Lamellar Inclusion Bodies Research Articles

Use of physicochemical calculation of pKa and CLogP to predict phospholipidosis-inducing potentialA case study with structurally related piperazines

Several cationic amphiphilic compounds are known to induce phospholipidosis, a condition primarily characterized by excessive accumulation of phospholipids in different cell types, giving the affected cells a finely foamy appearance. Excessive storage of lamellar membranous intralysosomal inclusion bodies is the hallmark for phospholipidosis on the electron microscopic level. In case of alveolar phospholipidosis, foamy macrophages accumulate within the alveolar spaces of the lung. Based on such findings in a one-year toxicity study with gepirone in rats, we studied the molecular properties of this compound and compounds suspected of being phospholipidosis inducers by means of physicochemical calculations. Physicochemical molecular calculations of molecular weight, ClogP (partition coefficient octanol/water), logD at pH 7.4, and pKa were performed, for the cationic amphiphilic compounds chlorpromazine, amiodarone, imipramine, propranolol and fluoxetine, and for the structurally related compounds 1-phenylpiperazine (1-PHP), gepirone (and its major metabolites, 3-OH-gepirone and 1-pyrimidinylpiperazine [1-PP]), and buspirone. ClogP and calculated pKa cluster differently for the amphiphilic drugs compared to the chemical series of piperazines. In line with this analysis, lamellar inclusion bodies were found in an in vitro validation experiment in the human monoblastoid cell line U- 937, incubated for 96 h at 10 μg/mL with cationic amphiphilic drugs (amiodarone, imipramine, or propranolol). No such lamellar inclusion bodies were seen for any of the compounds from the chemical series of piperazines including gepirone and its metabolites. The data presented support the use of simple physicochemical calculations of ClogP and pKa to discriminate rapidly between compounds suspected of being phospholipidosis inducers. Finally, the discriminative power of these physicochemical ClogP and pKa calculations to predict phospholipidosis-inducing potential was further validated by extension of the set of compounds.

Recent advances in drug-induced neuropathies.

Peripheral neuropathy is a common neurotoxic effect of medications. When medications are used to treat life-threatening illnesses, balancing the toxic effects of peripheral neuropathy with the therapeutic benefits of the drug can be difficult. This article examines recent research into the cellular mechanisms associated with neuropathy after treatment with medications to treat cancer, and HIV, and to prevent transplant rejection. Cisplatin and suramin induce a length, dose, and time-dependent axonal sensorimotor polyneuropathy. Cisplatin and suramin both result in apoptosis in dorsal root ganglion neurons that may partially explain the neuropathy that develops with treatment. In contrast, nerve growth factor prevents initiation of the programmed cell death associated with cisplatin neurotoxicity. Suramin causes accumulation of lamellar inclusion bodies in dorsal root ganglion neurons related to dose of administration and severity of the neuropathy. Nucleoside reverse transcriptase inhibitors affect mitochondrial function and lead to depletion of the nerve's mitochondrial DNA and inhibition of DNA polymerase. These effects on the mitochondrion may be related to the polyneuropathy that develops in these patients. In contrast to the axonal neuropathies, tacrolimus and rarely suramin can result in a demyelinating neuropathy that may mimic Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy. Many of these neuropathies can be reversed by early recognition of the symptoms or by using sensitive electrophysiological testing. In certain instances, specific therapies may ameliorate the neuropathy. Glutamine may reduce paclitaxel-induced toxicity, while some patients with tacrolimus or suramin-induced demyelinating neuropathy may respond to intravenous immunoglobulin or plasmapheresis. Improved understanding of neurotoxic mechanisms in the peripheral nervous system associated with chemotherapeutic and anti-HIV medications, coupled with early improved diagnosis, promises to help limit neurotoxicity associated with these medications.

Time-dependent effects of maternal continuous propranolol on fetal lung development in rats.

