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.

Phenobarbital (PB) alters expression of numerous hepatic genes, including genes of cytochrome P450 2B1 and 2B2 (CYP2B). However, the intracellular mechanisms remain to be fully elucidated. The present study investigated the involvement of mitogen-activated protein kinases (MAPKs) in rat hepatocytes in primary culture. We showed that PB induced an early, dose-dependent activation of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 MAPKs. Regarding the PB (1mM) induction of CYP2B mRNA expression, while chemically inhibiting JNK had no effect, specific inhibitors of the ERK (U0-126) and p38 (SB-203580) pathways up- and down-regulated this expression, respectively. However, although such a regulation was confirmed when testing the effect of a dominant negative mutant of the ERK pathway on the CYP2B2 enhancer-promoter activity, no such transcriptional role was found with the p38 pathway. Moreover, upon arrest of transcription, the stability of CYP2B mRNA remained unaffected by SB-203580. In conclusion, we show that the ERK pathway negatively regulates CYP2B2 enhancer-promoter activity and that, despite p38 activation upon PB exposure, the sensitivity of CYP2B mRNA expression to SB-203580 appears to be unrelated to this kinase.

Expression of the mouse glutathione transferase Alpha 4 (mGSTA4) has been studied during hepatocyte isolation and in cultured hepatocytes. Transient mGSTA4 induction during liver disruption correlated to strong oxidative stress and induction of the Jun N‐terminal kinase (JNK) pathway. Similarly, tumor necrosis factor α induced both JNK phosphorylation and mGSTA4 expression while specific JNK inhibitor JNKI1 prevented these two events and JNK activator anisomycin strongly induced mGSTA4 expression. We also found that endogenous JNK and mGSTA4 co‐immunoprecipitate. A second mGSTA4 induction occurred 2 days after cell seeding concomitantly to DNA replication and was prevented by treatment with mitogen‐activated protein kinase (MEK) inhibitor U0126. Our data demonstrate that mGSTA4 is strongly increased during oxidative stress possibly via JNK pathway and during proliferation via MEK/extracellular signal‐regulated kinase pathway, and suggest that mGSTA4 might be an endogenous regulator of JNK activity by direct binding.

The ubiquitous environmental pollutants polycyclic aromatic hydrocarbons are responsible for important carcinogenic and apoptotic effects, whose mechanisms are still poorly understood, owing to the multiplicity of possible cellular targets. Among these mechanisms, alterations of ionic homeostasis have been suggested. In this work, the effects of benzo(a)pyrene [B(a)P] on pHi were tested in the rat liver F258 epithelial cell line, using the fluoroprobe carboxy-SNARF-1. After a 48-h treatment, B(a)P (50 nM) induced an alkalinization, followed by an acidification after 72 h and the development of apoptosis. Determinations of pH(i) recovery following an acid load showed an increased acid efflux at 48 h. Cariporide inhibited both the early alkalinization and the increased acid efflux, thus suggesting the involvement of Na+/H+ exchanger 1 (NHE1). Besides, alpha-naphtoflavone (alpha-NF), an inhibitor of CYP1A1-mediated B(a)P metabolism, prevented all pH(i) changes, and NHE1 activation was blocked by the antioxidant thiourea, which inhibited CYP1A1 metabolism-dependent H2O2 production. Regarding B(a)P-induced apoptosis, this was prevented by alpha-NF and bongkrekic acid, an inhibitor of mitochondria-dependent apoptosis. Interestingly, apoptosis was significantly reduced by cariporide. Taken together, our results indicate that B(a)P, via H2O2 produced by CYP1A1-dependent metabolism, induces an early activation of NHE1, resulting in alkalinization; this appears to play a significant role in mitochondria-dependent B(a)P-induced apoptosis.

A bstract : How pH i changes, more specifically alkalinization, affect the apoptotic cascade has yet to be determined. The aim of the present work was to test the involvement of mitochondria in the apoptotic cascade triggered by benzo( a )pyrene [B(a)P] and to determine the role of pH i changes and p53 relative to mitochondria. Our results indicate that B(a)P‐induced apoptosis might rely upon a p53‐dependent and a pH‐sensitive mitochondrial dysfunction.

