Comparative study of the effects of cigarette smoke versus next-generation tobacco and nicotine product extracts on inflammatory biomarkers of human monocytes.

Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. As an alternative to smoking, next-generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine (nic). Treatment with 3R4F reduced cell viability in a dose-dependent manner, whereas exposure to alternative smoking products showed no difference to control. At the highest non-lethal dose of 3R4F (20%), the following notable mRNA expression changes were observed for 3R4F, HTP, and e-cig respectively, relative to control; HMOX1 (6-fold, < 2-fold, < 2-fold), NQO1 (3.5-fold, < 2-fold, < 2-fold), CCL2 (4-fold, 3.5-fold, 2.5-fold), IL1B (4-fold, 3-fold, < 2-fold), IL8 (5-fold, 2-fold, 2-fold), TNF (2-fold, 2-fold, < 2-fold) and ICAM1 was below the 2-fold threshold for all products. With respect to protein expression, IL1B (3-fold, < 2-fold, < 2-fold) and IL8 (3.5-fold, 2-fold, 2-fold) were elevated over the 2-fold threshold, whereas CCL2, TNF, and ICAM1 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however, NGP responses were typically lower than 3R4F. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs overall showed lower responses relative to controls than THP-1 cells exposed to 3R4F AqE.

Comparative study of the effects of cigarette smoke versus next generation tobacco and nicotine product extracts on endothelial function.

Tobacco smoking and hemodynamic forces are key stimuli for the development of endothelial dysfunction. As an alternative to smoking, next generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their potential pro-inflammatory and atherogenic effects on the endothelium. In this study, we analyzed key parameters of endothelial function after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine. All experiments were performed under atheroprotective high laminar or atherogenic low flow with primary human endothelial cells. Treatment with 3R4F, but not alternative smoking products, reduced endothelial cell viability and wound healing capability via the PI3K/AKT/eNOS(NOS3) pathway. Laminar flow delayed detrimental effects on cell viability by 3R4F treatment. 3R4F stimulation led to activation of NRF2 antioxidant defense system at nicotine concentrations ≥0.56 µg/ml and increased expression of its target genes HMOX1 and NQO1. Treatment with HTP revealed an induction of HMOX1 and NQO1 at dosages with ≥1.68 µg/ml nicotine, whereas e-cig and nicotine exposure had no impact. Analyses of pro-inflammatory genes revealed an increased ICAM1 expression under 3R4F treatment. 3R4F reduced VCAM1 expression in a dose-dependent manner; HTP treatment had similar but milder effects; e-cig and nicotine treatment had no impact. SELE expression was induced by 3R4F under static conditions. High laminar flow prevented this upregulation. Stimulation with laminar flow led to downregulation of CCL2 (MCP-1). From this downregulated level, only 3R4F increased CCL2 expression at higher concentrations. Finally, under static conditions, all components increased adhesion of monocytes to endothelial cells. Interestingly, only stimulation with 3R4F revealed increased monocyte adhesion under atherosclerosis-prone low flow. In conclusion, all product categories activated anti-oxidative or pro-inflammatory patterns. NGP responses were typically lower than in 3R4F exposed cells. Also, 3R4F stimulation led to an impaired endothelial wound healing and induced a pro-inflammatory phenotype compared to NGP treatment.

Impact of cigarette versus electronic cigarette aerosol conditioned media on aortic endothelial cells in a microfluidic cardiovascular model.

Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0-1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.

An Adverse Outcome Pathway for Decreased Lung Function Focusing on Mechanisms of Impaired Mucociliary Clearance Following Inhalation Exposure.

Adverse outcome pathways (AOPs) help to organize available mechanistic information related to an adverse outcome into key events (KEs) spanning all organizational levels of a biological system(s). AOPs, therefore, aid in the biological understanding of a particular pathogenesis and also help with linking exposures to eventual toxic effects. In the regulatory context, knowledge of disease mechanisms can help design testing strategies using in vitro methods that can measure or predict KEs relevant to the biological effect of interest. The AOP described here evaluates the major processes known to be involved in regulating efficient mucociliary clearance (MCC) following exposures causing oxidative stress. MCC is a key aspect of the innate immune defense against airborne pathogens and inhaled chemicals and is governed by the concerted action of its functional components, the cilia and airway surface liquid (ASL). The AOP network described here consists of sequences of KEs that culminate in the modulation of ciliary beat frequency and ASL height as well as mucus viscosity and hence, impairment of MCC, which in turn leads to decreased lung function.

