A randomised,crossover study on an electronic vapour product,a nicotine inhalator and a conventional cigarette. Part B: Safety and subjective effects

An Electronic Vapour Product (EVP) has been evaluated for short-term safety parameters and subjective effects in a 2-part study, in smokers. Part 1 compared the EVP with unflavoured (UF) and flavoured (FL) e-liquid at 2.0% nicotine to a conventional cigarette (CC; JPS Silver King Size, 0.6 mg) and a licensed nicotine inhalator (Nicorette^®, 15 mg). Part 2 assessed the effect of increasing concentrations of nicotine in the e-liquid used with the EVP (0%, 0.4%, 0.9%, 2.0%). The study was designed as a randomised, controlled, crossover trial. Outcomes included adverse events (AEs), vital signs, exhaled carbon monoxide (CO), clinical laboratory parameters, smoking urges and withdrawal symptoms. In both study parts, only mild non-serious AEs were reported. No major differences were observed in AEs between the EVPs and Nicorette^®. Exhaled CO levels only increased for CC. All products appeared to decrease smoking urges and nicotine withdrawal symptom scores to a similar extent. The EVP had a similar short-term safety profile to Nicorette^® and relieved smoking urges and nicotine withdrawal symptoms to a similar extent as Nicorette^® and CC. Unlike nicotine replacement therapies, the EVP may offer an alternative for those finding it difficult to quit the behavioural and sensorial aspects of smoking.     

A framework for in vitro systems toxicology assessment of e-liquids

Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an in vitro systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air–liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust in vitro assessment of e-liquids and their aerosols.     

The in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter with reduced toxicant yields

Tobacco smoke contains more than 5600 constituents, of which approximately 150 are toxicants. This paper describes the activities in the Neutral Red uptake (NRU) assay, the Salmonella mutagenicity test (SAL), the mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT) of Particulate Matter (PM) obtained from experimental cigarettes (ECs), designed to produce reduced levels of toxicants. The designs included tobacco substitute sheet (TSS) containing glycerol, which dilutes toxicants in smoke, or the incorporation of blend-treated (BT) tobacco to reduce the levels of nitrogenous toxicant precursors and some polyphenols. All samples were cytotoxic in the NRU, however TSS reduced PM cytotoxicity in this assay. All PMs were mutagenic in the SAL, MLA and IVMNT. Reductions in bacterial mutagenicity were observed in the SAL, for cigarettes with BT tobacco, compared with their respective controls. The quantitative changes in bacterial mutagenicity could be explained by analytical chemistry data on smoke generated from the ECs used in the study. These observations, and the absence of consistent qualitative differences in the activities of the experimental, control and reference cigarettes, suggest that reduced toxicity cigarettes, as measured by the tests described in this paper, may be developed without introducing any additional cytotoxic or genotoxic hazards, but the impact of this on human health risks remains unknown.     

Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke

Electronic cigarettes (E-cigarettes) are a potential means of addressing the harm to public health caused by tobacco smoking by offering smokers a less harmful means of receiving nicotine. As e-cigarettes are a relatively new phenomenon, there are limited scientific data on the longer-term health effects of their use. This study describes a robust in vitro method for assessing the cytotoxic response of e-cigarette aerosols that can be effectively compared with conventional cigarette smoke. This was measured using the regulatory accepted Neutral Red Uptake assay modified for air–liquid interface (ALI) exposures. An exposure system, comprising a smoking machine, traditionally used for in vitro tobacco smoke exposure assessments, was adapted for use with e-cigarettes to expose human lung epithelial cells at the ALI. Dosimetric analysis methods using real-time quartz crystal microbalances for mass, and post-exposure chemical analysis for nicotine, were employed to detect/distinguish aerosol dilutions from a reference Kentucky 3R4F cigarette and two commercially available e-cigarettes (Vype eStick and ePen). ePen aerosol induced 97%, 94% and 70% less cytotoxicity than 3R4F cigarette smoke based on matched EC₅₀ values at different dilutions (1:5 vs. 1:153 vol:vol), mass (52.1 vs. 3.1?μg/cm²) and nicotine (0.89 vs. 0.27?μg/cm²), respectively. Test doses where cigarette smoke and e-cigarette aerosol cytotoxicity were observed are comparable with calculated daily doses in consumers. Such experiments could form the basis of a larger package of work including chemical analyses, in vitro toxicology tests and clinical studies, to help assess the safety of current and next generation nicotine and tobacco products.     

