Propagation Of Free Radical Reactions Research Articles

Propagation of free-radical reactions in concentrated protein solutions

The reactions of proteins with biologically relevant oxidants have been widely studied, although most of the work has been performed in diluted homogenous solutions conditions that differ from those in intracellular environments. Cellular compartments represent highly crowded milieu in which high concentrations of biomolecules are present, unspecific intermolecular interactions are promoted, and physicochemical properties of constituents are modified. In this work, we propose that the high concentration at which proteins are present inside cells favours radical oxidative reactions between polypeptides which propagate in an oxygen-dependent process similar to membrane lipid peroxidation. The results presented herein show that highly concentrated solutions of bovine serum albumin (BSA) exposed to peroxynitrite, or metmyoglobin/H2O2, initiate the formation and propagation of protein peroxyl radicals, as evidenced by oxygen consumption, fluorescence spectroscopy, chemiluminescence, and electron paramagnetic resonance studies. Moreover, peroxyl radicals are capable of converting nitrite to nitrogen dioxide, which can oxidise amino acid residues to further assist radical-mediated protein oxidation. In addition, we also show that nitrone spin traps stop these propagation reactions in proteins, in line with the previously reported antioxidant role of these compounds in vivo. In summary, our results suggest that in crowded environments such as cellular compartments radical chain reactions propagate protein oxidative damage, highlighting a previously under recognised mechanism of cellular nitroxidative stress.

In-vitro antioxidative potential of methanolic aerial extracts from three ethnomedicinal plants of Assam: A Comparative Study

Antioxidants are the substances that neutralize free radicals or their actions. The traditional antioxidant phytochemicals from plants have been reported to inhibit the propagation of free radical reactions and to protect the human body from disease. The use of synthetic antioxidants has always been questioned because of their toxicity therefore researches are being carried out on plants nowadays to bring out the potent natural antioxidants. With this intent three ethnomedicinal plants of Assam viz., Calamus leptospadix Griff., Heliotropium indicum Linn., Lasia Spinosa Lour. were explored with a view to evaluate their in-vitro antioxidative potentials. The methanolic aerial extracts were studied for phytochemical analysis and antioxidative properties by different in-vitro assays: DPPH radical scavenging method, Nitric oxide radical scavenging activity, Hydrogen peroxide radical scavenging activity, Total phenolic content and Total flavonoid content. The percentage of inhibition was calculated by comparing the absorbance values of the control and test samples. The study conducted revealed that methanolic extract of Calamus leptospadix (CL) exhibited the highest DPPH radical scavenging activity (IC50 value of 29 ± 3.24 μg/ml), Nitric oxide radical scavenging activity (233.33 ± 3.54 μg/ml), Total phenolic content (8.02 ± 0.32 μg/ml expressed as gallic acid equivalents) and Total flavonoid contents (61.71 ± 1.16 μg/ml expressed as quercetin equivalents). Methanolic extract of Lasia spinosa (LS) showed highest hydrogen peroxide radical scavenging activity (IC50 value of 221.89 ± 2.14 μg/ml). The results obtained in the present study indicate that aerial parts of Calamus leptospadix have potent, Lasia spinosa have moderate and Heliotropium indicum (HI) have mild antioxidant and free radical scavenging activity.

Identification of a Lipid Peroxidation Product as the Source of Oxidation-specific Epitopes Recognized by Anti-DNA Autoantibodies*

Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases, including systemic lupus erythematosus (SLE). We have found that bovine serum albumin incubated with peroxidized polyunsaturated fatty acids significantly cross-reacted with the sera from MRL-lpr mice, a representative murine model of SLE. To identify the active substances responsible for the generation of autoantigenic epitopes recognized by the SLE sera, we performed the activity-guiding separation of a principal source from 13-hydroperoxy-9Z,11E-octadecadienoic acid and identified 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids, as the source of the autoantigenic epitopes. When the age-dependent change in the antibody titer against the ONE-modified protein was measured in the sera from MRL-lpr mice and control MRL-MpJ mice, all of the MRL-lpr mice developed an anti-ONE titer, which was comparable with the anti-DNA titer. Strikingly, a subset of the anti-DNA monoclonal antibodies generated from the SLE mice showing recognition specificity toward DNA cross-reacted with the ONE-specific epitopes. Furthermore, these dual-specific antibodies rapidly bound and internalized into living cells. These findings raised the possibility that the enhanced lipid peroxidation followed by the generation of ONE may be involved in the pathogenesis of autoimmune disorders.

