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 cytotoxi city have not been fully elucidated, although they are considered to b e mediated by the formation and effects of reactive oxygen metabolites . In the present study, the cytotoxic effects of alloxan/cysteine at h igh and low concentrations were investigated on a model system of cult ured J-774 cells. Viability was estimated by the trypan blue dye exclu sion 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) hydr ogen peroxide, readily diffusing through cellular membranes and produc ed extracellularly in large amounts by alloxan/cysteine at high concen trations, 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 catalytica lly active lysosomal iron is present. This would result in lysosomal m embrane damage followed by leakage of lysosomal contents to the cell s ap and cell degeneration. (b) Alloxan/cysteine at low concentrations i nduced production of superoxide and hydrogen peroxide in low amounts w hich caused almost no lysosomal damage and appeared to be non-toxic un less there was some plasma membrane-associated iron. Consequently, cel ls initially allowed to endocytose iron during culture, or briefly exp osed to iron just before exposure to alloxan and cysteine, showed grea tly enhanced sensitivity. In this case iron, in combination with super oxide and hydrogen peroxide, is believed to give rise to plasma membra ne-associated hydroxyl radical production (Fenton reaction) with resul tant loss of membrane integrity. We thus propose that reasons for pron ounced 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 exocytos is of this transition metal.