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.

Ultrastructural changes of neutrophils following IL-2 treatment in vivo.

An ultrastructural analysis of peripheral blood cells of cancer patients following recombinant interleukin-2 (rIL-2) treatment at high doses (rIL-2, 18 x 10(6) I.U./m2/day) has been performed. Characteristic patterns of intense phagocytosis (neutrophils with large vaculoes containing electron-dense bodies) were observed in granulocytes of the treated patients with respect to the basal situation. These characteristics were particularly evident in the advanced stages of the therapy (24 and 48 h). The state of neutrophil activation was sometimes accompanied by the appearance of a close net of cell membranes, probably derived from lamellopodia.

Characterization of the metabolism of perinecrotic cells in solid tumors by enzyme histochemistry.

Hypoxic tumor cells resist most therapies and cause tumor regrowth when their environment improves. Identifying the adaptation strategies to hypoxia would help develop better tailored cancer therapies. Ehrlich carcinomas implanted on mice were analyzed histochemically for the following enzyme activities: lactate, succinate and glucose-6-phosphate dehydrogenases, dihydrofolate reductase, purine nucleoside phosphorylase, xanthine oxidoreductase, and acid phosphatase. With the exception of xanthine oxidoreductase, which was not active in tumor cells, and of succinate dehydrogenase the activity of which was not significatively altered, all other activities were much higher in perinecrotic cells with respect to cells close to blood vessels. These data suggest the integration of metabolic paths allowing purine and lipid biosyntheses. Degradation products from the necrosis are presumed to be employed as surrogates of blood-borne nutritive substances by cells distant from the vascularization.

Stroma formation in Ehrlich carcinoma. I. Oedema phase. A mitosis burst as an index of physiological reoxygenation?

Tumor stroma induction has been shown closely to resemble wound repair process, both involving the replacement of a fibrin gel by vascularized connective tissue. In such a process the initial phase of hyperpermeability of blood vessels leads to diffuse oedema. It is here reported that cell loosening and a remarkably high mitotic burst were observed in Ehrlich carcinoma in regions in contact with the exudate, particularly at the perinecrotic (hypoxic) region. This suggests both an enhanced cell detachment from the tumour parenchyma and an improvement of the microenvironment, the exudate thus appearing as beneficial to the malignant cells contributing to the reoxygenation of formerly hypoxic regions. The temporary and well-localized concentration of mitoses in inner tumour areas has perhaps been disregarded by the pathologists engaged in mitosis counting for tumour grading. Peripheric and intraparenchymal concentrations of mast cells, lipid pools and platelets were seen in apparently key geometric disposition for controlling fibrin deposition and angiogenesis. Hypoxia is known to cause resistance to oxygen-dependent treatments and to facilitate cell detachment; normal fibroblasts respond and survive under hypoxic conditions by exhibiting features of the malignant phenotype. During reoxygenation, gene instability, cellular heterogeneity and increased drug resistance and metastatic spread have been reported. A reoxygenation process can also be deduced from several other histochemical and morphological patterns observed in this study. The findings here reported thus suggest that the oedema phase is a crucial phase regulating growth, invasion and dissemination of tumour cell populations, that should be specifically addressed therapeutically.

Tumor interstitial fluid: misconsidered component of the internal milieu of a solid tumor.

The tumor interstitial fluid (TIF) is a fluid phase present in the extracellular space of all tumors whose importance in oncology is seldom recognized. In order to stimulate other researchers to give it the due importance, a review of the available data (including our own) is provided. An hypothesis is presented for the genesis, fate and role of the TIF in the processes of invasion, growth and metastatization. Open questions regarding the TIF's role in tumor response to therapy are raised.

Histochemical probes for the detection of hypoxic tumour cells.

