Gap Junctional Intercellular Communication and Connexins.

Ambient air pollution and smoking are well-documented risk factors for male infertility. Prevalent air pollutants and cigarette smoke components, polycyclic aromatic hydrocarbons (PAHs), are environmental and occupational toxicants that act as chemicals disrupting endocrine regulation and reproductive potential in males. Testicular gap junctional intercellular communication (GJIC) is critical for normal development and function of testicular tissue, thus we assessed GJIC as a process potentially targeted by PAHs in testes. Lower MW PAHs with a bay or bay-like region rapidly dysregulated GJIC in Leydig TM3 cells by relocalization of major testicular gap junctional protein connexin 43 (Cx43) from plasma membrane to cytoplasm. This was associated with colocalization between Cx43 and ubiquitin in intracellular compartments, but without any effect on Cx43 degradation rate or steady-state Cx43 mRNA levels. A longer exposure to active PAHs decreased steady-state levels of full-length Cx43 protein and its 2 N-truncated isoforms. Inhibition of GJIC by PAHs, similarly to a prototypic GJIC-inhibitor TPA, was mediated via the MAP kinase-Erk1/2 and PKC pathways. Polycyclic aromatic hydrocarbon-induced GJIC dysregulation in testes was cell-type-specific because neither PAH dysregulated GJIC in Sertoli TM4 cells, despite PAHs were rapidly taken up by both Leydig TM3 as well as Sertoli TM4 cells. Because TPA effectively dysregulated GJIC in both testicular cell types, a unique regulator of GJIC targeted by PAHs might exist in Leydig TM3 cells. Our results indicate that PAHs could be a potential etiological agent contributing to reproductive dysfunctions in males through an impairment of testicular GJIC and junctional and/or nonjunctional functions of Cx43.

Cigarette smoking and metabolic syndrome components: a cross-sectional study from Maracaibo City, Venezuela.

Background: A growing body of evidence suggests that cigarette smoking can cause the onset of metabolic syndrome prior to cardiovascular diseases. Therefore, the objective of this study was to evaluate the relationship between smoking habit and metabolic syndrome components in an adult population from Maracaibo city, Venezuela. Methods: The Maracaibo City Metabolic Syndrome Prevalence Study is a descriptive, cross-sectional study with random and multi-stage sampling. In this sub-study, 2212 adults from both genders were selected. On the basis of their medical background, they were classified as smokers, non-smokers and former smokers. Metabolic syndrome was defined according to Harmonizing 2009 criteria, using population-specific abdominal circumference cut-off points. The association between risk factors was evaluated using a logistic regression model. Results: In the studied population, 14.8% were smokers, 15.4% were former smokers. In the multivariate analysis, the presence of metabolic syndrome (smokers: OR, 1.54; 95% CI, 1.11-2.14; p=0.010) and its components were related to cigarette smoking, with the exception of hyperglycemia. High blood pressure was inversely associated with current smoking status (smokers: OR, 0.70 (0.51-0.95); p=0.025). Conclusion: Cigarette smoking represents a related factor with metabolic syndrome, being associated with low high-density lipoprotein-cholesterol, increased abdominal circumference and elevated triacylglyceride levels. Former smokers did not present a greater risk for developing this metabolic disease when compared to non-smokers. The effect of avoiding this habit should be evaluated in future studies in our population.

