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 (NO) 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. We found that dehydroepiandrosterone (DHEA), a compound with known chemopreventive activity, marked decreased the level of iNOS in both cultured fibroblasts and in macrophages isolated from mice treated with DHEA in vivo. Although the formation of NO has been elucidated, the inactivation of NO is not well understood but has been the focus of considerable research activity because NO may be an important endogenous carcinogen. Although nitrosoproline (NPRO) is known to be formed from NO and excreted in the urine, its formation has not been considered a major route for NO detoxification because NPRO is formed from PRO only at extremely acid conditions (pH less than 4). We found that pyrroline-5-carboxylate, the precursor and degradative product of proline readily reacts with NO to form NPRO at physiologic pH. Identification of NPRO from this reaction was made by Gas chromatography-Mass spectrometry. Since pyrroline-5-carboxylate not only is an intracellular intermediate but also a diet-dependent constituent of human plasma, it may be a diet-dependent and readily available molecule for scavenging and inactivating excess NO.
