Agents In Cigarette Smoke Research Articles

The amelioration of nicotine-induced reproductive impairment in male mouse by Sambucus ebulus L. fruit extract.

Nicotine as a toxic agent in cigarette smoke impairs the reproductive system. Sambucus ebulus extract (SEE) is shown to have some beneficial effects such as antioxidant properties. The aim of this study was to evaluate the effects of SEE on the hormones of the pituitary-gonadal axis, lipid peroxidation index, antioxidant enzymes, spermatogenesis, and epididymal sperm parameters in male mice treated with nicotine. Adult male mice were divided into five groups; A: normal saline, B: 1 mg/kg nicotine, C: 1 mg/kg nicotine and 10 mg/kg SEE, D: 1 mg/kg nicotine and 50 mg/kg SEE, D: 1 mg/kg nicotine and 100 mg/kg SEE. Treatments lasted for 35 days. The spermicidal activity of SEE was tested in vitro. Sperm count, motility and morphology were assessed for fertility. Serum testosterone, prolactin and luteinizing hormone (LH) were measured, using ELISA. Serum malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) activity were measured, using colorimetric assays. Spermatogenesis was evaluated by Johnsen’s score and morphometry in histological slides. SEE at different doses did not have any spermicidal activity. Sperm parameters were reduced in the nicotine-treated group, compared with controls (P<0.01). Nicotine reduced testosterone and LH levels (P<0.01) and increased prolactin (P<0.01). A hike in MDA and a reduction in SOD activity without change on CAT, were observed in the nicotine group. Nicotine caused hypospermatogenesis. SEE improved most of the above-mentioned parameters, especially in the doses of 50 and 100 mg/kg. Beneficial effects of SEE in the doses of 50 and 100 mg/kg on male reproduction impairment, induced by nicotine might be partly attributed to the reduction of oxidative stress and changes in the hormones of the pituitary-gonadal axis.

Epigenetic Changes in Alveolar Type II Lung Cells of A/J Mice Following Intranasal Treatment with Lipopolysaccharide.

Lipopolysaccharide (LPS) is a bacterial endotoxin present in cigarette smoke. LPS is known to induce inflammation and to increase the size and the multiplicity of lung tumors induced by tobacco-specific nitrosamines. However, the means by which LPS contributes to pulmonary carcinogenesis are not known. One possible mechanism includes LPS-mediated epigenetic deregulation, which leads to aberrant expression of genes involved in DNA repair, tumor suppression, cell cycle progression, and cell growth. In the present work, epigenetic effects of LPS were examined in alveolar type II lung cells of A/J mice. Type II cells were selected because they serve as progenitors of lung adenocarcinomas in smoking induced lung cancer. A/J mice were intranasally treated with LPS, followed by isolation of alveolar type II cells from the lung using cell panning. Global levels of DNA methylation and histone acetylation were quantified by mass spectrometry, while genome-wide transcriptomic changes were characterized by RNA-Seq. LPS treatment was associated with epigenetic changes including decreased cytosine formylation and reduced histone H3K14 and H3K23 acetylation, as well as altered expression levels of genes involved in cell adhesion, inflammation, immune response, and epigenetic regulation. These results suggest that exposure to inflammatory agents in cigarette smoke leads to early epigenetic changes in the lung, which may collaborate with genetic changes to drive the development of lung cancer.

Therapeutic Potential of Cytosolic PLA2 Isoform–Specific Inhibitor Arachidonyl Trifluromethyl Ketone in Cigarette Smoke Condensate–Induced Pathological Conditions in Alveolar Type I and II Epithelial Cells

