Eel Hepatocytes Research Articles

Cellular and molecular mechanisms of NiONPs toxicity on eel hepatocytes HEPA-E1: An illustration of the impact of Ni release from mining activity in New Caledonia.

Anthropic activities such as open pit mining, amplify the natural erosion of metals contained in the soils, particularly in New Caledonia, leading to atmospheric emission of nickel oxide nanoparticles (NiONPs). These particles are produced during extraction end up in aquatic ecosystems through deposition or leaching in the rivers. Despite alarming freshwater Ni concentrations, only few studies have focused on the cellular and molecular mechanisms of NiONPs toxicity on aquatic organisms and particularly on eels. Those fish are known to be sensitive to metal contamination, especially their liver, which is a key organ for lipid metabolism, detoxification and reproduction. The objective of this study was to assess in vitro the cytotoxic effects of NiONPs on Anguilla japonica hepatocytes, HEPA-E1. HEPA-E1 were exposed to NiONPs (0.5-5μg/cm2) for 4 or 24h. Several endpoints were studied: (i) viability, (ii) ROS production, SOD activity and selected anti-oxidant genes expression, (iii) inflammation, (iv) calcium signalling, (v) mitochondrial function and (vi) apoptosis. The results evidenced that NiONPs induce a decrease of cell viability and an increase in oxidative stress with a significant superoxide anion production. An increase of mitochondrial calcium concentration and a decrease of mitochondrial membrane potential were observed, leading to apoptosis. These results underline the potential toxic impact of NiONPs on eels living in mining areas. Therefore, eel exposure to NiONPs can affect their migration and reproduction in New Caledonia.

Release of Nionps from Mining Activity in New Caledonia: Cellular and Molecular Mechanisms of Toxicity on Eel Hepatocytes Hepa-E1

Release of Nionps from Mining Activity in New Caledonia: Cellular and Molecular Mechanisms of Toxicity on Eel Hepatocytes Hepa-E1

Involvement of oxidative stress and calcium signalling in NiONPs - induced toxic effects on eel hepatocytes and endothelial cells

Involvement of oxidative stress and calcium signalling in NiONPs - induced toxic effects on eel hepatocytes and endothelial cells

Lipid peroxidation and its control in Anguilla anguilla hepatocytes under silica-coated iron oxide nanoparticles (with or without mercury) exposure

Having multidisciplinary applications, iron oxide nanoparticles can inevitably enter aquatic system and impact inhabitants such as fish. However, the studies in this context have ignored the significance of obvious interaction of iron oxide nanoparticles with other persistent co-contaminants such as mercury (Hg) in the modulation of the toxicity and underlying mechanisms of iron oxide nanoparticles and Hg alone, and concomitant exposures. This study aimed to evaluate lipid peroxidation (LPO) and its control with glutathione (GSH) and associated enzymes (such as glutathione reductase, GR; glutathione peroxidase, GPX; glutathione sulfo-transferase, GST) in European eel (Anguilla anguilla L.) hepatocytes exposed to stressors with following schemes: (i) no silica-coated iron oxide nanoparticles functionalized with dithiocarbamate (Fe3O4@SiO2/Si DTC, hereafter called 'FeNPs'; size range 82 ± 21 to 100 ± 30nm) or Hg, (ii) FeNPs (2.5μgL(-1)) alone, (iii) Hg (50μgL(-1)) alone and (iv) FeNPs + Hg concomitant condition during 0 to 72h. The exhibition of a differential coordination between GSH regeneration (determined as GR activity) and GSH metabolism (determined as the activity of GPX and GST) was perceptible in A. anguilla hepatocytes in order to control FeNPs, Hg and FeNPs + Hg exposure condition-mediated LPO. This study revealed the significance of a fine tuning among GR, GPX and GST in keeping LPO level under control during FeNPs or Hg alone exposure, and a direct role of total GSH (TGSH) in the control of LPO level and impaired GSH metabolism under the concomitant (FeNPs + Hg) exposure. An interpretation of the fish risk to FeNPs in a multi-pollution state should equally consider the potential outcome of the interaction of FeNPs with other contaminants.

