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Jatropha curcas is a medicinal plant whose dead leaves are particularly used in arterial hypertension and diabetes treatment. The purpose of this study is to conduct an acute and subacute toxicity study of Jatropha curcas leaves aqueous extract (JCLAE) using OECD method. For acute toxicity, dose limits of 2000 and 5000 mg/kg were used. In subacute toxicity study 4 batches were constituted including a control batch that received distilled water for 28 days and the other 3 batches, JCLAE doses of respectively 200, 400, 800 mg/kg for the same duration. The LD50 was determined and the hematological, biochemical and histological parameters were analyzed in mice. The LD50 is greater than 5000 mg/kg. HDL-C is the only biochemical parameter that has experienced significant rise variation. Hematological analysis showed a decrease in mean platelet volume and Platelets number. The histological study revealed cases of hepatic cellular apoptosis and kidneys tubular necrosis among animals treated with highest dose. JCLAE is less toxic than Jatropha curcas leaves aqueous extract and high-dose JCLAE also has a moderate toxic effect on thrombocyte line and a protective effect on cardiovascular system.

The aim of the present study was to evaluate the effect of long-term argan oil administration on vascular responsiveness and susceptibility to H 2O2. 16 Wistar rats were divided into 2 groups; control group and treated group receiving 5 ml/kg/day of argan oil. After 8 weeks of treatment, vascular contractility was measured on isolated aortic rings from both groups by addition to the organ bath of cumulative concentration of vasoactive drugs (phenylephrine, acetylcholine and sodium nitroprussiate). In parallel, these isolated aortic rings were exposed to increasing doses of H 2O2 (0, 1, 5 and 10 mM) for 20 minutes and the residual vascular response to phenylephrine (PE 10-6 M) and acetylcholine (Ach 10-6 M) was evaluated. Our results show that no significant differences between control and argan oil group were observed. Maximal contraction response to 10-6 M PE was 1.68 ± 0.20 g and 2.14 ± 0.20 g for control and argan oil groups, respectively. In precontracted isolated aortic rings, endothelium dependent relaxation to acetylcholine and endothelium-independent relaxation were not influenced by the diet. Maximal relaxation in response to 10-6 M of Ach was 54 % for control group and 47 % for argan oil group. Addition of hydrogen peroxide (H 2O2) to the organ bath for 20 minutes led to a dose dependent alteration of vascular reactivity, which was characterized by similar decrease in both the PE-induced contraction and the Ach-induced relaxation in the two experimental groups. These results suggest that argan oil treatment does not influence vascular reactivity nor its sensitivity to the oxidative stress.

Alpha-linolenic acid is one of the two essential fatty acids in humans. Epidemiological studies and dietary trials strongly suggest that this fatty acid is important in relation with the pathogenesis (and prevention) of coronary heart disease. Like other n-3 fatty acids from marine origin, it may prevent cardiac arrhythmias and sudden cardiac death. The optimal dietary intake of alpha-linolenic acid seems to be about 2 g per day or 0·6‐1% of total energy intake. Obtaining an optimal ratio of the two essential fatty acids, linoleic and alpha-linolenic acids — i.e. a ratio of about 4 to 1 in the diet — is a major issue. The main sources of alpha-linolenic acid for the European population should be canola oil (and canola-oil-based margarine if available), nuts (English walnut), ground linseeds and green leafy vegetables such as purslane.

Reactive oxygen species (ROS) such as superoxide anion (O2-*) and hydrogen peroxide (H2O2) can be produced by vascular endothelium and smooth muscle cells under diverse physiological and pathophysiological situations. These species are known to exert various deleterious effects by which they might induce changes in vascular reactivity. The aim of the present study was to evaluate the evolution of vascular susceptibility to H2O2 during aging in rats. Catalase activity was assessed in aortas from young adult (4 months) and aged (24 months) Wistar rats. In parallel experiments, isolated rings from both age groups were exposed to increasing doses of H2O2 (0, 0.1, 1, 5, or 10 mM) for 20 min and the residual vascular response to phenylephrine (PE = 10(-6) M) and acetylcholine (ACh = 10(-6) M) was evaluated. Our results indicate that aging increases aortic catalase activity (4 months: 0.20 +/- 0.02 IU/mg prot versus 24 months: 0.46 +/- 0.06 IU/mg prot, p0.001) while it exacerbates vascular sensitivity to H2O2. These results suggest that the observed increased H2O2-induced alterations of vascular reactivity during aging in rats might be due to increased sensitivity of the vasculature to ROS rather than to a decrease in the defense systems against these species.

Ageing is associated with an increase in myocardial susceptibility to ischemia and a decrease in post-ischemic recovery of function. In the present study, we have examined the effects of ageing on (i) myocardial ischemic contracture, (ii) the reperfusion syndrome and lipid peroxidation upon reperfusion, and (iii) the activity of enzymes involved in reactive oxygen species elimination. Hearts from male Wistar rats aged 4 (adults), 16 (old) or 24 months (senescent) were subjected to 20-min zero flow ischemia and 30-min reperfusion ex vivo. Cardiac activity of superoxide dismutase, catalase, and glutathione peroxidase, as well as cardiac content of thiobarbituric acid reactants were assessed in frozen heart samples. The effects of ageing on ischemic contracture of the sarcomeres were assessed on electromicrographs of tissues taken at the end of ischemia. In our experimental conditions, ischemic contracture of the sarcomeres increased progressively during ageing. In contrast, the severity of the reperfusion syndrome increased between 4 and 16 months of age, and then decreased up to 24 months of age. We propose that the peak of susceptibility of the myocardium to reperfusion observed during moderate ageing might be related to a decrease in the ability of cardiomyocytes to dismutate hydrogen peroxide as suggested by the observed decrease in catalase activity. Finally, the better resistance to the reperfusion syndrome exhibited by senescent rats compared to old rats might be due to a natural selection of a subpopulation of rats which is particularly resistant to oxidative stress.

