Smokeless tobacco mortality risks: an analysis of two contemporary nationally representative longitudinal mortality studies.

BACKGROUND: Assessments supporting smokeless tobacco (SLT) disease risk are generally decades old. Newer epidemiological data may more accurately represent the health risks associated with contemporary US-based SLT products, many of which contain lower levels of hazardous and potentially hazardous chemicals compared to previously available SLT products. METHODS: Data from two longitudinal datasets (National Longitudinal Mortality Study-NLMS, and the National Health Interview Survey-NHIS) were analyzed to determine potential associations between SLT use and/or cigarette smoking and all-cause and disease-specific mortality. Mortality hazard ratios (HR) were estimated using a Cox proportional hazards regression model applied to various groups, including never users of any tobacco or SLT product, and current and former SLT users and/or cigarette smokers. RESULTS: The two datasets yielded consistent findings with similar patterns evident for the specific causes of death measured. All-cause mortality risk for exclusive SLT users was significantly lower than that observed for exclusive cigarette smokers and dual SLT/cigarette users. Similar trends were found for mortality from diseases of the heart, chronic lower respiratory diseases, and malignant neoplasms. Mortality risk for lung cancer in exclusive cigarette smokers was increased by about 12-fold over never-tobacco users but was rarely present in exclusive SLT users in either survey (NHIS, < 5 cases/1,563 observations; NLMS, 3 cases/1,863 observations). While the data in the surveys are limited, SLT use by former cigarette smokers was not associated with an increase in the lung cancer risk HR compared to that by former cigarette smokers who never used SLT. CONCLUSIONS: Emerging epidemiological data provides a new perspective on the health risks of SLT use compared to risks associated with cigarette smoking. HR estimates derived from two current US datasets, which include data on contemporary tobacco products, demonstrate a clear mortality risk differential between modern SLT products and cigarettes. Cigarette smokers had an increased overall mortality risk and risk for several disease-specific causes of death, while SLT users consistently had lower mortality risks.

Cigarettes with different nicotine levels affect sensory perception and levels of biomarkers of exposure in adult smokers.

INTRODUCTION: Few clinical studies involving cigarettes have provided a comprehensive picture of smoke exposure, test article characterization, and insights into sensory properties combined. The purpose of these pilot studies was to determine whether cigarettes with different levels of nicotine but similar tar levels would affect sensory experience or smoking behavior so as to significantly alter levels of selected biomarkers of exposure (BOE). METHODS: In 2 confined, double-blind studies, 120 adult smokers switched from Marlboro Gold cigarettes at baseline to either 1 of 2 lower nicotine cigarettes or 1 of 2 higher nicotine cigarettes and then to the other cigarette after 5 days. Urinary excretion of exposure biomarkers (nicotine equivalents [NE], total and free 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL], 1-hydroxypyrene, and 3-hydroxypropyl mercapturic acid) as well as carboxyhemoglobin and plasma cotinine were measured at baseline, Day 5, and Day 10. Daily cigarette consumption was monitored and sensory characteristics were rated for each cigarette. RESULTS: With higher nicotine yield, urine NE, urine total NNAL, and plasma cotinine increased while nonnicotine BOE decreased without changes in cigarette consumption. In contrast, with lower nicotine yield, urine NE, urine total NNAL, and plasma cotinine dropped while nonnicotine BOE and cigarettes per day increased. Higher nicotine cigarettes were rated harsher and stronger than at baseline while lower nicotine cigarettes were less strong. All 4 test cigarettes were highly disliked. CONCLUSIONS: These studies demonstrate that abrupt increases or decreases in nicotine and the resulting sensory changes impact BOE through changes in intensity or frequency of smoking.

A comprehensive evaluation of the toxicology of different cut widths of tobacco in experimental cigarettes.

CONTEXT: Literature suggests that the width of tobacco strips in cigarettes may affect the smoke chemistry and toxicology of such products. OBJECTIVE: A comprehensive analysis of smoke from experimental cigarettes can be used to determine whether different cut widths of tobacco result in different toxicological activity. MATERIALS AND METHODS: A battery of tests was used to compare the chemistry and in vitro and in vivo toxicology of smoke from experimental cigarettes made with tobacco cut to different widths. RESULTS: Different cut widths of tobacco did not elicit consistent and significant differences in cigarette smoke chemistry, responses in in vitro mutagenicity or cytotoxicity assays or most endpoints in 90-d rat inhalation studies. Of note, however, were atypical in-life observations and slightly depressed body weights observed in two rat inhalation studies. CONCLUSION: Most of our data indicate that different cut widths of tobacco used in cigarettes are unlikely to change the toxicity of mainstream cigarette smoke; however, without additional investigation, the atypical in-life observations and depression in body weights cast doubt on the toxicological acceptability of cutting the tobacco into wider shreds.

Sources of and technical approaches for the abatement of tobacco specific nitrosamine formation in moist smokeless tobacco products.

