Nutrient Deprivation of Cultured Rat Hepatocytes Increases the Desferrioxamine-available Iron Pool and Augments the Sensitivity to Hydrogen Peroxide

Abstract

Primary cultures of rat hepatocytes were subjected to amino acid and serum deprivation for 4 h. This treatment augmented the sensitivity to ensuing hydrogen peroxide exposure for 30 min. The by nutrient deprivation-increased autophagocytosis was confirmed by transmission electron microscopy and uptake of the lysosomotropic weak base acridine orange within the intracellular acidic vacuolar apparatus. The desferrioxamine-available pool of iron increased 2.5-fold during deprivation, compared with control cells. Furthermore, amino acid deprivation increased the cellular protein turnover, measured by radioactive labeling with [ 3 H]Leu. Exposure to 40 M ascorbic acid specifically decreased the turnover of ferritin, as estimated by enzyme-linked immunosorbent assay, and prevented an increase of the desferrioxamine-available iron pool, resulting in protection against hydrogen peroxide-induced cell killing.Thus, hepatocytes with nutrient deprivation-enhanced autophagocytosis contain a larger pool of catalytically active iron than control cells. This iron pool is mainly derived from the turnover (autophagocytosis) of cytosolic ferritin and is probably situated in the lysosomes. Furthermore, nutrient-deprived cells show augmented sensitivity to hydrogen peroxide-induced oxidative stress, since the enhanced availability of iron in low molecular weight form results in an increased potential of intralysosomal Fenton chemistry, that may cause lysosomal rupture with release of potent hydrolytic enzymes.

Release of iron from ferritin requires lysosomal activity

“…Our findings are consistent with that of the following studies implicating lysosomal proteases in ferritin iron release: Konijn and Vaisman et al (18,46) for erythroid cells producing hemoglobin; Ollinger and Roberg (30) for primary hepatocytes responding to serum and amino acid deprivation by autophagocytosis of ferritin, and an increase in cytoplasmic DFOchelatable iron pools; Pourzand et al (32) showing in fibroblasts that release of DFO-available iron from ferritin by UV exposure was inhibited by leupeptin and chymostatin; and Kwok and Richardson (20), who showed in melanoma cells that leupeptin, chymostatin, or chloroquine had the same effect as doxorubicin, greatly increasing the amount of 59 Fe retained in ferritin. Like we, they also found that high concentrations of lactacystin (10 and 25 M) promoted retention of iron by ferritin, as did MG132, another proteasomal (but also lysosomal) inhibitor (35).…”

“…In K562 cells, the same inhibitors reduced ferritin losses and the rate of recovery of the "labile iron pool" depleted by treatment with the chelator SIH (18). Others showed that serum deprivation of rat hepatocytes promoted the transfer of cytosolic ferritin to lysosomes, and that this resulted in an enlargement of the labile iron pool (30). More recently and while we were carrying out our own work, Kwok and Richardson (19), followed up on their finding that the anticancer drug, doxorubicin, increased the amount of iron retained in ferritin.…”

Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells

“…Several previous studies showed that ferritin can be degraded by autophagy (Asano et al, 2011;De Domenico et al, 2009;Kidane et al, 2006;Ollinger and Roberg, 1997), and iron depletion can induce autophagy (De Domenico et al, 2009) (supplementary material Fig. S4).…”

Enhancement of iron toxicity in L929 cells by d-glucose: accelerated(re-)reduction

“…In mammalian cells, however, these measurements have only been performed in homogenates and not in whole cells. During sample preparation for these measurements (homogenization followed by freezing in liquid nitrogen as described in [3,[28][29][30]), an artificial oxidation of ferrous iron is very likely. In addition, neither the observation of rapid changes in iron valence nor measurements in living cells are possible with EPR spectroscopy.…”

“…The major part of the cellular iron is safely bound in ferritin as well as in haem-or iron-sulphurcluster-containing proteins (enzymes). However, a small part (0.2-3 % [1][2][3][4][5]) is loosely attached to proteins or lipids or weakly bound to low-molecular-mass ligands like phosphates or citrate, forming a transit iron pool that keeps iron available for the synthesis of iron-containing proteins [1]. This pool of iron is, because of its methodological definition, also often referred to as ' chelatable ' iron.…”