Oxyradicals have been implicated as a possible cause of reperfusion-arrhythmias (RA). However, the use of diverse exogenous oxyradical scavengers designed to reduce RA has given contradictory results. The aim of the present study was to determine whether enhancing the activity of the main endogenous enzyme involved in peroxide elimination in cardiac cells, namely glutathione peroxidase, may limit RA in isolated heart preparations by increasing their antioxidant status. For this purpose, a group of 15 male Wistar rats received a selenium enriched diet for ten weeks (1.5 mg Se/kg diet). Control animals (n = 15) received a standard diet containing 0.05 mg Se/kg diet. The incidence of early ventricular arrhythmias was investigated during the reperfusion period following 10 min regional ischemia induced ex-vivo by left coronary artery ligation. Our results show that selenium-supplementation significantly increased the global selenium status of the animals. In the isolated heart preparations, the selenium supplementation induced a significant reduction of the severity of RA as assessed by the arrhythmia score and the limitation of the incidence of both ventricular tachycardia (control: 91% vs selenium: 36%, p < 0.05) and irreversible ventricular fibrillation (control: 45% vs selenium: 0%, p < 0.05). These effects were associated with a significant increase in cardiac mitochondrial and cytosolic glutathione peroxidase activities in both the left and the right ventricles. These results illustrate the potential protective effect of selenium against ischemia-reperfusion injury and suggest that peroxides might play a key role in the genesis of some aspects of the reperfusion syndrome.

Adriamycin (doxorubicin; ADR) is an antineoplastic drug used to treat various cancers; however, its chronic us is unfortunately accompanied by cardiotoxicity. Previous results suggested that a free radical mechanism may contribute to ADR toxicity. Because it is often reported that cancer patients are deficient in selenium (Se), we hypothesised that ADR toxicity might be reduced by antioxidant agents such as vitamin E and Se. ADR-induced cardiotoxicity was examined in rats maintained on a Se-supplemented diet. The animals were kept on either a standard (0.22 mg/kg) or a Se-supplemented (2.5 mg/kg) diet starting 4 weeks prior to the first ADR treatment. ADR, or its excipient, was administered intraperitoneally in six equal injections over a period of 3 weeks giving a cumulative dose of 15 mg/kg body weight. Blood was collected in the thoracic cavity and the heart was subjected to a sequence of perfusion/partial ischemia/reperfusion ex vivo. Se status, GPx activities, vitamins E and C and MDA contents were determined on RBC and cardiac homogenates. ADR-treated rats showed a significant decrease in RBC Se (-29%) and in RBC Se-GPx (-34%) compared to the placebo group, and a significant increase in RBC MDA content (+2000%). This latter increase was attenuated by the Se supplementation (+1600%). In parallel, RBC vitamin E decreased markedly in the ADR-treated group (-50%) and was totally restored by the Se supplementation. Cardiac biochemical analyses confirmed the blood results. The present data confirm that a free radical mechanism does contribute to ADR toxicity, and show the importance of balancing the Se levels in ADR-treated subjects to limit its harmful myocardial action. Adecrease in ADR toxicity with no concomitant decrease in its antineoplastic activity would be of considerable value by improving the therapeutic benefit of the drug.

The effects of EUK-8, a synthetic, catalytic scavenger of reactive oxygen species, on isolated iron-overloaded rat hearts submitted to ischemia-reperfusion were studied. In the absence of EUK-8, functional parameters (systolic and diastolic pressures, oxygen consumption as estimated by the product heart rate times left ventricular diastolic pressure) were severely impaired 1 minute and 15 minutes after reperfusion following a 15 minute ischemic episode. Dimethylthiourea (10 mM), a hydroxyl radical scavenger, had a minimally protective effect. In contrast, EUK-8 at a concentration of 50 microM in the perfusion medium maintained these parameters at close to their preischemia values. Electron microscopic analysis of heart tissues after 15 minutes ischemia followed by 15 minutes reperfusion showed extensive damage to mitochondria and sarcomeres in untreated hearts, while the extent of damage was significantly lower in EUK-8-treated hearts. The functional and structural protection afforded by EUK-8 were significantly better than those induced by dimethylthiourea. These data suggest that EUK-8 may be therapeutically useful in preventing heart damage induced by ischemia-reperfusion, for example, during thrombolytic treatment of myocardial infarction.

Oxygen-derived free radicals are currently suspected to be widely involved in the aetiology of several clinical disorders. In animals as well as in man, antioxidant trials are often undertaken to prevent oxidative stress. Among antioxidant molecules selenium has been largely studied. This study shows that plasma Se level is not a good index of Se status in the organism, at least at high levels of selenium. Red blood cell Se seems to be a more reliable index of Se status and could replace plasma Se level in the supplementation trials both in animals and humans. Se supplementation did not result in a significant decrease in oxidative stress markers as evaluated by blood and tissue malondialdehyde contents in healthy animals. Furthermore, heart function was altered and plasma Alanine aminotransferase activity was significantly increased in the selenium-supplemented group, which could reflect a slight subtoxic effect of selenium supplementation at the level used here. In view of the results presented, the maximum selenium content in animal diet in selenium supplementation experiments should not be higher than 2 mg/kg.

International audience; Leptin is known to exert cardiodepressive effects and to induce left ventricular (LV) remodelling. Nevertheless, the autocrine and/or paracrine activities of this adipokine in the context of post-infarct dysfunction and remodelling have not yet been elucidated. Therefore, we have investigated the evolution of myocardial leptin expression following myocardial infarction (MI) and evaluated the consequences of specific cardiac leptin inhibition on subsequent LV dysfunction. Anaesthetized rats were subjected to temporary coronary occlusion. An antisense oligodesoxynucleotide (AS ODN) directed against leptin mRNA was injected intramyocardially along the border of the infarct 5 days after surgery. Cardiac morphometry and function were monitored by echocardiography over 11 weeks following MI. Production of myocardial leptin and pro-inflammatory cytokines interleukin (IL)-1β and IL-6 were assessed by ELISA. Our results show that (1) cardiac leptin level peaks 7 days after reperfused MI; (2) intramyocardial injection of leptin-AS ODN reduces early IL-1β and IL-6 overexpression and markedly protects contractile function. In conclusion, our findings demonstrate that cardiac leptin expression after MI could contribute to the evolution towards heart failure through autocrine and/or paracrine actions. The detrimental effect of leptin could be mediated by pro-inflammatory cytokines such as IL-1β and IL-6. Our data could constitute the basis of new therapeutic approaches aimed to improve post-MI outcome.