Chelation of Mitochondrial Iron Prevents Seizure-Induced Mitochondrial Dysfunction and Neuronal Injury

Chelatable iron is an important catalyst for the initiation and propagation of free radical reactions and implicated in the pathogenesis of diverse neuronal disorders. Studies in our laboratory have shown that mitochondria are the principal source of reactive oxygen species production after status epilepticus (SE). We asked whether SE modulates mitochondrial iron levels by two independent methods and whether consequent mitochondrial dysfunction and neuronal injury could be ameliorated with a cell-permeable iron chelator. Kainate-induced SE resulted in a time-dependent increase in chelatable iron in mitochondrial but not cytosolic fractions of the rat hippocampus. Systemically administered N,N'-bis (2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid (HBED), a synthetic iron chelator, ameliorated SE-induced changes in chelatable iron, mitochondrial oxidative stress (8-hydroxy-2' deoxyguanosine and glutathione depletion), mitochondrial DNA integrity and hippocampal cell loss. Measurement of brain HBED levels after systemic administration confirmed its penetration in hippocampal mitochondria. These results suggest a role for mitochondrial iron in the pathogenesis of SE-induced brain damage and subcellular iron chelation as a novel therapeutic approach for its management.

Communicated by A.A. Maradudin on 15 August 2005

Antioxidants inhibit the propagation of free radical reactions. Because of their widespread use as preservatives in processed foods, their biochemical effects have been investigated. Early feeding studies indicated that butylated hydroxytoluene (BHT) increased liver weight, induced proliferation of smooth endoplasmic reticulum, and elevated several hepatic microsomal mono-oxygenase activities typical of phase 1 metabolism. In addition to phase1, antioxidants also induce phase2 xenobiotic-metabolizing enzymes. Phase2 enzymes detoxify activated electrophilic metabolites of xenobiotics via conjugation of endogenous substrates such as glutathione (GSH). Antioxidants act to directly terminate the propagation of free radical reactions, and increase the activity of enzymes those readily metabolize and aid in the elimination of potential cytotoxic chemicals. Dietary administration of antioxidants induced phase 2 xenobiotic-metabolizing enzymes. Induction of phase 2 enzymes by antioxidants was found in many organs and tissues, such as liver, lung, kidney, small intestine, colon, and spleen, thereby affording protection at many anatomical sites .This chapter presents evidence for several newly identified antioxidant-inducible genes, describes the proposed mechanisms of antioxidant signal transduction leading to enhanced expression of these enzymes, and complements the information presented in related reviews concerning the mechanisms and consequences of induction of phase 2 xenobiotic-metabolizing enzymes by antioxidants. The chapter discusses the genes induced by antioxidants—cytochrome P450s, Glutathione, S-transferases, NAD(P)H: quinone reductase, UDP-glucuronosyltransferases, microsomal epoxide hydrolase, aflatoxin BI-aldehyde reductase, dihydrodiol dehydrogenases, aldehyde dehydrogenases, enzymes of glutathione and reduced nicotinamide metabolism, and other proteins and enzymes. The chapter delves into the mechanisms of gene induction by antioxidants—the antioxidant-response element (ARE), proteins binding and signal transduction through the ARE—and the consequences of antioxidant gene induction. The regulation of antioxidant-inducible genes helps understand the signals leading to cellular transformation and carcinogenesis. Antioxidants can be used to decipher the encrypted signal transduction pathways that prevent cell transformation and carcinogenesis.

Vitamin E: action, metabolism and perspectives

Natural vitamin E includes four tocopherols and four tocotrienols. RRR-alpha-tocopherol is the most abundant form in nature and has the highest biological activity. Although vitamin E is the main lipid-soluble antioxidant in the body, not all its properties can be assigned to this action. As antioxidant, vitamin E acts in cell membranes where prevents the propagation of free radical reactions, although it has been also shown to have pro-oxidant activity. Non-radical oxidation products are formed by the reaction between alpha-tocopheryl radical and other free radicals, which are conjugated to glucuronic acid and excreted through the bile or urine. Vitamin E is transported in plasma lipoproteins. After its intestinal absorption vitamin E is packaged into chylomicrons, which along the lymphatic pathway are secreted into the systemic circulation. By the action of lipoprotein lipase (LPL), part of the tocopherols transported in chylomicrons are taken up by extrahepatic tissues, and the remnant chylomicrons transport the remaining tocopherols to the liver. Here, by the action of the "alpha-tocopherol transfer protein", a major proportion of alpha-tocopherol is incorporated into nascent very low density lipoproteins (VLDL), whereas the excess of alpha-tocopherol plus the other forms of vitamin E are excreted in bile. Once secreted into the circulation, VLDL are converted into IDL and LDL by the action of LPL, and the excess of surface components, including alpha-tocopherol, are transferred to HDL. Besides the LPL action, the delivery of alpha-tocopherol to tissues takes place by the uptake of lipoproteins by different tissues throughout their corresponding receptors. Although we have already a substantial information on the action, effects and metabolism of vitamin E, there are still several questions open. The most intriguing is its interaction with other antioxidants that may explain how foods containing small amounts of vitamin E provide greater benefits than larger doses of vitamin E alone.

