Cultured Mouse Hepatocytes Research Articles

Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes

Most of the experiments studying acetaminophen (APAP) induced hepatotoxicity were performed using moue as model specie, right because its high sensitivity. While the toxic responses can be called forth easily in mice, the human relevancy of these results is questionable. In this study human, rat, and mouse primary hepatocytes were treated with increasing concentrations of APAP, and cell viability was measured by MTT cytotoxicity assay. Pronounced interspecies differences were obtained in cell viability following 24h of APAP treatment starting at 24h after seeding (EC50: 3.8mM, 7.6mM, and 28.2mM, in mouse, rat, and human hepatocyte culture, respectively). The longer time of culturing highly increased the resistance of hepatocytes of all species investigated. In rat hepatocyte culture EC50 values were 6.0mM, 12.5mM, and 18.8mM, when starting APAP treatment after 24, 48, and 72 h of seeding. Although N-acetylbenzoquinoneimine, a minor metabolite of APAP, which is mainly formed by CYP2E1 at high APAP concentration in every species studied, is thought to initiate the toxic processes, no correlation was found between CYP2E1 activities and hepatocyte sensitivity of different species. We conclude that the toxicity induced by APAP overdose highly depends on the animal model applied.

Synergism of Glucocorticoid Hormone with Growth Hormone for Female-Specific MouseCyp3a44Gene Expression

CYP3A44 and CYP3A41 are female-specific CYP3A in the mouse liver. In primary cultured mouse hepatocytes, dexamethasone concentration-dependently induced CYP3A44 mRNA, and the highest response was seen at 10(-5) M. In contrast, CYP3A41 mRNA expression was highest at lower concentrations (10(-7) or 10(-6) M). At submicromolar concentration (10(-7) M), the induction of CYP3A44 mRNA was very slight, but strongly enhanced induction was observed by the simultaneous addition of growth hormone (GH). Similar enhancement was also observed in CYP3A41 mRNA expression. Continuous exposure to GH, which mimics female-type secretion from the pituitary gland, was effective to enhance the expression of both mRNAs, but discontinuous exposure (male-type) was not. This synergistic induction of CYP3A44 mRNA was further enhanced by the transfection of glucocorticoid receptor (GR) expression plasmid or by the cotransfection of pregnane X receptor (PXR) and retinoid X receptor (RXR) alpha expression plasmids. Similar synergistic induction was seen in CYP3A41 mRNA by the transfection of GR expression plasmid but was not enhanced by cotransfection of PXR and RXR expression plasmids. These observations suggest that functional cross-talk between signaling pathways of female-type GH secretion and glucocorticoid hormone might be involved in the female-predominant expression of both genes. Additionally, one or more nuclear receptors mediating induction by glucocorticoid hormone are employed for collaboration with GH.

Bioactive Constituents from Chinese Natural Medicines. XXXI. Hepatoprotective Principles from Sinocrassula indica: Structures of Sinocrassosides A8, A9, A10, A11, and A12

-The methanolic extract from the whole plant of Sinocrassula indica (Crassulaceae) was found to show hepatoprotective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes. From the methanolic extract, five new acylated flavonol glycosides, sinocrassosides A 8 (1), A 9 (2), A 10 (3), An (4), and A 12 (5), were isolated together with 25 compounds. The structures of 1-5 were elucidated on the basis of chemical and physicochemical evidence. Among the isolated compounds, sinocrassosides A 1 (6), A 2 (7), and B 2 (14) were found to show potent hepatoprotective effect.

