Preparation Of Smokeless Tobacco Extract

Standardized smokeless tobacco (chewing tobacco) was purchased from the University of Kentucky, Tobacco and Health Research Institute (Lexington, KY).29 Quantities of smokeless tobacco were mixed with 5 volumes (5ml/g) of 0.10M phosphate buffer, pH 7.0, and stirred at room temperature for 24h. The pH of the extracts was readjusted as needed to pH 7.0 after 1h of stirring to ensure a physiological pH, and the extracts were centrifuged at 40,000g for 1h. The extracts were reconstituted in phosphate buffer at a concentration of 1.0 mg freeze-dried material per milliliter. The STE was standardized from batch to batch by quantitating the nicotine content in a Perkin Elmer 200 gas chromatograph (Perkin Elmer Corp., Norwalk, CT) equipped with a hydrogen flame ionization detector and a 15-*0.32-mm inside-diameter fused silica capillary column. The instrument was operated in a split mode, and the injector and detector temperatures were 225 and 227°C, respectively. Helium was used as the carrier gas at 25psi.29


The mechanism by which smokeless tobacco constituents produce genetic damage and cause tissue damage is not clear. Lipid peroxidation is a common endpoint for assessing tissue damage due to the production of reactive oxygen species (ROS). The production of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation was determined according to Buege and Aust30 and Bagchi and Stohs.31 Malondialdehyde, a common lipid peroxidation product, was used as the standard, with a molar extinction coefficient of 1.56*105M-1cm-1 at 535nm. The effects of an aqueous smokeless tobacco extract at doses of 0, 125, 250, and 500mg STE/kg in female Sprague-Dawley rats on the induction of hepatic mitochondrial and microsomal lipid peroxidation were examined at 24h posttreatment.29 Dose-dependent increases of 1.8-, 2.3-, and 4.4-fold in mitochondrial and 1.5-, 2.1-, and 3.6-fold in microsomal lipid peroxidation occurred at 125, 250, and 500mg STE/kg, respectively, relative to control values. In order to determine whether STE could induce lipid peroxidation based on direct contact, hepatic mitochondria and microsomes from untreated female Sprague-Dawley rats were incubated at 37°C for 60min in the presence of 0 to 500^g STE/ml. Dose-dependent increases of 1.1- to 2.4-fold occurred in 35 mitochondria and microsomes.32 These results strongly suggest that STE induces oxidative stress, which increases the production of ROS.

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