ALLOXAN CYTOTOXICITY IS HIGHLY POTENTIATED BY PLASMA MEMBRANE-ASSOCIATED AND LYSOSOMAL-ASSOCIATED IRON - A STUDY ON A MODEL SYSTEM OF CULTURED J-774 CELLS

Pancreatic islet beta cells, and some other cell types, are sensitive to the damaging effects of alloxan. The mechanisms behind the cytotoxicity have not been fully elucidated, although they are considered to be mediated by the formation and effects of reactive oxygen metabolites. In the present study, the cytotoxic effects of alloxan/cysteine at high and low concentrations were investigated on a model system of cultured J-774 cells. Viability was estimated by the trypan blue dye exclusion test, plasma membrane permeability by a modified microfluometric fluorescein diacetate technique and lysosomal membrane stability by a microfluorometric acridine orange method. The results showed: (a) hydrogen peroxide, readily diffusing through cellular membranes and produced extracellularly in large amounts by alloxan/cysteine at high concentrations, enters the secondary lysosomes if not previously degraded by cellular anti-oxidant systems. Intralysosomal Fenton reactions, with the formation of hydroxyl radicals, may be induced provided catalytically active lysosomal iron is present. This would result in lysosomal membrane damage followed by leakage of lysosomal contents to the cell sap and cell degeneration. (b) Alloxan/cysteine at low concentrations induced production of superoxide and hydrogen peroxide in low amounts which caused almost no lysosomal damage and appeared to be non-toxic unless there was some plasma membrane-associated iron. Consequently, cells initially allowed to endocytose iron during culture, or briefly exposed to iron just before exposure to alloxan and cysteine, showed greatly enhanced sensitivity. In this case iron, in combination with superoxide and hydrogen peroxide, is believed to give rise to plasma membrane-associated hydroxyl radical production (Fenton reaction) with resultant loss of membrane integrity. We thus propose that reasons for pronounced alloxan sensitivity may include lysosomal damage to cells with weak anti-oxidative defense systems or especially vulnerable secondary lysosomes or iron-catalysed plasma membrane damage following exocytosis of this transition metal.