Differential sensitivity of human hepatoma cell line and primary rat hepatocyte culture to benzo(a)pyrene-induced unscheduled DNA synthesis and adduct formation.

We studied the genotoxic potential of a carcinogen in the human hepatoma cell line, HepG2 and in primary rat hepatocyte culture. HepG2 is a well differentiated human hepatoblastoma cell line with biotransforming capacity. Rat hepatocytes were obtained by the standard two-step in situ perfusion technique. Following benzo(a)pyrene treatment, both HepG2 and primary rat hepatocyte culture showed unscheduled DNA synthesis with different sensitivity. In ³²P-postlabelling analysis, the chromatogram revealed quantitative and qualitative differences between HepG2 and primary rat hepatocyte cultures when treated with 10 μM benzo(a)pyrene for 18 hr. The results have demonstrated that the HepG2 cell line may be used in addition to primary rat hepatocytes in risk assessment for detection of environmental carcinogens.     

Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium.

A series of simian virus 40 (SV40)-immortalized hepatocyte cell lines were characterized for albumin production, the regulation of albumin production, and the expression of other liver-specific genes. This series of cell lines is particularly useful for studying the regulation of hepatocyte gene expression because the cell lines express liverlike levels of a number of liver-specific functions and do so while growing in a chemically defined medium. SV40-immortalized hepatocyte cell lines were derived from colonies of albumin-producing epithelial cells that arose after primary hepatocytes maintained in chemically defined medium were transfected with SV40 DNA. Some cell lines secreted albumin at levels equal to or greater than those secreted by freshly plated primary hepatocytes, and all but one line continued to produce albumin for more than 20 passages. The variation in albumin secretion among cell lines reflected differences in the amount of albumin produced per cell and not in the percentage of albumin-producing cells in each line. The characterization of selected cell lines showed that albumin production was regulated by cell density during the growth cycle. Albumin production in most cell lines was also regulated by dexamethasone; however, one cell line continued to produce high levels of albumin when the cells were grown in medium lacking dexamethasone, demonstrating that although glucocorticoid can induce albumin production in some cell lines, it is not required for high levels of albumin production by all cells in culture. Regulation of albumin production measured at the level of protein secretion was paralleled by changes in steady-state levels of a 2.3-kilobase albumin RNA. Albumin-producing SV40-immortalized hepatocytes secreted a variety of other plasma proteins, including transferrin, hemopexin, and the third component of complement. These cells also expressed tyrosine aminotransferase activity that was inducible by dexamethasone. Alpha-fetoprotein production was not detected in any of the cell lines examined.     

Adipogenic activity produced by hepatocyte-derived cell lines and by normal hepatocytes in primary culture.

Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh-7, PLC/PRF/5, and SK-Hep-1 do not contain adipogenic activity. Conditioned medium from HepG2 cells also stimulated differentiation of 3T3-L1 cells and of rat epididymal adipocyte precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treatment at 100 degrees C, with protease, with 2-mercaptoethanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity. In contrast, medium conditioned by primary culture of nonhepatocyte cell also isolated from liver was deprived of this activity. The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.     

Ethanol inhibits hormone stimulated hepatocyte DNA synthesis

Insulin, glucagon, and epidermal growth factor (EGF) addition stimulated DNA synthesis in primary hepatocyte cell cultures prepared from adult rat liver. The addition of ethanol (20-200mM) to the culture medium resulted in a substantial reduction in DNA synthesis as measured by 3H-thymidine incorporation and autoradiography. This effect was specific for differentiated hepatocytes compared to fibroblasts and two other human hepatoma cell lines. These studies demonstrate in a cell culture system that one of the major properties of ethanol is the inhibition of hepatocyte DNA synthesis.     

