Mordechai Chevion

A site-specific mechanism for free radical induced biological damage: The essential role of redox-active transition metals

The metal-mediated site-specific mechanism for free radical-induced biological damage is reviewed. According to this mechanism, cooper- or iron-binding sites on macromolecules serve as centers for repeated production of hydroxyl radicals that are generated via the Fenton reaction. The aberrations induced by superoxide, ascorbate, isouramil, and paraquat are summarized. An illustrative example is the enhancement of double-strand breaks by ascorbate/copper. Prevention of the site-specific free radical damage can be accomplished by using selective chelators for iron and copper, by displacing these redox-active metals with other redox-inactive metals such as zinc, by introducing high concentrations of hydroxyl radicals scavengers and spin trapping agents, and by applying protective enzymes that remove superoxide or hydrogen peroxide. Histidine is a special agent that can intervene in free radical reactions in variety of modes. In biological systems, there are traces of copper and iron that are at high enough levels to catalyze free-radical reactions, and account for such deleterious processes. In the human body Fe/Cu = 80/1 (w/w). Nevertheless, both (free) copper and iron are soluble enough, and the rate constants of their reduced forms with hydrogen peroxide are sufficiently high to suggest that they might be important mediators of free radical toxicity.

Advanced Glycation End Product-Induced Activation of NF-κB is Suppressed by α-Lipoic Acid in Cultured Endothelial Cells

Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxida-tive stress led to the activation of the transcription factor NF-KB and thus promoted the upregulation of various NF-KB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antiox-idative defense with the natural occurring antioxidant α-lipoic acid before AGE albumin induction completely prevented the AGE albumin–dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-KB activation was also reduced in a time- and dose-dependent manner as long as α-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since α-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-KB. Western blots further demonstrated that α-lipoic acid inhibited the release and translocation of NF-KB from the cytoplasm into the nucleus. As a consequence, α-lipoic acid reduced AGE albumin-induced NF-KB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered α-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.

Redox-active iron mediates amyloid-β toxicity

Abstract While amyloid-β toxicity is mediated by oxidative stress and can be attenuated by antioxidants, the actual biochemical mechanism underlying neurotoxicity remains to be established. However, since aggregated amyloid-β can interact with transition metals, such as iron, both in vitro and in vivo, we suspected that bound iron might be the mediator of toxicity such that holo- and apo-amyloid would have differential effects on cellular viability. Here we demonstrate that when amyloid-β is pretreated with the iron chelator deferoxamine, neuronal toxicity is significantly attenuated while conversely, incubation of holo-amyloid-β with excess free iron restores toxicity to original levels. These data, taken together with the known sequelae of amyloid-β, suggest that the toxicity of amyloid-β is mediated, at least in part, via redox-active iron that precipitates lipid peroxidation and cellular oxidative stress.

The use of cyclic voltammetry for the evaluation of antioxidant capacity.

Low-molecular weight antioxidants (LMWAs) play a major role in protecting biological systems against reactive oxygen-derived species and reflect the antioxidant capacity of the system. Cyclic voltammetry (CV), shown to be convenient methodology, has been validated for quantitation of the LMWA capacity of blood plasma, tissue homogenates, and plant extracts. Analysis of the CV tracing yields the values of (i) the biological oxidation potential, E and E(1/2), which relate to the nature of the specific molecule(s); (ii) the intensity (Ia) of the anodic current; and (iii) the area of the anodic wave (S). Both Ia and S relate to the concentration of the molecule(s). LMWA components of human plasma and animal tissues were identified and further validated by reconstruction of the CV tracing and by high-performance liquid chromatography-electrochemical detection. To reflect the oxidative stress status, the use of an additional parameter, R, has been proposed. R represents the level (%) of oxidized ascorbate (compared with total ascorbate) and is measured by high-performance liquid chromatography-electrochemical detection. All these parameters were monitored in healthy human subjects as well as in chronic (diabetes mellitus) and acute care patients (subjected to total body irradiation before bone marrow transplantation). The electroanalytical methodologies presented here could be widely employed for rapid evaluation of the status of subjects (in health and disease) for monitoring of their response to treatment and/or nutritional supplementation as well as for screening of specific populations.

Copper and zinc levels in normal and malignant tissues.

The copper and zinc levels in 53 malignant and 47 normal human tissue samples were measured. In the malignant tissues, the mean copper concentration was 46% higher (P < 0.001) than in the normal ones. Analysis of the individual organs showed this increment to be statistically significant in malignancies of the large bowel, stomach, urinary bladder and female reproductive organs, while in cancer of the breast, kidney and testis, the increase in copper level was not significant. The mean zinc concentration in the malignant tissues was not significantly different from that in the corresponding normal tissue specimens (‐11%; P < 0.2). In breast cancer, however, tissue zinc levels were increased by 72% (P < 0.01), and decreased markedly in carcinoma of the kidney (‐73%). A hypothesis for the possible mechanism involving elevated tissue copper levels in biological damage (previously caused by free radicals) which may be responsible for the malignant process, is presented and discussed.