Background: Cigarette smoking is responsible for various lung pathologies including chronic lung inflammation, emphysema, chronic obstructive pulmonary disease (COPD), cancer, and annually causes almost 10 million deaths globally. During smoke exposure, most affected cells are the alveolar epithelial cells where as a repair mechanism, activation of cytosolic phospholipase A2 enzymes takes place. High free radicals and cPLA2 activity due to continuous exposure of smoke exposure leads to elevated levels of secondary metabolites and various pathophysiologic conditions such as chronic inflammation, oxidative stress and cancer. To reduce the burden of chronic inflammation as well as oxidative stress, and higher levels of secondary metabolites whose role is well defined in progression of cancer, we checked the therapeutic potential of cPLA2 inhibitor arachidonyl trifluromethyl ketone (ATK) by pharmacologically targeting the most expressible cPLA2 during continuous exposure of cigarette smoke. Aim: To check the therapeutic potential of cytosolic PLA2 isoform specific inhibitor arachidonyl trifluromethyl ketone in cigarette smoke condensate–induced pathologic conditions in alveolar type I and II epithelial cells. Methods: Effect of cPLA2 inhibitor on CSC-induced cPLA2 activity were checked using colorimetric assay, cell viability using MTT assay, FDA uptake assay using fluorescence microscopy, ROS levels and apoptosis markers through flow cytometry, and ERK levels using ELISA, in both type of alveolar epithelial cells. Results: ATK significantly mimicked CSC-induced cPLA2 activity, free radicals, primary apoptosis, ratio of apoptotic/apoptotic proteins and levels of ERK whereas protected cells from loss of cell viability and membrane integrity. Conclusion: Current observations revealed cPLA2s as a potential therapeutic target and their inhibitor ATK as a potential therapeutic agent in Cigarette smoke induced pathological conditions in alveolar type I and II epithelial cells.

Linking Cigarette Smoking/Tobacco Exposure and Cluster Headache: A Pathogenesis Theory.

To propose a hypothesis theory to establish a linkage between cigarette smoking and cluster headache pathogenesis. Cluster headache is a primary headache syndrome grouped under the trigeminal autonomic cephalalgias. What distinguishes cluster headache from all other primary headache conditions is its inherent connection to cigarette smoking. It is undeniable that tobacco exposure is in some manner related to cluster headache. The connection to tobacco exposure for cluster headache is so strong that even if an individual sufferer never smoked, then that individual typically had significant secondary smoke exposure as a child from parental smoking behavior and in many instances both scenarios exist. The manner by which cigarette smoking is connected to cluster headache pathogenesis is unknown at present. If this could be determined this may contribute to advancing our understanding of cluster headache pathophysiology. Hypothesis statement. The hypothesis theory will include several principles: (1) the need of double lifetime tobacco exposure, (2) that cadmium is possibly the primary agent in cigarette smoke that leads to hypothalamic-pituitary-gonadal axis toxicity promoting cluster headache, (3) that the estrogenization of the brain and its specific sexually dimorphic nuclei is necessary to develop cluster headache with tobacco exposure, and (4) that the chronic effects of smoking and its toxic metabolites including cadmium and nicotine on the cortex are contributing to the morphometric and orexin alterations that have been previously attributed to the primary headache disorder itself.

Nicotine exposure alters human vascular smooth muscle cell phenotype from a contractile to a synthetic type

Objective: Cigarette smoking is a known risk factor for arteriosclerosis. In atheromatous plaques, vascular smooth muscle cells (VSMCs) display a phenotype that is different from the contractile type under normal conditions. Nicotine is the major pharmacological agent in cigarette smoke. However, any direct effect of nicotine on VSMCs remains uncertain. Because nicotine promotes VSMC migration, its phenotype may change due to nicotine. Approach and results: We used human aorta primary smooth muscle cells (HuAoSMCs), differentiated with transforming growth factor-β, to investigate changes in the protein levels of differentiation markers and in the activity of mitogen-activated protein kinases (MAPKs) after exposure to 0.1 μM of nicotine for 48 h. After nicotine exposure, the protein levels of myosin II 10 (2.93-fold) and β-actin (1.66-fold), synthetic type markers, were increased. In contrast, the levels of the contractile type markers, myosin II 11 (0.63-fold), high-molecular-weight caldesmon (0.40-fold) and SM22 (0.66-fold), which concern differentiated VSMC, were decreased. Moreover, nicotine exposure induced enhanced activation of p38 MAPK (1.30-fold) and extracellular signal-regulated kinase (1.91-fold). These results indicated that the phenotype of HuAoSMCs had changed to a synthetic-like type because of nicotine exposure. Thus, nicotine is one factor that can alter protein expression of differentiation markers in VSMCs. Besides, the increase of intracellular Ca2+ levels suggested that these effects of nicotine were mediated through nicotinic acetylcholine receptors. Conclusion: Nicotine has already been reported to promote VSMC migration from the tunica media to atheromatous plaques in the vascular intima. This phenomenon may occur because nicotine directly induces VSMC transformation from contractile type to synthetic-like type via nicotinic acetylcholine receptors and G protein-coupled receptors.

Galium verum aqueous extract strongly inhibits the motility of head and neck cancer cell lines and protects mucosal keratinocytes against toxic DNA damage.