Nicotine induces a dual effect on the beige-like phenotype in adipocytes

Nicotine, the main component of cigarette smoke, affects white/brown adipocytes. Few studies have concentrated on beige adipocytes. In this study, 3T3-L1 cells were differentiated in the presence of nicotine (25, 50 and 100 ?mol/L) during early differentiation and maintenance stages. Cell viability and the state of lipid droplets were assessed by the MTT assay and Oil Red O, respectively, and the expression of beige-related genes and proteins was examined by RT-qPCR, Western blotting and flow cytometry. Nicotine did not alter adipocyte differentiation; however, it increased the expression of peroxisome proliferator- activated receptor gamma (PPAR?) protein during early differentiation and maintenance. Nicotine treatment during early differentiation downregulated gene and protein expression of PPAR? coactivator 1-alpha (PGC-1?), uncoupling protein 1 (UCP1) and cluster of differentiation 137 (CD137), and gene expression of Cbp/p300 interacting transactivator with Glu/ Asp rich carboxy-terminal domain 1 (Cited1), transmembrane protein 26 (Tmem26), and short stature homeobox 2 (Shox2). Nicotine treatment during the maintenance stage upregulated these beige-related genes/proteins. Nicotine treatment of immature adipocytes damaged beige function through a decrease in PGC-1?/UCP1 expression, but nicotine treatment of mature adipocytes or both immature and mature cells enhanced beige functioning. Nicotine induced beige-like phenotype dysfunction in 3T3-L1 adipocytes. This process may affect thermogenesis in adipose tissue and cause a dysfunction in fat metabolism.

Sesame Extract Attenuates the Degradation of Collagen and Elastin Fibers in the Vascular Walls of Nicotine-administered Mice.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by the weakening of the vascular walls and the progressive dilation of the abdominal aorta. Nicotine, a primary component of cigarette smoke, is associated with AAA development and rupture. Nicotine induces AAA development by weakening vascular walls. However, little is known about preventive methods using functional food factors for nicotine-induced vascular destruction. Sesamin and sesamolin are functional food factors that are fat-soluble lignans found in Sesamum indicum seeds. Previous reports indicated that sesamin and sesamolin have anti-oxidative and anti-inflammatory effects. In this study, we evaluated the effects of sesamin and sesamolin-rich sesame extract on the weakening of vascular walls in nicotine-administered mice. Sesame extract attenuated the degradation of collagen and elastin fibers caused by nicotine. In addition, sesame extract decreased the area positive for matrix metalloproteinase 12 (MMP-12) and oxidative stress in the vascular walls. These results suggest that sesame extract may decrease the weakening of vascular walls by suppressing the nicotine-induced degradation of collagen and elastin fibers. Sesame extract may be effective in preventing AAA development by decreasing both, MMP-12 expression and oxidative stress in vascular walls.

Tobacco Exposure Enhances Human Papillomavirus 16 Oncogene Expression via EGFR/PI3K/Akt/c-Jun Signaling Pathway in Cervical Cancer Cells.

High-risk human papillomavirus (HR-HPV) infection is not a sufficient condition for cervical cancer development because most infections are benign and naturally cleared. Epidemiological studies revealed that tobacco smoking is a cofactor with HR-HPV for cervical cancer initiation and progression, even though the mechanism by which tobacco smoke cooperates with HR-HPV in this malignancy is poorly understood. As HR-HPV E6/E7 oncoproteins overexpressed in cervical carcinomas colocalize with cigarette smoke components (CSC), in this study we addressed the signaling pathways involved in a potential interaction between both carcinogenic agents. Cervical cancer-derived cell lines, CaSki (HPV16; 500 copies per cell) and SiHa (HPV16; 2 copies per cell), were acutely exposed to CSC at various non-toxic concentrations and we found that E6 and E7 levels were significantly increased in a dose-dependent manner. Using a reporter construct containing the luciferase gene under the control of the full HPV16 long control region (LCR), we also found that p97 promoter activity is dependent on CSC. Non-synonymous mutations in the LCR-resident TPA (12-O-tetradecanoylphorbol 13-acetate)-response elements (TRE) had significantly decreased p97 promoter activation. Phosphoproteomic arrays and specific inhibitors revealed that CSC-mediated E6/E7 overexpression is at least in part reliant on EGFR phosphorylation. In addition, we showed that the PI3K/Akt pathway is crucial for CSC-induced E6/E7 overexpression. Finally, we demonstrated that HPV16 E6/E7 overexpression is mediated by JUN. overexpression, c-Jun phosphorylation and recruitment of this transcription factor to TRE sites in the HPV16 LCR. We conclude that acute exposure to tobacco smoke activates the transcription of HPV16 E6 and E7 oncogenes through p97 promoter activation, which involves the EGFR/PI3K/Akt/C-Jun signaling pathway activation in cervical cancer cells.

