The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis.

Gap junctions are channels in plasma membrane composed of proteins called connexins. These channels are organized in special domains between cells, and provide for direct gap junctional intercellular communication (GJIC), allowing diffusion of signalling molecules <1 kD. GJIC regulates cell homeostasis and notably the balance between proliferation, cell cycle arrest, cell survival and apoptosis. Here, we have investigated benzo[a]pyrene (B[a]P) effects on GJIC and on the subcellular localization of the major protein of gap junction: connexin-43 (Cx43). Our results showed that B[a]P increased GJIC between mouse hepatoma Hepa1c1c7 cells via translocation of Cx43 from Golgi apparatus and lipid rafts into gap junction plaques. Interestingly, inhibition of GJIC by chlordane or small interference RNA directed against Cx43 enhanced B[a]P-induced apoptosis in Hepa1c1c7 cells. The increased apoptosis caused by inhibition of GJIC appeared to be mediated by ERK/MAPK pathway. It is suggested that B[a]P could induce transfer of cell survival signal or dilute cell death signal via regulation of ERK/MAPK through GJIC.

Modulation of alkaline phosphodiesterase I in cultured rat hepatocytes.

Alkaline phosphodiesterase I activity was measured in adult and foetal rat hepatocytes maintained in primary culture under various conditions. This enzyme was found to be expressed in both cell populations and could be resolved into two bands having apparent molecular weights of 130,000 and 250,000, respectively. Alkaline phosphodiesterase I activity was already at high levels in 15 day foetal liver and, as early as the 19th day of gestation, it reached adult levels. Alkaline phosphodiesterase I levels were well maintained during culture. In the absence of serum, its level continued to increase with time in foetal cells. It dramatically increased by days 4 and 5, in adult cells maintained on fibronectin and plastic, respectively. Dexamethasone stimulated alkaline phosphodiesterase I activity after a lag phase of 8 h, with a maximum reached after 40 h. As this induction was prevented by addition of actinomycin D or cycloheximide, it could be concluded that it required RNA and protein synthesis. Only the major Mr 250,000 form responded to dexamethasone and was sensitive to serum.

Chronic iron overload inhibits protein secretion by adult rat hepatocytes maintained in long-term primary culture.

Short-term pure cultures and long-term cocultures of adult rat hepatocytes with rat liver epithelial cells, presumably derived from primitive biliary cells, were used to define in vitro models of iron overloaded hepatocytes in order to understand the molecular mechanism responsible for liver damage occurring in patients with hemochromatosis. In vitro iron overload was obtained by daily addition of ferric nitrilotriacetate to the culture medium. A concentration of 20 microM ferric salt induced hepatocyte iron overload with minimal cytotoxicity as evaluated by cell viability, morphological changes of treated cells and cytosolic enzyme leakage into the culture medium. The effects of iron overload on protein biosynthesis and secretion were studied in both short-term pure cultures and long-term cocultures of hepatocytes. The amounts of intracellular and newly synthesized proteins were never modified by the iron treatment. Furthermore, neither the relative amounts of transferrin and albumin mRNAs nor their translational products were altered by iron overload. Moreover, no change in the transferrin isomeric forms were observed in treated cells. In contrast, a prolonged exposure of cocultured hepatocytes to 20 microM ferric salt led to a significant decrease in the amount of proteins secreted in the medium. This decrease included the two major secreted proteins, namely albumin and transferrin, and probably all other secreted proteins. These results demonstrate that iron loading alters neither the total nor the liver specific protein synthesis activity of cultured hepatocytes. They suggest that chronic overload may impede the protein secretion process.

Immunohistological analysis of glutathione transferase A4 distribution in several human tissues using a specific polyclonal antibody.

We examined the cellular distribution of glutathione transferase A4 (GSTA4) in various human tissues by indirect immunoperoxidase using a specific polyclonal antibody raised in rabbit. This enzyme was localized in hepatocytes, bile duct cells, and vascular endothelial cells in liver, upper layers of keratinocytes and sebaceous and sweat glands in skin, proximal convoluted tubules in kidney, epithelial cells of mucosa and muscle cells in colon, muscle cells in heart, and neurons in brain. Staining was increased in pathological situations such as cirrhosis, UV-irradiated skin, and myocardial infarction and was strongly decreased in hepatocellular carcinoma. These results strongly support the view of a close correlation between cellular GSTA4 localization and the formation of reactive oxygen species in the tissues investigated.

