Smokeless Tobacco Extract Research Articles

Initial Characterization of the Complement Activating Compounds in Extracts of Smokeless Tobacco

Aqueous extracts of smokeless tobacco (ST) have been shown to be potent activators of complement. However, the mechanisms by which smokeless tobacco activates complement are not well understood. This study was undertaken to identify the complement activating compounds in ST extracts. The approximate molecular size of the activating agent(s) in smokeless tobacco was determined by dialyzing aqueous extracts of loose leaf chewing tobacco (1S1), dry snuff (1S2), and moist snuff (1S3). Following dialysis (total dilution effect of 1:10 9), using a membrane with a molecular weight retention limit of 12-14 kDa, all extracts retained full capacity to activate serum complement as determined by a hemolytic assay. Fractionation of the extracts by gel filtration chromatography revealed that the complement activating agents in ST were high molecular weight compounds that eluted between 400 kDa and the void volume (1500 kDa) of a Sephacryl S300 column. The high molecular weight complement-activating peak was isolated and found to be a more potent complement activator than the unfractionated extract. The chemical nature of the complement activating compounds was determined by subjecting the extracts to boiling for 30 min, an organic extraction with chloroform/methanol 2:1, or treatment with a DNAse/RNAse enzyme cocktail. None of these treatments destroyed the capacity of ST extracts to activate complement, suggesting that the activating agents may be carbohydrate-like. Finally, an extraction protocol designed to remove polyphenols significantly diminished the complement activating capacity of the ST extracts. These results clearly demonstrate that the complement activating substances in smokeless tobacco extracts may be large (>400 kDa) polyphenol-containing compounds (i.e. tannins). Identification of this agent(s) will be important for distinguishing the mechanism of smokeless tobacco-induced complement activation.

Dexamethasone attenuates acute macromolecular efflux increase evoked by smokeless tobacco extract.

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.

Smokeless tobacco, oxidative stress, apoptosis, and antioxidants in human oral keratinocytes

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 μl;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 μM), vitamin E (75 μM), a combination of vitamins C & E (75 μM each), and a novel grape seed proanthocyanidin (IH636) extract (GSPE) (100 μg/ml) against STE induced oxidative stress and tissue damage were also determined. Following treatment of the cells with 300 μg 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 μg 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.

Evaluation of the effect of smokeless tobacco purified products and extracts on latent Epstein–Barr virus

Numerous chemical tumor promoters induce latent Epstein–Barr virus (EBV) to active replication. The effect of smokeless tobacco purified products N-nitrosonornicotine (NNN), 4-( N-methyl- N-nitrosamine)-1-3-pyridinyl)-1-butanone (NNK), benzo( a)pyrene (BaP), and smokeless tobacco extracts (dry snuff, moist snuff, and loose leaf tobacco) was tested for induction of latent EBV in Raji cells using fluorescence-activated cell sorter flow cytometry detection of the restricted component of EBV early antigen (EA-R). Concentrations of smokeless tobacco purified products or preparations were used that have carcinogenic effects in animal cell lines. There was no discernible effect for the 6–7-day duration of treatment on viability of Raji cells, or on induction of latent EBV in Raji cells. Results were comparable using paraformaldehyde- or acetone-fixed cells. There does not appear to be an in vitro effect of smokeless tobacco constituents on EBV-infected lymphocytes that may contribute to development of oral cancers.

Smokeless tobacco and nicotine bring about excessive cytokine responses of murine memory T-cells