Comparison of the effects of e-cigarette vapor and cigarette smoke on indoor air quality

Context: Electronic cigarettes (e-cigarettes) have earned considerable attention recently as an alternative to smoking tobacco, but uncertainties about their impact on health and indoor air quality have resulted in proposals for bans on indoor e-cigarette use.

Objective: To assess potential health impacts relating to the use of e-cigarettes, a series of studies were conducted using e-cigarettes and standard tobacco cigarettes.

Methods and materials: Four different high nicotine e-liquids were vaporized in two sets of experiments by generic 2-piece e-cigarettes to collect emissions and assess indoor air concentrations of common tobacco smoke by products. Tobacco cigarette smoke tests were conducted for comparison.

Results: Comparisons of pollutant concentrations were made between e-cigarette vapor and tobacco smoke samples. Pollutants included VOCs, carbonyls, PAHs, nicotine, TSNAs, and glycols. From these results, risk analyses were conducted based on dilution into a 40 m³ room and standard toxicological data. Non-cancer risk analysis revealed “No Significant Risk” of harm to human health for vapor samples from e-liquids (A-D). In contrast, for tobacco smoke most findings markedly exceeded risk limits indicating a condition of “Significant Risk” of harm to human health. With regard to cancer risk analysis, no vapor sample from e-liquids A-D exceeded the risk limit for either children or adults. The tobacco smoke sample approached the risk limits for adult exposure.

Conclusions: For all byproducts measured, electronic cigarettes produce very small exposures relative to tobacco cigarettes. The study indicates no apparent risk to human health from e-cigarette emissions based on the compounds analyzed.     

Multiple endpoint in vitro toxicity assessment of a prototype heated tobacco product indicates substantially reduced effects compared to those of combustible cigarette

This study aimed to compare the aerosol chemistry and in vitro toxicological profiles of two prototype Heated Tobacco Product (p-HTP) variants to the 1R6F Reference Cigarette. In the neutral red uptake screen the p-HTPs were 37–39-fold less potent than 1R6F, in the micronucleus assay, responses to the p-HTPs were 8–22-fold less, and in the Ames test mutagenicity was weak or removed compared to 1R6F. The cardiovascular scratch wound assay revealed 58-fold greater wound healing impairment following exposure to 1R6F smoke extracts than the p-HTPs. Furthermore, in seven cell stress-related high content screening endpoints (cell count, cytochrome c release, mitochondrial membrane potential, GSH depletion, NFkB translocation, phosphorylation of c-jun and phosphorylation of H2AX), at 4 and 24 h, responses were substantially greater to 1R6F smoke extracts at comparable nicotine levels. The reduced in vitro effects of the p-HTPs were attributed to substantial reductions (90–97%) in selected HPHCs measured compared to in 1R6F smoke. The multiple endpoint in vitro assessment approach provides greater mechanistic insight and the first reported toxicological characterisation of these p-HTPs in the literature. Overall, the findings contribute to the growing weight of evidence that HTPs may offer a reduced harm mode of nicotine delivery to adult smokers.     

Smoke chemistry, in vitro and in vivo toxicology evaluations of the electrically heated cigarette smoking system series K

The Electrically Heated Cigarette Smoking System Series K (EHCSS) produces smoke through the controlled electrical heating of tobacco. Evaluation of the EHCSS was accomplished by comparison with commercial and reference cigarettes, using International Organization for Standardization (ISO) and alternative puffing regimens based on nicotine exposures measured in a short-term clinical study. Using the alternative puffing regimen and compared with conventional cigarettes on a per cigarette basis, the EHCSS had 50–60% reductions in tar and nicotine; at least 90% reductions in carbon monoxide, nitrogen oxides, 1,3-butadiene, isoprene, acrylonitrile, polyaromatic hydrocarbons, hydrogen cyanide, aromatic amines, tobacco specific nitrosamines, and phenol; and least a 40% reduction in 2-nitropropane. Other important smoke constituents in EHCSS smoke were reduced as well. The in vitro studies showed similar large reductions in biological activity. Ames mutagenicity of total particulate matter (TPM) from the EHCSS was reduced by 70–90%; cytotoxicity of the TPM was reduced by approximately 82% and 65% for the gas–vapor phase. In vivo testing under ISO smoking conditions in the mouse skin painting assay demonstrated later dermal tumor onset, lower dermal tumor incidence, reduced dermal tumor multiplicity, and a lower proportion of malignant dermal tumors in EHCSS smoke condensate-exposed mice. Thirty-five day and 90-day nose-only inhalation studies in rats showed reductions in pulmonary inflammation and other biological activity, including histopathological endpoints. We conclude that under the conditions of these in vitro and in vivo studies, the EHCSS demonstrated significantly lower biological activity compared to conventional cigarettes, and may suggest the potential for reductions in human smokers.     