Ultrastructural indirect immunolocalization of transferrin in cultured rat hepatocytes permeabilized with saponin.

Transferrin was localized in 48-hr cultured adult rat hepatocytes by indirect immunoperoxidase following paraformaldehyde--glutaraldehyde fixation and the use of saponin as a membrane permeabilizing agent. The protein, present in all the parenchymal cells in variable amounts, was found to be specifically located in the endoplasmic reticulum and Golgi apparatus. These results are consistent with recent reports claiming that all adult hepatocytes may synthesize a given liver plasma protein at a given time. The procedure used in this study should be particularly useful for the detection of intracellular antigens in various intact cell types.

A procedure for light and electron microscopic intracellular immunolocalization of collagen and fibronectin in rat liver.

Experimental conditions have been designed that permit both extracellular and intracellular immunolocalization of various collagen types and fibronectin in rat liver. The procedure involves paraformaldehyde fixation by perfusion of the organ, use of saponin as a membrane permeabilizing agent, and visualization of the matrix components by indirect immunoperoxidase. Intracellular demonstration of collagens was particularly sensitive to the composition of the fixative and the duration of fixation. Hepatocytes contained fibronectin and types I and IV collagen, whereas fat-storing and endothelial cells evidenced type III collagen in addition. All the components were specifically located in the endoplasmic reticulum and/or the Golgi apparatus.

Immunocytochemical evidence for the maintenance of cytochrome P-450 isozymes, NADPH cytochrome C reductase, and epoxide hydrolase in pure and mixed primary cultures of adult human hepatocytes.

Specific polyclonal antibodies were used to investigate the distribution of two cytochrome P-450 isozymes (5 and 8), NADPH cytochrome c reductase, and epoxide hydrolase in adult human hepatocytes cultured alone or co-cultured with rat liver epithelial cells. The enzymes were localized by the indirect immunoperoxidase technique following fixation with a paraformaldehyde-glutaraldehyde mixture and membrane permeabilization with saponin. The pattern of distribution of the four enzymes after 24 hr in culture was similar to that found in vivo. Virtually all the hepatocytes exhibited nearly homogeneous positive staining for cytochrome P-450-8, whereas only 60-80% were positive for cytochrome P-450-5. Nearly homogeneous staining was also observed in all hepatocytes for NADPH cytochrome c reductase and epoxide hydrolase. During the first 12 days in pure culture, the intensity of staining, as well as the number of positively stained cells, decreased slightly except for epoxide hydrolase, which did not show any obvious change. In contrast, even after 15 days in co-culture the extent of staining for all the enzymes decreased less than in pure culture. These results indicate that adult human hepatocytes continue to express specific drug-metabolizing enzymes for several days in culture and provide further evidence that those cells are more stable than rodent hepatocytes in primary culture.

Intracellular pH alterations induced by tacrine in a rat liver biliary epithelial cell line.

1. The effects of tacrine (THA) on intracellular pH (pH(i)) were examined in a rat liver biliary epithelial cell line (RLEC) in HEPES-buffered medium. pH(i) was recorded using the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphtorhodafluor). 2. In the steady state, short-term exposures to THA resulted in alkalinization and re-acidification at 0.1 and 0.25 mM. Following a 24 h-treatment, no significant difference in pH(i) could be detected at 0.1 and 0.25 mM THA, whereas at 0.05 mM, pH(i) was slightly more acid (7.17+/-0. 02, n=16 versus 7.21+/-0.02, n=24 [control]). 3. In control and short-term treated cells, intracellular intrinsic buffering power (beta(i)) increased roughly linearly as pH(i) decreased. This dependence was not seen following long-term treatment. In all cases, beta(i) was increased by THA (by 1.6 to 3.5 fold). 4. Following an acid load (induced by 20 mM NH(4)Cl removal), pH(i) recovery in RLEC relied upon Na(+)/H(+) exchange. A short-term treatment (0.25 mM THA) did not affect total acid extrusion. In contrast, a 24 h-treatment with 0.05 mM THA reduced it (by approximately 36% at a pH(i) of 6.73) while at 0.25 mM, a large increase was detected (by approximately 109% at a pH(i) of 6.75). In Na(+)-free medium, THA (0. 25 mM) still induced an alkalinization in the steady state. Following an acid load, THA stimulated a Na(+)-independent acid efflux in a dose-dependent manner, inhibitable by alpha-cyano-4-hydroxy cinnamate (CHC, 4 mM) but not by quercetin (0. 125 mM). 6. In conclusion, this work demonstrates that THA affects pH(i) in RLEC, through a decrease in Na(+)/H(+) exchange and an increase in beta(i). Stimulation of a CHC-inhibitable, Na(+)-independent acid efflux is also detected.

