Biliary excretion of iron from hepatocyte lysosomes in the rat. A major excretory pathway in experimental iron overload.

In these experiments, we assessed the role of hepatocyte lysosomes in biliary excretion of iron. We loaded rats with iron by feeding 2% carbonyl iron and collected bile for 24 h via bile fistulae from iron-loaded and control rats. In additional rats, bile was collected before and after the administration of colchicine. Rats were then killed and their livers were homogenized and fractionated for biochemical analyses or processed for electron microscopy and x-ray microanalysis. Inclusion of 2% carbonyl iron in the diet caused a 45-fold increase (P less than 0.001) in hepatic iron concentration compared with controls (1,826 +/- 159 vs. 38 +/- 6.7 micrograms/g liver, mean +/- SE). Electron microscopy with quantitative morphometry and x-ray microanalysis showed that the excess iron was sequestered in an increased number of lysosomes concentrated in the pericanalicular region of the hepatocyte. Iron loading was also associated with a twofold increase in biliary iron excretion (4.06 +/- 0.3 vs. 1.75 +/- 0.1 micrograms/g liver/24 h; P less than 0.001). In contrast, the biliary outputs of three lysosomal enzymes were significantly lower (P less than 0.0005) in iron-loaded rats compared with controls (mean +/- SE) expressed as mU/24 h/g liver: N-acetyl-beta-glucosaminidase, 26.7 +/- 4.6 vs. 66.2 +/- 13.4; beta-glucuronidase, 10.1 +/- 1.3 vs. 53.2 +/- 17.9; beta-galactosidase, 8.9 +/- 1.0 vs. 15.4 +/- 2.3. In iron-loaded rats but not in controls, biliary iron excretion was coupled to the release into bile of each of the three lysosomal hydrolases as assessed by linear regression analysis (P less than 0.001). In contrast, no relationships were found between biliary iron excretion and the biliary outputs of a plasma membrane marker enzyme (alkaline phosphodiesterase I) or total protein. After administration of colchicine, there was a parallel increase in biliary excretion of iron and lysosomal enzymes in iron-loaded rats, but not controls. We interpret these data to indicate that, in the rat, biliary iron excretion from hepatocyte lysosomes is an important excretory route for excess hepatic iron.

Pulmonary alveolar phospholipoproteinosis: experience with 34 cases and a review.

A retrospective review of Mayo Clinic records through 1983 revealed 84 patients (24 male and 10 female; mean age, 41 years) with the diagnosis of pulmonary alveolar phospholipoproteinosis. The major clinical features were dyspnea, cough, fever, and chest pain. Chest roentgenograms usually showed bilateral symmetric alveolar infiltrates, but asymmetric, unilateral, and chronic patchy patterns were also noted. Diagnosis was established by thoracotomy-lung biopsy in 26 patients. Histologic analysis revealed uniform filling of the alveoli by periodic acid-Schiff-positive material and maintenance of normal alveolar architecture. Electron microscopy showed enlarged alveolar macrophages with lamellar osmiophilic inclusions, dense granules, and myeloid bodies. Of the 21 patients who underwent therapeutic bronchoalveolar lavage, 13 had no recurrence of the disease during a mean follow-up of 8.8 years. In patients who underwent pulmonary function testing both before and after lavage, significant restrictive dysfunctions present before the procedure were alleviated afterward. Three deaths occurred among the 34 patients. Pulmonary alveolar phospholipoproteinosis may result from defective clearance of phospholipids by the alveolar macrophages, excessive production of phospholipids by type II pneumocytes, or both. It is likely a nonspecific response to a variety of injuries to the alveolar macrophage or type II pneumocyte or both, including exposure to certain dusts and chemicals and occurrence of hematologic diseases or infections. The uncommon occurrence of this disorder suggests individual susceptibility.

Pulmonary alveolar microlithiasis. A review including ultrastructural and pulmonary function studies.