Reperfusion of acutely ischemic cardiac tissue is associated with several characteristic pathophysiological changes that are generally referred to as "reperfusion injury." It has been hypothesized that some of these changes are mediated by oxygen-derived free radicals. Indapamide, a nonthiazide diuretic, has been shown to exert free-radical scavenging properties comparable to that of alpha-tocopherol. The purpose of the present work was to investigate whether indapamide (IDP) may limit ultrastructural signs of reperfusion injury in an experimental model of myocardial ischemia and reperfusion in isolated rat hearts. Rats received a chronic oral administration of IDP (7 days at 3 mg/kg body weight/day) before excision of the heart. IDP was also added to the perfusion fluid at a final concentration of 10(-4) M. Isolated hearts were perfused under control conditions for 20 min and then submitted to 15 min of global no-flow ischemia, before being reperfused for 15 min. Hearts were fixed by glutaraldehyde perfusion fixation and left ventricular ultrastructure was studied on ultra-thin sections by electron microscopy. Micrographs were taken following a random procedure to obtain a representative overview of the whole section. In the untreated group, marked ultrastructural alterations were observed including contraction bands, disrupted membranes, and swollen mitochondria. In the indapamide-treated group, the degree of morphological injury was significantly lessened. It is concluded that indapamide protects the ultrastructure of ventricular myocytes against reperfusion injury. This effect might be related to the oxygen free-radical scavenging property of the drug.

International audience; Growing evidence indicates that the Mediterranean diet is beneficial to human health. Many epidemiological and research studies have reported that this diet pattern is able to limit the development and progression of coronary heart disease, one of the leading cause of morbidity and mortality in both developed and developing countries worldwide. There is now a large consensus about recommending Mediterranean diet to reduce atherosclerosis and coronary artery disease and to limit the risk of fatal complications such as sudden cardiac death and heart failure. This review underlines the role of two of the specific components of the Mediterranean diet, namely marine omega-3 fatty acids and wine, and the link between moderate wine consumption and fatty acid profiles.

International audience; In the mid-1960s, a small number of scientists postulated the role of oxidative stress and oxygen-derived free radicals in the pathophysiological mechanisms underlying ischemic heart disease. However, because of the technical difficulty of measuring free radicals and quantitating oxidative damage, it was very difficult to prove that free radicals could contribute to cell pathology. The role of oxidative stress in biological systems was not definitely recognized until the early 1980s when measurement of short-lived oxygen-derived reactive species was made possible by the advent of sophisticated techniques such as EPR spectroscopy or fluorescent probes. These enabled both the study of free radical biochemistry and the acquisition of useful information about the nature and consequences of free radical-induced protein and lipid oxidation. The hypothesis that reactive oxygen species mediate cellular damage produced upon reperfusion of ischemic myocardium has gained considerable support during the past 10-15 years. Several experimental studies indicated that the administration of antioxidant enzymes or non-enzymatic antioxidants offers a significant degree of protection against ischemic damage, improving functional recovery and reducing morphological alterations to cardiomyocytes. In this context, selenium, as an essential component of glutathione peroxidase, plays a critical role in protecting aerobic tissues from oxygen radical-initiated cell injury.

A marked increase in plasma TNF-α has been described in patients with chronic heart failure (CHF). Nevertheless, little is known about the direct role of this cytokine early after myocardial infarction (MI) and its possible effects on the subsequent development of CHF. Wistar rats were subjected to permanent in vivo coronary artery ligation. At 5, 7, and 9 days after MI, cardiac function, passive compliance of the left ventricle (LV), and cardiac geometry were evaluated. The same model was used to perform pharmacological studies 7 days and 10 wk after MI in rats treated with monomeric recombinant human soluble TNF-α receptor type II (sTNF-RII, 40 μg/kg iv) or a placebo on day 3. Maximal alterations of cardiac function and geometry occurred 7 days after MI, which correlated chronologically with a peak of cardiac and serum TNF-α, as shown by immunohistochemistry and ELISA, respectively. sTNF-RII improved LV end-diastolic pressure under basal conditions and after volume overload 7 days and 10 wk after MI. Moreover, a significant leftward shift of the pressure-volume curve in the sTNF-RII-treated group 7 days after MI indicated a preservation of LV volume. Infarct expansion index was also significantly improved by sTNF-RII 7 days after MI ( P < 0.01). Nevertheless, 10 wk after MI, geometric indexes and passive pressure-volume curves were not significantly improved by the treatment. In conclusion, TNF-α plays a major role in cardiac alterations 7 days after MI in rats and contributes to hemodynamic derangement, but not to cardiac remodeling, in subsequent CHF.

International audience; 1. The early stage of insulin resistance, also termed the 'prediabetic state', is characterized by the development of hyperinsulinaemia, which maintains normoglycaemia under fasting conditions. The metabolic disorders induced in myocardial cells during this stage of the disease may constitute a basis for an alteration of the tolerance of the heart to ischaemia and reperfusion. 2. To test this hypothesis, male Wistar rats were fed a 66% fructose diet for 4 weeks, inducing a prediabetic state. Rats were then subjected to in vivo left coronary artery ligation followed by reperfusion. Blood samples were collected for plasma lipid profile determination. 3. The prediabetic state significantly increased the severity of ischaemia-induced arrhythmias (arrhythmia score 1.4 +/- 0.2 vs 2.0 +/- 0.0 in control and fructose-fed rats, respectively; P < 0.05) and the size of infarction (infarct size 41.2 +/- 3.0 vs 56.0 +/- 2.0% in control and fructose-fed rats, respectively; P < 0.01). This alteration of the tolerance to in vivo ischaemia/reperfusion may be the consequence of an increase in mono-unsaturated fatty acids and a decrease in omega3 polyunsaturated fatty acids in fructose-fed-rats. 4. In conclusion, because it is known that the prediabetic state increases the incidence of cardiovascular diseases by promoting coronaropathy, our study suggests that this metabolic disorder may also affect the prognosis of heart disease by decreasing the tolerance of cardiomyocytes to ischaemic insults.

International audience; The mechanisms through which the consumption of alcoholic beverages, in particular wine, protects against cardiac and vascular diseases remain largely unexplored. New methods are needed to investigate that crucial medical and scientific question. Several groups are now beginning to use animal models of myocardial ischemia and reperfusion to explore whether certain nutrients, including ethanol and non-ethanolic components of wine, may have a specific protective effect on the myocardium, independently from the classical risk factors involved in vascular atherosclerosis and thrombosis. Concepts used in experimental cardiology, such as preconditioning and stunning, are now entering the field of nutrition, and this will undoubtedly lead to considerable improvements in the prevention and treatment of cardiovascular diseases.