Previous studies have demonstrated a role for the beta-adrenergic system in the maturation of the fetal alveolar epithelium. Chronic blockade of beta-adrenergic binding sites has been shown to adversely effect physiologic and biochemical indices of fetal lung maturation. In the present study timed-pregnant female Sprague-Dawley rats were treated with a continuous 0.5 mg/hr dose of propranolol HCl, or saline, via an osmotic pump. The treatment periods were days 18-21, or 20-23 of gestation. Fetal body weights were obtained, and the morphology of the fetal lungs studied by light and electron microscopy. Cytoplasmic volume densities of lamellar inclusion bodies and glycogen within developing type II alveolar epithelial cells were also determined. In addition, total phospholipids (as phosphorus) and glycogen content were determined biochemically. The fetuses from females treated from day 20-23 demonstrated no differences between saline-treated and propranolol-treated groups, in either fetal weight or the morphologic appearance of the developing lung. In contrast, the fetuses from mothers treated from day 18-21 with propranolol were significantly smaller, and their lungs appeared less mature than saline-treated counterparts. The glycogen content of developing type II alveolar epithelial cells was significantly more abundant (as judged by stereologic and biochemical analyses) in the propranolol-treated fetuses. In addition, total phospholipids were decreased in the propranolol-treated 21-day fetuses. The results of the present study suggest that the development of the alveolar epithelium is sensitive to continuous beta-adrenergic blockade by propranolol during a critical time late in gestation.

Spinosad Insecticide: Subchronic and Chronic Toxicity and Lack of Carcinogenicity in CD-1 Mice

The potential toxicologic and oncogenic effects of spinosad, a natural fermentation product with insecticidal properties, were investigated. The 13-week toxicity study consisted of groups of 10 CD-1 mice/sex provided diets containing 0, 0.005, 0.015, 0.045, or 0.12% spinosad (Study 1). The 0.12% group was terminated on Test Day 44 due to mortality and overt clinical signs of toxicity. An 18-month chronic oncogenicity study consisted of groups of 50 CD-1 mice/sex provided diets containing 0, 0.0025, 0.008, or 0.036% spinosad (Study 2). Two interim groups of 10 mice/sex/group were terminated after 3 and 12 months. Females given 0.036% were terminated on Day 455 due to markedly lower body weights and feed consumption, as well as excessive mortality. Because of the early termination of the female high-dose group, additional groups of 10 male and female mice (12-month interim necrospy) and 50 male and female mice (18-month necropsy) were provided diets containing 0, 0.0008, or 0.024% spinosad (Study 3) to fully assess potential chronic toxicity and oncogenicity. Standard toxicologic parameters were evaluated consistent with existing regulatory guidelines. The primary effect in the 13-week and 18-month studies was intracellular vacuolation of histiocytic and epithelial cells in numerous tissues and organs at doses of > or = 0.015%. The histological vacuolation corresponded to ultrastructural lysosomal lamellar inclusion bodies. This alteration was consistent with phospholipidosis, a condition that results from accumulation of polar lipids in lysosomes. Lesions with no apparent direct relation to vacuolation were hyperplasia of the glandular mucosa of the stomach, skeletal muscle myopathy, bone marrow necrosis, and anemia with associated splenic hematopoiesis. The incidence of tumors in mice given spinosad was not increased relative to controls at any dose level. The no observed effect level for the 13-week study was 0.005% (6 mg/kg/day) spinosad, and for the chronic toxicity/oncogenicity study was 0.008% (11 mg/kg/day) spinosad for male and female CD-1 mice.

Surfactant-like Materials(SF-phospholipids) in Fallopian Tubal Secretions and Their Physiologic Role in Reproduction.

