Effect Of Smokeless Tobacco Extract Research Articles

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 alters gene expression in human dermal fibroblasts

Information on the cellular responses to smokeless tobacco products is very limited. Since skin is the first barrier of defense against wound and disease, and smokeless tobacco is in direct contact with oral cavity skin, this study focused on the effects of smokeless tobacco extract (STE) on biomarkers for signal transduction pathways in normal adult human dermal fibroblasts (HDFa). Gene expression (PCR) array analyses showed that 1% STE from 2S3 reference smokeless tobacco altered levels of several genes including: early growth response 1 (egr1), heat shock factor 1 (hsf1), v-myc, tumor suppressor p53 (tp53), bone morphogenetic protein 4 (bmp4), cellular retinol binding protein 1 (rbp1), and transferrin receptor (p90, CD71) (tfrc). Increases in egr1, hsf1, bmp4 and tfrc occurred within 24 hrs of treatment, and in some cases, gene expression declined below control levels at 48 hrs. Levels of tp53 were reduced between 12 and 48 hrs. Future studies will address the changes in gene expression in response to tobacco smoke and smokeless tobacco for identifying biomarkers that predict harm from tobacco usage and assessing the relative risk of tobacco products. GEM is a RJR Leon Golberg Fellow.

Effect of smokeless tobacco extract on catecholamine metabolic enzymes in rat brain: “Dippers” are getting only half of the bang

Effect of smokeless tobacco extract on catecholamine metabolic enzymes in rat brain: “Dippers” are getting only half of the bang

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.

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.

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.

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.

The effects of smokeless tobacco extract on bone nodule formation and mineralization by chick osteoblasts in vitro

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.

Smokeless tobacco contains an inhibitor of prolyl hydroxylase activity

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.