The aim of this study was to evaluate the effect of organ oil on isolated heart function before and after ischemia and on the activity of cardiac antioxidant enzymes. 16 Wistar rats were divided into 2 groups; control group and treated group receiving 5 mL/kg/day of organ oil. After 8 weeks of treatment, hearts were perfused and subjected to a global ischemia followed by reperfusion. Activity of cardiac antioxidant enzymes was assessed in freeze-clamped hearts at the end of reperfusion. Results showed that organ oil induces: 1--damage to heart function during the preischemic period, 2--decreased functional recovery during reperfusion and 3--significant increase in catalase activity. It seems that, in our experimental conditions, organ oil increases heart sensitivity to ischemia and reperfusion. However, the mechanism involved has yet to be understood.

Oxygen-derived free radicals (O2•-, H2O2, and •OH) that are produced during postischemic reperfusion are currently suspected to be involved in the pathogenesis of tissue injury. Another reactive oxygen species, the electronically excited molecular oxygen (1O2), is of increasing interest in the area of experimental research in cardiology. In this review are discussed the main potential sources of singlet oxygen in the organism, particularly in the myocardium, the various cardiovascular cytotoxic effects induced by this reactive oxygen intermediate, and the growing evidence of its involvement in ischemia/reperfusion injury.

Oxygen radical scavengers have been shown to prevent the development of ischemic preconditioning, suggesting that reactive oxygen species (ROS) might be involved in this phenomenon. In the present study, we have investigated whether direct exposure to ROS produced by photoactivated Rose Bengal (RB) could mimic the protective effects of ischemic preconditioning.In vitro generation of ROS from photoactivated RB in a physiological buffer was first characterised by ESR spectroscopy in the presence of 2,2,6,6-tetramethyl-1-piperidone (oxoTEMP) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In a second part of the study, isolated rat hearts were exposed for 2.5 min to photoactivated RB. After 5 min washout, hearts underwent 30 min no-flow normothermic ischemia followed by 30 min of reperfusion.The production of singlet oxygen (1O2) by photoactivated RB in the perfusion medium was evidenced by the ESR detection of the nitroxyl radical oxoTEMPO. Histidine completely inhibited oxoTEMPO formation. In addition, the use of DMPO has indicated that (i) superoxide anions (O2*-) are produced directly and (ii) hydroxyl radicals (HO*) are formed indirectly from the successive O2*- dismutation and the Fenton reaction. In the perfusion experiments, myocardial post-ischemic recovery was dramatically impaired in hearts previously exposed to the ROS produced by RB photoactivation (1O2, O2*-, H2O2 and HO*) as well as when 1O2 was removed by histidine (50 mM) addition. However, functional recovery was significantly improved when hearts were exposed to photoactivated RB in presence of superoxide dismutase (10(5) IU/L) and catalase (10(6) IU/L). Further studies are now required to determine whether the cardioprotective effects of Rose Bengal in presence of O2*- and H2O2 scavengers are due to singlet oxygen or to other species produced by Rose Bengal degradation.

Two anomeric analogues of glucose labelled with 123 iodine in position 6, proposed as tracers of glucose transport in vivo, have been synthesized: alpha- and beta-methyl-6-deoxy-6-iodo-D-glucopyranoside (alpha MDIG and beta MDIG). The aim of this study was to determine whether these molecules interact with the glucose transporter and whether they could be used as tracers of glucose transport in vivo. The biodistribution of alpha MDIG and beta MDIG was studied in the mouse in vivo. To determine if these two anomers enter the cell via the glucose transporter, their uptake was measured in isolated perfused rat hearts, in human erythrocytes in suspension, and in cardiomyocytes of neonatal rat in culture. Both alpha MDIG and beta MDIG had similar repartitions in the mouse: myocardial uptake averaged 7% of the injected dose/g of organ at 2 min postinjection and alpha MDIG competed with D-glucose to enter the cells. Insulin produced a 123% increase of its uptake in isolated perfused rat hearts and a 100% increase in cardiomyocytes of neonatal rat in culture. alpha MDIG uptake was lowered in the presence of glucose transport inhibitors in each experimental model. An interaction between beta MDIG and glucose transporters was observed only in human erythrocytes in suspension. Only alpha MDIG interacts with the glucose transporter, and thus could be used to estimate glucose transport in vivo.

Analogues of glucose labeled with 123 iodine in positions 1, 2 or 3 have been synthesized. The aim of this study was to examine their biological behavior in four experimental models in order to assess whether they could be used to evaluate the uptake of glucose with single photon emission computed tomography (SPECT). The results obtained have shown that none of these molecules enters the cell using the glucose transporter. Therefore, they cannot be used as tracers of glucose uptake.

Numerous studies have shown that ischaemic damage and the so called reperfusion syndrome of the myocardium are, at least in part, associated to a burst of oxygen-derived free radicals (1). The involvement of reactive oxygen species (ROS) in post-ischaemic contractile dysfunction is supported by several studies underlying the benefit of the use of antioxidant catalytic agents such as Superoxide dismutase and/or catalase during experimental cardiac reperfusion (2,3). Endogenous catalytic ROS scavengers, namely Superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPx) and glutathione reductase (GRx) play key roles in the cellular defence systems against ROS under pathophysiological conditions (3–5). The myocardial activity of these enzymes is therefore determinant to protect the heart during post-ischaemic reperfusion. It is now well established that ageing is associated to an increase in cardiac susceptibility to ischaemia (6) and reperfusion (7) in rats. The present study was designed to define the relationship between the endogenous antioxidant status and cardiac susceptibility to ischaemia and reperfusion during ageing.

For an iodinated analogue of glucose to be useful for evaluating glucose uptake using single-photon emission computed tomography (SPECT), it must enter the cell via the same transporter as glucose and accumulate within the cell without being degraded. The biological behavior of the iodinated tracer must therefore be similar to that of 2-deoxy-d-[1- 14 C]-glucose (2-DG). In the present study, four experimental models (biodistribution in mouse, isolated rat heart, human erythrocytes in suspension and cultured neonatal rat cardiomyocytes) have been chosen and protocols have been set up which allow for the examination of small quantities of iodinated analogues of glucose. The uptakes of 2-DG and of l-[1- 14 C]-glucose have been measured in these models to establish reference values which will be compared with uptake values for iodinated analogues of glucose.

