Mechanisms and potential therapeutic targets for folic acid in cardiovascular disease

Folic acid (FA) is a member of the B-vitamin family with cardiovascular roles in homocysteine regulation and endothelial nitric oxide synthase (eNOS) activity. Its interaction with eNOS is thought to be due to the enhancement of tetrahydrobiopterin bioavailability, helping maintain eNOS in its coupled state to favor the generation of nitric oxide rather than oxygen free radicals. FA also plays a role in the prevention of several cardiac and noncardiac malformations, has potent direct antioxidant and antithrombotic effects, and can interfere with the production of the endothelial-derived hyperpolarizing factor. These multiple mechanisms of action have led to studies regarding the therapeutic potential of FA in cardiovascular disease. To date, studies have demonstrated that FA ameliorates endothelial dysfunction and nitrate tolerance and can improve pathological features of atherosclerosis. These effects appear to be homocysteine independent but rather related to their role in eNOS function. Given the growing evidence that nitric oxide synthase uncoupling plays a major role in many cardiovascular disorders, the potential of exogenous FA as an inexpensive and safe oral therapy is intriguing and is stimulating ongoing investigations.     

Inhibition of lipid peroxidation by S-nitrosoglutathione and copper

The antioxidant properties of S -nitrosoglutathione, a nitric oxide-derived product were studied in different experimental systems. By using the crocin bleaching test, S -nitrosoglutathione, in the presence of copper ions, shows an antioxidant capacity about six times higher than that of Trolox c and referable to the interception of peroxyl radicals by nitric oxide. Copper alone shows a modest inhibitory action, which is about seven times lower than that of Trolox c. S -nitrosoglutathione prevents lipid peroxidation induced by the well-known Fe 2+ /ascorbate system (IC 50 =450 μM) and the inhibitory effect is strongly reinforced by the presence of copper ions (IC 50 =6.5 μM). In addition, cumene hydroperoxide-induced lipid peroxidation is markedly decreased by S -nitrosoglutathione, provided that copper ions, maintained reduced by ascorbate, are present. Decomposition of S -nitrosoglutathione through metal catalysis and/or the presence of reducing agents and the consequent release of nitric oxide are of crucial importance for eliciting the antioxidant power. In this way, copper ions and/or reducing species with low antioxidant potency are able to promote the formation of an extremely strong antioxidant species such as nitric oxide.     

Inhibition of Lipid Peroxidation by S -nitrosoglutathione and Copper

The antioxidant properties of S -nitrosoglutathione, a nitric oxide-derived product were studied in different experimental systems. By using the crocin bleaching test, S -nitrosoglutathione, in the presence of copper ions, shows an antioxidant capacity about six times higher than that of Trolox c and referable to the interception of peroxyl radicals by nitric oxide. Copper alone shows a modest inhibitory action, which is about seven times lower than that of Trolox c. S -nitrosoglutathione prevents lipid peroxidation induced by the well-known Fe 2+ /ascorbate system (IC 50 =450 &#119 M) and the inhibitory effect is strongly reinforced by the presence of copper ions (IC 50 =6.5 &#119 M). In addition, cumene hydroperoxide-induced lipid peroxidation is markedly decreased by S -nitrosoglutathione, provided that copper ions, maintained reduced by ascorbate, are present. Decomposition of S -nitrosoglutathione through metal catalysis and/or the presence of reducing agents and the consequent release of nitric oxide are of crucial importance for eliciting the antioxidant power. In this way, copper ions and/or reducing species with low antioxidant potency are able to promote the formation of an extremely strong antioxidant species such as nitric oxide.     

Nitric oxide as an adaptive protection factor in hypoxia

Recent data concerning intracellular aspects of nitric oxide at hypoxia conditions and correction means pathologic states by it. On the basis of data obtained publications and own investigations about nitric oxide influence on mitochondrial respiration and oxidative phosphorylation possible mechanism its action is discussed. We conclude that breach of macroergs output at hypoxia connect with active oxygen forms, antioxidant enzymes activity and individual peculiarities physiologic reactivity of organism.     

Nitric Oxide and Low-Density Lipoprotein Oxidation

Nitric oxide can have both pro-oxidant and antioxidant effects on low-density lipoprotein. Nitric oxide does not appear to react directly with components of LDL. However, in the presence of oxygen (through NO₂ and N₂O₃ formation) or superoxide (through peroxynitrite formation) nitric oxide may cause oxidation of the lipid, protein and antioxidant components of LDL. Conversely, nitric oxide is a potent inhibitor of LDL oxidation when initiated by copper ions or by azo-initiators. The possible implications of these observations to vascular pathology are discussed.     