The presence of TSNA has been suggested as a potentially important cancer risk factor for moist smokeless tobacco (MST) products. We describe studies of the impact of tobacco agronomic and production practices which influence TSNA formation. TSNA were measured at points in the MST production chain from the farm to the finished product at the end of shelf life. Analyses were conducted to define points at which TSNA may occur, the factors related to the magnitude of occurrence, and actions which may be taken to mitigate such occurrence. Weather conditions during the curing season can have a dramatic impact on TSNA levels in tobacco, with wet seasons markedly increasing TSNA levels in cured tobacco. TSNA levels in MST do not increase beyond levels in cured tobacco when production practices limit the presence of nitrate reducing bacteria. Therefore, TSNA in such products are a function of the agronomic practices and conditions under which tobacco is produced at the farm level. Regional and annual variation in TSNA levels results from the stochastic nature of agronomic factors related to TSNA formation during tobacco growing and curing.

A comprehensive evaluation of the toxicology of cigarette ingredients: cocoa-derived ingredients.

CONTEXT: Cocoa-derived ingredients are used in cigarette tobacco. OBJECTIVE: A battery of tests was used to compare toxicity of mainstream smoke from experimental cigarettes containing different added levels of cocoa-derived ingredients. MATERIALS AND METHODS: Five cocoa-derived ingredients chocolate (CH), cocoa (COC), cocoa-grand prix black (CGPB), cocoa nibs tincture (CNT) and cocoa shells extract (CSE) were added individually to experimental cigarettes at three different levels. Smoke from each of the experimental cigarette types was evaluated using analytical chemistry; in vitro cytotoxicity and mutagenicity testing were performed for four of the five compounds. For CH, COC and CNT, 90-day smoke inhalation studies were performed with 6-week recovery periods. RESULTS: No consistent changes were found in the analytical chemistry results. Results of the cytotoxicity and mutagenicity were unaffected by any of the ingredients. Two of the three inhalation studies showed very few differences between the groups. The inhalation study with COC showed several increases in mean histopathology severity scores in groups exposed to different levels of COC, compared with the controls. These apparent effects of COC on histopathology lesion severity scores were only present in a single sex and none were dose-related, which is not consistent with a true increase in biological activity. Also there were effectively no differences in the patterns of recovery for any of the compounds. CONCLUSIONS: Even at high inclusion levels there was a lack of toxicological response in these COC derived ingredients.

Mouse B cell activation is inhibited by CD44 cross-linking.

Lymphocyte activation and trafficking are indispensable to the immune system. CD44 is an adhesion molecule with known importance in T cell activation, lymphocyte trafficking, and tumor metastasis. Although CD44 has been shown to participate in the activation, rolling and adhesion, and homing of T cells, the role of CD44 on B cells is relatively unknown. The effects of CD44 cross-linking on murine B cell activation via CD40L was explored using the anti-CD44 mAbs RK3G9 and IM7. When immobilized on a plate, both RK3G9 and IM7 were found to strongly inhibit B cell proliferation and Ig production, especially at lower cell input concentrations. IgE inhibition was especially prominent. In contrast, soluble RK3G9 added to the B cell cultures had no effect. The inhibitory effect of anti-CD44 on B cell activation was not influenced by the addition of the anti-FcgammaRII, indicating that Fc cross-linking did not play a role in this inhibition. As Ig production requires several days for both B cell proliferation and differentiation to occur, the effects of delayed addition of immobilized anti-CD44 mAbs were studied, and the results indicated no inhibition after 96 hrs of culture. Finally, B cells were activated by either LPS or anti-IgM F(ab')2. While LPS-induced B cell activation was inhibited by immobilized anti-CD44 mAbs, anti-IgM activation was refractory. Interestingly, addition of both anti-IgM and CD40L or LPS resulted in some modulation of the inhibitory activity. These results suggest that CD44 cross-linking could control polyclonal B cell activation by CD40L, but allow sIgM/CD40L activation to continue.

Aryl hydrocarbon receptor-dependent induction of loss of mitochondrial membrane potential in epididydimal spermatozoa by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant known to exhibit toxic effects on the male reproductive system, including the epididymus and spermatozoa. However, the mechanism(s) that mediate dioxin toxicity in spermatozoa remain unclear. The aim of the present study was to investigate whether exposure to TCDD would cause a loss in mitochondrial membrane potential (Deltapsi(m)) in spermatozoa and whether such an effect is mediated by the Ah receptor (AhR). Exposure of C57BL/6 male mice to TCDD at concentrations of 0.1-50 microg/kg for 24 h caused a dose-dependent loss of Deltapsi(m) in epididymal spermatozoa compared to spermatozoa from vehicle-treated mice. However, this effect was not apparent in spermatozoa from AhR knockout (KO) mice. Exposure of spermatozoa from C57BL/6 mice to 1 nM or 5 nM TCDD in vitro also induced loss of Deltapsi(m). TCDD-exposed C57BL/6 mice failed to exhibit changes in the morphology of testes and epididymus, and did not show any increase in number of apoptotic germ cells. In addition, comparison of reactive oxygen species (ROS) production in spermatozoa from vehicle- and TCDD-treated mice indicated that exposure to TCDD resulted in elevated ROS levels in the spermatozoa from TCDD-treated mice. Moreover, blockade of ROS production by pretreatment with ROS scavenger N-acetylcysteine (NAC) mitigated the loss of Deltapsi(m) following TCDD exposure. Taken together, these data suggest that direct exposure of spermatozoa to TCDD triggers loss of Deltapsi(m) that is mediated by AhR-dependent production of ROS.

