Glutathione and Ascorbate During Ischemia and Postischemic Reperfusion in Rat Brain

Thirty minutes of total cerebral ischemia (decapitation) decreased total glutathione (GSH + GSSG) by 7% but had no detectable effect on the concentration of oxidized glutathione (GSSG), reduced ascorbate, or total ascorbate. In a model of reversible, bilateral hemispheric ischemia (four-vessel occlusion) no changes in glutathione or ascorbate were detected after 30 min of ischemia. During 24 h of reperfusion following such an insult no detectable change in total ascorbate, reduced ascorbate, or oxidized glutathione was noted; however, total brain glutathione declined by 25%. The findings are discussed in relation to the hypothesis that the deleterious effects of ischemia are due to an increase in free radical production which in turn leads to increased lipid peroxidation.     

Magnesium (and trace substance) deficiencies in the pathogenesis of cancer

Except for a few experimental models of magnesium (Mg)-deficiency-induced neoplasms, less attention has been paid in the past quarter century in the Western world to this macromineral than to the trace elements; e.g., selenium (Se) and zinc (Zn), and to vitamins, deficiencies of which are each considered probable factors in oncogenesis. Although early epidemiologic studies showed an inverse correlation between the amount of Mg in soil and water and the incidence of (gastric) cancer, and several animal studies supported the premise that Mg has a prophylactic effect against induction of cancer, other studies showed that Mg supplementation increased the growth of established experimental tumors. Thus, enthusiasm for this approach subsided. The early epidemiologic findings have since been confirmed, and there have been studies demonstrating the importance of Mg in maintaining immunocompetence, and others indicating that immunodeficiencies increase susceptibility to the development of cancer. Evidence has now accrued that indicates that Mg deficiency increases susceptibility to chemical oncogens. The abnormal metabolism of tryptophan (yielding a carcinogenic metabolite) that indicates functional or absolute pyridoxine deficiency is an indirect clue to Mg deficiency. Vitamin B₆-activated enzymes require Mg as a cofactor. However, the early warnings against the use of Mg as part of an antineoplastic program against established cancer were justified, since rapidly metabolizing cells (such as cancers) are dependent on Mg. There are similarities between experiences with Mg and with Se and Zn. All three are required for normal metabolism; Se also protects against free radicals in the environment. Mg and Zn have increased established tumor growth, and their depletion has been applied to antineoplastic programs, with risks comparable to those of using antimetabolic agents.     

Free Radical Enhancer Xenobiotic is an Inducer of Cataract in Rabbit

Free radical enhancers, diquat, paraquat, plumbagin and juglone were used to study the oxy radical-induced damage to the rabbit lens in vitro and in vivo. Each compound caused a 6–8 fold increase in malondialdehyde (MDA) and a 30–55% decrease in reduced glutathione of the lens in vim. These peroxidative and oxidative changes were potentiated in the presence of 100% 0., abolished by N, and prevented by desferal-Mn (III) (DF-Mn) or liposomal superoxide dismutase (LSOD) indicating the involvement of O₂^?.

Diquat injected intravitreally as a single dose (300nmole in 30μl of isotonic saline) in the right eye of a 5-wk-old Dutch belted rabbit, induced early cataract after 24–72h. The lens of the contralateral control eye injected with isotonic saline had no change. In the right eye, O₂,^? and OH -productions were significantly (P < 0.01) higher; O₂-, was about 16 fold higher in the aqueous humor and vitreous humor, and 5 fold in the lens and retina, and OH. was 35 fold higher in the aqueous humor, 2 fold in vitreous humor and 5 fold in the lens and retina as compared to the respective tissues of the control eye. Enhanced lipid peroxidation in the lens was apparent from the higher levels of MDA and formation of aminophospholipid-MDA Schiff-base conjugates.

We propose that cyclic oxidation-reduction of xenobiotics coupled to the endogenous redox systems in the eye, could generate oxy radicals in excessive amounts, triggering cataractogcnesis.     

Transient Iron-Overload with Bleomycin-Detectable Iron Present During Cardiopulmonary Bypass Surgery

Extracorporeal circulation of blood during cardiopulmonary bypass surgery exposes cells to non-physiological surfaces and shear stress which can activate several regulatory cascades, and neutrophils to release superoxide and hydrogen peroxide. Shear stresses generated by pumps and suction systems cause lysis of red blood cells and the release of haemoglobin. Together the release of reactive forms of oxygen and haemoglobin can lead to the appearance of low molecular mass chelatable iron (bleomycin-detectable iron). All patients undergoing open heart surgery appear to release iron to plasma transferrin, increasing its iron saturation. In 13% of patients, however, the transferrin became fully iron-saturated, and by the end of open-heart surgery we could detect bleomycin-chelatable iron in the plasma. Saturation of transferrin with iron eliminates its iron-binding antioxidant properties, which can result in a stimulation of iron-dependent radical damage to selected detector molecules.     

