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Introduction: Snus is a low-nitrosamine smokeless product that appears to be safer than other smokeless products. Evidence indicates that snus has been used as an effective smoking cessation aid in Scandinavia. No randomized controlled trial has directly tested the efficacy of snus for smoking cessation., Methods: This randomized, double-blind, placebo-controlled multicentre trial tested the efficacy of snus for smoking cessation. Of the 250 subjects, 125 were randomized to active or placebo snus sachets. Subjects were followed up through 28 weeks after randomization. In total, 5 clinical visits and 8 telephone contacts were scheduled. Primary outcome measure was biologically verified continuous smoking abstinence from Week 6 through 28., Results: The continuous abstinence rate during Weeks 6-28 in the snus and placebo groups was 4.0% and 1.6% (odds ratio [OR]: 2.5, 95% CI: 0.4-27), respectively. The point prevalence abstinence rate at 6 weeks was 18.4% in the snus group versus 8.8% in the placebo group (OR: 2.3, 95% CI: 1.1-5.0, p = .03). At Week 28, the difference in favor of the snus group was not statistically significant (12.8% vs. 7.2%, OR: 1.9, 95% CI: 0.8-4.4). Snus was generally well tolerated. Treatment-related adverse events that were more common in the snus group were generally mild and included nausea, dyspepsia, gingivitis, hiccups, and dizziness., Conclusions: Although the cessation rates generally were low and, at 28 weeks, did not differ between active and placebo, early quit rates suggested that snus was superior and with similar effect sizes to those with nicotine replacement. These results suggest that snus needs to be further researched as a smoking cessation treatment.

Use of smokeless tobacco (ST) (chewing tobacco and snuff) can lead to a number of consequences detrimental to health. ST rapidly delivers high doses of nicotine, which can lead to dependence and is also a source of carcinogenic nitrosamines. Changes usually develop in the mouth area where the ST is most often placed. Non-malignant oral lesions include leuko-oedema, hyperkeratotic lesions of the oral mucosa and localised periodontal disease. Oral premalignant lesions are leukoplakia, erythroplakia, submucosal fibrosis and lichen planus. Betel chewing, with or without tobacco, may increase the incidence of oral cancer. There is conflicting evidence with regard to snuff users about the risk of oral and gastro-oesophageal cancer. ST use is a risk factor for pancreatic cancer and may increase the risk of fatal myocardial infarction and ischemic stroke. During pregnancy, ST is associated with an increase in pre-eclampsia, preterm delivery and stillbirth. Nicotine replacement therapy and bupropion reduce withdrawal symptoms and tobacco craving during ST cessation. However, they have not been shown to help long-term abstinence. Information concerning the potential hazards of ST products should be incorporated into educational programmes to discourage its use and to help users to quit. Smokeless tobacco is not recommended to help smoking cessation., (Copyright © 2011 SPLF. Published by Elsevier Masson SAS. All rights reserved.)

Unlabelled: The adverse health effects of cigarette smoking are well established including the increased risk of various types of cancer. In this study, the direct effects of ethanol, pure nicotine, cigarette smoke extract and Swedish type smokeless tobacco (Snus) extract on normal cells were investigated., Materials and Methods: Primary normal adult human endothelial cells and fibroblasts at early passage were used. Upon exposure to pure nicotine, cigarette smoke extract, Snus extract and ethanol, these cells were assessed for DNA synthesis, gene expression profile and cellular morphology., Results: Normal human fibroblasts and endothelial cells have unique gene expression profiles. The effects of treatment with ethanol and nicotine from different sources was more prominent in endothelial cells than fibroblasts. The combination of alterated gene expressions and strongly inhibited DNA synthesis was only detected in cells exposed to smoke extract. In the presence and absence of ethanol, pure nicotine and Snus extract induced abnormalities in the cytoplasm without any significant degree of cell death. With similar doses of nicotine and ethanol, the additional components in smoke extract had a dominant effect. The smoke extract induced vast cellular abnormalities and massive cell death., Conclusion: Cigarette smoke induced massive cell death and various abnormalities at cellular and molecular levels in surviving endothelial cells and fibroblasts. The combination of genomic alterations and the chronic inflammatory microenvironment induced from massive cell death, will potentially promote tumourigenesis and various diseases in cigarette smokers.

Understanding the molecular pathways perturbed in smokeless tobacco- (ST) associated head and neck squamous cell carcinoma (HNSCC) is critical for identifying novel complementary agents for effective disease management. Activation of nuclear factor-kappaB (NF-κB) and cyclooxygenase-2 (COX-2) was reported in ST-associated HNSCC by us [Sawhney,M. et al. (2007) Expression of NF-kappaB parallels COX-2 expression in oral precancer and cancer: association with smokeless tobacco. Int. J. Cancer, 120, 2545-2556]. In search of novel agents for treatment of HNSCC, we investigated the potential of guggulsterone (GS), (4,17(20)-pregnadiene-3,16-dione), a biosafe nutraceutical, in inhibiting ST- and nicotine-induced activation of NF-κB and signal transducer and activator of transcription (STAT) 3 pathways in HNSCC cells. GS inhibited the activation of NF-κB and STAT3 proteins in head and neck cancer cells. This inhibition of NF-κB by GS resulted from decreased phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha the inhibitory subunit of NF-κB. Importantly, treatment of HNSCC cells with GS abrogated both ST- and nicotine-induced nuclear activation of NF-κB and pSTAT3 proteins and their downstream targets COX-2 and vascular endothelial growth factor. Furthermore, GS treatment decreased the levels of ST- and nicotine-induced secreted interleukin-6 in culture media of HNSCC cells. In conclusion, our findings demonstrated that GS treatment abrogates the effects of ST and nicotine on activation of NF-κB and STAT3 pathways in HNSCC cells that contribute to inflammatory and angiogenic responses as well as its progression and metastasis. These findings provide a biologic rationale for further clinical investigation of GS as an effective complementary agent for inhibiting ST-induced head and neck cancer.

Background: Epidemiological association of head and neck cancer with smokeless tobacco (ST) emphasizes the need to unravel the molecular mechanisms implicated in cancer development, and identify pharmacologically safe agents for early intervention and prevention of disease recurrence. Guggulsterone (GS), a biosafe nutraceutical, inhibits the PI3K/Akt pathway that plays a critical role in HNSCC development. However, the potential of GS to suppress ST and nicotine (major component of ST) induced HNSCC remains unexplored. We hypothesized GS can abrogate the effects of ST and nicotine on apoptosis in HNSCC cells, in part by activation of PI3K/Akt pathway and its downstream targets, Bax and Bad., Methods and Results: Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins--Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway. ST and nicotine treatment also resulted in induction of Bad and Bax phosphorylation, increased the association of Bad with 14-3-3ζresulting in its sequestration in the cytoplasm of head and neck cancer cells, thus blocking its pro-apoptotic function. Notably, GS pre-treatment inhibited ST/nicotine induced activation of PI3K/Akt pathway, and inhibited the Akt mediated phosphorylation of Bax and Bad., Conclusions: In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST/nicotine on PI3K/Akt pathway in head and neck cancer cells. These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST/nicotine associated head and neck cancer.