2,3,7,8-tetrachlorodibenzo-p-dioxin enhances negative selection of T cells in the thymus but allows autoreactive T cells to escape deletion and migrate to the periphery.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental pollutant, has been shown to cause thymic atrophy and apoptosis. However, whether TCDD alters the process of T-cell selection in the thymus is not clear. To this end, we investigated the effects of TCDD in the context of the HY-T-cell receptor (TCR) transgenic (Tg) mouse model. We noted that negatively selecting male HY-TCR Tg mice were significantly more sensitive to the thymotoxic effects of TCDD relative to positively selecting female HY-TCR Tg mice, including increased reduction in cellularity and increased induction of apoptosis. TCDD exposure also altered the thymocyte subset composition in HY-TCR Tg male but not female mice. In addition, TCDD treatment resulted in increased extracellularly regulated kinase phosphorylation and lymphocyte-specific protein tyrosine kinase expression in thymocytes of HY-TCR Tg male but not female mice. The increase in proportion of CD8+ mature thymocytes noted in HY-TCR Tg male mice was reflected in the periphery, with TCDD-exposed HY-TCR Tg male mice having increased numbers of CD8+ T cells. Finally, we noted that the proliferative response of HY-TCR Tg male T cells to HY(self)-Ag was enhanced after exposure to TCDD, whereas that of HY-TCR Tg female mice was decreased. Taken together, these data suggest that TCDD alters the process of thymic selection, possibly by enhancing negative thymocyte selection, whereas at the same time allowing autoreactive T cells to escape deletion in the thymus and immigrate to the periphery.

Combined screening of thymocytes using apoptosis-specific cDNA array and promoter analysis yields novel gene targets mediating TCDD-induced toxicity.

We have used pathway-specific cDNA arrays coupled with analysis of gene promoter regions to identify novel genes that may mediate the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the thymus. C57BL/6 mice were injected ip with 50 microg/kg TCDD, and 3, 6, or 24 h later, RNA was extracted from the thymus and subjected to microarray analysis. Several members of the TNF and TNFR family were induced following TCDD exposure, including receptor/ligand pairs Ltbeta-R/LIGHT, OX40/OX40L and TNF-alpha/TNFR1. In addition, Fas and CD30 were also upregulated. Pro-apoptotic bcl-2 gene family members Bax and Hrk, among others, were also induced, as were pro-survival bcl-2 family genes Bcl-x and Bcl-w. Cell-cycle regulator p21Cip1 was also induced. In addition, we analyzed the promoter regions of genes induced by TCDD for the presence of dioxin-responsive elements (DREs). The Fas and LIGHT gene promoters were found to contain DREs as analyzed by Matinspector Web-based search algorithm. Furthermore, binding of the aryl hydrocarbon receptor (AhR) to the DREs present on these genes was confirmed by chromatin immunoprecipitation. Given that several of the genes, including Fas, LIGHT, and CD30 are involved in negative selection of T cells in the thymus, our studies suggest that TCDD-induced upregulation of these genes may enhance negative selection leading to thymic atrophy.

Role of CD44 in activation-induced cell death: CD44-deficient mice exhibit enhanced T cell response to conventional and superantigens.

T cells upon activation are known to up-regulate CD44 expression. However, the precise function of CD44 on activated T cells is not clear. In this report, we demonstrate that signaling through CD44 plays an important role in activation-induced cell death (AICD). CD44 knockout (KO) mice had an elevated in vivo primary and in vitro secondary response to challenge with conalbumin, anti-CD3 mAb and staphylococcal enterotoxin A (SEA), which correlated with reduced AICD when compared to CD44 wild-type mice. In addition, CD44 KO mice exhibited increased delayed-type hypersensitivity response to dinitrofluorobenzene. In a model examining in vitro AICD, splenocytes from CD44 KO mice showed resistance to TCR-mediated apoptosis when compared to splenocytes from CD44 wild-type mice. In addition, signaling through CD44 led to increased apoptosis in TCR-activated but not resting T cells from CD44 wild-type mice without affecting Fas expression. Injection of SEA into mice deficient in CD44 and Fas (CD44 KO/lpr) led to an increased primary response when compared to mice that expressed CD44 but not Fas (CD44 WT/lpr), suggesting that the enhanced response to SEA was dependent on CD44 but not Fas expression. Administration of anti-CD44 mAb into CD44 wild-type mice caused a significant decrease in antigen-specific T cell response. Together, these data implicate CD44 as an important regulator of AICD in T cells. Furthermore, targeting CD44 in vivo may constitute a novel approach to induce apoptosis in activated T cells, and therefore to treat autoimmune diseases, allograft rejection and graft versus host disease.