Antigenotoxic effect of hyperoside against Mitomycin C and hydrogen peroxide-induced genotoxic damage on human lymphocytes

Hyperoside is a flavonol glycoside isolated from various plant genera such as Hypericum and Crataegus. It has an important place in the human diet and is used medically to relieve pain and ameliorate cardiovascular functions. However, a comprehensive profile of the genotoxic and antigenotoxic effects of hyperoside is not known. The current study aimed to investigate the genotoxic and antigenotoxic effects of hyperoside against genetic damages induced by two genotoxins (MMC and H₂O₂) using chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) assays in human peripheral blood lymphocytes in vitro. Blood lymphocytes were incubated with 7.8–62.5 μg/mL concentrations of hyperoside alone and simultaneously with 0.20 μg/mL Mitomycin C (MMC) or 100 μM Hydrogen peroxide (H₂O₂). Hyperoside did not exhibit genotoxic potential in the CA, SCE, and MN assays. Moreover, it did not cause a decrease in mitotic index (MI) which is an indicator of cytotoxicity. On the other hand, hyperoside significantly decreased CA, SCE, and MN (except for MMC treatment) frequencies induced by MMC and H₂O₂. Hyperoside, increased mitotic index against both mutagenic agents at 24-h treatment when compared to positive control. Our results demonstrate that hyperoside exhibited antigenotoxic effects rather than genotoxic in vitro human lymphocytes. Therefore, hyperoside may be a potential preventive agent in inhibiting chromosomal and oxidative damage induced by genotoxic chemicals.     

Smoking history, nicotine dependence, and changes in craving and mood during short-term smoking abstinence in alcohol dependent vs. control smokers

Objective

The goal of this study was to compare lifetime cigarette smoking, severity of nicotine dependence, and subjective effects of short-term tobacco abstinence in abstinent alcohol dependent (AD) and control smokers.

Method

AD (n = 119) and control (n = 55) ever-smokers were compared on tobacco use history and nicotine dependence. Negative affect and craving to smoke were examined in a subsample of currently smoking AD (N = 34) and control (N = 19) participants during a 6-h period of tobacco abstinence using the Profile of Mood States (POMS) and the Questionnaire on Smoking Urges-Brief (QSU-B).

Results

Although AD smokers did not differ from controls on heaviness of smoking, they were more likely to meet lifetime criteria for nicotine dependence. AD smokers also reported more withdrawal symptoms and were more likely to endorse withdrawal-related depressed mood during past smoking reduction or abstinence periods. During short-term abstinence, AD smokers were more likely to report high craving to smoke for negative affect relief within the first 150 min of tobacco abstinence, but did not differ from controls on overall craving to smoke or withdrawal-related negative affect on the POMS.

Conclusions

Results support previous findings that AD smokers have a greater prevalence of nicotine dependence and more severe nicotine withdrawal, with a greater propensity toward withdrawal-related depressed mood. These results, along with our novel finding that greater craving to smoke in abstaining smokers with AD is specific to negative affect-related craving, suggest that negative reinforcement may be a particularly salient factor in the maintenance of tobacco use among individuals with AD.     

Evaluation of the Genotoxic and Cytotoxic Potential of Mainstream Whole Smoke and Smoke Condensate from a Cigarette Containing a Novel Carbon Filter

