Application of coomassie brilliant blue staining to cultured hepatocytes

Coomassie brilliant blue staining developed by Pena (1980) was applied to cultured hepatocytes of adult rats with some modifications. Many of organelles in the cytoplasms were clearly visible as blue granules by this method. Various cytoskeletal elements were also visualized clearly. Because of its simplicity, Coomassie blue staining proved to be a very powerful tool for study of morphological changes of cell organelles and cytoskeletal systems of cultured hepatocytes.     

Assessment of the function of the betaC-subunit of activin in cultured hepatocytes

We assessed the function of the beta(C)-subunit of activin in hepatocytes. We studied the effect of conditioned medium of Chinese hamster ovary (CHO) cell line stably expressing the beta(C) gene (CHO-beta(C)) on growth of AML12 hepatocytes. We also examined the effect of recombinant activin C and transfection of the beta(C) gene by using adenovirus vector. CHO-beta(C) secreted activin C, a homodimer of the beta(C), as well as precursors of the beta(C). The conditioned medium of CHO-beta(C) increased both [(3)H]thymidine incorporation and the cell number in AML12 cells. It also supported survival of AML12 cells in a serum-free condition. Recombinant human activin C also increased both [(3)H]thymidine incorporation and the number of AML12 cells. Transfection of AML12 cells with the beta(C)-subunit led to the stimulation of [(3)H]thymidine incorporation. Analysis of the conditioned medium revealed that the beta(C)-subunit formed a heterodimer with the endogenous beta(A), the formation of which was dependent on the amount of beta(C) expressed. Recombinant activin C did not affect the binding of (125)I-activin A to its receptor or follistatin. These results indicate that activin C stimulates growth of AML12 cells. The beta(C)-subunit modifies the function of the beta(A)-subunit by multiple mechanisms.     

Ultrastructural analysis of asialoglycoprotein receptor mediated endocytosis in cultured newborn hepatocytes

We examined the receptor-mediated endocytosis of asialoglycoproteins in thick sections of cultured hepatocytes of newborn rats by high voltage electron microscope. The organelles involved in endocytosis are revealed ultrastructurally using colloidal gold particles coupled to lactosylated bovine serum albumin. Keeping the cells at 4 degrees C the marker binds to the cell surface showing microvilli, and is not internalized. Increasing the temperature to 37 degrees C, we observed that within 5-15 min. the marker enters the intracellular endocytic organelles close to the cell surface. After 60 min. the marker is found in the larger and deeper endocytic organelles and in large lisosome-like vesicles. We find that the process of endocytosis in newborn cultured hepatocytes is similar to that found in cultured cells of adult rats, but the process of internalization is slower.     

Inherent toxicity of organ preservation solutions to cultured hepatocytes

Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 °C), intermediate (21 °C) and physiological (37 °C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 °C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 °C: HTK 76 ± 2%, Euro-Collins 78 ± 17%, HL 81 ± 15%; control: Krebs-Henseleit buffer 20 ± 6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions.     

Morphology and function of cultured hepatocytes isolated from rats with experimental toxic hepatitis

The goal of the study was to examine the morphology and function of primary hepatocytes isolated from rats with toxic hepatitis induced by a combination of CCl₄ and ethanol. Fluorescent immunocytochemical analysis demonstrated that normal and pathologic hepatocytes in culture formed actin cytoskeleton, cell-cell, and cell-matrix contacts. In this investigation, the morphology of mitochondria and their localization in hepatocytes was assayed with Rhodamine 123 staining. Glycogen and DNA contents in cultured hepatocytes were determined by fluorescent cytometry. It was found that the ploidy of hepatocytes isolated from normal and injured livers were different. Cells were maintained in culture for 5 days and no changes in ploidy distribution were observed. The glycogen content was 50% higher in the experimental group than the control one; it was decreased in control and cirrhotic hepatocytes treated with collagenase. Intact hepatocytes accumulated glycogen within 3 days; the glycogen level remained low in pathologic hepatocytes.     