Objective: Male fertility is affected by a variety of lifestyle habits that include tobacco use. A large population of Indian men is addicted to tobacco chewing. The objective of our study was to assess the relationship between tobacco chewing in these Indian men--who were part of an infertile couple--and their sperm characteristics., Design: Retrospective study., Setting: Private infertility clinic., Patient(s): Six hundred thirty-eight male patients undergoing infertility evaluations were grouped according to the frequency of their tobacco chewing habit: mild (< 3 times/day, n = 177), moderate (3-6 times/day, n = 264), and severe (> 6 times/day, n = 197)., Intervention(s): None., Main Outcome Measure(s): Sperm characteristics (concentration, motility, morphology, and viability)., Result(s): Sperm concentration, percentage motility, morphology, and percentage viability were significantly higher in the mild group vs. the moderate group and in the moderate group vs. the severe group. The percentage of men with azoospermia rose with the level of addiction (1%, 3%, and 14%) as did the percentage of men with oligoasthenoteratozoospermia (2%, 8%, and 29%), although the differences were not statistically significant., Conclusion(s): In our study, use of chewing tobacco by a group of Indian men who were undergoing infertility evaluation was strongly associated with a decrease in sperm quality and to a lesser extent with oligoasthenozoospermia or azoospermia. Infertile men should be counseled about the adverse effects of tobacco chewing on sperm quality.

Purpose: The inhalation of nasal snuff (powdered tobacco) is a common addiction in the Indian subcontinent. In the western world, there is a resurgence of interest in nasal snuff because it does have the morbidity associated with smoked tobacco. Very few studies have reported the long-term effects of snuff on nasal mucosa. The objective of the present study was to investigate the effect of long-term use of snuff on the nasal mucosa., Materials and Methods: We conducted a retrospective study on 29 snuff users. We investigated the reasons for initiation of this particular form of addiction along with the clinical signs and symptoms of long-term snuff usage. At the time of the study, all patients complained of one or more nasal symptoms. Nasal obstruction and nasal discharge taken together were reported by 62.5% of patients. Gross mucosal edema of the septum and turbinates was the main finding on nasal examination. The absolute eosinophil count and total serum immunoglobulin E were elevated in 62.5% and 66.7% of patients, respectively. On skin prick test, 41% of patients reacted positively to snuff and 25% to tobacco. Histopathologic examination of the turbinates (16 patients) showed squamous metaplasia, capillary proliferation, capillary and venous dilatation, inflammatory cell reaction, subepithelial edema, and fibrosis., Conclusions: Much has been written about the advantages of nasal snuff over products that deliver tobacco smoke. Our study shows that snuff users, after long-term abuse, develop a form of chronic rhinitis, as a consequence of which they develop blocked and stuffy noses. We conclude that nasal snuff is not a suitable substitute for smoked tobacco because it does not avoid ill health.

Smokeless tobacco (ST) is widely used as chewing tobacco and as oral snuff in the world. Also, in Kahramanmaraş, a city in Southern Turkey, ST used as 'oral powder' or 'Maraş Powder' is consumed widely instead of cigarette smoking. The aim of this study was to search the effect of ST use on serum total sialic acid (TSA) and to compare the serum TSA levels in smokers and ST users. The study was performed at the Department of Chemistry-Biochemistry, University of Kahramamaraş Sutcu Imam, Turkey, in 2003. Serum samples obtained from smokers (Group I), Maraş powder users (Group II), and healthy control subjects (Group III) who were nonsmokers and nonusers ST. Individuals who were smokers and ST users were classified into subgroups with respect to amount of consumed cigarette or oral powder. Serum TSA was measured with the Denny's colorimetric method. The TSA concentrations were significantly higher in the sera of smokers (p < 0.001) and Maraş powder users (p < 0.001) than those of control subjects. The mean serum TSA level was found to be lowest in the control group and highest in the Maraş Powder users. But, there was no significant difference in serum TSA levels between smokers and Maraş powder users (p > 0.05). We can conclude from the results obtained that serum TSA was affected by ST use as seen in smokers. This finding may be an indication of harmful effects of ST use as Maraş powder as well as cigarette smoking.

In Turkey, "Maras Powder," which is a kind of powder yielded from the shields of tobacco, is widely used as smokeless tobacco through buccal mucosa or together with cigarette. Maras powder, composed of ash and a plant named Nicotiana Rustica Linn, is sometimes used to give up smoking. The present study was aimed to investigate the effects of Maras powder use on respiratory functions of healthy subjects who do not have any chronic disease. We found statistically significant differences in percentage of forced expiratory volume in the first second (FEV(1)%) (p = 0.001), the ratio of FEV(1) to forced vital capacity (FEV(1)/FVC) (p = 0.024), percent of maximum expiratory flow rate (FEF(25-75)%) (p = 0.002) and percent of peak expiratory flow (PEF%) (p = 0.037) between cigarette smokers with Maras powder use (n = 23) and control subjects (n = 24). Likewise, when cigarette smokers (n = 24) and control subjects were compared, the differences for all these parameters were significant (p = 0.022, p = 0.048, p = 0.011 and p = 0.047, respectively). Only FEV(1)% and FEF(25-75)% were significantly lower in cigarette smokers with Maras powder use than in Maras powder users (n = 28) (p = 0.011 and p = 0.022, respectively). There was a negative correlation between forced vital capacity and Maras powder use (r = -0.315, p = 0.03). The present study suggests that Maras powder does not cause serious bronchial obstruction. This may be due to usage of the smokeless tobacco through buccal mucosa but not through inhalation as in case of cigarette smoking.

Maras Powder (MP) is a special kind of smokeless tobacco widely used in the southeast region of Turkey especially in Kahramanmaras and Gaziantep and other southeastern cities. It is obtained from a tobacco species, Nicotiana rustica L and ash of oak or grapevine wood. Tobacco may increase oxidative stress, which is related to the products of the oxygen metabolism taking place in all cells. Cellular antioxidants, e.g. catalase (CAT), superoxide dismutase (SOD) and glucose 6-phosphate dehydrogenase (G6PD) protect the cell against oxidative damage. An imbalance between the ROS and antioxidants in favour of ROS is described as oxidative stress. In this study, we aimed to investigate the effect of MP on antioxidant enzyme levels and lipid peroxidation. We measured malondialdehyde (MDA), CAT, SOD and G6PD levels in blood of 68 MP users and 30 healthy controls who did not use MP. CAT, SOD and G6PD levels were lower in MP users than in the controls. On the other hand, lipid peroxidation levels (MDA), one of the best indicators of cytological damage, was increased in MP users compared with the controls. The present study showed that MP increases oxidative stress, which may cause many systemic disorders, including arteriosclerosis., (Copyright 2004 John Wiley & Sons, Ltd.)

