When cultured hepatocytes were incubated in cell culture medium at 4 degreesC for up to 30 h and then returned to 37 degreesC, blebbing of the plasma membrane, cell detachment, chromatin condensation and margination, enhanced nuclear stainability with Hoechst 33342, ruffling of the nuclear membrane, and DNA fragmentation occurred. Similar to hepatocytes, cultured liver endothelial cells exhibited blebbing, chromatin condensation and margination, marked nuclear condensation, and increased stainability with Hoechst 33342 when exposed to hypothermia/rewarming. In both cell types, the occurrence and extent of these alterations were dependent on the duration of the cold incubation period. This cold-induced apoptosis was inhibited by hypoxia, by an array of free radical scavengers/antioxidants, and by iron chelators. However, the extent of the protection by the different antioxidants was different in the two cell types: iron chelators provided complete protection in liver endothelial cells but only partial protection in hepatocytes, whereas lipophilic antioxidants such as alpha-tocopherol provided complete protection in both cell types. During cold incubation, and especially during rewarming, lipid peroxidation occurred. These results suggest that the formation of reactive oxygen species (ROS) is a key mediator of cold-induced apoptosis, with ROS formation being completely iron-mediated in liver endothelial cells and partially iron-mediated in hepatocytes.
A very small, predominantly cytosolic pool of iron ions plays the central role in the cellular iron metabolism. It links the cellular iron uptake with the insertion of the metal in iron storage proteins and other essential iron-containing molecules. Furthermore, this transit ('labile') pool is essentially involved in the pathogenesis of a number of diseases. Due to its high physiological and pathophysiological significance, numerous methods for its characterization have been developed during the last five decades. Most of these methods, however, influence the size and nature of the transit iron pool artificially, as they are not applicable to viable biological material. Recently, fluorescence spectroscopic methods for measurements within viable cells have become available. Although these methods avoid the artifacts of previous methods, studies using fluorescent iron indicators revealed that the 'intracellular transit iron pool', which is methodically assessed as 'chelatable iron', is substantially defined by the method and/or the iron-chelating indicator applied for its detection, since the iron ions are bound to a large number of different ligands in different metabolic compartments. A more comprehensive characterization of the nature and the role of the thus not uniform cellular transit iron pool therefore requires parallel employment of different indicator molecules, which clearly differ in their intracellular distribution and their physico-chemical characteristics.
The intracellular pool of chelatable iron is considered to be a decisive pathogenetic factor for various kinds of cell injury. We therefore set about establishing a method of detecting chelatable iron in isolated hepatocytes based on digital fluorescence microscopy. The fluorescence of hepatocytes loaded with the fluorescent metal indicators, phen green SK (PG SK), phen green FL (PG FL), calcein, or fluorescein desferrioxamine (FL-DFO), was quenched when iron was added to the cells in a membrane-permeable form. It increased when cellular chelatable iron available to the probe was experimentally decreased by an excess of various membrane-permeable transition metal chelators. The quenching by means of the ferrous ammonium sulfate ؉ citrate complex and also the ''dequenching'' using 2,2Ј-dipyridyl (2,2Ј-DPD) were largest for PG. We therefore optimized the conditions for its use in hepatocytes and tested the influence of possible confounding factors. An ex situ calibration method was set up to determine the chelatable iron pool of cultured hepatocytes from the increase of PG SK fluorescence after the addition of excess 2,2Ј-DPD. Using this method, we found 9.8 ؎ 2.9 mol/L (mean ؎ SEM; n ؍ 18) chelatable iron in rat hepatocytes, which constituted 1.0% ؎ 0.3% of the total iron content of the cells as determined by atomic absorption spectroscopy. The concentration of chelatable iron in hepatocytes was higher than the one in K562 cells (4.0 ؎ 1.3 mol/L; mean ؎ SEM; n ؍ 8), which were used for comparison. This method allowed us to record time courses of iron uptake and of iron chelation by different chelators (e.g., deferoxamine, 1,10-phenanthroline) in single, intact cells. (HEPATOLOGY 1999;29: 1171-1179.)
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