High Concentrations Of Nicotine Research Articles

Nicotine derived genotoxic effects in human primary parotid gland cells as assessed in vitro by comet assay, cytokinesis-block micronucleus test and chromosome aberrations test

Genotoxic effects of nicotine were described in different human cells including salivary gland cells. Based on the high nicotine concentration in saliva of smokers or patients using therapeutic nicotine patches, the current study was performed to evaluate the genotoxic potential of nicotine in human salivary gland cells.Therefore, primary salivary gland cells from 10 patients undergoing parotid gland surgery were exposed to nicotine concentrations between 1μM and 1000μM for 1h in the absence of exogenous metabolic activation. The acinar phenotype was proven by immunofluorescent staining of alpha-amylase. Genotoxic effects were evaluated using the Comet assay, the micronucleus test and the chromosome aberration test. Cytotoxicity and apoptosis were determined by trypan blue exclusion test and Caspase-3 assay.Nicotine was able to induce genotoxic effects in all three assays. The chromosome aberration test was the most sensitive and increases in numerical and structural (chromatid-type and chromosome-type) aberrations were seen at ⩾1μM, whereas increases in micronuclei frequency were detected at 10μM and DNA damage as measured in the Comet assay was noted at >100μM. No cytotoxic damage or influence of apoptosis could be demonstrated.Nicotine as a possible risk factor for tumor initiation in salivary glands is still discussed controversially. Our results demonstrated the potential of nicotine to induce genotoxic effects in salivary gland cells. These results were observed at saliva nicotine levels similar to those found after oral or transdermal exposure to nicotine and suggest the necessity of careful monitoring of the use of nicotine in humans.

Snus (Swedish smokeless tobacco) use and risk of stroke: pooled analyses of incidence and survival

Snus is a moist smokeless tobacco product with high nicotine content. Its use has a short-term effect on the cardiovascular system, but the relationship between snus use and stroke is unclear. The aim of this study was to assess the associations between use of snus and incidence of and survival after stroke, both overall and according to subtypes. Pooled analyses of eight Swedish prospective cohort studies were conducted, including 130485 men who never smoked. We estimated hazard ratios (HRs) with 95% confidence intervals (CIs) of incidence and death after diagnosis using Cox proportional hazard regression models and case fatality and survival using logistic regression and Kaplan-Meier methods, respectively. No associations were observed between the use of snus and the risk of overall stroke (HR 1.04, 95% CI 0.92-1.17) or of any of the stroke subtypes. The odds ratio (OR) of 28-day case fatality was 1.42 (95% CI 0.99-2.04) amongst users of snus who had experienced a stroke, and the HR of death during the follow-up period was 1.32 (95% CI 1.08-1.61). Use of snus was not associated with the risk of stroke. Hence, nicotine is unlikely to contribute importantly to the pathophysiology of stroke. However, case fatality was increased in snus users, compared with nonusers, but further studies are needed to determine any possible causal mechanisms.

Some Responses of Two Nicotiana tabacum L. Cultivars Exposed to Vanadium

The effect of exposure to different vanadium (V) concentrations was studied in Nicotiana tabacum L. (cv. ‘Burley’ and cv. ‘Virginia’). In both cultivars, the vanadium did not affect the percentage of germination but root and shoot growth was inhibited, especially with the 80 μM dose. An altered root morphogenesis in vanadium growth plants was observed at the end of the experiment. The highest V accumulation was found in roots and the results showed a direct correlation between V content and V supply (P ≤ 0.01). The V concentrations did not modify nicotine content in the cv. ‘Burley’, but in the cv. ‘Virginia’ the highest nicotine content was found in vanadium exposed plants (P ≤ 0.05). The results suggest that these V concentrations may have a possible elicitor effect on alkaloid synthesis in N. tabacum L. cv. ‘Virginia’, and that this cultivar has a higher V sensitivity than the cv. ‘Burley’.