Snuff is a locally irritative agent causing hyperkeratinization and hyperplasia of the oral epithelium. The aim of the study was to investigate the effects of snuff on epithelial cell growth and differentiation in vitro. Three-dimensional HaCaT cell cultures were grown for 6, 12, 14, and 18 days in the presence of 1% snuff extract. Ki-67, p53 and cytokeratins (Cks) 5, 13, 10, 19, 18, involucrin and filaggrin were studied by means of immunohistochemistry. Ki-67 indices were assessed, and the results analyzed statistically. Marked morphologic changes were seen with advanced culture time in the snuff group, probably as a result of increased toxic effects. Snuff exposure decreased the percentage of Ki-67 positive cells on days 6, 12, and 14, suggesting that snuff does not stimulate proliferation activity in this in vitro model. Cornification-related Ck 10 decreased after snuff exposure, indicating disturbances in the epithelial differentiation process.

Antigen-presenting cells (APC) provide two essential signals, e.g., antigenic peptides as well as costimulatory molecules for T-cell activation. Small molecules of smoke tobacco extracts (SM-STE) inhibited antigen presentation of A20 to OVAp-specific T-cell hybridomas. Pretreatment of A20 but not T hybridomas abrogates the APC function. Viability of APC and levels of MHCII, CD40 and B7 of APC were not affected by this treatment. The active principle, inhibiting APC was reproduced with pure tobacco polyphenols, quercetin and its glycoside, rutin. Antioxidant activity of rutin is relevant since rutin downregulated levels of reactive oxygen species (ROS) in phorbol ester-stimulated A20; moreover, another antioxidant, N-acetyl cysteine (NAC) also inhibited antigen presentation, albeit at a higher concentration. Other types of APC, such as bone marrow-derived mast cells (BMMC), MHCII-transfected fibroblast, and splenocytes are affected by tobacco polyphenols. We propose that polyphenols may affect redox-sensitive signal transduction pathway since APC function of PD 98059, MEK inhibitor-pretreated A20 were similarly abrogated. Taken together, we propose that maintaining appropriate intracellular redox of APC is crucial for its antigen-presenting function.

With cigarette smoking declining in the modern world, the tobacco industry has to look for other products that can keep the old customers and attract new ones. Different forms of smokeless tobacco are currently massively promoted and are gaining in importance. Dry nasal snuff--the oldest known form of tobacco in Europe--is one of them. The health risks associated with it are different to those attributed to smoking and oral wet snuff. The nicotine contained leads to dependency. Its resorption rate is similar to that of smoking, so it could be seen as an adequate substitutional therapy. The risk for cardiovascular diseases is lower, compared to that for smokers. Chronic abuse leads to morphological and functional changes in the nasal mucosa. Although it contains substances that are potentially carcinogenic, at present, there is no firm evidence, relating the use of nasal snuff to a higher incidence of head and neck or other malignancies.

Background: Tobacco exposure reduction may be an alternative treatment approach for those tobacco users who are unwilling or unable to quit tobacco use. However, very little information is available on the feasibility of this type of intervention, especially in the area of oral moist snuff tobacco (ST). This pilot study examined whether reducing ST use using various methods can be achieved and whether this reduction results in lower exposure to carcinogens., Methods: Moist snuff users (N=40 males) were randomly assigned to 4 mg nicotine gum, non-tobacco mint snuff, brand switching, or elimination of ST use in specific situations. These approaches were used to reduce ST use or nicotine exposure by at least 25% for the first 2 weeks and 50% the subsequent 6 weeks of treatment. Follow-up sessions occurred at 12 and 26 weeks., Results: Significant reductions were observed in tins per week and cotinine levels across all conditions. Among the intent-to-treat population, the abstinence rate was 15% at 26 weeks. Reduction in nicotine exposure was associated with reduction in exposure to nitrosamines., Conclusion: Reduction in ST use may be a viable approach for those oral moist ST users with no immediate quit plans. Future research in this area is needed.

Objectives: To compare the morphological changes and quantitative distribution of mitochondria in the hamster cheek pouch (HCP) epithelium treated with smokeless tobacco (ST)., Materials and Methods: Archives of experimental material from previously published studies (Ashrafi et al., 1992) were utilized. Animals in experimental group received moist ST (snuff) in their right pouch, 5 days weekly for 24 months, while no snuff was given to control group. After 24 months, the epithelial tissues were processed for electron microscopy study. Volume densities of mitochondria were assessed by morphometry., Main Outcome Measures: Mitochondrial volume densities in the two groups, experimental vs control., Results: In both control and experimental groups mitochondria were concentrated between the nucleus and basal cell plasma membrane. A decrease in the mean mitochondrial volume density (Vvmit) was observed from the basal layer to the more superficial layers in both groups. The experimental HCP displayed more mitochondria than control, and the granular epithelial cell layer in experimental group showed significantly a higher mean Vvmit than the control group (P = 0.03). It was concluded that greater numbers of mitochondria were retained in ST-treated granular cells of the hyperplastic epithelia than in the normal epithelium.

Current understanding of the preparation and use of yopo, a hallucinogenic snuff made from the ground seeds of the Anadenanthera peregrina tree, has departed little from the accounts of scientists and travelers made over a century ago. Schultes and others have made refinements to these early accounts. While several scholars have drawn attention to the fact that little ethnographic work has been conducted to assess the ethnobotanical diversity and cultural framework of the snuff hallucinogen complex, few subsequent studies deal with botanical variations in preparation and use. This article contrasts historical accounts of yopo preparation with ethnographic data I have recently collected among the Piaroa of southern Venezuela to demonstrate one way in which yopo preparation and use deviates from the basic model established by Humboldt, Spruce and Safford. Piaroa shamans include B. caapi cuttings in the preparation of yopo and consume doses of B. caapi prior to snuff inhalation concomitant with the strength of visions desired for particular tasks. I argue that the combined use of yopo and B. caapi by Piaroa shamans is pharmacologically and ethnobotanically significant, and substantiates claims of the use of admixtures in snuff; further ethnographic investigation of the snuff hallucinogen complex is necessary.