Cadmium-induced hepatotoxicity in cultured rat hepatocytes as evaluated by morphometric analysis

Summary Freshly isolated hepatocytes from neonatal rats were cultured for approximately 24 h; incubated for 5, 30, or 60 min in solutions containing 0, 50, 100, or 200 μM cadmium; embedded in plastic; and sectioned for optical microscopy. The exeent of cadmium-induced hepatotoxicity was evaluated by double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of threedimensional information) whereby hepatocytes were classified on the basis of the severity of morphologic damage at the optical level. Both time and concentration effects were studied. Cultures exposed to 200 μM cadmium, for various intervals of time from 5 to 60 min, showed statistically significant reductions in the relative volume percent of normal hepatocytes, elevations (then reductions) in the relative volume percent of slightly damaged hepatocytes, increases in the relative volume percent of moderately damaged cells, and increases in the relative volume percent of severely damaged liver cells. As the concentration of cadmium was increased from 50 to 200 μM cadmium (during both 30 and 60-min exposures), significant trends were observed in cellular distribution patterns based on relative volume percent. Morphologically normal cells decreased, both slightly damaged and moderately damaged cells increased, and severely damaged cells remained unchanged. These results indicated that morphometric analysis at the optical level provided quantitative estimates for the evaluation of time- and concentration-effects of cadmium on cultured hepatocytes. This work was supported by a grant from the Johns Hopkins University Center for Alternatives to Animal Testing.     

Cadmium-induced hepatotoxicity in cultured rat hepatocytes as evaluated by morphometric analysis

Freshly isolated hepatocytes from neonatal rats were cultured for approximately 24 h; incubated for 5, 30, or 60 min in solutions containing 0, 50, 100, or 200 microM cadmium; embedded in plastic; and sectioned for optical microscopy. The extent of cadmium-induced hepatotoxicity was evaluated by double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information) whereby hepatocytes were classified on the basis of the severity of morphologic damage at the optical level. Both time and concentration effects were studied. Cultures exposed to 200 microM cadmium, for various intervals of time from 5 to 60 min, showed statistically significant reductions in the relative volume percent of normal hepatocytes, elevations (then reductions) in the relative volume percent of slightly damaged hepatocytes, increases in the relative volume percent of moderately damaged cells, and increases in the relative volume percent of severely damaged liver cells. As the concentration of cadmium was increased from 50 to 200 microM cadmium (during both 30 and 60-min exposures), significant trends were observed in cellular distribution patterns based on relative volume percent. Morphologically normal cells decreased, both slightly damaged and moderately damaged cells increased, and severely damaged cells remained unchanged. These results indicated that morphometric analysis at the optical level provided quantitative estimates for the evaluation of time- and concentration-effects of cadmium on cultured hepatocytes.     

Sialyltransferase activities in cultured rat hepatocytes

Previous studies on the age and sex dependency of the ganglioside patterns in rat liver in vivo and the concomitant determination of the activities of some enzymes involved in these pathways revealed the prominent role of the sialylation of GM3 to GD3 in determining the flow to the mono (a)- and polysialo (b)-series, respectively. Here, the influence of hormones on the activities of GM3 and GD3 synthases in isolated hepatocytes was studied. The combination of several factors (insulin, glucagon, epidermal growth factor, glucocorticoids) was found to be necessary for maintaining in vivo activity levels of GD3- but not of GM3-synthase.     

