Effect of in vivo heart irradiation on coronary reactivity in the rat.

The frequent exposure of the heart to radiation during thoracic tumor radiotherapy often results in chronic impairment of myocardial function. The aim of the present investigation was to evaluate the effect of irradiation on coronary vascular tone in rat hearts exposed in vivo to a single dose of 20 Gy gamma rays. The ability of rat hearts to respond to changes in coronary reactivity was analyzed 1, 15, 30 and 60 days following cardiac irradiation, using the Langendorff model, after perfusion of either L-nitro-arginine (LNA), an inhibitor of nitric oxide synthetase or SIN 1, a nitric oxide donor drug. LNA-induced vasoconstriction and SIN 1-induced vasodilation were lost respectively 15 days and 30 days after irradiation, and associated with smooth muscle cell alterations observed in microscopy, but without any changes in myocardial MDA levels. Thus, our results suggest that 1) endothelium may represent an early and specific radiation target, characterized by radiation-induced vascular tone dysfunctions, with no detectable microscopical changes; 2) alterations are progressive, resulting first from endothelial damage, followed by smooth muscle cell injuries. In conclusion, a local cardiac irradiation induced cellular dysfunction, characterized by a loss of coronary reactivity without changes of the lipid peroxidation index in the hearts.

[Genetically modified animal models in cardiovascular research]. / Modelos animales genéticamente modificados en investigación cardiovascular.

It is a basic tenet of molecular and clinical medicine that specific protein complements underlie cell and organ function. Since cellular and ultimately organ function depend upon the polypeptides that are present, it is not surprising that when function is altered changes in the protein pools occur. In the heart, numerous examples of contractile protein changes correlate with functional alterations, both during normal development and during the development of numerous pathologies. Similarly, different congenital heart diseases are characterized by certain shifts in the motor proteins. To understand these relationships, and to establish models in which the pathogenic processes can be studied longitudinally, it is necessary to direct the heart to stably synthesize, in the absence of other peliotropic changes, the candidate protein. Subsequently, one can determine if the protein's presence causes the effects directly or indirectly with the goal being to define potential therapeutic targets. By affecting the heart's protein complement in a defined manner, one has the means to establish both mechanism and the function of the different mutated proteins of protein isoforms. Gene targeting and transgenesis in the mouse provides a means to modify the mammalian genome and the cardiac motor protein complement. By directing expression of an engineered protein to the heart, one is now able to effectively remodel the cardiac protein profile and study the consequences of a single genetic manipulation at the molecular, biochemical, cytological and physiologic levels, both under normal and stress stimuli.

Manipulating the contractile apparatus: genetically defined animal models of cardiovascular disease.

Within the last 10 years via gene targeting and transgenesis, numerous models of cardiovascular disease have been established and used to determine if a protein's presence or absence causes cardiovascular disease. By affecting the heart's protein complement in a defined manner, the function of the different mutated proteins or protein isoforms present in the contractile apparatus can be determined and pathogenic mechanism(s) explored. We can now remodel the cardiac protein profile and effect replacement of even the most abundant contractile proteins. Precise genetic manipulation allows exploration of the structure-function relationships which underlie cardiac function, and the consequences of defined mutations at the molecular, biochemical, cytological and physiologic levels can be determined.

Antioxidant properties of aminoguanidine.

It is well known that aminoguanidine (AG) can diminish advanced glycosylation of proteins, which might be beneficial in preventing chronic diabetic complications. Recent reports suggested an inter-relationship between glycosylation of protein and free radical damage. In the present study, we examined the free radical scavenging properties of AG. Electron paramagnetic resonance using the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was performed to determine the superoxide and hydroxyl radical scavenging abilities of AG. These experiments revealed that AG was an effective hydroxyl radical scavenger even though it expressed a direct inhibitory effect on the xanthine oxidase activity at high concentrations (AG > or = 5 mM). In the second part of the study, allophycocyanin was used as an indicator of free radical mediated protein damage. In the assay, 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) was used as a peroxyl radical generator, and the loss of allophycocyanin fluorescence was monitored. The antioxidant effect of AG was expressed in oxygen-radical absorbing capacity (ORAC), where one ORAC unit equals the net protection produced by 1 microM Trolox (a water soluble analogue of vitamin E) as a control standard. AG exhibited a significant dose-dependent effect against free radical damage. These radical scavenging properties of AG may contribute to protective effects during glycation and explain the prevention of diabetic complications.