To determine the influence of smokeless tobacco (ST) and nicotine on the cytokine phenotype of memory T-cells, splenic mononuclear cells (SPM) were exposed to 1:10 2 or 1:10 3 dilutions of ST extract (ST-SPM), 10 or 100 μg/ml nicotine (NIC-SPM), or medium (CON-SPM) during 4 days of stimulation with anti-CD3. SPM were then washed extensively to remove residual ST or nicotine and restimulated with anti-CD3 and anti-CD28 in the absence of ST or nicotine. Expression of IL-2, IL-4, IL-10 and IFN- γ protein and mRNA levels after 4 days of primary stimulation and after 24 and 48 h of restimulation was evaluated using ELISA and RT-PCR, respectively. After 4 days of primary stimulation, SPM exposed to 100 μg/ml nicotine sustained expression of IL-2, IFN- γ, IL-10 and IL-4 mRNA as opposed to CON-SPM. Restimulation of CON-SPM resulted in maximum re-expression of cytokine mRNA at 24 h and a decline by 48 h. Restimulated NIC-SPM in the absence of nicotine delayed maximal re-expression of IL-2, IFN- γ, IL-10 and IL-4 mRNA until 48 h. Heightened expression of cytokine mRNA at 48 h was paralleled by a small but significant increase in production of IFN- γ, IL-4 and IL-10 protein by NIC-SPM as measured by ELISA. In contrast, ST-SPM did not exhibit residual expression of cytokine mRNA after 4 days of primary stimulation. Like NIC-SPM, however, restimulated ST-SPM exhibited maximum IL-2, IL-4, IFN- γ and IL-10 mRNA at 48 h. Heightened re-expression of cytokine mRNA at 48 h by ST-SPM was paralleled by increased production of IL-2, IFN- γ, IL-4 and IL-10 protein. These results indicate that exposure of T-cells to nicotine, but not ST, during a primary immune response result in inordinate cytokine expression after 4 days. In addition, memory T-cells initially exposed to nicotine or ST during a primary immune response, exhibit excessive cytokine expression when T-cells are restimulated in the absence of nicotine or ST.

Role of nitric oxide in the induction of apoptosis by smokeless tobacco extract.

Smokeless tobacco usage is a growing public health concern in the United States. Lesions of the oral cavity have been clearly linked to smokeless tobacco use. The objective of this study was to determine the biochemical effects of smokeless tobacco extract (STE) exposure upon hamster cheek pouch cell (HCPC-1) cultures. HCPC-1 cells were exposed to a 5 -fold dose-range of STE (0.5, 1.0 and 2.5%) over a time-course of 24-96 h. Following each exposure we measured various biochemical parameters of cell proliferation and cell death. Cell viability, cell cycle progression and S-phase DNA synthesis were measured as markers of cell proliferation. We measured lactate dehydrogenase leakage as a marker of cell membrane damage and cell death due to necrosis. No significant alterations were observed in cell cycle progression and cell proliferation as a result of exposure to STE. LDH measured colorimetrically indicated no significant effect with the lower doses(0.5, 1.0 and 2.5% STE). Apoptosis measured as the A0 peak and by the TUNEL procedure revealed that STE caused significant rates of apoptosis. Maximal apoptosis was noted between 48-96 h. In order to probe the mechanism further we measured the levels of nitrites as an indicator of nitric oxide (NO) in the media. NO levels were significantly elevated at the doses that caused an induction of apoptosis. The results from this study indicate that STE causes a dose-dependent induction of apoptosis and that this is mediated by nitric oxide.

Smokeless Tobacco Extracts Activate Complementin Vitro:A Potential Pathogenic Mechanism for Initiating Inflammation of the Oral Mucosa

The use of smokeless tobacco has been linked to an increased incidence of inflammation of the buccal and gingival mucosa. However, the mechanisms by which smokeless tobacco initiates inflammation are not well understood. The complement cascade is a ubiquitous source of proinflammatory molecules and can be activated rapidly by a wide variety of agents. Therefore, the effect of smokeless tobacco on complement was investigated as a potential pathogenic mechanism for triggering inflammation of the oral mucosa. Aqueous extracts of loose leaf chewing tobacco (1S1), dry snuff (1S2), and moist snuff (1S3), added to normal human serum, depleted complement hemolytic activity in a dose-dependent manner. Experiments utilizing sera deficient in one specific complement component indicated that the smokeless tobacco-induced depletion of hemolytic activity was due largely to consumption of C3. Furthermore, assays designed to test the activity of the alternative pathway of complement clearly showed that all three extracts depleted the hemolytic activity of this pathway. Finally, all three smokeless tobacco extracts activated the alternative pathway since significantly elevated levels of the cleavage fragments iC3b and Bb were detected in extract-treated serum. High quantities of the classical pathway cleavage fragment C4d also were detected in serum treated with moist snuff (1S3). The results clearly demonstrate that smokeless tobacco extracts activate the alternative pathway and also suggest some measure of classical pathway activation. Activation of complement by smokeless tobacco may be a mechanism for initiating inflammation of the oral mucosa.