The aim of the present study was to assess whether an 8-wk oral selenium supplementation (standard food enriched with 2500 micrograms Se/kg) in rats might prevent the cardiotoxicity of adriamycin (ADR) treatment. ADR was administered at a dose of 2.5 mg/kg body wt intraperitoneally twice weekly for 3 wk. One week after the end of ADR treatment, rats (n = 10 per group) were killed and their hearts were perfused on a Langendorff mode and subjected to a 30-min period of low-flow ischemia (residual flow = 0.1 ml/min) followed by reperfusion (15 min). The results were as follows: 1) selenium supplementation significantly increased the activity of cardiac mitochondrial glutathione peroxidase (GPx) in ADR-treated rats (control: 206 +/- 17.4 IU/g protein; Se: 277 +/- 24.5 IU/g protein, p0.05); 2) selenium supplementation reduced myocardial malondialdehyde content in ADR-treated rats (control: 1220 +/- 49.1 nmol/g protein; Se: 1010 +/- 75.9 nmol/g protein; p0.05); and 3) ADR treatment significantly increased the degree of reperfusion-induced structural alterations to sarcomeres compared to untreated hearts. Again, this phenomenon was abolished by selenium supplementation. In conclusion, this study demonstrates that selenium supplementation is able to limit ADR cardiotoxicity in isolated rat hearts submitted to a sequence of ischemia/reperfusion.

Adriamycin (doxorubicin) is an antineoplastic drug used to treat various cancers; however, its chronic use is accompanied by cardiotoxicity. Previous results suggested that free radical formation may contribute to Adriamycin toxicity. Adriamycin-induced cardiotoxicity was examined in rats maintained on either standard or selenium-supplemented diets. Adriamycin was administered intraperitoneally; the cumulative dose was 15 mg/kg body weight. Selenium status, glutathione peroxidase activities, vitamin E, and MDA contents were determined on red blood cell (RBC) and cardiac homogenates. RBC selenium levels and selenium glutathione peroxidase activity were significantly increased in selenium-supplemented animals compared with control or placebo groups. However, Adriamycin-treated rats showed a significant decrease in RBC selenium and selenium glutathione peroxidase compared with the placebo group. Moreover, treatment with Adriamycin increased RBC MDA content, which was attenuated by the selenium supplementation. In parallel, RBC vitamin E decreased markedly in the Adriamycin-treated group and was totally restored by selenium supplementation. Cardiac biochemical analyses confirmed the blood results. Thus, a free radical mechanism does contribute to Adriamycin cardiotoxicity, and shows the importance of balancing selenium levels in Adriamycin-treated subjects to limit Adriamycin's action. A decrease in Adriamycin cardiotoxicity with no concomitant decrease in its antineoplastic activity would considerably improve the therapeutic benefit of the drug.

We have recently shown that iron overload may increase the susceptibility of isolated rat hearts to ischemia and reperfusion, an effect that could be related to an iron-dependent activation of the Fenton reaction, thus producing cytotoxic hydroxyl radicals. The purpose of our study was to investigate the influence of EUK8, a new catalytic scavenger of superoxide anions that is similar to superoxide dismutase, on cardiac ultrastructural alterations associated with reperfusion. Experiments were carried out ex vivo in iron-overloaded rat hearts perfused at a constant flow rate (11 ml/min) with (EUK8) or without (control) EUK8 (50 mumol/L) throughout the perfusion. Hearts were submitted to 15 min global normothermic no-flow ischemia and then reperfused for 15 min before examination. For each heart, six micrographs at a magnification of x 11,300 were used to quantitate ultrastructural alterations. In our experimental model, EUK8 prevented some of the ultrastructural modifications associated with ischemia and reperfusion in iron-overloaded rat hearts. This beneficial effect could be related to the limitation of reperfusion-induced oxygen free radical production under the effect of EUK8.

[Bis (N-etoxy, N ethyl dithiocarbamato) nitrido] 99mTc (V) (TcN-NOET) is a new neutral lipophilic myocardial imaging agent proposed for clinical use for detecting coronary artery disease. We studied the relation between myocardial retention of TcN-NOET and myocardial blood flow (MBF) in a canine model.A wide range of MBF was induced by partial regional coronary occlusion and dipyridamole infusion (protocols 1,2 and 3). Myocardial activity of TcN-NOET was determined by in vitro tissue counting at 15 or 90 min postinjection. Tracer activity was correlated with radiolabeled microspheres using linear regression analysis.There was a linear correlation between myocardial TcN-NOET activity and microspheres in protocol 1 (r = 094, 15 min postinjection, protocol 2 (r = 0.94, 15 min postinjection after dipyridamole) and protocol 3 (r = 0.91, 90 min postinjection after dipyridamole). When arterial occlusion was discontinued (protocol 4), there was no longer a close linear correlation (r = 0.26). The first-pass myocardial extraction action of TcN-NOET was 75.5% +/- 4% under basal conditions and 85% +/- 2% under hyperemic conditions (p0.01).Up to 90 min after injection, the relationship between TcN-NOET myocardial retention and blood flow is excellent over a wide range of flows. After reflow, TcN-NOET redistributes almost completely within 90 min.

Although early reperfusion of cardiac tissue is now considered to be the only intervention capable of restoring the various cellular functions altered by ischaemia and preventing progression towards necrosis of myocardial cells, reperfusion is frequently accompanied by various manifestations grouped under the heading of reperfusion syndrome or reperfusion injury. Functional recovery is therefore not immediate, but usually appears after a certain delay following a period of contractile dysfunction (myocardial stunning) lasting for several hours or even days after the start of reperfusion. Characteristics reperfusion-induced arrhythmias are also observed. The cellular mechanisms underlying the reperfusion syndrome may involve cellular calcium overload, over-production of oxygen-derived free radicals, acidosis and/or development of an inflammatory reaction. Numerous pharmacological studies have been conducted over several years designed to limit such reperfusion injury and, consequently, prevent stunning and/or reperfusion-induced arrhythmias. A number of experimental studies in this field have demonstrated that trimetazidine exerts direct anti-ischaemic effects, limiting calcium accumulation and acidosis, inflammation and oxygen-derived free radical production following reperfusion. This agent therefore appears to be particularly promising clinically in the treatment of reperfusion lesions, for example in combination with thrombolysis during the acute phase of myocardial infarction.

