Aqueous Extract Of Smokeless Tobacco Research Articles

Aqueous Extract of Smokeless Tobacco (gutkha) Deregulates Tumor Suppressor and DNA Repair Response in a Murine Model of Smokeless Tobacco Use.

The effect of smokeless tobacco (gutkha) was investigated by treating male and female Swiss Albino mice with an aqueous extract of smokeless tobacco (AEST). AEST was administered at a dose of 25 mg kg-1 body weight per day for different time periods (6, 12, 16, and 24 weeks), and control animals were provided only drinking water without AEST for the same period. Control and AEST-treated mice were observed for different oxidative stress parameters, nitric oxide (NO) release, and myeloperoxidase (MPO) release, and they were evaluated for alterations in tumor suppressor and DNA repair responses in the liver and spleen. Both male and female mice treated with AEST showed significant increase in lipid peroxidation, protein carbonylation, and NO and MPO release in the liver and spleen compared to age- and gender-matched controls. The significant decline in tumor suppressor p53 protein levels, likely mediated by concomitantly upregulated levels of Mdm2, was observed. We also observed a significant decline in the levels of DNA repair proteins Brca2 and Ape-1 compared to the respective controls. Thus, AEST induces oxidative stress, inflammation, and significantly lowers tumor suppressor and DNA repair responses. These factors may work in conjunction to increase the risk for certain diseases, including cancer.

Resveratrol and pterostilbene ameliorate the metabolic derangements associated with smokeless tobacco in estrogen deficient female rats

The impact of oral administration of resveratrol and pterostilbene daily for 60 days in estrogen deficient female rats that were simultaneously exposed to oral aqueous extract of smokeless tobacco (AEST) (100 mg/kg/day) was investigated. Estrogen deficiency was induced in rats by 30 days daily exposure to 4-vinylcyclohexene diepoxide (80 mg/kg, i.p.). The plasma nicotine and cotinine levels were quantified by HPLC. AEST exposure in estrogen deficient rats caused significant increase in body weight, changes in anthropometrical parameters, fasting serum glucose and insulin levels that indicated impaired glucose tolerance with insulin resistance. Serum AST, ALT, and ALP were elevated and activity of Na+K+ATPase in the skeletal muscle was decreased. Importantly, AEST exposure in rats impaired the hepatic and gastrocnemius muscle gene expressions of SIRT1, PGC-1α, PPAR-α, TFAM, NRF-1 and decreased the mitochondrial DNA content. Resveratrol and pterostilbene reversed these metabolic perturbations thus averting induction of metabolic syndrome in this milieu.

Resveratrol and pterostilbene attenuated smokeless tobacco induced cardiovascular aberrations in estrogen deficient female rats.

This study evaluated the impact of resveratrol (RSV) and pterostilbene (PT) on the aqueous extract of smokeless tobacco (AEST) induced cardiovascular aberrations in estrogen deficient female Sprague-Dawley rats. Exposure to 4-vinylcyclohexene diepoxide (VCD) (80 mg kg-1, i.p.) for 30 days induces estrogen deficiency. The rats were administered AEST alone or AEST along with resveratrol and/or pterostilbene. Several markers of cardiovascular health were estimated to evaluate the repercussion of the exposures. RSV and PT per se and in combination significantly reversed the derangements caused by AEST. RSV decreased the atherogenic index and systolic blood pressure and normalized ECG. RSV and PT treatment markedly decreased aortic collagen, cardiac-carbonylated proteins, serum creatine-kinase, cholesterol, LDH, LDL, VLDL, CRP and TNF-α levels. Conversely, they increased serum nitrate-nitrite and HDL levels. The drugs improved the gene expression of SIRT1, PGC-1α, PPAR-α, TFAM, NRF-1 and mtDNA in the cardiac tissue. However, the expression of SIRT1 was not modified by PT. These favorable effects were comparable to those of estradiol therapy. Histopathological outcomes also corroborated these benefits. Thus, resveratrol and pterostilbene abrogated the deleterious effects of AEST on cardiovascular parameters in estrogen deficient female rats.