Galium verum, also known as Lady's Bedstraw, is an herbaceous plant native to Europe and Asia, and has been used in traditional medicine as an anticancer medicine applied in most cases as a decoction. The influence of a Galium verum decoction on the head and neck cancer cell lines HLaC78 and FADU was analyzed and proved to be toxic in high doses on both cell lines. Cytotoxicity appeared to be influenced by expression of p-glycoprotein (MDR-1) in the carcinoma cell lines. Mucosal keratinocytes, although void of MDR-1 expression, showed only low sensitivity against high Galium concentrations. Sublethal doses of Galium extract acted as strong inhibitors of motility, as shown by a spheroid-based invasion analysis on Matrigel-coated surfaces. Inhibition of invasion was significantly more pronounced in the invasive HLaC78 cell line. mRNA expression analysis of matrix metalloproteinases MMP-2 and MMP-9 and their inhibitors TIMP-1/-2 revealed significant TIMP-1 upregulation after an 8-h Galium exposition in FADU cells. Gelatinolytic activity, however, was not influenced by Galium extract in HLaC78, in the FADU cells MMP-2/-9 activity was slightly increased after incubation with Galium extract. In primary mucosal keratinocytes, Galium decoction protected DNA against benz[a]pyrene, one of the most DNA toxic agents in cigarette smoke. In conclusion Galium extract may be useful as a preventive and/or a concomitant therapeutic approach in head and neck cancer.

New validated LC-MS/MS method for the determination of three alkylated adenines in human urine and its application to the monitoring of alkylating agents in cigarette smoke.

A highly specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of urinary N(3)-methyladenine (N(3)-MeA), N(3)-ethyladenine (N(3)-EtA), and N(3)-(2-hydroxyethyl)adenine (N(3)-HOEtA). Chromatographic separation was achieved on a hydrophilic interaction liquid chromatography column, with a mobile phase gradient prepared from aqueous 10 mM ammonium formate-acetonitrile (5:95 v/v, pH 4.0). Quantification of the analytes was done by multiple reaction monitoring using a triple-quadrupole mass spectrometer in positive-ionization mode. The limits of quantification were 0.13, 0.02, and 0.03 ng/mL for N(3)-MeA, N(3)-EtA, and N(3)-HOEtA, respectively. Intraday and interday variations (relative standard deviations) ranged from 0.6 to 1.3 % and from 3.7 to 7.5 %. The recovery ranges of N(3)-MeA, N(3)-EtA, and N(3)-HOEtA in urine were 80.1-97.3 %, 83.3-90.0 %, and 100.0-110.0 %, respectively. The proposed method was successfully applied to urine samples from 251 volunteers including 193 regular smokers and 58 nonsmokers. The results showed that the levels of urinary N(3)-MeA, N(3)-EtA, and N(3)-HOEtA in smokers were significantly higher than those in nonsmokers. Furthermore, the level of urinary N(3)-MeA in smokers was found to be positively correlated with the level of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (r = 0.48, P < 0.001, N = 192). This method is appropriate for routine analysis and accurate quantification of N(3)-MeA, N(3)-EtA, and N(3)-HOEtA. It is also a useful tool for the surveillance of alkylating agent exposure.

LDH enzyme activity in human saliva: The effect of exposure to cigarette smoke and its different components

ObjectiveAldehydes and reactive nitrogen species (RNS) are important chemically active agents in cigarette smoke (CS). Salivary lactate dehydrogenase (LDH) originates predominantly from oral epithelium and was identified as an oral state marker. Its activity in saliva decreases after CS exposure. The aims of the current study were to identify the specific damaging agents in CS responsible for this activity reduction and to understand the mechanisms participating in CS oxidative damage to the salivary enzymes. MethodsPurified and salivary LDH samples were exposed to different levels of CS, pure acrolein, acetaldehyde, peroxynitrite and RNS donors. Each response of the isolated agent to the exposure was examined by a spectrophotometric enzyme activity assay and a Western blot. ResultsCS exposure caused a 34% reduction in LDH activity. Isolated treatment with unsaturated-aldehydes (acrolein, 10μmol) caused a 61% reduction, while saturated-aldehydes (acetaldehyde, 200μmol), peroxynitrite (200μM) and RNS donor (SIN-1, 2mM) caused no substantial effect. All five LDH isoenzymes reacted similarly. The carbonyl immunoblotting assay revealed a fourfold increase in carbonyl content when treated with CS and a sevenfold increase when treated with acrolein. Conclusionα,β-Unsaturated-aldehydes were identified as the main CS ingredient responsible for salivary LDH activity diminution. The effect of saturated-aldehydes and RNS donors was negligible. Unsaturated-aldehydes are capable of introducing carbonyl group into proteins, causing their dysfunction. This provides a molecular explanation for a decrease in LDH enzymatic activity in saliva.