It is currently believed that reactive oxygen species are produced in the heart post-ischemia reperfusion, causing pathophysiological disorders. Studies reported in the literature dealing with this subject have generated contradictory findings. The aim of this study was to assess the catalytic activity of the superoxide anion-producing enzyme xanthine oxidase, and the level of lipid peroxides in isolated rat heart muscle undergoing ischemia of varying duration and severity followed by reperfusion. Three levels of ischemia were investigated: total, and partial at either 0.10 or 0.35 ml/min (residual flow rate). Three different periods of ischemia were examined in each case. After each period of ischemia, followed by 10 min of reperfusion, the heart was frozen in liquid nitrogen. Xanthine oxidase activity and lipid peroxide levels were assayed in the cardiac homogenate and in the centrifuged supernatant, respectively. In the different experimental protocols studied here, both cardiac xanthine oxidase and lipid peroxide levels remained statistically unchanged compared to the continuously perfused control hearts. Moreover, in a recent study (Boucher et al., FEBS Lett. 203, 261-264, 1992), we were unable to detect reactive oxygen species in perfusate upon reperfusion of ischemic rat hearts. These results suggest that changes in xanthine oxidase activity during myocardial ischemia-reperfusion, and lipid peroxidation, as assessed by measuring thiobarbituric acid reactants and lipid hydroperoxides, are not predominant phenomena in ischemia-reperfusion-induced injury, at least in the experimental model used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Although early reperfusion of cardiac tissue is now considered to be the only intervention capable of restoring the various cellular functions altered by ischaemia and preventing progression towards necrosis of myocardial cells, reperfusion is frequently accompanied by various manifestations grouped under the heading of reperfusion syndrome or reperfusion injury. Functional recovery is therefore not immediate, but usually appears after a certain delay following a period of contractile dysfunction (myocardial stunning) lasting for several hours or even days after the start of reperfusion. Characteristic reperfusion-induced arrhythmias are also observed. The cellular mechanisms underlying the reperfusion syndrome may involve cellular calcium overload, over-production of oxygen-derived free radicals, acidosis and/or development of an inflammatory reaction. Numerous pharmacological studies have been conducted over several years designed to limit such reperfusion injury and, consequently, prevent stunning and/or reperfusion-induced arrhythmias. A number of experimental studies in this field have demonstrated that trimetazidine exerts direct anti-ischaemic effects, limiting calcium accumulation and acidosis, inflammation and oxygen-derived free radical production following reperfusion. This agent therefore appears to be particularly promising clinically in the treatment of reperfusion lesions, for example in combination with thrombolysis during the acute phase of myocardial infarction.

The objective of this study was to investigate the effect of dietary zinc on endogenous production of lipid peroxides, and on myocardial infarct size in rats. Male rats were fed a zinc-deficient diet containing 4 ppm zinc, or a standard diet containing 60 ppm zinc. After 3 weeks of diet, half of the animals underwent occlusion of the left coronary artery. The remaining animals underwent sham operation without occlusion. Forty-eight hours later, the hearts were sampled and lipid peroxide levels and infarct size were evaluated. Coronary occlusion was associated with an increase in cardiac lipid peroxide levels which were more pronounced in the zinc deficient group. However, infarct size appeared to be independent from zinc deficiency, despite the free radical-mediated lipid peroxide augmentation reported here. The pharmacological limitation of infarct size in rats with permanent coronary occlusion is discussed.

It has been suggested that iron might play a pivotal role in the development of reperfusion-induced cellular injury through the activation of oxygen free radical producing reactions. The present study examined the effects of myocardial iron overload on cardiac vulnerability to ischemia and reperfusion. Moreover, the effect of the iron chelator deferoxamine in reversing ischemia-reperfusion injury was studied. Animals were treated with iron dextran solution (i.m. injection, 25 mg every third day during a 5 week period). The control group received the same treatment without iron. Isolated rat hearts were perfused at constant flow (11 ml/min) and subjected to a 15 minute period of global normothermic ischemia followed by reperfusion for 15 minutes. The effects of iron overload were investigated using functional and biochemical parameters, as well as ultrastructural characteristics of the ischemic-reperfused myocardium compared with placebo values. The results suggest that (a) a significant iron overload was obtained in plasma and hepatic and cardiac tissues (x2.5, x16, and x8, respectively) after chronic intramuscular administration of iron dextran (25 mg); (b) during normoxia, iron overload was associated with a slight reduction in cardiac function and an increase in lactate dehydrogenase (LDH) release (x1.5); (c) upon reperfusion, functional recovery was similar whether the heart had been subjected to iron overload or not. However, in the control group left ventricular end-diastolic pressure remained higher than in preischemic conditions, an effect that was not observed in the iron-overloaded group. Moreover, LDH release was markedly increased in the iron-loaded group (x4.2); (d) iron overload was associated with a significant worsening of the structural alterations observed during reperfusion, particularly at the mitochondrial and sarcomere level; (e) after 15 minutes of reperfusion, the activity of the anti-free-radical enzyme, glutathione peroxidase (GPX), was significantly reduced in iron-overloaded hearts, whereas catalase activity was increased; (e) the overall modifications observed in the presence of iron overload were prevented by deferoxamine. In conclusion, this study underlines the possible role of cardiac iron in the development of injury associated with ischemia and reperfusion, and the possible importance of the use of an iron-chelating agent in anti-ischemic therapy.

