A range of new analogues of the promising iron chelator pyridoxal isonicotinoyl hydrazone was prepared and assessed for activity in reducing hepatocyte iron, mechanism of action and potential in iron-chelation therapy. A total of 45 compounds were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde) with various acid hydrazides prepared by systematic substitutions on the benzene ring or by the replacement of the ring with an acetyl, pyridyl, furoyl or thiophene moiety. The effects of these compounds on 59Fe uptake and intracellular distribution in hepatocytes in culture and on 59Fe mobilization from prelabeled hepatocytes were assessed. Toxicity, lipophilicity and the ability to chelate plasma transferrin-bound 59Fe were also evaluated. Several compounds were much more active than pyridoxal isonicotinoyl hydrazone and may have clinical potential. These included pyridoxal benzoyl hydrazone, pyridoxal p-methoxybenzoyl hydrazone, pyridoxal m-fluorobenzoyl hydrazone and pyridoxal 2-pyridyl hydrazone. All were more effective at reducing iron uptake than mobilizing hepatocyte iron; they also may act primarily on the transit iron pool rather than on storage iron. Other compounds (e.g., salicylaldehyde p-t-butyl-benzoyl hydrazone) redistributed ferritin-59Fe to different intracellular sites but had little net effect on hepatocyte iron levels.
The regulation of transferrin and iron release from the liver was studied using adult rat hepatocytes in primary monolayer culture. The cells were prelabeled by incubation with rat transferrin doubly labeled with iodine-125 and iron-59. Approximately 50% of the 125I-transferrin but only 10% of the iron-59 taken up by the cells was released during reincubation for 24 h. Less than 10% of the refluxed transferrin was catabolized as indicated by the protein-free iodine-125 values. These results suggest that at least part of iron uptake by hepatocytes is mediated by the reversible binding of transferrin in a manner comparable with erythroid cells and placenta. However, several iron chelators mobilized hepatic iron, in contrast to erythroid cells. Apotransferrin and desferrioxamine released a maximum of about 20% iron-59 with little effect on transferrin binding. A greater proportion of the iron-59 was available for chelation after shorter uptake times (1-2 h) than longer times. Hence, there are at least three iron compartments in hepatocytes in culture: rapidly refluxing iron that may be transferrin bound, a fixed pool, and a chelatable pool that may represent iron in transit between plasma transferrin and ferritin.
The mechanisms of iron (Fe) and transferrin (Tf) uptake by the human melanoma cell line, SK-MEL-28, have been investigated using chelators and metabolic probes. These data provide evidence for two saturable processes of Fe uptake from Tf, namely, specific receptor-mediated endocytosis and a second nonspecific, non-receptor-mediated mechanisms which saturated with respect to Fe uptake at a Tf concentration of approximately 0.3 mg/ml. In contrast to Fe uptake, Tf uptake increased linearly up to at least 1 mg/ml. Furthermore, under the culture conditions used, the second nonspecific, non-receptor-mediated mechanism was the most important process in terms of quantitative Fe uptake. Two concentrations of Tf-125I-59Fe (0.01 and 0.1 mg/ml) were used in order to characterise the specific and nonspecific Fe uptake pathways. Membrane permeable chelators were equally effective at both Tf concentrations, whereas membrane impermeable chelators were significantly (P < 0.001) more effective at reducing the internalisation of Fe at the higher Tf concentration, consistent with a mechanism of Fe uptake which occurred at a site in contact with the extracellular medium. The oxidoreductase inhibitor, amiloride, only slightly inhibited Fe uptake at the higher Tf concentration, suggesting that the second nonspecific process was not mediated by a diferric Tf reductase. Three lysosomotrophic agents and the endocytosis inhibitor, phenylglyoxal, markedly reduced Fe uptake at both Tf concentrations, and it is concluded that a saturable process consistent with receptor-mediated endocytosis of Tf occurred at the lower Tf concentration, while the predominant mechanism of Fe uptake at high Tf concentrations was a second saturable process consistent with adsorptive pinocytosis.
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