Styrene hepatotoxicity in rats treated by inhalation or intraperitoneally: a structural investigation.

The purpose of this study was to investigate the toxicity of styrene in the liver of adult rats treated either by inhalation of styrene vapour (300 ppm, 6 h/d, 5 d/wk, for 2 wk) or intraperitoneally with different styrene doses (4, 40, 400 mg/Kg) for 3 consecutive days. Using a light microscope, some alterations of liver parenchyma and sinusoid dilation were noticed, more marked in the group treated with the intraperitoneal administration of the chemical. Using an electron microscope, some additional changes were observed (once again, more marked in the latter group of rats): a) an increase in the content of lipids inside hepatocytes, and b) the rise of intracytoplasmic, intercellular and perisinusoidal collagen fibres. Therefore, cell damage and functional disturbance of sinusoids due to perisinusoidal fibrosis are apparent in the liver of both groups of rats exposed to styrene treatment, but these changes are definitely more significant in those subjected to intraperitoneal administration.

Evaluation of a possible styrene-induced damage to the haematopoietic tissues in the rat.

The aim of this study was to determine a possible risk of malignancies in the haematopoietic tissues of rats treated with styrene, either by injection or by inhalation. Two experiments were carried out: in the first (acute treatment), 12 male rats were treated intraperitoneally with different doses of styrene (40 mg/Kg/2 ml/die and 400 mg/Kg/2 ml/die) for 3 consecutive days. A control group (6 rats) was administered corn oil 2 ml/kg/die for the same period of time. In the second experiment (chronic treatment), 12 male rats were exposed to styrene inhalation (300 ppm/6 hour day/5 days a week for 2 weeks) for 3 weeks and sacrificed at the end of the experiment or after 3 weeks. The rats treated with the highest doses of styrene by injection (400 mg/kg, acute treatment) showed a hyperactivity of the erythropoietic series while the granulocytopoietic series was at the normal value. The rats exposed to 300 ppm styrene vapour (chronic treatment) showed a statistically significant increase in erythropoietic cells (basophilic, polychromatophilic and orthochromatic erythroblasts). A temporary block of immature cells of the granulocytopoietic series was also evident. These results indicate an alteration of the erythropoietic series independently of method of administration. However, these findings do not show a possible risk of preleukemic or leukemic disorders in rats exposed or treated with styrene.

Establishment and characterization of two cell lines derived from human glioblastoma multiforme.

We established and characterized two cell lines derived from glioblastoma multiforme. Both cell lines exhibited tumor cell morphology and growth kinetics and showed variable expression of glial fibrillary acidic protein (GFAP), S-100, fibronectin and vimentin. Cytofluorimetrical analysis of tumor samples showed a diploid DNA distribution, whereas permanent culture cells evolved to the hyperdiploid DNA content. Karyotype studies revealed cytogenetical abnormalities described in glial tumors including gain of chromosome 7, loss of chromosome 10 and presence of double minutes (DMs). Enhanced expression of Ha-ras and c-myc genes resulted from high p-21 and p-62 levels. The contemporary presence of TGF-alpha and EGF-Rc transcripts suggested an autocrine mechanism in the cell lines growth.

Leucine aminopeptidase activity in blood and hemopoietic organs of different vertebrates. A histochemical comparative study.

Leucine aminopeptidase distribution was examined in the cells of peripheral blood and hemopioetic organs of different Vertebrate classes using histoenzymatic methods. Various degrees of staining intensity were observed in leukocytes of the distinct Vertebrates: in particular, leukocytes of fresh water Osteichthyes and Mammals looked strongly positive, whereas leukocytes of Amphibians, Reptiles, and Birds were barely reactive without great differences among species. The observations should be related to enzyme molecular structure and to kinetics and substrate specificity.

Tetrahydrofolate dehydrogenase in blood cells and in hematopoietic organs of different vertebrate species.

Some general aspects in cytochemical demonstration of the tetrahydrofolate dehydrogenase, concerning the final reaction product, are studied. Steady and variable factors are detected in a comparative study of Vertebrate hemopoiesis: the enzyme exhibits peculiar features in different cell types. The reactivity progressively decrease in the erythropoietic series, in concomitance with hemoglobin synthesis. Conversely an increase in the intensity of reaction is found in the granulocytopoietic series; the correspondence of positive material with the specific (eosinophil and heterophil) granulations can be discussed. The thrombocytopoietic series is also labelled by this reaction.

Cytochemical study of K+-dependent p-nitrophenyl-phosphatase activity in the urinary bladder of Salamandra salamandra.

