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To learn more about pathological iron storage in the liver, two sorts of lysosomes were isolated from rat livers in Percoll - sucrose or sucrose gradients: siderosomes (= iron-loaded terminal lysosomes) and light lysosomes (secondary and terminal). Such cell fractions were obtained from acutely iron-loaded and control rat livers. After lysis with Triton X-100 the preparations were assayed for proteolytic activity against rat liver ferritin (RLF) and denatured bovine hemoglobin (DBH), for buffer-soluble ferritin protein content, total protein and non-heme iron. At pH 3.6 both fractions displayed considerable proteolytic activity (cathepsin D activity) against DBH and endogenous proteins but little activity against RLF. By contrast, proteolytic activity against RLF was maximal at the highest pH tested, 6.5, at which DBH was practically insusceptible. The behavior of proteolytic activity against ferritin at pH 6.5 makes it likely that a single enzyme was involved that acted by Michaelis-Menten kinetics. However, no more than 2.5% of endogenous ferritin protein in the organelles was buffer-soluble. 41 to 89 hours after an intramuscular dose of 50 mg Fe, given as iron dextran, the non-heme iron content of light lysosomes and siderosomes had increased markedly and the ratio of non-heme Fe to buffer-soluble ferritin protein also became much elevated in the organelles; but the ratio of buffer-soluble ferritin to total protein did not rise significantly. The rise in organellar non-heme Fe exceeded iron saturation of rat liver ferritin and thus reflected conversion of ferritin to hemosiderin, which is buffer-insoluble.(ABSTRACT TRUNCATED AT 250 WORDS)

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To investigate storage of ferritin and its transition to hemosiderin under conditions of iron overload, rats were either given multiple injections of iron dextran over 4 to 5 weeks or fed a diet containing 1.3% Fe as ferric ammonium citrate for 60 days. Then, preparations of liver siderosomes (heavily iron-laden lysosomes) were examined for content of buffer-soluble ferritin and buffer-insoluble, ferritin-related protein, total nonheme iron and protein, cathepsin D activity, and ability to incorporate 14C-leucine into ferritin. Total liver nonheme iron, ferritin protein and iron, and cathepsin D activity were also determined. Although parenteral iron loading produced higher total nonheme iron in livers than dietary loading, the iron content of ferritin was approximately 20% in both groups, reflecting saturation of ferritin with iron. Siderosome nonheme iron content was greater than 40% in relation to protein. The siderosomes contained little buffer-soluble ferritin; on isoelectric focusing this was composed of isoferritins present also in cytosol ferritin. Buffer-insoluble ferritin protein, identified in siderosomes by immunofluorescence, was solubilized and found to contain immunoreactive material corresponding to L and H subunits of buffer-soluble ferritin. Transmission electron microscopy indicated the presence of relatively large quantities of "ferritin" in siderosomes, and it is argued that this was mostly buffer insoluble (denatured) or represented ferritin [FeOOH]x cores divested of protein shells. Although siderosomes had substantial cathepsin D activity, the known resistance of ferritin to this and other proteases makes it unlikely that proteolysis is an early event in the decomposition of ferritin in siderosomes. Heavily iron-laden siderosomes did not take up newly labeled ferritin or ferritin protein or 14C-precursor within 24 hours of labeling, when 14C-labeled ferritin was abundant in cytosol. The author proposes a sequence of steps leading from sequestration of buffer-soluble cytosol ferritin to storage of insoluble "hemosiderin."

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As judged from 2-h blood level curves, adult female rats absorbed more FeII per cm2 of gross duodenal mucosa than adult male rats. By contrast, the 2-h blood level curves per cm2 of mucosa of proximal jejunum did not differ significantly in male and female rats although in both sexes, iron was absorbed more efficiently from the duodenum.

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Cultures of Kupffer cells and of hepatocytes, prepared from single rat livers, synthesized ferritin protein equally efficiently. In culture but not in suspension, both sorts of cells responded significantly to stimulation with iron by increased ferritin synthesis. As determined by isoelectric focusing, the isoferritin profiles of newly synthesized 14C-labeled Kupffer cell and hepatocyte ferritin were identical, each having three bands. However, unlabeled ferritin, extracted from nonparenchymal liver cells (mainly Kupffer and endothelial cells) of iron-loaded rats, contained an acidic isoferritin that was not present in hepatocyte ferritin. Investigation of ferritin synthesis in cultured peritoneal and alveolar macrophages yielded similar results. The isofocusing profile of newly synthesized peritoneal macrophage ferritin was indistinguishable from the profile of fresh Kupffer cell or hepatocyte ferritin. Thus, the three isoferritins common to Kupffer cells, hepatocytes, and extrahepatic macrophages are neither cell- nor tissue-specific. However, modifications on intracellular storage may affect the isofocusing properties. The findings, although consistent with the LnH24-n subunit model of ferritin protein, indicate identical restrictive genomic control of the H:L ratios in these sorts of cells. Further, they make it probable that Kupffer cell ferritin iron, originating by endogenous synthesis, is the principal source of Kupffer cell hemosiderin iron.

