Herbert M. Schulman

The kinetics of iron and transferrin incorporation and rabbit erythroid cells and the nature of stromal-bound iron

Abstract An in vitro system is described in which iron incorporation into rabbit reticulocytes and bone marrow cells from pure transferrin is qualitatively comparable to that found in vivo. The amount of stromal-bound iron reproducibly represents 2% and 12% of the total iron incorporated respectively into reticulocytes and bone marrow cells after 2.5 h of incubation. After 60 min of incubation only 40% of the reticulocyte stromal-bound iron behaved like a metabolic intermediate. All the metabolically active, membrane-bound iron could be accounted for a transferrin iron. Of the transferrin found in washed cells, 50–60% was recovered in the cytoplasmic fraciton, and was shown by isoelectric focusing to be undergraded. It is likely that it represents intracellular transferrin. The rate of hemoglobin synthesis was linear for at least 2.5 h in both reticulocytes and bone marrow cells, while bone marrow cells showed z greater iron incorporation into heme than reticulocytes. There was no detectabel low molecular weight iron in the cell supernatant of reticulocytes or bone marrow cells and hemoglobin-iron accounted for more than 92% of the total iron incorporated into the reticulocytes.

Iron utilization in rabbit reticulocytes: A study using succinylacetone as an inhibitor of heme synthesis

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-14C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 microMM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not delta-aminolevulinic acid, partly overcame the inhibition of 59Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits delta-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 microM 0.1 and 1 mM, inhibited 59Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte 59Fe uptake by about 25-30%. In succinylacetone-treated cells 59Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and 59Fe-transferrin-pretreated reticulocytes results in the transfer of 59Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.

Effect of pyridoxal isonicotinoyl hydrazone and other hydrazones on iron release from macrophages, reticulocytes and hepatocytes

A model consisting of 59Fe-labelled macrophages was developed for screening potential iron-chelating drugs. Mouse peritoneal macrophages, induced by previous intraperitoneal injections of 3% thioglycollate, were labelled in vitro by their exposure to immune complexes of 59Fe-transferrin-antitransferrin antibody. Optimal conditions for macrophage labelling and subsequent 59Fe release were established. Sixty-two aromatic hydrazones, the majority of which had iron binding structures similar to pyridoxal isonicotinoyl hydrazone, were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde, 2-hydroxy-1-naphthylaldehyde and 2-furaldehyde) with various acid hydrazides prepared by systematic substitutions on the benzene ring. These compounds were examined for their potential to stimulate 59Fe release from 59Fe-labelled macrophages and also from reticulocytes and hepatocytes loaded with non-heme 59Fe. The majority of hydrazones derived from pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde seemed to be equally effective in both the macrophage and reticulocyte testing systems. However, the pyridoxal hydrazones were much more active in hepatocytes than the other groups of hydrazones. Several compounds proved to be very potent in mobilizing 59Fe. These included hydrazones derived from 2-hydroxy-1-naphthylaldehyde and benzoic acid hydrazide, p-hydroxybenzoic acid hydrazide, 2-thiophenecarboxylic acid hydrazide, and also pyridoxal benzoyl hydrazone, pyridoxal m-fluorobenzoyl hydrazone and pyridoxal 2-thiophenecarboxyl hydrazone.

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) protects plasmid pUC-18 DNA against OH-mediated strand breaks

Pyridoxal isonicotinoyl hydrazone (PIH) has previously been studied for use in iron chelation therapy in iron-overload diseases. It is an efficient in vitro antioxidant due to its Fe(III) complexing activity (Schulman, H. M., et al. Redox Report 1:373-378; 1995). Pathologies associated with iron-overload include hepatic and other cancers. Since oxidative alterations of DNA can be linked to the development of cancer, we decided to study whether PIH protects DNA against in vitro oxidative stress. We report here that pUC-18 plasmid DNA is damaged by *OH radicals generated from Fe(II) plus H2O2 or from Fe(II) plus hypoxanthine/xanthine oxidase. The DNA damage was quantified by determining the diminution of supercoiled DNA forms after oxidative attack using agar gel electrophoresis. Micromolar amounts of PIH (20-30 microM) were able to half-protect DNA from iron (1-7.5 microM)-mediated *OH formation. The antioxidant capacity of PIH was significantly higher than that of some of its analogs and desferrioxamine. PIH and some of its analogues could also inhibit the oxidative degradation of 2-deoxyribose caused by Fenton reagents. Since we observed that PIH enhances the Fe(II) autoxidation rate, measured by the ferrozine technique, PIH may limit *OH formation and consequently DNA damage by decreasing the amount of Fe(II) available to catalyze Fenton reactions.