Replication across Regioisomeric Ethylated Thymidine Lesions by Purified DNA Polymerases

Causal links exist between smoking cigarettes and cancer development. Some genotoxic agents in cigarette smoke are capable of alkylating nucleobases in DNA, and higher levels of ethylated DNA lesions were observed in smokers than in nonsmokers. In this study, we examined comprehensively how the regioisomeric O(2)-, N3-, and O(4)-ethylthymidine (O(2)-, N3-, and O(4)-EtdT, respectively) perturb DNA replication mediated by purified human DNA polymerases (hPols) η, κ, and ι, yeast DNA polymerase ζ (yPol ζ), and the exonuclease-free Klenow fragment (Kf(-)) of Escherichia coli DNA polymerase I. Our results showed that hPol η and Kf(-) could bypass all three lesions and generate full-length replication products, whereas hPol ι stalled after inserting a single nucleotide opposite the lesions. Bypass conducted by hPol κ and yPol ζ differed markedly among the three lesions. Consistent with its known ability to efficiently bypass the minor groove N(2)-substituted 2'-deoxyguanosine lesions, hPol κ was able to bypass O(2)-EtdT, though it experienced great difficulty in bypassing N3-EtdT and O(4)-EtdT. yPol ζ was only modestly blocked by O(4)-EtdT, but the polymerase was strongly hindered by O(2)-EtdT and N3-EtdT. LC-MS/MS analysis of the replication products revealed that DNA synthesis opposite O(4)-EtdT was highly error-prone, with dGMP being preferentially inserted, while the presence of O(2)-EtdT and N3-EtdT in template DNA directed substantial frequencies of misincorporation of dGMP and, for hPol ι and Kf(-), dTMP. Thus, our results suggested that O(2)-EtdT and N3-EtdT may also contribute to the AT → TA and AT → GC mutations observed in cells and tissues of animals exposed to ethylating agents.

Cigarette Smoke‐Induced Myocardial Inflammation and Contractile Dysfunction Is Mediated by Unsaturated Aldehydes and TRPV1

Cigarette smoking triggers proinflammatory response through stimulating the capsaicin‐sensitive transient receptor potential cation channel TRPV1. Recent evidence suggests that smoking is associated with an overtly increased prevalence of heart diseases. This study was designed to examine the effect of 2 α,β‐unsaturated aldehydes present in cigarette smoke, namely crotonaldehyde and acrolein, on myocardial insulin sensitivity and contractile function. Our results revealed that in vitro short‐term exposure of crotonaldehyde, and acrolein provoked myocardial inflammatory response and dampened insulin signaling manifested as accumulation of the cytokines TNF‐α and NFκB as well as reduced insulin‐stimulated glucose uptake. These responses were accompanied with compromised cardiomyocyte contractile function including depressed peak cardiomyocyte contractile capacity, prolonged duration of relaxation, decreased intracellular Ca2+ release and slowed intracellular Ca2+ clearing. Activation of TRPV1 was enhanced in response to aldehyde exposure. Interestingly, the crotonaldehyde and acrolein‐induced pro‐inflammatory response, loss of insulin sensitivity and cardiomyocyte contractile function were significantly attenuated or mitigated by the aldehyde scavenger glutathione and the TRPV1 antagonist capsazepine. Both aldehydes and capsaicin increased the activity of Nox3 NADPH oxidase activity and STAT1. Inhibition of Nox3 and STAT1 independently blunt the crotonaldehyde and acrolein‐induced cardiomyocyte dysfunction. Inhibition of TRPV1 protected against the aldehyde cytotoxicity. Taken together, our data suggest a role of α,β‐unsaturated aldehydes as the main causative agents in cigarette smoke in the pathogenesis of cigarette smoke‐induced heart diseases possibly via a Nox3/STAT1‐ and TRPV1‐dependent mechanism.

Effects of perinatal nicotine exposure on the development of albino rat’s alveoli with special reference to the role of the vascular endothelial growth factor

BackgroundNicotine is one of the most toxic and addictive agents in cigarette smoke. Maternal cigarette smoking may affect lung development and maturation of the fetus. Recently, it has been reported that blood vessels promote alveolar growth during development and contribute toward the maintenance

Cigarette Smoking and Lung Cancer: Pediatric Roots.