Human hepatocytes express trifluoroacetylated neoantigens after in vitro exposure to halothane.

Biotransformation of anaesthetic halothane by cytochrome P450-dependent monooxygenases resulted in the production of reactive intermediate trifluoroacetyl (TFA) halide, capable of covalently binding to hepatocyte proteins. TFA-modified liver proteins can act as antigens and are implicated in the pathogenesis of halothane hepatitis in humans. The aim of this study was to investigate the formation of TFA-neoantigens in halothane-treated primary cultures of adult human hepatocytes and to evaluate the usefulness of this in vitro model for studying immune-mediated halothane hepatotoxicity. Cultured human hepatocytes were incubated with halothane under constant temperature, atmosphere and anaesthetic concentration conditions. The results obtained show that halothane-treated hepatocytes isolated from seven different donors produced TFA-antigens as detected by immunocytochemical and western immunoblot analysis using rabbit anti-TFA antiserum. TFA-adducts were localized mainly in the endoplasmic reticulum and in small amounts on the plasma membrane of parenchymal cells. By immunoblotting, several neoantigens, with molecular masses from 42 to 100 kDa, were detected in halothane-exposed hepatocytes. These observations are consistent with the formation of TFA-adducts through metabolism of the anaesthetic and suggest that primary cultures of human hepatocytes represent a suitable in vitro model to study the pathogenesis of immune-mediated halothane hepatotoxicity.

Irniine, a pyrrolidine alkaloid, isolated from Arisarum vulgare can induce apoptosis and/or necrosis in rat hepatocyte cultures.

The effects of irniine, a pyrrolidine alkaloid extracted from the tubers of Arisarum vulgare, on rat hepatocyte primary cultures and rat liver epithelial cell line (RLEC) were studied. Cytotoxicity was first evaluated by LDH release, MTT and NR tests and MDA production, while cellular alterations were visualized by electron microscopy and DNA gel-electrophoresis. In hepatocyte and RLEC cultures, a major toxicity appeared at 40 microM of irniine and was demonstrated by an increase in LDH release and decreases in MTT reduction and NR uptake while concentrations lower than 40 microM did not induce significant changes in these parameters. However, we observed an increase in MDA production at 30 microM. Important alterations of the nuclei and mitochondria were also visualized by electron microscopy in cells treated with 50 microM. Using DNA gel-electrophoresis, we demonstrated that irniine at 40 and 50 microM induced DNA damage. All together these results demonstrate that: (1) Irniine induces a significant hepatotoxicity. (2) Irniine toxicity is not mediated by a metabolic derivative since RLEC, which do not contain a monooxygenase system, were also affected by this compound. (3) Irniine induces a significant DNA damage and oxidative stress which leads to cell death by necrosis and/or by apoptosis. Moreover, our data suggest that the alkaloid irniine contained in A. vulgare may be involved in the toxic symptoms observed after medicinal use or consumption of the plant tubers as food both by humans and animals.

Essential fatty acid pattern of glycerolipids in rat hepatocytes in primary culture and in coculture with rat liver epithelial cells.