Differential regulation of insulin-like growth factor-II receptors in rat hepatocytes and hepatoma cells

This study compares the regulation of IGF-II receptors in three rat hepatoma lines, HTC, H-35 and 5123tc, and primary rat hepatocytes. In all cell types [125I]IGF-II bound solely to a species of approximately 250 kDa. Cell surface IGF-II receptors in hepatoma cells had slightly lower affinities (1-2 liters/nmol) than in hepatocytes (4 liters/nmol), but slightly higher IGF-I cross-reactivity (2-4% compared to 1% in hepatocytes). In confluent cultures, the three hepatoma lines expressed 5- to 15-fold more cell-surface receptors per cell than hepatocytes. However, while hepatocyte receptors showed marked inverse density-dependence, increasing over 6-fold between dense (3 x 10(5) cells/3.8 cm2) and sparse (0.16 x 10(5) cells/3.8 cm2) cultures, receptors in all hepatoma lines remained at a constant high level regardless of culture density. These distinct regulatory patterns resemble those described for growth-related functions in hepatocytes and hepatoma cells, and are thus consistent with a role for IGF-II receptors in liver cell proliferation.     

Hepatic progenitor cells

Liver diseases are associated with a marked reduction in the viable mass of hepatocytes. The most severe cases of liver disease (liver failure) are treated by orthotopic liver transplantation. One alternative to whole organ transplantation for patients with hepatic failure (and hereditary liver disease) is hepatocyte transplantation. However, there is a serious limitation to the treatment of liver diseases either by whole organ or hepatocyte transplantation, and that is the shortage of organ donors. Therefore, to overcome the problem of organ shortage, additional sources of hepatocytes must be found. Alternative sources of cells for transplantation have been proposed including embryonic stem cells, immortalised liver cells and differentiated cells. One other source of cells for transplantation found in the adult liver is the progeny of stem cells. These cells are termed hepatic progenitor cells (HPCs). The therapeutic potential of HPCs lies in their ability to proliferate and differentiate into hepatocytes and cholangiocytes. However, using HPCs as a cell therapy cannot be exploited fully until the mechanisms governing hepatocyte differentiation are elucidated. Here, we discuss the fundamental cellular and molecular elements required for HPC differentiation to 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.     

Enhanced DNA synthesis in rat hepatoma cells by conditioned media from Kupffer cells incubated with supernatants of tumor necrosis factor-alpha-pretreated hepatocytes.

The effects of tumor necrosis factor-alpha (TNF-alpha) on DNA synthesis in AH66 rat hepatoma cells and rat hepatocytes were analysed by means of [3H]thymidine incorporation. DNA synthesis in AH66 cells was suppressed when AH66 cells were directly incubated with TNF-alpha. When primary culture of rat Kupffer cells was incubated with hepatocyte conditioned media pretreated with TNF-alpha (0-200 U/ml), and AH66 cells were then treated with these hepatocyte/Kupffer cell-conditioned media, TNF-alpha used in the pretreatment caused a dose-dependent increase in DNA synthesis in AH66 cells with a maximum effect amounting to a more than 10-fold increase. In contrast, DNA synthesis in primary culture of rat hepatocytes was not stimulated by the TNF-alpha-pretreated hepatocyte/Kupffer cell conditioned media. These results suggest that TNF-alpha-mediated hepatocyte-Kupffer cell interaction selectively promotes proliferation of rat hepatoma cells.     

The effects of transformation and ZnT-1 silencing on zinc homeostasis in cultured cells