Absorption of blue light by cigarette smoke components is highly toxic for retinal pigmented epithelial cells.

Lesion to the retinal pigment epithelium (RPE) is a crucial event in the development of age-related macular degeneration (AMD), the leading cause of blindness in industrialized countries. Tobacco smoking and high-energy visible blue (HEV; 400-500nm) light exposure are major environmental risk factors for AMD. Individually, they have been shown to cause damage to the RPE. Tobacco smoke contains toxic polycyclic aromatic hydrocarbons (PAH) that can accumulate in RPE and which absorb HEV light. It can thus be postulated that the interaction between both factors in RPE cells can have a synergic toxic effect to the RPE. To test this hypothesis, cultured human RPE cells (ARPE19) were treated with nanomolar concentrations of benzo[a]pyrene (BaP) or indeno[1,2,3-cd]pyrene (IcdP), then exposed to HEV light using an irradiation system that mimics the solar spectrum normally transmitted to the retina through the human ocular media. Using mitochondrial network morphology changes and key features of AMD-related RPE defects such as apoptotic cell death and oxidative stress, we demonstrate that a synergistic phototoxicity is generated when nanomolar concentrations (≤ 500nM) of IcdP interact with sub-lethal amounts of HEV light. Indeed, we found IcdP to be at least 3000 times more toxic for RPE cells when irradiated with HEV light. This synergy translates into disruption of mitochondrial network, ROS enhanced accumulation and apoptosis of RPE cells. Our results underline an important interplay between two environmental risk factors involved in AMD progression and strongly indicate that IcdP, upon interaction with HEV light, may initiate the biological mechanisms underlying the association between cigarette smoking and AMD-related RPE degeneration.

Continuous exposure of nicotine and cotinine retards human primary pterygium cell proliferation and migration.

Pterygium is a triangular-shaped hyperplastic growth, characterized by conjunctivalization, inflammation, and connective tissue remodeling. Our previous meta-analysis found that cigarette smoking is associated with a reduced risk of pterygium. Yet, the biological effect of cigarette smoke components on pterygium has not been studied. Here we reported the proliferation and migration properties of human primary pterygium cells with continuous exposure to nicotine and cotinine. Human primary pterygium cells predominantly expressed the α5, β1, and γ subunits of the nicotinic acetylcholine receptor. Continuous exposure to the mixture of 0.15 μM nicotine and 2 μM cotinine retarded pterygium cell proliferation by 16.04% (P = 0.009) and hindered their migration by 11.93% ( P = 0.039), without affecting cell apoptosis. SNAIL and α-smooth muscle actin protein expression was significantly downregulated in pterygium cells treated with 0.15 μM nicotine-2 μM cotinine mixture by 1.33- ( P = 0.036) and 1.31-fold ( P = 0.001), respectively. Besides, the 0.15 μM nicotine-2 μM cotinine mixture also reduced matrix metalloproteinase (MMP)-1 and MMP-9 expressions in pterygium cells by 1.56- ( P = 0.043) and 1.27-fold ( P = 0.012), respectively. In summary, this study revealed that continuous exposure of nicotine and cotinine inhibited human primary pterygium cell proliferation and migration in vitro by reducing epithelial-to-mesenchymal transition and MMP protein expression, partially explaining the lower incidence of pterygium in cigarette smokers.