Effect of TBT and PAHs on CYP1A, AhR and Vitellogenin Gene Expression in the Japanese Eel, Anguilla japonica.

Gene expressions of cytochrome P4501A (CYP1A), aryl hydrocarbon receptor (AhR) and vitellogenin (Vg) by endocrine disruptors, benzo[α]pyrene (B[a]P) and tributyltin (TBT) were examined in cultured eel hepatocytes which were isolated from eels treated previously with B[a]P (10 mg/kg) or estradiol-17β (20 mg/kg) in vivo, and the relationship between CYP1A, AhR and Vg genes were studied. When the cultured eel hepatocytes were treated with B[a]P (10-6-10-5 M) the gene expressions of CYP1A and AhR were enhanced in a concentration-dependent manner. However, when treated with TBT (10-9-10-5 M) the gene expressions of CYP1A and AhR were suppressed at high concentrations (10-6-10-5 M), while having no effects at low concentrations (10-9-10-7 M). Gene expression of Vg was also suppressed by TBT in a concentration-dependent manner in cultured eel hepatocytes which was previously treated in vivo with estradiol-17β.

Studies on Eel Liver Functions Using Perfused Liver and Primary Cultured Hepatocytes

Perfused eel livers, isolated eel hepatocytes, and cultured eel hepatocytes are used to investigate eel liver functions such as gluconeogenesis, glycogen synthesis, and lipoprotein synthesis and methods for preparation are described. A novel phosphoenolpyruvate (PEP) synthesis pathway from pyruvate in gluconeogenesis in eel liver was elucidated and PEP synthesis pathways in eel, rat, and pigeon livers were compared. Glycogen synthesis from pyruvate, lactate, and glucose was investigated by using cultured eel hepatocytes. It was found that 10 –6 or 10 –7 M glucagon didn’t stimulate glycogen degradation in the presence of pyruvate but stimulated glycogen degradation in the presence of lactate. Glycogen synthesis from pyruvate was observed even when 10 –6 or 10 –7 M glucagon was present. The characteristics of the lipoprotein synthesized and secreted by cultured eel hepatocytes are clarified. Thyroxine and eel serum high-density lipoprotein (HDL) stimulated the lipoprotein synthesis by cultured eel hepatocytes. In the presence of estradiol-17β, eel serum HDL stimulated vitellogenin synthesis in eel hepatocytes. HDL specifically bound to eel hepatocytes and the ligand of HDL receptor in the plasma membrane of eel hepatocytes was identified to be ganglioside GM4 of eel serum HDL.

Ganglioside from eel serum high density lipoprotein (HDL) and its role as a ligand for HDL binding protein

First, we attempted to isolate glycosphingolipids from eel serum HDL. A single ganglioside containing N-acetylneuraminic acid (NeuAc), which is positive with resorcinol and orcinol reactions, was purified. The mobilities of the purified ganglioside and its lyso-form on high performance TLC were similar as those of authentic GM4 and its lyso-form, respectively. The mass of the purified ganglioside was determined by TOF mass spectrometer, and the mass of its oligosaccharide was the same as that of authentic GM4 from human brain consisting of disaccharide of NeuAc and galactose. The ganglioside from eel HDL was not hydrolyzed by recombinant endoglycoceramidase II, which cannot hydrolyze between galactose and ceramide of gangliosides, but hydrolyzes between glucose and ceramide. We concluded from these results that the ganglioside purified from eel serum HDL is GM4. Second, we investigated the effects of the ganglioside on binding of HDL labeled with fluorescein isothiocyanate (FITC-HDL) to cultured eel hepatocytes and on FITC-HDL ligand blotting by using plasma membrane proteins of the hepatocytes. Stimulatory effect of GM4 on FITC-HDL binding to the hepatocytes and FITC-HDL ligand blotting suggests strongly that GM4 is a ligand for HDL binding protein of eel hepatocytes.