The physiologic roles of the tubal endothelium and tubal secretions are as yet poorly understood. In this study, we examined the tubal surfactant-like materials (SF-phospholipids) found in the tubal endothelium and lumen in order to elucidate their roles in the maintenance and development of the zygote. Evaluation of tubal infertility based on the presence or absence of SF-phospholipids in human tubal endothelium and tubal secretions was carried out in order to determine the degree to which tubal function was intact. By using fluorescing phosphin E, we found fluorescence in both the endothelium and the tubal secretions throughout the entire length of the tubes. In electron microscopic observations of the tubal endothelium, we were able to identify high electron density, lamellar inclusion bodies, either scattered or clustered in the areas close to the tubal lumen, in both ciliated and secretory endothelial cells. These lamellar granules in the tubal secretions appear to be secreted by a merocrine process and are observed in the secretions as a dispersion. Tubes from the proliferatory phase and the secretory phase were evaluated with the following results. The constituents of the tubal secretions were found to be phospholipids and proteins. The phospholipids were most abundant at or around the time of ovulation. This material caused capacitation of sperm by inducing an acrosomal reaction in the hamster sperm penetration test.

Surfactant protein A and surfactant protein D in health and disease

Surfactant protein (SP) A and SP-D are collagenous glycoproteins with multiple functions in the lung. Both of these proteins are calcium-dependent lectins and are structurally similar to mannose-binding protein and bovine conglutinin. Both form polyvalent multimeric structures for interactions with pathogens, cells, or other molecules. SP-A is an integral part of the surfactant system, binds phospholipids avidly, and is found in lamellar bodies and tubular myelin. Initially, most research interest focused on its role in surfactant homeostasis. Recently, more attention has been placed on the role of SP-A as a host defense molecule and its interactions with pathogens and phagocytic cells. SP-D is much less involved with the surfactant system. SP-D appears to be primarily a host defense molecule that binds surfactant phospholipids poorly and is not found in lamellar inclusion bodies or tubular myelin. Both SP-A and SP-D bind a wide spectrum of pathogens including viruses, bacteria, fungi, and pneumocystis. In addition, both molecules have been measured in the systemic circulation by immunologic methods and may be useful biomarkers of disease. The current challenges are characterization of the three-dimensional crystal structure of SP-A and SP-D, molecular cloning of their receptors, and determination of their precise physiological functions in vivo.

Antioxidant activity in alveolar epithelial type 2 cells of rats during the development of bleomycin injury.

Bleomycin (BLM) induces lung inflammation and subsequent fibrosis in human and in animal models. Alveolar epithelial type 2 cells (T2 cells) are known to play a crucial role in the repair process after BLM injury. We hypothesized that resistance of T2 cells to BLM-damage was associated with an increase in their antioxidant system activity. We developed an animal model of lung lesions preceding fibrosis, using daily intraperitoneal administration of BLM (1.5 mg/day over 7 and 14 days). We observed a body weight stabilization in BLM-treated rats from the third day. After 14 days of BLM treatment, the number of cells recovered by bronchoalveolar lavage was significantly increased (p < 0.05), with a dramatic increase (p < 0.01) in the percentage of neutrophils associated with a decrease in macrophage percentage (p < 0.01) No evidence of fibrosis was seen by microscopic studies at this time. However, T2 cells in 14-day-treated rats were swollen with enlarged lamellar inclusion bodies. Biochemical study of freshly isolated T2 cells displayed a significant decrease of lactate dehydrogenase (LDH) released by these cells when isolated from 14-day-treated rats as compared with 7-day. By contrast, BLM induced an increase in superoxide dismutase (SOD) and glutathione peroxidase activities. Cell content of glutathione was decreased and gamma-glutamyl transpeptidase activity was markedly increased. These results show that BLM induces changes in the antioxidant system of T2 cells, particularly in the glutathione system.

Effects of almitrine detriazinyl metabolite upon lysosomal alterations on rat cultured macrophages.