Concentration Dependency in Nicotine Skin Penetration Flux from Aqueous Solutions Reflects Vehicle Induced Changes in Nicotine Stratum Corneum Retention

This study sought to understand the mechanism by which the steady state flux of nicotine across the human skin from aqueous solutions is markedly decreased at higher nicotine concentrations. Nicotine's steady state flux through human epidermis and its amount in the stratum corneum for a range of aqueous nicotine solutions was determined using Franz diffusion cells, with the nicotine analysed by high performance liquid chromatography (HPLC). Nicotine's thermodynamic activity in the various solutions was estimated from its partial vapour pressure and stratum corneum hydration was determined using a corneometer. The amount of nicotine retained in the stratum corneum was estimated from the nicotine amount found in individual stratum corneum tape strips and a D-Squame determined weight for each strip. The observed steady state flux of nicotine across human epidermis was found to show a parabolic dependence on nicotine concentration, with the flux proportional to its thermodynamic activity up to a concentration of 48% w/w. The nicotine retention in the stratum corneum showed a similar dependency on concentration whereas the diffusivity of nicotine in the stratum corneum appeared to be concentration independent. This retention, in turn, could be estimated from the extent of stratum corneum hydration and the nicotine concentration in the applied solution and volume of water in the skin. Nonlinear dependency of nicotine skin flux on its concentration results from a dehydration induced decrease in its stratum corneum retention at higher concentration and not dehydration induced changes nicotine diffusivity in the stratum corneum.

Abstract B16: The effect of e-cigarette exposure on airway epithelial cell gene expression and transformation.

Abstract Lung cancer is the leading cause of cancer-related mortality in the United States. Despite a strong correlation between cigarette smoking and the onset of lung cancer, the prevalence of smoking still remains high. Strategies to eliminate cigarette smoking have led to the emergence of new tobacco-related products as alternatives for cigarette smoking or tools for smoking cessation. The electronic cigarette (ECIG) is a battery-powered electronic nicotine delivery system (ENDS) designed to deliver nicotine without combusting tobacco. Since nicotine is widely considered the addictive component in tobacco with limited ability to initiate cancer, ECIGs have been advertised to be a safer alternative to tobacco cigarettes (TCIGs). However, the toxicity and potential carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H3mut-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer. The epithelial cells were exposed to both a low and high concentration of nicotine in the ECIG vapor- or TCIG smoke-conditioned media. The lower nicotine concentration was selected to mimic the average plasma nicotine levels in ENDS users and did not demonstrate toxic or anti-proliferative effects on the cells. The higher concentration was chosen to represent the anticipated nicotine levels to which the epithelial cells of smokers are actually exposed. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the H3mut-P53/KRAS cells following a 10-day treatment with the high nicotine ECIG- and TCIG-conditioned media compared to the untreated and low nicotine treatment groups. We next assessed the effect of ECIG and TCIG exposure on cell invasion using a three-dimensional air-liquid interface (ALI) model. At baseline, H3mut-P53/KRAS cells exhibit invasive behavior in the ALI model, due to the downstream effects of P53 silencing and KRAS activation. Treatment of H3mut-P53/KRAS cells with low nicotine ECIG- and TCIG-conditioned media did not further enhance the degree of invasion observed in the untreated group. We will next examine the effects of high nicotine conditioned media on cell invasion. Finally, gene expression studies show 263 differentially expressed genes following in vitro exposure to ECIG-conditioned media for 96hrs. The high nicotine ECIG-conditioned media induced a gene expression pattern similar to TCIG- conditioned media and whole cigarette smoke exposure in the H3mut-P53/KRAS cells. Preliminary analyses indicate the observed ECIG-specific gene expression changes were concordantly changed following TCIG-conditioned media exposure. We will next compare the ECIG-induced gene expression signature to carcinogenicity-related gene signatures established in previous and ongoing clinical investigations and test ECIG-altered candidate genes for their ability to drive the malignant transformation of airway epithelial cells. These studies will determine the impact of ECIG exposure on lung carcinogenicity and provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs. These studies were supported by funding from the following: NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW, SJP), NCI #U01CA152751-AS (SMD, KK), NCI #T32-CA009120-36 (SMD, SJP, PCP), NIH/NHLBI #T32HL072752 (SMD, EL), University of California Tobacco-Related Disease Research Program (TRDRP) #18FT-0060 (TCW), TRDRP #20KT-0055 (TCW), Lung Cancer SPORE P50CA70907 (JDM, JEL) Citation Format: Stacy J. Park, Tonya C. Walser, Catalina Perdomo, Teresa Wang, Paul C. Pagano, Elvira L. Liclican, Kostyantyn Krysan, Jill E. Larsen, John D. Minna, Marc E. Lenburg, Avrum Spira, Steven M. Dubinett. The effect of e-cigarette exposure on airway epithelial cell gene expression and transformation. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B16.

