Pharmacologically induced ischemia-reperfusion syndrome in the rat small intestine.

BACKGROUND: Reports of ischemia-reperfusion (I/R) injury in vivo describe experiments in which lesions are induced by the physical procedure of clamping, (I/RIC). We compare this procedure with a pharmacologic technique in which I/R injury is induced by drug superfusion (I/RID). MATERIALS AND METHODS: We used rat intestine to determine whether the responses provoked by I/RIC, such as changes in reactive oxygen species (ROS) and nitric oxide levels, are also provoked by I/RID. To this end, rats were treated with allopurinol, SOD, catalase, L-NAME, and L-arginine. In both I/R models ischemia was maintained for 60 min, followed by 30 min of reperfusion. RESULTS: In both ischemia models, we observed significant differences in Evans blue (vascular permeability) and LDH (tissue injury) concentrations during the reperfusion period compared with the control group. I/RIC always induced greater injury. However, proportionally, the degree of protection was similar in the two models for the different treatments assayed. This indicates that the pathophysiologic mechanisms are the same. CONCLUSIONS: Our I/RID model induces a significant intestinal alteration during the reperfusion period and, also in general terms, this alteration is prevented or worsened in a similar and proportional way to that observed when using the classic I/RIC model. The I/RID model helps to explain the development and evolution of pathologies characterized by the induction of intermittent vasospasms that produce transitory reductions in vascular perfusion, which in turn can generate ROS though an I/R mechanism.

Role of oxidative stress and adenosine nucleotides in the liver of aging rats.

We studied the response of several parameters related to oxidative stress in the liver of aging rats. Male Wistar rats aged 1.5, 3, 18 and 24 months were used. Livers showed an increase in superoxide anion (O(2)(-)) concentration at 1.5 and 18 months of age compared to the 3-month-old group; a decrease in superoxide dismutase (SOD) was seen at 1.5 months and catalase concentrations remained unaltered throughout the aging process. Nitric oxide (NO) progressively declined with age; a significant decrease was particularly apparent at 18 and 24 months of age. Thiobarbituric acid reactive substances (TBARS) decreased significantly at 1.5 months, whereas it increased at 18 and 24 months of age. Concentrations of prostaglandin E(2) (PGE(2)), and adenine nucleotides, and their metabolites, remained unchanged throughout the aging process. Although the mitochondrial damage caused by oxidative stress can result in reduced ATP production and compromised cell function, our results on adenosine nucleotides and their metabolites support the notion that the integrity of mitochondria and enzymatic activity remain mostly unchanged with aging. In conclusion, we observed a significant decrease in the levels of NO in the older groups of rats and hence in its antioxidant activity. This could explain the observed increase in lipid peroxides which suggests an important role for NO in oxidative stress in the liver of older rats.

Toxicity and antioxidant activity in vitro and in vivo of two Fucus vesiculosus extracts.

The consumption of seaweeds has increased in recent years. However, their adverse and beneficial effects have scarcely been studied. Two extracts from the brown seaweed Fucus vesiculosus containing 28.8% polyphenols or 18% polyphenols plus 0.0012% fucoxanthin have been obtained and studied to determine their toxicity in mice and rats and also their antioxidant activity. Both extracts were shown to lack any relevant toxic effects in an acute toxicity test following a 4 week daily treatment in rats. The extracts exhibited antioxidant activity in noncellular systems and in activated RAW 264.7 macrophages, as well as in ex vivo assays in plasma and erythrocytes, after the 4 week treatment in rats. Our ex vivo results indicated that compounds from extract 2 may be more easily absorbed and that the antioxidants in their parent or metabolized form are more active. These findings support the view that the daily consumption of F. vesiculosus extract 2 (Healsea) would have potential benefits to humans.

