Effects of alloxan and reducing agents on macrophages in culture

The diabetogenic effect of the quinonoid compound alloxan is not understood in detail although it supposedly involves reactions mediated by alloxan and oxygen radicals. These reactive species may form extra- or intracellularly and cause cell damage through a variety of complex interactions with several macromolecules. The purpose of this study was to elucidate early (less than or equal to 60 min) effects of alloxan and reducing agents (cysteine and ascorbic acid) on cultured macrophages, as assayed by the trypan blue dye exclusion test and the sensitive fluorescein diacetate and propidium iodide (FDA/PI) double staining technique. During the reactions between alloxan and reducing agents, oxygen was consumed as a sign of superoxide anion radical formation. When alloxan alone was added to two different culture media without serum, oxygen was still consumed, indicating formation of oxygen radicals due to the occurrence of reducing substances in cell culture media. This finding demonstrated the necessity of performing further studies in solutions without reducing capacity, e.g. in phosphate-buffered saline. The experiments showed that exposure of normal and malignant macrophages to alloxan and reducing substances resulted in rapidly occurring plasma membrane damage and ensuing cell death. Separate addition of catalase, desferrioxamine or superoxide dismutase resulted in evident, slight and no protection, respectively. The combinations of (i) catalase and desferrioxamine, and (ii) catalase, desferrioxamine and superoxide dismutase, however, inhibited cell damage in a pronounced and complete way, respectively. The results are interpreted as indicating cell damage due to the extracellular formation of hydrogen peroxide and hydroxyl radicals. The latter in close proximity to the cells and acting on the plasma membrane, while the former, after diffusing into the cell, may have several intracellular targets. The FDA/PI technique proved its value as a quantifiable method for the evaluation not only of cell death but also of cell damage with computer-based fluorometry.