Statistical analysis plan for "A randomised, controlled study to evaluate the effects of switching from cigarette smoking to using a tobacco heating product on health effect indicators in healthy subjects".

Tobacco harm reduction strategies aim to substitute smoking with potentially reduced risk products (PRRPs) such as e-cigarettes and tobacco-heating products (THPs). The health benefits of switching from smoking to PRRPs is unknown. A randomised controlled trial is being conducted to increase understanding of the health effects of switching from smoking to a THP in a 12-month long ambulatory study (ISRCTN81075760). Here we describe the study endpoints and the statistical analysis plan. Endpoints are divided into biomarkers of exposure (BoE) to tobacco smoke constituents and health effect indicators related to risk of lung cancer, cardiovascular and obstructive lung disease. These have been selected on the basis of extensive literature evidence. Three primary endpoints, augmentation index (risk factor for cardiovascular disease), total NNAL (linked to lung cancer) and 8-Epi-PGF2α type III (indicator of oxidative stress linked to various diseases), and multiple secondary endpoints will be analysed at 90, 180, and 360 days. Changes from baseline will be compared between study arms by specific contrasts in mixed models. Study wise multiple comparisons adjustments will be performed to account for multiplicity of timepoints and comparisons within timepoints. Generalisability of outcomes will be tested by a sensitivity analysis adjusting for age and gender. Importantly, an ancillary analysis will be performed to assess product compliance during the study based on plasma levels of CEVal, a surrogate marker for acrylonitrile exposure. The rationale underlying the selection of BoEs and health effect indicators, coupled with the statistical analysis plan will be central to understanding the potential health effects of replacing smoking with THP use for one year.

Evaluating the effects of switching from cigarette smoking to using a heated tobacco product on health effect indicators in healthy subjects: study protocol for a randomized controlled trial.

Tobacco heating products (THPs) are a potentially safer alternative to combustible cigarette smoking. Through continued use, THPs may reduce smoking-related disease risk, whilst maintaining the sensorial experience and nicotine delivery sought by smokers. While literature evidence of the biological effects of THP aerosol exposure is increasing, there remains a knowledge gap with respect to substantiation of THP reduced risk potential in longer term real-life use. This randomized, multi-centre, controlled clinical study will test the hypotheses that following a switch from combustible cigarettes to a THP for 1 year, participants will experience a sustained reduction in exposure to tobacco-related toxicants that will lead to favourable changes in health effect indicators associated with smoking-related disease development. Changes in such indicators will be contextualized against smoking cessation and never-smoker cohorts. Up to 280 participants who do not intend to quit smoking will be randomized to continued combustible smoking (arm A, up to n = 80) or a commercially available THP (arm B n = 200). Furthermore, up to 190 participants with a high intent to quit smoking will undergo smoking cessation (arm D), and 40 never smokers will serve as a control group (arm E). Recruitment numbers were determined to be sufficient to achieve n = 50 in arms A, B and D, at study end. Enrolment started in March 2018 and the trial is scheduled to be completed in March 2020. Data from this study will be a valuable addition to the growing body of evidence in the field of understanding the individual and public health impact of THPs.Clinical Trial Registration: https://www.isrctn.com/ISRCTN81075760.

Cigarette smoke constituents cause endothelial nitric oxide synthase dysfunction and uncoupling due to depletion of tetrahydrobiopterin with degradation of GTP cyclohydrolase.