Loss of adaptation to oxidative stress asa mechanism for aortic damage in aging rats

Cells are armed with a vast repertoire of antioxidant defence mechanisms to preventthe accumulation of oxidative damage. The cellular adaptive response is animportant antioxidant mechanism against physiological and pathophysiologicaloxidative alterations in a cell’s microenvironment. The aim of this paper was to study,in the rat aorta, whether this adaptive response and the inflammation associated withoxidative stress were expressed throughout the aging process. We examined the rataorta, as it is a very sensitive tissue to oxidative stress. Male Wistar rats of 1.5, 3, 12,18 and 24 months of age were used. Superoxide anion (O2-) generation; levels of twoantioxidant enzymes, superoxide dismutase (SOD) and catalase; and the levels ofprostaglandin E2 (PGE2), an inflammatory marker, were measured. The results forrats at different ages were compared with those for 3 months of age. A balancebetween production of O2- and SOD activity was found in the aorta of rats from 1.5to 12 months old. Oxidative stress was present in the aorta of old animals (18-24months), due to a failure in the mechanisms of adaptation to oxidative stress. Theobserved increase in PGE2 levels in these rats reflected an inflammatory response. Alltogether suggest that vascular oxidative stress and the inflammatory process observedin the old groups of rats could be closely related to vascular aging. Our results alsoremark the importance of the adaptative response to oxidative stress (AU)

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Loss of adaptation to oxidative stress as a mechanism for aortic damage in aging rats.

Cells are armed with a vast repertoire of antioxidant defence mechanisms to prevent the accumulation of oxidative damage. The cellular adaptive response is an important antioxidant mechanism against physiological and pathophysiological oxidative alterations in a cell's microenvironment. The aim of this paper was to study, in the rat aorta, whether this adaptive response and the inflammation associated with oxidative stress were expressed throughout the aging process. We examined the rat aorta, as it is a very sensitive tissue to oxidative stress. Male Wistar rats of 1.5, 3, 12, 18 and 24 months of age were used. Superoxide anion (O2(-)) generation; levels of two antioxidant enzymes, superoxide dismutase (SOD) and catalase; and the levels of prostaglandin E2 (PGE2), an inflammatory marker, were measured. The results for rats at different ages were compared with those for 3 months of age. A balance between production of O2(-) and SOD activity was found in the aorta of rats from 1.5 to 12 months old. Oxidative stress was present in the aorta of old animals (18-24 months), due to a failure in the mechanisms of adaptation to oxidative stress. The observed increase in PGE2 levels in these rats reflected an inflammatory response. All together suggest that vascular oxidative stress and the inflammatory process observed in the old groups of rats could be closely related to vascular aging. Our results also remark the importance of the adaptative response to oxidative stress.

A flavonoid-rich diet increases nitric oxide production in rat aorta.

1. Red wine intake is associated with a low risk of cardiovascular disease. This effect has been partly attributed to the action of polyphenolic compounds, which decrease the oxidation of plasma low density lipoproteins. Moreover, nitric oxide ((*)NO) is a vasodilator and polyphenolic compounds induce endothelium-dependent vasorelaxation in vitro. 2. Here we studied whether a diet rich in dealcoholated red wine (DRW) increases acetylcholine-induced vasorelaxation and whether ingestion of DRW-, quercetin- or catechin-rich diets modifies the (*)NO-cyclic guanosine-3',5'-monophosphate (cyclic GMP) pathway and superoxide anion (O2(.-)) release in aorta in a resting state in rats fed semi-purified diets containing either 35% (v w(-1)) DRW, 0.3% (w w(-1)) quercetin or 0.3% (w w(-1)) catechin for 10 days. 3. (*)NO-mediated vasorelaxation induced by acetylcholine was greater in rats fed the DRW-rich diet than in those that received the control diet. 4. Expression of endothelial (*)NO synthase (eNOS) was similar in the four dietary groups. The aortic rings of rats fed either the DRW-, quercetin-, or catechin-rich diets showed higher NOS activity, (*)NO production and cyclic GMP content than those of rats fed the control diet. No changes were observed in O2(.-) production. 5. In summary, diets rich in either DRW, quercetin or catechin induced endothelium-dependent vasorelaxation in rat aorta in a resting state through the enhancement of (*)NO production, without modifying O2(.-) generation, thus the bioavailability of (*)NO was increased. The increase in the (*)NO-cyclic GMP pathway explains the beneficial effect of flavonoids at vascular level.

