Determination of nicotine absorption from multiple tobacco products and nicotine gum.

INTRODUCTION: Snus is a smokeless tobacco product traditionally used in Scandinavia and available in pouched or loose forms. The objective of this study was to determine nicotine absorption for current pouched and loose snus products in comparison with a cigarette and an over-the-counter nicotine gum. METHODS: We conducted an open-label, randomized, 6-way, crossover study involving 20 healthy snus and cigarette users. One of 6 products (2 pouched snus, 2 weights of loose snus, a cigarette, and a nicotine gum) was administered at each of 6 visits. Blood samples were taken at intervals over 120 min and sensory perception assessed by questionnaire. RESULTS: For the 4 smokeless tobacco products and the nicotine gum, blood plasma levels of nicotine were ranked according to total nicotine content as follows: loose snus (27.1 mg nicotine) > pouched snus (14.7 mg nicotine) > loose snus (10.8 mg nicotine) = pouched snus (10.7 mg nicotine) > nicotine gum (4.2 mg nicotine). The area under the plasma concentration-time curve (AUC) and maximum plasma concentration (C(max)) of nicotine ranged from 26.9 to 13.1 ng.h/ml and 17.9 to 9.1 ng.h/ml, respectively across all the products. Nicotine was absorbed more rapidly from the cigarette but systemic exposure was within the range of the smokeless tobacco products (AUC = 14.8 ng.h/ml; C(max) = 12.8 ng.h/ml). CONCLUSIONS: This study has generated new information on comparative nicotine absorption from a cigarette, loose snus, and pouched snus typical of products sold in Scandinavia. The similar nicotine absorption for 1 g portions of loose and pouched snus with approximately 11 mg of nicotine indicate that absorption kinetics were dependent on quantity of tobacco by weight and total nicotine content rather than product form.

Smoking intensity before and after introduction of the public place smoking ban in Scotland.

A study was performed to determine whether cigarettes were smoked more intensely outside of public venues in Scotland, compared to indoors, after introduction of the public place smoking (PPS) ban. It was conducted in three waves: before the ban, immediately after and 6 months after introduction. The study included 322 regular smokers of four cigarette brand variants. Filter analysis measurements were used to estimate the human-smoked yields of tar and nicotine from cigarettes smoked predominantly inside (before the ban) or outside (after the ban) public venues. Self-reported cigarette consumption data were also collected. Numbers of cigarettes smoked indoors in public places fell dramatically after the ban. There was a corresponding rise in smoking incidence in outdoor public locations. The ban did not significantly affect the total number of cigarettes smoked by the subjects over the weekends investigated. Human-smoked yields of tar and nicotine decreased slightly after the introduction of the ban and some reductions were significant. Therefore, smoking outdoors at public venues, following the PPS ban, did not increase smoking intensity. Any changes in smoking behaviour that may have occurred had little effect on mainstream smoke exposure or cigarette consumption for those that continued to smoke.

Cigarette smoke total particulate matter increases mucous secreting cell numbers in vitro: a potential model of goblet cell hyperplasia.

Cigarette smoking is associated with chronic obstructive pulmonary disease (COPD)--a term encompassing chronic lung inflammation, chronic bronchitis and emphysema. Goblet cell hyperplasia is a characteristic feature of the lung epithelium in COPD patients contributing to the overproduction of airway mucus, including mucin MUC5AC. Using an in vitro model of differentiated lung epithelium we have investigated morphological and cellular changes in response to interleukin (IL)-13 (2.5-20 ng/ml), cigarette smoke total particulate matter (TPM; 0.31-20 microg/ml) and three mainstream cigarette smoke constituents: acrolein, formaldehyde and acetaldehyde (all at 0.001-1 microM) over 28 days during differentiation (agents replaced daily Monday-Friday). Control cultures of human bronchial epithelial cells (HBECs) underwent mucociliary differentiation producing a columnar epithelium containing goblet, ciliated and basal cells. Non-cytotoxic doses of IL-13 induced a significant increase in the percentage of MUC5AC positive cells. Using both flow cytometry and immunocytochemical techniques for identification of MUC5AC positive cells, TPM treatment induced an increase in MUC5AC positive cells, becoming maximal at 5 microg/ml. Acrolein treatment also increased the percentage of MUC5AC positive cells. However, formaldehyde or acetaldehyde had little effect. This study demonstrates that lung toxicants can induce a profound effect on cellular differentiation in an in vitro model of the human lung epithelium.

