Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes

Abstract

A series of fluorescent iron chelators has been synthesized such that a fluorescent function is covalently linked to a 3-hydroxypyridin-4-one. In the present study, the fluorescent iron chelators were loaded into isolated rat hepatocytes. The intracellular fluorescence was not only quenched by an addition of a highly lipophilic 8-hydroxyquinoline-iron(III) complex but also was dequenched by the addition of an excess of the membrane-permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The time course of uptake of iron and iron chelation in single, intact cells was recorded on-line by using digital fluorescence microscopy. Intracellular concentrations of various fluorescent iron chelators were determined by using a spectrofluorophotometer subsequent to lysis of probe-loaded cells and were found to depend on their partition coefficients; the more hydrophobic the compound, the higher the intracellular concentration. An ex situ calibration method was used to determine the chelatable iron pool of cultured rat hepatocytes. CP655 (7-diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromen-3-carboxamide), which is a moderately lipophilic fluorescent chelator, was found to be the most sensitive probe for monitoring chelatable iron, as determined by the intracellular fluorescence increase induced by the addition of CP94. The concentration of the intracellular chelatable iron pool in hepatocytes was determined by this probe to be 5.4+/-1.3 microM.

Roles of hormone replacement therapy and iron in proliferation of breast epithelial cells with different estrogen and progesterone receptor status

“…This observation prompted us to investigate whether combined exposure of Fe and E2 could enhance breast cell proliferation. Indeed, we have shown an additive effect on the growth of ER + breast cancer MCF-7 cells at high doses of E2 and Fe and synergistic effect at low doses, which are physiologically relevant 47,48 (Fig. 4).…”

Lysosomal iron liberation is responsible for the vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes

“…Thus, rapid lysosomal liberation of internalized IONPs in microglial cells is likely to generate large amounts of ferrous iron, which causes ROS formation and cell damage. This view is strongly supported by the ability of the membrane permeable ferrous iron chelator 2,2 0bipyridyl, but not its non-chelating analog 4,4 0 -bipyridyl (Kinnunen et al, 2002;Ma et al, 2006), to lower ROS generation and prevent toxicity in IONP-treated microglial cells. The residual ROS formation in microglial cells in the presence of 2,2 0bipyridyl might result from ROS production on the IONP-surface (Voinov et al, 2011) as the Fe 2+ -2,2 0 -bipyridyl complex is not Fenton reactive (Pierre & Fontecave, 1999).…”

Iron, Oxidative Stress and Early Neurological Deterioration in Ischemic Stroke

“…hemoglobin, myoglobin and iron-sulfur proteins, the metal has to be released from transport and storage proteins to a pool of "free iron" within the cell. In the healthy state, only trace amounts of "free iron" can be detected in the cell, estimated between 0.2 and 1.25 µM [134]. LIP seems to play a key role in intracellular iron homeostasis as a sensor for the IRE-IRP regulatory mechanism [135,136].…”