Effects of oxidative stress and antioxidant treatments on eicosanoid synthesis and lipid peroxidation in long term human umbilical vein endothelial cells culture.

We analyzed the influence of oxidative stress and agents that modify its effect in human umbilical vein endothelial cell cultures (HUVEC). The parameters analyzed were PGI2, TXA2, PGI2/TXA2 ratio, lipid peroxidation and cell viability. Oxidative stress was induced by H2O2. The agents (treatments) that were tested are: antioxidant enzymes (superoxide dismutase and catalase), oxygen free radical scavenger (vitamin E) and eicosanoids of the series 2 and 3 (Arachidonic acid, Eicosapentanoic acid). In this study we show, in long term endothelial cell cultures, the effects of different levels of oxidative stress alone or in combination with the different treatment agents, over the analyzed parameters. With induced oxidative stress alone the results obtained indicate that it has a harmful effect over cell function and viability, and that this effect is dose and time dependent. In absence of oxidative stress in basal situation, none of the treatments assayed showed significant differences compared to control cultures in the different analyzed parameters. When oxidative stress increased, antioxidant enzymes reduced cell damage and had a protective function, whereas Eicosapentanoic acid and vitamin E presented a lower level of protection. No beneficial effect was observed with arachidonic acid treatments. A significant increase in cell survival was observed in culture cells with oxidative stress when they were treated with antioxidant enzymes.

Free radical production in aortic rings from rats fed a fish oil-rich diet.

To study the effect of the degree of unsaturation of dietary fatty acids on the production of free radicals and on the vascular smooth muscle tone in rings of the aorta, Sprague-Dawley rats were fed a semipurified diet containing 5% lipids from either corn oil (CO) or menhaden oil (MO) for 8 wk. The MO diet did not change the basal or NADPH-dependent superoxide anion (O2-*) release. There were no significant differences in phenylephrine-induced contractions between the two groups in intact rings. However, these contractions increased in endothelium-intact aortic rings from the MO group after addition of the nitric oxide (*NO) synthase inhibitor NG-nitro-L-arginine and in endothelium-denuded rings, both indicating a greater endothelial basal *NO production in rats fed with the MO diet. Endothelium-dependent relaxations in response to acetylcholine were more prominent in rings from the MO group. These differences were not due to an increased smooth muscle response to.NO, because relaxations were the same using an exogenous *NO donor. Our results indicate that dietary MO did not modify O2-* release by the vessel wall or relaxation due to the cyclooxygenase pathway, but it potentiated endothelial production of *NO.

Protective effect of ozone treatment on the injury associated with hepatic ischemia-reperfusion: antioxidant-prooxidant balance.

The effects of ozone treatment on the injury associated to hepatic ischemia-reperfusion (I/R) was evaluated. Ozone treatment (1 mg/kg daily during 10 days by rectal insufflation) is shown to be protective as it attenuated the increases in transaminases (AST, ALT) and lactate levels observed after I/R. I/R leads to a decrease in endogenous antioxidant (SOD and glutathione) and an increase in reactive oxygen species (H2O2) with respect to the control group. However, ozone treatment results in a preservation (glutathione) or increase (SOD) in antioxidant defense and maintains H2O2 at levels comparable to those in the control group. The present study reports a protective effect of ozone treatment on the injury associated to hepatic I/R. The effectiveness of ozone could be related to its action on endogenous antioxidants and prooxidants balance in favour of antioxidants, thus attenuating oxidative stress.

The structural and electronical factors that contribute affinity for the time-dependent inhibition of PGHS-1 by indomethacin, diclofenac and fenamates.

