Blond and blood juice supplementation in high fat diet fed mice: effect on antioxidant status and DDAH/ADMA pathway.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease spread throughout the world. The most frequent causes of death in NAFLD patients are due both to liver and cardiovascular damage. Several pathways, including the dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway, are involved in the pathogenesis of NAFLD. It has been reported that ADMA plasmatic levels are increased in patients with hepatic dysfunction such as NAFLD. Although many studies demonstrated that some foods are effective in the treatment of NAFLD, few studies have evaluated their effects with respect to the prevention of the disease. It has been reported that sweet orange juice (OJ) consumption may be associated with potential health benefits. However, some varieties of sweet orange are more effective than others. The aim of the present paper was to investigate the effect of blond and blood sweet orange juice in prevention of NAFLD by evaluating its ability to improve liver steatosis in mice with diet-induced obesity, reducing oxidative stress and affecting the DDAH/ADMA pathway. Results obtained in our experimental conditions evidenced that blood orange juice rather than blond orange juice was more effective. Blood orange juice or blond orange juice enriched in anthocyanins may represent a promising dietary option for the prevention of fatty liver disease.

Oxidative profile in patients with colon cancer: effects of Ruta chalepensis L.

AIM: To verify the involvement of free radicals in tumor progression and to investigate the effects of an ethanolic extract of Ruta Chalepensis L. and of rutin in blood of patients with colon cancer. MATERIALS AND METHODS: Leaves of Ruta Chalepensis L. were collected in the area around Catania (Italy). For the preparation of the ethanol extract of leaves, an exhaustive extraction of 100 g of the drug was carried out in Soxhlet with 800 ml of 95% ethanol. Fifty-six patients with colorectal cancer were randomly selected for this study; among these, 34 were affected by an early stage (T1 N0 M0 according to scale), while 22 were affected by an advanced stage (T4, N1-2, M0) of cancer. Data obtained from these patients were compared with those of a control group consisting of 20 healthy subjects. Plasma of each sample was used for determining non-proteic antioxidant capacity, thiol groups, lipid hydroperoxides and nitrite/nitrate levels, evaluated by spectrophotometric tests. In addition, percentage of haemolysis was evaluated incubating (for 2 hours at 37 degrees C) erythrocyte suspension with a free radical donor (50 mM 2,2'-azobis-amidino propane chloridrate), in the presence or absence of ethanolic extract of Ruta Chalepensis L. (250 microg/ml) or rutin (1 mM). RESULTS: Non-proteic antioxidant capacity was significantly lower in cancerous patients than in healthy subjects (p < 0.001). This decrease was stage-related. In fact, non-proteic antioxidant capacity resulted lower in advanced than in early colorectal cancer (p < 0.001). The same significant stage-related decrease was observed in plasma thiol groups (p < 0.001). Coherently with the decrease in non-proteic antioxidant capacity and thiol groups, higher levels of lipid hydroperoxides and nitrite/nitrate were observed in patients with colorectal cancer with respect to healthy subjects (p < 0.001) and the increase in these markers of oxidative stress was related to the cancer stadiation. Neoplastic patients also showed an increased percentage of oxidative hemolysis respect to controls and the haemolytic damage was correlated with the stage of colon cancer. Both the extract of Ruta Chalepensis L. and rutin were able to protect erythrocytes from oxidative stress induced by the free radical donor, but the extract of Ruta Chalepensis L. was more effective than rutin. This protective effect was significant only in erythrocytes from patients with early colorectal group, whereas no significant modification was induced by Ruta Chalepensis L. or rutin in red blood cells from advanced colorectal cancer patients exposed to the same experimental conditions. CONCLUSION: Oxidative stress correlates with colon cancer stadiation and both the extract of Ruta chalepensis and rutin are able to protect red blood cells from radical-induced damage. However, their effects are significant in early stages of cancer. So these natural antioxidants might be usefull to prevent carcinogenesis and/or tumor progression.

Pharmacological induction of heme oxygenase-1 inhibits iNOS and oxidative stress in renal ischemia-reperfusion injury.