Regulation of apo-A-I processing in cultured hepatocytes

Apo-A-I, the major protein component of high density lipoproteins, appears intracellularly as an intermediate precursor (pro-apo-A-I) with a hexapeptide extension (RHFWQQ) at its amino terminus. Proteolytic processing of pro-apo-A-I to apo-A-I has been shown to occur extracellularly in cell and organ cultures from rat and human tissues. Recently, however, intracellular conversion has been detected in chickens. To determine what distinguishes and regulates these two processing methods, the proteolytic processing and secretion of apo-A-I was studied by metabolic labeling in chick hepatocytes and in Hep-G2 cells (derived from a human hepatocellular carcinoma). The proportions of intracellular and secreted pro-apo-A-I and apo-A-I were measured by sequencing NH2-terminal portions of the proteins and determining the location of radio-labeled amino acids. Chick hepatocytes cultured in the absence of hormones or fetal bovine serum secreted primarily processed apo-A-I (83%). In the presence of serum these cells secreted only pro-apo-A-I, whereas incubation with a combination of hormones (insulin, triiodothyronine, dexamethasone) resulted in secretion of a nearly equal mixture of the pro- and processed forms of the protein. In contrast, Hep-G2 cells, maintained in the absence of serum, secreted only pro-apo-A-I; when grown in the presence of serum these cells secreted a mixture of pro- and processed apo-A-I. Under conditions in which chick hepatocytes and Hep-G2 cells secreted both forms of the protein, a mixture of pro- and processed apo-A-I was also found intracellularly; when only the pro-form was secreted, the cells likewise contained only pro-apo-A-I. Under all the above conditions, the secreted apo-A-I exhibited similar isoform patterns in two-dimensional gel electrophoresis. These data show that both chick hepatocytes and human hepatoma cells are capable of intracellularly processing pro-apo-A-I to apo-A-I, and that the extent of intracellular processing is controlled by the cell's hormonal environment.     

Secretion of alpha-tocopherol from cultured rat hepatocytes

Primary cultures of rat hepatocytes and rat liver perfusions were used to study hepatic secretion of alpha-tocopherol. The secretion of alpha-tocopherol from hepatocytes in culture was linear with time for 4 h. Ultracentrifugation of the medium revealed that 89.4 +/- 2.1% of alpha-tocopherol secreted during 4 h incubation was associated with the very-low density lipoprotein fraction (VLDL, d less than 1.006 g/ml). Oleic acid had no significant effect on the secretory rate of alpha-tocopherol, whereas eicosapentaenoic acid reduced the amount of alpha-tocopherol secreted to 48.4 +/- 12.7% of the control value after 20 h incubation (P less than 0.01). Monensin, a known inhibitor of VLDL secretion, reduced the secretion of alpha-tocopherol to 14.1 +/- 4.3% of the control value (P less than 0.02). Colchicine and chloroquine inhibited the secretion of alpha-tocopherol in the same order of magnitude as monensin. Hepatic perfusion after intravenous injection of in vivo labeled alpha-[3H]tocopherol lymph, showed that about 75% of the secreted radioactivity was in the VLDL fraction. From these results we conclude that most alpha-tocopherol is secreted from the liver associated with nascent VLDL in rats.     

The use of cultured hepatocytes to investigate the mechanisms of drug hepatotoxicity

In the course of biotransformation reactions catalyzed both by cytochrome P450 and by conjugating enzymes, drug-derived reactive metabolites and active oxygen species can appear that may escape the detoxification process, initiating radical chain reactions (e.g., lipid peroxidation), covalently binding to macromolecules (proteins, DNA), or impairing the energetic balance of cells. This is usually followed by alterations of ion homeostasis that precede irreversible biochemical changes and cell death. There are, however, cellular mechanisms of defense that prevent, or repair, the damage caused by these reactive intermediates. Ultimately it is the balance between bioactivation, detoxification, and defense mechanisms that determines whether a compound will or will not elicit a toxic effect. Cultures of hepatocytes, including those of human origin, can be used to elucidate the mechanisms of drug toxicity. This is illustrated in the study of the mechanism of hepatotoxicity by diclofenac. Much less cytotoxicity is observed in nonmetabolizing hepatomas than in hepatocytes. The observed cell dysfunction parallels the biotransformation of the drug, and particularly the formation of the minor metabolite N,5-dihydroxydiclofenac by hepatocytes. This compound is able to inhibit mitochondrial ATP synthesis in hepatocytes.     

Responsiveness of RNA degradation to amino acids in cultured rat hepatocytes: comparison with isolated rat hepatocytes.

The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6-14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 +/- 0.4%/h in the absence of amino acids (0x). The catabolism of RNA was decreased to basal level (1.5 +/- 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation-induced response (0x minus 10x), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 x 10(6) cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3-methyl-adenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids.     