PGHS-1 and PGHS-2 are the targets of nonsteroidal anti-inflammatory drugs (NSAIDs). It appears that the high degree of selectivity for inhibition of PGHS-2 shown by certain compounds is the result of two mechanisms (time-dependent and time-independent inhibition), by which they interact with each isoform. The fenamic acids can be divided into competitive inhibitors of substrate binding and competitive inhibitors that cause time-dependent losses of cyclooxygenase activity. The cyclooxygenase activity was measured by oxygen consumption following preincubation of the enzyme and the inhibitor for increasing periods of time. The rate constants associated with binding inhibition kinetics and structure-activity relationships were calculated for a large number of fenamates, diclofenac and indomethacin. The K1* values are similar but the individual rate constants are markedly different: K1 is two-fold lower, and k2 is six-fold slower for diclofenac than for indomethacin. All the active time-dependent compounds show MEPs with a negative conical surface, with their vertex on the minimum of the carboxyl group, which extends around the first aromatic ring to the central region. The conical surface keeps an open angle of 61 degrees or larger, and a close contact surface with the residues Ala527, Ileu523, Val349, and Ser530, in the zones surrounding the bridging amino group and the chlorine atoms for meclofenamate and diclofenac, or in the region around the carbonyl group for indomethacin. The K1* and IC50 values indicate that the interactions that promote the slow binding kinetics must be examined in relation to the reaction energies of formation (delta Hr) of an ionic bond between the deprotonated carboxylic acid group of acid NSAIDs with the monocationic guanidinum group of Arg120, the free energies of solvation in aqueous solution, and the molecular volumes measured. Presumably indomethacin, diclofenac and meclofenamate cause the enzyme to undergo a subtle conformational change to a form that binds compounds even more tightly, with some slight structural changes confined to reorientations of the Arg277 and Gln358 side chains. These results show that the model has reliably chosen regions of biological significance consistent with both the X-ray crystallographic and kinetic results.

Evidence that inhibitory neurotransmission differs between the proximal and distal segments of guinea-pig taenia caeci.

The effect of atropine (1 microM) and N(G)-nitro-L-arginine (L-NOARG, 10 microM) on electrical field stimulation induced relaxation in proximal and distal segments of guinea-pig taenia caeci in the presence of guanethidine (4 microM) was studied. The frequency-dependent relaxations were lower in proximal than in distal segments both in the presence and in the absence of atropine. The effect of L-NOARG (an inhibitor of nitric oxide (NO) synthase) on relaxation in the presence of atropine depended on the frequency of electrical stimulation and the segment used; the effect of L-NOARG was greater in proximal segments than in distal segments. In the absence of atropine, the inhibitory effect of L-NOARG was the same in both segments at all frequencies tested. This study demonstrates differences between the opposite extremes of guinea-pig taenia caeci in relaxations induced by electrical stimulation. Our data also show a role of NO that is dependent on the integrity of cholinergic transmission.

Influence of aging on thromboxane A2 and prostacyclin levels in rat hippocampal brain slices.

We have investigated the influence of age (3, 18, 24 months) on Thromboxane A2 (TXA2) and Prostacyclin (PGI2) levels in hippocampal slices from F344/NHSD rats. A significant increase in TXA2 and PGI2 levels was observed in 18 and 24 months old compared to 3 months old animals. A significant reduction in the ratio TXA2/PGI2 produced by a higher increase in PGI2 was observed in 24 month old animals. The reduction in the TXA2/PGI2 ratio has been related to vasodilatory and antiaggregating effects that may contribute to protect the brain against neuronal damage.

Oxygen-free radicals and nitric oxide are involved in the thrombus growth produced by iontophoresis of ADP.

Thrombi have been induced by iontophoretic application of ADP on the venules of the mesentery of male Wistar rats (250-350 g). We determined the thrombus growth which is a reflection of platelet recruitment. We have demonstrated the ability of two oxygen-free radical scavengers, superoxide dismutase (SOD), a superoxide anion scavenger, and catalase, a hydrogen peroxide scavenger, to reduce thrombus growth. Imidazole, a thromboxane synthase inhibitor also reduces the thrombus growth. Dimethylthiourea, a hydroxyl radical scavenger, does not alter the thrombus size. The administration of a NO synthase inhibitor, Ng-nitro-L-arginine methyl ester (L-NAME), sharply increased the volume of the thrombus. Our results show the implication of superoxide anion, hydrogen peroxide and nitric oxide in platelet recruitment. (c) 1998 The Italian Pharmacological Society.

Role of antioxidants in gastric mucosal damage induced by indomethacin in rats.