Physiological and Biochemical Characterization of a Novel Nicotine-Degrading Bacterium Pseudomonas geniculata N1

Management of solid wastes with high nicotine content, such as those accumulated during tobacco manufacturing, poses a major challenge, which can be addressed by using bacteria such as Pseudomonas and Arthrobacter. In this study, a new species of Pseudomonas geniculata, namely strain N1, which is capable of efficiently degrading nicotine, was isolated and identified. The optimal growth conditions for strain N1 are a temperature of 30°C, and a pH 6.5, at a rotation rate of 120 rpm min−1 with 1 g l−1 nicotine as the sole source of carbon and nitrogen. Myosmine, cotinine, 6-hydroxynicotine, 6-hydroxy-N-methylmyosmine, and 6-hydroxy-pseudooxynicotine were detected as the five intermediates through gas chromatography-mass and liquid chromatography-mass analyses. The identified metabolites were different from those generated by Pseudomonas putida strains. The analysis also highlighted the bacterial metabolic diversity in relation to nicotine degradation by different Pseudomonas strains.

Abstract C58: The gene expression and functional impact of E-cigarette exposure on pulmonary epithelial transformation

Abstract Lung cancer is the leading cause of cancer deaths in the United States. Despite a strong correlation between cigarette smoking and the onset of lung cancer, the prevalence of smoking still remains high. Significant reduction or elimination of the use of combustible tobacco products would substantially lower tobacco-caused morbidity and mortality. The electronic cigarette (ECIG) is a battery-powered electronic nicotine delivery system (ENDS) designed to deliver nicotine without combusting tobacco. ECIGs are widely believed to be a safer alternative to tobacco cigarettes (TCIG), however, the toxicity and potential carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H3mut-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer. The epithelial cells were exposed to a low and high nicotine concentration of ECIG vapor- or TCIG smoke-conditioned media. The lower nicotine concentration was selected to mimic plasma nicotine levels in ENDS users and did not demonstrate toxic or anti-proliferative effects on the cells. The higher concentration was chosen to represent the anticipated nicotine levels to which the epithelial cells of smokers are actually exposed. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the H3mut-P53/KRAS cells following a 10-day treatment with the high nicotine concentration of ECIG- and TCIG-conditioned media compared to the untreated and low nicotine treatment groups. We next assessed the effect of ECIG and TCIG exposure on cell invasion using three-dimensional air-liquid interface (ALI) models. While treatment with the low nicotine concentration of ECIG-conditioned media did not induce invasion-associated morphological changes, cells treated with the low nicotine concentration of TCIG-conditioned media exhibited slightly invasive behavior by breaching the modified basement membrane and invading through the field of fibroblasts/collagen. At baseline, H3mut-P53/KRAS cells exhibit invasive behavior in the ALI model, due to the downstream effects of P53 silencing and KRAS activation. Treatment of H3mut-P53/KRAS cells with the low nicotine concentration of ECIG- and TCIG-conditioned media did not further enhance the degree of invasion observed in the untreated group. We will next examine the effect of the high nicotine concentration on cell invasion. In addition, we are currently evaluating gene expression profiles by microarray of H3mut-P53/KRAS cells exposed to high and low nicotine concentrations of ECIG-conditioned media. This will directly evaluate the impact of ECIGs on carcinogenicity-related gene expression signatures established in previous and ongoing clinical investigations. These studies will determine the impact of ECIG exposure on lung carcinogenicity, which will provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs. These studies were supported by the NIH/NCI #U01CA152751-S1 (SMD, AS, MEL, TCW) and NIH/NHLBI #T32HL072752 (SJP). Citation Format: Stacy J. Park, Tonya C. Walser, Catalina Perdomo, Paul P. Pagano, Daniel Brass, Elvira L. Liclican, Kostyantyn Krysan, Marc E. Lenburg, Avrum Spira, Steven M. Dubinett. The gene expression and functional impact of E-cigarette exposure on pulmonary epithelial transformation. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C58.

