Free radicals and environmental toxins

Some chemicals that contaminate our environment exert their toxic effects by virtue of their ability to form free radicals. In the absence of sufficient quenching reactions, these reactive radicals can attack biomolecules, resulting in their oxidative degradation. Biological membranes which contain polyunsaturated fatty acids are most susceptible to oxidative degradation (lipid peroxidation), although oxidation of DNA may have more severe biological consequences. Free radicals species can be generated by at least two mechanisms in vivo. The first, of which carbon tetrachloride (CCl4) is the classic example, is the biotransformation of the chemical to a free radical species. Metabolism of CCl4 to the trichloromethyl radical by the hepatic mixed-function oxidase system results in the initiation of lipid peroxidation, protein-lipid cross linkages, and trichloromethyl adducts with DNA, protein, and lipid. The second mechanism for forming free radicals involves their reduction to less stable free radical intermediates which are oxidized by molecular oxygen to give superoxide (O2-.). In the presence of transition metals, such as iron, O2-. can be converted to other oxygen radical species, such as the hydroxyl radical (.OH), an extremely powerful oxidant capable of cleaving DNA, oxidizing protein, and initiating lipid peroxidation. Under many conditions, lipid peroxidation appears not to be initiated by .OH, but rather by an iron-oxygen complex. Regardless of the identity of the initiating species, transition metals are required for most of the deleterious reactions of oxygen. Superoxide and certain organic radicals have been found to release iron from ferritin.     

Free radicals in digestive diseases

Free radicals have recently been implicated in a number of biochemical and chemical reactions in the body. Lipid peroxidation induced by free radical reaction is believed to be one of the major causes of cell damage and injuries in cell membranes. In recent years, reports have appeared citing the contribution of free radicals and active oxygen species in the etiology of various digestive diseases. For example, gastric mucosal injuries and the increases in thiobarbituric acid-reactive substances in the gastric mucosa induced by ischemia or ischemia/reperfusion were significantly inhibited by treatment with superoxide dismutase and catalase. It has been suggested that superoxide radical or hydroxyl radical may be the major oxygen radicals contributing to ischemia or ischemia/reperfusion injury in the stomach, small intestine, and liver. There reactive species can attack and damage important biological molecules. Within cellular membranes, hydroxyl radical can initiate a free radical chain reaction known as lipid peroxidation, in which polyunsaturated fatty acids are broken down into water soluble products and toxic lipid peroxides are produced with the consequent destruction of membrane integrity. The major source of active oxygen species produced after ischemia or ischemia/reperfusion seems to be the enzymatic xanthine oxidase and activated polymorphonuclear leukocytes (PMN). In the large intestine which has little activity in xanthine oxidase, PMNs are a more important source of active oxygen species and play a role in the pathogenesis of the inflammatory bowel diseases. The above information suggests that oxygen-derived free radicals are involved in the fundamental mechanism of tissue injury in various disorders of the digestive system.     

Stunned myocardium and oxygen free radicals--sarcolemmal membrane damage due to oxygen free radicals.

Reperfusion after reversible ischemia has been shown to result in prolonged depression of contractile function ("myocardial stunning"). Recent studies suggest that oxygen free radicals may mediate postischemic dysfunction. Since heart sarcolemmal membranes, which contain several types of enzymes, ion channels and receptors play important roles to maintain cell functions, the present study was undertaken to examine the effects of oxygen free radicals on heart sarcolemmal membrane functions in vitro. In the presence of a superoxide anion radical-generating system (2mM xanthine plus 0.03 U/ml xanthine oxidase), sarcolemmal Ca(2+)-stimulated ATPase activity and ATP-dependent Ca2+ accumulation were inhibited in an incubating time-dependent manner. Both lipid peroxidation (r = 0.82) and sulfhydryl group content (r = 0.95) showed significant correlations with Ca(2+)-stimulated ATPase activity. ATP-independent Ca2+ bindings were increased upon treating the membranes with xanthine plus xanthine oxidase. Voltage-dependent Ca(2+)-channels were also affected by oxygen free radicals. The maximal number of binding sites (Bmax) for [3H]-nitrendipine binding was depressed without any changes in dissociation constant (Kd). The effects of oxygen free radicals on adrenergic receptors were more complex. Bmax for [3H]-dihydroalprenolol (DHA) binding (beta-receptor) was increased whereas Bmax for [3H]-prazosin binding [alpha 1-receptor) was decreased after incubating the membrane with xanthine plus xanthine oxidase. Kd for [3H]-DHA or [3H]-prazosin binding was increased. Superoxide dismutase showed protective effects on the changes in these membrane functions due to xanthine plus xanthine oxidase. It is suggested that oxygen free radicals damage heart sarcolemmal membrane functions which may lead to cardiac dysfunction in the stunned myocardium.     