A vast array of data suggests that early age of smoking onset enhances the risk for development of lung cancer in adulthood. Initiation of smoking at a young age may influence the development of lung cancer because of its effect on duration of smoking. Early onset of smoking also may serve as an independent risk factor. It may increase the likelihood that smoking occurs during a critical period of development that enhances susceptibility to the adverse effects of cancer causing agents in cigarette smoke, thereby facilitating the initiation of the carcinogenic process. While evidence for the latter hypothesis derives from a variety of sources, definitive proof has proven elusive. Whether or not early age of smoking serves as an independent risk factor for lung carcinogenesis, the consensus of the public health community is that prevention of smoking onset at a young age and early cessation are keys to stemming the current lung cancer pandemic. Population approaches to tobacco prevention and control, such as measures contained in the World Health Organization Framework Convention Tobacco Control Treaty, offer the best opportunity, on the scale needed, to create a smoke-free world and bring an end to the pandemic of tobacco-related disease.

January 2012: This Month in JoVE

The Journal of Visualized Experiments (JoVE) begins 2012 by publishing its 1500th article, which focuses on a method for analyzing the free radical composition of cigarette smoke. Although tobacco tar is thought to be the most carcinogenic agent in cigarette smoke, free radicals are well known cancer-causing molecules, and the level of these carcinogens contained in cigarette smoke is not currently known. The method employed in this article combines the use of a single-port smoking device, which simulates cigarette puffing, with electron spin resonance (ESR) spectroscopy, a technique, which measures the spin energy emitted by the unpaired electrons in free radicals. One important modification of the single port smoking device used in this article, is the introduction of a liquid nitrogen trap in front of the spin trap. Liquid nitrogen removes water vapor from cigarette smoke and greatly improves ESR spectra as a result. In addition to measuring the free radical concentration found in cigarette smoke, plant antioxidants are introduced into modified cigarette filters and the ESR spectra of cigarette smoke passed through these filters is obtained. Grape seed extract and lycopene were observed to have the most significant scavenging effects on free radicals, and if cost effective means of including these molecules into cigarette filters can be developed, a less harmful cigarette could be created. In Clinical and Translational Medicine, JoVE presents an analytical method for determining the local Gyrification Index (lGI) - a measurement of the extent of cortical folding. Cortical folds develop during the first few post natal months, and adverse events during this time can lead to abnormalities in the brain's surface anatomy, which may ultimately correlate with neuropsychiatric disorders. The analysis tool developed for calculating the local Gyrification Index is Freesurfer, which can convert T1 weighted MRI scans into 3D reconstructions of the cortex. Separate reconstructions are created for the grey/white mater and grey/pial interfaces, which are manually adjusted before computating the lGI. Following a smoothing operation, circular regions of the pial and grey/white surfaces are compared, in order to compute the lGI for thousands of points over the entire brain. Freesurfer makes analysis of gyrification straightforward and accessible to clinicians and brain scientists, who are interested in comparing the extent of cortical folding between different individuals in normal and disease states. JoVE's Immunology and Infection article for the month of January continues the journal's tradition of publishing cutting edge methodology in vector born disease research. Specifically, this article adds to JoVE's comprehensive collection of protocols for malaria research by extending our library to cerebral malaria - a devastating condition with high susceptibility in children. Briefly, this protocol involves exposing a monolayer of human brain endothelial cells, passaged onto petri dishes, to Plasmodium falciparum-infected red blood cells. Following washing and enriching steps, high yields of parasites, which carry the potential to infect the brain, can be isolated and further characterized. In Neuroscience, JoVE showcases a method for studying the migration of neural crest cells. These cells are a transient, multipotent, developmental cell type that can, in addition to becoming neuronal tissue, differentiate into smooth muscle, chromaffin cells, cartiliage, and bone. Chick embryos are carefully prepared, so that the trunk region can be isolated and the neural tube excised for overnight culture on fibronectin-coated coverslips, which allows neural crest cells to emigrate from the neural tube. Following removal of the neural tube, coverslips containing the cultured neural crest cells are transferred to a modified Zigmond chamber - a chamber which enables a concentration gradient of putative chemotactic agent to be established across the culture. Timelapes imaging followed by analysis of cell trajectories, is used to assess the chemotactic potential of molecules loaded into the zigmund chamber, providing insight into the molecular cues that guide migration of this important developmental cell type. These featured articles comprise four of fifty video-protocols for release in January. Other notable articles include methods for visualizing the cuticle structures of c. elegans, thermal imaging of delayed onset muscle soreness, culturing the ventral midbrain in organotypic slices, and inducing atherosclerosis in mice.