Cigarette smoking (CS) is a well-established risk factor for cardiovascular disease (CVD). Endothelial dysfunction (ED) with loss of nitric oxide (NO) production is a central mechanism leading to the advent of CVD. Despite many prior studies of this major health problem, the exact mechanism by which CS induces ED is not well understood. This study examines the mechanism by which CS induces ED with altered endothelial NO synthase (eNOS) function in aortic endothelial cells (AECs). Exposure of AECs to cigarette smoke extract (CSE) resulted in a marked decrease in NO production with concomitant increase in superoxide (O2.-) generation and accumulation of 4-hydroxy-2-nonenal protein adducts. CSE exposure led to depletion of the essential eNOS cofactor tetrahydrobiopterin (BH4) as well as total biopterin levels and decreased the expression level of guanosine triphosphate cyclohydrolase (GTPCH), the rate limiting enzyme in BH4 biosynthesis. Moreover, exposure of AECs to CSE increased the level of ubiquitinated proteins and increased 26 S proteasomal activity in a concentration-dependent manner. Pre-treatment with MG132, a 26 S proteasome inhibitor, partially prevented CSE-induced loss of BH4, total biopterin, GTPCH, and increased NO production following CSE exposure, indicating a role of the ubiquitin-proteasome system in CSE-induced eNOS dysfunction. In conclusion, CSE-induced eNOS dysfunction and uncoupling occurs due to BH4 depletion with BH4de novo synthesis limited by diminished GTPCH expression.

Assessing modified risk tobacco and nicotine products: Description of the scientific framework and assessment of a closed modular electronic cigarette.

Cigarette smoking causes many human diseases including cardiovascular disease, lung disease and cancer. Novel tobacco products with reduced yields of toxicants compared to cigarettes, such as tobacco-heating products, snus and electronic cigarettes, hold great potential for reducing the harms associated with tobacco use. In the UK several public health agencies have advocated a potential role for novel products in tobacco harm reduction. Public Health England has stated that "The current best estimate is that e-cigarettes are around 95% less harmful than smoking" and the Royal College of Physicians has urged public health to "Promote e-cigarettes widely as substitute for smoking". Health related claims on novel products such as 'reduced exposure' and 'reduced risk' should be substantiated using a weight of evidence approach based on a comprehensive scientific assessment. The US FDA, has provided draft guidance outlining a framework to assess novel products as Modified Risk Tobacco Products (MRTP). Based on this, we now propose a framework comprising pre-clinical, clinical, and population studies to assess the risk profile of novel tobacco products. Additionally, the utility of this framework is assessed through the pre-clinical and part of the clinical comparison of a commercial e-cigarette (Vype ePen) with a scientific reference cigarette (3R4F) and the results of these studies suggest that ePen has the potential to be a reduced risk product.

A comparative assessment of e-cigarette aerosols and cigarette smoke on in vitro endothelial cell migration.

Cigarette smoking is a risk factor for several diseases. There has been a steep increase in the use of e-cigarettes that may offer a safer alternative to cigarette smoking. In vitro models of smoking-related diseases may provide valuable insights into disease mechanisms associated with tobacco use and could be used to assess e-cigarettes. We previously reported the application of a 'scratch wound' assay, measuring endothelial cell migration rate following artificial wounding, in the presence or absence of cigarette smoke extracts. This study reports the comparative effects of two commercial e-cigarette products (Vype ePen and Vype eStick) and a scientific reference cigarette (3R4F) on endothelial migration in vitro. Puff-matched extracts were generated using the Health Canada Intense (HCI) regime for cigarettes and a modified HCI for e-cigarettes. Exposure to 3R4F extract (20h) induced concentration-dependent inhibition of endothelial cell migration, with complete inhibition at concentrations >20%. E-cigarette extracts did not inhibit migration, even at double the 3R4F extract nicotine concentration, allowing cells to migrate into the wounded area. Our data demonstrate that e-cigarettes do not induce the inhibition of endothelial cell migration in vitro when compared to 3R4F. The scratch wound assay enables the comparative assessment between tobacco and nicotine products in vitro.

A novel hybrid tobacco product that delivers a tobacco flavour note with vapour aerosol (Part 2): In vitro biological assessment and comparison with different tobacco-heating products.

This study assessed the toxicological and biological responses of aerosols from a novel hybrid tobacco product. Toxicological responses from the hybrid tobacco product were compared to those from a commercially available Tobacco Heating Product (c-THP), a prototype THP (p-THP) and a 3R4F reference cigarette, using in vitro test methods which were outlined as part of a framework to substantiate the risk reduction potential of novel tobacco and nicotine products. Exposure matrices used included total particulate matter (TPM), whole aerosol (WA), and aqueous aerosol extracts (AqE) obtained after machine-puffing the test products under the Health Canada Intense smoking regime. Levels of carbonyls and nicotine in these matrices were measured to understand the aerosol dosimetry of the products. The hybrid tobacco product tested negative across the in vitro assays including mutagenicity, genotoxicity, cytotoxicity, tumour promotion, oxidative stress and endothelial dysfunction. All the THPs tested demonstrated significantly reduced responses in these in vitro assays when compared to 3R4F. The findings suggest these products have the potential for reduced health risks. Further pre-clinical and clinical assessments are required to substantiate the risk reduction of these novel products at individual and population levels.

