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

Abstract

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 (3×10⁶/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 μg/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°C for 60 min in the presence of 0–500 μg/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 μg/ml), catalase (100 μg/ml), SOD + catalase (100 μg/ml each), mannitol (1.25 mmol/ml), and allopurinol (100 μg/ml) inhibited STE (200 μg/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.