Toxicological evaluation of smokeless tobacco: 2-year chronic toxicity and carcinogenicity feeding study in Wistar Han rats

A comprehensive 2-year oral chronic toxicity/carcinogenicity study was conducted with smokeless tobacco using modern toxicological test methods and well-accepted standards. The study included a 1-year interim subgroup to assess toxicity at that intermediate time point. Test groups consisted of a tobacco blend (B) used in snus, and an aqueous tobacco extract of that tobacco blend (E) administered at 0.2, 2, or 5mgnicotine/kg body weight/day via dosed feed to male and female Wistar Han rats. The dosages were selected to simulate potential exposure in humans ingesting smokeless tobacco or an aqueous extract of smokeless tobacco (the latter intended to simulate a snus extract, to enable bridging these data to snus epidemiology data). The following endpoints were evaluated: clinical observations, body weights, feed consumption (FC), ophthalmic exams, toxicokinetics, clinical pathology, gross pathology, and histopathology. During the 2-year study, clear treatment-related, dose-responsive effects included: (1) increases in plasma nicotine and cotinine (indicating that animals were appropriately exposed to levels relevant to human exposure) and (2) decreases in body weights with some alterations in FC. At the 2-year time point, two tumor types (in the highest B doses) displayed statistically significantly increased incidence trends vs. controls: (1) uterine carcinoma in females and (2) epididymal mesothelioma in males. Three tumor types displayed statistically significantly decreased incidence trends: (1) mammary gland adenomas in females, (2) skin basal cell carcinomas in females, and (3) thyroid follicular cell adenomas in males. These increases (and decreases) in tumor trends were interpreted as not being treatment-related because: (1) there were no preneoplastic or related non-neoplastic histopathological findings in the treated rats at the 1-year or 2-year time points to suggest that any of these neoplastic findings were treatment-related and (2) the tumor morphologies and incidences were generally within the expected range of historical controls for Wistar Han rats. Findings from this study indicate that chronic exposure of male and female Wistar Han rats to either a tobacco blend used in snus, or a tobacco extract of that blend does not lead to increased toxicity or carcinogenicity, based on the specified outcomes measured.

Resveratrol attenuated smokeless tobacco–induced vascular and metabolic complications in ovariectomized rats

The aim of this study was to evaluate the effect of smokeless tobacco extract on ovariectomized female rats and to investigate the role of resveratrol in alleviating associated vascular and diabetic complications. Thirty-six female Wistar rats (8 wk old) were subjected to bilateral ovariectomy (OVX) or sham operation and randomly assigned to six groups: sham operation; OVX; OVX + aqueous extract of smokeless tobacco (AEST); OVX + AEST + 17β-estradiol; OVX + AEST + resveratrol 25 mg/kg/day PO; and OVX + AEST + resveratrol 50 mg/kg/day PO. All treatments were given for 60 days. Various vascular and metabolic markers (such as serum glucose, triglycerides, cholesterol, insulin, estradiol, glycosylated hemoglobin, glucose tolerance), ex vivo vascular reactivity of aortic ring, and aortic collagen levels were estimated after the treatments. Oral exposure to smokeless tobacco extract in ovariectomized female rats triggered a significant increase in metabolic markers (viz, serum triglycerides, cholesterol, glucose, insulin, glycosylated hemoglobin), and aortic collagen levels. It also led to decreased serum nitrate-nitrite levels and vascular reactivity. Resveratrol 50 mg/kg/day PO attenuated detrimental changes in aortic reactivity and aortic collagen levels, improved glucose tolerance, and reversed the deleterious effects on other serum parameters comparable to 17β-estradiol. Resveratrol treatment for 60 days abrogates the deleterious effects of smokeless tobacco on ovariectomized female rats. Resveratrol in adequate doses can be effectively used as an alternative to estrogen therapy for smokeless tobacco-induced vascular and diabetic complications.

