Biochem J 2001 May 15;356(Pt 1):61-9

Subcellular distribution of chelatable iron: a laser scanning microscopic study

Petrat F, de Groot H, Rauen U

Institut fur Physiologische Chemie, Universitatsklinikum, Hufelandstrasse 55, D-45122 Essen, Germany.

The pool of cellular chelatable iron ('free iron', 'low-molecular-weight iron', the 'labile iron pool') is usually considered to reside mainly within the cytosol. For the present study we adapted our previously established Phen Green method, based on quantitative laser scanning microscopy, to examine the subcellular distribution of chelatable iron in single intact cells for the first time. These measurements, performed in isolated rat hepatocytes and rat liver endothelial cells, showed considerable concentrations of chelatable iron, not only in the cytosol but also in several other subcellular compartments. In isolated rat hepatocytes we determined a chelatable iron concentration of 5.8+/-2.6 mM within the cytosol and of at least 4.8 mM in mitochondria. The hepatocellular nucleus contained chelatable iron at the surprisingly high concentration of 6.6+/-2.9 mM. In rat liver endothelial cells, the concentration of chelatable iron within all these compartments was even higher (cytosol, 7.3+/-2.6 mM; nucleus, 11.8+/-3.9 mM; mitochondria, 9.2+/-2.7 mM); in addition, chelatable iron (approx. 16+/-4 mM) was detected in a small subpopulation of the endosomal/lysosomal apparatus. Hence there is an uneven distribution of subcellular chelatable iron, a fact that is important to consider for (patho)physiological processes and that also has implications for the use of iron chelators to inhibit oxidative stress.

PMID: 11336636, UI: 21234876

Subject: NTBI/free/idiopathic

Blood 1988 Oct;72(4):1416-9

Nontransferrin-bound iron in plasma from hemochromatosis patients: effect of phlebotomy therapy.

Aruoma OI, Bomford A, Polson RJ, Halliwell B

Department of Biochemistry, University of London, United Kingdom.

Plasma from patients with iron overload resulting from idiopathic hemochromatosis contains nontransferrin-bound iron, measurable by the bleomycin, assay. During venesection therapy, the concentration of bleomycin iron declines in a way highly correlated with plasma ferritin concentrations. Even when patients had been venesected to give very low total plasma iron concentrations and high transferrin iron-binding capacity, bleomycin-detectable iron was still present at low concentrations. Bleomycin-detectable iron can stimulate damaging free radical reactions, and its persistence in plasma even after prolonged venesection might contribute to the tissue damage that results from iron overload.

PMID: 2458783, UI: 89001210

Subject: LIP/free radicals/oxidative stress

Blood 2000 May 1;95(9):2960-6

Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: a study of L5178Y mouse lymphoma cell lines differentially sensitive to H(2)O(2).

Lipinski P, Drapier JC, Oliveira L, Retmanska H, Sochanowicz B, Kruszewski M

Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, Poland. lipinkskip@rocketmail.com

The redox properties of iron make this metal a key participant in oxygen-mediated toxicity. Accordingly, L5178Y (LY) mouse lymphoma cell lines, which display a unique inverse cross-sensitivity to ionizing radiation (IR) and hydrogen peroxide (H(2)O(2)), are a suitable model for the study of possible differences in the constitutive control of intracellular iron availability. We report here that the level of iron in the cytosolic labile iron pool (LIP), ie, potentially active in the Fenton reaction, is more than 3-fold higher in IR-resistant, H(2)O(2)-sensitive (LY-R) cells than in IR-sensitive, H(2)O(2)-resistant (LY-S) cells. This difference is associated with markedly greater content of ferritin H-subunits (H-Ft) in LY-S than in LY-R cells. Our results show that different expression of H-Ft in LY cells is a consequence of an up-regulation of H-Ft mRNA in the LY-S mutant cell line. In contrast, posttranscriptional control of iron metabolism mediated by iron-responsive element-iron regulatory proteins (IRPs) interaction is similar in the 2 cell lines, although IRP1 protein levels in iron-rich LY-R cells are twice those in iron-deficient LY-S cells. In showing that LY cell lines exhibit 2 different patterns of intracellular iron regulation, our results highlight both the role of high LIP in the establishment of pro-oxidant status in mammalian cells and the antioxidant role of ferritin. (Blood. 2000;95:2960-2966)

PMID: 10779446, UI: 20243325

Subject: LIP/DNA/virus

J Biol Chem 2001 Apr 11;

Intracellular chelation of iron by bipyridyl inhibits DNA virus replication: Ribonucleotide reductase maturation as a probe of intracellular iron pools.

Romeo AM, Christen L, Niles EG, Kosman DJ

Department of Biochemistry, SUNY at Buffalo, Buffalo, NY 14214.

