The Effects of Vitamin E and Selenium on The Nitric Oxide Production of Macrophages

Abstract • 171

Macrophages are phagocytic cells which play a crucial role in clearance of microorganisms, antigen presentation, cytokine production, and anti-tumor activity. Their anti-tumor activity is facilitated by their production and release of both tumor necrosis factor-α and the toxic free-radical, nitric oxide (NO). Paradoxically, the toxic activity of NO not only destroys the membranes of tumor cells but can also damage and induce dysfunction in the macrophages, themselves, as well as in surrounding normal cells. The present study was performed to evaluate the role of the free radical scavengers, vitamin E and selenium for their ability to protect the macrophage cell membrane from auto-oxidation induced by NO production during endotoxin stimulation. A micro-ELISA assay employing the mouse monocyte macrophage cell line (TIB69) obtained from ATCC (Rockville, MD) was utilized in which Geiss reagent was used to determine the production and release of NO and 3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), to assess cell viability. The cells were cultured in dextrose minimal essential media (DMEM) with 10% fetal calf serum in 96-well microtiter plates. Endotoxin-stimulated macrophages were treated with various concentrations of vitamin E and selenium which were tested both separately and in combination. The results indicated that neither Vitamin E nor Selenium alone caused an increased production of nitric oxide, although an increase in cell viability was seen. In contrast, a combination of both free-radical scavengers resulted in a marked increase in the production of NO and cell viability. Dose response studies showed that the optimal concentration of Selenium and Vitamin E was 60µg/ml and 100 µg/ml, resp., slightly above the Recommended Daily Allowances (RDA) levels. Some toxicity was noted at higher concentrations of selenium, but no toxicity was ever detected using vitamin E. The results of this study indicate that using a combination of vitamin E and selenium, increases the viability of the macrophages and their production of NO. Collectively, these data suggest the potential importance of the use of free-radical scavengers in the prevention and treatment of a variety of diseases whose pathogenesis involves the toxic effects of free radicals, e.g., arteriosclerosis and cancer.