Smokeless Tobacco Extract (STE)-Induced Toxicity in Mammalian Cells is Mediated by the Disruption of Cellular Microtubule Network: A Key Mechanism of Cytotoxicity

Smokeless tobacco usage is a growing public health problem worldwide. The molecular mechanism(s) underlying smokeless tobacco associated tissue damage remain largely unidentified. In the present study we have tried to explore the effects of aqueous extract of smokeless tobacco (STE) on tubulin-microtubule, the major cytoskeleton protein that maintains cells morphology and participates in cell division. Exposure to STE resulted in dose-dependent cytotoxicity in a variety of mammalian transformed cell lines such as human lung epithelial cells A549, human liver epithelial cells HepG2, and mouse squamous epithelial cells HCC7, as well as non-tumorogenic human peripheral blood mononuclear cells PBMC. Cellular morphology of STE-treated cells was altered and the associated disruption of microtubule network indicates that STE targets tubulin-microtubule system in both cell lines. Furthermore it was also observed that STE-treatment resulted in the selective degradation of cellular tubulin, whereas actin remains unaltered. In vitro, polymerization of purified tubulin was inhibited by STE with the IC50 value∼150 µg/ml and this is associated with the loss of reactive cysteine residues of tubulin. Application of thiol-based antioxidant N-acetyl cysteine (NAC) significantly abrogates STE-mediated microtubule damage and associated cytotoxicity in both A549 and HepG2 cells. These results suggest that microtubule damage is one of the key mechanisms of STE-induced cytotoxity in mammalian cells.

Smokeless tobacco extract impairs iron homeostasis in rats and human hepatoma, HepG2 cells

Tobacco exposure may alter homeostasis of iron (Fe), one of the most abundant and essential transition metals in the body. In this study, the effect of aqueous extract of smokeless tobacco was evaluated on Fe homeostasis in rats and human hepatoma, HepG2 cells. Our findings suggested that tobacco consumption even at low doses impairs Fe homeostasis leading to Fe deficiency anemia. Significant alterations were noted with respect to hematological parameters and expression patterns of selected intestinal Fe-transporters, Fe-binding proteins, and Fe-regulatory hormone, hepcidin. Impairment in the hepatic and renal antioxidant defense system was also observed in the treated rats. Histopathological studies revealed cirrhosis of liver and goblet cell hyperplasia of small intestine. Further, analysis of hepcidin promoter and its expression along with ferritin (expression and ELISA) in HepG2 cells demonstrated an enhanced expression of both the genes resulting in sequestration of Fe in treated cells, thus indicating systemic Fe deficiency.

Involvement of Various Molecular Events in Cellular Injury Induced by Smokeless Tobacco

Smokeless tobacco (ST) consumption is implicated in the pathogenesis of oral diseases, including cancer. However, its pathological effect in other organs is not well understood. In the present study, the effect of aqueous extract of smokeless tobacco (AEST) prepared from "gutkha" (a form of ST) on the xenobiotic drug-metabolizing enzymes, histopathological changes, and damage to the genetic material in lung, liver, and kidney of rats was evaluated. Animals were orally administered AEST at a low dose (L-AEST, 96 mg/kg body wt/day) for 2 (L-AEST(2)) and 28 weeks (L-AEST(28)) and at a high dose (H-AEST, 960 mg/kg body wt/day) for 2 weeks (H-AEST(2)). Real-time PCR and immunohistological studies showed that administration of L-AEST(2) did not induce the expression of phase I cytochrome P450s (CYP1A1, 1A2, and 2E1) and phase II mu-glutathione-s-transferase (GST-mu) drug-metabolizing enzymes in lung, liver, and kidney. Although H-AEST(2) administration significantly induced both gene and protein expression of CYP1A1, 1A2, and 2E1 in all of the above organs, it mildly expressed the phase II detoxifying enzyme, GST-mu, in type I and type II epithelial cells of lung and in proximal tubular cells of kidney. L-AEST(28) enhanced the gene and protein expression of CYP1A1, 1A2, and 2E1 in lung, liver, and kidney in a differential manner and induced the expression of GST-mu in lung and kidney. L-AEST(28) induced the micronuclei formation in the peripheral blood mononuclear cells, TNF-alpha in plasma, and myeloperoxidase activity in the organs. L-AEST(28) significantly enhanced Bax, p53, and NF-kappaB and decreased Bcl-2 gene expressions differentially in an organ-specific manner. The differential changes in these organs due to AEST might be due to their different physiological functions and variable sensitivities toward the metabolites of AEST, which create a microenvironment favorable for AEST-induced pathogenesis. This study broadens the insight into the different molecular mechanisms in various organs, which appear to be deregulated due to AEST. Understanding these processes may help in clinical treatment planning strategies for tobacco-related diseases.