Reduction of lipid peroxidation in reperfused isolated rabbit hearts by diltiazem

The calcium-channel inhibiting agent, diltiazem, has been shown to enhance salvage of reperfused myocardium independent of effects on coronary blood flow or myocardial work. Because lipid peroxidation may be a mediator of reperfusion injury and modifiable by calcium-sensitive pathways, we evaluated the effects of diltiazem on the formation of malondialdehyde (MDA), a product of lipid peroxidation, in isolated rabbit hearts perfused with buffer under control conditions or after 60 minutes of ischemia with or without 3 minutes of reperfusion. Diltiazem (5 x 10(-7)M) reduced tissue MDA content in seven reperfused hearts compared with levels measured in 14 hearts reperfused without drug (1.54 +/- 1.09 [SD] compared with 3.57 +/- 1.88 nmol/g, p less than 0.05). Superoxide dismutase and catalase were ineffective in reducing tissue MDA content in reperfused hearts (n = 8; MDA concentration, 3.88 +/- 2.82 nmol/g) although they were effective in preventing lipid peroxidation in separate studies in which oxygen-centered free radicals were generated directly by an infusion of xanthine oxidase and hypoxanthine. These results suggest that the salutary effects of diltiazem in the setting of reperfusion may be mediated by reduction of lipid peroxidation at a locus not accessible to scavengers of oxygen-centered free radicals or by a mechanism not mediated by free radical pathways.     

Oxygen free radicals and cardiac reperfusion abnormalities.

Oxygen free radicals are highly reactive compounds causing peroxidation of lipids and proteins and are thought to play an important role in the pathogenesis of reperfusion abnormalities including myocardial stunning, irreversible injury, and reperfusion arrhythmias. Free radical accumulation has been measured in ischemic and reperfused myocardium directly using techniques such as electron paramagnetic resonance spectroscopy and tissue chemiluminescence and indirectly using biochemical assays of lipid peroxidation products. Potential sources of free radicals during ischemia and reperfusion have been identified in myocytes, vascular endothelium, and leukocytes. In several different experimental models exogenous free radical-generating systems have been shown to produce alterations in cardiac function that resemble the various reperfusion abnormalities described above. Injury to processes involved in regulation of the intracellular Ca2+ concentration may be a common mechanism underlying both free radical-induced and reperfusion abnormalities. Direct effects of free radicals on each of the known Ca(2+)-regulating mechanisms of the cell as well as the contractile proteins and various ionic membrane currents have been described. Free radicals also inhibit critical enzymes in anaerobic and aerobic metabolic pathways, which may limit the metabolic reserve of reperfused myocardium and contribute to intracellular Ca2+ overload. Inhibiting free radical accumulation during myocardial ischemia/reperfusion with free radical scavengers and inhibitors has been demonstrated to reduce the severity of myocardial stunning, irreversible injury, and reperfusion arrhythmias in many, but not all, studies. This evidence strongly implicates free radical accumulation during myocardial ischemia/reperfusion as an important pathophysiological mechanism of reperfusion abnormalities, although many issues remain unresolved.     

Free radical and granulocyte-mediated injury during myocardial ischemia and reperfusion

The success of thrombolytic/reperfusion therapy in limiting the extent of myocardial infarction may be limited by reperfusion injury. Damage from acute ischemia is not due solely to the interruption of blood flow; rather, ischemia initiates a cascade of reactions involving partially reduced oxygen, inflammatory mediators, mechanical capillary obstruction by granulocytes and other events that lead to irreversible injury. A surprising consequence is that reperfusion by delivering oxygen and granulocytes may counteract some of the benefits of restoring flow. Mechanisms of neutrophil and free radical injury include superoxide radical formation and lipid peroxidation, progressive leukocyte capillary plugging and capillary no-reflow, and edema. The interaction of various specific mechanisms of injury in the heart (i.e., xanthine oxidase, mitochondrial superoxide leak, neutrophil superoxide, degranulation and capillary plugging, and neutrophil-derived vasoconstrictors) deserves further study.     

Comparison of the efficacy of mechanistically different antioxidants in the rat hemorrhagic shock model.

Four pharmacological mechanisms for antagonizing free radical generation or reactions were compared in terms of their efficacy in attenuating hemorrhagic shock in rats. These included opposing superoxide generation by xanthine oxidase (e.g., oxypurinol), inhibiting arachidonic acid oxidation by cyclooxygenase (e.g., ibuprofen), chelating iron (e.g., desferal), and inhibiting lipid peroxidation (e.g., tirilazad mesylate [U-74006F] and U-78517G). Animals were hemorrhaged to a mean arterial pressure (MAP) of 43-45 mmHg where they were held for 2 hr. Five minutes prior to the end of the hemorrhage period, either vehicle, U-74006F (10 mg/kg), U-78517G (10 mg/kg), oxypurinol (10 or 25 mg/kg), desferal (10 or 25 mg/kg), or ibuprofen (10 mg/kg) was administered i.v., followed by the reinfusion of shed blood. In vehicle-treated animals, MAP declined progressively over the 2 hr post-reinfusion. Ibuprofen, desferal, and oxypurinol treatments each failed to attenuate this decline. In contrast, both U-74006F and U-78517G resulted in a significantly improved maintenance of MAP. Evidence of shock-induced lipid peroxidation was observed in terms of a 73.8% depletion in liver vitamin E content at 2 hr post-reinfusion in vehicle-treated rats. This decrease was prevented by both U-74006F and U-78517G. Inhibition of free radical-induced lipid peroxidation appears more effective for attenuating free radical pathophysiology in hemorrhagic shock that attempting to inhibit specific pathways of oxygen radical generation.     