The linoleic acid content of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and triglyceride (TG) rapidly fell in rat hepatocytes in primary culture up to four days and in coculture with liver epithelial cells up to eight days. At the same time, the level of polyunsaturated fatty acids (PUFA), especially arachidonic acid, remained constant in PE, slightly decreased in PC and dropped in TG. There was no variation of the nonessential PUFA, 20:3n-9. Linoleic acid supplementation of cultures 24 hr before the harvest induced a rise in the linoleic acid level of the three lipid classes. Arachidonic acid remained constant in TG and only slightly decreased in PE and PC at day 4 of primary culture and day 8 of coculture. The level of 20:3n-9 increased in PE and PC and much more in TG. This net increase in the arachidonic acid and 20:3n-9 levels in TG could not be explained only by a transfer from the phospholipid pools of PUFA because the phospholipid content of hepatocytes and PUFA levels of phospholipids did not vary under linoleic supplementation. The low percentage of arachidonic acid in epithelial cells rules out any participation of these cells in the increase of arachidonic acid in supplemented cocultures. Triglycerides may act as a storage pool for plasma PUFA up to four days of primary culture and eight days of coculture. Besides, coculture seems more potent than primary culture to maintain the phospholipid level, to spare the essential PUFA in PE and to increase the TG synthesis in response to linoleic acid supplementation.

Ultrastructural localization of the major components of the extracellular matrix in normal mouse liver.

We have investigated the extracellular and intracellular distribution of the major components of the hepatic extracellular matrix: types I and IV collagen (CI and CIV), type III procollagen (PCIII), fibronectin (FN) and laminin (LM) in the normal mouse liver by electron microscopy, using the indirect immunoperoxidase method. Extracellular localization of CI and PCIII revealed the typical collagen fibrils and small bundles in portal tracts and Disse's space. Faint extracellular deposition of CIV was detectable in basement membranes of portal structures and as discontinuous precipitates in Disse's space. FN strongly reacted in Disse's space and formed a continuous layer as well as strands between hepatocytes and endothelial cells. Moderate LM reaction was seen along the sinusoids in discrete spots and in basement membranes surrounding bile ducts and blood vessels. The cellular source of these components was demonstrated to be of mixed origin. Hepatocytes synthesized very little CI and large amounts of FN. Additionally, CI was produced by periportal fibroblasts. Fat-storing cells clearly participated in the production of PCIII, CIV and LM. LM synthesis was also found in bile duct cells and the cells of Hering's canal.

Replication of hepatitis B virus in differentiated adult rat hepatocytes transfected with cloned viral DNA.

The possibility of obtaining expression of human hepatitis B virus (HBV) genes and production of virus particles in normal liver cells from heterologous species like normal adult rat hepatocytes, by transfecting the complete HBV genome, was investigated. Various techniques for hepatocyte transfection were assayed including the usual calcium-phosphate coprecipitation technique, the Pasco and Fagan modified calcium-phosphate procedure, and the lipofection technique. Transfection efficiency was determined by measuring the production of HBV surface antigen under various culture conditions. Transfection was the most efficient when assayed 1 or 2 days after hepatocyte plating at low density. Few variations in the efficiency were observed between the different transfection procedures. We show that under these culture conditions, replication of HBV can be achieved in differentiated adult rat hepatocytes. Synthesis of relaxed circular and single-stranded DNA forms and of viral transcripts including pregenome RNA occurred in the cells whereas viral antigens and mature and immature viral particles were released into the culture medium. The production of viral proteins was always higher in hepatocytes cocultivated with rat liver epithelial cells and maintained at a low density. In contrast, viral replication was not obtained by transfecting undifferentiated rat liver epithelial cells. These results demonstrate that replication of HBV can occur in hepatocytes from mammalian species non-closely related to primates and strongly support the idea that attachment of the virus and its penetration into the cells are critical steps in the host-specificity of the infection process and that hepatic-specific regulating factors could be essential for viral replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of hepatocyte co-cultures in the assessment of drug toxicity from chronic exposure.

1. In rat hepatocyte cultures, clonidine and malotilate were cytotoxic at 170 microM but did not induce changes at lower concentrations during 24 h exposure. Amitriptyline induced cell injury at 20 microM but was ineffective at 2 microM. 2. In co-culture of rat or human hepatocytes with rat liver epithelial cells, 2 microM amitriptyline was cytotoxic after 7 days treatment whereas 70 to 100 microM clonidine or malotilate gave no significant effect. 3. These results suggest that co-cultured hepatocytes which retain their differentiated state for several days or weeks, represents a promising tool for studying hepatotoxicity from chronic treatment in vitro.

Use of hepatocyte cultures for the study of hepatotoxic compounds.