Reactive oxygen species play a role in the formation of gastric lesions induced by nonsteroidal antiinflammatory drugs. The present study was undertaken to determine whether endogenous antioxidants in gastric mucosa can protect it against the damaging effects of oxygen free radicals. This study examine oxygen free radical production (superoxide anions and hydrogen peroxide), gastric mucosa antioxidant defense mechanisms (glutathione, catalase, superoxide dismutase), the lesion-inducing effects of the generated oxygen free radicals (vascular permeability, lipid peroxidation) and gastric ulceration in rats treated orally with indomethacin at 10 mg/kg at 2 and 6 h after drug administration. Two hours after administration of the antiinflammatory drug, there was a sharp increase in production of oxygen free radicals in the gastric mucosa with no alteration in other parameters examined. Six hours after indomethacin administration the production of oxygen free radicals returned to basal levels, but there was a high degree of gastric ulceration and a significant increase in lipid peroxidation and vascular permeability together with decreases of 45% in glutathione concentration and 30% in catalase relative to the control group. These results suggest that like plasma, the gastric mucosa has an antioxidant capacity and only when this capacity is exhausted are the lesive effects of the oxygen free radicals manifested.

Iron metabolism and oxidative stress during acute and chronic phases of experimental inflammation: effect of iron-dextran and deferoxamine.

Iron overload induces a rise in lipid peroxidation, but there are no data on the effects of iron administered in vivo on the production of free radicals by inflammatory cells. Further, there is lack of agreement about the benefits of deferoxamine (Dfx) in the treatment of anemia and oxidative stress during inflammation and chronic diseases. In this study, iron-dextran (Fe-dextran) or Dfx was administered subcutaneously during the acute and chronic phases of carrageenan-induced granuloma. Several parameters related to iron metabolism, inflammatory cell activity, and lipid peroxidation were measured in liver, plasma, and the inflammatory exudate. Treatment with Fe-dextran increased iron content in plasma and in stores, increased production of superoxide anion (O2-) by inflammatory cells and lipid peroxidation, and also altered the inflammatory process. Dfx mobilized iron from stores without modifying essential parameters related to anemia or to the level of lipid peroxidation induced by inflammation. We conclude that treatment with Fe-dextran had a beneficial effect on recovery from the anemia of inflammation. Nevertheless, the high levels of loosely-bound iron found after Fe-dextran treatment in plasma and in exudate contribute to the increase in oxidative stress. Dfx treatment had no effect on anemia or on lipid peroxidation.

Prostacyclin, thromboxane, and oxygen free radicals and postoperative liver function in human liver transplantation.

The aim of this prospective study is to evaluate prostanoid (prostacyclin and thromboxane) and lipid peroxide levels at the portal and hepatic veins, and their relation to immediate postoperative liver function. Nineteen patients with liver cirrhosis undergoing orthotopic liver transplantation were prospectively studied. Blood samples were obtained within 5 min and 1 and 2 hr after reperfusion of the new liver, through a catheter placed at the portal vein in the recipient and another at the left hepatic vein in the donor liver. Plasma prostacyclin and thromboxane were analyzed by HPLC and RIA. The formation of lipid peroxides was determined and expressed in terms of thiobarbituric acid-reacting substances. Immediate postoperative liver function was evaluated using the transaminase levels within the first 48 hr and the early postoperative graft function score, as described previously. After reperfusion, only determinations at 5 min were related with liver function. Either prostacyclin (R = -0.61, P = 0.004) levels at the hepatic vein or prostacyclin production (subtraction between hepatic and portal vein levels) (R = -0.47, P = 0.04) correlated significantly with the early postoperative graft function score. Besides, there was a significant relationship between lipid peroxide production as measured by thiobarbituric acid-reacting substances and a worse early postoperative graft function score (R = 0.61, P = .005). These results suggest that prostacyclin released after liver grafting attenuates preservation and reperfusion damage of the liver, supporting the hypothesis that there is an imbalance of prostanoids within the microvasculature in patients with a compromised postoperative liver function. Our results agree with the involvement of some degree of lipid peroxidation products in the damage of hepatocytes during anoxia and reperfusion.

Dietary lipid and iron status modulate lipid peroxidation in rats with induced adjuvant arthritis.