Pulmonary alveolar microlithiasis is a rare disease of unknown cause in which calcium phosphate microliths are deposited throughout the lungs. These deposits are of sufficient density to be almost diagnostic on chest roentgenograms. The Mayo Clinic experience with 8 patients is added to the approximately 120 cases reported in the world literature. The age range of all patients is from newborn to 80 years, with a mean age at diagnosis of about 35 years. No sexual predominance has been noted, but in about half of the reported cases a familial pattern has been found. The progression of the disease is generally very slow, some patients having been followed up for more than 30 years without evidence of change. No specific treatment is available. Pulmonary function studies demonstrate a tendency toward a restrictive pattern. Technetium-99m scanning and scanning and transmission electron microscopy are useful procedures for analysis of pulmonary alveolar microliths.

Analytical assessment of Broviac catheter occlusion.

Eight of 92 consecutive silastic central venous catheters used for home parenteral nutrition occluded. Six of the eight had patency restored by the instillation of urokinase or streptokinase into the catheter. The thrombus in one of the two catheters that was not reopened with thrombolytic agents was studied in detail by electron microscopy, x-ray dispersive analysis, solubility in isopropyl alcohol-diethyl ether (1:1, v:v), and thin-layer chromatography of extracted lipids. Electron microscopy found the clot to be an amorphous mass without features to suggest crystalline properties. The x-ray dispersive analysis showed that the only elements which were significantly increased were chloride and silicon and the silicon detected was likely from the underlying catheter. Treatment with isopropyl alcohol-diethyl ether left an insoluble, flaky residue that resembled protein from a thrombus. Thin-layer chromatography detected a lipid profile suggestive of circulating endogenous fat instead of the fat that was infused through the catheter.

Biological availability of nickel arsenides: toxic effects of particulate Ni5As2.

To determine if fugitive nickel arsenides from an oil shale retort could pose a threat to living organisms, we studied the effects of particulate Ni5As2 on cultured mammalian cells. Culture growth rate was greatly reduced at the lowest suspension concentration tested (10 microM), even though much of the Ni5As2 powder remained insoluble. FCM analysis indicated Ni5As2 arrested cell-cycle traverse in G1 and G2. Cell survival studies indicated cells could overcome this toxicity if exposure levels were low (less than or equal to 25 microM in suspension) and restricted to less than or equal to 24 h. At higher powder levels, survival was greatly reduced. Transmission electron microscopy (TEM) demonstrated that cells exposed to less than or equal to 100 microM powder did not phagocytize the Ni5As2 particles. At higher concentrations, TEM X-ray microanalysis demonstrated that As was preferentially extracted from the Ni5As2 particle surface and free Ni was deposited inside the cell. These observations suggest that the toxicity of Ni5As2 particles may be caused by some soluble product of Ni5As2.

Action of rotenone and related respiratory inhibitors on mammalian cell division. 1 Cell kinetics and biochemical aspects.

Inhibitors of mitochondrial respiration, phosphorylation inhibitors, and uncoupling agents have been reported to delay or inhibit mitosis in cultured mammalian cells. Although the molecular mechanism by which mitosis is delayed in the presence of most respiratory inhibitors presumably involves lowered ATP production for mitotic requirements, one respiratory inhibitor, rotenone, was determined to arrest mitosis by an unrelated mechanism. Cell cycle kinetics studies, oxygen consumption measurements, and viscosity assays indicate that rotenone arrests cultured mammalian cells in mitosis by inhibiting spindle microtubule assembly by a mechanism analogous with colchicine, Colecemid and related antimitotic drugs. Amytal, which blocks electron transport at the same site as does rotenone, failed to arrest cell progression at mitosis. Rotenone delayed cell progression in all phases of the cell cycle, apparently as a direct result of respiration inhibition. Thus, rotenone appears to exert a dual function on events of the cell cycle.

Action of rotenone and related respiratory inhibitors on mammalian cell division. 2 Ultrastructural studies.