The present study investigated the effects of two herbal components (BACCOFF and DIPSTOP) of a commercially available smokeless tobacco treatment program for reducing subjective withdrawal symptoms during deprivation. One component, BACCOFF, is a non-nicotinic chew. The second component, DIPSTOP, is a liquid containing the alkaloid lobeline, which to some extent mimics peripheral nicotinic effects. All participants (N = 22 males) were placed in four conditions: BACCOFF + DIPSTOP, BACCOFF + placebo control, DIPSTOP, and placebo control only. The conditions involved 48 h of deprivation, and subjects were exposed to one condition per week for 4 weeks. Withdrawal measures were taken at baseline, 24 h, and 48 h of deprivation. Individuals were randomly assigned, and conditions were counterbalanced. Results showed that BACCOFF, as compared with DIPSTOP, significantly reduced withdrawal symptoms but not craving. These data suggest that behavioral/sensory substitutes' influence on withdrawal might be routed through the product's ability to approximate the preferred moist snuff.

Modulation of IFN-gamma production from T cells by smokeless tobacco extract (STE) could be a factor in periodontal disease. The major inducer of IFN-gamma from T cells is bioactive IL-12 (p70), a heterodimeric protein composed of p35 and p40 subunits, while homodimeric IL-12 p40 antagonizes bioactive IL-12. Both p70 and p40 are produced by macrophages in response to lipopolysaccharide (LPS), IFN-gamma and/or CD40 ligation. To determine the impact of STE on IL-12 p40, p70 and IFN-gamma, splenic T cells were stimulated with anti-CD3 while splenic macrophages were stimulated with LPS in the presence or absence of STE. Production of IL-12 p40 and p70 from LPS-stimulated splenic macrophages and IL-12 p40, p70 and IFN-gamma from LPS/anti-CD3-stimulated T cells and macrophages was decreased by STE. To determine the impact of STE on macrophage IL-12 production alone, splenic or peritoneal macrophages were enriched and then stimulated. STE significantly diminished production of IL-12 p40 and p70 from LPS-stimulated peritoneal macrophages, LPS/IFN-gamma-stimulated peritoneal and splenic macrophages, but increased production of IL-12 p40 and p70 from IFN-gamma/CD40-stimulated splenic macrophages or IFN-gamma-stimulated peritoneal macrophages. None of the effects of STE on IL-12 was due to nicotine, rutin or chlorogenic acid. In contrast to STE, nicotine at 100 microg/ml significantly elevated production of IL-12 p40 and p70 from splenic macrophages stimulate by IFN-gamma/LPS. The results indicate that STE has a significant overall effect upon IL-12 production. It suppresses p40 and p70 production during responses to LPS or LPS/IFN-gamma but augments p40 and p70 production during responses to IFN-gamma without LPS. This affect could have a major impact on diseases associated with excessive production of IL-12.

The oral use of chewing tobacco has greatly increased in recent years, and this usage is associated with cancers of the mouth, lip, nasal cavities, esophagus and gut. Oral cancer accounts for 3% of all cancers in U.S.A. and is the seventh most common cancer. Previous studies in our laboratory have demonstrated the protective abilities of a novel IH636 grape seed proanthocyanidin extract (GSPE) against reactive oxygen species both in vitro and in vivo models, and provided significantly better protection as compared to vitamins C, E and beta-carotene. In the recent past, we have demonstrated smokeless tobacco (STE)-induced oxidative stress, apoptotic cell death in a primary culture of normal human oral keratinocytes (NHOK), and have compared the protective abilities of vitamins C and E, singly and in combination, and GSPE in this pathobiology [Free Rad. Biol. Med., 26, 992-1000 (1999)]. In the present study, we have assessed the protective role of vitamins C and E, and GSPE against STE-induced modulation of intracellular oxidized states in NHOK cells as demonstrated by laser scanning confocal microscopy. Approximately 11%, 26%, 28% and 50% protection were observed following incubation with vitamin C, vitamin E, a combination of vitamins C plus E, and GSPE, respectively. DNA fragmentation was assessed as an index of oxidative DNA damage and similar results were observed. Furthermore, the cellular viability and functional roles of Bcl-2, p53 and c-myc genes were assessed in STE-induced oxidative stress in NHOK cells. NHOK cells were treated with STE (0-200 micrograms/ml) for 24 h and changes in the expression of Bcl-2, p53 and c-myc genes were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the protective effect of GSPE was assessed. Approximately a 2.0-fold increase in p53 gene expression was observed following incubation of the oral keratinocytes with 100 micrograms/ml of STE, beyond which the expression of p53 decreased, confirming increased apoptotic cell death with a higher concentration of STE as reported earlier. GSPE significantly modulated STE-induced changes in p53. The expression of antiapoptotic Bcl-2 gene decreased with STE treatment and the expression of Bcl-2 gene increased significantly following preincubation with GSPE. No significant change in the expression of transcription factor c-myc gene responsible for cell cycle growth was observed following incubation with STE and/or GSPE. Thus, c-myc may not be involved in STE-induced cytotoxicity towards NHOK cells. These results suggest that antioxidant protection of STE-induced cellular injury is associated with alterations in Bcl-2 and p53 expression.

Habitual use of smokeless tobacco leads to accumulation of inflammatory leukocytes at the site of placement, which may contribute to tissue damage. Recruitment of leukocytes is facilitated by the endothelial lining of blood vessels, which can be activated to express adhesion molecules and to produce chemoattractants. The ability of aqueous extracts of chewing tobacco, dry snuff, and moist snuff to stimulate such changes was investigated using cultured human umbilical vein endothelial cells (HUVEC). All three extracts caused HUVEC to express the adhesion molecule E-selectin and to produce the chemokines interleukin-8 and monocyte chemoattractant protein-1. Neutrophils migrated avidly across HUVEC monolayers that had been previously exposed to the extracts, whereas migration across unstimulated monolayers was negligible. The smokeless tobacco extracts contained relatively high concentrations of bacterial lipopolysaccharide (LPS). Although LPS appeared to be the major stimulatory component in extracts of chewing tobacco, it accounted for only part of the activity found in extracts of moist and dry snuffs. These observations suggest that smokeless tobacco may induce inflammatory changes in vivo by activating endothelium in a manner that promotes recruitment of leukocytes.

