Isolated Mouse Hepatocytes Research Articles

A 43 kDa protein from the herb Cajanus indicus L. protects thioacetamide induced cytotoxicity in hepatocytes

The aim of this study was to investigate the role of a hepatoprotective protein isolated from the herb Cajanus indicus L. on thioacetamide (TAA) induced toxicity in isolated mouse hepatocytes. In vitro cell viability, lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and total protein leakage were measured as the indicator of cell damage. The amount of glutathione (GSH) and lipid peroxidation were also measured to determine the oxidative status of the cells. The reduced cell viability in TAA treated hepatocytes was almost completely recovered upon protein treatment. LDH, ALT and total protein secretion outside the cells after TAA treatment confirmed the cell membrane damage. Incubation of hepatocytes with the protein prior to TAA administration significantly prevented the cell membrane damage as revealed from less LDH, ALT and total protein leakage. TAA depleted endogenous antioxidant GSH and increased membrane lipid peroxidation in hepatocytes. The protein had very prominent effect in altering the GSH level and lipid peroxidation. The protein exhibited all these cytoprotective effects in a dose-dependent manner. Besides, measurement of DPPH radical scavenging activity showed that the protein could scavenge free radicals. In addition, the protein resisted TAA induced alterations of various effects when applied in combination with TAA. The cytoprotective activity of the protein was found to be comparable with α-tocopherol, a well-known antioxidant. Results suggest that the protein from C. indicus can act as a hepatoprotector and primary antioxidant against TAA-induced cytotoxicity in mouse hepatocytes.

In Vivo Engineering of Metabolically Active Hepatic Tissues in a Neovascularized Subcutaneous Cavity

Recent success in clinical hepatocyte transplantation therapy has encouraged further investigation into bioengineering hepatic tissues in vivo. Engineering tissues in the subcutaneous space is an attractive method; however, hepatocyte survival has been transient due to insufficient vascular network formation. To establish a vascularized cavity, we created a polyethylene terephthalate mesh device coated with poly(vinylalcohol) that allowed for the gradual release of basic fibroblast growth factor (bFGF), a potent angiogenic factor. The efficacy of the bFGF-releasing device in inducing vascular network formation in the subcutaneous space was observed in mouse and rat studies. Isolated mouse hepatocytes transplanted into newly vascularized subcutaneous cavities allowed for persistent survival up to 120 days. In the absence of a vascularized compartment, the survival of the transplanted hepatocytes was markedly diminished. Functional maintenance of the engineered hepatic tissues was confirmed by high expression of liver-specific mRNAs and proteins. These engineered hepatic tissues have the ability to take up inoculated compounds and express strong induction of drug-metabolizing enzymes, demonstrating functional relevance as a metabolic tissue. In conclusion, we have created a novel technology to engineer functionally active hepatic tissues in the subcutaneous space, which will likely facilitate hepatocyte-based therapies.

Mechanisms of acetaminophen-induced hepatotoxicity: role of oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes.

Freshly isolated mouse hepatocytes were used to determine the role of mitochondrial permeability transition (MPT) in acetaminophen (APAP) toxicity. Incubation of APAP (1 mM) with hepatocytes resulted in cell death as indicated by increased alanine aminotransferase in the media and propidium iodide fluorescence. To separate metabolic events from later events in toxicity, hepatocytes were preincubated with APAP for 2 h followed by centrifugation of the cells and resuspension of the pellet to remove the drug and reincubating the cells in media alone. At 2 h, toxicity was not significantly different between control and APAP-incubated cells; however, preincubation with APAP followed by reincubation with media alone resulted in a marked increase in toxicity at 3 to 5 h that was not different from incubation with APAP for the entire time. Inclusion of cyclosporine A, trifluoperazine, dithiothreitol (DTT), or N-acetylcysteine (NAC) in the reincubation phase prevented hepatocyte toxicity. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress. Tetramethylrhodamine methyl ester perchlorate fluorescence decreased during the reincubation phase indicating a loss of mitochondrial membrane potential. Inclusion of cyclosporine A, DTT, or NAC decreased oxidative stress and loss of mitochondrial membrane potential. Confocal microscopy studies with the dye calcein acetoxymethyl ester indicated that MPT had also occurred. These data are consistent with a hypothesis where APAP-induced cell death occurs by two phases, a metabolic phase and an oxidative phase. The metabolic phase occurs with GSH depletion and APAP-protein binding. The oxidative phase occurs with increased oxidative stress, loss of mitochondrial membrane potential, MPT, and toxicity.