Metabolic Arrest and Its Regulation in Anoxic Eel Hepatocytes

Some vertebrates depress overall metabolism in an abrupt and reversible fashion when challenged with anoxia, ensuring stabilization of cellular [ATP] and long-term survival, but little is known about the eliciting stimuli (e.g., change in O2, adenylates) and downstream effectors responsible for metabolic arrest. Accordingly, eel (Anguilla anguilla) hepatocytes were treated with inhibitors of putative components of the oxygen/metabolite-sensing pathway(s) and exposed to anoxia (Po2=0 mmHg). Anoxia in untreated cells caused a remarkable 85-fold decrease in ATP production rate, but cellular ATP levels stabilized following an initial steep drop. Reoxygenation of cells after 4 h of anoxia caused a fast metabolization of accumulated lactate and reestablishment of preanoxic ATP levels. Unlike physiological anoxia, pharmacological inhibition of the electron transport chain in the presence of oxygen caused extensive cellular ATP depletion, though no loss in viability. In contrast, cellular lactate (i.e., ATP) production rate was affected similarly by either treatment, suggesting that anaerobic glycolysis is regulated by a stimulus other than oxygen tension per se, whereas the continuous matching of ATP consumption and a rapidly ceasing mitochondrial ATP supply require a physiological relevant change in oxygen tension. Protein kinases, notably kinase C (PKC) and A (PKA), have been proposed as key downstream regulators of stress-induced defense mechanisms, but anoxic cell viability, metabolic rate, and [ATP] were not significantly affected by inhibitors of PKC and PKA. Likewise, inhibition of the upstream PKC-activating enzymes phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI 3-K) had no effect on recorded parameters. Anoxic cell survival in complex organisms may, in vivo, also depend on stress hormones released from distant oxygen-sensing cells. Accordingly, adrenaline elevated anaerobic energy production but, apparently, also elevated ATP consumption because cellular ATP levels during oxygen deprivation were slightly lowered by adrenergic stimulation.

Participation of high-density lipoprotein in vitellogenesis in Japanese eel hepatocytes

To investigate effect of estradiol-17β (E 2) treatment in vivo on binding of eel hepatocytes to HDL, we developed hepatocytes binding assay. When hepatocytes were incubated with 200 times excess of eel HDL, the binding of hepatocytes to HDL precoated on wells was inhibited competitively. This indicates that eel hepatocytes bound specifically to HDL. E 2 treatment in vivo induced vitellogenin (VTG) synthesis. Hepatocytes prepared from the same E 2 treatment eel showed 3-fold higher ability of binding to HDL compared to hepatocytes prepared from ells without E 2 treatment. We also examined effects of E 2 and HDL on VTG induction in cultured hepatocytes. VTG, determined by enzyme-linked immunosorbent assay (ELISA), induction was about two-times higher in the presence of both 10 −5 M of E 2 and 400 μg of HDL than in the presence of 10 −5 M E 2 alone. At concentrations below 10 −6 M E 2, VTG was not induced in the presence or absence of HDL. By SDS–PAGE and immunoblotting, VTG was detected only in the presence of both 10 −5 M of E 2 and HDL. Our findings strongly support the idea that HDL correlates with vitellogenesis in eel liver.

Lipoprotein metabolism in a coculture system with eel skeletal muscle cells and hepatocytes

: Eel muscle contains lipid as much as 15–20% of wet weight of muscle. However, it is uncertain whether lipid is synthesized in muscle or transported from another tissue such as liver. We prepared isolated muscle cells to investigate the synthetic ability of lipid by muscle cell itself and studied lipoprotein metabolism in coculture with eel hepatocytes. Isolated eel muscle cells were prepared by collagenase digestion of skeletal muscle and their length and width were approximately 2.2 mm and 5–20 µm, respectively. The length corresponded to that of myotome between myocommata. Cultured muscle cells synthesized lipid and protein from 14C-acetate, but the ability of lipid synthesis by muscle cells was below 1/100 that of eel hepatocytes. In the absence of lipoprotein lipase (LPL), the highest component among 14C-lipid changed from 14C-phospholipid in muscle cells cultured alone to 14C-triacylglycerol (14C-TG) in cocultured muscle cells. These results suggest that cocultured muscle cells incorporate the very low-density lipoprotein (VLDL)-like lipoprotein secreted by cocultured hepatocytes, of which the main lipid is 14C-TG. In the presence of LPL in the coculture system, 14C-lipid in muscle cells increased 1.6-fold during 48 h incubation compared to that without LPL. 14C-Free fatty acids to total 14C-lipid in muscle cells increased from 1.1 to 32.6% by addition of 6 U/mL of LPL. Furthermore, the secreted VLDL-like lipoprotein was decreased and a gel filtration column separated formed high-density lipoprotein (HDL)-like lipoprotein. These results suggest that 14C-free fatty acids produced by LPL digestion of 14C-VLDL-like lipoprotein were transported to muscle cells. We concluded that there are two metabolisms of the secreted lipoprotein dependent or independent on LPL.