The cigarette smoke components induced the cell proliferation and epithelial to mesenchymal transition via production of reactive oxygen species in endometrial adenocarcinoma cells

Cigarette smoke (CS) causes about 480,000 deaths each year worldwide and is well-known to have harmful effects on the human body, leading to heart disease, stroke, lung cancer, and cardiovascular problems. In the present study, the effects of acrylonitrile (AN), benzo(a)pyrene (B(a)P), formaldehyde (FOR), isoprene (ISO), nicotine-derived nitrosamine ketone (NNK), which are the main components of CS, on the proliferation, invasion, and the epithelial-mesenchymal transition (EMT) process of human Ishikawa endometrial adenocarcinoma cells were investigated. Treating Ishikawa cells with CS components resulted in increased cell growth and altered expression of cell cycle-related genes: the protein expression of cyclin D & E increased, while the levels of p21 & p27 were reduced following treatment of these five CS components. In addition, CS components increased the invasion capacity of Ishikawa cells. The expression of the epithelial markers, E-cadherin and occludin, were significantly decreased, while the expression of the mesenchymal marker, N-cadherin, was significantly increased by CS components. In dichloro-dihydro-fluorescein diacetate (H2DCF-DA) assay, ROS production increased by treatment of CS components. The CS components activated the ROS-p38 MAPK-EMT pathway by increasing the level of phosphorylated p38 MAPK and p44/42 (ERK1/2), and by up-regulating Snail and Slug, the transcription factors for EMT. Taken together, these results indicate that CS components can promote progression of endometrial adenocarcinoma via increasing cell proliferation and the ROS-mediated EMT process.

Smoking-induced genetic and epigenetic alterations in infertile men.

Male fertility rates have shown a progressive decrease in both developing and industrialised countries in the past 50years. Clinical and epidemiological studies have demonstrated controversial results about the harmful effects of cigarette smoking on seminal parameters. Some studies could not establish a negative effect by tobacco smoking on sperm quality and function, whereas others have found a significant reduction in sperm quality and function. This study reviews the components in cigarette smoke and discusses the effects of smoking on male fertility by focusing extensively on smoking-induced genetic and epigenetic alterations in infertile men. Chromosomal aneuploidies, sperm DNA fragmentation and gene mutations are discussed in the first section, while changes in DNA methylation, chromatin remodelling and noncoding RNAs are discussed in the second section as part of epigenetic alterations.

Nicotine induces endothelial dysfunction and promotes atherosclerosis via GTPCH1.

Smoking is a major preventable risk factor for atherosclerosis. However, the causative link between cigarette smoke and atherosclerosis remains to be established. The objective of this study is to characterize the role of GTP cyclohydrolase 1 (GTPCH1), the rate‐limiting enzyme for de novo tetrahydrobiopterin (BH4) synthesis, in the smoking‐accelerated atherosclerosis and the mechanism involved. In vitro, human umbilical vein endothelial cells were treated with nicotine, a major component of cigarette smoke, which reduced the mRNA and protein levels of GTPCH1 and led to endothelial dysfunction. GTPCH1 overexpression or sepiapterin could attenuate nicotine‐reduced nitric oxide and ‐increased reactive oxygen species levels. Mechanistically, human antigen R (HuR) bound with the adenylateuridylate‐rich elements of the GTPCH1 3′ untranslated region and increased its stability; nicotine inhibited HuR translocation from the nucleus to cytosol, which downregulated GTPCH1. In vivo, nicotine induced endothelial dysfunction and promoted atherosclerosis in ApoE−/− mice, which were attenuated by GTPCH1 overexpression or BH4 supplement. Our findings may provide a novel and promising approach to atherosclerosis treatment.