Subchronic effects of smokeless tobacco extract (STE) on hepatic lipid peroxidation, DNA damage and excretion of urinary metabolites in rats

The oral use of moist smokeless tobacco products (snuff) is causally associated with cancer of the mouth, lip, nasal cavities, esophagus and gut. The mechanism by which smokeless tobacco constituents produce genetic and tissue damage is not known. Recent studies in our laboratories have shown that an aqueous extract of smokeless tobacco (STE) activates macrophages with the resultant production of reactive oxygen species (ROS), including nitric oxide. Furthermore, the administration of acute doses of STE (125–500 mg/kg) to rats induces dose dependent increases in mitochondrial and microsomal lipid peroxidation, enhances DNA single strand breaks, and significantly increases the urinary excretion of the lipid metabolites malondialdehyde, formaldehyde, acetaldehyde and acetone. Since the use of tobacco is a chronic process, the effects of an aqueous extract of STE in rats following low dose exposure were examined. Female Sprague–Dawley rats were treated orally with 25 mg STE/kg every other day for 105 days. The effects of subchronic treatment of STE on hepatic microsomal and mitochondrial lipid peroxidation and the incidence of hepatic nuclear DNA damage were assessed. Lipid peroxidation increased 1.4- to 3.3-fold in hepatic mitochondria and microsome with STE treatment between 0 and 105 days with respect to control animals while hepatic DNA single strand breaks increased up to 3.4-fold. Maximum increases in lipid peroxidation and DNA single strand breaks occurred between 75 and 90 days of treatment. Urinary excretion of the four lipid metabolites malondialdehyde, formaldehyde, acetaldehyde and acetone was monitored by high pressure liquid chromatography (HPLC) with maximum increases being observed between 60 and 75 days of treatment. The results clearly indicate that low dose subchronic administration of STE induces an oxidative stress resulting in tissue damaging effects which may contribute to the toxicity and carcinogenicity of STE.

In vitro effects of smokeless tobacco extract on tumor necrosis factor- α (TNF- α) and interleukin-1 β (I l-1 β) production, and on lymphocyte proliferation

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- α (TNF- α), interleukin -1 β (I L-1 β), and the proliferation of lymphocytes. A macrophage cell line (J774-A1) was used to measure the effects of STE on tumor necrosis factor- α (TNF- α) and interleukin -1 β (I L-1 β) 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- α and I L-1 β. 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.

Nicotine and smokeless tobacco effects on gingival and peripheral blood mononuclear cells.

The pathogenesis of tobacco-related periodontal diseases is not well understood. The purpose of this study was therefore to investigate smokeless tobacco extract (ST) and nicotine effects on prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) secretion by peripheral blood mononuclear cells (PBMC, consisting of monocytes and lymphocytes) and gingival mononuclear cells (GMC). Both peripheral blood and gingival tissue adjacent to the alveolar crest were taken from non-smoking adult periodontitis patients. Gingival tissue was treated with collagenase and deoxyribonuclease and GMC and PBMC were isolated by Ficoll-Hypaque centrifugation. GMC and PBMC (100,000 cells/200 microl) were cultured for 24 hours in supplemented RPMI 1640 alone (control), or in supplemented RPMI 1640 containing 1% ST, 100 microg/ml nicotine, 1 microg/ml Porphyromonas gingivalis LPS, or 1 microg/ml P. gingivalis LPS and either 100 microg/ml nicotine or 1% ST. Enzyme immunoassays were used to quantify PGE2 and IL-1beta. Treatments were compared by repeated measures ANOVA. 100 microg/ml nicotine (7-fold, p<0.02) and 1% ST (3.5-fold, p<0.004) significantly increased secretion of PGE2 by PBMC relative to control cultures. 100 microg/ml nicotine and 1% ST, however, had no effect on IL-1beta secretion by PBMC. Enhanced PGE2 secretion also was seen when PBMC were treated with P. gingivalis LPS+ 100 microg/ml nicotine relative to P. gingivalis LPS alone (p<0.007). In contrast, 100 microg/ml nicotine significantly downregulated IL-1beta secretion by GMC relative to medium alone (p<0.008) and had no effect on PGE2 secretion by GMC. These data indicate that while nicotine and ST can stimulate PBMC to secrete PGE2, they cannot activate further mononuclear cells extracted from gingiva, possibly due to maximal previous stimulation in the periodontitis lesion.

