Effect of epidermal growth factor on cultured adult rat hepatocytes

When adult rat hepatocytes were cultured in plastic Petri dishes in a medium containing insulin and glucagon, supplementation with epidermal growth factor (EGF) had a pronounced effect on their viability, morphology, and biochemical integrity. Transmission and scanning electron microscopic studies showed that after 1 week cells denied EGF accumulated numerous non-electron-dense bodies and filamentous whorls, had irregular nuclei, and exhibited atypical cell surfaces. In contrast, cells grown for 2-3 weeks in the presence of EGF had well-preserved cellular organelles and remained as an epithelial-like monolayer. After 3 weeks EGF-exposed cultures were still inducible for liver-specific tyrosine aminotransferase, and both rat albumin and rat transferrin were recoverable from the culture medium. Virtually no viable cells were present at 3 weeks in EGF-deprived cultures.     

Neonatal rat hepatocytes in primary tissue culture free from density-dependent regulation of growth

Summary Studies employing [³H]thymidine and radioautography as well as colchicine and Feulgen staining of DNA showed that up to 19-fold increases in the degree of cell crowding in vitro, i.e. from 1.45 to 27.55×10⁴ cells per specimen, did not change the rates of entry into DNA synthesis and mitosis of cultivated primary neonatal rat hepatocytes.     

Receptor-mediated endocytosis of galactose and mannose exposing ligands: an electron microscopic study on adult and neonatal cultured rat hepatocytes.

We compared the receptor-mediated endocytosis for galactose and mannose exposing ligands in primary cultures of hepatocytes from newborn and adult rats. The endocytic pathway was revealed ultrastructurally using colloidal gold particles coupled to lactosylated bovine serum albumin and invertase. The binding activity on the cell surfaces is observed by keeping the cells at 4 degrees C. For both ligands used, the binding capacity for hepatocytes from adult rats was greater than for neonatal cultured cells. Increasing the temperature to 37 degrees C, we observed that the protein-gold complexes entered the intracellular endocytic organelles. Within 5-15 min, the marker was confined in vesicles close to the cell surface and in the endosome, while after 60 min, the marker is found in lysosome-like compartments. We found that the process of endocytosis is similar for galactose and mannose exposing ligands. The organelles involved in the process of endocytosis in newborn cultured hepatocytes are not different in shape from those of cultured cells of adult rats, but the process of internalization is slower.     

Calcium amelioration of cadmium-induced cytotoxicity in cultured rat hepatocytes

Summary Parenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium with or without calcium, and processed for scanning electron microscopy. To assess the severity of cadmium-induced changes, exposed hepatocytes were categorized based upon the extent of morphological damage. Differences in surface blebbing, alterations in microvilli, variations in the degree of swelling, and changes in cell shape were used to categorize the severity of cell damage. A double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information) of cellular changes was conducted for each exposure time and for each concentration of cadmium in the presence or absence of calcium. Significant decreases occurred in the percent relative volume of normal, flattened cells present in cultures exposed for 30 min to 50 or 100 μM cadmium in the absence of calcium. In contrast, the percent relative volume of severely damaged spherical cells was significantly increased after exposure to solutions containing 50 or 100 μM cadmium and lacking calcium. Percent relative volume of intermediate cells (which were slightly swollen and showed changes in microvillar number) was significantly increased following a 30 min exposure to all cadmium concentrations in the absence of calcium. The examination of hepatocytes exposed for 60 min showed that the degree of cadmium-induced cytotoxicity was more severe in the absence of calcium than was the case for the hepatocyte cultures exposed for 30 min: approximately 30% more spherical cells and 30% fewer flattened cells were present if cultures were exposed in the absence of calcium for 60 min compared to those exposed for 30 min. The degree of blebbing was significantly greater at all cadmium concentrations in the absence of calcium. The presence of calcium, therefore, reduced cadmium-induced cytotoxicity in primary cultures of rat hepatocytes subjected to morphometric analysis after scanning electron microscopy.     

Hormonal modulation of hepatic plasma membrane lactate transport in cultured rat hepatocytes

Hormonal modulation of hepatic plasma membrane lactate transport was studied in primary cultures of isolated hepatocytes from fed rats to examine the mechanism for the known enhancement of lactate transport in starvation and diabetes. Total cellular lactate entry was increased by 14% in the presence of dexamethasone; this was accounted for by an approximately 40% increase in the carrier-mediated component of entry with no effect on diffusion. A trend of similar magnitude was evident with glucagon. The effects of dexamethasone and glucagon on lactate transport constitute an additional potential mechanism for enhancement of gluconeogenesis by these hormones.     

