Alloxan Toxicity to macrophages and Insulinoma Cells

• Alloxan induces damage and death of pancreatic islet B-cells in severalexperimental animal models, thus causing insulin-dependent diabetes mellitus (IDDM or type I diabetes). This unique cytotoxicity of alloxan has been studied for more than fifty years. The mechanisms behind the cytotoxicity of alloxan have, however, never been fully understood, although an increasing number of authors now suggest formation of reactive oxygen species, targeting the plasma membrane, mitochondria and DNA. In the present study, we have investigated: (i) the production of superoxide and hydrogen peroxide during reactions between alloxan and reducing agents such as cysteine, reduced glutathione, and ascorbic acid; (ii) the cytotoxic effects of alloxan in the absence of reducing agents, on model systems of cultured macrophages and insulinoma cells; (iii) the cytotoxicity of alloxan together with the reducing agents on these cultured cells; (iv) the cytotoxicity of hydrogen peroxide, used at concentrations similar to those formed during the reactions of alloxan with reducing agents; (v) the influence of iron and the iron-chelator, desferrioxamine, on the alloxan-induced cytotoxicity; and (vi) the influence of starvation-induced autophagocytosis on the sensitivity of cells to hydrogen peroxide-induced oxidative stress. Cell viability was estimated by a delayed trypan blue dye exclusion test and plasma membrane permeability by a modified microfluorometric combined fluorescein diacetate-propidium iodide staining technique. Lysosomal membrane stability was microfluorometrically assayed by acridine orange and neutral red relocalization techniques. The intracellular amounts of iron, reduced glutathione, antioxidant enzymes, and ATP were biochemically and cytochemically studied under a variety of conditions. The results showed that: (i) superoxide artion radicals and hydrogen peroxide were produced by reactions between alloxan and several reducing agents (e.g. cysteine, reduced glutathione and ascorbic acid). Hydrogen peroxide readily diffused through cellular membranes into the lysosomes if it was not previously degraded by the cellular antioxidative defence systems. Hydroxyl radicals might be produced by intralysosornal Fenton reactions, if reactive iron was present, resulting in lysosomal membrane damage followed by a leakage of lysosomal lytic enzymes with ensuing cell degeneration and eventually cell death. (ii) If iron was adsorbed to plasma membranes, extracellularly produced superoxide anion radicals and hydrogen peroxide might cause the plasma membrane damage due to Fenton reactions. (iii) Preincubation with desferrioxamine, or the presence of catalase inhibited the cytotoxicity induced by alloxan and reducing agents. (iv) The antioxidative defence activity of insulinoma cells was low. (v) Starvation in PBS enhanced the sensitivity of both macrophages and insulinoma cells to oxidative stress induced by hydrogen peroxide mediated through increased activity of autophagocytotosis. Thus, the amount of intralysosomal reactive iron consequently resulted from the degradation of various iron-containing metallo-proteins. We conclude that the exposure of cells to alloxan together with a reducing agent created cellular oxidative stress through extracellular formation of superoxide anion radicals and hydrogen peroxide. The latter compound easily penetrated plasma and lysosomal membranes, reaching the lysosomal interior. If enough reactive iron was present within lysosomes and the hydrogen peroxide was not degraded by catalase or glutathione peroxidase before entering the acidic vacuolar apparatus hydroxyl radicals could be produced via intralysosomal Fenton reactions.The hydroxyl radicals, in turn, would attack and damage the lysosomalmembranes, causing a leakage of lysosomal enzymes to the cytosol and eventually leading to cell death. The sensitivity of cells to alloxan-induced cytotoxicity in the presence of reducing agents was therefore a function of (i) the rate of hydrogen peroxide production, (ii) the cellular antioxidative defence systems, (iii) the lysosomal amount of reactive iron, and (iv) the capacity of autophagocytosis.

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