Light and electron microscopic examination of cultured mammalian cells treated with the respiratory inhibitor rotenone revealed that chromosome, spindle, and centriole configurations were virtually identical to that of mitotic cells arrested with Colcemid, a microtubule assembly inhibitor. The chromosomes of cells arrested in mitosis with either drug were grouped in a spherical mass near the cell centre and centrioles failed to opposite mitotic poles. Spindle microtubules were observed in limited numbers near some chromosome kinetochores and the centrioles. The outer portions of the cell cytoplasm were devoid of microtubules. Scanning and transmission electron microscopy revealed that cells did not progress beyond early stages of mitosis in the presence of rotenone or Colcemid. The ultrastructure of cells harvested from cultures grown in amytal was similar to that of untreated cells. These observations suggest that rotenone arrests mitosis in mammalian cells by inhibition of spindle microtubule assembly.

Effect of chloroquine on the form and function of hepatocyte lysosomes. Morphologic modifications and physiologic alterations related to the biliary excretion of lipids and proteins.

In these experiments, we tested the hypothesis that chloroquine, a lysosomotropic agent which modifies protein and lipid metabolism by hepatocyte lysosomes, would alter the biliary excretion of lipids and lysosomal enzymes. We treated male rats for 5 days with intraperitoneal chloroquine (50 mg/kg body wt, n = 9) or saline (n = 8) and collected bile for 6 h via bile fistulas; rats were then killed and livers homogenized for biochemical analyses or processed for electron microscopy. Chloroquine markedly increased the biliary excretion of three lysosomal enzymes (mean +/- SEM) expressed as milliunits of activity per gram liver: N-acetyl-beta-glucosaminidase (24.4 +/- 2.7 vs. 12.5 +/- 1.4, p less than 0.01), beta-glucuronidase (26.4 +/- 4.7 vs. 10.9 +/- 1.4, p less than 0.01), and beta-galactosidase (9.8 +/- 1.7 vs. 5.5 +/- 0.8, p less than 0.05). In contrast, biliary outputs of enzymes associated with other organelles (e.g., alkaline phosphodiesterase I and lactic dehydrogenase) were unaffected by chloroquine treatment. Biliary cholesterol secretion was decreased after chloroquine administration (0.28 +/- 0.02 mumol/g liver vs. 0.39 +/- 0.03 mumol/g liver, p less than 0.01), but bile acid and phospholipid secretion were not altered; as a result, cholesterol saturation of bile decreased by 22% (p less than 0.05). Hepatic activities of all three lysosomal enzymes were increased after chloroquine administration (p less than 0.04); activities of enzymes associated with mitochondria, plasma membrane, endoplasmic reticulum, and cell sap were not altered. Morphometric analysis of electron micrographs of rat livers demonstrated a marked increase (p less than 0.001) in the number of lysosomelike vesicles and autophagic vacuoles in the vicinity of bile canaliculi after chloroquine administration; also, the number of canalicular microvilli decreased (p less than 0.003) after chloroquine treatment. We conclude that altered hepatic lysosomal morphology and function after chloroquine is accompanied by marked changes in outputs of lipids and lysosomal enzymes into bile. These findings call attention to a possible role for hepatic lysosomes in modulating biliary protein and lipid secretion.

Ultrastructural and biochemical studies of guinea pig seminal vesicle during postnatal development and after castration.

Final structural and biochemical probes were combined in a study of guinea pig seminal vesicle epithelium. Tissues from intact neonatal, juvenile, and adult, and castrated adult animals were examined using transmission electron microscopy. Thin sections revealed a general increase in secretory activity during normal development which steadily declined during the 90 days after castration. The ultrastructure of the epithelium from the long-term castrate differed significantly from that of the prepubertal intact animal. Protein content of the seminal vesicle epithelium and epithelial cell number decreased dramatically after castration. Other findings are presented which also indicate that the seminal vesicle epithelial cell of the juvenile animal is different from that of the long-term castrated adult.

Ultrastructural surface alterations of guinea pig seminal vesicle epithelium during postnatal development and after castration.