Nitric oxide synthase/PGE(2) cross-talk in rat submandibular gland

It is known that nitric oxide modulates the prostaglandin generation. However, little is known about the regulatory action of prostaglandin on nitric oxide production. There is a molecular cross-talk between nitric oxide and prostaglandin. Here, we examined biochemical signalling pathways coupled to the prostaglandin E(2) (PGE(2)) receptor related to nitric oxide synthase stimulation in rat submandibular gland. PGE(2) through the stimulation of its own receptor, triggered activation of phosphoinositide turnover (IPs), translocation of protein kinase C (PKC), stimulation of nitric oxide synthase activity (NOS) and increased production of cyclic GMP (cGMP). PGE(2) stimulation of NOS and cGMP production was blunted by agents interfering with calcium influx, calcium/calmodulin and phospholipase C (PLC) activities; while PKC inhibitor was able to stimulate PGE(2) effects. PGE(2) did not evoke amylase release, indicating that NOS/ cGMP pathway were not associated with this enzyme secretion. Our results suggest that this prostanoid could act as vasoactive chemical mediator through its ability to activate NOS-cGMP pathway via own gland membrane receptor.     

Inhibition of gastric cancer cell proliferation by resveratrol: role of nitric oxide

Resveratrol is a dietary phytochemical that has been shown to inhibit proliferation of a number of cell lines, and it behaves as a chemopreventive agent in assays that measure the three stages of carcinogenesis. We tested for its chemopreventive potential against gastric cancer by determining its interaction with signaling mechanisms that contribute to the proliferation of transformed cells. Low levels of exogenous reactive oxygen (H(2)O(2)) stimulated [(3)H]thymidine uptake in human gastric adenocarcinoma SNU-1 cells, whereas resveratrol suppressed both synthesis of DNA and generation of endogenous O(2)(-) but stimulated nitric oxide (NO) synthase (NOS) activity. To address the role of NO in the antioxidant action of resveratrol, we measured the effect of sodium nitroprusside (SNP), an NO donor, on O(2)(-) generation and on [(3)H]thymidine incorporation. SNP inhibited DNA synthesis and suppressed ionomycin-stimulated O(2)(-) generation in a concentration-dependent manner. Our results revealed that the antioxidant action of resveratrol toward gastric adenocarcinoma SNU-1 cells may reside in its ability to stimulate NOS to produce low levels of NO, which, in turn, exert antioxidant action. Resveratrol-induced inhibition of SNU-1 proliferation may be partly dependent on NO formation, and we hypothesize that resveratrol exerts its antiproliferative action by interfering with the action of endogenously produced reactive oxygen. These data are supportive of the action of NO against reactive oxygen and suggest that a resveratrol-rich diet may be chemopreventive against gastric cancer.     

Nitric oxide decreases superoxide anion generation by microsomes from soybean embryonic axes

Experiments were carried out to evaluate the effects of exposure to nitric oxide on the ability by NADPH‐dependent microsomal electron transfer to generate oxygen radicals. Such interactions could play a role in the potential antioxidant action of nitric oxide (NO). Isolated microsomes from soybean ( Glycine max [L.] Merr. cv. Hood) embryonic axes were exposed to an exogenously added source of nitric oxide (NO) (S‐nitrosoglutathione + dithiothreitol). The O₂⁻ generation rate by microsomes exposed to NO decreased significantly as compared to the rate measured in microsomes incubated in the absence of NO. The exposure of the microsomes to the NO donor did not alter the microsomal rate of hydroxyl radical generation. Preincubation of the microsomes with the NO donor affected neither iron reduction rate nor activity of cytochrome c reductase. However, cytochrome P₄₅₀ activity was significantly inhibited after exposure to NO. This inhibition was completely prevented by hemoglobin. The data are consistent with the hypothesis that NO exhibits a potential antioxidant role in the plant cell by decreasing the rate of generation of superoxide anion. Since endogenous NO was detected in homogenates of soybean embryonic axes by EPR studies, this interaction between NO and cytochrome P₄₅₀ in soybean embryonic axes could be a factor of relevance for the control of oxidative stress in vivo.     

Iron as an inducer of nitric oxide formation in the animal body

Citrate iron complex injections to mice or rats resulted in the nitric oxide formation detected by nitric oxide binding to iron-diethyldithiocarbomate complexes. The mononitrosyl iron complexes formed were paramagnetic and EPR active. The maximal nitric oxide concentrations in rat livers were 15-20 nm per gram of tissue. Phenosan-K (an antioxidant) inhibited partly the iron capacity to nitric oxide formation in animal organisms. The nitric oxide formation was proposed to be due to some endogenic amino groups oxidation by active oxygen agents or products of lipid or non-saturated fatty acid production under the prooxidant action of the iron.     