Protective Effects of Tea Polyphenols against Oxidative Damage to Red Blood Cells

Tea polyphenols (TPP) from black and green teas were evaluated for their antioxidant effects on normal red blood cells (RBC) and beta-thalassemic RBC membranes challenged with exogenous oxidants in vitro. The TPP of both types protected RBC against primaquine-induced lysis; they also protected the whole cells and the membranes against H2O2-induced lipid peroxidation so that about 80% protection was reached at [TPP] = 10 microg/mL. TPP from black tea at the same concentration protected normal RBC from morphological alterations caused by the peroxide treatment. The mechanism of the effects of TPP was investigated using a chemical system generating .OH (iron + ascorbic acid). TPP from both black and green teas inhibited the .OH fluxes in a concentration-dependent manner, indicating the possibility of iron chelation by TPP. Spectrophotometric titration revealed that TPP could stoichiometrically bind ferric iron to form a redox-inactive Fe-TPP complex. Quantitative analysis suggests that one or more major catechins from the TPP preparations are the likely iron-binding compounds accounting for the antioxidant effects of TPP on RBC.

The potential of desferrioxamine-gallium as an anti-Pseudomonas therapeutic agent

The opportunistic pathogen Pseudomonas aeruginosa causes infections that are difficult to treat by antibiotic therapy. This bacterium can cause biofilm infections where it shows tolerance to antibiotics. Here we report the novel use of a metallo-complex, desferrioxamine-gallium (DFO-Ga) that targets P. aeruginosa iron metabolism. This complex kills free-living bacteria and blocks biofilm formation. A combination of DFO-Ga and the anti-Pseudomonas antibiotic gentamicin caused massive killing of P. aeruginosa cells in mature biofilms. In a P. aeruginosa rabbit corneal infection, topical administration of DFO-Ga together with gentamicin decreased both infiltrate and final scar size by about 50% compared to topical application of gentamicin alone. The use of DFO-Ga as a Trojan horse delivery system that interferes with iron metabolism shows promise as a treatment for P. aeruginosa infections.

Anoxia pretreatment protects soybean cells against H2O2-induced cell death: possible involvement of peroxidases and of alternative oxidase

Anoxia followed by reoxygenation causes extensive damage to cellular components through generation of reactive oxygen intermediates. We examined cellular responses to oxidative stress after anoxia in cultured soybean or human fibroblast cells. Anoxia pretreatment protected soybean but not fibroblasts against H2O2 concentrations that induced programmed cell death in normoxic cells. H2O2 removal in anoxia‐pretreated soybean cultures was faster. Protection was associated with increased action of alternative oxidase (AOX) and peroxidases. AOX inhibitors abolished the protective effect, while induction of AOX protected normoxic cells against H2O2. We propose that during anoxia, plant cells can prepare for reoxygenation injury by up‐regulating their antioxidant capacity, and that AOX is involved in this process.

Irradiation-induced damage to the salivary glands: the role of redox-active iron and copper.

The mechanism of irradiation-induced hypofunction of the salivary glands is a process that is not fully understood. Here we examine the hypothesis that intracellular and redox-active ions of iron and copper, which are associated with the secretion granules, play a catalytic role in the irradiation-induced damage. Rats were subjected to head and neck irradiation (15 Gy X rays) and allowed to recover for 2 months. The function of the parotid and submandibular glands was then determined by pilocarpine-stimulated salivary secretion. A 45% decrease in the function of both glands was obtained when compared to nonirradiated controls. Treatment prior to irradiation (90 min) with cyclocytidine (200 mg/kg) led to a massive degranulation of the parotid gland and yielded nearly complete protection from radiation-induced damage. In contrast, pilocarpine stimulation prior to irradiation led to a marginal degranulation of the parotid gland and yielded only 13% protection. Neither agent caused degranulation of the submandibular gland mucous cells or yielded functional protection of this gland. Treatment with both agents yielded a marked increase in iron, copper and manganese levels in the parotid gland saliva. An analogous marked increase in the redox activity of iron and copper ions was recorded for the parotid saliva stimulated by pilocarpine and cyclocytidine. Pilocarpine-stimulated submandibular gland saliva contained metal levels similar to those of the parotid gland saliva. However, no redox activity and no increase in metal mobilization could be demonstrated in the submandibular gland saliva stimulated by both agents. The correlation between the patterns of gland degranulation, mobilization of redoxactive metals and the protection of gland function, for both parotid and submandibular glands, focuses attention on the catalytic roles played by transition metal ions in promoting free radical reactions, which likely participate in the process of injury to the tissue.

Hydroxyl radical generation in β-thalassemic red blood cells☆

Abstract To provide more experimental evidence for the proposed role of oxygen free radicals in red blood cell (RBC) damage in β-thalassemia, hydroxyl radical generation was studied in thalassemic (Th) vs. normal (N) RBC. ḃ OH fluxes were quantified by the conversion of salicylic acid (SA) into its hydroxylated products, 2,3- and 2,5-dihydroxybenzoic acids (DHBA) and catechol, assayed with HPLC coupled to electrochemical detection. No significant difference in spontaneous ḃ OH generation between N-RBC and Th-RBC was found. Ascorbic acid (0.5–3.0 mM) induced many-fold increases in SA hydroxylation in a dose-dependant manner in both types of cells. In the presence of ascorbate (1.0 mM), the SA hydroxylated products were determined in Th-RBC vs. N-RBC as follows (nmol/ml): 2,5-DHBA, 1.45 ± 0.06 vs. 1.81 ± 0.05 ( p = 0.001); 2,3-DHBA, 1.89 ± 0.21 vs. 1.15 ± 0.08 ( p = 0.008) and catechol, 0.87 ± 0.13 vs. 0.38 ± 0.05 ( p = 0.006). The results showed significant increase in the total SA hydroxylation in Th-RBC as compared to N-RBC with a tendency to form 2,3-DHBA and catechol at the expanse of 2,5-DHBA. The excessive · OH generation in Th-RBC is attributed to the abnormally high content of redox active iron in the cytosolic and/or membrane compartments of these cells.