Hypoxia is thought to be a major cause of failure in cancer treatment. In this paper, we report methods transposable to clinical practice, for identifying hypoxic tumour cells. They consist of histochemical tests for revealing lactate dehydrogenase activity, endogenous lactate and accumulation of neutral fat. An ascites tumour (Yoshida hepatoma) and a solid tumour (Ehrlich carcinoma) were used as the experimental models. A gel film technique was used for visualizing lactate dehydrogenase and "nothing dehydrogenase" (or endogenous lactate). The fluorescent dyes Nile Red and Acridine Orange were used to demonstrate lipid accumulation and to visualize the tumour morphology, respectively. Tumour cells at the edge of areas of necrosis and at a distance of about 130 microns from a blood vessel were presumed to be hypoxic and showed the following features: 1) a dark blue granular pattern of lactate dehydrogenase (LDH) activity, ascribed to intense activity of the LDH5 and/or LDHk isoenzymes bound to membranous structures; 2) an intense granular positivity of "Nothing Dehydrogenase" due to high concentrations of endogenous lactate; 3) neutral lipid droplets emitting an intense yellow fluorescence in Nile Red-stained preparations; 4) a yellow cytoplasmic fluorescence in Acridine Orange-stained sections, attributable to a low cellular RNA content. Electron microscopy revealed moderately osmiophilic lipid globules in close association with damaged mitochondria. Better oxygenated cells showed: (a) a reddish-blue diffuse pattern of LDH, ascribed to moderately active soluble LDH isoenzymes containing H subunits; (b) almost no "Nothing Dehydrogenase" positivity; (c) no cytoplasmic lipid droplets; and (d) an intense orange-red fluorescence in the cytoplasm of Acridine Orange-stained specimens, due to high concentrations of cellular RNA. Nile Red fluorescence showed that the lipids of the solid tumour membranes were more hydrophobic than in the normal surrounding tissue. This suggests that there are abnormal domains of neutral lipids in the tumour cell membranes. In solid tumours, cells with the characteristics attributable to hypoxia were usually observed on the edge of necrosis of cuff-like formations. In very advanced growth stages, however, they were also seen surrounding (and occasionally clogging) blood vessels, or in tentacular formations coming from a necrosis border and polarized towards the vessels. Lipid-loaded cells were also seen in blood vessels distant from the tumour. These observations point towards a chemotactic process of hypoxic cells towards better environments.

Nuclear changes and morphology of the epidermis in the hibernating frog.

Cytochemical changes of chromatin and DNA in frog epidermal cells were correlated with some morphological features to investigate the skin physiology during hibernation in comparison with the active period. The epidermal cells of hibernating frogs showed less condensed chromatin in all the layers; a greater loss of DNA was found during the transition from the middle to the superficial layer. In the germinative layer, a lesser frequency of hyperdiploid cells and a remarkably low amount of mitoses were detected; this is accompanied by the increase of epidermal thickness and the presence of two layers of cornified cells. The slowing of tissue differentiation and cell renewal kinetics during hibernation can be related to lowered activity of the frog skin. Further, the smaller intercellular spaces as well as the scarcity of puffed ER and vacuoles may be indicative of a lower ion transport in epidermal cells during hibernation.

Morphohistochemical changes in hepatocytes during the life cycle of the European eel.

The comparative analysis of morphological, histochemical and cytochemical patterns of eel (Anguilla anguilla L.) hepatocytes reveals clear differences between two stages of its life cycle, i.e. the trophic stage (yellow eel) and reproductive stage (silver eel). The storage of glycogen prevails in the yellow eel, whilst lipids appear to be remarkably increased in the silver eel, in which some hepatocytes also show glycogen-rich areas. Generally, in the silver eel dehydrogenase and acid phosphatase activities seem greater and different distribution of the reaction products is present; on the contrary, a lower G6PDH activity is observed. The electron microscopy characteristics and distribution of both cellular organelles and reserve materials reflect the modifications found at light microscopy. The ultrastructural patterns provide further evidence for the heterogeneity of liver parenchyma in silver eel. In particular, the coexistence of nuclei showing a different degree of chromatin compactness is also accounted for by the quantitative cytochemical data on the nuclear DNA after Feulgen reaction and intercalation with propidium iodide at low and high concentrations. With regard to the DNA content, the hepatocytes in the silver eel as well as in the yellow eel are mainly 2c. However, some 4c values are also found, which according to the literature can be ascribed to cells in G2 phase. The present data may express the onset of different functional requirements during the reproductive stage in comparison with the trophic one. Moreover, our results are consistent with modifications found by other authors as a consequence of interruption of nourishment and during gonad maturation, i.e. two phenomena characterizing the transition from yellow to silver eel.

The action of cisplatinum (cis-DDP) on the postnatal growth of the rat liver. Cytokinetics and ultrastructural studies.

The early and late effects of cis-DDP treatment on liver cell kinetics were analyzed after its intraperitoneal injection into 17-day old rats. Frequency of binucleate hepatocytes, cellular DNA content distribution, 3H-thymidine labelling and ultrastructure of the nuclei were analyzed. Two days after treatment, a block of mononucleate hepatocytes in the S phase and in the G2 phase was demonstrated by the increase of intermediate 2c-4c and 4c DNA values in the absence of changes in 3H-thymidine labelling; 8c binucleate cells, which are essential for the formation of tetraploid mononucleate cells, were not found. In cell nuclei, large areas of more condensed chromatin appeared, perhaps providing further evidence for a G2 block. Seven days after treatment, there was a tendency to catch up with the normal situation, as shown by the unblocking of the S phase in mononucleate cells indicated by both cytophotometry and autoradiography. The presence of 8c binucleate cells and 4c-8c mononucleate cells indicates that 4c mononucleate hepatocytes are either diploid cells in the G2 phase or true tetraploid G1 cells. The decrease in the heterochromatin areas and the appearance of hypertrophic nucleoli demonstrate an increase in the metabolic activity of the nuclei. Thirty six days after treatment, the incidence of different DNA hepatocyte classes and the 3H-thymidine labelling were already similar in control and in treated rats.