Nitric oxide (NO), produced by nitric oxide synthase, is implicated in the pathophysiology of renal ischemia/reperfusion (I/R) injury. This study sought to elucidate the impact of pharmacological induction of heme oxygenase-1 (HO-1) on renal I/R injury. Rats were subjected to 45 minutes of renal ischemia followed by various times of reperfusion (30 minutes, 1 hour, or 3 hours). Plasma from sacrificed rats was obtained, and the kidneys processed for the expression of iNOS, cleaved caspase-3, p38MAPK and for immunohistochemical analysis. Furthermore, we determined renal and plasma levels of lipid hydroperoxides, total thiol groups, and plasmatic NO2-/NO3- formation. Our results showed a time-dependent increase in iNOS expression, which was also confirmed by increased plasma formation of NO2-/NO3-. Interestingly, this effect was reversed by pretreatment (12 hours) with SnCl2, a potent and specific inducer of renal HO-1 expression and activity, or by intraperitoneal injection of biliverdin (10 mg/kg). Furthermore, we observed a concomitant reduction in plasma and renal LOOH formation, a normalization of renal total thiol content, a reduction of caspase-3-mediated apoptosis, and a significant increase in p38MAPK phosphoration. Taken together, these results suggested that HO-1 and its byproduct biliverdin play major roles in the pathophysiological cascade leading to renal I/R injury.

Imidazole derivatives as antioxidants and selective inhibitors of nNOS.

The reperfusion of ischemic tissue often delays its physiological and functional recovery; this paradoxical effect is ascribed to increased release of free radicals including O(2)(-) and NO. For these reasons, scavenging reactive oxygen species or inhibition the NO synthesis has been shown to result in an enhanced neuronal survival after cerebral ischemia. Many authors believe that therapy for stroke patients would be a cocktail of drugs with various mechanisms of action. Combination therapy is a difficult and complicated avenue for drug development because of the possibility of drug-drug interactions. An alternative approach would be to combine multiple activities within the same compound. In consideration of the free-radical scavenging and inhibitory effect on NOS of various natural and synthetic compounds, the aim of this study was to analyze the antioxidant properties of some imidazole derivatives previously synthesized in our laboratory. Results obtained in the present study provide evidence that tested compounds exhibit interesting antioxidant properties, expressed either by their capacity to scavenge free radicals or their ability to reduce lipid peroxidation. In particular, compounds A and B represent chemical structures which can be easily modified to improve the observed antioxidant properties and to provide new therapeutic strategies focused on multiple downstream events.

Effects of norepinephrine depletion in rats during cerebral post-ischemic reperfusion.

The present paper reports the effects of norepinephrine depletion in rats, after treatment with N-(2-chloroethyl)-N-ethyl 2-bromobenzylamine (DSP-4) neurotoxin, on partial cerebral ischemia and reperfusion. Histological observations made under experimental conditions of noradrenergic (NA)-depletion demonstrated that neuronal lesions were not exacerbated; in fact, in DSP-4-treated ischemic animals, a minor number of neurons appeared damaged. Our results suggest that neuronal recovery after post-ischemic reperfusion is not affected by NA-depletion. DSP-4 neurotoxin does not induce 5-hydroxy-triptamine (5-HT) depletion.

Novel inhibitors of neuronal nitric oxide synthase.

Selective inhibitors of neuronal nitric oxide synthase (nNOS), which are devoid of any effect on the endothelial isoform (eNOS), may be required for the treatment of some neurological disorders. In our search for novel nNOS inhibitors, we recently described some 1-[(Aryloxy)ethyl]-1H-imidazoles as interesting molecules for their selectivity for nNOS against eNOS. This work reports a new series of 1-[(Aryloxy)alkyl]-1H-imidazoles in which a longer methylene chain is present between the imidazole and the phenol part of molecule. Some of these molecules were found to be more potent nNOS inhibitors than the parent ethylenic compounds, although this increase in potency resulted in a partial loss of selectivity. The most interesting compound was investigated to establish its mechanism of action and was found to interact with the tetrahydrobiopterin (BH(4)) binding site of nNOS, without interference with any other cofactors or substrate binding sites.

Progress in the development of selective nitric oxide synthase (NOS) inhibitors.

