PROTECTIVE EFFECTS OF FREE-RADICAL SCAVENGERS AND ANTIOXIDANTS AGAINST SMOKELESS TOBACCO EXTRACT (STE)-INDUCED OXIDATIVE STRESS IN MACROPHAGE J774A.1 CELL-CULTURES

Previous studies have demonstrated that an aqueous smokeless tobacco e xtract (STE) administered in an acute oral dose to rats induces an enh anced induction of hepatic mitochondrial and microsomal lipid peroxida tion, hepatic nuclear DNA single strand breaks, enhanced excretion of urinary lipid metabolites, including malondialdehyde, formaldehyde, ac etaldehyde and acetone, and increased production of nitric oxide (NO) by peritoneal macrophage cells. These observations indicate that STE i nduces the production of oxygen free radicals. We have therefore exami ned the in vitro incubation of cultured J774A.1 macrophage cells with STE on the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular membrane damage and cytotoxicity. Th e amount of LDH released by STE was both concentration- and time-depen dent. The cytotoxicity of STE to macrophage J774A.1 cells in culture w as further determined from percent viability after various periods of incubation. The addition of 250 mu g STE/ml to the cultured J774A.1 ce lls resulted in a 2.9-fold increase in the release of LDH. individual coincubation with superoxide dismutase (SOD), catalase, mannitol, and allopurinol had no significant effect on the release of LDH into the c ulture medium, while a combination of the four free radical scavengers resulted in a 59% decrease in the STE-induced release of LDH. At 75 m u M concentrations of vitamine E and vitamin E succinate, approximatel y 28% and 41% inhibitions were observed in STE-induced LDB leakage, re spectively. Taken together with previous studies, the results indicate that STE activates macrophage cells, resulting in the production of r eactive oxygen species. These oxygen free radicals may be responsible for tissue damaging effects including membrane damage, and selected ox ygen free radical scavengers and antioxidants can attenuate these tiss ue damaging effects.