We studied the effect of dietary lipids on iron metabolism and lipid peroxidation during induced adjuvant arthritis and/or iron overload in rats. We compared a control diet containing corn oil and rapeseed oil with a diet devoid of polyunsaturated fatty acids containing only tripalmitin as lipids. Four subgroups of rats were used with each diet: without further treatment, with induction of adjuvant arthritis, with iron overload, and with induction of adjuvant arthritis and iron overload. The profile of fatty acids present in plasma and in microsomes changed in rats fed the tripalmitin diet. The level of tetra- and pentaunsaturated fatty acids was reduced, and the level of monounsaturated fatty acids and iron stores was increased with respect to control rats. Thus, ingestion of a tripalmitin diet reduced the substrate for lipid peroxidation, as shown by the decrease in thiobarbituric acid-reactive substances in plasma and conjugated dienes in hepatic microsomal fraction. Adjuvant arthritis and iron overload had a synergistic effect on lipid peroxidation and iron storage in liver. Further, in the hepatic microsomal fraction, tripalmitin reduced the levels of cytochrome P-450, and arthritis reduced the levels of cytochrome P-450 and Ca2+ sequestration. Our results suggest that rats fed tripalmitin showed a reduction of lipid peroxidation induced by inflammation or by iron overload, because of the lack of polyunsaturated fatty acids in the diet, although tripalmitin usually increases the iron stores in the body and causes hepatic alterations.

Prevention of experimental cyclosporine nephrotoxicity by dietary supplementation with LSL 90202, a lysine salt of eicosapentaenoic acid. Role of thromboxane and prostacyclin in renal tissue.

Cyclosporine (CsA) nephrotoxicity is partially mediated by renal vasoconstriction due to an imbalance between vasodilator and vasoconstrictor eicosanoids. LSL 90202 is a purified lysine salt of eicosapentaenoic acid which is a known inhibitor of renal eicosanoid synthesis. The aim of the present work was to determine if chronic dietary supplementation with LSL 90202 prevented CsA nephrotoxicity and to establish the role of thromboxane and prostacyclin in renal tissue. Thirty-three male Sprague-Dawley rats were divided into 4 groups: group 1, CsA in olive oil (n = 10); group 2, isovolumetric olive oil (n = 7); group 3, CsA in olive oil plus LSL 90202 (n = 8); group 4, isovolumetric olive oil plus LSL 90202 (n = 8). CsA and LSL 90202 were given at 20 mg/kg/day. Weight and creatinine clearance (CrCl) were determined before and on days 14 and 30. On day 30 whole-blood CsA was determined and renal tissue processed for renal malondialdehyde, thromboxane B2 and 6-keto-PGF1 alpha measurement and for conventional histology. CrCl was severely reduced in the CsA in olive oil group compared to olive oil and LSL 90202 control groups. On day 30, CrCl in the CsA in olive oil plus LSL 90202 group showed a slight decrease, but the mean CrCl was significantly higher than in the CsA in olive oil group. Trough whole blood CsA levels were not significantly different in both groups given the drug. No morphological differences were found between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Antioxidant defenses and its modulation by iron in carrageenan-induced inflammation in rats.

The concentration of endogenous antioxidants has been studied in rats with a carrageenan-induced granuloma. This animal model of inflammation allowed us to study the antioxidant defenses and the oxidative stress in plasma and in the site of inflammation (exudate) and their modulation by the levels of iron in the organism after iron-dextran or desferrioxamine administration. In inflamed rats without supplementary treatment, an interrelation between urate, ascorbate and vitamin E levels has been observed and it appears to be an important mechanism to prevent the depletion of the antioxidants. Further, the sulphydryl groups, caeruloplasmin and retinol also contribute to the defense in this experimental model. Iron overload increases the production of malondialdehyde and decreases some antioxidants such as ascorbic acid and SH groups but, on the other hand, it raises the levels of urate and caeruloplasmin. However, the protective effect of desferrioxamine has not been observed, and in our conditions this may be due to the induced mobilization of iron. Our results show that antioxidants have an important role in the prevention of lipid peroxidation by free radicals produced during inflammatory processes. This protective effect depends on the stage of inflammation.