E-cigarette aerosols induce lower oxidative stress in vitro when compared to tobacco smoke.

Tobacco smoking is a risk factor for various diseases. The underlying cellular mechanisms are not fully characterized, but include oxidative stress, apoptosis, and necrosis. Electronic-cigarettes (e-cigarettes) have emerged as an alternative to and a possible means to reduce harm from tobacco smoking. E-cigarette vapor contains significantly lower levels of toxicants than cigarette smoke, but standardized methods to assess cellular responses to exposure are not well established. We investigated whether an in vitro model of the airway epithelium (human bronchial epithelial cells) and commercially available assays could differentiate cellular stress responses to aqueous aerosol extracts (AqE) generated from cigarette smoke and e-cigarette aerosols. After exposure to AqE concentrations of 0.063-0.500 puffs/mL, we measured the intracellular glutathione ratio (GSH:GSSG), intracellular generation of oxidant species, and activation of the nuclear factor erythroid-related factor 2 (Nrf2)-controlled antioxidant response elements (ARE) to characterize oxidative stress. Apoptotic and necrotic responses were characterized by increases in caspase 3/7 activity and reductions in viable cell protease activities. Concentration-dependent responses indicative of oxidative stress were obtained for all endpoints following exposure to cigarette smoke AqE: intracellular generation of oxidant species increased by up to 83%, GSH:GSSG reduced by 98.6% and transcriptional activation of ARE increased by up to 335%. Caspase 3/7 activity was increased by up to 37% and the viable cell population declined by up to 76%. No cellular stress responses were detected following exposure to e-cigarette AqE. The methods used were suitably sensitive to be employed for comparative studies of tobacco and nicotine products.

Changes in levels of biomarkers of exposure and biological effect in a controlled study of smokers switched from conventional cigarettes to reduced-toxicant-prototype cigarettes.

BACKGROUND: Development of cigarettes that reduce exposure to harmful smoke constituents is a suggested tobacco harm reduction strategy, but robust methods for measurement of change are required. We investigated whether changes in biomarkers of exposure (BoE), effective dose (BoED) and biological effect (BoBE) could be detected after switching from conventional cigarettes to a reduced-toxicant-prototype cigarette (RTP). METHODS: Regular smokers of 6-8mg ISO tar yield cigarettes were recruited in Hamburg, Germany, and supplied with a conventional 7mg ISO tar yield cigarette for 2weeks then switched to the same cigarette with a different tipping paper (control) or the RTP for 6months. Subjects smoked mostly at home and attended five residential clinic visits where urine and blood samples were collected for analysis. Primary endpoints were changes in specific biomarker levels compared with non-smoker background levels. Changes in daily cigarette consumption were also investigated. RESULTS: BoE levels in controls generally increased over the study period, whereas most BoE and all BoED significantly declined in RTP smokers. Most BoBE data were similar across groups and/or too variable within individuals to detect changes. Increased daily cigarette consumption was affected by supply of free cigarettes, perceived shorter smoking time per cigarette than usual brands, and perceived reduced harm. CONCLUSIONS: Despite increased cigarette consumption, reductions in BoE and BoED were detectable.

A cross-sectional analysis of candidate biomarkers of biological effect in smokers, never-smokers and ex-smokers.

CONTEXT: Biomarkers of biological effect (BOBE) have been proposed as potential tools to assess tobacco product use, toxicity and disease risk. OBJECTIVE: To determine if candidate BOBE can distinguish between smokers, never-smokers and former smokers. METHODS: Biomarker levels were compared from 143 smokers, 61 never-smokers and 61 ex-smokers. RESULTS: In total, 27 candidate biomarkers were assessed, 14 were significantly different between smokers and never-smokers (p < 0.01) and of these 14 biomarkers, 12 were able to distinguish between smokers and former smokers (p < 0.05), which indicates the potential for reversibility. CONCLUSIONS: A total of 12 of 27 BOBE are potentially useful tools for future product assessment.