Dear Sir, Xanthine oxidase-superoxide production has been implicated in the initiation of the peroxidative process in the myocardium under certain pathophysiological conditions [1]. In line with this hypothesis are reports which show that allopurinol [4-hydroxypyrazolo-(3,4d) pyrimidine], a xanthine oxidase inhibitor, can prevent or significantly reduce myocardial reperfusion injury [2]. In most of these studies, however, the doses of allopurinol required are very high, and in all cases, they are far above those used in humans for the treatment of gout. Different reports have shown that overdosing with allopurinol can exert certain toxic effects in the rat [3,4]. In a previous study [4] we have shown that chronic administration of allopurinol (20 mg/kg twice a day for 5 days) fails to exert any cardioprotective effect in rats submitted to permanent coronary artery ligation. Moreover, our results suggested the occurrence of a toxic effect of the treatment, as shown by an increase in tissue edema in the nonischemic portion of the myocardium in treated animals. In the present study we examined the effects of chronic administration of allopurinol (according to the protocol previously described [4]) on (1) myocardial malondialdehyde (MDA) content, taken as an index of lipid peroxidation, (2) superoxide dismutase (SOD) and catalase activities, which are the main enzymatic systems involved in the clearance of oxygen free radicals, and (3) xanthine oxidase activity, which is supposed to be inhibited by the treatment. The experiment was carried out on male adult Wistar rats (220-250 g). Allopurinol (Sigma Chemical Company), suspended in an aqueous solution of gum arabic (20 g/l), was administered orally at 20 mg/kg twice a day for 5 days. Control animals received a similar treatment using vehicle only (gum arabic at 2%). Volumes of administration were adjusted to 1 ml for 100 g body weight. On the last day of treatment, animals (n = 9/group) were anesthetized with sodium pentobarbital (40 mg/kg i.p., 1 ml/kg) and heparin was administered via a femoral vein (100 IU/100 g). The hearts were cut out and perfused via the aorta for 4 minutes to remove any blood from the vascular beds. The perfusate was a Krebs-Henseleit buffer (Ca 2÷ = 2.4 mM; K ÷ = 5.6 mM; glucose 11 mM) equilibrated with a gas mixture of 95% 02 and 5% C02 at 37°C, pH 7.4. Following this perfusion, the hearts were quickly frozen in liquid nitrogen. MDA myocardial content, as well as SOD, catalase, and xanthine oxidase activities, were measured using conventional assays (using the method of Ohkawa et al. [5] for MDA measurement, of Marklund [6] for SOD activity, of Beers and Sizer [7] for catalase activity, and of Sugiura et al. [8] for xanthine oxidase activity). Results were expressed as mean _+ standard error of the mean (SEM) and compared using Student's t test following analysis of variance by Fisher's test. Treatment with allopurinol is likely to be associated with stimulation of a peroxidative process, shown by the elevation of myocardial MDA content and by the increase of SOD and catalase activities. Moreover, the activity of xanthine oxidase that has the potential to generate superoxide anions appears paradoxically to be increased in the myocardium of treated animals. Allopurinol is both a substrate and an inhibitor of xanthine oxidase [9]; during the reaction it is transformed into alloxanthine (oxypurinol). This metabolite is itself an inhibitor of the enzyme and is slowly cleared by the kidney. Suzuki and Sudo [3] have reported an elevation of both MDA content and xanthine oxidase activity in the kidney of rats 3 days after the onset of treatment with allopurinol (100 mg/kg b.w./ day, per os). These authors suggested that allopurinol could lead, in the early phase of the treatment, to an elevation of superoxide anion production because of the oxidation of allopurinol to oxypurinol. In a second phase, oxypuriliol produced by the reaction could reach a sufficient level to inhibit the enzyme. However, there is still no experimental evidence showing that either longer durations of treatment with allopurinol or oxypurinol administration can lead to an inhibition of xanthine oxidase-dependent superoxide production. Thus, the mechanism of the toxic effect of allopurinol remains unclear. In the light of our results it appears that, in our experimental conditions, allopurinol toxicity might involve an overproduction of oxy

The aim of this work was to assess the catalytic activity of xanthine oxidase, the level of lipid peroxides and enzymic antioxidant systems in isolated rat heart muscle subjected to a globally partial ischemia followed by varying durations of reperfusion. After 40 min of globally partial ischemia (residual perfusion flow rate: 0.1 ml/min), four different durations of reperfusion were investigated (0, 20, 40, and 60 min). After each experimental ischemia/reperfusion sequence, the heart was frozen in liquid nitrogen. Lipid peroxides were assayed in the cardiac homogenate and the catalytic activity of xanthine oxidase and enzymic antioxidant systems (glutathione peroxidase, superoxide dismutase and catalase) were determined in the centrifuged supernatant. In the different experimental protocols studied in this work, there was no significant increase in the activity of cardiac xanthine oxidase or in the level of lipid peroxides when compared to the non reperfused or to the continuously perfused hearts. Indeed, enzymic antioxidant systems were also not significantly modified in the different periods of reperfusion when compared to control hearts (continuously perfused hearts). These results suggest that xanthine oxidase is apparently not a major source of free radicals in the course of an ischemia-reperfusion sequence in heart muscle, in particular, if we consider the early phases of reperfusion. The process of lipid peroxidation, assessed by assaying thiobarbituric acid reactants, is not a predominant phenomenon of reperfusion-induced injury, at least in the experimental model used here. However, enzymic antioxidant systems investigated in this study do not seem modified. This could mean that the small quantity of oxygen free radicals produced does not overwhelm the enzymic antioxidant systems of myocardium which is in agreement with peroxidatized lipid results.

Adriamycin (doxorubicin) is an antineoplastic drug used to treat various cancers; however, its chronic use is unfortunately accompanied by cardiotoxicity. This toxicity can be reduced by antioxidant agents such as selenium, and it is particularly interesting that cancer patients are usually deficient in this trace element, which suggests that its supplementation could contribute to beneficial treatment. We have examined the effect of adriamycin on chronic cardiotoxicity in 6-week selenium deficiency in rats. Selenium-deficient rats showed a considerable reduction of selenium levels and of selenium-containing glutathione peroxidase. Cardiac lipid peroxides increased slightly in the deficient rats, whereas plasma and heart lipid peroxides increased markedly in adriamycin-treated rats. This increase was greatly accentuated in selenium deficiency. These results suggest that free radical mechanism may be contributing to adriamycin toxicity, and above all show the importance of balancing the selenium levels in adriamycin-treated subjects to limit its harmful myocardial action. A decrease in adriamycin cardiotoxicity with no concomitant decrease in its antineoplastic activity would be of considerable value by improving the therapeutic benefit of the drug.