Smoking-induced satellite associations in a rural population of south India: An in vitro study

Background:Genotoxic carcinogens in cigarette smoke interact with DNA, causing cytotoxicity. Cytogenetic damage therefore seems to be an excellent biomarker for determining the effect of exposure to chromosome-damaging agents in cigarette smoke.Purpose:To study the utility of measurement of frequency of satellite associations (SA) as a biomarker for chromosomal damage using cytogenetic assay in peripheral blood lymphocytes.Materials and Methods:This study was conducted on 30 smokers and 30 nonsmokers drawn from a rural population of South India. Smokers were divided into three groups of ten each based on their smoking index (SI) (group I: SI < 150, group II: SI 150–300, and group III: SI > 300) and the frequency of SAs was studied.Results:The frequency of SAs was significantly greater in smokers than in nonsmokers and the frequency of SAs among the smokers was also seen to increase with increase in SI.Conclusion:The results of this study indicate that the genotoxic effect of cigarette smoke on chromosomes increases with smoking intensity. These findings can be used to support smoking cessation interventions.

Abstract 4697: Liquid chromatography-electrospray ionization tandem mass spectrometry analysis of 7-ethylguanine in human urine

Abstract Considerable evidence in the literature demonstrates the exposure of humans to an unknown ethylating agent. Previous studies have demonstrated the presence of 3-ethyl-Ade in urine, 7-ethylguanine (7-ethyl-Gua) in urine and human liver DNA, 7-ethyldGuo and O4-ethyl-dThd in human lung DNA, and ethylvaline in hemoglobin. Some studies also report higher levels of ethyl adducts in smokers compared to nonsmokers, and there is convincing evidence for an uncharacterized ethylating agent in cigarette smoke. To further investigate this question and evaluate the potential role of ethylating agents in smoking-induced cancer we have developed a liquid chromatography-electrospray ionization tandem mass spectrometry-selected reaction monitoring (LC-ESI-MS/MS-SRM) method for analysis of 7-ethyl-Gua in human urine. A two-dimensional chromatography approach was used with [15N5]7-ethyl-Gua as the internal standard. Human urine was diluted with 15 mM ammonium acetate and partially purified by a solid-phase extraction cartridge (Strata-X 33 µm, 30 mg/1 mL, Phenomenex) and a mixed mode cation exchange reversed-phase extraction cartridge (Oasis MCX, 30 mg/cartridge, Waters). The fraction from this last purification step containing the analyte (30% MeOH, 5% NH4OH) was dried and redissolved in 80% acetonitrile, 0.1% formic acid and purified by HPLC on a YMC Polyamino II column (150 × 1.0 mm I.D. 5 μm). The fraction containing 7-ethyl-Gua was collected, dried, dissolved in H2O and analysed by LC-ESI-MS/MS-SRM using a Thermo Hypersil Gold PFP column (150 × 0.5 mm I.D. 3 μm). The analysis was carried out using the transition m/z 180 [M + H]+ → m/z 152 [Gua+ H]+ for 7-ethyl-Gua and m/z 185 → m/z 157 for the internal standard. Recovery was about 50%. The detection limit was approximately 10 fmol on column. The peaks attributed to 7-ethyl-Gua and the labeled internal standard had identical retention time and were observed in most of the human urine samples analyzed. The identity of the peak was confirmed by re-injecting the samples after spiking with a standard solution of 7-ethyl-Gua. Twenty-one human urine samples (1 mL) from 11 smokers and 10 nonsmokers were analyzed. 7-Ethyl-Gua was clearly detected in 8 out of 11 samples from smokers and 5 out of 10 samples from nonsmokers. These results strongly indicate that 7-ethyl-Gua is present in human urine and demonstrate that it can be detected and quantified. Further investigation is required to clarify its relationship to cigarette smoking. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4697.

