Original Contribution
Lipofuscin-bound iron is a major intracellular source of oxidants: Role in senescent cells

https://doi.org/10.1016/j.freeradbiomed.2010.01.030Get rights and content

Abstract

Aging is accompanied by an intracellular accumulation of lipofuscin, a hydrophobic yellow-brownish material that accumulates especially in the lysosomal compartment, where it can be neither degraded nor exocytosed from the cell. The intracellular effects of accumulating lipofuscin are still a subject of speculation. In addition to the demonstrated inhibition of the proteasome, it was proposed that lipofuscin is cytotoxic because of its ability to incorporate transition metals such as copper and iron, resulting in a redox-active surface, able to catalyze the Fenton reaction. This characteristic of lipofuscin may contribute to an increased level of radical formation and oxidatively modified cellular components such as proteins, lipids, and RNA/DNA, which has been shown to be extensive in aging cells. In this study for the first time the lipofuscin-mediated formation of oxidants and the role of iron in this process are directly shown in a model of senescent fibroblasts, as well as in vitro with artificial lipofuscin. We demonstrate that this oxidant production is independent of mitochondria and has cytotoxic effects. The ability of lipofuscin to produce oxidants is dependent on the amount of transition metals incorporated. Although the amount of oxidants formed by cellular lipofuscin turned out to be moderate, it is chronic and thus lipofuscin is able to catalyze its own formation.

Section snippets

Materials

If not indicated differently all chemicals were obtained from Sigma (Deisenhofen, Germany) and the cell culture materials were purchased from Biochrom (Berlin, Germany). The caspase-3 assay kit from BD Bioscience (Pharmingen, San Diego, CA, USA) was used.

Cell culture

Experiments were performed using human dermal fibroblasts obtained from skin tissue. Cells were cultured in Dulbecco's MEM supplemented with 10% fetal calf medium, 1% penicillin/streptomycin, and 1% Glutamax at 37°C in a humidified 5% CO2

Formation of free radicals in young and SIPS cells with and without inhibition of the respiratory chain

Cellular senescence is often considered to be accompanied by an enhanced formation of oxidants. Therefore, we tested whether our SIPS fibroblasts also have this phenotype. As shown in Fig. 1A, the amount of free radicals in SIPS cells, quantified via DCF fluorescence, is significantly increased (up to 310%) compared to unexposed young human fibroblasts. After inhibition of the respiratory chain (with antimycin A and rotenone for 24 h) the amount of DCF fluorescence is still significantly

Discussion

Keeping the cytosol clear of the consequences of oxidative damage is one main function of the lysosomal and proteasomal systems. The capacity of these systems decreases over time in postmitotic cells, in several pathologies, and during oxidative stress [12]. The mitochondria are the main intracellular source of oxidative stress [29], contributing about 50%, whereas other main enzymes are xanthine- and NADPH-oxidase [43]. The main primary radical generated by the three systems mentioned is the

Acknowledgment

TG was supported by the DFG.

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