Influence of rat strain on P-glycoprotein expression in cultured hepatocytes

The amount and activity of the multi-drug transport P-glycoprotein (Pgp) have been measured in cultured hepatocytes derived from different rat strains. A marked increase in Pgp, as revealed by Western blotting, occurred 48 h after seeding in hepatocytes from Sprague-Dawley, Wistar and Fischer 344 rats, the last showing the highest value. The addition of dexamethasone (DEX) to culture medium delayed Pgp overexpression in all the strains, proportionally to the protein amount in the absence of hormone. The R-123 functional test for Pgp showed that Fischer 344 hepatocytes had the lowest ability to extrude the fluorescent dye as compared with Sprague-Dawley or Wistar rats. These results suggest that the Fischer 344 rat is more prone than other strains to culture stressing conditions, leading to an overexpression of Pgp that is not necessarily functional.Abbreviations DEX dexamethasone - FCS fetal calf serum - HRP horseradish peroxide - Pgp P-glycoprotein     

Expression and induction of drug-metabolizing enzymes in cultured fetal rat hepatocytes

Anin vitro experimental model, fetal rat hepatocytes in culture, was metabolically characterized. Several enzymatic activities were expressed in these hepatocytes, namely, testosterone hydroxylations. Hepatocytes cultured up to 3 weeks in the presence of dexamethasone and phenobarbital still expressed some drug-metabolizing enzyme activities (e.g., ECOD). The enzymatic activities were measured both directly on monolayers during culture and on the corresponding harvested and homogenized cells. The results correlate perfectly with each other. The on cell procedure allows us to repeat the assay or to measure several activities on the same cells at different time intervals. The presence of dexamethasone in the culture medium allows the expression and the induction of several cytochrome P450 isoenzymes, namely, those hydroxylating testosterone. This makes the model particularly attractive for induction experiments as well as for metabolic or toxicological studies needing longer treatments.Abbreviations BA benzanthracene - CLO clofibric acid - DEXA dexamethasone - DMSO dimethylsulfoxide - ECOD ethoxycoumarin-O-dethylase - PB phenobarbital - RER rough endoplasmic reticulum     

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture

Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.     

Effect of sodium butyrate on primary cultures of adult rat hepatocytes

Summary Sodium butyrate, at millimolar concentrations, seems to mediate or initiate multiple effects on many mammalian cells in culture. Although many transformed cell lines respond to butyrate treatment with acquisition of normal cellular characteristics, the effect of butyrate on a normal cell type, the parenchymal hepatocyte, has not been studied. Serum-free primary cultures of adult rat hepatocytes maintain many adult characteristics, yet after several days in culture a loss of adult characteristics occurs while fetal characteristics are often reexpressed. Therefore, we investigated whether butyrate treatment would improve the morphologic and biochemical characteristics of cultured hepatocytes. Exposure to 5 mM butyrate for 3 d did not affect hepatocyte viability or morphology but retarded the progressive decline in cytochrome P-450 levels and 5′-nucleotidase activity. The spontaneous increase in alkaline phosphatase activity was reduced and the induction of tyrosine aminotransferase was inhibited after 3 d in culture. The fetal liver characteristic, gamma glutamyltranspeptidase, was not affected by butyrate treatment. Results of this study suggest that butyrate represents a nontoxic compound capable of improving the maintenance of cell culture characteristics of adult rat hepatocytes.     