Patterns and behaviors of snus consumption in Sweden.

INTRODUCTION: Snus is an oral snuff consisting of moist finely ground tobacco which is available in a loose form or with portions of the tobacco sealed in small sachets termed "pouches." The product has a long history of use in Sweden. Currently, there is very little published information on levels of consumption and usage behaviors for snus in Sweden. The objective of this study was to obtain data on the frequency and duration of loose and pouched snus consumption in Sweden and investigate usage behaviors. METHODS: Telephone surveys of snus users randomly selected from telephone directories in all regions of Sweden were conducted in 2007 and 2008. In total, 2,914 respondents answered questions on snus usage, including the types of products used and the quantity and frequency of use. RESULTS: The majority of respondents (96%) used either pouched or loose snus alone. A minority (12.6%) reported dual use of smokeless and combustible tobacco products. Average daily consumption was 11-12 g for pouched snus and 29-32 g for loose snus. The typical duration of use of each pouch/portion was 60-70 min. DISCUSSION: This survey has provided new insights into contemporary snus use in Sweden, such as the marked differences in daily consumption between loose and pouched snus, length of time that snus users typically keep pouches in the mouth, differential patterns of use in males and females, and the simultaneous use of multiple pouches in a small proportion of users.

Agents associated with lung inflammation induce similar responses in NCI-H292 lung epithelial cells.

The aim of this study was to investigate an in vitro lung epithelial model for assessment of potential inhalation toxicity. The selected NCI-H292 lung carcinoma cell line is sensitive to cigarette smoke, responds in a similar manner to primary human lung epithelial cells and produces airway mucins. The following agents associated with inhalation toxicity were tested in the model: cigarette smoke total particulate matter, fly ash, bleomycin, lipopolysaccharide, vanadyl sulphate, diesel exhaust particles and carbon black. Polystyrene, poly-methylmethacrylate and dimethyl sulphoxide were used as negative controls. Response markers were chosen on the basis of reported injurious effects of lung toxicants in humans, and included pro-inflammatory cytokines, matrix metalloprotease-1, the airway mucin MUC5AC and heparin-binding epidermal growth factor-like growth factor. Markers were quantified at the mRNA and/or protein level in control and treated cells. Many of the selected markers were regulated in a similar manner by cigarette smoke and the other toxic substances in the H292 cell model. By comparison, the negative control agents were largely ineffective. We conclude that, with further validation, this assay may form part of a tiered strategy for toxicological assessment of inhaled agents prior to more complex primary cell models and animal inhalation studies.

Human bronchial epithelial cell transcriptome: gene expression changes following acute exposure to whole cigarette smoke in vitro.

Cigarette smoke is a complex mixture of more than 4,000 constituents. Its effects on cell biology are poorly understood, partly because whole smoke exposure in vitro is technically challenging. To investigate the effects of smoke on cell signaling and function, a three-dimensional air-liquid interface model of tracheobronchial epithelium, grown from primary human lung epithelial cells, was exposed to air or whole mainstream cigarette smoke for 1 h in a purpose-designed chamber. Gene expression profiles were then determined at 1, 6, and 24 h postexposure using Affymetrix HGU133-2 Plus microarrays. Cells from three different donors were used in the study, and the experiment was performed in triplicate for each donor. Genes significantly regulated by smoke, compared with the air control, in all experiments were determined. Genes exhibiting differential expression were assigned to functional categories and mapped to signaling pathways. Effects were observed on many cellular processes including xenobiotic metabolism, oxidant/antioxidant balance, and DNA damage and repair. Notably, there was marked downregulation of the transforming growth factor-beta pathway, which has not been previously reported. This study provides important data on the acute effects of whole cigarette smoke on mucociliary epithelium and may be used to gain a greater understanding of smoke toxicity.

Expression of genes involved in oxidative stress responses in airway epithelial cells of smokers with chronic obstructive pulmonary disease.