Efficient replication of large DNA viruses requires dNTPs supplied by a viral ribonucleotide reductase. Viral ribonucleotide reductase is an early gene product of both vaccinia and herpes simplex virus. For productive infection, the apo-protein must scavenge iron from the endogenous, labile iron pool(s). The membrane-permeant, intracellular Fe2+ chelator , 2,2'-bipyridine (bipyridyl, BIP), is known to sequester iron from this pool. We show here that BIP strongly inhibits the replication of both vaccinia and herpes simplex virus (HSV-1). In a standard plaque assay, 50 mM BIP caused a 50% reduction in plaque forming units with either virus. Strong inhibition was observed only when BIP was added within 3 hours post infection (hpi). This time dependence was observed also in regards to inhibition of viral late protein and DNA synthesis by BIP. BIP did not inihibit the activity of vaccinia RR, its synthesis nor its stability indicating that BIP blocked the activation of the apo protein. In parallel with its inhibition of vaccinia RR activation, BIP treatment increased the RNA binding activity of the endogenous iron response protein, IRP1, by 1.9-fold. The data indicate that the diiron prosthetic group in vaccinia RR is assembled from iron taken from the BIP-accessible, labile iron pool that is sampled also by ferritin and the iron regulated protein found in the cytosol of mammalian cells.

PMID: 11301321

Subject: LIP

Blood 1999 Sep 15;94(6):2128-34

The cellular labile iron pool and intracellular ferritin in K562 cells.

Konijn AM, Glickstein H, Vaisman B, Meyron-Holtz EG, Slotki IN, Cabantchik ZI

Department of Human Nutrition, Faculty of Medicine, Institute of Life Sciences, Hebrew University of Jerusalem and Nephrology Unit, Jerusalem, Israel.

(HOME) The labile iron pool (LIP) harbors the metabolically active and regulatory forms of cellular iron. We assessed the role of intracellular ferritin in the maintenance of intracellular LIP levels. Treating K562 cells with the permeant chelator isonicotinoyl salicylaldehyde hydrazone reduced the LIP from 0.8 to 0.2 micromol/L, as monitored by the metalo-sensing probe calcein. When cells were reincubated in serum-free and chelator -free medium, the LIP partially recovered in a complex pattern. The first component of the LIP to reappear was relatively small and occurred within 1 hour, whereas the second was larger and relatively slow to occur, paralleling the decline in intracellular ferritin level (t1/2= 8 hours). Protease inhibitors such as leupeptin suppressed both the changes in ferritin levels and cellular LIP recovery after chelation . The changes in the LIP were also inversely reflected in the activity of iron regulatory protein (IRP). The 2 ferritin subunits, H and L, behaved qualitatively similarly in response to long-term treatments with the iron chelator deferoxamine, although L-ferritin declined more rapidly, resulting in a 4-fold higher H/L-ferritin ratio. The decline in L-ferritin, but not H-ferritin, was partially attenuated by the lysosomotrophic agent, chloroquine; on the other hand, antiproteases inhibited the degradation of both subunits to the same extent. These findings indicate that, after acute LIP depletion with fast-acting chelators, iron can be mobilized into the LIP from intracellular sources. The underlying mechanisms can be kinetically analyzed into components associated with fast release from accessible cellular sources and slow release from cytosolic ferritin via proteolysis. Because these iron forms are known to be redox-active, our studies are important for understanding the biological effects of cellular iron chelation .

PMID: 10477743, UI: 99408754

Mol Biochem Parasitol 1999 Jun 25;101(1-2):43-59

Chelation of iron within the erythrocytic Plasmodium falciparum parasite by iron chelators .

Loyevsky M, John C, Dickens B, Hu V, Miller JH, Gordeuk VR

Department of Medicine, The George Washington University Medical Center, Washington, DC 20037, USA. loyevsky@gwis2.circ.gwu.edu

To examine the site of action of antimalarial iron chelators , iron ligands were added to control erythrocytes and to erythrocytes parasitized with Plasmodium falciparum, and the concentration of intracellular labile iron was monitored with the fluorescent probe, calcein. The fluorescence of calcein quenches upon binding iron and increases upon releasing iron. The chelators included desferrioxamine B, 2',2'-bipyridyl, and aminophenol II, a compound that is being newly reported as having anti-plasmodial properties. Calcein-loaded parasitized cells displayed fluorescence predominantly within the cytosol of both rings and trophozoites. The addition of chelators to both control and parasitized erythrocytes led to significant increases of fluorescence (P < 0.001). Fluorescence was observed to increase within the parasite itself after addition of iron chelators , indicating that these agents bound labile iron within the plasmodium. The relative increases of fluorescence after addition of chelators were greater in control than parasitized erythrocytes (P < 0.05) as were the estimated labile iron concentrations (P < or = 0.001). These results suggest that (i) the anti-malarial action of iron chelators might result from the ability to reach the infected cell's parasite compartment and bind iron within the parasite cytosol, and (ii) the labile iron pool of the host red cell may be either utilized or stored during plasmodial growth.

PMID: 10413042, UI: 99339387