P. Poňka

A study of intracellular iron metabolism using pyridoxal isonicotinoyl hydrazone and other synthetic chelating agents

Abstract 1. 1. Rabbit reticulocytes with a high level of non-heme radioiron induced by preincubation with isonicotinic acid hydrazide and transferrin-bound 59Fe, were reincubated with various synthetic chelating agents and the amount of radioiron released from the cells was determined. Some substances, especially derivatives of pyridoxal or 2-hydroxybenzaldehyde and isonicotinic acid hydrazide or benzhydrazide, were found to mobilize significantly iron from 59Fe-labeled reticulocytes. The effectiveness of the compounds tested decreases in the following order: pyridoxal isonicotinoyl hydrazone, pyridoxal benzoyl hydrazone, 2-hydroxybenzal isonicotinoyl hydrazone, 2-hydroxybenzal benzoyl hydrazone, pyridoxal-valine Schiff base, pyridoxal. The efficiency of these compounds exceeded the ability of common chelators such as desferrioxamine, 2,2′-bipyridine, nitrolotriacetic acid, etc., to mobilize iron from reticulocytes. 2. 2. Iron mobilization from reticulocytes by pyridoxal isonicotinoyl hydrazone requires ATP to be produced by cells and is completely blocked by low temperature (4°C). Although the effect of desferrioxamine is also prevented by low temperature, modest iron mobilization due to this chelator seems to occur independently of ATP production in reticulocytes. 3. 3. Pyridoxal isonicotinoyl hydrazone mobilizes iron mainly from mitochondria and in part also from ferritin. Although 2,2′-bipyridine seems to enter reticulocyte mitochondria and bind iron there, this chelator is not able to release iron either from mitochondria or from the cells. 4. 4. Reticulocytes with a high level of non-heme radioiron are envisaged as a useful system for testing biological effectiveness of various iron chelators. 5. 5. Pyridoxal isonicotinoyl hydrazone was shown to be an effective in vivo chelator since its administration to mice decreased 59Fe radioactivity in liver, spleen and kidney.

Study of intracellular iron distribution in rabbit reticulocytes with normal and inhibited heme synthesis

Abstract Iron compartments in which iron accumulates during the inhibited heme synthesis after treatment with isonicotinic acid hydrazide were studied in rabbit reticulocytes. A great accumulation of 59Fe radioactivity was found in mitochondria, low molecular weight iron compounds and non-hemoglobin proteins, especially ferritin. In a chase experiment, approximately 50% of the 59Fe radioactivity accumulated in mitochondria and low molecular weight iron compounds was re-utilized for the synthesis of hemoglobin. Although some iron is incorporated into ferritin, it apparently is not utilized for heme synthesis. In control reticulocytes, only traces of 59Fe radioactivity were detected in mitochondria and only a minute amount of 59Fe radioactivity was detected in low molecular weight iron compounds. The release of low molecular weight iron from 59Fe-transferrin occured in intact reticulocytes to a larger extent than in the stroma-free hemolysate. An attempt to establish the possible pathway of iron transport inside the reticulocyte was made. It is suggested that an iron-transferrin complexes enters the reticulocyte cytoplasm, and the majority of released iron is taken up by mitochondria for heme synthesis. When protoporphyrin IX is not available, iron accumulates inside the mitochondria.

Biliary iron excretion in rats following pyridoxal isonicotinoyl hydrazone.

Summary Biliary excretion of iron after administration of pyridoxal isonicotinoyl hydrazone (PIH), a recently identified effective iron‐chelating agent, was investigated in rats. PIH administered both intraperitoneally and orally was shown to increase significantly 59Fe excretion into bile of rats which had previously been injected with 59Fe‐transferrin to label hepatic parenchymal cells. 59Fe‐PIH appears in bile as early as 15 min after chelator administration and the peak of 59Fe‐radioactivity in bile is seen 1–5 h following intraperitoneal PIH injection. PIH, administered intraperitoneally, 125–250 mg/kg, increased 24 h biliary radioiron excretion about 35 times and in addition increased urinary and faecal iron excretion. When PIH was given immediately before 59Fe‐transferrin, 24 h cumulative biliary 59Fe excretion was even higher. PIH was also demonstrated to increase biliary excretion of radioiron released from 59Fe‐haemoglobin catabolysed in reticuloendothelial cells. The effect of PIH was confirmed by estimation of biliary iron concentration using the method of atomic absorption spectrophotometry. Repeated PIH administration to rats decreased 59Fe radioactivity in liver and kidney and increased urinary and faecal iron excretion.

Evidence for the presence of free and protein-bound nonhemoglobin heme in rabbit reticulocytes

Abstract In the hemolysate of reticulocytes incubated with 59 Fe or [2– 14 C]glycine a radioactivity is found also in nonhemoglobin heme. About 96% of the radioactivity of nonhemoglobin heme is associated with proteins and approx. 4% is in a free form. Labeled free heme is removed from the reticulocyte hemolysate on the column of Sephadex G-25. Retained heme is eluted from the column of Sephadex G-25 by means of the special eluting solution containing serum albumin. Protein-bound nonhemoglobin heme radioactivity is recovered in hermin isolated from nonhemoglobin proteins separated by means of the chromatography on CM-Sephadex. Isonicotinic acid hydrazide which specifically inhibits heme synthesis prevents the incorporation of 59 Fe into hemoglobin heme. In the hemolysate of cycloheximide incubated reticulocytes the radioactivity in both free and protein-bound nonhemoglobin heme increases essentially. The accumulation of 59 Fe-labeled heme in the nonhemoglobin proteins in reticulocytes incubated with cycloheximide provides evidence for an existence of an increased pool of heme which is not incorporated into hemoglobin or catalase. The finding of the existence of free heme in reticulocytes might contribute to recent theories of regulation of hemoglobin synthesis.