We hypothesized that glutathione transferases could be induced and may participate to cellular defenses against the oxidative stress occurring during liver regeneration. Here, we evidenced that murine GSTA1 (mGSTA1), A4, Pi, and Mu are up-regulated during mouse liver regeneration, exhibiting a biphasic pattern of induction correlating early G(1) phase and G(1)/S transition of the cell cycle. Using confocal microscopy Immunolocalization and subcellular fractionation, mGSTA4 was demonstrated in both mitochondria and cytosol and found preferentially increased in cytosol during liver regeneration. In addition, mGSTA4 was induced in vivo and in cultured hepatocytes by tumor necrosis factor a (TNFalpha), interleukin-6 (IL-6), and epidermal growth factor (EGF), factors that play crucial roles in hepatocyte survival and proliferation during liver regeneration. However, the mitogenic effect of EGF was not responsible for the induction of mGSTA4. In transient transfections, IL-6 and EGF, but not TNFalpha, transactivated the human GSTA4 (hGSTA4) promoter cloned upstream of the luciferase reporter gene suggesting that IL-6 and EGF up-regulated hGSTA4 at a transcriptional level, whereas TNFalpha could rather act at a post-transcriptional level. The inhibition of phosphoinositide 3-kinase, p38 MAPK, and MEK/ERK signaling pathways, using specific inhibitors, prevented EGF-dependent induction of mGSTA4 and transactivation of hGSTA4 promoter. Altogether, these data favor the conclusion that, in regenerating hepatocytes, several GST isoforms are induced and that cytokines TNFalpha and IL-6 and survival factor EGF positively regulate mGSTA4 via survival signaling pathways.

We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.

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.

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.

Tacrine (THA), used in the treatment of Alzheimer's disease, is known to induce hepatotoxicity, the mechanisms of which remain to be fully established. We have previously shown that THA reduced intracellular glutathione concentration in rat hepatocytes in primary culture, thus pointing to a possible role for oxidative stress in THA toxicity. To test this, the effects of antioxidant molecules, namely, the flavonoids silibinin, silibinin dihydrogensuccinate, and silymarin, were evaluated on the toxicity of THA in cultured rat hepatocytes. This toxicity was investigated after a 24-h treatment over a concentration range from 0 to 1 mM, in the presence or absence of antioxidant (1 and 10 microM). We found that simultaneous treatment of hepatocytes with any of the antioxidants and THA remained ineffective on the lactate dehydrogenase release induced by THA. Then, the production of lipid-derived radicals (to estimate lipid peroxidation) was measured in THA (0.05-0.50 mM)-treated cells using a spin-trapping technique coupled to electron paramagnetic resonance (EPR) spectroscopy. No increase of the EPR signal was observed over the period of 30 min to 24 h. In contrast, treatment of cells with the spin label 12-doxyl stearic acid followed by EPR spectroscopy showed that THA (0.05 and 0.25 mM) rapidly increased hepatocyte membrane fluidity. Extracellular application of GM1 ganglioside (60 microM) both reversed this increase in fluidity and partially reduced lactate dehydrogenase release on THA exposure. In conclusion, this work indicates that early alterations of membrane fluidity, not resulting from lipid peroxidation, are likely to play an important role in the development of THA toxicity.

Cytochrome P4502E1 (CYP2E1) plays a key role in the metabolism of numerous drug substrates, mostly in mammalian liver. Both the apoprotein and mRNA levels are increased in response to interleukin 4 (IL-4) in primary human hepatocyte cultures. We developed a human hepatoma cell model that faithfully reproduces the responsiveness of the CYP2E1 gene to IL-4 at least in part through transcriptional activation, upon treatment with 150 U/ml of IL-4. As expected, IL-4 induced tyrosine phosphorylation of the STAT6 transcription factor, an effect prevented by the tyrosine kinase inhibitor tyrphostin A25. However, this inhibitor as well as genistein (another inhibitor of tyrosine kinases) had no effect on the IL-4 induction of CYP2E1. Similarly, protein kinase A activators (forskolin and dibutyryl-cAMP) and inhibitor (H89) did not influence the response to IL-4. However, PKC inhibitors (H7 and calphostin C) strongly blocked any induction of the gene, as well as the IL-4-dependent translocation of PKCζ. Taken together, our results show that IL-4 coordinately induces CYP2E1 transcription, mRNA and apoprotein levels in human hepatoma cells in a PKC-dependent manner, potentially through the activity of the PKCζ isoform.

