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
M. Bagchi et al., 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, Archives of environmental contamination and toxicology, 30(3), 1996, pp. 418-422
The possible role of reaction oxygen species in the toxicity of smokel
ess tobacco was explored. In order to determine possible sources of re
active 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 incubat
ed with an aqueous smokeless tobacco extract (STE) (200 mu g/ml). STE
resulted in rapid increases in chemiluminescence with maximum increase
s occurring at approximately 6 min for the macrophages and 8 min for m
itochondria and microsomes. Maximum increases in chemiluminescence of
1.4-, 3.2-, and 3.1-fold relative to control values occurred for macro
phages, mitochondria, and microsomes, respectively. Hepatic mitochondr
ia and microsomes (1 mg protein/ml) from female Sprague-Dawley rats we
re incubated at 37 degrees C for 60 min in the presence of 0-500 mu g/
ml STE. Potential tissue damage was measured as lipid peroxidation, an
d dose-dependent increases of 1.1-2.4-fold occurred in mitochondria an
d microsomes. Pre-incubation with various oxygen free radical scavenge
rs including superoxide dismutase (SOD) (100 mu g/ml), catalase (100 m
u g/ml), SOD + catalase (100 mu g/ml each), mannitol (1.25 mmol/ml), a
nd allopurinol (100 mu g/ml) inhibited STE (200 mu g/ml) induced lipid
peroxidation by 15% to 70%. Previous studies in our laboratories stro
ngly suggest that STE induces the production of oxygen free radicals w
hich cause tissue-damaging effects. We therefore examined the cytotoxi
city of STE by incubating cultured human oral epidermal carcinoma (KB)
cells with STE, and assessing the release of the enzyme lactate dehyd
rogenase (LDH) into the media as an indicator of cellular membrane dam
age. The amount of LDH released by STE was both concentration- and tim
e-dependent. The results demonstrate that oral cells, peritoneal macro
phages, and hepatic mitochondria and microsomes produce reactive oxyge
n species following in vitro incubation with an aqueous extract of smo
keless tobacco. Tissue damage in response to STE may occur as the resu
lt of reactive oxygen species production.