Intranuclear iron deposition in hepatocytes and renal tubular cells in mice treated with ferric nitrilotriacetate.

Cytoplasmic and intranuclear iron depositions were observed in the livers and kidneys of male and female ddY mice treated for 4-12 weeks with ferric nitrilotriacetate (Fe-NTA), a known renal carcinogen that acts through the production of free radicals. The intranuclear iron deposition consisted of a spherical aggregation of ferritin particles of approximately 10 nm diameter, as revealed by immunohistochemical staining and electron microscopy. Although the incidence of renal tumours was greater in the males than in the females, the incidence of iron depositions did not differ with gender. The most abundant intranuclear iron depositions were observed in the animals treated with Fe-NTA for the longest duration (12 weeks). These findings suggest that the intranuclear production and propagation of free radical reactions are prevented by the trapping of iron in a chemically inert iron form of ferritin.

Antioxidant-Inducible Genes

Antioxidants inhibit the propagation of free radical reactions. Because of their widespread use as preservatives in processed foods, their biochemical effects have been investigated. Early feeding studies indicated that butylated hydroxytoluene (BHT) increased liver weight, induced proliferation of smooth endoplasmic reticulum, and elevated several hepatic microsomal mono-oxygenase activities typical of phase 1 metabolism. In addition to phase1, antioxidants also induce phase2 xenobiotic-metabolizing enzymes. Phase2 enzymes detoxify activated electrophilic metabolites of xenobiotics via conjugation of endogenous substrates such as glutathione (GSH). Antioxidants act to directly terminate the propagation of free radical reactions, and increase the activity of enzymes those readily metabolize and aid in the elimination of potential cytotoxic chemicals. Dietary administration of antioxidants induced phase 2 xenobiotic-metabolizing enzymes. Induction of phase 2 enzymes by antioxidants was found in many organs and tissues, such as liver, lung, kidney, small intestine, colon, and spleen, thereby affording protection at many anatomical sites .This chapter presents evidence for several newly identified antioxidant-inducible genes, describes the proposed mechanisms of antioxidant signal transduction leading to enhanced expression of these enzymes, and complements the information presented in related reviews concerning the mechanisms and consequences of induction of phase 2 xenobiotic-metabolizing enzymes by antioxidants. The chapter discusses the genes induced by antioxidants—cytochrome P450s, Glutathione, S-transferases, NAD(P)H: quinone reductase, UDP-glucuronosyltransferases, microsomal epoxide hydrolase, aflatoxin BI-aldehyde reductase, dihydrodiol dehydrogenases, aldehyde dehydrogenases, enzymes of glutathione and reduced nicotinamide metabolism, and other proteins and enzymes. The chapter delves into the mechanisms of gene induction by antioxidants—the antioxidant-response element (ARE), proteins binding and signal transduction through the ARE—and the consequences of antioxidant gene induction. The regulation of antioxidant-inducible genes helps understand the signals leading to cellular transformation and carcinogenesis. Antioxidants can be used to decipher the encrypted signal transduction pathways that prevent cell transformation and carcinogenesis.

Iron binding to alpha-tocopherol-containing phospholipid liposomes

Tocopherols (vitamin E) located in the hydrophobic domains of biological membranes act as chain breaking antioxidants preventing the propagation of free radical reactions of lipid peroxidation. The naturally occurring form, d-alpha tocopherol is an exquisite molecule in that it is intercalated in the membrane in such a way that the hydrophobic tail anchors the molecule positioning the chromanol ring containing the hydroxyl group, which is the essence of its antioxidant function, at the polar hydrocarbon interface of phospholipid membranes. The interaction of this group with water soluble substances is not very well understood. In the present study, an investigation was made of the interaction of ascorbate and ferrous ions (Fe +2) initiators of lipid peroxidation with alpha tocopherol. The results show that tocopherol increases membrane associated iron. The formation of a tocopherol iron complex in the presence of phospholipid liposomes and ascorbate in its reduced form is indicated. These results suggest a new way in which tocopherols act to inhibit lipid peroxidation.

Brain lipid peroxidation induced by postischemic reoxygenation in vitro: effect of vitamin E.

The aerobic incubation of brain after a period of ischemia induced lipid peroxidation. The effect was greatest in vitamin E-deficient rats, intermediate in vitamin E-normal rats, and least in animals supplemented with vitamin E. In contrast, nitrogen incubation following ischemia produced a small effect only in the vitamin E-deficient animals. It appears that reoxygenation is required for lipid peroxides to accumulate in the brain. However, a trace of oxygen remaining during extreme ischemic hypoxia may be sufficient to cause slow propagation of free radical reactions when the vitamin E level is low.