Phospholipidosis-inducing effects of difluorobenzhydrylpiperadine (DFBP) on cultured rat per-itoneal macrophages were studied as compared with those of several amphiphilic drugs. At 24 hr of exposure to DFBP as well as other amphiphilic drugs, intracytoplasmic acid hematein-stained inclusion bodies were dose-dependently produced in macrophages. Electron microscopy showed lamellar inclusion bodies in macrophages exposed to DFBP. Effective concentrations for 30% positivity of treated cells were: 0.43μM/diethylamino-ethoxyhexestrol, 1.9 μM/perhexiline, 2.8μM/quinacrine, 4.3μM/DFBP, 11μM/chlorcyclizine, and 14 μM/ propranolol. Neither almitrine nor aspirin induced the cytoplasmic inclusion bodies.

Induction of Alveolar Type II Cell Differentiation in Fetal Tracheal Epithelium by Grafted Distal Lung Mesenchyme

Normal branching morphogenesis and cytodifferentiation of the lung require a specific interaction between the epithelial rudiment and pulmonary mesenchyme. Previous studies in the mouse have shown that glandular stage pulmonary mesenchyme can elicit a lung-like pattern of branching morphogenesis when grafted onto tracheal epithelium of the same age that has been denuded of its own mesenchyme. We have examined whether this pattern of branching is accompanied by changes in epithelial cytodifferentiation. Purified pulmonary mesenchyme was isolated from the distal tips of Day 13-14 fetal rat lungs and grafted onto a stretch of tracheal epithelium from which the mesenchyme had been removed. The grafts were cultured on semisolid 0.5% agarose in Waymouth's medium containing 10% serum for 5 days and then for an additional 2 days in the same medium containing 10-6M cortisol. Unoperated or operated-ungrafted tracheae and intact lung explants served as controls. Grafting distal tip pulmonary mesenchyme onto the tracheal epithelium induced a pattern of branching identical to that seen in control lung explant cultures, while ungrafted control tracheal cultures formed cystic structures. Light microscopy of the induced tracheal epithelium showed that the constituent cells exhibited a morphology virtually identical to cells in control lung explants. Ultrastructural analysis demonstrated that the induced tracheal epithelial cells contained lamellar inclusion bodies, and the lumina of the induced acinar structures contained tubular myelin figures. Reverse transcription polymerase chain reaction analysis of the induced tracheae revealed the presence of mRNAs for surfactant protein A (SP-A) and SP-B, as well as SP-C, which is a specific marker for type II cell differentiation in the adult rat. Control tracheal cultures contained mRNAs only for SP-A and SP-B. Immunocytochemistry demonstrated that many cells within the induced tracheal epithelium were positive for SP-A and SP-C proteins. Further studies demonstrated that the ability of mesenchyme to induce tracheal epithelium varied within the pulmonary tree, and that the competence of the tracheal epithelium to respond to the influence of distal tip mesenchyme was temporally restricted. Our results demonstrate that pulmonary mesenchyme can act instructively on the tracheal epithelium to induce a program of type II cell differentiation.

Cytochrome-P450 2B1 Immunoreactivity in Bronchiolar and Alveolar Epithelial Cells after Exposure of Rats to Ozone

Pulmonary cytochrome P450 (P450) plays an important role in the metabolism of xenobiotics. We have previously demonstrated that exposure to ozone (O3), a major oxidant of photo-chemical smog, increases P450 content and xenobiotic metabolizing activities in the rat lung. Therefore, it is conceivable that O3 exposures modify the pattern and potency of xenobiotic metabolism. The aim of this study is to examine the effect of O3 exposure on localization of P450 in the lung. After exposure of rats to 0.4 ppm O3 for 14 days, the concentration of P450 2B1, the main isozyme of P450 in the lung, increased to 214% of the control value. By immunohistochemical analyses, O3 exposure caused hypertrophy and hyperplasia of P450 2B1-positive nonciliated bronchiolar epithelial (Clara) cells, showing an increase in the cell number to 128% of the control level. At the proximal alveoli, O3 exposure induced hypertrophic epithelial cells that presented immunoreactivity to P450 2B1 the morphological characteristics of both type 1 and type 2 cells. These hypertrophic cells had lamellar inclusion bodies, microvilli, and extended cytoplasm. Typical type 2 cells did not react with anti-P450 2B1 antibody even after O3 exposure. These data suggest that O3 exposure may activate the P450-mediated xenobiotic metabolism in the pulmonary epithelial cells, and that expression of P450 2B1 in the pulmonary epithelial cells might be regulated by the factors involved in the process of the differentiation from type 2 to type 1 cells.