Medicinal Flowers. XXIV. Chemical Structures and Hepatoprotective Effects of Constituents from Flowers of Hedychium coronarium

The 80% aqueous acetone extract from the flowers of Hedychium coronarium was found to show a protective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes. On the other hand, two new labdane-type diterpene glycosides, coronalactosides I (1) and II (2), and a new labdane-type trinorditerpene, coronadiene (3), were isolated together with 8 known compounds from the extracts, which were obtained with chloroform and 80% aqueous acetone from the flowers of H. coronarium. The structures of new constituents were elucidated on the basis of chemical and physicochemical evidence. In addition, the principal constituents, coronaririn C and 15-hydroxylabda-8(17),11,13-trien-16,15-olide, displayed hepatoprotective effects, which were stronger than that of the hepatoprotective agent, silybin.

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.

Glutathione Depletion Down-regulates Tumor Necrosis Factor α-induced NF-κB Activity via IκB Kinase-dependent and -independent Mechanisms

Reduced glutathione (GSH) plays a crucial role in hepatocyte function, and GSH depletion by diethyl maleate was shown previously to inhibit expression of NF-kappaB target genes induced by tumor necrosis factor alpha (TNFalpha) and sensitize primary cultured mouse hepatocytes to TNF-mediated apoptotic killing. Here we demonstrate in the same system that GSH depletion down-regulates TNF-induced NF-kappaB transactivation via two mechanisms, depending on the extent of the depletion. With moderate GSH depletion (approximately 50%), the down-regulation is IkappaB kinase (IKK)-independent and likely acts on NF-kappaB transcriptional activity because TNF-induced IKK activation, IkappaBalpha phosphorylation and degradation, NF-kappaB nuclear translocation, NF-kappaB DNA binding in vitro, and NF-kappaB subunit RelA(p65) recruitment to kappaB sites of target gene promoters all appear unaltered. On the other hand, with profound GSH depletion (approximately 80%), the down-regulation also is IKK-dependent, and a timeline is established linking the inhibition of polyubiquitination of receptor-interacting protein 1 in TNF receptor 1 complex to partial blockage of IKK activation, IkappaBalpha phosphorylation and degradation, and NF-kappaB nuclear translocation. Of note, pretreatment with antioxidant trolox protects against the inhibitory effect of profound GSH depletion on IKK activation and NF-kappaB nuclear translocation but fails to restore expression of NF-kappaB target genes, revealing both IKK-dependent and -independent inhibition. These findings provide new insights into the complex effects of oxidative stress and redox perturbations on the NF-kappaB pathway.

HYPOXIA ACTIVATES c-JUN N-TERMINAL KINASE VIA RAC1-DEPENDENT REACTIVE OXYGEN SPECIES PRODUCTION IN HEPATOCYTES

The earliest events after the induction of hemorrhagic shock (HS) are complex and poorly understood. We have recently demonstrated that decreased tissue perfusion and hypoxia during HS lead to an increased phosphorylation of c-Jun N-terminal kinase (JNK) in vivo. The purpose of these investigations was to test the hypothesis that hypoxia activates JNK via Rac1-dependent reactive oxygen species (ROS) signaling. Mice subjected to HS and resuscitated with Ringer's ethyl pyruvate solution (REPS) or N-acetylcysteine (NAC), two scavengers of ROS, demonstrated decreased levels of phosphorylated JNK. Exposure of primary mouse hepatocytes in culture to 1% oxygen led to increased production of ROS and phosphorylation of JNK. The duration of hypoxia correlated with the level of generation of ROS and JNK activation. The phosphorylation of JNK was attenuated in the presence of ROS scavengers or the nicotinamide adenosine dinucleotide phosphate [NDA(P)H] oxidase inhibitor, diphenyleneiodonium (DPI). In addition, hypoxia increased activation of Rac1. Inhibition of Rac1 activation by adenoviral gene transfer of dominant-negative Rac1 (AdRac1) attenuated both ROS formation and JNK activation. Together, these data suggest that ROS generation during hypoxia in the liver directly leads to JNK activation in a Rac1-dependent process.

Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[ a]anthracene, β-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo- p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression.