A novel carbon filter has been developed which primarily reducesthe amount of certain vapor phase constituents of tobacco smokewith greater efficiency than the charcoal filters of cigarettescurrently in the market In vitro indicators of genotoxic andcytotoxic potential were used to compare the cigarette smokecondensate (particulate phase) or whole cigarette smoke (vaporphase and particulate phase) from cigarettes containing thenovel carbon filter with smoke condensate or whole smoke fromcommercial or prototype cigarettes not containing the novelcarbon filter. Ames bacterial mutagenicity, sister chromatidexchange (SCE) in Chinese hamster ovary (CHO) cells, and neutralred cytotoxicity assays in CHO cells were utilized to assessthe genotoxic and cytotoxic potential of the cigarette smokecondensates. SCE and neutral red cytotoxicity assays were utilizedto assess the genotoxic and cytotoxic potential of the wholesmoke. As expected, the novel carbon filter did not significantlyaffect the genotoxic or cytotoxic activity of the smoke condensate,although we did observe that the use of low-nitrogen tobaccoreduced the mutagenicity of the condensate in Salmonella typhimuriumstrain TA98. However, the whole smoke from cigarettes containingthe novel carbon filter demonstrated significant reductionsin genotoxic and cytotoxic potential compared to cigaretteswithout the novel carbon filter. The toxicity of the smoke wascorrelated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCEinduction) to the aggregate mass of several vapor phase components(acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene,ammonia, NO_x, HCN, benzene, isoprene, and formaldehyde) in thesmoke of the cigarettes utilized in this study. In conclusion,this novel carbon filter, which significantly reduced the amountof carbonyls and other volatiles in mainstream cigarette smoke,resulted in significant reductions in the genotoxic and cytotoxicactivity of the smoke as measured by these assays.     

Genotoxicity evaluation of Mequindox in different short-term tests

Quinoxaline-1,4-dioxides (QdNOs) are the potent heterocyclic N-oxides with interesting biological properties such as antibacterial, anticandida, antitubercular, anticancer and antiprotozoal activities. Here, we tested and compared the mequindox (MEQ) for mutagenic abilities in a battery of different short term tests according to OECD guidelines. When compared with the controls, a strong mutagenicity of MEQ and carbadox (CBX) was observed with an approximate concentration-effect relationship in Salmonella reverse mutation test, chromosome aberration test, unscheduled DNA synthesis assay and HGPRT gene mutation test, in the absence and presence of S₉-mix. In in vivo micronucleus test, CBX produced significant increase in the proportion of micronucleus formation than MEQ in mice bone marrow cells. From these results, we can conclude that MEQ had a strong genotoxic potential to mammalian cells in vitro as well as in vivo and its mutagenicity is slightly higher than CBX. Our results, for the 1st time, discuss the genotoxic potential of MEQ. These results not only confirm the earlier findings about CBX but also extend the knowledge and awareness about the genotoxic risk of QdNO derivatives.     

The genotoxic effect of nicotine on chromosomes of human fetal cells: The first report described as an important study

Context: Recent studies have suggested a direct contribution of nicotine ? the addictive component of tobacco and tobacco smoke ? to human carcinogenesis, and it remains the most common harmful substance to which pregnant women are exposed. Also, it has deleterious effects on the fetus. The sperm of smoking fathers and newborns of smoking mothers have elevated frequencies of chromosome translocations and DNA strand breaks.

Objective: We tried to understand the genotoxic effect of nicotine in pregnancies of active or passive smoking mothers. For this reason, we provide the evidence that nicotine exposure in vitro has detrimental effects on fetal cells.

Materials and methods: We examined the effect of nicotine sulphate on amniotic cells by designing an experimental setting consisting fetal cells grown in nicotine containing medium (25?ng/mL) in study group and fetal cells grown in control medium, which did not contain nicotine.

Results: According to our findings, there is a significant difference of chromosomal aberrations (CAs) between nicotine containing medium grown cells and control medium grown cells, determined by the χ2 test (P <0.001). We found CAs in 21.5% of cells analyzed. The 19.4% of the all cells had numerical aberrations. Chromosomes 21, 22, 8, 15 and 20 related numerical abnormalities were found to be the most frequent numerical abnormalities.

Conclusion: Results of this study confirm that the nicotine leads to significant direct genotoxic effects in human fetal cells in vitro. We speculate that there is an association between prenatal exposure to cigarette smoke and in utero aneuploidies.     

Genotoxicity testing of a Hoodia gordonii extract

Hoodia gordonii extract consists of a mixture of steroid glycosides, fatty acids, plant sterols and alcohols. As part of the overall safety assessment H. gordonii extract was assessed for genotoxicity in two assays in vitro: a bacterial mutation assay; and a gene mutation assay using mouse lymphoma cells. H. gordonii extract showed no evidence of genotoxic activity in either of these assays. In addition, H. gordonii extract was assessed for mutagenic activity in a bone marrow micronucleus (MN) assay in the mouse, with 400 mg/kg selected as the high-dose group, based on observations in a dose-range-finding study. The group mean frequencies of micronucleated polychromatic erythrocytes of treated animals were similar to those of the vehicle control group, indicating H. gordonii extract to be non-genotoxic under the conditions of this test. All assays were performed in compliance with the Good Laboratory Practice Regulations and in accordance with standard guidelines for genotoxicity tests. H. gordonii extract was shown to be non-genotoxic in 3 independent assays (a bacterial mutation test, a gene mutation assay using mouse lymphoma cells and a bone marrow micronucleus assay in the mouse).     