Human and animal hepatocytes in primary culture are widely used in pharmacotoxicological research. They represent a unique in vitro model since they retain both phase I and phase II enzyme activities as well as their inducibility by xenobiotics. Hepatocyte cultures are used for drug screening, identification of the lesions induced by toxic compounds and determination of mechanisms by which xenobiotics exert liver injury.

Cell types involved in the expression of foetal aldolases during rat azo-dye hepatocarcinogenesis.

Cellular and subcellular immunolocalization of aldose isozymes and alpha-foetoprotein (AFP) was performed in rat liver during the different stages of carcinogenesis induced by 3'-methyl-4-dimethylaminoazobenzene. During the early stages, double-labelling experiments showed that oval and transitional cells that expressed foetal aldolases did not contain adult aldolase B; this isozyme was only found in small and "normal' hepatocytes. AFP was present in transitional cells and in small hepatocytes. During hyperplastic nodule development, neither foetal aldolases nor AFP were located in hepatocytes. These foetal proteins were still observed in transitional cells. In hepatocellular carcinomas, both foetal proteins (aldolase isozymes and AFP) and adult aldolase B were present in malignant cells. Moreover, during the different stages foetal aldolases were also found in sinusoidal cells. These results indicate that, during azo-dye hepatocarcinogenesis, (a) several cell types synthesize foetal aldolases: oval and transitional cells, hepatoma cells and sinusoidal cells; (b) only hepatoma cells and not hepatocytes located in hyperplastic nodules can express both foetal and adult aldolases. This suggests that in primary, as in transplanted, hepatoma the resurgence of foetal isozymes is the consequence of a disturbance of control gene expression.

Halothane-induced cytotoxicity to rat centrilobular hepatocytes in primary culture is not increased under low oxygen concentration.

BACKGROUND: Halothane can be metabolized by both oxidative and reductive pathways in the liver. This anesthetic can induce direct liver injury preferentially localized in centrilobular areas, probably in relation with lower oxygen tension. The reductive pathway has been related to liver damage; however, a correlation between lower oxygen concentration in centrilobular areas, the extent of reductive metabolism of halothane, and the degree of liver injury has not yet been demonstrated. This study was designed to better evaluate the toxicity of the reduced metabolites by using centrilobular and periportal rat hepatocyte subpopulations. METHODS: Adult rat hepatocytes, either as whole cell preparations or after separation in centrilobular and periportal cell subpopulations, were placed in primary culture and exposed to either 2% or 4% halothane under various oxygen concentrations. The enriched centrilobular hepatocyte subpopulations isolated by the digitonin-collagenase method were characterized by immunolocalization of glutamine synthetase. Three oxygen concentrations were tested: 5%, 20%, and 95%, and the main parameters measured were cell viability and fluoride ion formation. RESULTS: Viability of centrilobular hepatocytes was similar under 5% and 20% O2, but the unpurified hepatocyte population was more susceptible to 5% O2 (P < 0.01). Significantly higher cytochrome P-450 content was found in whole hepatocyte populations under 5% versus 20% oxygen, indicating that centrilobular hepatocytes that contained higher cytochrome P-450 monooxygenase activities were less sensitive to low oxygen concentrations. Halothane toxicity to centrilobular hepatocytes was enhanced under 95% versus 20% O2 (P < 0.05). By contrast, no significant difference was observed when the cells were maintained under 5% O2, although fluoride ions, indicative of reductive metabolism of halothane, were found in much higher amounts in the culture medium. Moreover, under 20% O2, halothane toxicity was significantly greater in centrilobular versus unpurified hepatocytes (P < 0.05). CONCLUSIONS: Isolated centrilobular hepatocytes appear to be more sensitive to halothane than their periportal counterparts in vitro. However, the authors' results support the conclusion that increased reductive metabolism of halothane induced by decreasing oxygen concentration is not a critical parameter for the occurrence of liver damage in these cells.

Toxic effects of tacrine on primary hepatocytes and liver epithelial cells in culture.