Involvement of Glucocorticoid Receptor and Pregnane X Receptor in the Regulation of Mouse CYP3A44 Female-Predominant Expression by Glucocorticoid Hormone

The role of the glucocorticoid receptor (GR) and pregnane X receptor (PXR) in the regulation of female-predominant expression of mouse CYP3A44 by glucocorticoid hormones was evaluated using a primary culture of female mouse hepatocytes, as the expression was suppressed in adrenalectomized female mice, restored by dexamethasone (DEX) treatment and was not detected in male mouse livers. Glucocorticoid hormones, such as DEX, hydrocortisone, and corticosterone, 11beta-[4-dimethylamino] phenyl-17beta-hydroxy-17-[1-propynyl]estra-4,9-diene-3-one (RU486), antagonists for GR and an agonist for PXR, and rifampicin, an agonist for PXR, were chosen to investigate the relationship of GR/PXR activation and Cyp3a44 gene expression. Glucocorticoid-inducible expression of CYP3A44 was not suppressed but rather was increased by RU486. Treatment of GR expression plasmid-transfected hepatocytes with DEX concentration dependently enhanced the expression of PXR as well as CYP3A44 mRNAs. A synergistic effect of DEX at submicromolar concentrations and rifampicin is observed. Furthermore, transfection of PXR and retinoid X receptor-alpha (RXRalpha) also showed prominent induction of CYP3A44 mRNA by DEX. These results suggest that DEX plays a dual role in CYP3A44 expression: first, direct activation of the Cyp3a44 gene by the PXR-RXRalpha complex, and, second, indirect activation of the Cyp3a44 gene through the induction of PXR gene expression by the GR pathway.

Role of apoptosis in acetaminophen hepatotoxicity

Acetaminophen overdose causes liver injury by mechanisms involving glutathione depletion, oxidative stress and mitochondrial dysfunction. The role of apoptosis in acetaminophen-induced cell killing is still controversial. Here, our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen-induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. Acetaminophen (10 micromol/L) induced necrotic killing in approximately 50% of hepatocytes after 6 h and cyclosporin A (CsA), MPT inhibitor, temporarily decreased necrotic killing after 6 h, but cytoprotection was lost after 16 h. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization at approximately 4.5 h after acetaminophen. CsA delayed these changes indicative of the MPT to about 11 h after acetaminophen. TUNEL labeling and caspase 3 activation also increased after acetaminophen. Fructose (20 mmol/L, an ATP-generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis. In conclusion, acetaminophen induces the MPT and ATP-depletion-dependent necrosis or caspase-dependent apoptosis as determined, in part, by ATP availability from glycolysis.

Reduction of Low Molecular Weight Protein-tyrosine Phosphatase Expression Improves Hyperglycemia and Insulin Sensitivity in Obese Mice

To investigate the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in glucose metabolism and insulin action, a specific antisense oligonucleotide (ASO) was used to reduce its expression both in vitro and in vivo. Reduction of LMW-PTP expression with the ASO in cultured mouse hepatocytes and in liver and fat tissues of diet-induced obese (DIO) mice and ob/ob mice led to increased phosphorylation and activity of key insulin signaling intermediates, including insulin receptor-beta subunit, phosphatidylinositol 3-kinase, and Akt in response to insulin stimulation. The ASO-treated DIO and ob/ob animals showed improved insulin sensitivity, which was reflected by a lowering of both plasma insulin and glucose levels and improved glucose and insulin tolerance in DIO mice. The treatment did not decrease body weight or increase metabolic rate. These data demonstrate that LMW-PTP is a key negative regulator of insulin action and a potential novel target for the treatment of insulin resistance and type 2 diabetes.

Purification, characterization and biological activity on hepatocytes of a polysaccharide from Flammulina velutipes mycelium

A water-soluble polysaccharide named as FVP2 has been isolated from Flammulina velutipes mycelium by hot water extraction, anion-exchange and gel-permeation chromatography. Structure study shows FVP2 is an α-(1→4)- d-glucan, with a single α- d-glucan at the C-6 position approximately every seven residues, along the main chain. The weight-average molecular weight is 1.89 × 10 4 Da. Its biological activity was tested on hepatocytes. In vitro study indicates FVP2 can enhance the livability of primary culture of mouse hepatocytes and decrease the release of ALT as well as apoptosis of hepatocytes after CCl 4 intoxication.