Effects of combined irradiation and doxorubicin treatment on cardiac function and antioxidant defenses in the rat.

Combined radiotherapy and chemotherapy have represented a major advance in the therapeutic management of cancer therapy. However, the combination of doxorubicin (DXR) and cardiac irradiation (IRR) could precipitate the unexpected expression of congestive heart failure. Oxidative lesions induced by IRR and DXR could represent one of the pathogenic factors of myocardial dysfunction. Our investigations were performed to evaluate in the rat: 1) cardiac functional changes, 2) cardiac and plasma peroxidative damage and antioxidant defenses variations, that occur 24 h (acute effects) and 30 d (middle term effects) following DXR treatment 1 mg/kg(-1)/day(-1) IP for 10 d and a 1 x 20 Gy cardiac gamma-irradiation. Our results showed that DXR affected heart reactivity as early as the end of its administration, although irradiation exerted no detectable effect. Antioxidant defenses disturbances in hearts of DXR treated rats were characterized by vitamins C and E decreases, catalase activity induction and an increase in lipid peroxidation. Moreover, plasma vitamin C consumption and the lower level of plasma lipid peroxidation attested to the efficient solicitation of antioxidant defenses that probably contributed to the preservation of cardiac function at 24 h. After 30 d, cardiac dysfunction became symptomatic at rest, resulting from DXR cardiac toxicity. In spite of the persistent activation of cardiac catalase activity, antioxidant deficiency and increased plasma and cardiac lipid peroxidation highlighted defenses overtaken. Thus, different physiopathological mechanisms are involved in heart disturbance at acute and middle terms, IRR and DXR acting on distinct targets without disclosing synergistic effects. After 30 d, cardiac and plasma biochemical abnormalities were emphasized by the combined DXR+IRR therapy, pointing out the severity of the damage. Oxidative damage to the heart induced both by irradiation and DXR, may be one of the pathogenic factors of myocardial dysfunction. There is the possibility that the deleterious effects might be limited by the use of pharmacologic antioxidant agents.

Studies by electron paramagnetic resonance of the importance of iron in the hydroxyl scavenging properties of ascorbic acid in plasma: effects of iron chelators.

Ascorbic acid is considered to be the most important antioxidant of plasma. Its oxidation leads to the ascorbyl free radical (AFR), detected by electron paramagnetic resonance (EPR) spectroscopy. The purpose of this study was to investigate by EPR the interaction of plasma AFR levels in different situations of oxidative stress. Our results showed that plasma AFR remains constant after rat feeding with vitamin C (5 mg or 50 mg per 100 g body weight). We also demonstrated that: (1) the ascorbyl free radical (AFR) level was increased after direct addition of iron Fe3+/EDTA to plasma, the optimal level was reached after addition of 8 microM Fe3+/EDTA (1:2); (2) this AFR production was associated with the formation of hydroxyl radicals. Iron chelators (deferrioxamine, a synthetic iron chelator and apotransferrine, a biological iron chelator) added just before the Fe3+/EDTA complex inhibited the increase of AFR signal induced by this complex. The scavenging effect of plasma was significantly correlated with the AFR production. Therefore, AFR, which is naturally present in plasma, could be used as an index of oxidative stress in which free radicals or adverse iron mobilisation are implicated.

Comparison of epirubicin and doxorubicin cardiotoxicity induced by low doses: evolution of the diastolic and systolic parameters studied by radionuclide angiography.