Administration of tacrine (THA) for the treatment of Alzheimer's disease results in a reversible hepatotoxicity in 30-50% of patients, as indicated by an increase in transaminase levels. However, the intracellular mechanisms underlying such a toxicity have not yet been elucidated. In this study, we performed short-term and long-term in vitro treatments on primary human and rat hepatocyte cultures as well as on nonparenchymal rat liver epithelial cells (RLEC), known as CYP1A-deficient cells. Cell ultrastructure was analyzed under different conditions and the release of lactate dehydrogenase (LDH) was used to evaluate cytotoxicity. The effects of THA on protein synthesis, intermediary metabolism and reduced glutathione (GSH) level were also determined in rat hepatocytes. THA induced dose-dependent toxic effects in liver parenchymal and nonparenchymal cells, with human hepatocytes being less sensitive. This toxicity appeared to be unrelated to metabolism of THA since similar effects were observed in rat hepatocytes and RLEC, in which THA metabolism was found negligible. Ribosome aggregation appeared only at high concentrations (> 1 mmol/L) and was not specific to hepatocytes. Therefore, the THA-induced decrease in protein synthesis observed at lower concentrations was likely not related to this alteration. ATP and glycogen levels as well as GSH content were reduced upon THA. However, while glycogen level decreased at THA doses similar to those inducing an increase in LDH release, the fall in ATP and GSH contents occurred at higher doses. Thus, glycogen level in hepatocytes appeared to be a more sensitive indicator of THA toxicity than were ATP and GSH levels. We also found that protein synthesis started to decrease at THA doses that were still ineffective on LDH release. This might suggest that the decrease in synthesis of one or several proteins upon THA treatment represents the early signal leading cells to death.

Distribution and cellular origin of collagen VI during development and in cirrhosis.

Collagen VI is a ubiquitous microfibrillar collagen that forms a network in most interstitial connective tissues, including soft organs and cartilage. The extracellular and intracellular distribution of collagen VI in human liver was studied by light and electron microscopy using the indirect immunoperoxidase method. In normal adult liver, collagen VI was seen mainly in portal spaces and formed a continuous layer in the sinusoids. Fetal liver contained more of collagen VI in the sinusoid than newborn and adult livers. In alcoholic fibrotic and cirrhotic livers, collagen VI antibodies intensely stained fibrous septa that invaded the lobule. Immunoelectron microscopy on normal liver showed that collagen VI antibodies labeled microfibrillar material and occasionally the surface of cells including hepatocytes. In both perinatal and fibrotic livers, electron-dense deposits were abundant in the space of Disse, intensely staining fibrils located around bundles of banded collagen. In both normal and fibrotic adult livers, collagen VI was abundant in the rough endoplasmic reticulum of Ito cells, while hepatocytes were constantly negative. In fetal livers, hepatocytes also contained collagen VI. These results suggest that collagen VI is a major constituent of the hepatic extracellular matrix. Furthermore, the cellular sources of collagen VI appear to be different in adult and developing livers.

Oval cell proliferation in early stages of hepatocarcinogenesis in simian virus 40 large T transgenic mice.

In transgenic mice bearing the Simian Virus 40 large T antigen under the control of the human antithrombin III regulatory sequences, a stepwise progression toward hepatocellular carcinoma is observed. We have used two monoclonal antibodies (A6 and G7) developed against a surface antigen expressed in oval cells from dipin-treated mice, to analyze the emergence of such preneoplastic populations in the livers of antithrombin III Simian Virus 40 T transgenic mice. We show that a unique population of small heterogeneous epithelial cells, which probably corresponds to oval and/or transitional cells according to their morphological features, consistently appears at approximately the 10th week after birth and proliferates thereafter. This oval cell-like population stained positively for A6 and G7 monoclonal antibodies. Furthermore, different subpopulations usually recognized as possible precursors of carcinoma cells including hyperplastic foci and neoplastic nodules as well as carcinoma cells, were also positive for A6 but not G7 monoclonal antibodies. Stimulation of cell proliferation by partial hepatectomy performed at the time of emergence of the oval-like cells resulted in a rapid increase in the number of oval/transitional A6-positive cells. Our findings support the view that a common mechanism may be involved in the development of carcinomas that are induced by chemical carcinogens and in transgenic mice expressing a potent oncogene under the control of a hepatic specific promoter. In addition, our findings demonstrate a specific precursor-product relationship between the appearance of the oval/transitional cells and the development of neoplastic hepatocytes in this transgenic model.