Ultrastructural characteristics of intercellular contacts and bile canaliculi in neonatal rat hepatocytes in primary culture

The ultrastructure of the cellular contacts and bile canaliculi was examined in cultured neonatal (day 5) rat hepatocytes to elucidate the development of cellular polarity. A new scanning electron microscopic technique for cultured hepatocytes allowed a view of cell-cell attachment and the entire cell surface, including the underside on plastic dishes. At 3 h after plating, neonatal hepatocytes were shown to be round, with loss of the preferential localization of cell organelles. After 6 h of culture, the cells had become oblong; they were aggregated in groups of several cells and the cellular contacts were not as rigid or as straight as those in adult hepatocytes. Transmission electron microscopy showed the biliary functional polarity to be like that in vivo. On the undersurfaces of adjacent neonatal heptocytes a hemicanalicular structure lined with microvilli was found, which probably corresponds to the ultrastructure of bile canaliculi in vivo. However, no canaliculi or orifices of bile channels were found in adult hepatocytes. These results suggest that in neonatal rat hepatocyts the formation of tight rigid cellular contacts was suppressed. Modulation of cell membranes appeared on the undersurfaces of neonatal hepatocytes in early culture stages. The difference in the development of cellular polality could be caused by the proliferating activity of neonatal hepatocytes.     

Age- and hormone-dependent ganglioside patterns of rat hepatocytes in primary culture

Using an improved procedure for the quantitative extraction of all glycolipids from small tissue samples the hepatic ganglioside pattern of rats was analysed during development. While this parameter remained fairly constant in adult animals, hepatocytes in primary culture showed drastic changes both in content and relative distribution among the various ganglioside species. The content of lipid-bound sialic acid increased several-fold during 6 days in monolayer and the pattern changed in favour of the higher sialylated forms. Dexamethasone delayed this transition and enhanced the content of GD1a and GM1 relative to GM3. The ganglioside content was also dependent on the density of hepatocytes in the primary culture. If the cell density was insufficient for formation of a confluent monolayer, higher ganglioside-sialic acid contents were found and the relative amount of GD3 increased after 3-4 days. These results support the notion that gangliosides are involved in cellular differentiation and cell-cell contact.     

Characteristics of taurine transport in rat hepatocytes in primary culture

Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2 degrees C was unsaturable, whereas that at 37 degrees C consisted of unsaturable and saturable processes. The saturable transport system was sodium-dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 microM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol-1) was competitively inhibited by 2,4-dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37 degrees C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy-dependent carrier-mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested.     

Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture

In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro. Most of the lymphocyte clones obtained are CD 8 + cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously. In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8+ or CD 4+ cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cell suspension. The activated cells were then coincubated with rat hepatocytes in primary culture. The killing capacity of the activated lymphocytes was monitored by light and electron microscopy and by measurement of lactic dehydrogenase (LDH)-release into the culture medium. It was found that cytotoxic CD 8 +, but not CD 4 + helper lymphocytes very effectively killed hepatocytes. The killing effect was dependent on the time of cocultivation and on effector-target (E/T) ratio. Total breakdown of the hepatocyte monolayer was achieved after 10–20 h coculture and at an E/T ratio of 10 to 1. As LDH-release in the culture medium reached about 80% of the total LDH-content, most of the hepatocytes were lysed by activated lymphocytes. Cytotoxic activity of clones obtained from different biopsies was comparable with that of clones from peripheral blood. Hepatocytes in primary culture seem to be very sensitive to the killing capacity of activated cytotoxic lymphocytes. Supported by DFG grants Ra 362/5-2 and SFB 311 A7 (G.R.) and A5 (H.P.D.)     

Investigation of rifampicin-induced hepatotoxicity in rat hepatocytes maintained in gel entrapment culture

Rifampicin-induced hepatotoxicity has been well recognized in animals and patients. However, it is undetectable in cultured hepatocyte monolayers in vitro at the equivalent toxic concentration in vivo. This study investigated the rifampicin-induced toxicity on rat hepatocytes in gel entrapment vs. in monolayer culture. Thiazolyl tetrazolium reduction and albumin secretion were routinely detected to identify the toxic responses of rat hepatocytes to rifampicin, while reactive oxygen species (ROS) accumulation and intracellular glutathione (GSH) content were assayed as biomarkers of oxidative stress. In addition, Nile red staining and malondialdehyde (MDA) generation were, respectively, used as endpoints for lipid accumulation and peroxidation. After treatment of hepatocytes for 96 h at a serum rifampicin concentration (12 μM), gel-entrapped rat hepatocytes showed significant cellular damage indicated by alternations of all parameters indicated above, while hepatocyte monolayers did not show severe responses. In contrast to a lack of protections by cytochrome P 450 inhibitors, the ROS scavenger (glycyrrhizic acid) and thiol compounds (N-acetylcysteine and GSH) significantly reduced rifampicin toxicity in gel-entrapped hepatocytes. It appears that gel-entrapped rat hepatocytes reflected significant hepatotoxicity of rifampicin in vivo, and this toxicity was most possibly associated with oxidative stress and lipid accumulation.     