Nitric oxide (NO), a molecular messenger synthesized by nitric oxide synthase (NOS) from L-arginine and molecular oxygen, is involved in a number of physiological and pathological processes in mammalians. Three structurally distinct isoforms of NOS have been identified: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). Although NO mediates several physiological functions, overproduction of NO by nNOS has been reported in a number of clinical disorders including acute (stroke) and chronic (schizophrenia, Alzheimer s, Parkinson s and AIDS dementia) neurodegenerative diseases, convulsions and pain; overproduction of NO by iNOS has been implicated in various pathological processes including septic shock, tissue damage following inflammation and rheumatoid arthritis. On the contrary, NO produced by eNOS has only physiological roles such as maintaining physiological vascular tone. Accordingly, selective inhibition of nNOS or iNOS vs eNOS may provide a novel therapeutic approach to various diseases; in addition selective inhibitors may represent useful tools for investigating other biological functions of NO. For these reasons, after the identification of N-methyl-L-arginine (L-NMA) as the first inhibitor of NO biosynthesis, design of selective NOS inhibitors has received much attention. In this article the recent developments of new molecules endowed with inhibitory properties against the various isoforms of NOS are reviewed. Major focus is placed on structure-activity-selectivity relationships especially concerning compounds belonging to the non-amino acid-based inhibitors.

Expression of bone morphogenetic protein-6 and transforming growth factor-beta1 in the rat brain after a mild and reversible ischemic damage.

We have examined the distribution of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-6 (BMP-6) in the brain of rats subjected to a mild and reversible ischemic damage produced by a 20-min occlusion of both carotid arteries without occlusion of the vertebral arteries. We have selected this model to study how the expression of trophic factor of the TGF-beta superfamily changes in neurons that recover from a transient insult. Immunocytochemical analysis showed a loss of TGF-beta1 in neurons of all hippocampal subfields immediately after the ischemic period, followed by a recovery of immunoreactivity in CA1 and CA3 neurons after reperfusion. BMP-6 immunoreactivity was also lost in most hippocampal neurons, but immunostaining became particularly intense in the interstitial space after both ischemia and reperfusion. An interstitial localization of BMP-6 was also observed in the cerebral cortex, particularly after reperfusion. Mild ischemia also induced substantial changes in the expression of TGF-beta1 and BMP-6 within the cerebellar cortex. In control animals, these factors appeared to be localized in granule cells (TGF-beta1) and Purkinje cells (both), whereas the molecular layer was not immunopositive. Both TGF-beta1 and BMP-6 were highly expressed in the interstitial spaces of the cerebellar cortex either 20 min after ischemia or 20 min after reperfusion. Taken collectively, these results suggest that a mild and reversible ischemia stimulates the release of BMP-6 from neurons into the interstitial space. We speculate that BMP-6, besides functioning during brain development, may also regulate neuronal resistance to insults of the adult brain.

Inhibition of neuronal nitric oxide synthase by N-phenacyl imidazoles.

Nitric oxide (NO) mediates a series of physiological processes, including regulation of vascular tone, macrofage-mediated neurotoxicity, platelet aggregation, learning and long-term potentiation, and neuronal transmission. Although NO mediates several physiological functions, overproduction of NO can be detrimental and play multiple roles in several pathological diseases. Accordingly, more potent inhibitors, more selective for neuronal nitric oxide synthase (nNOS) than endothelial NOS (eNOS) or inducible NOS (iNOS), could be useful in the treatment of cerebral ischemia and other neurodegenerative diseases. We recently described the synthesis of a series of imidazole derivatives. Among them N-(4-nitrophenacyl) imidazole (A) and N-(4-nitrophenacyl)-2-methyl-imidazole (B) were considered selective nNOS inhibitors. In the present study the action mechanism of compounds A and B was analyzed. Spectral changes observed in the presence of compound A indicate that this inhibitor exerts its effect without interaction with heme iron. Moreover compounds A and B, inhibit nNOS "noncompetitively" versus arginine, but "competitively" versus BH(4).

Fibroblast growth factor-2 and transforming growth factor-beta1 immunostaining in rat brain after cerebral postischemic reperfusion.

Several trophic factors are known to regulate the survival and growth of neurons in brain and peripheral tissues. Several findings suggest that basic fibroblast growth factor-2 (FGF-2) plays an important role in the "self-repair" responses that follow injuries such as trauma and brain ischemia and that FGF-2 contributes to the repair of damaged tissue. Transforming growth factor-beta (TGF-beta) is a potent growth-regulatory protein secreted by virtually all cells. In the present study, we used immunohistochemical techniques to investigate whether FGF-2 and TGF-beta1 participate in the healing of damaged tissue following partial brain ischemia. The profile of the observed immunoreactivities indicated that TGF-beta1 and FGF-2 release varies between the different cerebral areas subjected to ischemic insult. Moreover, the sectorial heterogeneity of immunocytochemical response suggests that, during postischemic reperfusion, neuronal recovery may be due not only to neuron-glia interaction but also to neurochemical conditions involving inhibitory interneurons.