The effects of tacrine (THA) on intracellular pH (pHi) were examined in a rat liver biliary epithelial cell line (RLEC) in HEPES‐buffered medium. pHi was recorded using the pH‐sensitive fluoroprobe, carboxy‐SNARF‐1 (carboxy‐seminaphtorhodafluor). 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 pHi could be detected at 0.1 and 0.25 mM THA, whereas at 0.05 mM, pHi was slightly more acid (7.17±0.02, n=16 versus 7.21±0.02, n=24 [control]). In control and short‐term treated cells, intracellular intrinsic buffering power (βi) increased roughly linearly as pHi decreased. This dependence was not seen following long‐term treatment. In all cases, βi was increased by THA (by 1.6 to 3.5 fold). Following an acid load (induced by 20 mM NH4Cl removal), pHi 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 ∼36% at a pHi of 6.73) while at 0.25 mM, a large increase was detected (by ∼109% at a pHi 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 α‐cyano‐4‐hydroxy cinnamate (CHC, 4 mM) but not by quercetin (0.125 mM). In conclusion, this work demonstrates that THA affects pHi in RLEC, through a decrease in Na+/H+ exchange and an increase in βi. Stimulation of a CHC‐inhibitable, Na+‐independent acid efflux is also detected. British Journal of Pharmacology (1999) 128, 1673–1682; doi:10.1038/sj.bjp.0702972

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.

Ribavirin, a guanosine analog, used in combination with interferon alpha (IFN-alpha) in the treatment of chronic hepatitis induced by hepatitis C virus (HCV) infection, has been shown to improve liver histology and to decrease transaminases even when administered alone. We analyzed the direct effects of ribavirin on the liver by using primary cultures of human and rat hepatocytes. Between 10 to 60 micromol/L, ribavirin was found to inhibit both the synthesis and secretion of whole proteins in a time- and dose-dependent fashion. Such an effect was confirmed by the measurement of albumin and haptoglobin secretion rates. [3H]-Thymidine incorporation was suppressed both in hepatocyte growth factor-stimulated human hepatocytes and in epidermal growth factor (EGF)-stimulated rat hepatocytes in the presence of ribavirin. The inhibitory effect on DNA synthesis was associated with a delayed progression to S phase of the cell cycle, as determined by flow cytometry and detection of cyclin A and cdc2 which are two proteins expressed during the S phase. The inhibition of DNA synthesis, caused by 50 micromol/L ribavirin, was completely restored by the addition of 80 micromol/L guanosine. These observations demonstrate that ribavirin at concentrations close to those found in plasma of treated patients can directly affect hepatic functions in vitro. Its effects could, however, be reduced in vivo by guanosine salvage supply.

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.

Background/Aims: Intra-hepatic bile ducts are the primary site of damage in several immunologically mediated liver diseases, However, immunological processes underlying biliary epithelial cell recognition by T lymphocytes are poorly understood, Therefore, a convenient in vitro model that could mimic these immunologic disorders would be of great interest.Methods: A human cell line (HuGB) was established from a metastasis of gallbladder adenocarcinoma in the liver, Intermediate filament expression was analysed by immunostaining, and gamma-glutamyl transpeptidase and albumin secretion mere measured, VLA integrin expression pattern, expression of HLA class I and II antigens and ICAM-1 protein were analysed by how cytometry and their modulation by interferon-gamma was quantitated using a QIFIKIT commercial kit.Results: Histological analysis showed high similarity between the initial gallbladder adenocarcinoma and the established cell line, Cytokeratins 8 and 19 and vimentin showed strong positive staining in the established cell line, Gamma-glutamyl transpeptidase was secreted by these cells while albumin expression was negative, HuGB cells also expressed VLA-alpha 2, VLA-alpha 3, VLA-alpha 6, VLA-beta 1, but not VLA-alpha(1), VLA-alpha 4 and NCAM, a pattern of adhesion molecule expression compatible with the biliary epithelium, Also, similar to the biliary epithelium found in normal liver, HuGB cells expressed abundant HLA class I but few HLA class LT antigens. We found that the expression increased during interferon-gamma treatment of HuGB cell line.Conclusions: Both phenotypic and morphological characteristics of HuGB cells suggested their biliary origin, Sensitivity of HuGB cells to interferon-gamma suggests that this new cell line could represent a suitable model to investigate the up-regulation of membrane antigens occurring in immune diseases involving biliary epithelial cells.