We have designed a topographic anatomic study using scanning electron microscopy to define surface changes in seminal vesicle epithelium during normal development and after bilateral orchiectomy. Scanning electron microscopy of seminal vesicles from 1-day, 4-week, and 3-month-old intact animals revealed a microvilli-studded luminal epithelial surface with high polyhedral cell profiles extending into the luminal area. Deep furrows outlined all intercellular junctions. Microvilli profiles ranged from short knob-like projections located preferentially along cell margins in 1-day-old animals to 1 mu-long microvilli wih random distribution in mature animals. The most striking finding was that the seminal vesicle epithelium in castrated animals did not revert to the prepubertal state. Rather, cell apices receded from the luminal surface to a level below that of the cell borders and microvilli generally remained somewhat longer in the castrate than in the prepubertal animal. These morphologic findings suggest that the seminal vesicle epithelium in the prepubertal animal and the long-term castrate are quite different.

1,25-Dihydroxyvitamin D3 rapidly alters the morphology of the duodenal mucosa of rachitic chicks: evidence for novel effects of 1,25-dihydroxyvitamin D3.

When rachitic chicks are given 1,25-dihydroxyvitamin D3 in amounts as low as 50 ng/bird, the appearance of the duodenal mucosa is altered within 2 h of the administration of the hormone. The changes are most readily apparent on scanning electron microscopy and include: a more plump appearance of villi with loss of furrows and pits on their surfaces, elongation of villi and a smoother, more uniform microvillus surface. These changes occur within 2 h of the administration of the hormone and persist for as long as 24 h. The morphological change precedes the increase in calcium absorption induced by 1,25-dihydroxyvitamin D3 in the intestine. These observations suggest that 1,25-dihydroxyvitamin D3 may play an important part in maintenance of the structure of the duodenal mucosa.

In vitro propagation of seminal vesicle epithelial cells.

The unique anatomic features of the guinea pig seminal vesicle allow establishment of nearly pure cultures of epithelial cells in vitro. Primary monolayer cultures can be maintained for up to 5 weeks and suspension cultures for 5 to 7 days. The rapidly proliferating cells in monolayer culture do not seem to be markedly sensitive to the presence of androgens or estrogens in the medium. Scanning and transmission electron microscopy indicates that the proliferating cells are epithelial in nature and are quite different from control seminal vesicle fibroblast cultures; transmission electron microscopy shows that these cells are markedly dedifferentiated vis-à-vis the native tissue in vivo. This is the first report of the successful cultivation of seminal vesicle epithelial cells in vitro.

The fine structural localization of testicular phosphatases in man: the control testis.

Electron microscopic cytochemistry was used to determine the localization of five phosphatase enzymes-glucose-6-phosphatase, inosine diphosphatase, thiamine pyrophosphatase, acid phosphatase, and adenosine triphosphatase-in control human testes. Glucose-6-phosphatase occurred in the endoplasmic reticulum and nuclear envelope of Sertoli cells, Leydig cells and primitive spermatogonia, but was not observed in more advanced spermatogenic cells. The presence of glucose-6-phosphatase activity paralleled the presence of glycogen in spermatogenic cells, i.e., both occurred in type AL and AD spermatogonia but not in type AP or B spermatogonia or in more advanced spermatogenic cells. Inosine diphosphatase activity was found in the endoplasmic reticulum, nuclear envelope, and Golgi complex of Sertoli cells and all spermatogenic cells except late spermatids. Additionally, inosine diphosphatase activity was localized at the junctions between Sertoli cells and late spermatids, but was not associated with any other plasma membrane. Thiamine pyrophosphatase reaction product was found in the Golgi bodies of Sertoli cells and in spermatogenic cells through immature spermatids. Neither inosine diphosphatase nor thiamine pyrophosphatase was observed in the Golgi bodies of spermatids during acrosomal formation. Acid phosphatase activity was found in lysosomes of spermatogonia, spermatocytes, and spermatids, in lysosomes of Leydig cells, and in lysosomes, lipofuscin bodies, and Golgi cisternae of Sertoli cells. It is thought that Sertoli lysosomes play a role in the phagocytosis of degenerating germ cells; however, the role of spermatogenic or Leydig lysosomes is unknown. Adenosine triphosphatase activity occurred at the interfaces between two spermatogonia, and between Sertoli cells and spermatogonia, but was not observed in the spaces between two Sertoli cells, two spermatocytes, two spermatids, or between Sertoli cells and spermatocytes, or between Sertoli cells and spermatids.

