Ceroid/Lipofuscin-loaded human fibroblasts show increased susceptibility to oxidative stress

Abstract

To test whether the possibly enhanced sensitivity of aged cells to oxidative stress may depend on their content of ceroid/lipofuscin, AG-1518 human fibroblasts with various amounts of the pigment accumulated due to prolonged cultivation under normobaric hyperoxia were exposed to acute oxidative stress (2.5 μM naphthazarin, 15 min) and then returned to standard culture conditions. Twenty-four hours after the naphthazarin treatment, 37% of the cells were still vital, whereas others had undergone oxidative stress-induced apoptosis with ensuing postapoptotic necrosis. The average amount of ceroid/lipofuscin within the surviving cells was only about half of that of the initial population of cells, as measured before the naphthazarin exposure. This finding suggests that ceroid/lipofuscin-rich cells have an increased sensitivity to oxidative stress. The ceroid/lipofuscin quantity strongly positively correlated with the size of the acidic compartment (as evaluated by uptake of the weakly basic lysosomotropic fluorochrome acridine orange) and with its content of the lysosomal protease cathepsin D, as assayed by immunocytochemistry. We hypothesize that the enhanced sensitivity of ceroid/lipofuscin-loaded cells to oxidative stress may be caused by the increased amounts of lysosomal enzymes, known as mediators of oxidative damage, and/or by catalysis of intralysosomal oxidative reactions by lipofuscin-associated iron.

Introduction

Lipofuscin (age pigment)—a recognized hallmark of aged postmitotic cells—is a nondegradable, autofluorescent, electron-dense, polymeric intralysosomal substance Strehler et al 1959, Sohal and Wolfe 1986, Harman 1989, Porta 1991. Practically the same properties are characteristics for ceroid—a pigment that forms under different pathological conditions Ghadially 1975, Armstrong and Koppang 1981, Goebel et al 1988, Wolfe et al 1988. Evidence suggests that oxidative modification of macromolecules undergoing intralysosomal degradation plays a key role in lipofuscinogenesis and in many instances of ceroidogenesis as well Sohal et al 1989, Brunk et al 1992, De Gritz and Rahko 1995, Terman and Brunk 1998a.

Little is known, however, about whether lipofuscin is only a harmless waste product, typical of old age, or if it can interfere with cellular functions, especially the function of the acidic vacuolar apparatus, and maybe even increase the probability of cell death. As we have hypothesized earlier (Brunk and Terman, 1999), large intralysosomal amounts of ceroid/lipofuscin may induce a compensatory production of new lysosomes and lysosomal enzymes, making up for the volume occupied by ceroid/lipofuscin and for the quantity of enzyme lost on a futile trial to degrade the undegradable pigment. An enlarged lysosomal compartment may promote cellular damage when lysosomal membranes are destabilized by pathogenic factors, such as oxidative stress, resulting in leak of lysosomal enzymes into the cytosol. Moreover, the iron present within ceroid/lipofuscin granules may sensitize lysosomes to oxidative damage by Fenton-type chemistry. There are, therefore, good reasons to believe that ceroid/lipofuscin accumulation is not only a harmless result of oxidative damage but may enforce an increased susceptibility of cells to oxidative injury, as well.

Recent studies indicated that oxidative damage to cultured fibroblasts, macrophage-like J-774 cells, cardiac myocytes, a variety of insulinoma cells, and Jurkat T cells results in lysosomal leak with ensuing apoptotic cell death (Brunk et al 1997, Hellquist et al 1997; Olejnicka et al., 1997; Roberg and Ollinger 1998, Brunk 1999). To clarify whether a heavy ceroid/lipofuscin accumulation makes cells more sensitive to oxidative stress, using cultured human fibroblasts as a model system, we studied: 1) the relationship between the amount of ceroid/lipofuscin and the susceptibility of cells to oxidant-induced apoptosis; 2) the relationship among the amount of ceroid/lipofuscin, the size of the acidic vacuolar compartment (evaluated by uptake of the lysosomotropic weak base acridine orange), and the content of the lysosomal aspartic protease cathespin D (assayed by immunocytochemistry); 3) early effect of the exposure of cells to naphthazarin on the stability of the acidic vacuolar compartment (evaluated by the uptake of the lysosomotropic weak base acridine orange and the intracellular redistribution of cathespin D).

Fibroblasts were loaded with ceroid/lipofuscin by a prolonged exposure of confluent cultures to normobaric hyperoxia. The pigment within the fibroblasts is here called ceroid/lipofuscin because both pathogenic factors and aging are involved in its formation in our model. Although for ceroid/lipofuscin loading of the cells we used a prolonged moderate oxidative stress (hyperoxia), apoptosis was induced by an acute, much more intensive, although not fully lethal oxidative stress (naphthazarin treatment). Naphthazarin—5,8-dihydroxy-1,4-naphthoquinone—is known to produce equimolar amounts of O₂^·− in a redox cycle, followed by conversion of O₂^·− into H₂O₂, spontaneously or enzymatically by superoxide dismutase (Öllinger and Brunmark, 1991).