We have previously demonstrated that reducing the availability of zinc with the extracellular chelator diethylenetriaminepentaacetic acid (DTPA) promotes efflux of (65)Zn from rat primary hepatocytes and pituitary cells, but increases retention of label in rat hepatoma (H4IIE) and anterior pituitary tumor (GH3) cell lines. To further understand this differential response between primary cells and the corresponding cancer cell lines, we investigated the effects of immortalizing primary cells on their zinc homeostasis. Rat primary hepatocytes were electroporated with the SV40 large T-antigen-coding plasmid pSV3-neo and selected for neomycin resistance. This resulted in cell division of the normally quiescent hepatocytes. When these cells were prelabeled with (65)Zn, DTPA decreased efflux of (65)Zn, similarly to hepatoma cells and differently from primary hepatocytes. This homeostatic change may be required to account for the greater zinc requirements of dividing cells and be mediated by alterations in the activity of zinc transporter ZnT-1, which is responsible for zinc efflux. To further understand the mechanism of DTPA-induced zinc retention, we down-regulated the expression of ZnT-1 in rat hepatoma cells using vector-based short hairpin RNA interference. Expression of ZnT-1 protein was reduced to approximately 50%. Down-regulation of ZnT-1 resulted in greater retention of (65)Zn in control cells. However, DTPA increased rather than decreased efflux of label from knockdown cells, suggesting that functional ZnT-1 is required for the decreased efflux in response to DTPA. We conclude that ZnT-1 expression is crucial for maintaining zinc homeostasis, in particular, for the enhanced retention of zinc in transformed cells when subjected to zinc deprivation.     

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two‐dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell‐specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three‐dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen‐coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver‐cell‐specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver‐specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real‐time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well‐preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF‐4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re‐established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO‐1‐positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes. Biotechnol. Bioeng. 2011; 108:141–150. © 2010 Wiley Periodicals, Inc.     

Mechanisms responsible for long-term survival of adult rat hepatocytes in the presence of phenobarbital in primary culture

The mechanisms, by which phenobarbital (PB) supports the survival of adult rat hepatocytes in primary culture, were investigated. PB altered the shape of rat erythrocytes to produce cup-formed cells and protected them from hypotonic hemolysis. Anesthetics (ketamine, lidocaine, mepivacaine, and bupivacaine) and an anti-inflammatory agent (indomethacin), which are also known to protect erythrocytes from hypotonic hemolysis by stabilizing their membranes, efficiently supported the survival of hepatocytes in primary culture. Furthermore, the well-known biological membrane stabilizers, such as cholesterol and vitamin E, also showed the maintenance effect on primary cultured hepatocytes. PB effectively reduced the leakage of lactate dehydrogenase from hepatocytes caused by chenodeoxycholic acid in primary culture. Rotenone and amobarbital, which act repressively on the PB-sensitive site in the respiratory chain and are known to inhibit the mitochondrial formation of active oxygen species with NAD-linked substances, effectively prolonged the hepatocyte survival in primary culture. Elevation of oxygen tension in primary culture remarkably decreased the hepatocyte survival rate, which was preserved by addition of antioxidant substances, such as vitamin C, vitamin E, bifemelane, selenite, and superoxide dismutase. On the other hand, in the presence of PB, the hepatocyte survival rate hardly changed with the elevation of oxygen tension. From these findings, it seems that PB stabilizes the hepatocyte membranes and reduces the mitochondrial formation of active oxygen species and that the stabilized functions of membrane and the reduction of oxidative stress result in the prolonged survival of hepatocytes in primary culture.     

Characterization of the effects of human placental HGF on rat hepatocytes.

Hepatocyte growth factor (HGF) also known as hepatopoietin A (HPTA) (Michalopoulos, FASEB J., 4:176-187, 1990) is a heparin-binding growth factor whose characterization and tissue distribution have been reported elsewhere. This growth factor was recently cloned and its amino acid sequence determined under the name of hepatocyte growth factor (HGF) (Miyazawa et al., Biochem. Biophys. Res. Commun., 163:967-973, 1989; Zarnegar et al., Biochem. Biophys. Res. Commun., 163:1370-1376, 1989; Nakamura et al., Nature, 342:440-443, 1989). Human placenta is one of the tissues that contains significant amounts of HGF. We isolated HGF from human placenta and characterized its biologic effect on rat hepatocytes. Human placenta HGF was isolated in high purity as a single chain molecule. Single chain HGF stimulated DNA synthesis in primary rat hepatocyte cultures in serum-free medium. The maximal effect was seen at 5-10 ng/ml. The maximal response occurred at 25-48 hours after plating of the hepatocytes. Protein synthesis was also stimulated by HGF in primary rat hepatocyte cultures. There were peak responses at 19-24 and 37-42 hours after plating of the hepatocytes. TGF beta 1 inhibited more than 95% of HGF-induced DNA synthesis but only 25% of HGF-induced protein synthesis. HGF interacted in an additive manner with EGF, a well-known hepatocyte mitogen. There was not an additive interaction between HGF and aFGF. Regenerating liver hepatocytes obtained from rats which underwent two-thirds partial hepatectomies (PHX) also responded to HGF in a dose-dependent manner as the hepatocytes from normal liver.     