Hypothermia Inhibits Fas-Mediated Apoptosis of Primary Mouse Hepatocytes in Culture

Apoptosis occurs during the isolation and even short-term storage and culture of hepatocytes, and in the pathogenesis of liver diseases, such as hepatic failure and hepatitis. Therapeutic hypothermia has beneficial effects in experimental models of fulminant hepatic failure. The mechanisms underlying the potential benefits of mild hypothermia on the liver have not been well investigated. We examined the effects of temperature on soluble Fas ligand-induced apoptosis in freshly isolated mouse hepatocytes. Decreasing the culture temperature from 37 degrees C to 32 degrees C produced significant suppression of Fas-mediated apoptosis in cultured hepatocytes over a 12-h period. This observation was supported by cell morphology, flow cytometry analysis of cellular DNA content, and Annexin V-FITC staining of membrane phosphatidylserine translocation. In hypothermic conditions, Fas-mediated cytochrome c release from mitochondria of hepatocytes and the proximate downstream activation of caspase-9 were suppressed under mild hypothermic conditions. Effector caspase-7 activity was also inhibited at 32 degrees C. In contrast, the activation of initiator caspase-8 and cleavage of Bid were not affected after Fas-ligand stimulation. These findings suggest that mild hypothermia suppresses Fas-mediated apoptosis of liver cells by the partial inhibition of signaling events including mitochondrial damage, cytochrome c release, and subsequent apoptosome formation and effector caspase activation.

Preferential proliferation of attenuated salmonella typhimurium within neuroblastoma

Background/purpose Attenuated Salmonella typhimurium, a facultative intracellular parasite that colonizes the liver, has been shown to accumulate within extrahepatic malignancies. The authors sought to define a mechanism for attenuated Salmonella accumulation within cancer cells compared with hepatocytes. Methods Invasion and intracellular proliferation of attenuated Salmonella was assessed through an in vitro assay performed on neuroblasoma, osteosarcoma, hepatoma, and colon adenocarcinoma cell lines and compared with freshly isolated mouse hepatocytes. Results The efficiency of attenuated S typhimurium invasion into hepatocytes was greater than any malignant cell line (3.8 v 0.46; P < .04). However, the intracellular proliferation of the bacteria was most abundant within neuroblastoma, exceeding the proliferation within hepatocytes (14.3 v 6.2; P < .01). Conclusions Attenuated S typhimurium may prove to be an effective in vivo immunotherapy for the local delivery of therapeutic proteins to neuroblastoma.

Optimisation of the comet genotoxicity assay in freshly isolated murine hepatocytes: detection of strong in vitro DNA damaging properties for styrene

While the comet assay is used to detect DNA damage in isolated cells following exposure to chemicals in vitro, few publications report the use of the procedure in liver cells isolated from mice. Our initial efforts to use the assay to assess DNA damage in mouse hepatocytes maintained on collagen-coated dishes were hampered by high levels of baseline damage in controls, which appeared to result from mechanical damage sustained during the dislodgement of adherent cells in the early stages of the assay protocol. Here we describe an efficient version of the comet assay in cultured mouse hepatocytes that involves careful recovery of cells using a “scraping” buffer supplemented with 10% high purity grade DMSO. Use of this buffer strongly diminished the frequency of false positives. Using the industrial reagent styrene as a positive control in the optimised procedure, non-cytotoxic concentrations of this substance (2.5–10 mM) significantly increased mean comet tail length, area, and moment. Co-incubation with the CYP inhibitor SKF-525A strongly attenuated these effects of styrene. Collectively, these findings confirm this method is highly suitable for the detection of DNA damage by bioactivation-dependent compounds in freshly isolated mouse hepatocytes.

EGF mediates protection against Fas‐induced apoptosis by depleting and oxidizing intracellular GSH stocks

Several pieces of evidence have demonstrated the importance of reduction/oxidation (redox) signaling in biological processes, including sensitivity toward apoptosis. In parallel, it was recently reported that growth factors induce the generation of reactive oxygen species (ROS). Therefore, we tested the hypothesis that the anti-apoptotic effect of epidermal growth factor (EGF) was mediated by changes in the redox state of hepatocytes through changes in GSH stocks. Isolated mouse hepatocytes were cultured and exposed to anti-Fas stimulation in order to induce apoptosis. Cell death by apoptosis was assessed by Hoechst 33258 staining and by measuring caspase-3 proteolysis activity. Cell treatment with EGF significantly decreased total (GSx) and reduced (GSH) glutathione levels in the presence and the absence of anti-Fas. Furthermore, glutathione reductase activity was lower in EGF-treated cultures (by 28%) as compared to untreated cultures which lead to a significant decline in GSH/GSx ratio. These effects were found to be EGF-receptor tyrosine kinase activity dependent. Co-stimulation of cells with anti-Fas and EGF attenuated caspase-3 activation and cell death by apoptosis by 70%. GSH monoethylester (GSHmee) significantly attenuated the effect of EGF on GSH and GSH/GSx ratio. It caused an increase in caspase-3 activation and in the percentage of apoptotic cells in anti-Fas + EGF-treated cells, thus resulting in a 53% decline in the protective effect of EGF. In conclusion, EGF induces a significant and specific depletion and oxidization of intracellular GSH, paralleled by a protection against Fas-induced apoptosis. GSH repenishment partly counteracted these effects suggesting that GSH depletion contributed to the protective effect of EGF against caspase-3 activation and cell death by apoptosis.