Determination of methyl-, 2-hydroxyethyl- and 2-cyanoethylmercapturic acids as biomarkers of exposure to alkylating agents in cigarette smoke

Alkylating agents occur in the environment and are formed endogenously. Tobacco smoke contains a variety of alkylating agents or precursors including, among others, N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acrylonitrile and ethylene oxide. We developed and validated a method for the simultaneous determination of methylmercapturic acid (MMA, biomarker for methylating agents such as NDMA and NNK), 2-hydroxyethylmercapturic acid (HEMA, biomarker for ethylene oxide) and 2-cyanoethylmercapturic acid (CEMA, biomarker for acrylonitrile) in human urine using deuterated internal standards of each compound. The method involves liquid/liquid extraction of the urine sample, solid phase extraction on anion exchange cartridges, derivatization with pentafluorobenzyl bromide (PFBBr), liquid/liquid extraction of the reaction mixture and LC–MS/MS analysis with positive electrospray ionization. The method was linear in the ranges of 5.00–600, 1.00–50.0 and 1.50–900 ng/ml for MMA, HEMA and CEMA, respectively. The method was applied to two clinical studies in adult smokers of conventional cigarettes who either continued smoking conventional cigarettes, were switched to test cigarettes consisting of either an electrically heated cigarette smoking system (EHCSS) or having a highly activated carbon granule filter that were shown to have reduced exposure to specific smoke constituents, or stopped smoking. Urinary excretion of MMA was found to be unaffected by switching to the test cigarettes or stop smoking. Urinary HEMA excretion decreased by 46 to 54% after switching to test cigarettes and by approximately 74% when stopping smoking. Urinary CEMA excretion decreased by 74–77% when switching to test cigarettes and by approximately 90% when stopping smoking. This validated method for urinary alkylmercapturic acids is suitable to distinguish differences in exposure not only between smokers and nonsmokers but also between smoking of conventional and the two test cigarettes investigated in this study.

Progress and challenges in selected areas of tobacco carcinogenesis.

Tobacco use continues to be a major cause of cancer in the developed world, and despite significant progress in this country in tobacco control, which is driving a decrease in cancer mortality, there are still over 1 billion smokers in the world. This perspective discusses some selected issues in tobacco carcinogenesis focusing on progress during the 20 years of publication of Chemical Research in Toxicology. The topics covered include metabolism and DNA modification by tobacco-specific nitrosamines, tobacco carcinogen biomarkers, an unidentified DNA ethylating agent in cigarette smoke, mutations in the K-RAS and p53 gene in tobacco-induced lung cancer and their possible relationship to specific carcinogens, secondhand smoke and lung cancer, emerging issues in smokeless tobacco use, and a conceptual model for understanding tobacco carcinogenesis. It is hoped that a better understanding of mechanisms of tobacco-induced cancer will lead to new and useful approaches for the prevention of lung cancer and other cancers caused by tobacco use.

Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry Analysis of 7-Ethylguanine in Human Liver DNA

Considerable evidence in the literature demonstrates the exposure of humans to an unknown ethylating agent. Previous studies have demonstrated the presence of 7-ethyl-Gua and 3-ethyl-Ade in urine, 7-ethyl-dGuo and O4-ethyl-dThd in human lung, and ethylvaline in hemoglobin. Some studies also report higher levels of ethyl adducts in smokers than in nonsmokers, and there is convincing evidence for an uncharacterized ethylating agent in cigarette smoke. To further investigate this question, we have developed a liquid chromatography-electrospray ionization tandem mass spectrometry-selected reaction monitoring method for analysis of 7-ethyl-Gua in human liver DNA. To our knowledge, there are no previous reports of MS analyses of 7-ethyl-Gua in human tissues. [15N 5]7-Ethyl-Gua was synthesized and used as the internal standard. Human liver DNA was heated to release 7-ethyl-Gua. After partial purification by solid-phase extraction, analysis was carried out using the transition m/z 180 [M+H]+-->m/z 152 [Gua+H]+ for 7-ethyl-Gua and m/z 185-->m/z 157 for the internal standard. The method was accurate and precise. The detection limit was approximately 8-9 fmol/micromol Gua, starting with 1-2 mg of DNA. Clear coeluting peaks for 7-ethyl-Gua and the internal standard were observed in the human liver DNA samples. Twenty-six human liver DNA samples (0.77+/-0.40 mg) were analyzed, and 25 were positive for 7-ethyl-Gua. The mean level of 7-ethyl-Gua was 42.2+/-43.0 fmol/micromol Gua (8.4+/-8.6 adducts per 10(9) nucleotides). These results demonstrate that 7-ethyl-Gua is a common DNA adduct in human liver with likely endogenous sources that require further investigation.

