The Role of Free Radicals in the Pathogenesis of Amiodarone Toxicity

Free Radicals and Amiodarone Toxicity. Introduction: In vitro and in vivo studies were performed to elucidate the pathogenesis of amiodarone toxicity. Methods and Results: Rats were treated with amiodarone alone (500 mg/kg body weight per day) or together with antioxidants (silibinin or MTDQ-DA: 50 mg/kg body weight per day) or with either antioxidanl alone. They received amiodarone for 30 days and antioxidant for 33 days (3 days pretreatment) In vitro, amiodarone induced a dose-dependent chemiluminescence signal, which was inhibited by the two dihydroquinolin-type antioxidants (MTDQ-DA, CH 402). Chemiluminometric results from liver homogenate demonstrated that simultaneous treatment with silibinin partially prevented the liver homogenate superoxide anion radical scavenger capacity decreasing effect of amtodarone. Amiodarone treatment caused a significant increase of NADPH and Fe³⁺ induced lipid peroxidalion in the liver microsomal fraction, which antioxidants (silibinin, MTDQ-DA) were unable to prevent. Light microscopy of the lung tissue in amiodarone-treated rats showed accumulation of foamy macrophages with thickening of the interalveolar septa, pneumonitis, and variable interstitial tibrosis. Antioxidant treatment did not prevent these changes. Electron micrographs of lung from amiodarone-treated ruts showed lysosomal phospholipoidosis, intralysosomal electron dense deposits, and increased lysosome number and size. In contrast to rats treated with amiodarone alone, those treated with both amiodarone and silibinin had significantly fewer lysosomes (P < 0.01); the lysosome size, shape, and internal characteristics remained he same. Simultaneous treatment with silibinin and amiodarone decreased lysosomal phospholipoidosis compared to amiodarone treatment alone. Simultaneous treatment with MTDQ-DA and amiodarone did not show any beneficial effect. Pulse radiolysis and cobalt 60-gamma (⁶⁰Co-γ) radiolysis studies showed that the main free radical product in a reducing environment was a very reactive aryl radical formed after the partial deiodination of the amiodarone molecule. The radioseasitizing effect of amiodarone was also verified in rat liver microsomal preparations using in vivo amiodarone with or without MTDQ-DA pretreatment and ⁶⁰Co-γ irradiation with or without the in vitro addition of antioxidants (CH 402, MTDQ-DA). In vivo, the MTDQ-DA treatment also had a radiosensitizing effect; however, the in vitro addition of both antioxidants resulted in a radio-protective effect. The aryl radical also may emerge in vivo during the metabolism of amiodarone. Conclusion: These observations suggest that amiodarone in vitro and in vivo generates free radicals that may play a role in the pathogenesis of amiodarone toxicity beside other well-established mechanisms, and antioxidants may have a partial protective effect against amiodarone toxicity.     

Effect of the platelet-activating factor antagonist RP 59227 (Tulopafant) on myocardial ischemia/reperfusion injury and neutrophil function

The effect of the platelet activating factor antagonist RP 59227 (Tulopafant) on myocardial infarct size was compared to that of vehicle-treated control group in barbital-anesthetized dogs subjected to 90 min of left circumflex coronary artery occlusion and 5 h of reperfusion. The myocardial region at risk and infarct size were determined by the triphenyltetrazolium histochemical technique and regional myocardial blood flow by radioactive microspheres. Myeloperoxidase (MPO) activity was used as an index of neutrophil infiltration into the ischemic-reperfused area. Vehicle (n=11) or RP 59227 (n=12, 2.5 mg/kg i.v.) were administered 15 min prior to occlusion. Hemodynamics and regional myocardial blood flow in the ischemic-reperfused areas did not differ between the two groups throughout the experiment. In contrast, the number of dogs which developed ventricular fibrillation during occlusion and reperfusion was significantly less in RP 59227-treated dogs (8 of 11 in the control group vs. 3 of 12 in the RP 59227-treated group, p<0.05). Myocardial infarct size expressed as a percent of the area at risk (control, 39.0±5.0; RP 59227, 24.8±3.4%) or as a percent of the left ventricle (control, 15.3±1.9; RP 59227, 9.0±1.3%) was significantly smaller in the RP 59227-treated group. Infarct size was inversely related to the collateral blood flow in the inner two thirds of the left ventricular wall of the infarct area, and this relationship was shifted downward in the RP 59227-treated group (p<0.05). MPO activity in the border zone immediately adjacent to infarcted tissue was reduced in the RP 59227-treated dogs (control, 7.4±0.5 U/g; RP 59227, 4.1±0.4 U/g). In additional in vitro studies, the addition of PAF was found to elicit a concentration-dependent potentiation in chemiluminescence produced by purified canine neutrophils, a measure of oxygen-derived free radicals, which was stimulated with a low concentration of opsonized zymosan. Preincubation of neutrophils with RP 59227 resulted in a concentration-dependent decrease in chemiluminescence produced by PAF primed cells. Taken together, these data demonstrate that RP 59227 effectively reduces myocardial infarct size and reduces the incidence of ischemia and reperfusion-induced arrhythmias in barbital-anesthetized dogs, and support the concept that PAF plays an important role in the pathogenesis of acute myocardial infarction. Received: 11 March 1998, Returned for 1. revision: 2 April 1998, 1. Revision received: 18 May 1998, Accepted: 18 May 1998     