Bioflavonoids as antiradicals, antioxidants and DNA cleavage protectors.

Flavonoids have recently aroused considerable interest because of their broad pharmacological activity. In fact, flavonoids have been reported to have antiviral, antiallergic, antiplatelet, anti-inflammatory and antitumoral activities. The pharmacological properties of bioflavonoids have been ascribed both to the concomitant inhibition of enzymes involved in the production of free radicals and to their free-radical scavenging and iron chelating capacity. However the antioxidant capacity of bioflavonoids due to free-radical scavenging and/or to iron chelating is still controversial. In this study, we have investigated the free-radical scavenging capacity of bioflavonoids (rutin, catechin, and naringin). In addition, the effects of these polyphenols on xanthine oxidase activity, spontaneous lipid peroxidation, and DNA cleavage were investigated. The bioflavonoids under examination showed a dose-dependent free-radical scavenging effect, a significant inhibition of xanthine oxidase activity, and an antilipoperoxidative capacity. In addition, they showed a protective effect on DNA cleavage.

L -propionyl-carnitine as superoxide scavenger, antioxidant, and DNA cleavage protector.

L-Propionylcarnitine, a propionyl ester of L-carnitine, increases the intracellular pool of L-carnitine. It exhibits a high affinity for the enzyme carnitine acetyltransferase (CAT) and, thus, is readily converted into propionyl-coenzyme A and free carnitine. It has been reported that L-propionylcarnitine possesses a protective action against heart ischemia-reperfusion injury; however, the antioxidant mechanism is not yet clear. L-Propionylcarnitine might reduce the hydroxyl radical production in the Fenton system, by chelating the iron required for the generation of hydroxyl radicals. To obtain a better insight into the antiradical mechanism of L-propionylcarnitine, the present research analyzed the superoxide scavenging capacity of L-propionylcarnitine and its effect on linoleic acid peroxidation. In addition, the effect of L-propionylcarnitine against DNA cleavage was estimated using pBR322 plasmid. We found that L-propionylcarnitine showed a dose-dependent free-radical scavenging activity. In fact, it was able to scavenge superoxide anion, to inhibit the lipoperoxidation of linoleic acid, and to protect pBR322 DNA from cleavage induced by H2O2 UV-photolysis.

Lipophilic methotrexate conjugates with antitumor activity.

Lipophilic methotrexate (MTX)-lipoamino acid conjugates coupled with amide or ester linkages (1a-1r) were synthesised. The inhibitory activity of the conjugates was evaluated on bovine liver DHFR. The in vitro growth inhibitory effect against MTX-sensitive human lymphoblastoid CCRF-CEM cells and an MTX-resistant sub-line (CEM/MTX), which displays defective intracellular transport of MTX, was determined under short-term and continuous (120-h incubation) exposure conditions. The alpha, gamma, or alpha,gamma amide conjugates showed different activity in inhibiting the growth of parent cells. CEM/MTX cells were much less susceptible than CCRF-CEM cells to inhibition by alpha or alpha,gamma-substituted lipoamino acid conjugates, whereas both cell lines were almost equally sensitive to the MTX-gamma conjugates. Although less potent than MTX, they could partially circumvent the impaired transport system. These findings confirm that lipophilic MTX conjugates may be good lead compounds on the drug development for the treatment of some MTX-resistant tumors. Ester-type conjugates displayed an interesting activity against parent CCRF-CEM cells, although they were less potent against the transport-resistant sub-line. Stability studies on these molecules indicated that they are not degraded into MTX in the culture medium, thus suggesting that they are not able to over-cross cell resistance despite of their lipophilicity.

Nitric oxide synthetase activity in cerebral post-ischemic reperfusion and effects of L-N(G)-nitroarginine and 7-nitroindazole on the survival.

Nitric Oxide (NO) mediates a series of physiological processes including regulation of vascular tone, macrophage-mediated cytotoxicity, platelet aggregation, learning and long-term potentiation, neuronal transmission. Although NO mediates several physiological functions, overproduction of NO can be detrimental and play multiple roles in the pathophysiology of focal cerebral ischemia. In the present study NOS activities were evaluated in cerebellum and cerebral cortex of ischemic and post-ischemic reperfused rats using an experimental model of partial cerebral ischemia; moreover, the effects of L-N(G)Nitroarginine (NA, nonselective NOS inhibitor) or 7-Nitroindazole (7-NI, selective neuronal NOS inhibitor) administration were assayed on percentage survival of ischemic rats. An increase of NOS activity in the cerebellum and in cerebral cortex of ischemic and post-ischemic reperfused rats was observed. NA administration failed to induce neuroprotective effects, by increasing percentage of mortality of treated ischemic rats with respect to control group. In contrast, the treatment with the selective neuronal NOS inhibitor, 7-NI, induced a significant neuroprotective effect.