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In the proximal tubular cells of rats or mice given a single, parenteral dose of lead, clusters of ferritin are frequently associated with characteristic cytoplasmic fibrillar bodies. To learn more about this relationship, we have investigated content and synthesis of ferritin protein and incorporation of iron into ferritin in rat kidneys 48 hours after a single parenteral dose of lead (10 microgram/g). By immunoradiometric assays, we found that the kidneys of female rats, whether treated with lead or not, contained significantly more ferritin protein than did kidneys of males of the same age and provenance. Administration of lead diminished (or did not significantly alter) the incorporation of 14C-amino acids into newly synthesized ferritin protein. Contrary to expectation, administration of lead tended to depress incorporation of 59Fe into kidney ferritin in rats maintained on standard rations and distilled water. Electron microscopy confirmed the presence of clusters of ferritin in close association with dense fibrillar bodies in the cytoplasm of proximal tubular cells of rats given lead. Considered together, the findings indicate that clustering of ferritin next to the dense fibrillar cytoplasmic lesions is a selective effect of lead that requires neither augmented synthesis of ferritin protein nor increased incorporation of iron into preexisting ferritin.

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A single intracardiac dose of lead acetate (40 microgram lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. The prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G2 phase. The injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. The incremental labeled cells may have originated from a small fraction of the G1 population that was ready to enter the S phase without the usual pre-synthetic delay.

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Cell fractions were prepared from ACI rat livers and from rat hepatoma cell clone M-5123-C1. Radioimmunoassays of ferritin and of its protein subunits in various cell fractions after biosynthetic labeling with [14C]leucine were done by means of ferritin-specific and subunit-specific rabbit antibody. In both ACI rat livers and M-5123-C1 hepatoma cells free polyribosomes synthesized approximately 81% of the protein subunits of ferritin, and membrane-bound polyribosomes synthesized the rest. In both polyribosomal fractions, [14C]leucine-labeled subunits were detected earlier than [14C]leucine-labeled ferritin and apoferritin (5 min as against 30 min after initiation of a pulse). Time sequence studies of the shifts of biosynthetically labeled subunits and ferritin through different cell compartments provided evidence for vectorial transport of subunits and of ferritin, the direction of transport being from the two polyribosomal systems to the smooth membrane compartment and to the cytosol.

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Ferritin and its protein subunits in rat hepatoma cell clone M-5123-C1 were biosynthetically labeled with [14C]leucine and 59Fe. Radioimmunoassays of ferritin/apoferritin and of protein subunits in the free polyribosome, membrane-bound polyribosome, smooth membrane, and cytosol fractions were done with ferritin-specific and subunit-specific rabbit IgG antibodies at various time intervals after pulsing. Much more 59Fe was bound by ferritin/apoferritin than by subunits in all of the cell fractions. Binding of iron to subunits may have been a random process. When hepatoma cells were simultaneously pulse-labeled with 59Fe and [14C]leucine, uptake of much of the 59Fe by ferritin occurred relatively early, in comparison to incorporation of [14C]leucine, in all of the cell fractions examined. Thus, 59Fe was readily incorporated into pre-existing ferritin. We conclude that most, if not nearly all, of the iron is incorporated after assembly of protein subunits.

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Characteristic cytoplasmic and intranuclear fibrillar bodies were produced, within 24 hours, in epithelial cells of the proximal convoluted renal tubules of rats and mice by injecting a single dose of lead acetate either intraperitoneally (100 mug Pb/g) or into the heart (10 mug Pb/g). The frequency of cytoplasmic fibrillar bodies (CFB) rose during the first 4 days following injection of lead and diminished thereafter. Ten days after intracardiac injection of lead no CFB were found; 10 days after intraperitoneal injection, they were still present, though probably in diminished number. Disappearance of CFB may be related to autophagocytosis. Intranuclear fibrillar bodies did not disappear, perhaps because nuclei lack a lysosomal apparatus. Within the first 3 days after injection of lead, clusters or paracrystalline arrays of ferritin molecules were frequently situated in the immediate vicinity of CRB or abutted against CFB; after the third day, little or no ferritin was found near CFB. Intramuscular injection of iron-dextran complex (50 mg Fe/ml) 24 hours prior to intraperitoneal administration of lead did not increase incidence or size of ferritin clusters in the vicinity of CFB in rats. The presence of ferritin near CFB may have been an indirect consequence of inhibition, by lead, of synthesis of heme prosthetic groups.