A novel neuroprotective agent with antioxidant and nitric oxide synthase inhibitory action

N(alpha)-vanillyl-N(omega)-nitroarginine (N - 1) that combines the active functions of natural antioxidant and nitric oxide synthase inhibitor was developed for its neuroprotective properties. N - 1 exhibited protective effects against hydrogen peroxide-induced cell damage and the inhibitory effect on nitric oxide 'NO' production induced by calcium ionophore in NG 108-15 cells. N - 1 inhibited the constitutive NOS isolated from rat cerebellar in a greater extent than constitutive NOS from human endothelial cells. Low binding energy (-10.2 kcal/mol) obtained from docking N - 1 to nNOS supported the additional mode of action of N - 1 as an nNOS inhibitor. The in vivo neuroprotective effect on kainic acid-induced nitric oxide production and neuronal cell death in rat brain was investigated via microdialysis. Rats were injected intra-peritonially with N - 1 at 75 micromol/kg before kainic acid injection (10 mg/kg). The significant suppression effect on kainic acid-induced NO and significant increase in surviving cells were observed in the hippocampus at 40 min after the induction.     

Biological chemistry and clinical potential of S-nitrosothiols

S-Nitrosothiols are endogenous metabolites of nitric oxide that have been detected in extra- and intracellular spaces. Many biological functions of S-nitrosothiols have been described that can be categorized as being due to one or more of the following: (i) nitric oxide release, (ii) transnitrosation, (iii) S-thiolation, and (iv) direct action. This emphasizes the fact that S-nitrosothiols are more than simply nitric oxide donors. Many of the biological functions that have been described for S-nitrosothiols have clinical correlates. This review describes the biological chemistry, biological actions, and clinical potential of these compounds.     

Synthesis and evaluation of in vitro antioxidant properties of novel 2,5-disubstituted 1,3,4-oxadiazoles

2,5-Disubstituted 1,3,4-oxadiazole compounds are one of the most attractive heterocyclic compounds for researchers due to their biological activities. In the undertaken research, a number of potential 2,5-disubstituted 1,3,4-oxadiazole analogues were synthesized through multi step reaction and characterized by FT-IR, ¹H NMR, mass spectra, and also by elemental analysis. Further benzophenone tagged indole acetohydrazides and 2,5-disubstituted 1,3,4-oxadiazoles were evaluated for antioxidant potential, through different in vitro models such as DPPH, nitric oxide and hydrogen peroxide methods. In the series of compounds some of them had shown good to moderate in vitro antioxidant potential compare to the standard drug ascorbic acid in all the above three methods.     

Melatonin: New Places in Therapy

The fact that the full extent of the function of the pineal gland has not yet been elucidated, has stimulated melatonin research worldwide. This review introduces melatonin’s mechanism of action, direct and indirect antioxidant actions as well as the antioxidant properties of its metabolites, 6-hydroxymelatonin (6-OHM) and N-acetyl-N-formyl-5-methoxykynurenamine (AFMK). At present the mechanism of action is proposed to be receptor-, protein- and nonprotein-mediated. From its popular role in the treatment of jetlag, melatonin is now implicated in the reduction of oxidative stess, both as a free radical scavenger and antioxidant. Melatonin’s direct scavenging action in respect of the following will be discussed: superoxide anions, hydrogen peroxide, hydroxyl radicals, singlet oxygen, peroxy radicals and nitric oxide/peroxy nitrite anions. In addition melatonin also possesses indirect antioxidant activity and the role of its metabolites, AFMK and 6-OHM will be presented. It is these free radical scavenging and antioxidant properties of melatonin that has shifted the focus from that of merely strengthening circadian rhythms to that of neuroprotectant: a new place in therapy.     