Combining ursodeoxycholic acid or its NO-releasing derivative NCX-1000 with lipophilic antioxidants better protects mouse hepatocytes against amiodarone toxicity

Nonalcoholic steatohepatitis (NASH) is a common and potentially severe form of liver disease. This study aimed to determine the effect of ursodeoxycholic acid and its NO-releasing derivative NCX-1000 alone or in combination with antioxidants on cultured mouse hepatocytes treated with amiodarone to mimic certain aspects of hepatocyte injury found in NASH. Isolated mouse hepatocytes were incubated with ursodeoxycholic acid or NCX-1000 (0-100 micromol/L) combined or not combined with the hydrophilic antioxidants butylated hydroxytoluene and ascorbic acid (0-100 micromol/L) or with the lipophilic antioxidant alpha-tocopherol (0-100 micromol/L) 15 min before adding amiodarone (50 micromol/L) to the culture medium. Twenty hours later, necrosis, apoptosis, superoxide anion production, and malondialdehyde levels were assessed in cultured cells. Amiodarone led to a dose-dependent decrease in cell viability with an LD50 of 50 micromol/L and increased production of superoxide anion and lipid peroxidation. NCX-1000 showed a better protective potential than ursodeoxycholic acid against the toxic effects of amiodarone. The hydrophilic antioxidants had no effect on the toxicity of amiodarone, whereas alpha-tocopherol at a concentration >100 micromol/L almost completely suppressed it. Ursodeoxycholic acid and NCX-1000 protection was additive only when they were combined with alpha-tocopherol, not with butylated hydroxytoluene or ascorbic acid. In addition, all the antioxidants tested reduced the superoxide anion detected, but only alpha-tocopherol prevented lipid peroxidation induced by amiodarone. The combination of lipophilic antioxidants with ursodeoxycholic acid or NCX-1000 enhances their protective potential and could represent an interesting therapeutic approach to explore for the treatment of NASH.

Bioactive Constituents from Chinese Natural Medicines. XXV. New Flavonol Bisdesmosides, Sarmenosides I, II, III, and IV, with Hepatoprotective Activity from Sedum sarmentosum (Crassulaceae)

-Four new flavonol bisdesmosides, sarmenosides I, II, III, and IV, were isolated from the whole plant of Sedum sarmentosum (Crassulaceae). Their structures were elucidated on the basis of chemical and physicochemical evidence. Among them, sarmenoside III was found to show potent hepatoprotective effect (IC 50 = 4.4 μM) on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes.

Bioactive Constituents from Chinese Natural Medicines. XXVI. Chemical Structures and Hepatoprotective Effects of Constituents from Roots of Rhodiola sachalinensis

The methanolic extract from the roots of Rhodiola sachalinensis was found to show a protective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes. From the methanolic extract, five new glycosides, two monoterpene glycosides, two flavonol bisdesmosides, and a cyanogenic glycoside, were isolated together with 34 known compounds. The structures of new constituents were elucidated on the basis of chemical and physicochemical evidence. In addition, the principal constituents, sachalosides III and IV, rhodiosin, and trans-caffeic acid, displayed hepatoprotective effects.

