Nitric oxide, enzymatically synthesized by nitric oxide synthases (NOSI, II and III) which are encoded by three distinct genes, has important functions in both physiology and the pathophysiology of tumorigenesis. The regulation of nitric oxide synthases, NOSII or inducible NOS (iNOS) in particular, is therefore of great interest to us. We have been studying the expression of iNOS, which is normally induced by interferon-g (IFN-g) and bacterial lipopolysaccharide (LPS), and its regulation by a variety of factors including the chemopreventive agent dehydroepiandrosterone (DHEA), tumor suppressor protein p53, diet-related metabolic intermediate pyrroline-5-carboxylate (P5C) and hypoxia. We found that DHEA, a compound with known chemopreventive activity, markedly decreased the level of iNOS in response to LPS plus IFN-gamma in both cultured fibroblasts and macrophages as well as in macrophages freshly isolated from mice treated with DHEA in vivo. Reduced responsiveness of iNOS induction, demonstrated by Western and Northern blots as well as by the Griess reaction, has been observed in cells missing both alleles of p53. Interestingly, although P5C may interact with NO directly to form nitroso-proline (NPRO) as discussed below, it may also play a novel role in the induction of iNOS under hypoxic condition. P5C synergetically induces iNOS along with hypoxia. The newly identified hypoxia-responsive element on iNOS gene presents this interaction the possibility of a potential novel pathway for iNOS induction. Although the formation of NO has been elucidated, the inactivation of NO is not well understood but is important because NO may be the source for endogenous carcinogens. Although 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 proline only at extremely acid conditions (pH < 4). We found that P5C, the precursor and degradative product of proline reacts with NO to produce NPRO at physiologic pH. Identification of NPRO from this reaction has been made by GC-MS. Since P5C not only is an intracellular intermediate but also a diet-dependent constituent of human plasma, it may be a molecule for scavenging and inactivating excess NO produced under certain pathophysiologic conditions, such as chronic infection and inflammation.