Effectiveness of Nicotinic Agonists as Desensitizers at Presynaptic 4 2- and 4 5 2-Nicotinic Acetylcholine Receptors

Nicotine interacts with nicotinic acetylcholine receptors (nAChRs) and modifies neuronal functions. The net result of nicotine exposure is difficult to assess because multiple nAChR subtypes exist and are expressed on multiple classes of neurons. Nicotine, unlike the natural agonist acetylcholine, remains in tissues for hours, and during this extended exposure nAChRs desensitize. Therefore, agonists can block the natural functions of nAChRs. Higher nicotine concentrations are required to desensitize α4β2-nAChRs containing the α5 subunit. The aim of these experiments was to determine if this property holds true for compounds other than nicotine. [(3)H]-dopamine release from crude mouse striatal synaptosomal preparations was used to measure activation and desensitization of the [(α4β2)2β2] and [(α4β2)2α5] nAChR subtypes. Affinity was measured by competition with [(125)I]-epibatidine. Nine compounds of varying affinity and efficacy were tested. All compounds partially desensitized both subtypes; concentration necessary for desensitization correlated with binding site affinity but not efficacy. All compounds showed a similar, significant shift in concentration necessary for a 50% effect when the α5 subunit was included (averaging 8-fold higher). The extent of desensitization produced by a 10-min exposure did not correlate with affinity or efficacy of compound. Full or partial nicotinic agonists used as medications may effectively desensitize α4β2-nAChRs. However, significantly higher concentrations of all compounds tested were required to elicit desensitization of α4α5β2-nAChRs than α4β2-nAChRs. If desensitization is the important property for a smoking cessation drug, basic screening at both subtypes may provide a mechanistic foundation for effectiveness.

Bimodal Concentration-Response of Nicotine Involves the Nicotinic Acetylcholine Receptor, Transient Receptor Potential Vanilloid Type 1, and Transient Receptor Potential Ankyrin 1 Channels in Mouse Trachea and Sensory Neurons

High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 µM (-)-nicotine, a maximum at 100 µM, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pHe-dependent, - five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 µM nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200-fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded.

Bird pollinators differ in their tolerance of a nectar alkaloid

Although the function of nectar is to attract and reward pollinators, secondary metabolites produced by plants as anti‐herbivore defences are frequently present in floral nectars. Greater understanding is needed of the effects of secondary metabolites in nectar on the foraging behaviour and performance of pollinators, and on plant–pollinator interactions. We investigated how nectar‐feeding birds, both specialist (white‐bellied sunbirds Cinnyris talatala) and generalist (dark‐capped bulbuls Pycnonotus tricolor and Cape white‐eyes Zosterops virens), respond to artificial nectar containing the alkaloid nicotine, present in nectar of Nicotiana species. Preference tests were carried out with a range of nicotine concentrations (0.1–300 μM) in two sucrose concentrations (0.25 and 1 M), and for bulbuls also in two sugars (sucrose and hexose). In addition, we measured short‐term feeding patterns in white‐bellied sunbirds that were offered nicotine (0–50 μM) in 0.63 M sucrose. Both nicotine and sugar concentrations influenced the response of bird pollinators to nicotine. The birds showed dose‐dependent responses to nicotine; and their tolerance of high nicotine concentrations was reduced on the dilute 0.25 M sucrose diet, on which they increased consumption to maintain energy intake. White‐bellied sunbirds decreased both feeding frequency and feeding duration as the nicotine concentration in artificial nectar increased. Of the three species, bulbuls showed the highest tolerance for nicotine, and sugar type (sucrose or hexose) had no effect. The indifference of bulbuls to nicotine may be related to their primarily frugivorous diet. However, the response of white‐eyes to nicotine in the dilute sucrose solution was very similar to that of sunbirds, even though white‐eyes are generalist nectar‐feeders. Additional testing of other avian nectarivores and different secondary metabolites is required to further elucidate whether generalist bird pollinators, which utilise dilute nectars in which secondary metabolites have stronger deterrent effects, are more tolerant of ‘toxic’ nectar.