The ultrastructural Na+-K+-ATPase localization in the salamander urinary bladder has been studied by the histochemical techniques of Ernst (1972) and Mayahara et al. (1980). Reaction products have been specially found on the basal and lateral membranes of granular and mitochondria-rich cells. The variable presence of artifacts, often accompanying Pb-capture phosphatase cytochemistry, is discussed. Additional data on the active ion transport have been obtained by using electrophysiological techniques.

Glycogen changes in the liver cells of young rats following isoprenaline treatment. (Histochemical and ultrastructural investigations).

Glycogen changes were investigated by light and electron microscopy in the liver cells of newborn rats following isoprenaline (IPR) treatment either for 1 day (2 intraperitoneal injections at 12 h interval), or for 8 days (2 intraperitoneal injections daily). On the day following the interruption of both treatments glycogen depletion was observed as compared to control rats, as evaluated by PAS reaction and confirmed by higher total phosphorylase activity. During this stage electron microscopy revealed mainly alpha-particles of glycogen associated to highly dilated smooth endoplasmic reticulum (SER) after 1-day-treatment, and to mostly increased SER after prolonged treatment. In the animals submitted to prolonged IPR administration and sacrificed at later times, glycogen masses intensely PAS-positive were strongly increased, while the activity of total phosphorylase proved accordingly lower than in the rats sacrificed at earlier times. Electron microscopy examination confirmed an increased amount of glycogen (alpha- and beta-particles) differently distributed: beta-particles were more numerous in the liver of the rats sacrificed on the last day of the experiment. At this time SER didn't appear modified as compared to control rats.

Comparison of K(+)-p-nitrophenyl phosphatase activity in the urinary bladder of the frog Rana esculenta during hibernation and active life.

We have studied effects of hibernation on the frog urinary bladder, an organ involved in water and ion transepithelial transport and taking part in osmoregulation. We have demonstrated K(+)-p-nitrophenyl phosphatase activity (an enzyme involved in ion and water transport) both in active and hibernating frogs. Most of the reaction product deposition was found on basolateral membranes of granular cells of the urinary bladder epithelium during all seasons. Therefore, it seems likely that this organ, unlike organs studied previously (skin, kidney and lung), maintains its function in the osmoregulatory process during hibernation.

Ultrastructural cytochemical investigations on adenyl cyclase activity in isoprenaline-treated rat liver.

The possibility to demonstrate with cytochemical methods (REIK et al. 1970, HOWELL and WHITFIELD 1972) the activity of adenyl cyclase in rat liver following isoprenaline (IPR) stimulation was evaluated. The drug proved effective as an activator of the enzyme, particularly after in vivo treatment for several days, since it led to the formation of many precipitates which could be ascribed to adenyl cyclase activity, mainly on hepatocyte plasma membranes facing sinusoidal areas. The findings obtained during the experiment, through consistent with the hypothesis of a stimulation of hepatic adenyl cyclase by IPR, pointed out some limitations of the cytochemical techniques now available.

Histochemical demonstration of oxidoreductase activities in the fat body and symbionts of Blattella germanica (Blattodea) following chlortetracycline-treatment.

The metabolic apport of prokaryotic symbionts in the fat body of Blattella germanica was investigated by histoenzymatic methods, using chlortetracycline-treated and normal strains. In the experimental insects, bacteriocytes showed a decreased oxidoreductase activity, whereas the staining intensity of the other cell types was generally unchanged. Electron microscopic observations showed that some bacteria were still present in the bacteriocytes of the treated insects, but exhibited degeneration patterns to a different extent; therefore, they are not likely to carry on any enzymatic activity. Hence, chlortetracycline, an antibiotic that blocks the transovaric transmission of the symbionts, is active also on the endocellular symbionts of the fat body.

Styrene-induced alterations in the respiratory tract of rats treated by inhalation or intraperitoneally.

Although exposure to styrene occurs primarily via inhalation, the action of this agent on the respiratory tract has scarcely been investigated. This article describes morphological and biochemical changes occurring in the respiratory tract of rats after either inhalation of styrene vapors (300 ppm, 6 h/d, 5 d/wk, for 2 wk) or systemic (ip) treatment with 40 or 400 mg/kg styrene for 3 consecutive days. Electron microscopy analysis showed diffuse cell damage involving the tracheal, bronchiolar, and alveolar epithelium. In the tracheal epithelium, several cell types were affected. Ciliated cells presented vacuolation, detachment of cilia, blebbing of the apical cytoplasm, and compound cilia. Most secretory cells showed scant secretory granules and blebbings. Dense bodies and fibrillary inclusions were seen in intermediate and basal cells. Styrene also caused alterations of cytoplasmic components in type II pneumocytes and bronchiolar cells as well as thickness of the alveolar wall. These abnormalities were accompanied by depletion of glutathione (GSH) in the lung tissue. Pneumotoxic effects of systemic administration of styrene were dose dependent and tended to be more severe than those seen in the animals exposed for longer periods to styrene by inhalation. Metabolic activation of styrene and subsequent cell damage induced by the reactive metabolite styrene oxide may be involved in the sequence of events culminating in the toxic insult to the respiratory tract.