Smokeless tobacco-exposed oral keratinocytes increase macromolecular efflux from the in situ oral mucosa.

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.

Angiotensin-converting enzyme and neutral endopeptidase modulate smokeless tobacco-induced increase in macromolecular efflux from the oral mucosa in vivo

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.

Purified ACE attenuates smokeless tobacco-induced increase in macromolecular efflux from the oral mucosa.

The purpose of this study was to determine whether purified angiotensin I-converting enzyme (ACE) attenuates smokeless tobacco extract (STE)-induced increase in macromolecular efflux from the in situ oral mucosa. By using intravital microscopy, we found that suffusion of an aqueous extract of smokeless tobacco elicited significant concentration-dependent leaky site formation and increase in clearance of fluorescein isothiocyanate-labeled dextran (mol mass, 70 kDa) from the hamster cheek pouch (P < 0.05). Suffusion of purified rabbit lung ACE significantly attenuated these responses in a concentration-dependent fashion (P < 0.05). These effects were specific because purified ACE also significantly attenuated the increase in macromolecular efflux elicited by bradykinin, which is produced in the cheek pouch during suffusion of STE, but did not attenuate the increase elicited by adenosine. Moreover, suffusion of heat-inactivated purified ACE and purified superoxide dismutase had no significant effects on STE- and bradykinin-induced responses. Collectively, these data suggest that exogenous ACE attenuates STE-induced increase in macromolecular efflux from the in situ oral mucosa, in part, by promoting local bradykinin catabolism.

The effect of smokeless tobacco extract on murine T cell cytokine production

Exposure of oral tissue to components of smokeless tobacco (ST) increases susceptibility to oral cancer and periodontal disease. Altered T cell cytokine expression patterns due to ST components could affect the course of these oral diseases. To determine if ST components bring about changes in cytokine expression, T cells in whole splenic mononuclear cell populations (SPM) and enriched T cells, costimulated with anti-CD28 were exposed to 1:102 to 1:104 dilutions of ST extract and stimulated with anti-CD3. IL-2, IL-4, IFN-γ and IL-10 were measured using enzyme immunoassays and RT-PCR. IFN-γ production by enriched T cells costimulated with anti-CD28, was decreased at all concentration of ST while IL-10 production were decreased at 72 h in cultures with 1:102. IL-2 production was significantly increased upon exposure of T cells to 1:102 ST extract. ST did not significantly impact IL-4 production. Overall the data indicate that expression of key cytokines, IFN-γ and IL-10, are consistently decreased upon exposure to ST while IL-2 is increased. Thus, exposure of T cells to physiological concentrations of ST can alter the T cell cytokine expression pattern as to potentially influence oral cancer and periodontal disease.

Mechanisms of smokeless tobacco-induced oral mucosa inflammation: role of bradykinin.

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.

Effect of nicotine on secretory component synthesis by secretory epithelial cells.

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.

The effect of nicotine on murine CD4 T cell responses

T cells were exposed to various concentrations of nicotine or smokeless tobacco extract (STE) during in vitro immune responses in order to examine effects upon expression of T cell costimulatory counter-receptors, CD28 and CTLA-4, and cytokine production. Splenic mononuclear cells (SPM) were exposed to 1:10 2 to 1:10 3 dilutions of STE or 1–100 μg/ml nicotine during 48 and 72h of stimulation with anti-CD3. Expression of CD28 and CTLA-4 was evaluated with flow cytometry and production and expression of IL-2, IL-4, IL-10 and IFN-γ were evaluated using ELISA and RT-PCR. The data here indicate that the percentage of CD4 + T cells expressing CD28 declined while percentage and intensity of CTLA-4 expression increased with exposure to a 1:10 2 dilution of STE during the T cell response. Exposure to nicotine decreased the percentage of CD4 + T cells expressing both CD28 and CTLA-4 and decreased the intensity of CD28 expression. Responding T cells exposed to nicotine produced significantly less Thl cytokines, IL-2 and IFN-γ, but significantly more Th2 cytokines, IL-4 and IL-10. Cytokine specific mRNA expression was only slightly affected by the exposure to nicotine. Thus, exposure of T cells to physiological concentrations of STE or nicotine can alter the T cell expression of CD28 and CTLA-4, and the CD4 T cell cytokine expression pattern.