Bioactive Constituents from Chinese Natural Medicines. XXIII. Absolute Structures of New Megastigmane Glycosides, Sedumosides A4, A5, A6, H, and I, and Hepatoprotective Megastigmanes from Sedum sarmentosum

The methanol-eluted fraction of the hot water extract from the whole plant of Sedum sarmentosum (Crassulaceae) was found to show hepatoprotective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes. From the active fraction, five new megastigmane glycosides, sedumosides A(4), A(5), A(6), H, and I, were isolated together with 22 megastigmane constituents. Their absolute stereostructures were elucidated on the basis of chemical and physicochemical evidence. Among them, sedumoside F(1) (IC(50)=47 microM), (3S,5R,6S,9R)-megastigmane-3,9-diol (61 microM), and myrsinionosides A (52 microM) and D (62 microM) were found to show the strong hepatoprotective activity.

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

New Flavanone Oligoglycosides, Theaflavanosides I, II, III, and IV, with Hepatoprotective Activity from the Seeds of Tea Plant (Camellia sinensis)

Four new flavanone oligoglycosides, theaflavanosides I, II, III, and IV, were isolated from the seeds of Camellia sinensis. The structures of theaflavanosides were elucidated on the basis of chemical and physicochemical evidence. Among them, theaflavanoside III was found to show hepatoprotective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes.

Stimulation of AMP-Activated Protein Kinase Is Essential for the Induction of Drug Metabolizing Enzymes by Phenobarbital in Human and Mouse Liver

Our previous studies have suggested a role for AMP-activated protein kinase (AMPK) in the induction of CYP2B6 by phenobarbital (PB) in hepatoma-derived cells (Rencurel et al., 2005). In this study, we showed in primary human hepatocytes that: 1) 5'-phosphoribosyl-5-aminoimidazol-4-carboxamide 1-beta-d-ribofuranoside and the biguanide metformin, known activators of AMPK, dose-dependently increase the expression of CYP2B6 and CYP3A4 to an extent similar to that of PB. 2) PB, but not the human nuclear receptor constitutive active/androstane receptor (CAR) ligand 6-(4-chlorophenyl)imidazol[2,1-6][1,3]thiazole-5-carbaldehyde, dose-dependently increase AMPK activity. 3) Pharmacological inhibition of AMPK activity with compound C or dominant-negative forms of AMPK blunt the inductive response to phenobarbital. Furthermore, in transgenic mice with a liver-specific deletion of both the alpha1 and alpha2 AMPK catalytic subunits, basal levels of Cyp2b10 and Cyp3a11 mRNA were increased but not in primary culture of mouse hepatocytes. However, phenobarbital or 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene, a mouse CAR ligand, failed to induce the expression of these genes in the liver or cultured hepatocytes from mice lacking hepatic expression of the alpha1 and alpha2 subunits of AMPK. The distribution of CAR between the nucleus and cytosol was not altered in hepatocytes from mice lacking both AMPK catalytic subunits. These data highlight the essential role of AMPK in the CAR-mediated signal transduction pathway.

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo

Ceramidases (CDases) play a key role in cancer therapy through enhanced conversion of ceramide into sphingosine 1-phosphate (S1P), but their involvement in hepatocarcinogenesis is unknown. Here, we report that daunorubicin (DNR) activated acid CDase post-transcriptionally in established human (HepG2 cells) or mouse (Hepa1c1c7) hepatoma cell lines as well as in primary cells from murine liver tumors, but not in cultured mouse hepatocytes. Acid CDase silencing by small interfering RNA (siRNA) or pharmacological inhibition with N-oleoylethanolamine (NOE) enhanced the ceramide to S1P balance compared to DNR alone, sensitizing hepatoma cells (HepG2, Hep-3B, SK-Hep and Hepa1c1c7) to DNR-induced cell death. DNR plus NOE or acid CDase siRNA-induced cell death was preceded by ultrastructural changes in mitochondria, stimulation of reactive oxygen species generation, release of Smac/DIABLO and cytochrome c and caspase-3 activation. In addition, in vivo siRNA treatment targeting acid CDase reduced tumor growth in liver tumor xenografts of HepG2 cells and enhanced DNR therapy. Thus, acid CDase promotes hepatocarcinogenesis and its antagonism may be a promising strategy in the treatment of liver cancer.