Free radicals in cellular pathology : Mechanisms behind lipofucin formation in cultured myocardial cells

Abstract: Lipofuscin, or age pigment, may be defined as a yellowish-brown,autofluorescent, protein and lipid containing pigment that accumulates inthe lysosomal vacuome of a variety of post-mitotic cell types in man andanimals during aging. Lipofuscin is the end product of peroxidation,fragmentation and polymerization of proteins and lipids. Protein andlipid containing materials are regularly sequestered to the lysosomal system by means of pinocytosis, phagocytosis and autophagocytosis. Lipid peroxidation involves the production of semistable peroxides from free radical intermediates produced by direct reaction of oxygen derived reactive metabolites with unsaturated lipids. The aim of the present project was to isolate the factors thought to be involved in the formation of lipofuscin by using a cell culture model of post-mitotic myocardial cells. Specifically the role of oxidative stress, transition metals, metal chelators, oxidants, antioxidants and lysosomal enzyme inhibitors. We introduced our cell culture model system and reported briefly theeffect of oxygen stress on lipofuscin accumulation (paper 1). Our resultsindicated that the level of autofluorescence, increases in direct relationship to age in culture and ambient oxygen concentration. The rapid increase of autofluorescence materials in the cells kept under 40% oxygen is thought to be due to the high production of superoxide anion radicals. Another study (paper ll) was aimed at the catalytic effect of metals onlipofuscin formation using the transition metal iron and the metal chelatordesferrioxamine on lipofuscin formation. Lipofuscin was quantified by microspectrofluorometry of individual cells. Augmentation of iron in the culture medium markedly increased the level of lipofuscin accumulationwhile desferrioxamine had the opposite effect. Both of these effects were more pronounced at higher oxygen tension. The effect of the hydrophobic-hydrophilic, peroxyle radical trapping, chain-breaking antioxidant vitamin E and some metal chelators e.g. EDTA, DTPA and Desferal was studied (paper Ill). All of them except EDTA decreased lipofuscin accumulation. In paper IV, inhibition of proteinases and lipases were studied underdifferent ambient oxygen concentrations for up to 14 days in culture. The results indicated that the rate of lipofuscin accumulation was enhanced by the lytic enzyme inhibitors as well as by increase in the ambient oxygen concentration. The finding shown that the specific lysosomal proteinase inhibitor, E-64, is a considerably more effective enhanced of lipofuscinogenesis than leupeptin, which also inhibits proteinases other than intralysosomal. Effects of Al, Cd, Cr, Cu, Hg, Pb and Zn chloride on lipofuscinogenesiswere examined on rat myocytes after 14 days in culture under 5, 20 and 40% oxygen concentrations. Addition of Al, Cd, Hg and Pb to the culture medium markedly increased the level of lipofuscin accumulation, whereas Cr, Cu and Zn had the opposite effect (paper V). Both effects were more pronounced at higher oxygen concentrations. Lipofuscin may, or may not, have influence on cellular metabolism, directly or via its influence on the function of the lysosomal vacuome. This question is so far completely unresolved, probably due to inadequate experimental conditions. Our cell culture model seems to be a suitable system also for further functional studies