Outcomes of developmental exposure to total particulate matter from cigarette smoke in zebrafish (Danio rerio)

The effects of prenatal exposure to cigarette smoke remain a subject of major interest, especially as it relates to neural development and adverse behavioral outcomes. Several studies have investigated the developmental toxicity of cigarette smoke components in a zebrafish model, showing that developmental exposure to total particulate matter (TPM; particulate phase of cigarette smoke) leads to adverse physiological aberrations and locomotor hyperactivity. Thus, the current study examines whether developmental TPM exposure of zebrafish embryos/larvae (F0) leads to physiological and behavioral alterations, and whether adverse effects are observed in adult fish and the next generation (F1; i.e. F0 offspring). We also examine whether behavioral effects are associated with changes in neural development, stress response, neurotransmitters, and bioenergetics. We demonstrate that TPM exposure during F0 development increased the incidence of deformities in F0 larvae, but F1 larvae did not exhibit any deformities. TPM exposure also resulted in swimming hyperactivity in F0 larvae and several behavioral changes were noted in F0 fish when they grew into adulthood. These behavioral changes were generally not associated with changes in markers of neural development in larvae, stress response in F0 adults, and concentration of neurotransmitters (acetylcholine, dopamine, and serotonin) in F0 adult brain. There were also no changes in F0 or F1 embryonic oxygen consumption rate (OCR; marker of bioenergetics and mitochondrial health); however, the OCR in the brain of F0 males was reduced with TPM. We conclude that developmental exposure to TPM affects larval physiology and induces hyperactive swimming behavior, but these effects do not persist in F1 larvae. Moreover, developmental TPM exposure leads to long-lasting sex-specific behavioral outcomes in the F0 adult fish.

Biomonitoring of Urinary Benzene Metabolite SPMA in the General Population in Central Italy

Background: Benzene is an important component of cigarette smoke and car exhaust. Products containing benzene in concentrations greater than 0.1% are prohibited in Europe, but 1% of benzene is still allowed in gasoline. The purpose of the study was to assess the levels of urine benzene biomarkers in a sample of the general population not occupationally exposed to benzene, resident in the period 2013–2014 in Central Italy, compared to other groups. Methods: The urinary levels of the benzene metabolites S-phenyl-mercapturic acid (SPMA) and cotinine (nicotine metabolite) were determined by means of HPLC with mass spectrometric detection in 1076 subjects. Results: The median SPMA value in smokers was 1.132 µg/g of creatinine while in non-smokers it was 0.097 µg/g of creatinine, and the 95th percentile results were seven times higher. Conclusion: The main source of benzene exposure in the studied population was active smoking, however, non-smokers were also exposed to airborne benzene concentrations. The concentration ranges found in this study can be used as a background reference for occupational exposure assessment to benzene by means of SPMA biomonitoring.

Targeting mitochondria with folic acid and vitamin B12 ameliorates nicotine mediated islet cell dysfunction.

Nicotine, one of the well-known highly toxic components of cigarette smoke, causes a number of adverse health effects and diseases. Our previous study has shown that nicotine induces reactive oxygen species (ROS) in islet cell and disrupts islet cell mitochondrial membrane potential (ΔΨm). However, supplementation with folic acid and vitamin B12 were found effective against nicotine induced changes in pancreatic islet cells. But the toxicological effects and underlying mechanisms of nicotine-induced mitochondrial dysfunction is still unknown. In this study, nicotine exposure decreases mitochondrial enzymes (pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, aconitase, malate dehydrogenase) activities by increasing cytosolic Ca2+ level which may contribute to increased mitochondrial ROS production by raising its flow to mitochondria. This in turn produces malondialdehyde and nitric oxide (NO) with a concomitant decrease in the activities of antioxidative enzymes and glutathione levels leading to loss of ΔΨm. Simultaneously, nicotine induces pancreatic islet cell apoptosis by modulating ΔΨm via increased cytosolic Ca2+ level, altered Bcl-2, Bax, cytochrome c, caspase-9, PARP expressions which were prevented by the supplementation of folic acid and vitamin B12 . In conclusion, nicotine alters islet cell mitochondrial redox status, apoptotic machinery, and enzymes to cause disruption in the ΔΨm and supplementation of folic acid and vitamin B12 possibly blunted all these mitochondrial alterations. Therefore, this study may help to determine the pathophysiology of nicotine-mediated islet cell mitochondrial dysfunction.