Involvement of androgens and growth hormone in the synthesis of vitellogenin in Japanese eel (Anguilla japonica)

Immature eels positively responded to estradiol-17β (E2) injection in terms of vitellogenin (Vg) synthesis but not to growth hormone (GH) or 17α-methyltestosterone (MT) injection. However, injection of MT or GH combined with E2 strongly increased Vg synthesis. In in vitro experiments, eel hepatocytes treated with E2, GH, or MT alone did not produce detectable amount of Vg, whereas the combination of E2 with GH or MT, or both greatly increased Vg synthesis in the hepatocytes.

G proteins immunodetection and adrenergic transduction pathways in the liver of Anguilla anguilla.

G proteins are members of a highly conserved superfamily of GTPases, which includes heterotrimeric (alpha, beta, gamma) proteins acting as critical control points for transmembrane signaling. In ectothermal vertebrates, knowledge about these proteins is scarce, and our work provides the first demonstration that G(s), G(q), and G(i) proteins are all present in the liver of a fish. G(q)alpha subunits of about 42 kDa have been identified in European eel (Anguilla anguilla) liver membranes, supporting previous reports about the existence of hormone transduction pathways coupled to inositol 1,4,5-trisphosphate/Ca(2+) enhancement in fish hepatocytes. Although two G(s)alpha proteins of about 45 and 52 kDa have been reported in mammals, a single isoform of approximately 45 kDa has been recognized in eel liver. G(s)alpha and G(q)alpha proteins are involved in the epinephrine transduction pathway, leading to cAMP and Ca(2+) intracellular increments, respectively. Interestingly, both messengers significantly stimulated glucose release from eel hepatocytes but with a different time course. In fact, the Ca(2+)-dependent glucose output preceded the cAMP-mediated release by about 7 min. G(i)alpha subunits of about 40 kDa were also immunodetected, suggesting the presence of hormone receptors leading to adenylyl cyclase inhibition in eel liver; however, alpha(2)- adrenoreceptor ligands were ineffective on both enzyme activity and glucose release.

Cd 2+ and Hg 2+ affect glucose release and cAMP-dependent transduction pathway in isolated eel hepatocytes

Isolated hepatocytes of the European eel ( Anguilla anguilla) have been used as experimental model to characterize the effects of Cd 2+ and Hg 2+ on either basal or epinephrine-stimulated glucose release. Cd 2+ strongly reduced glucose output from cells perifused in BioGel P4 columns and challenged with epinephrine, with a maximum inhibition of 95% reached at 10 μM (IC 50 0.04 μM). The epinephrine-stimulated glucose output was also reduced by Hg 2+, although a significant inhibition of about 60% was achieved only at 10 μM (IC 50 5 μM). The possible influence of Cd 2+ and Hg 2+ on adenylyl cyclase/cAMP transduction pathway has been investigated, since this system is known to play a pivotal role in the regulation of fish liver glycogen breakdown and consequent glucose release. Micromolar concentrations of both heavy metals significantly reduced the epinephrine-modulated cAMP levels in isolated eel hepatocytes, in good agreement with the reduction of glucose output. Cd 2+ and Hg 2+ also significantly reduced basal and epinephrine-stimulated adenylyl cyclase activity in liver membrane preparations. A competitive inhibition with respect to Mg 2+ was shown by Cd 2+ and Hg 2+, which significantly reduced the affinity of the allosteric activator for the adenylyl cyclase system. Apparent Km for Mg 2+ was 4.35 mM in basal conditions, and increased to 9.1 and 7.1 mM in the presence of 10 μM Cd 2+ and Hg 2+, respectively. These results indicate that Cd 2+ and Hg 2+ may impair a crucial intracellular transduction pathway involved in the adrenergic control of glucose metabolism, but also in several other routes of hormonal regulation of liver functions.