Sclerosing hemangioma of the lung: Pathological study and enzyme immunoassay for estrogen and progesterone receptors

Sclerosing hemangioma of the lung is a rare benign neoplasm of uncertain histogenesis. In this study, thirteen cases of sclerosing hemangiomas were analyzed by immunohistochemistry for various histochemical markers including surfactant apoprotein. The pale cells were shown to share several immunohistochemical markers with epithelial lining cells and were suggested to be immature cells differentiating toward epithelial lining cells. Electron microscopic study failed to characterize the exact nature of pale cells, but it disclosed the precise structure of large lamellar bodies, which are frequently present in the cystic spaces of sclerosing hemangioma. These large lamellar bodies had common morphological characteristics to lamellar inclusion bodies of type II pneumocytes or epithelial lining cells. Eight cases of sclerosing hemangioma obtained freshly at surgery had a quantitative analysis of estrogen and progesterone receptors performed by enzyme immunoassay. The value of estrogen receptors ranged from 0 to 7.8 fmol/mg protein. In addition to this quantitative analysis, the immunohistochemical study demonstrated estrogen receptors mainly in the pale cell nuclei of several sclerosing hemangiomas. Estrogen receptors may play an important role in growth, differentiation and surfactant production in the sclerosing hemangioma.

Effects of anti-mouse EGF antiserum on prenatal lung development in fetal mice.

To clarify the precise role of epidermal growth factor (EGF) on fetal lung development, rabbit anti-mouse EGF (anti-mEGF) antiserum was administered to pregnant mice from days 10 to 17 during late gestation. Control mice were administered either normal rabbit serum (NRS) or physiological saline (PS). Serum EGF was not detected in fetuses from anti-mEGF antiserum treated mothers, but the level in NRS treated control animals was 4.73 +/- 0.66 ng/mL. One day prior to birth, the fetuses were removed and their body and lung weights were measured. There was no difference between body weights and lung weights of anti-mEGF antiserum treated animals and NRS-treated control animals. On light microscopic morphometry, there was no obvious difference between pulmonary architecture of anti-mEGF antiserum treated animals and NRS treated control animals. On transmission electron microscopy, osmiophillic lamellar inclusion bodies were less prominent in the type II epithelial cells in anti-mEGF antiserum treated animals. Electron microscopic morphometric study revealed that the osmiophillic lamellar inclusion bodies in type II epithelial cells of anti-mEGF antiserum treated animals were fewer in number and had decreased area fraction. These findings support the previous finding that EGF promotes epithelial cell differentiation of the fetal lung without affecting body weight and lung weight.

Lung adenocarcinoma with type II pneumocyte characteristics

We report a case of primary lung adenocarcinoma with type II pneumocyte characteristics. Electron microscopic examination demonstrated that the tumour cells had well-developed microvilli and cytoplasmic lamellar inclusion bodies. These ultrastructural features are similar to those seen in type II pneumocytes of normal lung tissue. Western blot analysis, using monoclonal antibody against human surfactant protein A (SP-A), clearly demonstrated that the tumour cells expressed human SP-A, which is a major pulmonary surfactant protein produced by type II pneumocytes. These observations suggest that the tumour was derived from a type II pneumocyte.