Abstract 2222: Using rare variants to characterize lung cancer risk

Abstract The human genome is exposed to a variety of exogenous and endogenous assaults capable of inducing DNA damage and contributing to diseases like cancer. Multiple DNA repair mechanisms are tasked with identifying and repairing these aberrant nucleotides, but damage is occasionally missed giving rise to somatic mutations. As we age, the relative abundance of somatic mutations increases, which may increase our risk for developing cancer. In addition, inter-individual variation in somatic mutation prevalence suggests an inter-individual variation in (a) inherited DNA repair capacity and/or (b) exposure to environmental mutagens. Therefore, somatic mutation prevalence may serve as an end-point biomarker for cancer risk, as it provides critical information regarding both. While Next Generation Sequencing (NGS) has provided unparalleled access to the human genome and revolutionized the field of cancer genomics, its implementation into the clinical setting has been limited by technical errors introduced during library preparation and on the sequencing platform. This makes identification of rare variants below 10-4 mutations per nucleotide through NGS a challenge, and the use of somatic mutations to characterize cancer risk unfeasible. To address this issue, unique molecular indexes (UMI) were assigned to each genomic copy, enabling reliable identification of rare somatic mutations versus technical errors. We hypothesize that measurement of somatic mutation prevalence in normal bronchial epithelial cells (NBEC) will serve as an end-point biomarker for lung cancer risk by identifying both (a) inherited sub-optimal DNA repair/protection and (b) exposure to inhaled environmental mutagens including components of cigarette smoke, radon, and/or occupational hazards. In our initial pilot study, we used genomic DNA derived from an A549 cell line to optimize an ERCC5 gene-specific, dual-indexed PCR method for UMI assignment, followed by sequencing on the Illumina MiSeq platform. In parallel analyses, UMI correction using 25,000 genomic copies provided over 10-fold greater sensitivity in identifying somatic mutations compared to no UMI correction. Based on this pilot, we obtained three clinical NBEC specimens via cytology brush biopsy of grossly normal (non-cancerous) airway from (a) heavy smoker with lung cancer, (b) heavy smoker without lung cancer, and (c) non-smoking control. UMIs were assigned to 50,000 genomic copies at 16 loci, increasing our theoretical limit of detection to approximately 1 in 4x107 nucleotides. Results thus far support further application of this approach in studies to assess NBEC somatic mutation prevalence as a biomarker for cancer risk. Citation Format: Daniel J. Craig, Mazzin Elsamaloty, Thomas M. Blomquist, Erin L. Crawford, James C. Willey. Using rare variants to characterize lung cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2222.

Cigarette smoke alters lung vascular permeability and endothelial barrier function (2017 Grover Conference Series).

Smoking of tobacco products continues to be widespread, despite recent progress in decreasing use. Both in the United States and worldwide, cigarette smoking is a major cause of morbidity and mortality. Growing evidence indicates that acute respiratory distress syndrome (ARDS) is among the consequences of cigarette smoking. Based on the topic from the 2017 Grover Conference, we review evidence that cigarette smoking increases lung vascular permeability using both acute and longer exposures of mice to cigarette smoke (CS). We also review studies indicating that CS extract disrupts cultured lung endothelial cell barrier function through effects on focal adhesion contacts, adherens junctions, actin cytoskeleton, and microtubules. Among the potentially injurious components of CS, the reactive aldehyde, acrolein, similarly increases lung vascular permeability and disrupts barrier function. We speculate that inhibition of aldehyde-induced lung vascular permeability may prevent CS-induced lung injury.

Cigarette smoke directly impairs skeletal muscle function through capillary regression and altered myofibre calcium kinetics in mice.

