No abstract

SUMMARYNitric oxide (NO) plays an important role in inflammation and multiple stages of carcinogenesis. We investigated the effect of various tea polyphenols and caffeine on the induction of NO synthase (NOS) in thioglycollate-elicited and lipopolysaccharide (LPS)-activated peritoneal macrophages. Gallic acid (GA), (Ϫ)-epigallocatechin (EGC), and (Ϫ)-epigallocatechin-3-gallate (EGCG), the major tea catechin, were found to inhibit inducible NOS (iNOS) protein in activated macrophages. EGCG, a potent antitumor agent with anti-inflammatory and antioxidant properties, inhibited NO generation, as measured by the amount of nitrite released into the culture medium. Inhibition of NO production was observed when cells were cotreated with EGCG and LPS. iNOS activity in soluble extracts of lipopolysaccharide-activated macrophages treated with EGCG (5 and 10 M) for 6 -24 hr was significantly lower than that in macrophages without EGCG treatment. Western blot, reverse transcriptionpolymerase chain reaction, and Northern blot analyses demonstrated that significantly reduced 130-kDa protein and 4.5-kb mRNA levels of iNOS were expressed in lipopolysaccharideactivated macrophages with EGCG compared with those without EGCG. Electrophoretic mobility shift assay indicated that EGCG blocked the activation of nuclear factor-B, a transcription factor necessary for iNOS induction. EGCG also blocked disappearance of inhibitor B from cytosolic fraction. These results suggest that EGCG decreases the activity and protein levels of iNOS by reducing the expression of iNOS mRNA and the reduction could occur through prevention of the binding of nuclear factor-B to the iNOS promoter, thereby inhibiting the induction of iNOS transcription.NO is a short-lived bioactive molecule that participates in the physiology and pathophysiology of many systems (1). NO is synthesized in vivo from L-arginine by NOS with NADPH and oxygen as cosubstrates (2). Molecular cloning and sequencing analyses revealed the existence of at least three main types of NOS isoforms. NOS in the endothelium of blood vessels and brain neurons is constitutive and Ca 2ϩ / calmodulin dependent. This constitutive enzyme synthesizes small amounts of NO triggered by various agonists (e.g., bradykinin, acetylcholine) that increase intracellular Ca 2ϩ . NOS in macrophages and hepatocytes is inducible, not detectable, in unstimulated cells and requires protein synthesis for expression. iNOS produces large amounts of NO several hours after exposure to endotoxin and/or cytokines in macrophages, Kupffer cells, hepatocytes, and fibroblasts. NO has a wide biological role in modulating physiological and pathophysiological processes (1, 3), such as macrophage cytotoxicities, neurotransmissions, neurotoxicities, and regulation of blood pressure. Low concentrations of NO are sufficient, in most cases, to effect these functions. However, during infection and inflammation, in vivo formation of NO is increased, suggesting that NO concentrations are well above those found in normal physiological funct...

1 Resveratrol, naringenin and naringin are naturally occurring¯avonoids in grapes and grapefruits. The anti-in¯ammatory e ects of these¯avonoids have been well documented, but the mechanism is poorly characterized. High concentration of NO are produced by inducible NO synthase (iNOS) in in¯ammation, and the prevention of the expression of iNOS may be an important anti-in¯ammatory mechanism. In this study, the e ects of these¯avonoids on the induction of NO synthase (NOS) in RAW 264.7 cells activated with bacterial lipopolysaccharide (LPS, 50 ng ml 71 ) were investigated. 2 Resveratrol was found strongly to inhibit NO generation in activated macrophages, as measured by the amount of nitrite released into the culture medium, and resveratrol strongly reduced the amount of cytosolic iNOS protein and steady state mRNA levels. However, the inhibitory abilities of naringenin were lower, and the inhibitory abilities of naringin were almost negligible. 3 In electrophoretic mobility shift assays, the activation of NFkB induced by LPS for 1 h was inhibited by resveratrol (30 mM). Furthermore, in immunoblotting analysis, cells treated with LPS plus resveratrol showed an inhibition of phosphorylation as well as degradation of IkBa, and a reduced nuclear content of NFkB subunits. 4 The¯avonoids may be of value for inhibiting the enhanced expression of iNOS in in¯ammation through down-regulation of NFkB binding activity.

No abstract

Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. However, the mechanism is still unclear. In this study, we demonstrate that arsenite is able to induce apoptosis in a concentration- and time-dependent manner; however, arsenate is unable to do so. An increase of intracellular peroxide levels was accompanied with arsenite-induced apoptosis, as demonstrated by flow cytometry using DCFH-DA. N-Acetyl-L-cysteine (a thiol-containing antioxidant), diphenylene iodonium (an inhibitor of NADPH oxidase), 4,5-dihydro-1,3-benzene disulfonic acid (a selective scavenger of O2-), and catalase significantly inhibit arsenite-induced apoptosis and intracellular fluorescence intensity. In contrast, allopurinol (an inhibitor of xanthine oxidase), indomethacin (an inhibitor of cyclooxygenase), superoxide dismutase, or PDTC had no effect on arsenite-induced cell death. Activation of CPP32 activity, PARP (a DNA repair enzyme) degradation, and release of cytochrome c from mitochondria to the cytosol are involved in arsenite-induced apoptosis, and Bcl-2 antagonize arsenite-induced apoptosis by a mechanism that interferes in the activity of CPP32. These results lead to a working hypothesis that arsenite-induced apoptosis is triggered by the generation of hydrogen peroxide through activation of flavoprotein-dependent superoxide-producing enzymes (such as NADPH oxidase), and hydrogen peroxide might play a role as a mediator to induce apoptosis through release of cytochrome c to cytosol, activation of CPP32 protease, and PARP degradation.