Characteristics of rhizobia isolated from three legumes indigenous to the Canadian high arctic: Astragalus alpinus, Oxytropis maydelliana, and Oxytropis arctobia

SummaryForty-eight strains of rhizobia were isolated from the root nodules ofAstragalus alpinus (21),Oxytropis maydelliana (19) andOxytropis arctobia (8), three species of arctic legumes found in the Melville Peninsula, Northwest Territories, Canada. On the basis of 74 characteristics (cultural, physiological, biochemical and host nodulation range) the 48 arctic rhizobia could be divided into 11 distinct groups by numerical analysis techniques. All 48 arctic rhizobia were able to nodulate the three arctic legume species and also sainfoin (Onobrychis viciifolia), however, milkvetch (Astragalus cicer) was only nodulated by 33 strains. In general, the arctic rhizobia showed properties found in both Rhizobium and Bradyrhizobium. The adaptation of the arctic strains to low temperature is indicated by their ability to grow in liquid culture at 5°C.

Ferric pyridoxal isonicotinol hydrazone can provide iron for heme synthesis in reticulocytes

We have examined whether reticulocytes depleted of transferrin might incorporate 59Fe from 59Fe-labelled pyridoxal isonicotinoyl hydrazone (PIH). Transferrin-depleted reticulocytes showed a time-, temperature- and concentration-dependent incorporation of 59Fe when incubated with 20-200 microM 59Fe-PIH. The amount of 59Fe incorporated with 200 microM 59Fe-PIH is equal to or higher than that taken up from transferrin at 20 microM 59Fe concentration. After 60 min about 60% of the 59Fe taken up by the cells is recovered in heme while the remainder is probably still bound to PIH. 1 mM succinyl acetone (a specific inhibitor of heme synthesis) inhibits PIH-mediated incorporation of 59Fe into heme by about 70%, indicating that 59Fe from 59Fe-PIH is incorporated into de novo synthesized protoporphyrin. As is the case with transferrin, erythrocytes do not incorporate 59Fe from 59Fe-PIH. Pretreatment of reticulocytes with pronase does not inhibit their ability to incorporate 59Fe from 59Fe-PIH, suggesting that, unlike the uptake of Fe from transferrin, membrane receptors are not involved in the uptake of Fe-PIH by the cells.

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and its analogues prevent damage to 2-deoxyribose mediated by ferric iron plus ascorbate.

Iron chelating agents are essential for treating iron overload in diseases such as beta-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H(2)O(2) or Fe(III)-EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)-EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 microM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)-EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)-EDTA or Fe(III)-NTA) and forms a Fe(III)-PIH(2) complex that does not catalyze oxyradical formation.

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction.

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.

Transferrin and iron uptake by human lymphoblastoid and K-562 cells

Abstract Two human lymphoblastoid cell lines and K-562 cells were found to take up radioiodinated transferrin and transferrin-bound iron in amounts comparable to reticulocytes. These cell lines were also shown to possess transferrin receptors whose numbers and affinity for transferrin were similar to those of reticulocytes. However, unlike reticulocytes, in which at least 90% of the iron taken up is incorporated into heme, in the lymphoblastoid and K-562 cells only around 10% of the incorporated iron is found in heme. In addition, in contrast to the hemoglobin synthesizing cells, excess heme does not inhibit the removal of iron from transferrin by the lymphoblastoid and K-562 cells, suggesting that only during erythroid differentiation do cells acquire a specific mechanism for removing iron from transferrin which is subject to feedback inhibition by heme.

The reticulocyte-mediated release of iron and bicarbonate from transferrin: Effect of metabolic inhibitors

Abstract The effect of three groups of metabolic inhibitors on the incorporation of Fe and release of bicarbonate from transferrin by rabbit reticulocytes was measured. Inhibitors which affect reticulocyte Fe and transferrin uptake to the same extent (sodium arsenite, N-ethylmaleimide and iodoacetamide); those which inhibit reticulocyte Fe uptake to a greater extent than transferrin uptake (NaN3, NaF, NaCN, rotenone, oligomycin, 2,4-dinitrophenol and cycloheximide); and compounds which after reticulocyte heme synthesis (CoCl2, isonicotinic acid hydrazide and hemin) were used. In each case the effect on Fe incorporation and bicarbonate release was the same Thus, additional evidence has been obtained for the idea that the reticulocyte-mediated release of Fe and bicarbonate from transferrin are tightly coupled. The results are consistent with the hypothesis that an enzymatic attack on transferrin-bound bicarbonate is involved in the removal of Fe from transferrin by erythroid cells.