RATIONALE: The molecular mechanisms involved in airway oxidative stress responses reported in healthy smokers and in those with chronic obstructive pulmonary disease (COPD) are poorly understood. OBJECTIVES: To assess the expression of genes involved in oxidative stress responses in the bronchial epithelium of smokers with or without COPD and in relation to disease severity. METHODS: Global gene expression was assessed in bronchial brushings in 38 subjects with COPD, 14 healthy nonsmokers, and 18 healthy smokers. RESULTS: Gene expression analysis using Affymetrix arrays revealed mRNAs representing 341 out of 642 oxidative stress genes from two predefined gene sets to be differentially expressed in healthy nonsmokers when compared with healthy smokers, and 200 differentially expressed oxidative genes in subjects with COPD when compared with healthy smokers. Gene set enrichment analysis showed that pathways involved in oxidant/antioxidant responses were among the most differentially expressed gene pathways in smoking individuals, with further differences seen in COPD. Distinct, nonlinear gene expression patterns were identified across the severity spectrum of COPD, which correlated with the presence of certain transcription factor binding sites in their promoters. Significant changes in oxidant response genes observed in vivo were reproduced in vitro using primary bronchial epithelial cells from the same donors cultured at an air-liquid interface and exposed to cigarette smoke extract. CONCLUSIONS: Cigarette smoke induces significant changes in oxidant defense responses; some of these are further amplified, but not in a linear fashion, in individuals who develop COPD.

Exposure of bronchial epithelial cells to whole cigarette smoke: assessment of cellular responses.

Cigarette smoke is composed of approximately 5% particulate phase and 95% vapour phase by weight. However, routine in vitro toxicological testing of smoke normally only measures the activity of the particulate phase. This study describes a new system for exposing cells at an air-liquid interface to serial dilutions of gaseous smoke. Confluent monolayers of NCI-H292 human lung epithelial cells on semipermeable membranes were placed in a purpose-designed Perspex chamber at an air-liquid interface. The cells were exposed to dilute whole mainstream cigarette smoke for 30 minutes, followed by a 20-hour recovery period. Firstly, high and low delivery cigarettes were compared, and cytotoxicity was determined by using the neutral red uptake assay. Clear differential cytotoxic responses were observed with the two cigarette types, which correlated positively with the concentrations of components in smoke, and particularly compounds in the vapour phase, such as aldehydes. Secondly, low doses of smoke were found to up-regulate mRNA levels of the secreted mucin, MUC5AC, and to stimulate the production of interleukin (IL)-6, IL-8 and matrix-metalloprotease-1, but had no effect on growth-related oncogene alpha. This system will facilitate further investigations into the toxicological mechanisms of cigarette smoke components, and may be useful for studying other gaseous mixtures or aerosols.

Increased expression of p21(waf) cyclin-dependent kinase inhibitor in asthmatic bronchial epithelium.

Because the asthmatic bronchial epithelium is characterized by widespread damage, we postulated that this is associated with expression of cell cycle inhibitors that control proliferation. Using bronchial biopsies, the epithelium was the major site of expression of the cyclin-dependent kinase inhibitor, p21(waf). Immunostaining usually occurred in the cytoplasm of columnar cells; however, in severe asthma, nuclear staining was also evident in the proliferative, basal cell compartment. p21(waf) expression was significantly higher in asthmatic versus nonasthmatic epithelium and was unaffected by corticosteroid treatment; proliferating cell nuclear antigen was not significantly different in any group. p21(waf), but not p27(kip1), mRNA and protein were induced by treatment of bronchial epithelial cells in vitro with transforming growth factor (TGF)-beta or H2O2, but not by dexamethasone, which induced p57(kip2). TGF-beta and dexamethasone inhibited epidermal growth factor (EGF)-induced DNA synthesis, whereas low concentrations of H2O2 synergized with EGF; at higher doses, growth inhibition and induction of apoptosis occurred. TGF-beta caused p21(waf) to become nuclear, suggesting interaction with the replicative machinery; however, in oxidant-stressed cells, p21(waf) was predominantly cytoplasmic, where it has been linked to cell survival. We conclude that p21(waf) overexpression in asthma influences cell proliferation and survival. This may cause abnormal repair responses that contribute to airway inflammation and remodeling.

Asthmatic bronchial epithelium is more susceptible to oxidant-induced apoptosis.