Dihydrofolate reductase activity in the Purkinje neuron populations of some non-mammalian vertebrates.

The intensity and distribution of histochemically demonstrable dihydrofolate reductase (FH2-R EC 1.5.1.3.) in unfixed cryostat sections was studied in Purkinje neurons of adult vertebrates that have either simple neural circuits and cytoarchitectonics (Ictalurus nebulosus, Rana esculenta) or complex neural circuits and cytoarchitectonics (Coturnix coturnix japonica), compared with the rat as a control. The reaction was generally undetectable in Ictalurus nebulosus and in Rana esculenta; with positive reactions in only a few neurons. On the contrary, FH2-R in the Purkinje cell population of Coturnix coturnix japonica had several pattern (heterogeneity) as in the rat. These results suggest that the existence of FH2-R in Purkinje cell population may be correlated with the complexity of the neural circuits of the vertebrate's cerebellum and that the "heterogeneity" of the reaction may be related to the different functional states of the Purkinje cells within the cerebellum.

Immunohistochemistry of dihydrofolate reductase in methotrexate-sensitive and -resistant human cell lines by flow cytometry: a comparison with the cytochemical tetrazolium salt method.

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an enzyme involved in the metabolism of nucleic acids; it is also an important target for folate antagonists such as methotrexate (MTX). The distribution and expression of DHFR both in human HeLa BU-25 cell line and in methotrexate-resistant (MTX-R) variant, deriving from the human VA2-B cell line (having the DHFR gene amplified) was studied by tetrazolium salt method and by flow cytometric analysis. The immunohistochemical labelling of DHFR was achieved by using the streptavidinbiotinilated complex technique. DHFR activity was low in the human HeLa BU-25 cell line, while it was very high in the MTX-R cell line; the activity level increased with the increasing concentration of the MTX. The results obtained with cytochemical and immunohistochemical technique were compared. These findings showed that the hyperproduction of DHFR is strictly related with the cells having the DHFR gene amplified. Since MTX resistance is a common finding in the cells of patients with acute leukaemia, these studies may be extended to tumour-bearing patients at onset and following chemotherapy with methotrexate.

A qualitative and quantitative cytochemical assay of dihydrofolate reductase in erythroid cells.

The distribution and intensity of dihydrofolate reductase (DHFR) cytochemically demonstrable was studied in erythroid cells. Cells of normal human bone marrow, of human erythroleukaemia (M6), and cells of the Friend (MEL) clone 745A murine erythroleukaemia (also after differentiation with dimethylsulphoxide, DMSO) were stained according to Gerzeli and de Piceis Polver (1969) technique; quantification of the reaction product was made using a Vickers M86 microdensitometer. The enzyme activity progressively decreased during the normal differentiation of the erythropoietic series while persisted at high levels in erythroleukaemia cells. It can be suggested that in the 1st case, the cytochemical pattern of dihydrofolate reductase may be a useful added tool for studying the erythroid differentiation. In the 2nd case, the increased level of this enzyme may be related to an amplification of the gene of DHFR in the malignant transformation.

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.

Cytofluorometric study of the erythrocyte glycoconjugates content in different species of vertebrates.

The PAS-positive material was measured by cytofluorometry in peripheral circulating erythrocytes of 22 species from different classes of Vertebrates. The following parameters were considered: total fluorescence intensity per cell, concentration index, fluorescence fading. The highest variability of concentration indexes was found in Fishes with a minimum in the more active swimmers (Scylliorhinus and Salmo); this fact may be related to a more intense erythropoiesis. The values were more homogeneous in the Amphibians, Reptiles, and Birds, the difference never exceeding 1/2 between the maximum and minimum values. The lowest concentration indexes were found in Mammals, a class with very specialized and enucleated erythrocytes: the data appear rather heterogeneous with a minimum in Macaca. The patterns of fluorescence fading suggest a biochemical homogeneity of the PAS-positive erythrocyte material in the species considered. This fact might indicate the presence of a fundamental biochemical component possibly linked to different structures of the red cells.

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.

Cytochemical pattern of alpha-mannosidase, alpha-fucosidase and neutral maltase in normal blood cells.

The activity of alpha-mannosidase, alpha-fucosidase and neutral maltase was studied by cytochemical techniques in blood cells of 20 controls and of 4 T and B lymphocyte concentrates. All granulocytes, monocytes and platelets showed fine or coarse reaction product deposition, whereas lymphocytes were negative or showed various positivity patterns. A significant difference of the positivity between T and B subpopulations was observed only for the fucosidase reaction. It is possible that the different positivity patterns of the lymphoid cells are related to different functional activities. Further studies will probably confirm the interest of the alpha-fucosidase reaction for the characterization of normal and pathological lymphoid cells.

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.