A novel autoantibody test for the detection of pre-neoplastic lung lesions.

BACKGROUND: Atypical adenomatous hyperplasia (AAH) and squamous cell dysplasia (SCD) are associated with the development of malignant lesions in the lung. Accurate diagnosis of AAH and SCD could facilitate earlier clinical intervention and provide useful information for assessing lung cancer risk in human populations. Detection of AAH and SCD has been achieved by imaging and bronchoscopy clinically, but sensitivity and specificity remain less than satisfactory. We utilized the ability of the immune system to identify lesion specific proteins for detection of AAH and SCD. METHODS: AAH and SCD tissue was surgically removed from six patients of Chinese descent (3 AAH and 3 SCD) with corresponding serum samples. Total RNA was extracted from the tissues and a cDNA library was generated and incorporated into a T7 bacteriophage vector. Following enrichment to remove "normal" reactive phages, a total of 200 AAH related and 200 SCD related phage clones were chosen for statistical classifier development and incorporation into a microarray. Microarray slides were tested with an independent double-blinded population consisting of 100 AAH subjects, 100 SCD subjects and 200 healthy control subjects. RESULTS: Sensitivity of 82% and specificity of 70% were achieved in the detection of AAH using a combination of 9 autoantibody biomarkers. Likewise, 86% sensitivity and 78% specificity were achieved in the detection of SCD using a combination of 13 SCD-associated markers. Sequencing analysis identified that most of these 22 autoantibody biomarkers had known malignant associations. CONCLUSIONS: Both diagnostic values showed promising sensitivity and specificity in detection of pre-neoplastic lung lesions. Hence, this technology could be a useful non-invasive tool to assess lung cancer risk in human populations.

Lung cancer biomarkers for the assessment of modified risk tobacco products: an oxidative stress perspective.

Manufacturers have developed prototype cigarettes yielding reduced levels of some tobacco smoke toxicants, when tested using laboratory machine smoking under standardised conditions. For the scientific assessment of modified risk tobacco products, tests that offer objective, reproducible data, which can be obtained in a much shorter time than the requirements of conventional epidemiology are needed. In this review, we consider whether biomarkers of biological effect related to oxidative stress can be used in this role. Based on published data, urinary 8-oxo-7,8-dihydro-2-deoxyguanosine, thymidine glycol, F2-isoprostanes, serum dehydroascorbic acid to ascorbic acid ratio and carotenoid concentrations show promise, while 4-hydroxynonenal requires further qualification.

A cross-sectional investigation of biomarkers of risk after a decade of smoking.

Two groups of 20 healthy volunteers with cigarettes of different tar yield were compared with a group of 20 never smokers over 24 h for several biomarkers. All groups were of similar mean ages and the smokers had smoked for a homogeneous period of approximately 10 yr. The groups were assessed using routine medical parameters as well as biomarkers of recent smoke exposure and other biomarkers that were under evaluation as possible markers of risk for smoking-associated diseases. All biomarkers of exposure-carbon monoxide, nicotine plus its five major metabolites, and 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol (NNAL)-were significantly elevated in smokers. For biomarkers of potential risk evaluated in the blood, white cells and immunoglobulin (Ig) G showed a decrease related to smoking status (p < .01). Interleukin 6 levels were higher in smoker groups compared to never smokers, with a significant increasing trend across the groups (p < .05). Among the urinary biomarkers studied, 11-deydro-thromboxane B2, 2,3-dinor-thromboxane B2, and thymidine glycol showed significant increasing trends across the groups (p < .01). The results suggest that after the first decade or less of smoking, changes in inflammatory, immunological, and cardiovascular function can be observed. However, further studies on larger groups will be required to better understand the kinetics of these subtle effects observed early in smokers and their relationship with the potential risk of subsequent smoking-associated disease.

Evaluation of biomarkers of exposure and potential harm in smokers, former smokers and never-smokers.