The purpose of this study was to determine whether dexamethasone attenuates the acute increase in macromolecular efflux from the oral mucosa elicited by an aqueous extract of smokeless tobacco (STE) in vivo, and, if so, whether this response is specific. Using intravital microscopy, we found that 20-min suffusion of STE elicited significant, concentration-related leaky site formation and an increase in clearance of fluorescein isothiocyanate-labeled dextran (FITC-dextran; mol mass 70 kDa) from the in situ hamster cheek pouch (P < 0.05). This response was significantly attenuated by dexamethasone (10 mg/kg iv). Dexamethasone also attenuated the bradykinin-induced leaky site formation and the increase in clearance of FITC-dextran from the cheek pouch. However, it had no significant effects on adenosine-induced responses. Dexamethasone had no significant effects on baseline arteriolar diameter and on bradykinin-induced vasodilation in the cheek pouch. Collectively, these data indicate that dexamethasone attenuates, in a specific fashion, the acute increase in macromolecular efflux from the in situ oral mucosa evoked by short-term suffusion of STE. We suggest that corticosteroids mitigate acute oral mucosa inflammation elicited by smokeless tobacco.

We have investigated the effects of a smokeless tobacco extract (STE) on lipid peroxidation, cytochrome c reduction, DNA fragmentation and apoptotic cell death in normal human oral keratinocyte cells, and assessed the protective abilities of selected antioxidants. The cells, isolated and cultured from human oral tissues, were treated with STE (0-300 microl;g/ml) for 24 h. Superoxide anion production was determined by cytochrome c reductase. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, whereas apoptotic cell death was assessed by flow cytometry. STE-induced fragmentation of genomic DNA was also determined by gel electrophoresis. The comparative protective abilities of vitamin C (75 microM), vitamin E (75 microM), a combination of vitamins C & E (75 microM each), and a novel grape seed proanthocyanidin (IH636) extract (GSPE) (100 microg/ml) against STE induced oxidative stress and tissue damage were also determined. Following treatment of the cells with 300 microg STE/ml 1.5-7.6-fold increases in lipid peroxidation, cytochrome c reduction and DNA fragmentation were observed. The addition of the antioxidants to cells treated with STE provided 10-54% decreases in these parameters. Approximately 9, 29, and 35% increases in apoptotic cell death were observed following treatment with 100, 200, and 300 microg STE/ml, respectively, and 51-85% decreases in apoptotic cell death were observed with the antioxidants. The results demonstrate that STE produces oxidative tissue damage and apoptosis, which can be attenuated by antioxidants including vitamin C, vitamin E, a combination of vitamins C plus E and GSPE. GSPE exhibited better protection against STE than vitamins C and E, singly and in combination.

The use of smokeless tobacco (moist snuff) products is associated with mucosal lesions, gingival recession, attachment loss, and oral cancer. Despite numerous reports on the general toxic effects of smokeless tobacco extract, little information is available regarding the specific effects of smokeless tobacco on immune response. Inflammatory cytokines released as a result of smokeless tobacco-induced irritation may play a role in the development of oral mucosal lesions at habitual tobacco placement sites in smokeless tobacco users. Consequently, the purpose of this study was to determine whether an aqueous extract of smokeless tobacco (STE) affects the secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and the proliferation of lymphocytes. A macrophage cell line (J774-A1) was used to measure the effects of STE on tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) secretion. Mouse splenocytes were used to assess the effects of STE on lymphocyte proliferation. We found that STE at low concentrations enhanced the production of both TNF-alpha and IL-1beta. Furthermore, STE at similar concentrations enhanced mitogen-induced murine splenocyte proliferation. Overall, these data suggest that smokeless tobacco upregulated two key proinflammatory cytokines and also induces lymphocyte proliferation.

The purpose of this study was to determine whether supernatants of cultured human oral keratinocytes (HOK) exposed to an aqueous extract of smokeless tobacco (STE) increase macromolecular efflux from the oral mucosa in vivo and, if so, whether bradykinin mediates in part this response. Subconfluent monolayers of HOK were incubated with STE or media, and supernatants were collected 24, 48, and 72 h thereafter. Using intravital microscopy, we found that suffusion of supernatants of STE- but not media-exposed HOK elicited significant concentration- and time-dependent increases in efflux of fluorescein isothiocyanate-labeled dextran (mol mass 70 kDa) from the in situ hamster cheek pouch (P < 0.05). These effects were significantly attenuated by HOE-140 and NPC-17647 but not by des-Arg9, [Leu8]-bradykinin. Proteolytic activity was increased in supernatants of STE- but not media-exposed HOK. However, a mixture of leupeptin, Bestatin, and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid had no significant effects on HOK supernatant-induced responses. Collectively, these data suggest that oral keratinocytes modulate smokeless tobacco-induced increase in macromolecular efflux from the in situ oral mucosa in part by elaborating proteases that may account for local bradykinin production.

Fifteen male users of oral snuff participated in an experiment where we used an auditory-visual vigilance task to study nicotine effects on P300 and response parameters. Quantitative EEG was also studied. Fifteen male nonusers served as controls. We found some decrease of response times and a tendency towards improved signal detection. P300 parameters were not affected in this study. Quantitative EEG analysis indicated an expected increase of arousal, as the activity within the alpha band shifted towards higher frequencies.

A surgically created canal was used as a reservoir for instillation of snuff into the lower lip of rats for 10 weeks. DNA was isolated and digested with micrococcal nuclease/spleen phosphodiesterase. Polar DNA adducts were detected in all tissues examined when digests were fractionated by high performance liquid chromatography (HPLC), 32P postlabeled, 3' dephosphorylated and analyzed by two-dimensional thin layer chromatography (TLC). DNA adducts derived from aromatic carcinogens were not detected when digests were 32P postlabeled and analyzed by four-dimensional thin layer chromatography. These results suggest that non-aromatic agents initiate carcinogenesis following exposure to snuff. The adduction to DNA in organs of the gastrointestinal tract and the kidneys indicates that snuff usage results in systemic exposure to carcinogens and may contribute to the incidence of neoplasms in organs outside the oral cavity.

Smokeless tobacco elicits plasma exudation from the oral mucosa that is mediated by bradykinin, and it decreases the activity of tissue angiotensin-converting enzyme (ACE), a peptidase that cleaves and inactivates bradykinin. However, the mechanisms regulating bradykinin-induced responses during exposure to smokeless tobacco are uncertain. The purpose of this study was to begin to address this issue by determining whether inhibitors of ACE and neutral endopeptidase (NEP), a membrane-bound peptidase widely distributed in the oral mucosa that also cleaves and inactivates bradykinin, potentiate a smokeless tobacco-induced increase in macromolecular efflux from the oral mucosa in vivo. Using intravital microscopy, we found that suffusion of an aqueous extract of smokeless tobacco elicited a significant concentration-dependent increase in fluorescein isothiocyanate-labeled dextran (molecular mass 70 kd) leaky site formation in the hamster cheek pouch (p < 0.05). This response was significantly potentiated by captopril and lisinopril, two ACE inhibitors, and by phosphoramidon and thiorphan, two NEP inhibitors (p < 0.05). The effects of ACE and NEP inhibitors were additive. By contrast, a mixture of proteinase inhibitors consisting of leupeptin, Bestatin, and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid had no significant effects on smokeless tobacco extract-induced responses. Overall, these data suggest that ACE and NEP each play a role in modulating a smokeless tobacco-induced increase in macromolecular efflux from the in situ oral mucosa, in part by regulating local bradykinin catabolism.