Comparison of the Reinforcing Properties of Nicotine and Cigarette Smoke Extract in Rats

Tobacco dependence is difficult to treat, with the vast majority of those who try to quit relapsing within the first year. Improvements in smoking cessation therapies may be achieved by improving current preclinical research methods. However, most experimental tests in animals use nicotine alone, ignoring the 8000 other constituents found in tobacco smoke. To improve on this model, we have used self-administration to test the reinforcing properties of aqueous cigarette smoke extract (CSE) in rats, made by bubbling cigarette smoke through a saline solution. CSE is more potent than nicotine alone in both the acquisition and maintenance of self-administration, but did not exhibit higher progressive ratio responding. Mecamylamine and varenicline had similar potencies to block nicotine and CSE self-administration, indicating the involvement of nicotinic receptors in CSE reinforcement. Following extinction of responding, reinstatement was triggered by exposing animals to a pharmacological stressor, yohimbine (2.5 mg/kg, i.p.), alone and in combination with cues. Animals that self-administered CSE were significantly more sensitive to stress-induced reinstatement than those that self-administered nicotine. Ligand binding autoradiography studies showed nicotine and CSE to have similar affinities for different nicotinic receptor types. CSE significantly reduced MAO-A and MAO-B activities in vitro, whereas nicotine did not. Although CSE inhibition of MAO-A activity in vitro was found to be partially irreversible, irreversible inhibition was not observed in vivo. These experiments show that CSE is an effective reinforcer acting via nicotinic receptors. Furthermore, it better models MAO inhibition and is more sensitive to stress-induced reinstatement than nicotine alone, which is a potent trigger for relapse in smokers.     

The tobacco alkaloid nicotine demonstrates genotoxicity in human tonsillar tissue and lymphocytes.

Recent studies suggest a direct contribution of nicotine, the addictive component of tobacco and tobacco smoke, to human carcinogenesis. To assess the genotoxicity of nicotine, the DNA-damaging effect on human lymphocytes and target cells from lymphatic tissue of the palatine tonsils from 10 healthy patients was tested with the alkaline single-cell microgel electrophoresis (Comet) assay. The degree of DNA migration, a measure of possible DNA single strand breaks, alkali labile sites, and incomplete excision repair sites, was expressed as the Olive tail moment, the percentage of DNA in the tail, and the tail length. One hour exposure to nicotine at 0.125, 0.25, 0.5, 1, 2, and 4 mM induced a statistically significant dose-dependent increase of DNA migration up to 3.8-fold and 3.2-fold in tonsillar cells and lymphocytes, respectively. The lowest concentration eliciting significant DNA damage was 0.5 mM nicotine. The genotoxic effect was confirmed in a second series of experiments using nicotine of high purity from two different suppliers. There were no significant differences between the two series, excluding artifacts from the source of nicotine. Finally, DNA damage by nicotine was compared in cells incubated in medium strictly adjusted to neutral pH, with non-adjusted medium becoming alkaline with increasing nicotine concentrations. Again no differences in DNA migration were observed. The data indicate that nicotine expresses significant direct genotoxic effects in human target cells in vitro. However, no differences in DNA damage were observed in cells from smokers and nonsmokers incubated without nicotine. The lack of higher DNA damage in smokers compared to nonsmokers could be a question of nicotine dose, rapid DNA repair, or interactions with other smoke constituents. These results require further investigations on the contribution of nicotine to tobacco carcinogenesis.     

The resolving power of in vitro genotoxicity assays for cigarette smoke particulate matter

In vitro genotoxicity assays are often used to compare tobacco smoke particulate matter (PM) from different cigarettes. The quantitative aspect of the comparisons requires appropriate statistical methods and replication levels, to support the interpretation in terms of power and significance. This paper recommends a uniform statistical analysis for the Ames test, mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT); involving a hierarchical decision process with respect to slope, fixed effect and single dose comparisons. With these methods, replication levels of 5 (Ames test TA98), 4 (Ames test TA100), 10 (Ames test TA1537), 6 (MLA) and 4 (IVMNT) resolved a 30% difference in PM genotoxicity.     