Free Radicals and Ethanol-Induced Cytotoxicity in Neural Crest Cells

Associations between ethanol-induced cranial neural crest cell (NCC) damage in mammalian embryos and subsequent malformations as observed in human fetal alcohol syndrome have previously been illustrated. The vulnerability of NCCs to this teratogen may result, at least in part, from their sensitivity to free radical damage. To examine relationships between free radical generation and NCC cytotoxicity, primary culture of mouse NCCs was used. NCC viability was determined in both dose- and time-response studies involving ethanol exposure. After 48 hr of culture, cell viability was significantly diminished at all doses tested (i.e., 50,100,150, and 200 mM ethanol). At 100 mM ethanol (a dosage that is teratogenic in vivo and in whole embryo culture), cell viability decreased to ?50% of control values over the first 12 hr of culture, and decreased further, to ?20% by 48 hr. Using nitroblue tetrazolium as a probe, it was observed that exposure of NCCs to ethanol stimulated the production of superoxide anion radicals. Co-treatment of the ethanol-exposed NCCs with free radical scavengers including 300 units/ml of superoxide dismutase, catalase (500 units/ml), or ?-tocopherol (300 μM) significantly improved NCC viability. These results suggest that the ethanol-induced NCC injury is mediated, at least in part, through the generation of free radicals. To test this hypothesis further, NCCs were exposed in culture to xanthine/xanthine oxidase. Exogenous free radicals generated by the xanthine/xanthine oxidase system resulted in reduced NCC viability, the severity of which Increased in a time and enzyme concentration-related manner. Superoxide dismutase (300 units/ml) and catalase (500 units/ml) significantly reduced the effects of the xanthine/xanthine oxidase-generated free radicals on NCC viability. The similarity between the susceptibility of NCCs to ethanol and their susceptibility to exogenous free radicals in concert with the free radical scavenger-mediated amelioration of ethanol and exogenous free radical-induced NCC death strongly suggest that free radicals play a significant role in ethanol-induced NCC death.     

Enhanced suppression of myocardial slow action potentials during hypoxia by free fatty acids

Effects of free fatty acids (palmitate and linoleate) on myocardial contractility and slow action potentials (APs) were examined in Langendorff-perfused chick hearts. To study the slow APs exclusively, the fast Na+ channels were voltage-inactivated in elevated K+ (25 mM), and the concentration of Ca2+ ion was increased to 5.4 mM in order to induce slow APs. Palmitate (0.18, 0.54 or 0.72 mM) along with albumin (0.12 mM) was added to the perfusate. Albumin by itself did not affect contractility or the slow APs during normoxia and hypoxia. Under well oxygenated conditions, palmitate had no effect on contractility or the slow APs. However, palmitate accelerated the decline of contractility during hypoxia in a dose-dependent fashion. Hypoxia suppressed the slow APs, and palmitate and linoleate further exacerbated the suppression of slow APs produced by hypoxia. Nevertheless, palmitate and linoleate did not enhance the hypoxic reduction of the tissue high energy phosphate level. The present results suggest that free fatty acids elicit cardio-depressant effects on hearts through their direct action on the myocardial cell membrane (slow channels) rather than through any metabolic effects.     

The interrelation of glutathione reductase,catalase, glutathione peroxidase,superoxide dismutase,and glucose-6-phosphate in the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is the most common form of inflammatory arthritis, a systemic autoimmune disease characterized by chronic inflammation of the synovial joints, ultimately leading to joint destruction and permanent disability, affecting 1% of the world population. Oxidative stress in rheumatoid inflammation, due to the fact that antioxidant systems are impaired in RA and caused by fee radicals, might have an essential role in etiology of RA. This review includes the interrelation of antioxidants against free radicals in RA patients. There is much evidence that antioxidant team that covers glutathione reductase, catalase, glutathione peroxidase, superoxide dismutase, and glucose-6-phopshate destroy reactive oxygen species and other free radicals through enzymatic as well as nonenzymatic means. The change in relative levels of antioxidants vis-à-vis free radical formation and level could be used as indicators for effective and earlier diagnosis of RA.     