BACKGROUND: Previous studies have demonstrated that epirubicin (EPI) has a lower propensity to produce cardiotoxic effects than doxorubicin (DXR) at high doses. HYPOTHESIS: The aim of the study was to compare the cardiotoxicity induced by low doses of EPI and DXR in patients before and 1 month after the end of chemotherapy. METHOD: In a prospective study, 99 patients with a mean age of 51 +/- 12 years and without cardiac disease were studied before and 1 month after the end of chemotherapy. Group 1 included 38 patients receiving 246 +/- 96 mg/m2 of DXR and Group 2 included 61 patients receiving EPI with and equivalent dose of 219 +/- 92 mg/m2 of DXR. Ejection fraction (EF) of the left ventricle (LV), peak ejection rate (PER), and peak filling rate (PFR) [expressed in end-diastolic volume/s (EDV/s)] were evaluated by gated radionuclide angiography; PFR/PER were also calculated. RESULTS: Moderate and similar alterations of left ventricular ejection fraction were shown for low doses of anthracyclines. The EF of the LV decreased from 57 +/- 6% to 54 +/- 6% for DXR group (Group 1) (p = 0.005), and from 58 +/- 5% to 55 +/- 5% for the EPI group (Group 2)(p = 0.001). The PER of the left ventricle fell from 3.08 +/- 0.46 EDV/s to 2.79 +/- 0.49 in Group 1 (p = 0.004) and from 2.98 +/- 0.50 to 2.73 +/- 0.34 EDV/s in Group 2 (p = 0.001). In contrast, no significant alteration of PFR appeared in Group 2 (from 2.72 +/- 0.51 to 2.62 +/- 0.41 EDV/s) for the equivalent dose of anthracycline, while PFR of the LV dropped from 2.82 +/- 0.76 (EDV/s) to 2.41 +/- 0.55 after doxorubicin (p = 0.004). No difference was found between 1 and 12 months after the end of the treatment in 25 patients in Group 1 and 28 patients in Group 2. These results confirm the advantage of EPI over DXR in terms of cardiotoxicity and help explain the relationship of cellular damage mechanisms with the functional parameters of nuclear investigation. CONCLUSION: A possible explanation for specific alteration after DXR could be the increased production of semiquinone free radicals, which are known to induce membrane damage and, consequently, myocardial edema and diastolic alteration.

Plasma iron status and lipid peroxidation following thrombolytic therapy for acute myocardial infarction.

Free radical species have been implicated as important agents involved in myocardial ischemic and reperfusion injuries. Superoxide is capable of mobilizing iron from ferritin and the released iron can cause hydroxyl formation from H2O2. The aim of this study was to evaluate the time-dependent increase in lipid peroxidation assessed by plasma thiobarbituric acid reactive substances (TBARS) and the relationship between lipid-peroxidation and the iron status. Peripheral venous blood samples were obtained from 17 men with acute myocardial infarction (AMI) before thrombolytic treatment (T0) and 1, 2, 3, 4, 8, 12, 16, 20, 24 and 48 hours after commencing fibrinolytic treatment. The concentration of TBARS, the parameters of iron metabolism, serum myoglobin, creatine kinase, and creatine kinase-MB were measured. Early reperfusion was judged by regression of sinus tachycardia (ST) elevation and reduction of chest pain. Recanalization of coronary artery was evaluated by a late coronary angiography 24-96 hours after thrombolysis. After thrombolytic therapy, the TBARS level was raised from 2.98 +/- 0.80 (T0) to 4.57 +/- 1.24 (peak), and decreased to 2.96 +/- 0.40 nmol/mL plasma at T48 (T0 vs peak: P < 0.001, peak vs T48: P < 0.001, T0 vs T48: NS). The mean time of the peak was observed at 9.7 +/- 7.5 hours. The iron increased significantly from 0.67 +/- 0.34 (T0) to 1.15 +/- 0.52 mg/L (peak), and returned to the pre-reperfusion to levels: 0.53 +/- 0.28 UI/L at T48 (TO vs peak: P < 0.001, peak vs T48: P < 0.001, T0 vs T48: NS). The mean time of the peak was observed at 9.4 +/- 7.3 hours. In return, no correlation was found between the increase of plasma creatine-kinase activity, myoglobin and iron or between the biochemical markers and time of fibrinolytic therapy. The results confirmed the importance of the temporal relationship between lipid peroxidation and iron status after thrombolytic therapy. Our results are in agreement with the concept that antioxidant agents used in association with thrombolytic therapy might be useful.

Direct evidence of caeruloplasmin antioxidant properties.