Coloured and Toxic Nectar: Feeding Choices of the Madagascar Giant Day Gecko, Phelsuma grandis

AbstractColoured nectar is a rare phenomenon best known from islands and insular habitats. Islands are also known for lizard pollination, where coloured nectar potentially acts as a visual cue to attract pollinators, advertising the sweet reward. However, nectar may also contain secondary metabolites with toxic or deterrent effects. The aim of this study was to determine which factors are important as artificial nectar choice determinants to the Madagascar giant day gecko, Phelsuma grandis, an island pollinator: artificial nectar colour, artificial nectar colour saturation, artificial nectar conspicuousness and/or the presence of the alkaloid nicotine. Coloured artificial nectar and the darkest artificial nectar colour saturation were found to be important visual cues for the geckos, while the contrast between artificial nectar and petal colour was not. Geckos were deterred only by high nicotine concentrations (1000 μm in 0.63 m sucrose) and may even prefer low nicotine concentrations to sucrose‐only solutions. Given their overall fondness for sugar solutions, Madagascar giant day geckos are likely to be important pollinators of Malagasy plant species that produce enough nectar to attract them, and plants with coloured nectar and/or secondary metabolites may have evolved those traits to attract the geckos in particular.

Identification of nicotinic acetylcholine receptor subunit expression in early avian embryos

Most previous studies on nicotine used relatively high concentrations of nicotine, administered later in development. Using chick embryos as our model system, we have found a single low dose of nicotine (100 nM) administered during early development significantly retarded embryo growth, reduced heart rate, and increased the incidence of defects four days later. It is often assumed that nicotine's teratogenic effects are mediated by nicotinic acetylcholine receptors (nAChRs), whose expression at very early stages of embryonic development has not been thoroughly investigated. The objective was to identify which nAChR subunits are expressed during early avian embryogenesis. We performed RT‐PCR expression analysis for eleven nAChR subunits, including alpha1‐alpha7, alpha9, alpha10, beta3, and beta4, at 24‐, 48‐, 72‐, and 96‐hrs of development. Surprisingly, we find nAChR expression is widespread during early development, which suggests the teratogenic effects of nicotine were mediated by the early activation of nAChRs.Nebraska NIH‐INBRE Program; Nebraska Health and Human Services (LB595); Ferlic Undergraduate Summer Research Program, Creighton University College of Arts and SciencesGrant Funding Source: none current

Review Article: Prevalence and Health Effects of Water Pipe (Shisha) Tobacco Smoking

Every day approximately 100 million people smoke water pipe. The trend is increasing in Arabic countries, in Europe and the USA especially in adolescents. Studies revealed that the tobacco smoke from water pipe, depending on the used tobacco and charcoal, contains higher concentrations of nicotine, carbon monoxide, carboxy hemoglobin,heavy metals, carcinogens and fine dust particles compared to cigarette smoke. According to studies, water pipe tobacco smoking was significantly associated with lung cancer, chronic lung diseases, periodontal diseases and low birth-weight. Similar to cigarette smoking, water pipe tobacco smoking causes a severe health risk by environmental tobacco smoke, because the side-stream smoke of the water pipe contains higher concentrations of polycyclic aromatic hydrocarbons, aldehydes and carbon monoxide compared to the side-stream smoke of cigarettes.