Smokeless Tobacco Impairs the Antioxidant Defense in Liver, Lung, and Kidney of Rats

The present study was designed to evaluate the effects of long-term use of aqueous extract of gutkha (a form of smokeless tobacco) on the antioxidant defense status and histopathological changes in liver, lung, and kidney of male Wistar rats. Animals were orally administered aqueous extract of smokeless tobacco (AEST) at a low dose (96 mg/kg body weight per day) for 2 and 32 weeks, and at a high dose (960 mg/kg body weight per day) for 2 weeks. High-dose AEST for 2 weeks decreased the hepatic glutathione (GSH) and glutathione peroxidase (GPx), and increased lipid peroxidation (Lpx) by 17%, 19%, and 20%, respectively. Low-dose AEST for 32 weeks significantly decreased (p < 0.05) the antioxidant status in these organs. In liver, AEST decreased GSH levels and the activities of superoxide dismutase (SOD), catalase (CAT), and GPx by 34.6%, 29%, 17.1%, and 17.4%, respectively, but it increased Lpx by 64%. In kidney, GSH, SOD, CAT, and GPx were decreased by 26.6%, 23%, 33%, and 18%, respectively, with an increase of Lpx by 65%. AEST decreased the lung GSH, SOD, CAT, and GPx, and increased lung Lpx by 43%, 28.5%, 37%, 40%, and 24%, respectively. However, no change in the plasma levels of vitamins A, C, and E were observed with AEST treatment. Histopathological findings suggest that administration of AEST at the high dose for 2 weeks or at the low dose for 32 weeks could cause mild to moderate inflammation in liver and lungs. In conclusion, a decrease in the antioxidant defense system and long-term inflammation caused by smokeless tobacco may be risk factors for gutkha-induced pathogenesis.

Smokeless tobacco potentiates VIP-induced DNA synthesis and inactivates NEP 24.11 in oral keratinocytes.

The purpose of this study was to determine whether exposure of cultured chemically transformed hamster oral keratinocytes (HCPC-1) to an aqueous extract of smokeless tobacco (STE) potentiates DNA synthesis elicited by vasoactive intestinal peptide (VIP), an autocrine neuropeptide, and, if so, whether this response is associated with inactivation of neutral endopeptidase 24.11 (NEP 24. 11), an ectoenzyme that cleaves and inactivates VIP very effectively, in these cells. I found that STE and VIP each elicited a modest, albeit significant, increase in DNA synthesis in cultured HCPC-1 cells (P < 0.05). However, incubation of HCPC-1 cells with STE together with VIP evoked a significant, concentration- dependent increase in DNA synthesis that was mediated by VIP receptors. The effects of STE and VIP were synergistic. Maximal response was observed after a 48-h incubation. STE significantly attenuated NEP 24.11 activity in HCPC-1 cells at a time when VIP-induced DNA synthesis was maximal. Collectively, these data indicate that STE potentiates VIP-induced DNA synthesis in cultured oral keratinocytes, and that this response is temporally related to STE-induced inactivation of NEP 24.11 in these cells. I suggest that NEP 24.11 modulates the mitogenic effects of smokeless tobacco in the oral epithelium, in part, by inactivating VIP.

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.

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.

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.

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.

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.

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.

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.

Immunostimulatory potential of smokeless tobacco extract in in vitro cultures of murine lymphoid tissues.

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.