Uptake and intracellular transport of cationic ferritin in the bronchiolar and alveolar epithelia of the rat

Cationic ferritin was used as a marker to reveal the processes of endocytosis and intracellular transport in bronchiolar and alveolar epithelia. The marker was injected into the lung via the trachea, and ultrastructural observation of the distribution of ferritin particles in bronchiolar and alveolar epithelial cells was carried out at intervals of 5, 15, 30 and 60 min after the injection. The luminal surface of the airway and the alveolar epithelium showed diffuse labeling with cationic ferritin. In general, ferritin particles were observed in vesicles and vacuoles of the bronchiolar and alveolar epithelial cells within 5 min of injection; they appeared in multivesicular bodies within 15 min. Multivesicular bodies and secondary lysosomes containing ferritin particles, some of which showed a positive reaction for acid phosphatase, were seen in the basal cytoplasm within 30 min; ferritin particles appeared in the basal lamina below the Clara cells, ciliated cells and type 2 alveolar cells within 30 min. Ferritin particles were seen in ovoid granules of some Clara cells and in lamellar inclusion bodies of many type 2 alveolar cells. Brush cells and type 1 alveolar cells took up only a small quantity of ferritin particles.

Electron-microscopic observations on rat lungs after long term inhalation of diesel emissions--non-neoplastic lesions

Non-neoplastic lesions in the respiratory organs of rats (F344) were investigated after exposure to heavy and light duty diesel emissions for 30 months. Pathological examinations of the lungs after every 6 months of exposure were performed by light and electron microscopy. Anthracosis caused by the inhaled diesel soot was the main morphological change observed in the lung. Foci of alveoli filled with alveolar macrophages engorged with diesel particles were scattered in the lung parenchyma. Marked changes observed in these areas consisted of intake of diesel particles by type 1 epithelium, hypertrophy and glandular proliferation of type 2 epithelium with many extended microvilli and increased number and size of lamellar inclusion bodies, focal increase of collagen fibers in the interstitium, and infiltration of particle-laden macrophages, neutrophils, mast cells and plasma cells into the interstitium of alveolar septa. Within the alveolar lumina there was marked accumulation of particle-laden macrophages or neutrophils and their debris, as well as laminated or scrolled materials discharged from the type 2 epithelium. The most marked changes observed in the airways were focal shortening of cilia and protrusions of non-ciliated cells, which were attributable mainly to the effects of gaseous components in the diesel exhaust. These morphological changes appeared in all the groups exposed to particle concentrations greater than 1 mg/m3 after 6 months' exposure, and were more marked with increase in particle concentration and with time.

Culture of fetal alveolar epithelial type II cells in serum-free medium

A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle's minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistence of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.

Kinetics of biochemical, electrophysiological and morphological events (including lysosomal disorder) during the course of suramin-induced differentiation of the human colon-cancer cell clone HT29-D4

Suramin, a polysulfonated naphtylurea, has been shown to bind to a wide variety of tumor growth factors, and to exhibit anti-proliferative effects on several cell lines. We have followed the suramin-induced (100 micrograms/ml) evolution of morphological, biochemical and electrophysiological changes in HT29-D4 human colonic adenocarcinoma cell clone as a function of culture time. After 5 days of culture in the presence of the drug the cells were polarized and exhibited apical brush border and tight junctions. The polarization process of carcino-embryonic antigen (CEA) in the apical membrane domain was achieved after 8 days of treatment, while the correct localization of HLA class-I molecules in the basolateral membrane domain occurred after 14 days of culture in the presence of suramin. Spontaneous potential difference (PD) and transepithelial resistance (Rt) were recorded from the 9th day of treatment and reached maximum values at day 15 (PD = 3 mV; RT = 450 omega cm2), giving evidence that the differentiation process triggered by suramin concerned virtually all cells in the monolayer. Untreated cells were consistently found to be electrically inactive. Finally, from day 10 of suramin treatment, the lysosomal system was perturbed including accumulation of large autophagic vacuoles and, later, typical lamellar inclusion bodies. These structures were never seen when cells were induced to differentiate in suramin-containing serum-free medium. Moreover, similar perturbations of the lysosomal system could be obtained by adding BSA in the suramin-containing defined medium, suggesting that the lysosomal storage disorder occurring upon suramin treatment was due to endocytosis of suramin-BSA complexes. We conclude that lysosomal impairment due to the presence of BSA in the culture medium did not prevent HT29-D4 cells from differentiating and that it was not an early event which could be involved in the mechanism of action of suramin. However, this perturbation might account for some of the toxic effects occurring during chronic suramin treatment in humans.