Cigarette smoke components directly alter muscle fatigue resistance and intracellular muscle fibre Ca2+ handling independent of a change in lung structure. Changes in muscle vascular structure are associated with a depletion of satellite cells. Sarcoplasmic reticulum Ca2+ uptake is substantially impaired in myofibres during fatiguing contractions in mice treated with cigarette smoke extract. Cigarette smokers exhibit exercise intolerance before a decline in respiratory function. In the present study, the direct effects of cigarette smoke on limb muscle function were tested by comparing cigarette smoke delivered to mice by weekly injections of cigarette smoke extract (CSE), or nose-only exposure (CS) 5days each week, for 8weeks. Cigarette smoke delivered by either route did not alter pulmonary airspace size. Muscle fatigue measured in situ was 50% lower in the CSE and CS groups than in control. This was accompanied by 34% and 22% decreases in soleus capillary-to-fibre ratio of the CSE and CS groups, respectively, and a trend for fewer skeletal muscle actin-positive arterioles (P=0.07). In addition, fewer quiescent satellite cells (Nes+Pax7+) were associated with soleus fibres in mice with skeletal myofibre VEGF gene deletion (decreased 47%) and CS exposed (decreased 73%) than with control fibres. Contractile properties of isolated extensor digitorum longus and soleus muscles were impaired. In flexor digitorum brevis myofibres isolated from CSE mice, fatigue resistance was diminished by 43% compared to control and CS myofibres, and this was accompanied by a pronounced slowing in relaxation, an increase in intracellular Ca2+ accumulation, and a slowing in sarcoplasmic reticulum Ca2+ uptake. These data suggest that cigarette smoke components may impair hindlimb muscle vascular structure, fatigue resistance and myofibre calcium handling, and these changes ultimately affect contractile efficiency of locomotor muscles independent of a change in lung function.

Acrolein-induced autophagy–dependent apoptosis via activation of the lysosomal–mitochondrial pathway in EAhy926 cells

Acrolein, a highly reactive α,β-unsaturated aldehyde, is a toxic component of cigarette smoke. As a lipid peroxidation biomarker, acrolein plays an important role in a wide variety of disease states, such as neurodegenerative, Alzheimer's disease, diabetes and atherosclerosis. Endothelial cell injury is one of the initiating factors of atherosclerosis, but the underlying molecular mechanisms remain unclear. Our study primarily focused on acrolein-induced autophagy–dependent apoptosis and the possible molecular mechanism. The results showed that treatment with acrolein increased the number of intracellular GFP-LC3 II punctuates and the expression of autophagosome biomarker LC3-II, with the low dose (25 μM) or at the early stage of treatment (3 h). Following treatment of EAhy926 cells with acrolein for 6 h, lysosomal permeabilization changed, and cathepsin B (CB) was released. Additionally, acrolein induced the collapse of mitochondrial transmembrane potential, and cytochrome c was released. Furthermore, caspase-3 and caspase-9 activation showed that acrolein induced EAhy926 cell apoptosis. Autophagy inhibitor 3MA and CB inhibitor CA-074 Me (CA) attenuated acrolein-induced apoptosis. Collectively, our results suggested that acrolein-induced apoptosis is autophagy-dependent, occurring via injury to lysosomes and mitochondria. This study provides new mechanistic insight toward understanding the pathogenesis of acrolein-related disorders.

β-Carotene Supplementation and Lung Cancer Incidence in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study: The Role of Tar and Nicotine.

The Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study demonstrated that β-carotene supplementation increases lung cancer incidence in smokers. Further, cigarettes with higher tar and nicotine content are associated with a higher risk of lung cancer. However, no studies have examined whether the increased risk associated with β-carotene supplementation in smokers varies by the tar or nicotine content of cigarettes. The ATBC Study was a randomized, double-blind intervention trial conducted in southwest Finland. A total of 29 133 male smokers, aged 50-69 years, were enrolled and randomly assigned to one of four groups (α-tocopherol, β-carotene, both, or placebo). Cox proportional hazards models were used to estimate the hazard ratio (HR) and 95% confidence intervals (CI) of lung cancer risk by β-carotene trial assignment stratified by a priori categories of cigarette tar and nicotine content. The β-carotene supplementation group had significantly higher risk of developing lung cancer in all categories of tar content (yes vs. no β-carotene supplementation-ultralight cigarettes [≤7 mg tar]: HR = 1.31, 95% CI = 0.91 to 1.89; nonfiltered cigarettes [≥21 mg tar]: HR = 1.22, 95% CI = 0.91 to 1.64; p for interaction = .91). Similarly, there was no interaction with nicotine content (yes vs. no β-carotene supplementation-ventilated cigarettes [≤0.8 µg nicotine]: HR = 1.23, 95% CI = 0.98 to 1.54; nonfiltered cigarettes [≥1.3 µg nicotine]: HR = 1.22, 95% CI = 0.91 to 1.64; p for interaction = .83). These findings support the conclusion that supplementation with β-carotene increases the risk of lung cancer in smokers regardless of the tar or nicotine content of cigarettes smoked. Our data suggest that all smokers should continue to avoid β-carotene supplementation. Previous studies demonstrated that β-carotene supplementation increases risk of lung cancer in smokers. This study moves the field forward by examining the potential for modification of risk of lung cancer with different levels of tar and nicotine in cigarettes smoked, as interaction with carcinogens in these components of cigarette smoke is hypothesized to be the mechanism by which β-carotene increases risk. Our study provides evidence that the increased risk of lung cancer in smokers who take β-carotene supplements is not dependent upon the tar or nicotine level of cigarettes smoked and suggests that all smokers should continue to avoid β-carotene supplementation.

CONTRIBUTION OF ENVIRONMENTAL POLLUTANTS TO EPIGENETIC MODIFICATION IN RAT VASCULAR SMOOTH MUSCLE CELLS

Objective: More than a quarter of human diseases have been linked to exposure to environmental pollutants. These pollutants can exacerbate disease conditions through labile epigenetic modifications and directly result in changes in gene transcription. The most commonly reported epigenetic changes are acetylation and methylation of histone lysine. Contribution of acrolein, an environmental pollutant and a major component of cigarette smoke, to cardiovascular diseases and several neurological disorders has been reported. We have previously reported toxicity of acrolein in rat's vascular smooth muscle cells (VSMCs) and the effect of precursor of glutathione transferase, N-acetyl cysteine (NAC), in prevention of acrolein toxicity. In the present study, modification of histones and their effect on protein expression was further investigated in rat's VSMCs in the present/absent of acrolein and NAC. Design and method: VSMCs were treated with 3 μg/ml of acrolein for 6 and 24 hours in the present/absent of 0.2 mM NAC. At the end of the treatment, MTS essay, ELISA, western blot analysis, immunofluorescence, and LC/MS/MS analysis were used to check cells viability, superoxide dismutase (SOD) activity/expression, reactive oxygen species (ROS) generation, expression and cellular localization of H3K9 tri-methylation and acetylation, and identification of proteins affected by these changes. Results: Acrolein treated VSMCs exhibited the highest toxicity after 6 hours. Acrolein increased generation of ROS, reduced SOD activity and expression. There was 52% induction of acetylation and 62% in tri-methylation. Addition of N-acetyl cysteine reduced ROS, induced SOD (45%), reduced H3K9 acetylation by 102% and tri-methylation by 120%. These changes were confirmed with immunofluorescence staining. LC/MS/MS analysis revealed increased in actin protein and a significant decreased in annexin, heat shock cognate, and myosin 9 proteins. Conclusions: Based on our data we are concluding that effect of acrolein on VSCMs is partially due to alterations of H3K9 methylation/acetylation, resulting in overexpression of actin and significant reduction of annexin, heat shock cognate, and myosin 9 proteins. Addition of 0.2 mM NAC resulted in prevention of toxicity by acrolein by reducing ROS, improving SOD and preventing changes in H3K9 acetylation and methylation.