We have identified a liver-regulating protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes in rat testis, Liver-regulating protein was studied using monoclonal antibody L8 prepared from rat primitive biliary epithelial cells, This molecule was located in vivo at the interface of Sertoli cells and spermatocytes, and expressed in a stage-dependent manner (expression peaked on leptotene-zygotene spermatocytes), In vitro, the liver-regulating protein was found on Sertoli cell, spermatocyte and early spermatid membranes, Immunoaffinity procedures revealed two peptides of 85 and 73 kDa for Sertoli cells, while spermatocytes and spermatids displayed a single smaller peptide of 56 kDa, The involvement of the liver-regulating protein in cell interaction-mediated regulation of Sertoli cell was assessed in vitro by tracing Sertoli cell transferrin and inhibin secretion, as well as mRNA synthesis in spermatocyte-Sertoli cell cocultures and in rat liver biliary epithelial cell-Sertoli cell cocultures, performed in the presence or absence of monoclonal antibody L8. Inhibition of the spermatocyte- and liver biliary epithelial cell-stimulated secretion of transferrin and inhibin by Sertoli cells was observed in the presence of antibody, whereas spermatocyte adhesiveness was unchanged, Using northern blot analysis, the steady state levels of transferrin mRNA decreased when the anti-liver-regulating protein antibody was added to the Sertoli cell-spermatocyte cocultures or to the Sertoli cell-liver biliary epithelial cell cocultures, The data demonstrate the role of the liver-regulating protein in cell-cell contact-mediated regulation of Sertoli function by primary spermatocytes and the important implications of this cell contact-dependent control in testicular activity.

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.

Liver regulating protein (LRP) is an integral plasma membrane protein that plays a critical role in maintaining the differentiated phenotype of adult rat hepatocytes by mediating cell-cell interactions with rat liver epithelial cells. Using a specific monoclonal antibody (MAb L8) capable of inhibiting the interactions between these two cell types, the cellular distribution of LRP was analyzed in the liver. Various cell types, including hepatocytes and several sinusoidal cells, were found to be positive, whereas vascular endothelial cells and bile duct cells were consistently negative. This observation led us to question whether cells of nonhepatic origin would also express LRP. We show that MAb L8 immunoreactive material was detected in only three groups of tissues and corresponded to molecules similar to LRP but with different molecular weights. LRP-like molecules were demonstrated on acinar cells of the exocrine pancreas and on all hemopoietic cells regardless of their localization in the organism. LRP-like molecules were also expressed by germ cells and surrounding feeder cells in the testis and ovary in a stage-dependent manner. These results demonstrate the existence of a family of LRP proteins and strongly suggest a critical role for these molecules in regulating cell-cell communication in specific tissues.

Sertoli cells are a key element in the endocrine and paracrine regulation of the spermatogenic process. They are responsible for the constitution of the microenvironment required for germ cell division, differentiation, and metabolism (1, 2). It is also now well established that germ cells control this environment via the regulation of Sertoli cell activity (2, 3). The nature of the molecules and the mechanisms involved in this regulation are beginning to be understood. We have proposed that the germinal control of Sertoli cell function is exerted via germ cell-soluble factors, transfer of germ cell materials to the Sertoli cell (e.g., residual bodies), and plasma membrane molecules (2, 4, 5). Evidence exists that supports this hypothesis: (i) The purification and identification of some germ cell-soluble regulating proteins are in progress (6); (ii) morphologists have described in great detail the various devices involved in the transfer of spermatogonial and spermatid materials to the Sertoli cells (4, 7–9); and (iii) the involvement of residual bodies in the regulation of Sertoli cell IL-1α and IL-6 production has recently been established (10, 11). Furthermore, from recent in vivo studies, it appears that the different molecular families of cell adhesion molecules (CAMs)—Cadherins, integrins, selectins, and CAMs belonging to the immunoglobulin superfamily—are represented in rodent seminiferous tubules (5, 12, 13).