Smoke Extracts and Nicotine, but not Tobacco Extracts, Potentiate Firing and Burst Activity of Ventral Tegmental Area Dopaminergic Neurons in Mice

Nicotine prominently mediates the behavioral effects of tobacco consumption, either through smoking or when taking tobacco by snuff or chew. However, many studies question the exclusive role of nicotine in these effects. The use of preparations containing all the components of tobacco, such as tobacco and smoke extracts, may be more suitable than nicotine alone to investigate the behavioral effects of smoking and tobacco intake. In the present study, the electrophysiological effects of tobacco and smoke on ventral tegmental area dopaminergic (DA) neurons were examined in vivo in anesthetized wild-type (WT), β2-nicotinic acetylcholine receptor (nAChR) knockout (β2−/−), α4−/−, and α6−/− mice and compared with those of nicotine alone. In WT mice, smoke and nicotine had similar potentiating effects on DA cell activity, but the action of tobacco on neuronal firing was weak and often inhibitory. In particular, nicotine triggered strong bursting activity, whereas no bursting activity was observed after tobacco extract (ToE) administration. In β2−/− mice, nicotine or extract elicited no modification of the firing patterns of DA cells, indicating that extract acts predominantly through nAChRs. The differences between DA cell activation profiles induced by tobacco and nicotine alone observed in WT persisted in α6−/− mice but not in α4−/− mice. These results would suggest that tobacco has lower addiction-generating properties compared with either nicotine alone or smoke. The weak activation and prominent inhibition obtained with ToEs suggest that tobacco contains compounds that counteract some of the activating effects of nicotine and promote inhibition on DA cell acting through α4β2^*-nAChRs. The nature of these compounds remains to be elucidated. It nevertheless confirms that nicotine is the main substance involved in the tobacco addiction-related activation of mesolimbic DA neurons.     

Effects of nicotine,its metabolites and tobacco extracts on human platelet function in vitro

Cigarette smoking is a leading cause of cardiovascular disease. The cardiovascular effects of smoking are probably multifactorial, including effects on platelets. Previous reports investigating the effects of nicotine and tobacco on platelet function are inconsistent.The present study investigated in vitro effects of nicotine, its major metabolites, tobacco extracts and extract of tobacco-free snuff on human platelets.None of the metabolites cotinine, cotinine-N-oxide, nicotine-1′-N-oxide or trans-3′-hydroxycotinine (0.1–10 μM) affected platelet aggregation or P-selectin expression. Nicotine (10 μM) weakly increased platelet aggregation, whereas trans-3′-hydroxycotinine (0.1 μM) and nicotine-1′-N-oxide (1–10 μM) weakly inhibited adhesion to fibrinogen. To elucidate the influence of other tobacco compounds, we investigated the impact of moist tobacco and smoke extracts on platelet function. Filtered extracts of oral snuff, cigarette smoke and tobacco free snuff inhibited platelet adhesion concentration-dependently. The inhibitory effects of tobacco extracts on platelet adhesion were independent of nicotine content and the nitric-oxide-pathway and not mediated through a platelet-nicotine-receptor.Taken together, tobacco extracts inhibit platelet activation during short-term in vitro challenge. As only limited effects of nicotine and nicotine metabolites were seen, the tobacco-induced platelet inhibition are likely induced by other compounds present in tobacco and tobacco free snuff.     

Do smokers of specialty and conventional cigarettes differ in their dependence on nicotine?

Specialty cigarettes, bidis and kreteks, have commonly been viewed by adolescent users as being less harmful than conventional cigarettes. Biochemical studies, however, have shown that the concentration and delivery of nicotine from these tobacco products are not insignificant. The current study tested whether the diagnosis and symptoms for nicotine dependence differed among conventional-only smokers (n = 16 959), specialty-only smokers (n = 313), and poly-tobacco smokers (n = 1288) from the 2002 and 2003 National Surveys on Drug Use and Health. Compared with the specialty-only smokers, the conventional-only and poly-tobacco smokers were more dependent on nicotine, assessed by the Nicotine Dependence Syndrome Scale and a single item from the Fagerstrom Test for Nicotine Dependence. However, after accounting for differences in smoking frequency, the specialty-only smokers had significantly greater odds of being nicotine dependent than the conventional-only smokers. The reversed effect was primarily attributed to the specialty-only smokers who smoked less frequently, but reported a shorter time to their first cigarette. These findings suggest that the nicotine acquired from specialty cigarettes may be sufficient in yielding a sense of urgency to smoke.