Reducing-oxidizing reactions function not only in immune defense mechanisms but also in cellular signaling and regulation. Genotoxic events mediated by oxidants and the protection of antioxidants have been suggested in carcinogenesis and in the mechanisms of diet-dependent cancer prevention. In this context, nitric oxide may play an important role. Enzymatically synthesized by nitric oxide synthase, this molecule can form other reactive oxygen species and act in both pro-oxidant and antioxidant pathways. Therefore, the level of this enzyme and its regulation by dietary compounds and nutrients is of interest for carcinogenesis and cancer prevention. The inducible form of nitric oxide synthase (iNOS) is found in macrophages and can be induced by lipopolysaccharide and interferon gamma. We used two methods for estimating enzyme activity. First, in a direct cell-free assay system, the production of citrulline from precursor radiolabeled arginine was quantitated by recovering citrulline with ion-exchange chromatography. Second, an estimate of activity in intact cells was based on the accumulation of nitrite in tissue culture medium. Using these methods, we have studied several tissue culture cells using a variety of nutrients, cytokines, pro-oxidant and antioxidant effector molecules in order to identify interactions of dietary compounds and nutrients with this system of potential importance in carcinogenesis. After identifying regulatory effectors, we will use Western immunoblotting to characterize the enzyme protein. We will develop a cDNA probe to study the expression of iNOS and its regulation by dietary and nutritional factors.