It has been shown that allopurinol, an inhibitor of xanthine oxidase, may limit the extent of myocardial infarction in dogs. In the present work, we studied the effect of a chronic administration of allopurinol on myocardial infarct size measured histochemically 48 h after in situ left coronary artery ligation in the rat. Our results indicate that allopurinol pretreatment does not produce any limitation of the extent of necrosis, but induces a significant increase in the volume of the non-ischemic portion of the myocardium, accompanied by an increase in protein content. This phenomenon, which could be due to the development of an edema in the non-ischemic portion of the myocardium, may well explain some discrepancies reported in previous experimental studies in which the infarct size was conventionally expressed as a percentage of the total volume of ventricular tissue. We have also shown that allopurinol pretreatment failed to improve the residual cardiac function in rats after left coronary artery ligation. We conclude that the enzyme xanthine oxidase is probably not involved in the pathophysiology of myocardial infarction in the rat because of the absence of collateral vasculature in this species which prevents any oxygen supply to the ischemic zone. In most other mammals such as the dog, the existence of a collateral system maintains a residual blood flow and oxygen supply to the ischemic portion of ligated hearts, allowing the xanthine oxidase-induced production of superoxide anions to be activated, thereby initiating peroxidative lesions in membrane lipids.

Myocardial ischemia results from imbalance between the needs of cardiac tissue and oxygenated blood supplied by the coronary circulation. This imbalance causes a deficit in tissue supplies of oxygen and of metabolizable substrates which leads, to varying degrees according to the severity of the ischemic process, to a decrease in the production of energy. This production rapidly becomes insufficient to ensure normal function. Furthermore, the accumulation of metabolic waste products causes the development of progressive cellular acidosis with inhibitory functional and metabolic effects. These metabolic and functional changes in the cell are accompanied by changes in structure and arrhythmias, as well as modifications in all-energy dependent mechanisms, particularly clearly marked in relation to membrane systems responsible for ion transport. The degree of these various disturbances is dependent upon the severity of the ischemic process as well as its duration. Reversibility can be ensured if normal perfusion is restored early. If reperfusion occurs later, it is generally incapable of ensuring the recovery of normal cell function and the ischemic cell is then destined to necrosis. Among the possible factors responsible for irreversible ischemic injury, an increase in cellular free-calcium concentration or an over-production of oxygen free-radicals, have been largely suggested. A number of therapeutic measures (metabolic or pharmaceutical) have been suggested with the aim of prolonging the duration of tolerable ischemia before the development of irreversible changes which render reperfusion ineffective. This is particularly the case of anti-ischemic drugs, the prototype of which is for instance trimetazidine.

The Anthracycline antibiotic Adriamycin (Doxorubicin) is widely used in clinical medicine for cancer chemotherapy. Unfortunately, the clinical use of Adriamycin (ADR) is accompanied by side effects of which cardioto×icity is the most serious. The cumulative dose-dependent cardiomyopathy severely limits the total dose of ADR that can be administered in the treatment of neoplastic diseases(l). The prevention of ADR cardiotoxicity without interference with the drug’s antitumor activity would be of considerable advantage in achieving additional therapeutic benefit from this agent.

The aim of this study was to investigate the possibility of oxygen-free radical spin trapping with PBN, in models of isolated perfused hearts. Preliminary studies reported here demonstrate that (i) PBN may be precisely measured with UV spectroscopy, (ii) commercially available PBN does not show any ESR signal, (iii) PBN does not trap significant amounts of free radicals in a perfusion medium oxygenated for at least 3 h, and (iv) when added at 15 or 56 mM in the perfusion medium, PBN is a highly toxic compound, whereas no toxic effect was observed with 3mM-containing perfusate.

A number of cardioplegic solutions have been described for the reduction of cellular damage during ischemic cardiac arrest. Using an isolated working rat heart model, we have attempted to precise some of the factors affecting the post-ischemic recovery of myocardial tissue after a 30-min period of total ischemia at 37 °C. The results indicate that procaine (1MM) is able to afford some protection against normothermic ischemia while this protective effect remains consistently lower than that of the St. Thomas' Hospital solution (procaine + high K+ + high Mg2+; Jynge et al., 1977). On the other hand, hearts from rats of the Wistar strain consistently exhibit a significantly better degree of recovery than do hearts from rats of the Shermann strain. When hearts were perfused at different levels of preload (1 or 2 kPa) and afterload (8 or 10 kPa), post-ischemic recovery was better in hearts with lower levels of cardiac work. Glucose, insulin and dl-propranolol which have been shown to exert a protective...

The increasing use of cardioplegic protective infusates for reducing ischemic tissue injury requires that all infusates be carefully assessed for any protective or damaging properties. This study describes ultrastructural, enzymatic, and functional assessments of the efficacy of three infusates (Bretschneider, Kirsch, and St. Thomas' Hospital) in a rat heart model of cardiopulmonary bypass and ischemic cardiac arrest. The study reveals a close concordance of results as assessed by the three totally different indices of tissue damage. The results also indicate that, in the rat heart model, the St. Thomas' Hospital solution is an effective protective agent under all conditions studied but the Bretschneider solution is effective only under hypothermic conditions and the Kirsch solution is ineffective under all conditions studied and may exacerbate tissue injury. The studies further suggest that the potentially damaging effects of calcium-free myocardial infusates may be due to their induction of a "calcium paradox."

Glucose and fatty acid substrates had different effects on the cardiac output, heart work and rates of release of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) from isolated perfused working rat hearts with coronary artery ligation. Release of LDH or CPK was increased in hearts perfused with free fatty acids (FFA) instead of glucose. The addition of insulin decreased palmitate-induced release of LDH but did not increase cardiac output, while the addition of glucose to hearts perfused with fatty acid both increased cardiac output and depressed LDH release. In FFA-perfused hearts addition of both glucose and insulin gave lowest rates of enzyme release and increased cardiac output, heart rate and heart work. It is proposed that glucose and/or insulin protected the hearts from the deleterious effects of FFA.

Calcium-magnesium-free perfusions induced a large release of lactate dehydrogenase from isolated rat heart after returning to normal perfusion. This release generally appeared without irreversible damage and was quantitatively dependent on the duration and the temperature of the abnormal perfusion. Mechanical activity and the lack thereof are important factors in determining enzyme release. It is suggested that lactate dehydrogenase release does not result from a paradoxical effect of calcium but is dependent on the recovery of contraction in a damaged tissue.