Measurement of oxidatively induced DNA damage and its repair,by mass spectrometric techniques

Abstract

Oxidatively induced damage caused by free radicals and other DNA-damaging agents generate a plethora of products in the DNA of living organisms. There is mounting evidence for the involvement of this type of damage in the etiology of numerous diseases including carcinogenesis. For a thorough understanding of the mechanisms, cellular repair, and biological consequences of DNA damage, accurate measurement of resulting products must be achieved. There are various analytical techniques, with their own advantages and drawbacks, which can be used for this purpose. Mass spectrometric techniques with isotope dilution, which include gas chromatography (GC) and liquid chromatography (LC), provide structural elucidation of products and ascertain accurate quantification, which are absolutely necessary for reliable measurement. Both gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), in single or tandem versions, have been used for the measurement of numerous DNA products such as sugar and base lesions, 8,5’-cyclopurine-2’-deoxynucleosides, base-base tandem lesions, and DNA-protein crosslinks, in vitro and in vivo. This article reviews these techniques and their applications in the measurement of oxidatively induced DNA damage and its repair.     

Commentary Salicylate Trapping of -OH as a Tool for Studying Post-Ischemic Oxidative Injury in the Isolated Rat Heart

The use of salicylate as a chemical trap for -OH represents a simple and convenient alternative to the use of spin trapping techniques to study oxidative injury in isolated perfused organs. In these systems, salicylate is included in the perfusion buffer at concentrations ranging from 0.1 to 2mM depending on the detection apparatus employed. In our studies, we have used a coulometric detector, which has a theoretical efficiency of 100% as compared to 1-5% for the standard glassy carbon electrode. We have been able to generate reproducible results by inclusion of only 100 μM salicylate, a concentration demonstrated not to affect pre- or post-ischemic cardiac function. In initial studies, we observed an increase in perfusate 2,5-dihydroxybenzoic acid consistent with an early post-ischemic burst of -OH, not unlike that reported using spin trapping techniques. Since then we and others have used this technique to examine possible relationships between -OH formation and treatments that alter post-ischemic cardiac functional recovery. For example, preischemic loading of hearts with copper results in increases in postischemic dysfunction and LDH release that were associated with an increase in 2,5-dihydroxybenzoate and by inference, -OH formation. Alternatively, we have reported that the nitroxide spin label, TEMPO, reputed to be a superoxide dismutase mimetic, decreased post-ischemic arrhythmias and 2,5-dihydroxybenzoate formation. Most recently, we have observed that preischemic loading of hearts with zinc-bis-histidinate results in improved post-ischemic cardiac function and decreased LDH release; changes that were associated with decreased 2,5-dihydroxybenzoate formation. These studies indicate that under certain conditions, salicylate is a valuable alternative to spin trapping techniques to probe the role of -OH in cardiac oxidative injury, particularly when applied to the isolated perfused heart preparation.     

Bioreductive Activation of Quinones: Redox Properties and Thiol Reactivity

Redox properties and thiol reactivity are central to the therapeutic and toxicological properties of qui-nones. The use of other physicochemical parameters to establish predictive relationships for redox properties of quinones is discussed. and attention drawn to situations where such relationships may be unreliable. The rates of reaction of semiquinone radicals with oxygen, including those of chemotherapeutic agents such as mitomycin and the anthracyclines. can be predicted with reasonable confidence from the redox properties. The reactions of quinones with thiols such as glutathione produces reduced quinones and radicals. but the reactions are complex and all the features are not well understood     

Long‐lived electrochemiluminescence of ruthenium (II) complexes/tri‐n‐propylamine in aqueous solutions

Although the clinical use of immunoassays based on the oxidative‐reduction electrochemiluminescence (ECL) of tris(2,2′‐bipyridine)ruthenium (II)/tri‐n‐propylamine has been a great success, elucidation of the ECL generation mechanism still remains unsatisfactory. We report here our experimental observations of long‐lived luminescence that remains detectable for several seconds after termination of electrochemical heterogeneous oxidation. Long‐lived luminescence was observed in both a surfactant‐free buffer and a surfactant‐containing broadly used commercial buffer under different conditions. The slow decay of emission seems to have been unnoticed in previous ECL mechanistic studies. Within the frame of the reaction schemes so far proposed, its origin is inconclusively ascribed to the reductive‐oxidation process of ruthenium (II) complex, that is Ru(bpy)₃²⁺ → Ru(bpy)₃¹⁺ → Ru(bpy)₃²⁺* → Ru(bpy)₃²⁺ with the involvement of the tri‐n‐propylamine‐derived radical cation. It is anticipated that long‐lived ECL will suggest a research approach to separate some homogeneous reactions from the complicated reaction system and therefore help to resolve the mechanistic mystery.     

Free radicals act as effectors in the growth inhibition and apoptosis of iron-treated Burkitt's lymphoma cells

The addition of ferric citrate to Burkitt's lymphoma (BL) cell lines inhibits growth, leads to the accumulation of cells in the phase G₂/M of the cell cycle and to the modulation of translocated c-myc expression. The increase in the labile iron pool (LIP) of iron-treated BL cells leads to cytotoxicity. Indeed, intracellular free iron catalyzes the formation of highly reactive compounds such as hydroxyl radicals and nitric oxide (NO) that damages macromolecular components of cells, eventually resulting in apoptosis. In this report, we have investigated the possible involvement of free radicals in the response of Ramos cells to iron. When added to Ramos cells, iron increased the intracellular levels of peroxide/peroxynitrite and NO. Moreover, the addition of free radicals scavengers (TROLOX^® and Carboxy-PTIO) neutralized the effects of iron on Ramos cells while addition of an NO donor or hydrogen peroxide (H₂O₂) to cells generated effects which partially mimicked those induced by iron addition. Collectively, our results suggest the involvement of free radicals as effectors in the iron specific growth inhibition of BL cells observed in vitro.