The chain-breaking antioxidant potential of caeruloplasmin and bovine serum albumin (BSA) has been investigated in comparison with other well-established antioxidants. Their Oxygen Radical Absorbing Capacity (ORAC), was measured by using beta-phycocyanin (beta-PC) as a fluorescent indicator protein, 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) as a peroxyl radical generator and the water soluble vitamin E analogue, Trolox, as a reference standard. The relative peroxyl absorbing capacities/mole for Trolox, caeruloplasmin, heat-denatured caeruloplasmin (hCP), catalase, bovine serum albumin (BSA), superoxide dismutase (SOD), and deferoxamine were 1; 2.6; 3.3; 3.7; 1.2; 0.1; 0.2, respectively. Caeruloplasmin was far more effective as a peroxyl radical scavenger than SOD, deferoxamine and BSA, but slightly less effective than catalase. The peroxyl radical absorbing capacity of caeruloplasmin was enhanced by heat-denaturation of the protein. Electron paramagnetic resonance (EPR) spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trap, was applied in order to measure the scavenger abilities of caeruloplasmin on superoxide radical and hydroxyl radical production and the concentration required to inhibit by 50% oxygen free radical formation (IC50) was determined. The IC50 values of caeruloplasmin, hCP, and BSA for the superoxide radical were 12, 2, 260 microM and for the hydroxyl radical 15, 2, 200 microM. These results show that caeruloplasmin is an effective chain-breaking antioxidant for a variety of radicals, independently of its catalytic ferroxidase activity.

In vitro studies of interactions of NO. donor drugs with superoxide and hydroxyl radicals.

Nitric oxide (NO.) is a free radical characterized by a high spontaneous chemical reactivity with many other molecules including the superoxide radical (O2.-). This complex interaction may generate a peroxynitrite anion (ONOO-), which behaves as an important mediator of oxidative stress in many pathological states. In the present study, in vitro experiments were performed to assess directly the O2.- and hydroxyl (.OH) radical scavenging effects of various NO. donor drugs, i.e. sodium nitroprusside (SNP), sodium nitrite (NaNO2), molsidomine and SIN 1, at pH 7.4, 7 or 6. Concentrations of NO. in the incubation medium containing the different NO. donor drugs were measured by the assay based on the reaction of Fe-N-methyl-D-glucamine dithiocarbamate (MGD) with NO. that yields a stable spin-adduct measured by electron paramagnetic resonance (EPR). O2.- and .OH generation was characterized by EPR spin trapping techniques, using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). These free radicals were generated from the enzymatic system xanthine-xanthine oxidase, in phosphate buffer adjusted at pH 7.4, 7 and 6. Under these experimental conditions, SNP exhibited the strongest superoxide scavenging properties, characterized by IC50 values expressed in the micromolar range, which decreased at low pH. Addition of SNP (800 microM) to solution containing MGD and Fe2+ (5:1) at pH 7 4 produced a three line EPR spectrum which is identified to [(MGD)2-Fe2+-NO]. In control experiments no EPR signal was observed. We obtained the same results with NaNO2 and an augmentation of the spin-adduct level was noted with the prolongation of the incubation period. In return, molsidomine (2 mM) did not produce, in our conditions, a detectable production of NO.. NaNO2 displayed a significant superoxide scavenging effect only at pH 6, whilst neither molsidomine nor SIN 1 had any effect. Therefore, the superoxide scavenging properties of SNP, NaNO2, and molsidomine appeared to be closely related to their potential for NO release, which partially depends on the pH conditions. The behaviour of SIN 1 is more complicated, the speed of oxygen diffusion probably acting as a limiting factor in NO. formation in our conditions. The production of NO. was detected in presence of SIN 1. The intensity of the complex is comparable with the signal founded with NaNO2. By contrast, all molecules exhibited hydroxyl radical scavenging properties, highlighting the capacity of .OH to react with a wide range of molecules. In conclusion, considering the poor chemical reactivity of O2.-, the NO. donor drugs/O2.- interactions suggest a special relationship between these two radical species, which, in certain pathological states, could lead to the generation of cytotoxic end-products with strong oxidizing properties.