The medial habenula as a regulator of anxiety in adult zebrafish

OPINION article Front. Neural Circuits, 27 May 2013 | https://doi.org/10.3389/fncir.2013.00099

Assessments of the effects of nicotine and ketamine using tyrosine hydroxylase-green fluorescent protein transgenic zebrafish asbiosensors

Transgenic zebrafish are a common vertebrate model system for the study of addictive behavior. In the present study, plasmid constructs containing green fluorescent protein (GFP) and the promoter of tyrosine hydroxylase (TH), a key synthetic enzyme for catecholamines, were produced. The TH-GFP constructs were microinjected into zebrafish embryonic cells. Three days post-fertilization, GFP began expressing in distinct catecholaminergic areas. The TH-GFP transgenic zebrafish were employed as live biosensors to test the effects of the commonly abused drugs nicotine and ketamine. First, locomotion assays were used to study the general excitatory effects of the drugs. Maximal locomotor activity was obtained after treatment with a high concentration of nicotine (10μM), but with a much lower concentration of ketamine (0.1μM). Second, TH protein levels in zebrafish brains were assessed by Western blot. TH protein levels were significantly increased, with maximal protein levels found after treatment with the same drug concentrations that gave maximal locomotor activity. Importantly, analysis of GFP in the zebrafish catecholaminergic areas revealed the same expression patterns as was obtained by Western blot. The present results indicate that increased locomotor activity can be correlated to TH protein expression, as indicated by Western blot and expression of TH-GFP. We have shown that TH-GFP expression is a reliable method to show the effects of drugs on TH expression that may be employed as a novel high-throughput live biosensor for screening drugs ofabuse.

Trends in the Use and Advertising of Discount Versus Premium Snuff

The Conwood Company, a major producer of discount moist snuff, was awarded a $1 billion antitrust settlement in the year 2000 against its leading competitor, the U.S. Smokeless Tobacco Company. The objective of this study was to examine the trends in use and advertising of discount versus premium snuff since the Conwood settlement, a topic seldom addressed in the tobacco control literature. 2 sources of data were analyzed in 2011: (a) male snuff users from the 2002-2009 National Surveys on Drug Use and Health (N = 13,172) and (b) total advertisements of moist snuff identified from over 350 consumer magazines dated 2005-2009 (N = 861). For the survey data, demographic and tobacco-related measures were assessed as predictors of use of discount versus premium snuff in logistic regression models. For the advertising data, associations were examined between the snuff category and nicotine content, magazine youth readership, and year of magazine publication. The prevalence of discount and premium snuff use among males increased and decreased, respectively, from 2002 to 2009. Significant predictors of using discount versus premium snuff were being an adolescent, being an African-American, being a current or former smoker, living in a less populated region of the country, and using snuff frequently. Discount snuff advertising was associated with publication in magazines with a high youth readership. Discount snuff has grown in popularity among male adolescents who have been a target of advertising. The tobacco's cheap price and high nicotine content pose a public health problem because of the potential for long-term tobacco use and dependence.

Effect of nicotine on growth and metabolism of Streptococcus mutans

Streptococcus mutans is a key contributor to dental caries. Smokers have a higher number of caries-affected teeth than do nonsmokers, but the association among tobacco, nicotine, caries, and S. mutans growth has not been investigated in detail. Seven S. mutans strains--UA159, UA130, 10449, A32-2, NG8, LM7, and OMZ175--were used in the present study. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), planktonic cell growth, biofilm formation, metabolism, and structure (determined using scanning electron microscopy) of the seven strains treated with different concentrations of nicotine (0-32 mg ml(-1)) were investigated. The MIC, MBC, and MBIC were 16 mg ml(-1) (0.1 M), 32 mg ml(-1) (0.2 M), and 16 mg ml(-1) (0.1 M), respectively, for most of the S. mutans strains. Growth of planktonic S. mutans cells was significantly repressed by 2.0-8.0 mg ml(-1) of nicotine. Biofilm formation and metabolic activity of S. mutans was increased in a nicotine-dependent manner up to 16.0 mg ml(-1) of nicotine. Scanning electron microscopy revealed that S. mutans treated with a high concentration of nicotine a had thicker biofilm and more spherical bacterial cells. In summary, nicotine enhances S. mutans biofilm formation and biofilm metabolic activity. These results suggest that smoking can increase the development of caries by fostering increased formation of S. mutans biofilm on tooth surfaces.