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We have reinvestigated the association and dissociation of ferritin and apoferritin in phosphate buffer (pH 7.2, I = 0.05). When oligomer-enriched solutions of horse spleen ferritin were mixed with more concentrated, but unenriched solutions of horse spleen apoferritin, there was dissociation of the ferritin oligomers, as determined by polyacrylamide gel electrophoresis and from iron/protein ratios. Some evidence was also obtained for association of monomers in the mixture of ferritin and apoferritin after pelleting and redissolution of pellets in minimal volumes of the phosphate buffer. Monomer-enriched, biosynthetically labeled rat liver ferritin was pelleted, redissolved in minimal volumes of phosphate buffer, and separated by polyacrylamide gel electrophoresis; the fractions were isolated and counted. The results revealed that an association of monomers of the rat liver ferritin had taken place which doubled the concentration of dimers. However, our results also indicate that association by concentration was limited to a fraction of monomers.

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Using precipitating antibodies to ACI rat liver ferritin and to sodium-dodecyl-sulfate-dissociated protein subunits of ACI rat liver ferritin, we have demonstrated the presence of ferritin-positive sites and subunit-positive sites in situ in several rat hepatoma cell lines by immunofluorescence. Hepatoma cells from three transplantable rat hepatomas (Reuber H-139, Reuber H-35, and Morris 5123) were explanted and propagated. Rabbit antibodies specific for either protein subunits of ferritin or ferritin were prepared by affinity chromatography or by dissociation of antibody-antigen complexes with 0.1 M acetic acid followed by differential ultracentrifugation. Explants of Reuber H-139, Reuber H-35, and Morris 5123 hepatoma cells, grown either in ordinary McCoy's 5a medium or in such medium enriched with iron (0.002% Fe), gave positive immunofluorescence for subunits as well as ferritin. Exposure of a clonal strain of Morris 5123 hepatoma cells to iron-enriched culture medium for varying lengths of time of up to 24 hours resulted in progressive increase in the quantity of ferritin-specific immunofluorescent cytoplasmic material, which was at first present diffusely, and later in clumps. By contrast, during the initial 24-hour period, subunit-specific immunofluorescence remained at relatively low intensity, with diffuse distribution through the cytoplasma. Our findings indicate a) the presence, in the cytoplasm, of the three kinds of hepatoma cells, of unassembled or only partly assembled subunits of fragments of subunits as well as of ferritin, and b) rapid assembly of the protein subunits into apoferritin and ferritin after administration of iron, so that the concentration of subunits in the cytoplasm was not significantly increased.

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A single dose of lead (5 mug/g body weight), given as lead acetate by intracardiac injection, produces, within 8 hours, characteristic fibrillar intranuclear and intracytoplasmic inclusions in proximal tubular epithelial cells in mouse kidneys; after a dose of 40 mug/g body weight, such inclusion appeared within 6 hours. Their development was completely prevented by cycloheximide (one or more intraperitoneal injections of 20 mug/g body weight). The development of intranuclear inclusions was also noted in tubular epithelial cells explanted from normal mice and grown in vitro for 15 hours in a medium containing 20 mug lead/ml. Thus, the development of the characteristic fibrillar inclusion bodies depends upon de novo synthesis of inclusion body protein, induced by lead.

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Antisera raised in rabbits against protein subunits of rat liver ferritin contain antibodies (IgG) that precipitate subunits and other antibodies (IgG) that precipitate undissociated ferritin and apoferritin. Removal of the latter antibodies from IgG fractions of immune sera by repeated addition of ferritin leaves antibodies that specifically precipitate subunits. Using these subunit-specific antibodies, we have demonstrated the presence of subunit-positive sites in cells by immunofluorescence. Subunit-positive sites were best demonstrated in rats that had been loaded with iron. These sites were diffusely spread through the cytoplasm of hepatocytes and Kupffer cells in rat livers and of macrophages and fibroblasts in rat livers and hearts. With antibodies to ferritin, the presence of ferritin was demonstrated in the same kinds of cells. Although, by immunofluorescence, the intracellular localization of ferritin-positive and subunit-positive sites was similar, ferritin-positive immunofluorescence was most intense in Prussian blue-positive cytoplasmic granules in which high concentrations of ferritin were presumed to be present on the basis of electron microscopic studies. Our findings can be interpreted to indicate either the presence of unassociated subunits or of fragments of subunits in the cytoplasm of the several kinds of cells studied. It is also possible that partly assembled molecules of ferritin or apoferritin in these cells reacted with subunit-specific antibodies. Subunit-specific antibodies may be useful for localizing sites of assembly of ferritin molecules at the level of cell fine structure and for elucidation of the biosynthesis of ferritin.

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