Aqueous smokeless tobacco extract impairs endothelium-dependent vasodilation in the oral mucosa.

The purpose of this study was to determine whether an aqueous extract of smokeless tobacco (moist snuff) modulates vasomotor tone in the oral mucosa in situ and, if so, to determine the mechanisms that mediated these responses. Using intravital microscopy, we found that the extract had no significant effects on diameter of resistance (second-order) arterioles [44 +/- 5 (SD) microns] in the hamster cheek pouch. However, it significantly attenuated vasodilation elicited by two endothelium-dependent agonists, acetylcholine and bradykinin (P < 0.05). These effects were specific because smokeless tobacco extract had no significant effects on vasodilation elicited by nitroglycerin, an endothelium-independent agonist. Indomethacin, a cyclooxygenase inhibitor, and SQ-29548, a thromboxane A2/prostaglandin H2-receptor antagonist, abrogated the attenuating effects of smokeless tobacco extract on acetylcholine- and bradykinin-induced vasodilation. These data indicate that an aqueous extract of smokeless tobacco impairs endothelium-dependent vasodilation in the oral mucosa in situ in a specific fashion and that these effects are mediated, in part, by cyclooxygenase products of arachidonic acid metabolism that stimulate thromboxane A2/prostaglandin H2 receptors.

In Vitro Effects of a Smokeless Tobacco Extract on the Production of Reactive Oxygen Species by Human Oral Epidermal Cells and Rat Hepatic Mitochondria and Microsomes, and Peritoneal Macrophages

In Vitro Effects of a Smokeless Tobacco Extract on the Production of Reactive Oxygen Species by Human Oral Epidermal Cells and Rat Hepatic Mitochondria and Microsomes, and Peritoneal Macrophages

In vitro effects of a smokeless tobacco extract on the production of reactive oxygen species by human oral epidermal cells and rat hepatic mitochondria and microsomes, and peritoneal macrophages.

The possible role of reaction oxygen species in the toxicity of smokeless tobacco was explored. In order to determine possible sources of reactive oxygen species in response to smokeless tobacco, rat peritoneal macrophages (3 x 10(6)/ml) and hepatic mitochondria and microsomes (1 mg protein/ml) from untreated female Sprague-Dawley rats were incubated with an aqueous smokeless tobacco extract (STE) (200 micro g/ml). STE resulted in rapid increases in chemiluminescence with maximum increases occurring at approximately 6 min for the macrophages and 8 min for mitochondria and microsomes. Maximum increases in chemiluminescence of 1.4-, 3.2-, and 3.1-fold relative to control values occurred for macrophages, mitochondria, and microsomes, respectively. Hepatic mitochondria and microsomes (1 mg protein/ml) from female Sprague-Dawley rats were incubated at 37 degrees C for 60 min in the presence of 0-500 micro g/ml STE. Potential tissue damage was measured as lipid peroxidation, and dose-dependent increases of 1.1-2.4-fold occurred in mitochondria and microsomes. Pre-incubation with various oxygen free radical scavengers including superoxide dismutase (SOD) (100 micro g/ml), catalase (100 micro g/ml), SOD + catalase (100 micro g/ml each), mannitol (1.25 mmol/ml), and allopurinol (100 micro g/ml) inhibited STE (200 micro g/ml) induced lipid peroxidation by 15% to 70%. Previous studies in our laboratories strongly suggest that STE induces the production of oxygen free radicals which cause tissue-damaging effects. We therefore examined the cytotoxicity of STE by incubating cultured human oral epidermal carcinoma (KB) cells with STE, and assessing the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular membrane damage. The amount of LDH released by STE was both concentration- and time-dependent. The results demonstrate that oral cells, peritoneal macrophages, and hepatic mitochondria and microsomes produce reactive oxygen species following in vitro incubation with an aqueous extract of smokeless tobacco. Tissue damage in response to STE may occur as the result of reactive oxygen species production.