Simultaneous stressors: Interactive effects of an immune challenge and dietary toxin can be detrimental to honeybees

Recent large-scale mortality of honeybee colonies is believed to be caused by multiple interactions between diseases, parasites, pesticide exposure, and other stress factors. To test whether a dual challenge has an additive effect in reducing survival, we experimentally stimulated the immune system of caged Apis mellifera scutellata workers from six colonies by injecting saline or Escherichia coli lipopolysaccharides (LPS), and additionally fed them the alkaloid nicotine (0μM, 3μM and 300μM in 0.63M sucrose). Workers did not increase their sucrose intake to compensate for the immune system activation, and those injected with E. coli LPS decreased their intake on the highest nicotine concentration. In the single challenges, injection and high nicotine doses negatively affected survival. All injected worker groups showed reduced survival. Without nicotine, survival of the saline and E. coli LPS worker groups was similar, but survival of E. coli LPS-challenged workers dropped below that of the saline groups when additionally challenged by nicotine, with bees dying earlier at higher nicotine concentrations. In the dual challenge of saline injection and dietary nicotine, a reduced effect on survival was observed, with lower mortality than expected from the summed mortalities due to the single challenges. However, additive and synergistic effects on survival were observed in workers simultaneously challenged by E. coli LPS and nicotine, indicating that interactive effects of simultaneous pathogen exposure and dietary toxin are detrimental to honeybee fitness.

Toxicity of Extract Tobacco Leaves to The growth of Oreochromis niloticus

Tobacco can be used as natural pesticide since it contained high concentration of nicotine that may killed pest and fish diseases. This work aimed to know the influence and inhibiting concentration of tobacco leaves extract to the growth of Tilapia. The research was conducted on March to May 2007 at laboratory of Satker PBIAT Ngrajek, Magelang. Fish fry used were in between 1,74±0,105 g with the density of 10 were tested in 20 L fresh water, were tested using several concentration of tobacco leaves extract. Sub lethal toxicity tested in the laboratory using complete random design for 28 days. The result showed that LC50 -96 hours is 22,492 mg/L. It is shown that extract of tobacco leaves has a significant influence to the fish growth. The highest absolute biomass growth is at treatment K (3,72 g), followed by treatment A (3,29 g), B (2,96 g), C (2,75 g), and D (2,24 g). Whereas the highest daily growth rate is also at treatment K (3,93 %), followed by A (3,75 %), B (3,59 %), C (3,45 %), and D (3,06 %). The inhibiting concentration (IC 50) is 15,733 mg/L. Water quality during the experiment indicated a suitable condition for fish life medium. Key Words : Extract of tobacco leaves, Tilapia fry, Growth

Honeybees and nectar nicotine: Deterrence and reduced survival versus potential health benefits

Secondary metabolites produced by plants for herbivore defence are often found in floral nectar, but their effect on the foraging behaviour and physiological performance of pollinators is largely unknown. Nicotine is highly toxic to most herbivores, and nicotine-based insecticides may contribute to current pollinator declines. We examined the effects of nectar nicotine on honeybee foraging choices and worker longevity. Free-flying honeybee (Apis mellifera scutellata) workers from six colonies were given a choice between multiple nicotine concentrations (0–1000μM) in artificial nectar (0.15–0.63M sucrose). The dose-dependent deterrent effect of nicotine was stronger in lower sugar concentrations, but even the highest nicotine concentrations did not completely repel honeybees, i.e. bees did not stop feeding on these diets. Nicotine in nectar acts as a partial repellent, which may keep pollinators moving between plants and enhance cross-pollination. In the second part of the study, newly emerged workers from 12 colonies were caged and fed one of four nicotine concentrations (0–300μM) in 0.63M sucrose for 21days. Moderate (⩽30μM) nicotine concentrations had no significant detrimental effect, but high nicotine concentrations reduced the survival of caged workers and their nectar storage in the honey comb. In contrast, worker groups that survived poorly on sugar-only diets demonstrated increased survival on all nicotine diets. In the absence of alternative nectar sources, honeybees tolerate naturally occurring nectar nicotine concentrations; and low concentrations can even be beneficial to honeybees. However, high nicotine concentrations may have a detrimental effect on colony fitness.