卡维地洛对氧自由基所致心肌细胞L型钙电流异常的保护作用

目的　研究卡维地洛对氧自由基引起的豚鼠单个心室肌细胞L型钙电流异常的保护作用。方法　采用全细胞膜片钳技术 ,观察 0 5mmol/L的H2 O2 引起单个豚鼠心室肌细胞L型钙电流改变及预先应用 0 5 μmol/L卡维地洛对这种改变的影响。结果　 0 5 μmol/L卡维地洛对正常豚鼠心室肌细胞L型钙电流及其通道动力学影响不显著。 0 5mmol/LH2 O2 作用下 ,豚鼠心室肌细胞L型钙电流峰值明显降低 (P <0 0 0 1) ,电流 电压曲线上移 ,通道稳态激活曲线和稳态失活曲线左移 ,通道恢复时间明显延长 (P <0 0 0 1)。预先给予 0 5 μmol/L卡维地洛 ,明显减轻H2 O2 对L型钙电流的抑制作用 (P <0 0 1) ;并且可减轻H2 O2 对L型钙通道动力学的异常影响。结论　卡维地洛可减轻氧化应激对心肌细胞L型钙电流的影响 ,这可能是其治疗心力衰竭的机制之一     

Metabolic changes by acute insulin deficiency in rabbits injected with anti-insulin serum

Summary Acute insulin deficiency was produced in rabbits treated with anti-insulin guinea pig serum. The changes in the levels of blood sugar, free fatty acids, aceto-acetate, SGOT, SGPT, liver glutamicoxaloacetic and glutamic-pyruvic transaminases, and liver lactate dehydrogenase were examined. A significant increase of the blood glucose level and the free fatty acids as well as glycosuria was observed. The other examined parameters remained unchanged. The results confirm the statement that acute insulin deficiency causes a prompt impairment of glucose and fat metabolism.

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Zusammenfassung Ein akuter Insulin-Mangelzustand wurde in mit anti-Insulinserum vom Meerschweinchen behandelten Kaninchen hervorgerufen. Es wurden die Veraenderungen des Blutzuckerspiegels, der freien Fettsaeuren, des Azetoazetats, der SGOT, SGPT, der Glutaminsaeure-Oxalessigsaeure-Transaminase und der Glutaminsaeure-Pyruvat-Transaminase, sowie der LDH in der Leber untersucht. Es wurde eine signifikante Steigerung des Blutzuckers, der freien Fettsaeuren und auch des Harnzuckers festgestellt. Die anderen untersuchten Parameter blieben unveraendert. Die Resultate bestaetigen den Tatbestand, dass der akute Insulinmangelzustand eine sofortige Stoerung des Kohlenhydrat- sowie des Fettstoffwechsels ausloest.

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Resumen Una carencia insulínica aguda ha sido provocada en los conejos tratados con suero de cobayo anti-insulina. Han sido estudiadas las variaciones en los niveles de la glucemia, de los ácidos grasos libres, del acetoacetato, de la SGOT, de la SGPT, de las transaminasas glutámico-oxaloacética y glutámico-pirúvica hepáticas y de la láctico-dehidrogenasa hepática. Ha sido puesto de manifiesto un aumento significativo de la glucemia y de los ácidos grasos libres, y también de la glucosuria. Los otros parámetros examinados han quedado invariados. Los resultados ratifican el hecho de que la carencia aguda de insulina provoca una alteración inmediata en el metabolismo de los glúcidos y de los lípidos.

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Resume On a provoqué une carence en insuline aïgue chez des lapins traités au sérum de cobaye antiinsuline. On a étudié les variations de taux de glycémie, des acides gras libres, d'acéto-acétate, de SGOT, de SGPT, des transaminases glutamique oxalacétique et glutamico-pyruvique hépatiques ainsi que de la déshydrogénase lactique hépatique. On a observé une augmentation significative de la glycémie et des acides gras libres, et même de la glycosurie. Les autres paramètres étudiés sont restés inchangés. Les résultats confirment le fait que la carence aïgue en insuline provoque une perturbation immédiate du métabolisme des glucides et des lipides.

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Riassunto Una carenza insulinica acuta è stata provocata nei conigli trattati con siero di cavia anti-insulina. Sono state studiate le variazioni del livello della glicemia, degli acidi grassi liberi, dell'acetoacetato, della SGOT, della SGPT, delle transaminasi glutammico-ossalacetica e glutammico-piruvica epatiche e della lattico deidrogenasi epatica. E' stato rilevato un aumento significativo della glicemia e degli acidi grassi liberi, ed anche della glicosuria. Gli altri parametri esaminati sono rimasti invariati. I risultati confermano il fatto che la carenza acuta di insulina provoca un disturbo immediato del metabolismo glucidico e lipidico.