Lipid synthesis in permeabilized cultured rat hepatocytes

Hepatic lipid synthesis was verified and studied in lysolecithin-permeabilized cultured rat hepatocytes and compared to that of intact liver cells. Triacylglycerol synthesis in permeabilized cells incubated in the presence of glycerol 3-phosphate and long chain fatty acids approached that of intact hepatocytes. Similarly, phosphatidylcholine synthesis in permeable cells incubated in the presence of exogenous CDP-choline was similar to that of intact hepatocytes and at the expense of microsomal neutral lipid synthesis. Phosphatidic acid accumulation in lysolecithin-permeabilized liver cells was remarkably increased as compared to that of intact cells, and its synthesis was mostly accounted for by the activity of mitochondrial glycerol-3-phosphate acyltransferase. Mitochondrial-generated phosphatidate was found to migrate to the endoplasmic reticulum, thus establishing a novel lipid esterification pathway which begins in mitochondrial glycerol 3-phosphate acylation and results in microsomal triacylglycerol and phospholipid synthesis. The free access of permeabilized liver cells to substrates and modulators of lipid synthesis, while maintaining an overall synthetic pattern similar to that of intact hepatocytes, makes them a system of choice for studying hepatic lipid synthesis in general and the microsomal/mitochondrial distribution of fluxes in particular.     

Oxygen uptake rates in cultured rat hepatocytes

One potential treatment of acute liver failure involves the use of an extracorporeal device composed of functional hepatocytes. A major issue in the design of such a large-scale device is providing the hepatocytes with a sufficient supply of oxygen and other nutrients. In this study, we have designed and characterized a simple perfusion system hepatocytes using this system. The OUR of hepatocytes was determined during the first day after seeding on a single collagen gel and during the long-term stable culture after the addition of a top layer of collagen. The OUR increased to 20.7 +/- 0.57 pmol/sec/mug DNA during the first 13 hours of culture on a single collagen gel, while during the next 11 hours, the OUR declined to 10.6 +/- 1.5 pmol/sec/mug DNA. In parallel with the increase in OUR during the first 10 hours, we observed significant cell spreading, suggesting that the oxygen supply to the cells may be critical for the spreading and adaptation of the anchorage-dependent hepatocytes following isolation. Addition of a top layer of collagen to hepatocyte cultures for 24 hours of culture on a single collagen layer resulted in a stable OUR for 15 days. These results indicate that OUR of hepatocytes in culture may vary depending on the phase of culture (i.e., early vs. late) and on the extracellular environment. (c) 1992 John Wiley & Sons, Inc.     

A perifusion system for cultured hepatocytes.

A perifusion system for primary cultures of hepatocytes is described. The system accommodates 20 rotated petri dishes (60 mm) and allows individual medium composition and sampling for each dish. Cell number and insulin (15 pM to 7.7 nM) were stable in the system for at least 24 h. The dose-response relationship for induction by insulin of glucokinase and pyruvate kinase was shifted to the left by a factor of 9 and 5, respectively, as compared to conventional, stationary cultures. The system is useful for studies at low and/or constant concentrations of substrates, hormones, growth factors, etc., with monolayers of cells having a high metabolic capacity.     

Antioxidants prevent aluminum-induced toxicity in cultured hepatocytes

Cellular Al accumulation has been shown to alter iron metabolism and induce peroxidative injury. Therefore antioxidants could potentially reduce or prevent peroxidative injury in Al-loaded cells. To test this hypothesis we assessed the effect of the antioxidants N-acetyl cysteine (NAC), catalase, superoxide dismutase (SOD), and tetramethylpiperidine 1-oxyl (TEMPO) in abrogating Al-associated cell toxicity and melonyldialdehyde (MDA) accumulation in mouse hepatocytes. Mouse hepatocytes (MH) were grown in media containing the minimum toxic concentration of Al (100 microg/L as Al-transferrin). All antioxidants protected MH from injury as assessed by cell growth and enzyme leakage into media. The antioxidants did not affect Al uptake by MH, protect MH from lipid peroxidation or decrease the reactive iron content of MH. Although antioxidants protected Al loaded MH from injury the mechanisms of this effect are unknown.     

Synthesis of transcobalamin II by cultured human hepatocytes

Cultured HepG2 cells, derived from a human hepatoma synthesized and released unsaturated, immunoreactive transcobalamin II. Synthesis was confirmed by the blocking with inhibitors of protein synthesis and by incorporation of tritiated leucine into transcobalamin II.