Role of oxygen radicals in ischemia-induced lesions in the cat stomach

Ischemia in a stomach that contains acid may produce severe gastric mucosal injury. The extent to which oxygen-derived free radicals are involved in the pathogenesis of this injury was investigated in the present study. Local gastric ischemia was achieved by reducing celiac artery pressure to 30 mmHg for 1 h. Ischemic injury was assessed by recording the loss of 125I-albumin and 51Cr-red cells across the gastric mucosa. Cats were treated with a xanthine oxidase inhibitor (allopurinol), a superoxide radical scavenging enzyme (superoxide dismutase), and a scavenger of hydroxyl radicals (dimethyl sulfoxide). The damage associated with ischemia only occurred during reperfusion of the stomach and was worst in the antrum. The level of xanthine oxidase in the antrum was twice that of the corpus. Treatment with allopurinol, superoxide dismutase, and dimethyl sulfoxide reduced 51Cr-red cell loss to 15%, 25%, and 21% of control (untreated) animals, respectively. The data indicate that oxygen-derived free radicals play a role in ischemic injury to the stomach and that the hydroxyl radical, a secondary radical produced from the superoxide anion, appears to be the major oxygen radical contributing to ischemic damage.     

Role of oxygen radicals in cardiac injury due to reoxygenation

The ability of oxygen derived free radicals to induce irreversible cellular injuries during reoxygenation was studied on isolated potassium-arrested heart preparation. Enzymatic scavengers of hydrogen peroxide (H2O2) and superoxide anion (O-2), catalase and superoxide dismutase, were not effective in reversing the cardiac alterations induced by hypoxia. Cellular injuries induced by reoxygenation, 'Oxygen paradox', were partially prevented by scavengers of H2O2 (glutathione reduced form, catalase) and O-2 (superoxide dismutase). The 'oxygen paradox' was associated with a release of malonaldehyde. The inhibition of lipid peroxidation by alpha-tocopherol prevented the toxic effect of molecular oxygen on hypoxic hearts. The specific quenchers of singlet oxygen (histidine) and hydroxyl radical (mannitol) reduced the peroxidation of unsaturated lipids and the intensity of the 'oxygen paradox' phenomenon. The results indicate that in cardiac muscle (i) oxygen derived free radicals are important byproducts of abnormal oxidative metabolism present during the post hypoxic period; (ii) the 'oxygen paradox' phenomenon is related to the formation of lipid hydroperoxides leading to the cellular membrane disruption and to the irreversible alteration of cardiac integrity.     

Effect of superoxide generation on rat heart mitochondrial pyruvate utilization

Previous research has shown that heart mitochondria are able to produce reactive species of oxygen such as superoxide radicals, hydrogen peroxide and hydroxyl radicals [10, 11]. When these compounds are formed beyond a certain level they are not completely removed by the enzymatic and metabolic processes which neutralize their toxicity, and as a result they are able to produce structural and functional damages that impair mitochondrial function [5, 10]. In order to study the molecular mechanism/s by which the oxygen radicals may function as mediators of cellular injury a flow of these radicals by chemical, enzymatic or photochemical methods has been generated in vitro in the presence of cellular preparations. For example, the exposure of isolated subcellular particles to the enzymatic flow of oxygen radicals produced by the reaction of xanthine oxidase upon xanthine reduced both calcium uptake velocity and Ca2+-ATPase activity in sarcoplasmic reticulum [7], while it reduced Ca2+-stimulated ATPase activity in myofibrillar preparations [4]. In addition, incubation with the xanthine oxidase reaction produced an impairment of the respiratory functions associated with an increased lipid peroxidation in the isolated mitochondria [5, 10]. These negative effects were augmented in alpha-tocopherol-deficient mitochondria [3], but were opposed by the exogenous addition of superoxide dismutase [10]. This report shows that the superoxide radicals generated by the xanthine oxidase reaction reduced rat heart mitochondrial respiration induced by pyruvate. This negative effect was partially prevented by superoxide dismutase and catalase and by thiol protecting agents. Moreover, the generation of free radicals caused a significant reduction in the rate of (1-14C) -pyruvate decarboxylation, while it did not change the transport of pyruvate into mitochondria.