The purpose of this study was to determine whether an aqueous extract of smokeless tobacco (moist snuff) increases clearance of macromolecules from postcapillary venules in the in situ oral mucosa and, if so, whether bradykinin mediated this response. Using intravital microscopy, we found that 20-min suffusion of the extract elicited significant concentration-dependent leaky site formation and increase in clearance of FITC-dextran (molecular mass, 70 kDa) from the hamster cheek pouch (p < 0.05). These responses were associated with a significant increase in bradykinin-like immunoreactivity in the suffusate. Smokeless tobacco extract-induced leaky site formation and increase in clearance of FITC-dextran were significantly attenuated by NPC 17647 and Hoe 140 (p < 0.05), two bradykinin B2 receptor antagonists, but not by desArg9,[Leu8]bradykinin, a bradykinin B1 receptor antagonist. Both bradykinin B2 receptor antagonists had no significant effects on adenosine-induced responses. Indomethacin had no significant effects on smokeless tobacco extract-induced responses. Exposure to smokeless tobacco extract was associated with a significant decrease in angiotensin I-converting enzyme activity and a small, but significant, increase in neutral endopeptidase 24.11 activity in the cheek pouch, two peptidases widely distributed in the microcirculation that cleave and inactivate bradykinin (p < 0.05). Overall, these data suggest that smokeless tobacco elicits plasma exudation in the oral mucosa in vivo in a specific fashion, and that this response is mediated by bradykinin.

Previously, we reported that secretory component (SC), lactoferrin (LF), and lysozyme (LY) levels were significantly lower in saliva from smokeless tobacco (ST) users than in saliva from control non-tobacco users. However, the levels of salivary immunoglobulin A were significantly higher, albeit with an altered attachment of SC, in ST users than in control subjects. SC, LF, and LY are synthesized by secretory epithelial cells at mucosal sites adjacent to lymphocyte regions. In the present report, HT-29 human epithelial cells, cultured with various concentrations of an ST aqueous extract or pure nicotine (0 to 1 mg/ml) or cotinine (0 to 5 mg/ml), exhibited significantly lower levels of cell-associated cell lysate (CL) and secreted culture supernatant (CS) SC, LF, and LY than cells cultured without ST components. Nicotine significantly decreased (P < or = 0.05) the synthesis of SC by 20 to 100%, LF by 20 to 60%, and LY by 5 to 75% of CL and CS control values. Studies also indicated significant decreases (P < or = 0.05) in SC, LF, and LY levels in both CL and CS of cells cultured with ST aqueous extract or cotinine. Total cell numbers and metabolic activity significantly decreased primarily when cells were incubated with higher concentrations of ST extract, nicotine, or cotinine. The addition of human recombinant interleukin-4 or gamma interferon diminished the effects ST had on HT-29 cell synthesis of SC, LF, and LY. Our data indicate that nicotine, cotinine, and ST have an adverse effect on synthesis and secretion of SC, LF, and LY. These effects were below ST concentrations found to be cytotoxic for secretory epithelial cells. Furthermore, addition of interleukin-4 or gamma interferon reduced the suppressive effect of ST on synthesis or secretion of SC, LF, or LY.

Purpose: The carcinogens in smokeless tobacco have been identified as the tobacco-specific nitrosamines and the effect of one of these, N-Nitrosonornicotine (NNN), on the buccal mucosa of the Syrian hamster was studied., Materials and Methods: Buccal pouches of 36 Syrian hamsters were painted five times per week for 24 weeks with 10 mg/mL 98% pure NNN in suspension with mineral oil. Animals were killed at 6, 8, 12, and 24 weeks., Results: At 6 weeks, the buccal pouch mucosa of the experimental animals appeared clinically more hyperemic than those of the controls. From 12 weeks onward, all experimental animals showed epithelial hyperplasia and inflammation on histologic examination. Three animals killed at 24 weeks showed mild epithelial dysplasia., Conclusions: Exposure of Syrian hamster buccal mucosa to NNN, five times per week for 24 weeks, did not result in clinical or histologic cancerous changes. NNN may require other factors for cancer production, such as a cocarcinogen, a higher concentration, or a longer period of application.

Short-term exposure to smokeless tobacco extracts (STE) reportedly inhibits osteoblast metabolism. The objective of this study was to determine the effects of serial dilutions of a water-soluble extract of smokeless tobacco on osteoblast proliferation and their potential to form and mineralize bone nodules. STE significantly stimulated cell proliferation when diluted 10(2)-10(4) times; 10(3) and 10(4) dilutions produced the greatest effect. 10(2)-10(4) STE dilutions significantly increased alkaline phosphatase activity at day 7 but 10(6) STE dilutions significantly decreased it. 10(3) and 10(4) dilutions significantly increased bone nodule formation, but inhibited their mineralization. In contrast, 10(5) and 10(6) dilutions significantly decreased bone nodule formation, but increased their mineralization. Stimulation of in vitro bone nodule formation by STE was similar to that produced by 10(-7) M insulin-like growth factor 1 (IGF-1) in vivo. Heat and acid treatment of STE significantly reduced its beneficial effect on cell proliferation, suggesting that a peptide within STE may be responsible for enhancement of osteogenic cell proliferation. Thus, STE may contain a peptide capable of significantly stimulating osteoblast proliferation, differentiation and metabolism, similar to the effects of IGF-1. This peptide could have potential therapeutic benefits.

Background: Studies show sustained levels of nicotine among young males using smokeless tobacco, causing concern for subsequent cardiovascular risk. Also, there is little information on effects of nicotine replacement on cardiovascular risk in cessation programs. This study investigates the effects of nicotine gum replacement in smokeless tobacco cessation on cardiovascular risk factors., Methods: Smokeless tobacco users, ages 18-65, were randomly assigned in a double-blind fashion to 2-mg nicotine or placebo gum. At baseline, Week 4, and Week 8, dependent measurements, heart rate, blood pressure, and weight were recorded, and fasting lipoprotein profiles were drawn., Results: This paper focuses on the smokeless tobacco users who refrained from use during the study period (N = 56). The nicotine gum group weighed less (P = 0.033) than the placebo group throughout the study and weight increased at a significant rate between Weeks 4 and 8 for both groups as gum decreased. Triglycerides were higher for the nicotine gum group than the placebo group (P = 0.031), with triglycerides decreasing between Weeks 4 and 8, with a similar effect seen among nonabstinent smokeless tobacco users. There was no dose, time, or dose by time effect for the other dependent measures., Conclusions: Among smokeless tobacco users who were abstinent, weight increased, with subjects on nicotine gum weighing less throughout the study. The lipoprotein profile, heart rate, and blood pressure did not improve over time, contrary to smokers in whom HDL increases and heart rate decreases with cessation. This could relate to different routes of administration, pharmacokinetics, or by-products of tobacco smoking being absent in smokeless tobacco. In addition, nicotine gum appeared to have neither an adverse nor a positive effect on heart rate, blood pressure, LDL, HDL, or total cholesterol.