Dose-dependent enhancing effects of quinacrine on induction of preneoplastic glutathione S-transferase placental form positive liver cell foci in male F344 rats

Dose-dependent modifying effects of quinacrine on induction of preneoplastic liver cell foci were investigated in male F344 rats. Six week old animals were injected i.p. with N-nitrosodiethylamine (DEN) at a dose of 200 mg/kg, and starting 2 weeks later, rats were given quinacrine at dietary levels of 20, 100 and 500 p.p.m. for 6 weeks. Groups without either DEN or quinacrine treatment were used as controls. At week 3 following DEN administration, all animals were subjected to two-thirds partial hepatectomy, and after killing the animals at week 8, development of preneoplastic liver cell foci was investigated using the glutathione S-transferase placental form (GST-P) as a marker. The numbers and unit areas of GST-P-positive foci per cm2 were significantly increased in the DEN/quinacrine (500 p.p.m.) group as compared to DEN-alone group values. An increase in number was also evident in the 100 p.p.m. but not the 20 p.p.m. treated group, no lesions being induced by quinacrine alone (500 p.p.m.). Electron microscopic study confirmed that quinacrine dose-dependently induces lipidosis in hepatocytes, i.e. markedly myeloid lamellar cytoplasmic inclusion bodies were observed. The results thus demonstrated that quinacrine treatment enhances GST-P-positive liver cell foci development in a dose-dependent way, this effect presumably being related to the induction of lipidosis.

Suramin-induced changes in sympathetic neurons: Correlation between catecholamine fluorescence, tyrosine hydroxylase immunoreactivity and accumulation of pigment bodies

The effect of suramin, a strong inhibitor of lysosomal enzymes, was studied in rat superior cervical (SCG) and dorsal root ganglia (DRG). Ten days after administration, both types of ganglion showed increased intraneuronal pigment fluorescence; in DRG there were also accumulated pigments in Schwann cells. Ultrastructurally the pigment bodies were membranous or lamellar inclusion bodies. In SCG suramin-induced inclusion bodies were associated with decreased catecholamine fluorescence and tyrosine hydroxylase immunoreactivity, suggesting that altered activity of lysosomal enzymes and the formation of lysosomal inclusion bodies may be coupled with decreased synthesis of neurotransmitters in rat sympathetic neurons.

Histopathologic analysis of suspected amiodarone hepatotoxicity

This analysis of the morphology of suspected amiodarone (AD) liver disease is based on a study of liver specimens from 17 individuals. Changes similar to alcoholic liver injury were commonly seen. Steatosis, both macrovesicular and microvesicular, was the most frequent histopathologic feature. Ballooming of hepatocytes, Mallory bodies, and fibrosis were also common. Other changes included nuclear unrest, acidophilic bodies, foam cells, glycogenated nuclei, and portal inflammation. Characteristic lamellar lysosomal inclusion bodies representing phospholipidosis were found in two of 14 specimens studied ultrastructurally. These changes of pseudoalcoholic hepatitis and/or phospholipidosis were present in liver specimens from asymptomatic, anicteric patients with mild elevations in serum aminotransferase or alkaline phosphatase values with or without hepatomegaly, as well as in patients with clinically overt symptoms of hepatotoxicity. Phospholipidosis appears to be a generalized systemic effect of cationic amphophilic compounds, such as AD. The cytotoxic pseudoalcoholic changes appear to be an independent phenomenon in susceptible patients, whom we speculate may have been unable or less able to metabolize AD through normal pathways. The true incidence of hepatic injury from AD remains to be determined from prospective evaluations of pretreatment and follow-up liver biopsies.