The effect of oxygen tension on rat hepatocytes in short-term culture

Summary Cell viability, cytochrome P-450 content, cell respiration, and lipid peroxidation were all investigated as a function of oxygen tension in adult rat hepatocytes in short-term culture (less than 9 h). The various oxygen tensions used in this study were obtained by equilibrating culture medium with air, air + nitrogen, or air + oxygen. Cell viability, as assessed by trypan blue exclusion, was significantly greater at all time points tested when hepatocytes were cultured in Ham's F12 medium containing 132 μM O₂, as compared to medium equilibrated with air (220 μM O₂) or air + oxygen (298 μM O₂). Cells cultured in 220 μM O₂ (air) also exhibited a gradual loss of cytochrome P-450, so that by 9 h of incubation less than 60% of the active material remained. This loss of P-450 was minimized when cells were cultured in 163 μM O₂ and abolished when cells were cultured in 132 μM O₂. The 132 μM O₂ exposure conditions also maintained cell respiration at the 1 h incubation values, whereas there was a continuous loss in cell respiration over time when the cells were cultured in either 220 μM O₂ (air) or 298 μM O₂ (air:O₂). These cytotoxicity findings may be related to oxidative cell damage inasmuch as it was additionally demonstrated that lipid peroxidation (as measured by malondieldehyde equivalents) was consistantly lower in hepatocytes cultured in air:N₂ as compared to air or air:O₂. These results suggest that hepatocyte culture in low oxygen tension improves not only cell viability but also maintains other functional characteristics of the cell. This work was supported by a Biomedical Research Support Grant S-S07-RR 05448 awarded to the University of Minnesota School of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health, Bethesda, MD.     

Enzyme leakage due to change of membrane permeability of primary cultured rat hepatocytes treated with various hepatotoxins and its prevention by glycyrrhizin

Various hepatotoxins were added to the medium of primary cultures of adult rat hepatocytes and the release of the cytosolic enzymes lactic dehydrogenase, glutamic-oxaloacetic and glutamic-pyruvic aminotransferases were measured 24 h later. CCl₄ at low concentrations caused dose-dependent release of soluble enzymes into medium without appreciable cytolysis of the hepatocytes. Mitochondrial enzymes were not released under these conditions. At 5 mM CCl₄, both soluble and mitochondrial glutamic-oxaloacetic aminotransferase were found in the culture medium. Glycyrrhizin, a triterpenoid glycoside of licorice roots, prevented the enzyme release caused by CCl₄.Abbreviations CCl₄ carbon tetrachloride - GDH glutamic dehydrogenase - GOT glutamic-oxaloacetic aminotransferase - GPT glutamic-pyruvic aminotransferase - LDH lactic dehydrogenase     

Amino acid and energy requirements for rat hepatocytes in primary culture

Summary The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations, and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained—on a collagen substratum—for at least a week without any significant net loss of cells or cellular protein. The work was supported by grants from The Norwegian Cancer Society and from The Norwegian Council for Science and the Humanities. An erratum to this article is available at .     

Influence of medium composition and culture conditions on glutathione S-transferase activity in adult rat hepatocytes during culture

Summary Glutathione S-transferase (GST) activity was measured in adult rat hepatocytes during either pure culture or coculture with another rat liver cell type in various media. Addition of nicotinamide, selenium, or dimethylsulfoxide, deprivation of cyst(e)ie and the use of two complex media were tested. Whatever the conditions used, after a constant decrease during the first 24 h, GST remained active over the whole culture period (1–2 wk). However, various patterns were observed: GST activity either remained relatively stable to approximately 50% of the initial value or showed a moderate or strong increase. The highest values were found in pure hepatocyte cultures maintained in the presence of nicotinamide or dimethylsulfoxide. Similar changes were observed using 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene as substrates for GST. Addition of 10⁻⁴ M indomethacin resulted in 37 to 60% inhibition of enzyme activity. Thus, these results demonstrate that GST remained expressed during culture but its levels markedly varied depending on the medium composition and type and age of culture. Y. V. was supported by Instituut voor Wetenschappel?k Onderzoek in Landbouw en Nijverheid. This work was supported by INSERM.