Abnormal apoptotic mechanisms are associated with disease pathogenesis. Because the asthmatic bronchial epithelium is characteristically damaged with loss of columnar epithelial cells, we postulated that this is due to unscheduled apoptosis. Using an antibody directed toward the caspase cleavage product of poly(ADP-ribose) polymerase, immunohistochemistry applied to endobronchial biopsies showed higher levels of staining in the bronchial epithelium of subjects with asthma as compared with normal control subjects (% epithelial staining [median (range) = 10.5 (1.4-24.5) versus 0.4 (0.0-9.7)]; P < 0.001). Because we were unable to determine whether this difference was due to ongoing inflammation in vivo, cultures of normal and asthmatic bronchial epithelial cells were used to study apoptosis in vitro. In complete growth medium, these cells showed no difference in their rate of proliferation or viability. However, cells from subjects with asthma were more susceptible to the apoptotic effects of H2O2 than cells from normal control subjects (% apoptotic cells = 32.2 [8.8-54.9] versus 14.3 [6.4-24.7]; P < 0.05), even though both were similarly affected by treatment with actinomycin D. These data indicate that the susceptibility of asthmatic bronchial epithelium to oxidants is greater than normal. This susceptibility may contribute to the rising trends in asthma associated with air pollution and diets low in antioxidants.

Autocrine ligands for the epidermal growth factor receptor mediate interleukin-8 release from bronchial epithelial cells in response to cigarette smoke.

Airway neutrophilia is a prominent feature of chronic obstructive pulmonary disease. As cigarette smoke (CS) and epidermal growth factor (EGF) both cause release of interleukin-8 (IL-8) from epithelial cells in vitro, we investigated whether autocrine ligands for the EGF receptor (EGFR) are involved in this proinflammatory response to CS. NCI-H292 or primary bronchial epithelial cells were cultured with or without cigarette smoke extract (CSE) or EGF for 6-48 h. We then tested culture supernatants for lactate dehydrogenase activity to assess cell viability, and for IL-8 and EGFR ligands by ELISA; quantitative RT-PCR was used to measure IL-8 and EGFR ligand mRNA. EGF and low concentrations of CSE both promoted cell survival and caused enhanced transcription and release of IL-8. Similarly, levels of mRNA encoding transforming growth factor alpha (TGF-alpha), heparin-binding EGF-like growth factor, and amphiregulin (AR) were increased, as was shedding of TGF-alpha and AR protein into the culture medium. With the exception of AR gene transcription, the CS-induced responses were blocked by the EGFR-selective kinase inhibitor AG1478. Furthermore, ~ 45% of CS-induced IL-8 release was inhibited by a neutralising anti-EGFR. Our data indicate that secretion of IL-8 in response to CSE is dependent on EGFR activation and that autocrine production of TGF-alpha makes a substantial contribution to this response.

Cooperative effects of Th2 cytokines and allergen on normal and asthmatic bronchial epithelial cells.

In sensitized individuals, exposure to allergens such as Dermatophagoides pteronyssinus (Der p) causes Th2 polarization and release of cytokines, including IL-4 and IL-13. Because Der p extracts also have direct effects on epithelial cells, we hypothesized that allergen augments the effects of Th2 cytokines by promoting mediator release from the bronchial epithelium in allergic asthma. To test our hypothesis, primary bronchial epithelial cultures were grown from bronchial brushings of normal and atopic asthmatic subjects. RT-PCR showed that each culture expressed IL-4R(alpha), common gamma-chain, and IL-13R(alpha)(1), as well as IL-13R(alpha)(2), which negatively regulates IL-13 signaling; FACS analysis confirmed IL-13R(alpha)(2) protein expression. Exposure of epithelial cultures to either Der p extracts, TNF-alpha, IL-4, or IL-13 enhanced GM-CSF and IL-8 release, and this was partially suppressible by corticosteroids. Simultaneous exposure of the epithelial cultures to IL-4 or IL-13 together with Der p resulted in a further increase in cytokine release, which was at least additive. Release of TGF-alpha was also increased by TNF-alpha and combinations of IL-4, IL-13, and Der p; however, this stimulation was only significant in the asthma-derived cultures. These data suggest that, in an allergic environment, Th2 cytokines and allergen have the potential to sustain airway inflammation through a cooperative effect on cytokine release by the bronchial epithelium. Our novel finding that IL-4, IL-13, and allergen enhance release of TGF-alpha, a ligand for the epidermal growth factor receptor that stimulates fibroblast proliferation and goblet cell differentiation, provides a potential link between allergen exposure, Th2 cytokines, and airway remodelling in asthma.