Lead poisoning: rapid formation of intranuclear inclusions.

A single dose of lead (0.05 milligram per gram of body weight) induced characteristic intranuclear inclusions in the epithelium of proximal tubules in rat kidney within 1 to 6 days. The development of the intranuclear inclusions is thus an acute manifestation of lead poisoning, not a delayed one, as has been thought hitherto. Cytoplasmic structures resembling the intranuclear inclusions and situated in the vicinity of endoplasmic reticulum were regularly found in cells bearing the pathognomonic intranuclear inclusions. The latter and the cytoplasmic structures may be derived from a common precursor, perhaps a soluble protein-lead complex.

Studies of iron overload. Lysosomal proteolysis of rat liver ferritin.

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)

Evolution of cytoplasmic fibrillar bodies induced by lead in rat and mouse kidneys.

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.

Effects of cyclic starvation-feeding and of splenectomy on the development of hemosiderosis in rat livers.

The development of siderosis of liver and spleen was investigated in rats subjected alternately to periods of starvation and periods of feeding of diets rich in iron (0.71% or 1.23% Fe) or of control diets, during periods ranging up to 245 days. With 0.71% iron in the diet, cyclic starvation-feeding markedly enhanced the accumulation of iron in rat livers by comparison to feeding ad libitum even though rats fed ad libitum ingested far greater total amounts of iron than cyclically fed rats. With 1.23% iron in the diet, the concentration of iron in livers reached more or less the same plateau in cyclically starved-fed rats and in rats fed ad libitum (betwen 4 and 5 mg Fe/g wet weight); but the mean rate of accumulation of iron in the livers of cyclically starved and fed rats was more than twice that in rats fed ad libitum, whereas mean ingestion of iron per feeding day was only 16% higher in the former group. Surgical removal of the spleen enhanced the accumulation of iron in the liver in cyclically starved-fed rats and in rats fed ad libitum. Histologically, siderosis of the liver was moderate in rats fed the diet with 0.71% iron but was severe in rats fed the diet with 1.23% iron and most severe in those without spleens. Stainable iron was deposited in hepatocytes and in Kupffer cells. None of the rats developed cirrhosis of the liver. The data suggest that in rats a barrier to the absorption of iron from the gut, or to its later utilization, is surmounted if the concentration of iron in the food exceeds a certain limit value, somewhere between 0.71 and 1.23%. With iron in the food below this value, cyclic starvation-feeding markedly potentiates accumulation of iron in the liver in the course of several months, but siderosis is moderate. With iron in the food above the limit value, cyclic starvation-feeding and feeding ad libitum can equally lead to massive siderosis of the liver.

Studies of iron overload. Rat liver siderosome ferritin.

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."

Biosynthesis of ferritin in rat hepatoma cells and rat livers. I. Synthesis and assembly of protein subunits of ferritin.

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.

Biosynthesis of ferritin in rat hepatoma cells and rat livers. II. Binding of iron by ferritin protein.

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.

Isoferritins in rat Kupffer cells, hepatocytes, and extrahepatic macrophages. Biosynthesis in cell suspensions and cultures in response to iron.

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.

Electron microscopy of a strain of Bordetella bronchiseptica.

A strain of Bordetella bronchiseptica that had been isolated from a rat hepatoma cell culture was investigated by means of electron microscopy. Bacteria were examined after (i) negative staining with phosphotungstate or uranyl acetate, (ii) metal shadowing with platinum-palladium, and (iii) fixation with glutaraldehyde followed by embedding, sectioning, and staining. The multilayered bacterial cell walls appeared lobulated in negatively stained and in metal-shadowed specimens; the lobules were demarcated by grooves, 100 to 200 A in width, but without interruption of continuity in any layer of the cell wall. Cross sections of fixed material revealed wrinkled cell walls in many-but not all-preparations. Bacterial cell membranes and cytoplasm were similar to those of other gram-negative bacilli (e.g., Escherichia coli). Bacteria fixed in 1.5% glutaraldehyde contained intertwined or whorled fibrils, down to about 20 A in thickness. The flagella were peritrichous, measured about 200 A in width, and were composed of braided strands, about 20 A in width.

Cell proliferation in rat kidney induced by lead acetate and effects of uninephrectomy on the proliferation.

Effects of a single dose of lead (0.04 mg lead g body weight) on the proliferation of proximal tubular epithelium in rat kidneys were investigated by autoradiography over a period of 72 hours, using (3)H-thymidine as a label. The results demonstrate that cell proliferation was greatly stimulated within 2 days after lead was injected. The increase in DNA synthesis began about 20 hours after intraperitoneal injection of lead, reached a sharp peak at 30 hours, and declined rapidly thereafter. At the peak, the mean labeling activity was 40 times that observed in control rats. Cumulatively, an average of 14.5% of the proximal tubular epithelial cells were labeled 72 hours after lead was injected. When uninephrectomy was followed immediately by injection of lead, the stimulation of DNA synthesis in the remaining kidney was, on the average, greater than the sum of the separate effects of the two treatments. This indicates that the stimulatory effects of uninephrectomy and injection of lead on renal cell proliferation were additive.

Cell proliferation in rat kidneys after prolonged treatment with lead.

Effects of chronic lead intoxication on cellular proliferation in rat kidneys were investigated by autoradiography. The rats were given intraperitoneal injections of lead acetate in aqueous solution for 6 months. At the end of this period, the proliferative activity of proximal tubular epithelial cells was about 15 times greater in rats given lead than in untreated controls. In the leaded rats approximately 40% of the proximal tubular cells contained intranuclear inclusions, approximately 0.5% of proximal tubular epithelial cells were labeled and approximately 6% of labeled cells contained intranuclear inclusions. Thus, cells with intranuclear inclusions can replicate DNA. Effects of chronic lead poisoning on the replication of proximal tubular cells in rats subjected to left uninephretomy before inception of treatment with lead were substantially the same. No renal carcinomas were found after 6 months of treatment with lead, but there was epithelial hyperplasia in some proximal tubules, with occasional atypia. The presence of increased synthesis of DNA and epithelial hyperplasia in the kidneys of rats chronically poisoned with lead suggests that the renal carcinogenicity of lead, observed by others, is related to lead-induced stimulation of renal cell proliferation.

Microembolic renal disease in rats induced with sephadex.

Sephadex particles (20-80 mu in size) were injected into the abdominal aorta of 134 male Sprague-Dawley rats near the renal arteries. In 31 rats, the right kidney was then removed. The Sephadex particles lodged in glomerular capillaries, afferent glomerular arterioles and interlobular arteries, creating renal infarcts, some of which were grossly visible. Shortly after injection, arterial blood pressure rose significantly in most animals. The hypertension in uninephrectomized rats was not demonstrably different from that in rats with two Kidneys. Severity and duration of hypertension (up to 8 months) were positively correlated with the number of Sephadex particles in renal vessels, and there was also a positive correlation between the degree of hypertension and serum urea nitrogen levels, and between degree of hypertension and degree of cardiac hypertrophy. The vascular permeability in acutely hypertensive rats was abnormal, as judged from penetration of iron-dextran into vessel walls. This experimental model resembles atheromatous microembolic renovascular disease, which may play a significant role in the pathogenesis of unexplained hypertension in patients with advanced aortic atherosclerosis.

G2 sub-population in mouse liver induced into mitosis by lead acetate.

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.

Absorption of iron from gut into blood: sex- and time-related studies in rats.

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.

The monomers and oligomers of ferritin and apoferritin: association and dissociation.

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.

Biogenesis of intranuclear lead-protein inclusions in mouse kidney.

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.