Herbert de Groot

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.

Reactive oxygen species contribute decisively to a great variety of diseases. Flavonoids are benzo-gamma-pyrone derivatives of plant origin found in various fruits and vegetables but also in tea and in red wine. Some of the flavonoids, such as quercetin and silibinin, can effectively protect cells and tissues against the deleterious effects of reactive oxygen species. Their antioxidant activity results from scavenging of free radicals and other oxidizing intermediates, from the chelation of iron or copper ions and from inhibition of oxidases. For their free radical scavenging properties, scavenging of lipid- and protein-derived radicals is presumably of special importance. A non-radical reactive oxygen species effectively trapped by flavonoids is hypochlorous acid. In general, the antioxidative properties of flavonoids are favoured by a high degree of OH substitution. On the other hand, inhibition of enzymatic functions other than oxidases, e.g., inhibition of lipoxygenase and thus prevention of the formation of leukotrienes, may also participate in the cell and tissue protective properties of flavonoids.

Endogenous oxygen- and nitrogen-centered free radicals are considered to play a decisive role in a variety of diseases such as neurodegenerative disorders, atherosclerosis, or cancer. Directly operating antioxidants limit the action of freely diffusing radicals by scavenging the attacking, oxidizing radical and re-reducing the oxidized biomolecule, i.e., the biomolecule-derived radical. From textbooks of biochemistry it is understood that NAD(P)H acts as a hydride (hydrogen anion) donor in a variety of enzymatic processes. One example is the re-reduction of GSSG to GSH, catalyzed by glutathione reductase. Because of this reaction, NADPH has been suggested to also act as an indirectly operating antioxidant, thus maintaining the antioxidative power of glutathione. To the best of our knowledge, however, neither NADPH nor NADH has been considered to be directly operating antioxidants. Based on recently published data, new experiments, and theoretical considerations, we propose that NAD(P)H represents a decisive, directly operating antioxidant that should be considered of major importance in the mitochondrial compartment. NAD(P)H fulfills this task both by scavenging toxic free radicals and repairing biomolecule-derived radicals.

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.