Ferritin—a mediator of apoptosis?

Previously we have demonstrated an apoptosis inducing activity for a rat hepatocyte conditioned medium (CM) presumably mediated by acidic isoferritins. Here, we present support for this assumption since isoferritins purified from different rat hepatocyte CM significantly enhanced the frequency of apoptotic cells in primary rat hepatocytes, an effect completely inhibited by a neutralizing anti-H-ferritin antibody. The apoptosis induction appears to be related to a 43 kDa ferritin subunit contained in the isoferritins released from primary hepatocytes, presumably representing a ferritin heavy/light chain heterodimer. In addition, these isoferritins immunologically crossreact with antibodies raised against placental isoferritin p43-PLF (which also contains a 43 kDa ferritin subunit) and melanoma-derived H-chain ferritin, representing ferritin isoforms which reveal immunomodulatory properties. Furthermore, p53 and FasL are upregulated upon isoferritin treatment in a time dependent mode, and apoptosis induction can be suppressed by neutralizing anti-FasL antibodies. Proapoptotic Bid is upregulated too and translocated into mitochondria in primary hepatocytes exposed to the isoferritins purified from the CM. Finally, epidermal growth factor (EGF) and dexamethasone (DEX), which counteract proapoptotic mitochondrial signalling, almost completely abolished the proapoptotic effect of the hepatocyte derived isoferritins. In conclusion, our findings demonstrate that acidic isoferritins with homology to immunomodulatory ferritin isoforms (p43-PLF, melanoma-derived-H-chain ferritin) are released from hepatocytes in vitro, and are able to stimulate upregulation of p53 and mediate apoptosis involving Fas (CD95) signalling as well as addressing the intrinsic mitochondrial proapoptotic pathway.     

Cultivation of the exoerythrocytic stage ofPlasmodium berghei in primary cultures of mouse hepatocytes and continuous mouse cell lines

Summary Plasmodium berghei exoerythrocytic (EE) stages have been cultured in vitro in human continuous cell lines and primary cultures of both human and rat hepatocytes. Although the predominant experimental model of irradiated sporozoite-induced protective immunity is the mouse,P. berghei has not been cultivated in primary mouse hepatocytes or in continuous mouse lines. Because of this, target cells are not available for determining if these immunized mice produce cytotoxic T lymphocytes (CTLs) that recognizeP. berghei antigens expressed on hepatocytes in the context of class I major histocompatability (MHC) antigens. We report the development of methods for cultivating the (EE) stage ofP. berghei in murine hepatocytes and in two cell lines derived from the livers of BALB/c mice; one line produced from a primary hepatocyte culture and the other produced by fusion of mouse hepatocytes with a continuous rat liver line. Mature parasites were detected by microscopy and by DNA probe in both cell lines, each of which supported complete development ofP. berghei liver stages and production of infectious merozoites. Since class I MHC antigens are present on the surface of primary hepatocytes and the mouse X rat hybrid line, these cells can be used to detect cytotoxic T cells against liver stage parasites. This work was supported by the Naval medical Research and Development Command, Bethesda, MD, work unit no. 3M161102B510AK111, ONR contract N00014-83-C-0355, and by contract DPE-0453-C-00-3051-00 of the U.S. Agency for International Development, Washington, D.C. The opinions and assertions herein are not to be construed as official or as reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the “Guide for the Care and Use of Laboratory Animals”, Institute of Laboratory Animal Resources, National Research Council, DHHS, Pub. no. (NIH)85-23