FREE RADICALS, OXIDATIVE STRESS AND ANTIOXIDANTS

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FREE RADICALS, OXIDATIVE STRESS AND ANTIOXIDANTS

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Possible role of membrane gamma-glutamyltransferase activity in the facilitation of transferrin-dependent and -independent iron uptake by cancer cells.

BACKGROUND: The molecular mechanisms by which iron is physiologically transported trough the cellular membranes are still only partially understood. Several studies indicate that a reduction step of ferric iron to ferrous is necessary, both in the case of transferrin-mediated and transferrin-independent iron uptake. Recent studies from our laboratory described gamma-glutamyltransferase activity (GGT) as a factor capable to effect iron reduction in the cell microenvironment. GGT is located on the outer aspect of plasma membrane of most cell types, and is often expressed at high levels in malignant tumors and their metastases. The present study was aimed at verifying the possibility that GGT-mediated iron reduction may participate in the process of cellular iron uptake. RESULTS: Four distinct human tumor cell lines, exhibiting different levels of GGT activity, were studied. The uptake of transferrin-bound iron was investigated by using (55)Fe-loaded transferrin, as well as by monitoring fluorimetrically the intracellular iron levels in calcein-preloaded cells. Transferrin-independent iron uptake was investigated using (55)Fe complexed by nitrilotriacetic acid ((55)Fe-NTA complex). The stimulation of GGT activity, by administration to cells of the substrates glutathione and glycyl-glycine, was generally reflected in a facilitation of transferrin-bound iron uptake. The extent of such facilitation was correlated with the intrinsic levels of the enzyme present in each cell line. Accordingly, inhibition of GGT activity by means of two independent inhibitors, acivicin and serine/boric acid complex, resulted in a decreased uptake of transferrin-bound iron. With Fe-NTA complex, the inhibitory effect – but not the stimulatory one – was also observed. CONCLUSION: It is concluded that membrane GGT can represent a facilitating factor in iron uptake by GGT-expressing cancer cells, thus providing them with a selective growth advantage over clones that do not possess the enzyme

Cleavage of Bcl-2 in oxidant- and cisplatin-induced apoptosis of human melanoma cells

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Lipid peroxidation and antioxidant systems in the liver injury produced by glutathione depleting agents

The mechanisms of the liver damage produced by three glutathione (GSH) depleting agents, bromobenzene, allyl alcohol and diethylmaleate, was investigated. The change in the antioxidant systems represented by α-tocopherol (vitamin E) and ascorbic acid were studied under conditions of severe GSH depletion. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. The hepatic level of vitamin E was decreased whenever extensive lipid peroxidation developed. In the case of bromobenzene intoxication, vitamin E decreased before the onset of lipid peroxidation. Changes in levels of the ascorbic and dehydroascorbic acid indicated a redox cycling of vitamin C with the oxidative stress induced by all the three agents. Such a change of the redox state of vitamin C (increase of the oxidized over the reduced form) may be an index of oxidative stress preceding lipid peroxidation in the case of bromobenzene. In the other cases, such a change is likely to be a consequence of lipid peroxidation. Experiments carried out with vitamin E deficient or supplemented diets indicated that the pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. Animals fed a vitamin E supplemented diet had an hepatic vitamin E level double that obtained with a commercial pellet diet. In such animals, bromobenzene and allyl alcohol had only limited toxicity and diethylmaleate none in spite of comparable hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of the toxicity by GSH depleting agents. © 1990

Glutathione depletion: Its effects on other antioxidant systems and hepatocellular damage

1. The mechanisms of the liver damage produced by three glutathione (GSH)-depleting agents, bromobenzene, allyl alcohol and diethyl maleate, were investigated. 2. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. 3. Changes in antioxidant systems by αtocopherol (vitamin E) and ascorbic acid were studied. A decrease in the hepatic level of vitamin E, and a change in the redox state of vitamin C (increase in oxidized over reduced form) were evident whenever extensive lipid peroxidation developed. However, in the case of bromobenzene intoxication these alterations preceded lipid peroxidation, and may be an index of oxidative stress leading to subsequent membrane damage. 4. Experiments carried out with vitamin E-deficient or supplemented diets indicated that pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E-deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. In animals fed a vitamin E-supplemented diet, bromobenzene and allyl alcohol had only limited toxicity, and diethyl maleate none, in spite of similar hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of toxicity by GSH-depleting agents. © 1991 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

Oxidative stress, isoprostanes and hepatic fibrosis

An introduction to oxidative stress enlightening the spreading of interest in lipid peroxidation in the 60's and in the identification of cytotoxic aldehydes originating from it is given. The discovery of F2 -isoprostanes as specific markers of oxidative stress is described. Isoprostanes are also agonists of important biological effects. Since a relationship between oxidative stress and collagen hyperproduction has been previously suggested, and since lipid peroxidation products (aldehydes) have been proposed as possible mediators of liver fibrosis, we investigated whether collagen synthesis is induced by F2 -isoprostanes, which can posses receptors for signal transduction pathways. In a rat model of carbon tetrachloride-induced hepatic fibrosis, plasma isoprostanes were markedly elevated for the entire experimental period and hepatic collagen content was also increased. Moreover, when hepatic stellate cells (HSC) isolated from normal livers were cultured up to activation and then treated with F2 -isoprostanes (8-epiPGF2α ) in the concentration range found in the in vivo studies (10-9 to 10-8 M), a striking increase in DNA synthesis, in cell proliferation and in collagen synthesis was observed. F2 -isoprostanes also increased the production of transforming growth factor-ß1 by U937 cells, assumed as a model of Kupffer cells or liver macrophages. The hypothesis that F2 -isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen hyperproduction, seen in this experimental hepatic fibrosis, was reinforced by the demonstration, by using immunoblot analysis, that isoprostane receptors identical or analogous to those for thromboxane A2 (TxA2 r) are present in HSC. Immunocytochemical studies showed the major localization of TxA2 r in the perinuclear site and its colocalization with α-smooth muscle actin