Regulation of synthesis and secretion of the lipoprotein by cultured eel hepatocytes

Regulation of synthesis and secretion of the lipoprotein by cultured eel hepatocytes

High density lipoprotein binding protein of eel ( Anguilla japonica) liver with specificity of binding to apoAI as a ligand

High-density lipoprotein (HDL) binding protein (HBP) was isolated from the microsomal fraction of eel liver homogenate by affinity chromatography with a HDL-column. After SDS-PAGE and blotting, HBP on the PVDF membrane was detected by FITC-labeled HDL and apolipoprotein AI (apoAI) as a ligand. HBP in the microsomal fraction was most abundant among microsomal, mitochondrial and cytosolic fractions. The HBP isolated by a HDL-column consisted of at least three proteins with low molecular weights of 18.5, 14.5 and 13.5 kDa; the main component was 14.5 kDa. These proteins are not products of protease digestion, as the procedure was carried out in the presence of protease inhibitors including ( p-aminophenyl) methansulfonyl fluoride, 4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, aprotinin and EDTA. The HBP specifically bound to FITC-apoAI and faintly bound or did not bind to FITC-apoAII. Furthermore, binding of HDL labeled with lipophilic fluorescence to isolated eel hepatocytes was inhibited by the antibody to apoAI, but not inhibited by the antibody to apolipoprotein AII (apoAII). These results strongly suggest that the HBP isolated from the microsomal fraction is present on the plasma membrane of eel liver and plays important roles for the lipid transport through the interaction with HDL.

Characterization of [3H]CGP 12177 Binding to β-Adrenergic Receptors in Intact Eel Hepatocytes

The aim of this study was to characterize [3H]CGP 12177 (CGP) binding to β-adrenergic receptors in isolated hepatocytes of the European eel (Anguilla anguilla), in which the involvement of cAMP in epinephrine-induced glucose release has been previously observed. Specific binding of CGP was saturable, reversible, and linear as a function of cell number. Analysis of binding data suggested a single class of binding sites, with a Kd of 1.31 nM and a number of approximately 7000 β-adrenergic receptors per cell. The potency order of specific inhibition of [3H]CGP binding was CGP > propranolol ≥ alprenolol ⪢ butoxamine ≥ atenolol, while phentolamine and prazosin failed to significantly displace the tracer at concentrations up to 100 μM. The binding kinetics of CGP were closely related to its biological effect. In fact, the drug dose-dependently counteracted the enhancement of intracellular cAMP levels induced by epinephrine in isolated hepatocytes with a Kd of 1.06 nM. Moreover, it antagonized the hormone-induced stimulation of adenylyl cyclase activity in hepatic membranes as well as of glucose release from cells. These data clearly show that β-adrenergic receptors are coupled to the adenylyl cyclase/cAMP transduction pathway in eel liver.

Multiplicity, Structures, and Endocrine and Exocrine Natures of Eel Fucose-binding Lectins