Clonal variation of cadmium response in human tumor cell lines.

Subpopulations of human tumor-derived cell lines A101D, A204, and A549 were screened for Cd2+ cytotoxic response. Three of six A549, two of seven A101D, and four of seven A204 subpopulations were found to differ significantly from the parental line. A variant subpopulation of A101D (T3) was shown by flow cytometry to be comprised of cells having two distinct DNA histograms. One histogram, type 1, resembles that of normal human fibroblasts. The other, type 2, represents cells with one-third more DNA. Early passage T3 clonal populations were comprised primarily of type 1 cells. With passage, type 1 cells decreased relative to type 2 so that by passage 47 the culture was predominantly type 2. Correspondingly, the A101D T3 subpopulation became more Cd2+ sensitive with time in culture. Subclones having only type 1 DNA histograms were found to be Cd2+ resistant relative to subclones with type 2 histograms, and treatment of A101D T3 cultures having approximately equal amounts of type 1 and 2 cells with 2 microM Cd2+ resulted in the selection of type 1 cells. The enhanced Cd2+ resistance phenotype shown by A101D T3 type 1 cells correlated with reduced Cd2+ uptake and is not attributable to enhanced metallothionein synthesis.

Microtubule modulation of biliary excretion of endogenous and exogenous hepatic lysosomal constituents.

We tested the hypothesis that hepatocyte microtubules modulate the biliary excretion of endogenous and exogenous constituents of hepatocyte lysosomes. We collected bile via bile fistulas from male rats before and after acute administration of colchicine and vinblastine, agents known to bind to hepatocyte microtubules; rats were then killed and livers were homogenized for biochemical analyses or processed for electron microscopy. Colchicine caused biphasic, parallel alterations in the biliary excretion of three lysosomal enzymes compared with control rats given saline or lumicolchicine; a peak rise in enzyme outputs of approximately 175% at 45-60 min after colchicine administration was followed by a sustained fall to approximately 25% of control values, which persisted for 2-4 h. When hepatocyte lysosomes were prelabeled in vivo by administration of [3H]Triton WR-1339, a nonionic detergent that is sequestered in hepatic lysosomes, the biliary excretion of radiolabel in response to colchicine paralleled the biliary excretion of the three lysosomal enzymes. Vinblastine also induced a biphasic response in biliary lysosomal enzyme output that was similar to that produced by colchicine administration. Morphometric analysis of electron micrographs of rat livers demonstrated changes in the number of lysosomelike vesicles in the vicinity of bile canaliculi after colchicine and vinblastine administration; the initial increase in lysosomal enzyme secretion was associated with a significant decrease in the number of pericanalicular lysosomes after both agents, while the subsequent decrease in enzyme secretion coincided with an increase in the number of pericanalicular lysosomes after vinblastine.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear protein matrix of seminal vesicle epithelium.

Nuclear protein matrix was isolated from guinea pig seminal vesicle epithelium and liver. The two matrices were similar in fine structure as seen by transmission electron microscopy, in protein electropherograms, and in percent composition relative to protein, DNA, and RNA. Scanning electron microscopy was used to examine intact seminal vesicle nuclei, nuclei after treatment with Triton X-100 and DNAse I, and purified nuclear matrix. The matrix surface presented a 'porous' appearance by both scanning and transmission electron microscopy. The matrices of liver and seminal vesicle epithelium (SVE) and the intact nuclei of SVE were assayed for specific binding of free synthetic androgen, 17 alpha-methyltrienolone (R1881). Saturable specific binding was demonstrable for seminal vesicle matrix but not for liver matrix. Maximal binding of androgen occurred at a concentration of approximately 12 nM and was demonstrated to be 1.34 +/- 0.22 pmol of R1881 per mg of seminal vesicle matrix protein; the Kd was approximately 8 nM. The binding of labeled R1881 to matrix could be inhibited with low concentrations of unlabeled androgens, but not with estrogens or other steroids. Our data indicate that the binding of androgen to matrix could account for at least 21% of the binding to intact nuclei.