Accumulation of heme in mitochondria from rabbit reticulocytes with inhibited globin synthesis

Incubation of rabbit reticulocytes with cycloheximide and 59Fe bound to transferrin in plasma induces excessive non-hemoglobin 59Fe-labeled heme accumulation in mitochondria. During incubation of these mitochondria in vitro a part of 59Fe-labeled heme is released into the surrounding medium. The addition of globin or bovine serum albumin to the incubation mixture essentially increases the amount of heme released from mitochondria.

Changes In Stem Cell Compartments In Mice After Hydroxyurea

Hydroxyurea injection kills only approximately 10% of CFU, which are in the S phase of the cell cycle. In mice given a single injection of hydroxyurea CFUs in the femur decreased only about 50% in 2–4 days after hydroxyurea, and then started to return to normal levels with an overshoot evident after the eighth day after hydroxyurea. CFUs in spleens of mice given a single injection of hydroxyurea show an evident overshoot as early as 3 days after the drug, reaching levels which are about 300% of normal. CFUs from blood disappear rapidly, but equally rapidly return to normal values followed by a significant overshoot. the radioresistance of mice increased up to 4 days after hydroxyurea.

Problems of reduced heme synthesis in reticulocytes with impaired globin synthesis

SummaryThe mechanism by which heme synthesis is inhibited in reticulocytes with inhibited globin synthesis is not yet established. Evidence was found that the accumulated free heme does not inhibit the activity of δ-aminolaevulic acidsynthetase. The very early inhibition of heme synthesis may be caused by the effect of uncompleted globin chains or amino acids on the δ-aminolaevulic acidsynthetase or some other enzymes of the porphyrin biosynthetic chain. The accumulated free heme, the presence of which in reticulocytes with inhibited globin synthesis has been indirectly proved may reduce the iron entry to the reticulocytes which in turn can influence the synthesis of heme especially in the later periods of incubation. A feedback inhibitory effect of accumulated free heme at some critical site on the enzymes of the porphyrin biosynthetic chain cannot still be quite excluded.ZusammenfassungDer Mechanismus, mit dem die Häm-Synthese bei Retikulozyten mit gestörter Globulinsynthese gehemmt wird, ist noch nicht geklärt. Es wurden Anzeichen dafür gefunden, daß die akkumulierten freien Häme die Aktivität der δ-Aminolävulinsäuresynthetase nicht behindern. Die sehr frühe Hemmung der Häm-Synthese kann durch die Wirkung unvollständiger Globinketten oder Aminosäuren auf die δ-Aminolävulinsäuresynthetase oder einige andere Enzyme der biosynthetischen Porphyrinkette verursacht werden. Die akkumulierten freien Häme, deren Anwesenheit in Retikulozyten mit gehemmter Globinsynthese indirekt nachgewiesen wurde, können den Eintritt von Eisen in die Retikulozyten reduzieren, was wiederum die Synthese der Häme, besonders in den späteren Inkubationszeiten beeinflussen kann. Eine hemmende Rückkoppelungswirkung akkumulierter freier Häme an irgendeiner kritischen Stelle der Enzyme der biosynthetischen Prophyrinkette kann nicht ganz ausgeschlossen werden.

Decreased Sensitivity to Hydroxyurea and to [3H]Thymidine Suicide In the Middle of the S Phase

Pluripotent haemopoietic stem cells (CFUs) move synchronously through the cell cycle in hydroxyurea‐treated mice in a cohort 1–2 hr broad. Ten to fifteen hours after hydroxyurea they pass through S phase. DNA synthesis appears to be depressed 5–10 times when the cells are in the middle part of the S phase but does not seem to be completely interrupted. High concentrations of [3H]thymidine must be used for ‘suicide’ in order to achieve lethality for the cells with depressed DNA synthesis. At the time when DNA synthesis is depressed, the sensitivity of the cells to hydroxyurea also decreases. This may lead to a significant underestimation of the S phase fraction by the hydroxyurea method, because CFUs with low DNA synthesis rate are resistant to hydroxyurea although being in S phase.

Effect of some cytostatics on the haemopoietic stem cells (CFUs) in blood.

SummaryThe effect of some cell-cycle stage-specific cytostatics on the pluripotent stem cells (CFUs) present in the blood was investigated. The amount of these cells in the blood responds markedly and very quickly to a single administration of any of these drugs. Their effects on the circulating CFUs allowed the drugs tested to be divided into two groups: (1) Hydroxyurea and arabinosyl cytosine induced a profound decrease in the number of CFUs in the blood, which was followed by a return to the normal value and an overshoot that lasted several days; (2) Colchicine, vinblastine, vincristine, and methotrexate first induced an increase in the number of CFUs in the blood, which was followed by a decrease to below the normal values, and still later an overshoot that lasted several days.The preliminary data on the effect of hydroxyurea on the CFUs circulating in large quantities in the blood of mice with Friend virus leukaemia indicate that the tumorous stem cells (CFUs) might respond to these cytostatics in a similar way.