Fc-receptor-mediated intracellular delivery of Cu/Zn-superoxide dismutase (SOD1) protects against redox-induced apoptosis through a nitric oxide dependent mechanism

BACKGROUND: Using specific antibodies against bovine Cu/Zn-superoxide dismutase (EC 1.15.1.1, SOD1) we demonstrated that anti-SOD antibodies (IgG1) are able to promote the intracellular translocation of the antioxidant enzyme. The transduction signalling mediated by IgG1 immune complexes are known to promote a concomitant production of superoxide and nitric oxide leading to the production of peroxynitrites and cell death by apoptosis. The Fc-mediated intracellular delivery of SOD1 thus limited the endogenous production of superoxide. It was thus of interest to confirm that in the absence of superoxide anion, the production of nitric oxide protected cells against apoptosis. Study in greater detail clearly stated that under superoxide anion-free conditions, nitric oxide promoted the cell antioxidant armature and thus protected cells against redox-induced apoptosis. MATERIALS AND METHODS: The murine macrophage cell-lines J774 A1 were preactivated or not with interferon-gamma and were then stimulated by IgG1 immune complexes (IC), free SOD1 or SOD1 IC and superoxide anion, nitric oxide, peroxynitrite, and tumor necrosis factor-alpha (TNF-alpha) production was evaluated. The redox consequences of these activation processes were also evaluated on mitochondrial respiration and apoptosis as well as on the controlled expression of the cellular antioxidant armature. RESULTS: We demonstrated that SOD1 IC induced a Fcgamma receptor (FcgammaR)-dependent intracellular delivery of the antioxidant enzyme in IFN-gamma activated murine macrophages (the J774 AI cell line). The concomitant stimulation of the FcyR and the translocation of the SOD1 in the cytoplasm of IFN-gamma-activated macrophages not only reduced the production of superoxide anion but also induced the expression of the inducible form of nitric oxide synthase (iNOS) and the related NO production. This inducing effect in the absence of superoxide anion production reduced mitochondrial damages and cell death by apoptosis and promoted the intracellular antioxidant armature. CONCLUSIONS: To define the pharmacologic mechanism of action of bovine SOD1, we attempted to identify the second messengers that are induced by SOD1 IC. In this work, we propose that Fc-mediated intracellular delivery of the SOD1 that reduced the production of superoxide anion and of peroxynitrite, promoted a NO-induced protective effect in inducing the antioxidant armature of the cells. Taken together, these data suggested that specific immune responses against antigenic SOD1 could promote the pharmacological properties of the antioxidant enzyme likely via a NO-dependent mechanism.     

Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP+-dependent isocitrate dehydrogenase and superoxide dismutase

It has been reported that chronic alcohol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. Several studies have shown the importance of superoxide dismutase (SOD) in protecting cells against ethanol-induced oxidative stress. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. In this report, we demonstrate that ethanol induces the peroxynitrite-mediated cytotoxicity in HepG2 cells through inactivation of antioxidant enzymes such as ICDH and SOD. Upon exposure to 100mM ethanol for 3days to HepG2 cells, a significant decrease in the viability and activities of ICDH and SOD was observed. The ethanol-induced inactivation of antioxidant enzymes resulted in the cellular oxidative damage and modulation of redox status as well as mitochondrial dysfunction in HepG2 cells. The cytoxicity of ethanol and inactivation of antioxidant enzymes were effectively protected by manganeses(III) tetrakis(N-methyl-2-pyridyl) porphyrin, a manganese SOD mimetic, and N'-monomethyl-l-arginine, a nitric oxide synthase inhibitor. These results indicate that ethanol toxicity is mediated by peroxynitrite and the peroxynitrite-mediated damage to ICDH and SOD may be resulted in the perturbation of the cellular antioxidant defense systems and subsequently lead to a pro-oxidant condition.     

Caffeic acid phenethyl ester reduces neurovascular inflammation and protects rat brain following transient focal cerebral ischemia

Ischemic stroke is a neurovascular disease treatable by thrombolytic therapy, but the therapy has to be initiated within 3 h of the incident. This therapeutic limitation stems from the secondary injury which results mainly from oxidative stress and inflammation. A potent antioxidant/anti-inflammatory agent, caffeic acid phenethyl ester (CAPE) has potential to mitigate stroke's secondary injury, and thereby widening the therapeutic window. We observed that CAPE protected the brain in a dose-dependent manner (1-10 mg/kg body weight) and showed a wide therapeutic window (about 18 h) in a rat model of transient focal cerebral ischemia and reperfusion. The treatment also increased nitric oxide and glutathione levels, decreased lipid peroxidation and nitrotyrosine levels, and enhanced cerebral blood flow. CAPE down-regulated inflammation by blocking nuclear factor kappa B activity. The affected mediators included adhesion molecules (intercellular adhesion molecule-1 and E-selectin), cytokines (tumor necrosis factor-alpha and interleukin-1beta) and inducible nitric oxide synthase. Anti-inflammatory action of CAPE was further documented through reduction of ED1 (marker of activated macrophage/microglia) expression. The treatment inhibited apoptotic cell death by down-regulating caspase 3 and up-regulating anti-apoptotic protein Bcl-xL. Conclusively, CAPE is a promising drug candidate for ischemic stroke treatment due to its inhibition of oxidative stress and inflammation, and its clinically relevant wide therapeutic window.     

