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 extract (STE) administered in an acute oral dose to rats induces an enhanced induction of hepatic mitochondrial and microsomal lipid peroxidation, hepatic nuclear DNA single strand breaks, enhanced excretion of urinary lipid metabolites, including malondialdehyde, formaldehyde, acetaldehyde and acetone, and increased production of nitric oxide (NO) by peritoneal macrophage cells. These observations indicate that STE induces the production of oxygen free radicals. We have therefore examined 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. The amount of LDH released by STE was both concentration- and time-dependent. The cytotoxicity of STE to macrophage J774A.1 cells in culture was further determined from percent viability after various periods of incubation. The addition of 250 μg STE/ml to the cultured J774A.1 cells 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 culture medium, while a combination of the four free radical scavengers resulted in a 59% decrease in the STE-induced release of LDH. At 75 μM concentrations of viramine E and vitamin E succinate, approximately 28% and 41% inhibitions were observed in STE-induced LDH leakage, respectively. Taken together with previous studies, the results indicate that STE activates macrophage cells, resulting in the production of reactive oxygen species. These oxygen free radicals may be responsible for tissue damaging effects including membrane damage, and selected oxygen free radical scavengers and antioxidants can attenuate these tissue damaging effects.