Effects of isoprenaline treatment on the rat liver parenchyma. Ultrastructural investigations.

Ultrastructural modifications of liver cells were studied in adult rats treated with high doses of isoprenaline for 8 days and subsequently sacrificed at various times up to 25 days from the beginning of treatment. The most evident changes were observed at the earliest times after the end of treatment. They involved both the nucleus (changes in shape and chromatin organization) and cytoplasmic organelles such as the rough endoplasmic reticulum and the smooth endoplasmic reticulum, the latter exhibiting marked dilation and increase, while glycogen generally appeared to be decreased. At later times after the end of treatment, these changes tended to disappear, while there was an increase of peroxisomes and particularly of lysosomes, which exhibited a clearly polymorphic pattern.

Propidium iodide as a probe for the study of chromatin thermal denaturation in situ.

The possibility of using propidium iodide, a phenanthridinic fluorochrome specific for double-stranded nucleic acids, for the study of chromatin thermal denaturation in situ has been examined. Smears of lymphocytes and hepatocyte nuclei from 15-day-old rats were fixed in acetic acid--ethanol (1:3 v/v), treated with RNAse and submitted to different protein extraction procedures, namely, incubation with pepsin, trypsin and sodium chloride. Denaturation experiments were performed in Sörensen buffer at pH 7.4 containing 10% formamide at temperatures between 27 and 95 degrees C. The samples were stained with propidium iodide and mounted in buffer or glycerol. Measurements were performed with a microfluorometer at a wavelength of 446 nm. The results indicate a higher thermostability of lymphocytes as compared to hepatocytes. The denaturation pattern suggests a certain organization complexity of chromatin, better emphasized by the derivative curves which show the presence of at least three fractions with different melting points. After protein extraction, the denaturation curves exhibit a somewhat simplified pattern, with the disappearance of the most stable peak in the derivative curves. The samples mounted in glycerine exhibit a better stability of staining with time, and an increased quantum efficiency of the fluorochrome with regard to those mounted in buffer. These data confirm the importance of protein--DNA interactions in the organization of chromatin and point to some differences, depending on the cell type and on functional activity.

Cytochemical evidence for potassium-dependent p-nitrophenylphosphatase activity in pavement cells of Rana esculenta mesentery.

BACKGROUND: We previously reported that during hibernation in Rana esculenta, various organs (i.e., skin, urinary bladder, kidney) change their osmoregulatory activity. Here, we considered the possible role of the frog mesentery in the ion transport, evaluating morphological and cytochemical (K+-p-nitrophenylphosphatase activity) aspects. METHODS: Pieces of mesentery from Rana esculenta collected in their natural environment during April, June, October, and January were processed to reveal ultrastructural morphology and K+-p-NPPase activity, using cerium as capture agent. RESULTS: The mesenteric mesothelium contained three types of cells: pavement, mitochondria-rich, and ciliated. Only the pavement cells expressed intense reactivity on the basolateral membranes and in the adjacent pinocytotic vesicles; some reaction product also was found on the apical membranes. Moreover, morphological and cytochemical characteristics of the pavement cells appeared to be very seasonal. CONCLUSIONS: The presence of mitochondria-rich cells and ciliated cells, generally found in structures involved in the transport of liquids, as well as K+-p-NPPase activity and pinocytosis in pavement cells, is consistent with the hypothesis that frog mesentery may be involved in seasonally variable osmoregulation.

The sulfatides and some histochemical correlations of the lachrymal glands involved in salt secretion in Chelonia.

High concentrations of sulfolipids (four fractions having different hexose/sulfate ratio), intense enzyme activity (ATPase, oxoreductases) and evidence of mucines (staining with PAS and Alcian blue) in intercellular spaces were found in the lachrymal glands of Caretta caretta and Malaclemys terrapin adapted to sea water. In addition, the supranuclear region of the gland cells in Malaclemys terrapin is filled with mucin granules. These biochemical and histochemical observations indicate that these glands have a function in salt secretion in both species and are also consistent with a function of mucous secretion exclusively in Malaclemys terrapin. Limited signs of hypotrophy are not accompanied by changes in concentrations of sulfolipids in Malaclemys terrapin adapted to fresh water; only the reactions for enzyme activities are less intense. The mucous secretion is not affected, whereas, in correlation with changes in salt secretion, the change in ATPase activity is mot conspicuous. The correlations between the different components of the gland and salt secretion are compared with salt glands of birds and elasmobranchs.