In vitro antioxidant studies in leaves of Annona species

Antioxidant potential of leaves of three different species of Annona was studied by using different in vitro models eg., 1,1-diphenyl-2-picryl hydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothizoline-6-sulphonate) (ABTS), nitric oxide, superoxide, hydroxy radical and lipid peroxidation. The ethanolic extract of A. muricata at 500 microg/ml showed maximum scavenging activity (90.05%) of ABTS radical cation followed by the scavenging of hydroxyl radical (85.88%) and nitric oxide (72.60%) at the same concentration. However, the extract showed only moderate lipid peroxidation inhibition activity. In contrast, the extract of A. reticulata showed better activity in quenching DPPH (89.37%) and superoxide radical (80.88%) respectively. A.squamosa extract exhibited least inhibition in all in vitro antioxidant models excepting hydroxyl radical (79.79%). These findings suggest that the extracts of A. muricata possess potent in vitro antioxidant activity as compared to leaves of A. squamosa and A. reticulata suggesting its role as an effective free radical scavenger, augmenting its therapeutic     

Acute effects of a partially purified fraction from garlic on plasma glucose and cholesterol levels in rats: putative involvement of nitric oxide

Garlic has been extensively used as a medicinal plant. Most of its numerous beneficial effects such as antioxidant, antibacterial, antitumoral involve sulfur-derived amino acids. In the present work, we reevaluated the acute effects of aqueous extract of garlic on plasma glucose and cholesterol levels in normal rats. Control (vehicle H2O) or garlic extract-treated group at 100-120 mg protein/kg body wt were intraperitoneally injected (IP) and glucose, cholesterol, insulin and nitric oxide metabolites levels were determined after a short-term duration of 6 h. We confirmed that garlic contained an active fraction, exerting both glucose and cholesterol-lowering activity. The glucose-lowering effect was triggered by an increase in insulinemia. Preliminary study indicated that the active agent was different from S-allyl-cysteine-sulfoxide, the active principle implicated in hypoglycaemic and hypolipidemic effects of garlic or arginine. The mechanism of action seemed to involve nitric oxide (NO), which increased time and dose-dependently. The garlic effects were abolished by diphenyleneiodonium chloride (DPI = 1 mg/kg body wt), a specific inhibitor of NO production, suggesting the involvement of constitutive nitric oxide synthase.     

Nitric oxide as a mediator of inflammation?—You had better believe it

Nitric oxide has enigmatic qualities in inflammation. In order to appreciate the precise contributions of nitric oxide to a pathophysiological process, one must account for enzyme source, coproduction of oxidants and antioxidant defences, time, rate of nitric oxide production, cellular source, peroxynitrite formation and effects on DNA (mutagenesis/apoptosis). We contend that there is ample evidence to consider nitric oxide as a molecular aggressor in inflammation, particularly chronic inflammation. Therapeutic benefit can be achieved by inhibition of inducible nitric oxide synthase and not the donation of additional nitric oxide. Furthermore, there is growing appreciation that nitric oxide and products derived thereof, are critical components linking the increased incidence of cancer in states of chronic inflammation.     

Protective effect of resveratrol on acute endotoxemia-induced nephrotoxicity in rat through nitric oxide independent mechanism

Lipopolysaccharide (LPS) is a glycolipid component of the cell wall of gram negative bacteria inducing deleterious effects on the kidney. Endotoxemia-induced nephrotoxicity is characterized by disturbed intracellular redox balance and reactive oxygen species (ROS) accumulation leading to DNA, proteins and membrane lipid damages. Resveratrol (trans-3,5,4′-trihydroxystilbene) is a polyphenol displaying antioxidant and anti-inflammatory properties. This study investigated its effects on LPS-induced nephrotoxicity in rats. Resveratrol counteracted all LPS-induced changes in renal haemodynamic parameters. In the kidney resveratrol abrogated LPS-induced lipoperoxidation and antioxidant enzyme activities depletion as superoxide dismutase (SOD) and catalase (CAT) but not peroxidase (POD) activity. LPS increased plasma and urine nitric oxide (NO) level and resveratrol reversed them. More importantly, LPS-induced iron mobilization from plasma to kidney, which was also abolished by resveratrol treatment. All these results suggest that resveratrol exerted strong antioxidant properties against LPS-induced nephrotoxicity and that its mode of action seemed to involve iron shuttling proteins.