BACKGROUND: The objective of this study was to obtain baseline data on biomarkers of exposure (BoE) and biomarkers of potential harm (BoPH) in smokers, former smokers and never-smokers. METHODS: This was a cross-sectional study of 80 healthy male and female volunteers over 21 years old, self-selected for smoking status. Subjects were prescreened by medical staff at an independent clinical research unit, within 1 week prior to a single overnight residential visit and sample collection. RESULTS: All BoE were able to differentiate between the two smoking groups and smokers from all nonsmokers. There was a strong correlation between cigarettes smoked per day and total urinary nicotine equivalents (TNE; r=0.85). TNE correlated better with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol levels than cigarettes smoked per day (r=0.75 and r=0.56, respectively). Of the BoPH included in this study, seven (11-dehydro-thromboxane B2, 2, 3-dinorthrom-boxane B2, 8-epi prostaglandin F(2alpha), 8-hydroxy-2 deoxyguanosine, cis-thymidine glycol, low-density lipoprotein cholesterol and IgG) were significantly different between the group who smoked more cigarettes per day and never-smokers. These differences became more apparent and extended to the group who smoked 10 or less cigarettes per day, when total urinary recovery values were corrected for creatinine clearance. CONCLUSIONS: While BoE clearly differentiate between groups based on self-declared smoking status, most BoPH examined could not do so in a consistent manner. The dynamics of BoPH levels are not well understood. Future studies of BoPH should eliminate potential confounding factors and increase the number of subjects to allow the investigation of genetic polymorphism in metabolic pathways.

Cardiovascular biomarkers in groups of established smokers after a decade of smoking.

To investigate tools for evaluation of smoking-associated disease initiation and progression, we examined basic clinical parameters and biomarkers of cardiovascular disease risk, in a group of healthy volunteers with an average 10-year smoking history. A small cross-sectional study of never-smokers, moderate smokers and smokers was performed. Caucasians were recruited to match pre-defined cigarette tar yields and cigarettes smoked per day. For haematological parameters, significant differences between never-smokers and all female smokers combined were seen for haemoglobin concentration, haematocrit, total leucocyte count, neutrophil count and lymphocyte count. For all male smokers combined, only total leucocyte count was statistically different. Analysis of exhaled CO and other smoke exposure biomarker (nicotine and its metabolites) data showed a statistically significant increase in all groups of smokers with a trend related to the number of cigarettes smoked per day. Thromboxane urinary metabolites 11-dehydro-thromboxane B(2) and 2,3-dinor-thromboxane B(2) were statistically significantly elevated in smokers. Significant statistical differences between smokers with approximately 10 years of smoking history and non-smokers in white cells count, hemoglobin and thromboxane turnover were seen, although they did not reach levels associated with overt diseases. These data could provide insight into early biomarkers predictive of risk for coronary and vascular disease.

Smoking selectively accelerates carotid atherosclerosis in hypertensive patients.

BACKGROUND AND OBJECTIVES: Left ventricular hypertrophy, carotid atherosclerosis and renal dysfunction are indicators of target organ damage in hypertension, and independent risk factors for both fatal and non-fatal cardio- and cerebrovascular events. In the general population, smoking is associated with increases in left ventricular mass and carotid intima-media thickness (IMT), and impaired renal function. The aim of the present study was to evaluate whether smoking affects the development of target organ damage in patients with arterial hypertension. METHODS: 3192 hypertensive patients referred to the Hypertension Clinic of the "Federico II" University of Naples from January 2000 to July 2006 were retrospectively analysed. Subjects were aged from 18 to 75 years. Among these patients, 1391 were smokers and 1801 non-smokers. RESULTS: The duration and severity of hypertension was significantly shorter in smokers when compared with non-smokers. The maximum arterial IMT was significantly higher in smokers compared with non-smokers (1.7 ± 0.1 mm vs 1.5 ± 0.1, p < 0.0001), while left ventricular mass index was comparable between the two groups. In contrast, glomerular filtration rate was observed to be higher in smokers compared with non-smokers. Logistic regression analysis showed that smoking, age, sex, duration of hypertension, systolic blood pressure and diastolic blood pressure were significantly correlated with IMT. Furthermore, a strong correlation was found between the number of cigarettes smoked per day and IMT. CONCLUSIONS: Together, these data indicate that in hypertensive patients who have a high risk of developing atherosclerosis, smoking could potentiate the development of atherosclerotic plaques.