     

游离皮瓣移植修复肢体软组织缺损(附59例报告)

对 5 9例严重肢体软组织缺损伤患者 ,取自体组织皮瓣游离移植修复肢体缺损。根据不同缺损与缺损的范围应用不同的游离皮瓣。皮瓣面积最小 10 cm× 12 cm,最大 2 0 cm× 40 cm。其中背阔肌皮瓣 43例 ,股前外侧皮瓣 7例 ,侧胸皮瓣 6例 ,脐胸皮瓣 3例。结果 :完全成活 5 6例。因受区创面不新鲜感染 ,2例部分坏死 ,1例完全坏死。认为用游离自体皮瓣移植修复严重的肢体软组织缺损 ,可有效修复肢体创面 ,防止因皮肤缺损所致血管、神经和骨骼的进一步损伤或感染 ,提高伤肢修复的疗效和功能     

Melatonin prevents the free radical and MADD metabolic profiles induced by antituberculosis drugs in an animal model

The objective was to determine the effect of combined antituberculosis (anti-TB) drug therapy and an antioxidant, melatonin, on the free radical and organic acid profiles in an experimental rat model. A combined anti-TB drug, Rifater, consisting of 12.0 mg rifampicin, 0.8 mg isoniazid, and 23.0 mg pyrazinamide and 18.56 microg melatonin/kg body weight per day (corresponding to average physiological human intake) were orally administered to Sprague-Dawley rats. Hydroxyl radical production was monitored by quantifying 2,3-dihydroxybenzoic acid produced after intraperitonial sodium salicylate injections. Organic acid extractions and gas chromatography-coupled mass spectrometry analyses were performed on collected urine samples. The results show hydroxyl radicals (P = 0.0019) and organic acids (P-value range: 0.037 to <0.001), characteristic of a multiple acyl-CoA dehydrogenase defect (MADD), were elevated with Rifater treatment and these elevations were significantly lowered with melatonin pretreatment (P-value range: 0.031 to <0.001), probably because of its inherent antioxidant activity. We conclude that hydroxyl radical production and an increased organic acid profile induced by anti-TB medication indicates inhibition of the electron transport chain. We also conclude that free radicals leading to clinical symptoms associated with an MADD metabolic profile induced by anti-TB treatment could be alleviated by melatonin intervention.     

Deferoxamine infusion during coronary artery bypass grafting ameliorates lipid peroxidation and protects the myocardium against reperfusion injury: immediate and long-term significance.

AIMS: Previous reports have demonstrated enhanced myocardial protection and better post-ischaemic recovery using the oxygen free radical scavenger deferoxamine (DEF) during cardioplegia. The aim of this study was to test whether, in patients undergoing coronary artery bypass grafting (CABG), DEF i.v. infusion can reduce reperfusion injury on a short- and long-term basis. METHODS AND RESULTS: Forty-five consecutive male patients were randomly allocated to two groups: in group D (n=25, age 60.8+/-8.6 years), 4 g of DEF were infused for 8 h starting immediately after the induction of anaesthesia; in group C (n=20, age 62.2+/-6.4 years) dextrose solution was given for the same time as placebo. Haemodynamic monitoring and measurement of oxygen free radical production [by measuring thiobarbituric acid reactive substances (TBARS)] were carried out before and after CABG. Left ventricular ejection fraction (EF) and wall motion score index (WMSI) were measured before and after CABG and 12 months later. Haemodynamic measurements were similar in both groups before and after CABG. TBARS peaked at 4.8+/-1.1 nmol/mL in group C, but remained unchanged (2.4+/-0.9 nmol/mL) in group D (P=0.01). At baseline, both the EF and WMSI were similar between the groups. Following CABG, EF increased more in group D (8.8+/-8.4%) than in group C (1.3+/-6.7%), P=0.008, while WMSI decreased more in group D (-0.7+/-0.3) than in group C (-0.2+/-0.2), P=0.0001. Dividing group D according to the pre-operative median EF value (38%), we observed that after 1 year follow-up, DEF infusion conferred more protection in patients with a lower EF (EF increased by 19.3+/-6.2%, WMSI decreased by -1.1+/-0.2) than in those with a higher EF (EF increased by 7.7+/-4.5%, WMSI decreased by -0.8+/-0.2), P=0.001, respectively. CONCLUSION: In patients undergoing CABG, DEF i.v. infusion ameliorates oxygen free radical production and protects the myocardium against reperfusion injury. Patients with a lower EF seem to benefit more by DEF i.v. infusion.