Accumulation of radionuclide-labeled platelets and fibrinogen in paraquat-damaged rat lungs.

After intraperitoneal injection of paraquat, rats showed evidence of neurologic and respiratory damage and had a mortality rate of 41% in 3 days. The lungs quadrupled in weight between the third and the fifth day. Pulmonary edema and extravascular fibrin and platelets were identified by light and transmission electron microscopy. As early as 4 h after injection of the paraquat, 51Cr from labeled platelets began accumulating in the lungs. The peak was reached by 48 h; 125I from labeled fibrinogen also concentrated in the lungs of treated rats. Total complement was unchanged. The paraquat-treated rat is a suitable model for study of the behavior of fibrin and platelets in permeability pulmonary edema. Disturbances of copper metabolism deserve further investigation.

Triton WR-1339, a lysosomotropic compound, is excreted into bile and alters the biliary excretion of lysosomal enzymes and lipids.

In these experiments, we tested two hypothesis: first, that Triton WR-1339, a nonionic detergent which is sequestered in hepatocyte lysosomes, undergoes biliary excretion; and second, that Triton WR-1339, which also alters serum lipid levels and modifies hepatic catabolism of lipoproteins, affects the biliary output of proteins and lipids. When 3H-Triton WR-1339 was administered to rats, biochemical and morphologic studies showed that hepatocyte lysosomes sequestered Triton WR-1339: (i) the subcellular distribution of 3H was identical to that of lysosomal enzymes after liver fractionation by differential or isopycnic centrifugation, and (ii) lysosomes appeared engorged with Triton WR-1339 on electron microscopy. 3H was also excreted into bile in parallel to three lysosomal enzymes. Triton WR-1339 administration caused a coordinate increase in the biliary excretion of three lysosomal enzymes and also increased the biliary output of total protein, bile acids, and phospholipid. Triton WR-1339 administration did not affect bile flow or the biliary outputs of cholesterol, plasma membrane, and cytosolic enzymes, but did decrease biliary cholesterol saturation by 50%. These results demonstrate that an exogenous compound which is sequestered in hepatocyte lysosomes may be excreted directly into bile in parallel with endogenous lysosomal constituents. The data also show that such a lysosomotropic agent may also selectively modify the biliary excretion of proteins and lipids. The findings are consistent with the existence of a lysosome-to-bile hepatic excretory pathway and suggest that hepatocyte lysosomes may be important in modulating biliary protein and lipid secretion.

Morphologic features of chronic hepatitis associated with primary sclerosing cholangitis and chronic ulcerative colitis.

Histologic, ultrastructural, chemical, and statistical methods were used to study liver biopsy and autopsy specimens from 43 patients who had primary sclerosing cholangitis (PSC), with or without chronic ulcerative colitis (CUC), and from 19 patients who had CUC without PSC. In all study groups, essentially the same abnormalities were found in the hepatic parenchyma outside the major bile ducts, although nondiagnostic tissue samples were observed also. Specimens from patients with extrahepatic PSC were indistinguishable from those patients with combined extra- and intrahepatic PSC. Common findings included periductal fibrosis and inflammation, portal edema and fibrosis, focal proliferation of bile ducts and ductules, focal bile duct obliteration and loss of bile ducts, copper deposition, and cholestasis. Proliferation of bile ducts in some portal tracts and obliteration or absence of bile duct in others were the most characteristic changes. In most specimens, inflammatory changes appeared mild, yet biliary cirrhosis had developed in 34% of the patients. Specimens from patients with PSC, with or without CUC, more often contained bile and strikingly increased stainable copper (Grades 2 and 3) than did specimens from patients with CUC without PSC. Hepatic copper contents, measured by atomic absorption spectrophotometry, also were higher in specimens from patients with PSC. Study of PCS specimens by transmission electron microscopy and by energy-dispersive X-ray microanalysis revealed that most copper was sequestered in lipolysosomes. The recognition of strikingly similar morphologic features in many liver specimens from patients with either PSC or CUC or both suggests that the causes of these conditions are closely related.