The possible role of reactive oxygen species in the toxicity of smokeless tobacco (ST) was explored. The effects of an aqueous smokeless tobacco extract (STE) at doses of 125, 250 and 500 mg STE/kg in rats on the induction of hepatic mitochondrial and microsomal lipid peroxidation and the incidence of hepatic nuclear DNA damage 24 hours post treatment were examined. Dose-dependent increases of 1.8, 2.3 and 4.4-fold in mitochondrial and 1.5, 2.1 and 3.6-fold in microsomal lipid peroxidation occurred at 125, 250 and 500 mg STE/kg, respectively, relative to control values. At these same three doses of STE, 1.3, 1.4 and 2.7-fold increases in hepatic DNA single-strand breaks occurred relative to control values. STE administration also resulted in significant increases in excretion of urinary metabolites. Urinary excretion of the four lipid metabolites malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT) and acetone (ACON) was monitored by HPLC for 72 hours after treatment of rats with 125 and 250 mg STE/kg. Increases occurred in the excretion of the four lipid metabolites at every dose and time point with maximum increases in the excretion of all lipid metabolites being observed between 12 and 24 hours post treatment. The results suggest the involvement of an oxidative stress in the toxicity of STE.

Despite numerous studies on the general toxicologic effects of smokeless tobacco (ST) little immunotoxicologic information is available. As a first step in assessing the potential activity of ST on the immune system, the effects of an aqueous extract of ST was studied in in vitro cultures of mouse lymphoid cells. There was a significant increase in the proliferation of spleen cells cultured with different concentrations of ST extract. The polyclonal IgM antibody responses as determined by protein A plaque assay were also elevated in ST stimulated spleen cell cultures. Similar immunostimulatory results were seen in the mesenteric lymph node cell cultures also. ST extract was able to stimulate the spleen cells of the immune defective CBA/N mice. The mitogenic ability of ST extract may not be due to lipopolysaccharide (LPS) contamination as determined by its response in the LPS resistant C3H/HeJ mice spleen cells. ST extract was mitogenic not only to B cells but also to T cells. However the magnitude of response was less in T cells than in B cells. The proliferation of T cells was not accompanied by secretion of IL-2 or expression of IL-2 receptors on T cells. However there was an increase of IL-1 activity in spleen cells cultured with ST extract. Finally, activation of B or T lymphocytes by ST did not result in the elevation of intracellular calcium levels. Since ST is consumed orally, the chronic immunostimulation by ST in oral mucosal lymphoid tissues may be associated with the increased incidence of gingivitis, leukoplakia and oral cancer seen in human ST users.

Little is known about the effects of snuff use on health. We have investigated electrolyte levels, adrenocortical and calcium regulating hormones in three groups of healthy young men, including 18 non-tobacco users, 21 snuff users and 19 smokers with similar age and body mass index. Smoking and snuff use was positively associated with alcohol and coffee consumption and inversely related to physical activity. Compared to non-tobacco users, smokers had significantly increased levels of serum sodium and magnesium, plasma calcitonin, urinary cortisol and potassium levels and decreased serum sex hormone-binding globulin as well as serum and urinary creatinine values. However, only decreased sexual hormone-binding globulin and urinary creatinine and increased serum phosphate and urinary potassium levels were seen in snuff users. Among tobacco users we noted that smokers differed from snuff users in that they had higher serum sodium (1.4 mmol/l, p < 0.01), plasma calcitonin (3.3 pmol/l, p < 0.05) and urinary cortisol (41 nmol/24 h, p < 0.05) but lower serum creatinine (5.8 mumol/l, p < 0.01). We conclude that chronic snuff use appears to have less influence on hormone and electrolyte balance than does smoking, and that some of the abnormalities seen in smokers do not seem to be mediated by nicotine.

The inhalation of nasal snuff (powdered tobacco) is widely prevalent in the Indian sub-continent. The effect of snuff application on the nasal mucociliary clearance (NMMC) time has been suspected but not demonstrated before. In this study the saccharin test is used to demonstrate the rate of nasal mucociliary clearance among chronic snuff users. The study demonstrates the depressant action of snuff on the nasal mucociliary clearance rate. A pharmacological and pathological basis for the depressant action of snuff on the NMMC rate is hypothesised on the basis of the results obtained.

The hemodynamic effects during rest and exercise of oral snuff were investigated in an open, placebo-controlled study of nine habitual users of oral snuff. Blood pressure, heart rate, and central hemodynamics were measured noninvasively. Plasma concentrations of nicotine, cotinine, norepinephrine, and epinephrine, as well as neuropeptide Y-like immunoreactivity were measured before and after snuff intake during rest and exercise. Snuff intake induced a significant increase in heart rate and blood pressure and a decrease in stroke volume during rest. Hemodynamic changes were unrelated to nicotine or cotinine concentrations. Resting levels of norepinephrine and neuropeptide Y-like immunoreactivity were similar with or without snuff, whereas epinephrine was slightly increased 30 minutes after snuff intake. The exercise-induced increase in norepinephrine and neuropeptide Y-like immunoreactivity did not differ between the days subjects received snuff and the days they received placebo. In contrast, maximum work load was associated with a slight increase in circulating epinephrine after snuff intake. The findings suggest that snuff intake is associated with significant hemodynamic effects during rest but not during exercise. These effects could not be readily explained by activation of the sympathetic nervous system.

The effects of smokeless tobacco extract (STE) and various constituents of STE on prolyl hydroxylase activity were determined using enzyme extracted from chick embryos. STE inhibited prolyl hydroxylase activity in a concentration-dependent manner, but nicotine and anabasine had essentially no effect. Enzymatic activity was inhibited by zinc, but not by the other inorganic elements in STE; however, the zinc concentration in STE was not high enough to produce the observed inhibition. The inhibition by STE was diminished by increasing concentrations of 2-oxoglutarate, but not by increasing concentrations of other cofactors. Thus, STE contains an inhibitor of prolyl hydroxylase which may be competitive with 2-oxoglutarate.