Antioxidant systems in rat lens as a function of age: effect of chronic administration of vitamin E and ascorbate.

Oxidative damage occurring in the lenses of patients with senile cataract may be due to partially reduced forms of oxygen. We assayed the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Red), and glucose-6-phosphate dehydrogenase (G6PD) in rat lenses at different ages (1, 4, and 24 months), and also evaluated lens glutathione (GSH) levels and the effects of chronic administration of vitamin E and sodium ascorbate. We observed a significant age-related decrease in GSH-Px, GSH-Red and G6PD activities, but no age-related change in SOD activity. Chronic treatment with both vitamin E and sodium ascorbate failed to restore enzymatic activities to the levels of younger rats. An age-related reduction in GSH content was also observed; however, chronic administration of vitamin E, but not of sodium ascorbate, restored GSH levels to those of younger rats.

Effects of glutathione depletors on post-ischemic reperfusion in rat brain.

The present paper reports the effects of GSH depletion (diethylmaleate induced) on partial cerebral ischemia and reperfusion for 7 and 20 days. Our results confirm that there is a paradoxical protective effect of the GSH-depletor and suggest an improved neuronal trophism induced by diethylmaleate treatment.

GFAP, S-100 and vimentin proteins in rat after cerebral post-ischemic reperfusion.

In the present study astrocytes reactivity during cerebral post-ischemic reperfusion was evaluated immunocytochemically by using antibodies to vimentin, glial fibrillary acidic protein (GFAP) and S-100 protein. At the 7th day of post-ischemic reperfusion few GFAP-positive cells were observed in the hippocampus and cerebellum, the number of GFAP-positive cells increased slightly after 20 days of reperfusion. This poor GFAP-positivity may be due to the inhibition of GFAP polymerization by S-100; in fact, S-100 immuno-reactivity was already evident from the 7th day. Vimentin immuno-staining was evident both at the 7th and 20th day of reperfusion in microglial cells and in oligodendrocytes, suggesting that these cells are involved in the recovery of neurons following brain injury.

Ornithine decarboxylase activity in cerebral post-ischemic reperfusion damage: effect of methionine sulfoximine.

Excessive activation of glutamate receptors via the N-methyl-D-aspartate (NMDA) subtype appears to play a role in the sequence of cellular events which lead to irreversible ischemic damage to neurons. Furthermore, NMDA receptor activation induces a stimulation of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine (PA) biosynthesis. In order to better understand the role of PA we have measured ODC activity and the effect of methionine sulfoximine (MSO), a molecule able to stimulate ODC, on a model of transient cerebral ischemia. There was a significant increase in ODC activity in the rat cerebral cortex during post-ischemic reperfusion. The treatment with MSO induced a significant decrease in cerebral glutamine synthetase activity accompanied by a marked increase in ODC activity. In MSO-pretreated rats there was a significant decrease in the survival rate when compared to untreated ischemic rats.

MAP2, synaptophysin immunostaining in rat brain and behavioral modifications after cerebral postischemic reperfusion.

Plasticity in the central nervous system after cerebral ischemia is a controversial issue; focal cerebral ischemia produces an area of infarction that is surrounded by neurons that may respond to nearby damage by creating new synapses. In the present study the expression of the postsynaptic microtubule-associated protein 2 (MAP2) and the presynaptic marker protein, synaptophysin, was investigated by immunocytochemical techniques in the CA1 sector of hippocampus and in cerebellum of rats made ischemic by bilateral clamping of common carotid arteries and reperfused for 7 and 30 days. In addition, ischemia-induced behavioral alterations were also evaluated after 7 and 30 days of reperfusion. The present study demonstrates a decreased postsynaptic MAP2 immunoreactivity, representative of neuronal loss, particularly in CA1 sector of hippocampus and in cerebellum of ischemic rats reperfused for 7 days. After 30 days of reperfusion, MAP2 immunostaining was similar to control. In the same brain sections an increased presynaptic synaptophysin immunoreactivity has been observed only after 30 days of reperfusion. These data suggest compensatory regenerative changes associated with synaptic remodelling and are supported by behavioral recovery observed under the same experimental conditions.