Airway irritation and cough evoked by inhaled cigarette smoke: Role of neuronal nicotinic acetylcholine receptors

In a series of studies carried out in different experimental models, we investigated the type(s) of lung afferents and mechanism(s) underlying the cigarette smoke-induced airway irritation and cough. In healthy non-smokers, the intensity of airway irritation and cough evoked by cigarette smoke was markedly reduced after premedication with hexamethonium. A similar pattern of responses was also triggered by inhalation of nicotine aerosol. These studies in human subjects suggested nicotine as the primary causative agent in cigarette smoke that evokes airway irritation. Indeed, single-fiber recording experiments performed in anesthetized dogs showed that both C-fibers and rapidly adapting receptors in the lungs and airways were stimulated by inhalation of one puff of cigarette smoke, and the intensity of this stimulatory effect was related to the nicotine content in the cigarette and abolished by hexamethonium. To further study the direct effect of nicotine on these sensory nerves, we measured the change in intracellular calcium concentration ([Ca 2+] i) of pulmonary sensory neurons isolated from the nodose and jugular ganglia of adult rats. Our results showed that nicotine evoked an abrupt and transient increase in [Ca 2+] i in ∼34% of the 522 neurons tested, and 1,1-dimethyl-4-phenylpiperazinium, a selective agonist of the neuronal nicotinic acetylcholine receptors (NnAChRs), evoked a similar pattern of response as that of nicotine in these neurons. In conclusion, results of these studies show that nicotine exerts a direct stimulatory effect on vagal pulmonary sensory neurons. This stimulatory effect of nicotine is primarily responsible for the airway irritation and cough evoked by inhaled cigarette smoke, and is mediated through an activation of the NnAChRs.

Cilomilast

The central role of cyclic nucleotides as intracellular second messengers dates back almost 50 years. The importance of phosphodiesterase in regulating this system was recognized early, and the potential therapeutic role of phosphodiesterase inhibitors in modulating pathologic conditions was also suggested. At that time, the methylxanthines represented major pharmacologic agents capable of inhibiting cyclic nucleotides and were widely used in respiratory medicine. Initially, bronchodilator effects were considered their major mechanism of action, but subsequent studies suggested other potential roles including an anti-inflammatory one. A number of developments led to the decline in popularity of this class of agents, the foremost being their side-effect profile. The discovery of multiple phosphodiesterase isoforms paired with a better understanding of the physiologic and clinical properties of the phosphodiesterases has re-awakened interest in therapeutic agents in this area and in particular the potential for the development of selective phosphodiesterase inhibitors. Cilomilast is a systemically available, second- generation, selective phosphodiesterase-4 inhibitor. It retains the therapeutic activity of the first generation phosphodiesterase-4 inhibitors (such as rolipram) but is believed to have less of an emetic effect. Cilomilast causes a reduction of tissue cells considered central to the ongoing inflammatory process (macrophages and CD8+ lymphocytes) in patients with chronic obstructive pulmonary disease. Chronic obstructive pulmonary disease is now considered a chronic inflammatory disease of the lungs resulting from prolonged exposure to inflammatory agents in cigarette smoke and other environmental and occupational pollutants, and it is currently the principal target of cilomilast. It is characterized by progressive destruction of parenchymal tissue and punctuated by acute exacerbations. The inflammation is thought to begin in the peripheral airways and lung parenchyma. Chronic obstructive pulmonary disease is a progressive disease, leading to disability and eventual death despite conventional therapy. Cilomilast is completely absorbed following oral administration and has negligible first-pass metabolism. It exhibits low between-subject variability. Cilomilast is predominantly protein bound. Plasma clearance is almost entirely metabolic, through multiple parallel pathways. Its terminal elimination half-life is approximately 6.5 hours, and steady state is rapidly achieved. A dose of 15 mg twice daily has been found to be clinically effective. Smoking and age have no clinically relevant effects on cilomilast pharmacokinetics. Most drugs frequently used in patients with chronic obstructive pulmonary disease do not alter its side effect profile. Initial concerns of arteritis involving the gastrointestinal tract in rodent animal models have not been reported in clinical trials. Nausea, presumably of central origin, is the principal adverse reaction seen in healthy subjects taking cilomilast. It has not been associated with the serious cardiac or neurological adverse effects seen with theophylline. Preliminary clinical studies suggest a favorable clinical effect in chronic obstructive pulmonary disease. Cilomilast is generally well tolerated and has not generated safety concerns in reported clinical studies.