The effects of smokeless tobacco (ST) on psychophysiological response and performance were examined in two studies. Study 1 compared heavy and moderate ST users on psychomotor tasks (simple reaction time/anticipation time and choice reaction time/movement time) in ST (2 g amount) and no smokeless tobacco (NST) conditions. Moderate users performed significantly better than heavy users; there were no significant psychomotor performance differences comparing ST/NST conditions. Heart rate and blood pressure were elevated equally for both groups with the use of ST. Heavy users reported significantly less state anxiety in both ST and NST conditions than moderate users. Study 2 replicated the psychomotor performance results of study 1 when comparing nonusers and ST users who were given four dose amounts of ST (NST, 1/3 mean dose, mean dose, and 5/3 mean) over 4 d. For the math and Stroop tasks, ST use resulted in 12.1% and 10.4% better performance than the NST condition. Physiological measures showed significant elevations during the ST conditions. No differences between dose amounts of ST were apparent for any of the measures. These studies demonstrate that ST users perform better than nonusers when having to react to cognitively challenging task situations.

Tobacco products and alcohol are commonly used as nonmedicinal drugs by pregnant women, and both are known to cause various effects on the fetus and the newborn. The objective of this study was to examine the fetal effects of both drugs when administered individually and simultaneously to pregnant CD-1 mice at moderate dosages. Specifically, we wanted to determine whether or not the effect on the fetus of these two biologically active substances was additive, ameliorative, or synergistic. A total of 65 CD-1 dams were divided among four groups receiving either ST equivalent to 8 mg/kg nicotine, ethanol (ETOH) 1.8 g/kg, a combination of ST+ETOH in the same dosages, or D-glucose (controls and ST alone) to supply calories equivalent to the dose of ethanol. Mice were dosed three times per day on gestational days 6-15. On gestational day 17 all dams were killed, fetal and placental weights recorded, and the number of resorbed, dead, and malformed fetuses noted. The mean maternal plasma drug levels were: nicotine-321 ng/ml and ethanol-0.105 g%. No significant differences were observed in maternal weight gain, litter size, or in the incidence of resorptions, deaths and/or malformations. Fetal weights were reduced in all three treatment groups (P less than 0.05), with the greatest reduction (13% decrease) recorded in the ST group, followed by a 9% decrease in the ETOH group, and a 7% decrease in the ST+ETOH group. Placentas of the ST group weighed significantly less (P less than 0.05) than controls. Ossification of the fetal skeleton, observed in ten sites, was affected to the greatest extent in the ST group, followed by the ETOH and ST+ETOH groups. Craniofacial measurements were significantly affected (P less than 0.05) in all three treatment groups, compared to controls. We conclude that under these experimental conditions, in terms of fetal growth and ossification, ST had the greatest effect, followed by ETOH and ST+ETOH. The interaction of ST+ETOH was neither additive, synergistic, nor ameliorative.

The purpose of this investigation was to determine the effects of oral smokeless tobacco (OST) usage on oxygen uptake (VO2), cardiac output (Qc), stroke volume (SV), heart rate (HR), and plasma lactate concentration (Lc) during rest and exercise. Fifteen asymptomatic subjects were recruited from 18 to 33-yr-old male users of OST. Comparisons of the responses of VO2, Qc, SV, HR, and Lc were made between 2.5-g OST and placebo experimental conditions during rest and at 60% and 85% maximal VO2 treadmill exercise. Plasma nicotine concentrations (Nc) were determined by radioimmunoassay. There were significant increases in HR and Lc and a decrease in SV during rest and at 60% and 85% maximal exercise (P less than 0.05). Furthermore, there were no significant differences in maximal HR, Lc, and VO2 (P greater than 0.05). In conclusion, these data indicate that the increased Nc incurred by OST usage increases anaerobic energy production and produces an increased tachycardiac response to a given relative submaximal workload.

The effects of smokeless tobacco (snuff) on hamster cheek mucosa were studied by light microscopy, transmission (TEM) and scanning electron microscopy (SEM). Two grams of commercially available smokeless tobacco were placed into the blind end of the right cheek pouch of each experimental animal, once a day and five days a week for 24 months. The control animals did not receive smokeless tobacco. After 24 months treatment with smokeless tobacco, hamster cheek mucosal epithelium lost its translucency and had become whitish in color. By light microscopy hyperorthokeratosis, prominent granular cell layers with increased keratohyalin granules and hyperplasia were seen. At the ultrastructural level, wider intercellular spaces filled with microvilli, numerous shorter desmosomes, many thin tonofilament bundles, increased number of mitochondria, membrane coating granules and keratohyalin granules were seen in snuff-treated epithelium. The changes in the surface of the epithelium as seen by SEM were the development of an irregular arrangement of the microridges and the disappearance of the normal honeycomb pattern. The microridges were irregular, widened and surrounded the irregular elongated pits. Some smooth areas without microridges and pits were also seen. The long-term histological, TEM and SEM changes induced by smokeless tobacco treatment of the epithelium are well correlated with each other and were similar to those reported in human leukoplakia without dyskeratosis. They imply changes of pathological response resulting from topically applied snuff.

To determine the influence of varying doses of smokeless tobacco (moist snuff) on resting heart rate and blood pressure and the performance of brief, high-intensity exercise, 12 snuff users were examined at rest and while performing Wingate Anaerobic Tests after taking six different doses of snuff. Snuff caused an increase in heart rate; the larger the dose, the larger the response. Increases in systolic and diastolic blood pressure also occurred but were similar at all doses. Performance of brief, high-intensity exercise was unaffected, so that there was no benefit for such activities (e.g., sprinting bases in base ball).

Two studies examining nicotine intake in users of Swedish moist oral snuff are reported. Absorption form a single pinch (2 g) in ten users after overnight abstinence was fairly rapid. The increment in plasma nicotine concentrations averaged 9.9 ng/ml (SD 6.5) after 10 min and peaked at 14.5 ng/ml (SD 4.6) shortly after discarding at 30 min. Among groups of habitual snuff takers (n = 27) and cigarette smokers (n = 35) studied on a day of normal snuffing/smoking, peak blood nicotine levels after use were similar [averaging 36.6 ng/ml (SD 14.4) and 36.7 ng/ml (SD 16.1), respectively], but there was a tendency to higher cotinine levels in the snuffers (399.2 ng/ml versus 306.3 ng/ml). The snuff takers and cigarette smokers reported similar levels of subjective dependence on tobacco. Epidemiological study of Swedish snuff users could clarify whether the cardiovascular risks of tobacco are attributable to nicotine or to other smoke components, as in their case nicotine intake is not accompanied by combustion products.