Lectins, a group of proteins that bind to cell surface carbohydrates and play important roles in innate immunity, are widely used experimentally to distinguish cell types and to induce cell proliferation. Eel serum lectins have been useful as anti-H hemagglutinins and also in lectin histochemistry as fucose-binding lectins (fucolectins), but their structures have not been determined. Here we report the primary structures and the sites of synthesis of eel fucolectins. Eel serum fucolectins were separated by two-dimensional gel electrophoresis and sequenced. cDNA cloning, based on the amino acid sequence information, and Northern blot analysis indicated that 1) the fucose-binding lectins are secretory proteins and have unique structures among the lectins, exhibiting only weak similarities to frog pentraxin, horseshoe crab tachylectin-4, and fly fw protein; 2) there are at least seven closely related members; and 3) their messages are abundantly expressed in the liver and in significant levels in the gill and intestine. The lectin-producing hepatic cells were identified by immunostaining; in the gill, exocrine mucous cells were stained, suggesting that serum fucolectins derive from the liver. Using primary culture of eel hepatocytes, the message levels were shown to be increased by lipopolysaccharide, suggesting a role for fucolectins in host defense. SDS-polyacrylamide gel electrophoresis analysis showed that eel fucolectins have a SDS-resistant tetrameric structure consisting of two disulfide-linked dimers.

Distribution of apolipoproteins A-I and A-II in serum, secreted proteins by cultured hepatocytes, and the plasma membrane of hepatocytes in eel

SUMMARY: Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) plays a key role in cholesterol transport as a ligand of HDL binding proteins or HDL receptors and an acceptor of intracellular cholesterol. It has been known that the latter role of apoA-I is carried out by free apoA-I dissociated from HDL. We investigated the distribution of free apoA-I and apoA-II in eel serum and secreted proteins from cultured eel hepatocytes using monoclonal antibodies specific to apoA-I or apoA-II of eel serum HDL. Free apoA-I was found in eel serum and the secreted proteins, but free apoA-II was not. ApoA-I and apoA-II were also found in serum very low-density lipoprotein and chylomicron-like lipoprotein secreted by cultured eel hepatocytes. We observed the existence of apoA-I and apoA-II in the plasma membrane of the hepatocytes by immunological method after fixation of the cells. Furthermore, when the isolated hepatocytes treated with the antibody to apoA-II were incubated with HDL stained by a fluorescent lipophilic dye, the binding of the HDL to the hepatocytes was inhibited by 30%. However, we did not observe the inhibition by the hepatocytes treated with the antibody to apoA-I.

α-Adrenoceptor-mediated glucose release from perifused catfish hepatocytes

In fish liver catecholamines bind to β-adrenoceptors (AR) and increase glucose release via cAMP augmentation, α 1-AR have recently been shown to mediate IP 3 and Ca 2+ elevation in catfish and eel hepatocytes, although their coupling to a physiological response has remained doubtful. We have perifused isolated catfish hepatocytes in Bio-Gel P4 columns with epinephrine in the presence of prazosin and/or propranolol, α- and β-AR antagonists, respectively. Ten nM epinephrine stimulated glucose release approximately 3-fold, and this effect was completely antagonized by the simultaneous presence of both α- and β-AR blockers. The two AR antagonists separately inhibited about one-third and two-third of the total stimulation, respectively. Through α-AR occupancy, epinephrine provoked a significant increase of glucose release whereas no stimulation was detected in Ca 2+-depleted hepatocytes. Glucose release was strongly elevated by both ionomycin and dibutyryl cAMP. These results represent the first direct evidence that α-AR transduction pathway is involved in epinephrine-induced glucose release from fish hepatocytes.

Metabolic studies on eel ( Anguilla anguilla L.) hepatocytes in primary culture: effect of 17 β-estradiol and growth hormone

Previous studies demonstrated that native and recombinant growth hormone from mammalian and fish species potentiate the estrogenic induction of vitellogenin synthesis by cultured eel hepatocytes. In the present study, the metabolic competence (respiratory activity and estradiol catabolism) of cultured hepatocytes and their functional capacity to synthesize a specific protein, vitellogenin, in the presence of estradiol and/or bovine growth hormone was investigated. In addition, we examined the possible role of insulin-like growth factors as mediators of growth hormone. Hepatocytes retain a high level of metabolic activity under the primary culture conditions applied. Estradiol has a half life of several hours in the hepatocyte culture, and is metabolized into conjugated forms. Estradiol and/or growth hormone had no effects on respiratory activity of the cultured hepatocytes. Moreover, the estradiol catabolic parameters were not affected by growth hormone. Finally, human and trout recombinant insulin-like growth factors do not potentiate vitellogenin synthesis induced by estradiol.