UDP-glucuronosyltransferase-dependent bioactivation of clofibric acid to a DNA-damaging intermediate in mouse hepatocytes

Glucuronidation of a number of carboxyl-containing drugs generates reactive acyl glucuronide metabolites. These electrophilic species alkylate cell proteins and may be implicated in the pathogenesis of a number of toxic syndromes seen in patients receiving the parent aglycones. Whether acyl glucuronides also attack nuclear DNA is unknown, although the acyl glucuronide formed from clofibric acid was recently found to decrease the transfection efficiency of phage DNA and generate strand breaks in plasmid DNA in vitro. To determine if such a DNA damage occurs within a cellular environment, the comet assay (i.e. single-cell gel electrophoresis) was used to detect DNA lesions in the nuclear genome of isolated mouse hepatocytes cultured with clofibric acid. Overnight exposure to 50 μM and higher concentrations of clofibric acid produced concentration-dependent increases in the comet areas of hepatocyte nuclei, with 1 mM clofibrate producing a 3.6-fold elevation over controls. These effects closely coincided with culture medium concentrations of the glucuronide metabolite formed from clofibric acid, 1- O-β-clofibryl glucuronide. Consistent with a role for glucuronidation in the DNA damage observed, the glucuronidation inhibitor borneol diminished glucuronide formation from 100 μM clofibrate by 98% and returned comet areas to baseline levels. Collectively, these results suggest that the acyl glucuronide formed from clofibric acid is capable of migrating from its site of formation within the endoplasmic reticulum to generate strand nicks in nuclear DNA.

Nordihydroguaiaretic acid: hepatotoxicity and detoxification in the mouse

Larrea tridentata (Moc & Sess) Cov. (Zygophyllaceae) is an ethnobotanically important plant found in the American Southwest and northern Mexico. Although numerous beneficial effects have been attributed to this plant, several case reports have demonstrated high doses of Larrea-containing herbals induce hepatotoxicity and nephrotoxicity in humans. Nordihydriguaiaretic acid (NDGA) is a lignan found in high amounts (up to 10% by dry weight) in the leaves and twigs of L. tridentata. Previously, NDGA has been shown to induce cystic nephropathy in the rat, however, no reports have been made concerning this compound's hepatotoxic potential. Here, we report that intraperitoneal adminstration of NDGA is lethal in the mouse (LD 50=75 mg/kg). Administration is associated with a time and dose-dependent increase in serum alanine aminotransferase levels, which suggest liver damage. Indeed, freshly isolated mouse hepatocytes are more sensitive to NDGA than human melanoma cells. Furthermore, we have identified glucuronidation as a potential detoxification mechanism for NDGA. Both mono and diglucuronide conjugates of NDGA are formed after intravenous dosing. The monoglucuronide is also formed after incubation of NDGA with human hepatic microsomes; suggesting that glucuronide conjugation is important in the metabolism of NDGA by humans. In summary, this report indicates that NDGA may contribute to the hepatotoxicity of L. tridentata and provides preliminary information on NDGA metabolism.

Functional analysis of mouse hepatocytes differing in DNA content: volume, receptor expression, and effect of IFNgamma.

Polyploidy and binuclearity are characteristics of the mammalian liver. Increasing polyploidisation occurs with age and after administration of various drugs and chemicals. This study was designed to examine the function of ploidy by addressing several questions: (1) Does the increase in size of polyploid hepatocytes have any physiological function by altering surface receptor expression such as intercellular adhesion molecule-1 (ICAM-1, CD54) or IFNgammaR? and (2) Do polyploid cells respond differently to inflammatory cytokines such as interferon gamma (IFNgamma)? We have developed a method to accurately measure the volume of live isolated hepatocytes using confocal microscopy and image analysis. Using flow cytometry, we have shown that the expression of ICAM-1 increases with increasing DNA content and IFNgammaR is not detectable on isolated mouse hepatocytes. Diploid (2n), tetraploid (4n) and octoploid (8n) hepatocytes were found to be equally susceptible to IFNgamma-induced apoptosis in vitro. Although the function of polyploidy remains unanswered, we have described some of the characteristics of polyploidy in isolated hepatocytes and in vitro.

Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytesin Vitro and in Vivo

The asialoglycoprotein receptor (ASGPr) on hepatocytes plays a role in the clearance of desialylated proteins from the serum. Although its sugar preference (N-acetylgalactosamine (GalNAc) >> galactose) and the effects of ligand valency (tetraantennary > triantennary >> diantennary >> monoantennary) and sugar spacing (20 A 10 A 4 A) are well documented, the effect of particle size on recognition and uptake of ligands by the receptor is poorly defined. In the present study, we assessed the maximum ligand size that still allows effective processing by the ASGPr of mouse hepatocytes in vivo and in vitro. Here too, we synthesized a novel glycolipid, which possesses a highly hydrophobic steroid moiety for stable incorporation into liposomes, and a triantennary GalNAc(3)-terminated cluster glycoside with a high nanomolar affinity (2 nm) for the ASGPr. Incorporation of the glycolipid into small (30 nm) [(3)H]cholesteryl oleate-labeled long circulating liposomes (1-50%, w/w) caused a concentration-dependent increase in particle clearance that was liver-specific (reaching 85 +/- 7% of the injected dose at 30 min after injection) and mediated by the ASGPr on hepatocytes, as shown by competition studies with asialoorosomucoid in vivo. By using glycolipid-laden liposomes of various sizes between 30 and 90 nm, it was demonstrated that particles with a diameter of >70 nm could no longer be recognized and processed by the ASGPr in vivo. This threshold size for effective uptake was not related to the physical barrier raised by the fenestrated sinusoidal endothelium, which shields hepatocytes from the circulation, because similar results were obtained by studying the uptake of liposomes on isolated mouse hepatocytes in vitro. From these data we conclude that in addition to the species, valency, and orientation of sugar residues, size is also an important determinant for effective recognition and processing of substrates by the ASGPr. Therefore, these data have important implications for the design of ASGPr-specific carriers that are aimed at hepatocyte-directed delivery of drugs and genes.

Is hyperthermia the triggering factor for hepatotoxicity induced by 3,4-methylenedioxymethamphetamine (ecstasy)? An in vitro study using freshly isolated mouse hepatocytes.

The consumption of 3,4-methylenedioxymethamphetamine (ecstasy; MDMA) may cause hepatocellular damage in humans, a toxic effect that has been increasing in frequency in the last few years, although the underlying mechanisms are still unknown. The metabolism of MDMA involves the production of reactive metabolites which form adducts with intracellular nucleophilic sites, as is the case with glutathione (GSH). Also, MDMA administration elicits hyperthermia, a potentially deleterious condition that may aggravate its direct toxic effects. Thus, the objective of this study was to evaluate the extent of MDMA-induced depletion of GSH, induction of lipid peroxidation and loss of cell viability in freshly isolated mouse hepatocytes under normothermic conditions (37 degrees C) and to compare the results with the effects obtained under hyperthermic conditions (41 degrees C). By itself, hyperthermia was an important cause of cell toxicity. A rise in incubation temperature from 37 degrees C to 41 degrees C caused oxidative stress in freshly isolated mouse hepatocytes, reflected as a time-dependent induction of lipid peroxidation and consequent loss of cell viability (up to 40-45%), although the variations in GSH and GSSG levels were similar to those under normothermic conditions. MDMA (100, 200, 400, 800 and 1600 microM) induced a concentration- and time-dependent GSH depletion at 37 degrees C but had a negligible effect on lipid peroxidation and cell viability at this temperature. It is particularly noteworthy that hyperthermia (41 degrees C) potentiated MDMA-induced depletion of GSH, production of lipid peroxidation and loss of cell viability (up to 90-100%). It is therefore concluded that hyperthermia potentiates MDMA-induced toxicity in freshly isolated mouse hepatocytes.

Effects of a Novel Hepatoprotective Drug, ZNC-2381, on Fas-Induced Hepatocellular Caspase-3 Activity and Apoptosis in Mice

We examined the effects of ZNC-2381 (1-(4-aminophenyl)methyl-3-(3-nitrophenyl)-1,3-dihydroimidazo[4,5-b] pyridine-2-one), a new oral hepatoprotective agent, on hepatocellular caspase-3 activity and apoptosis induced by anti-mouse Fas antibody (anti-Fas ab) in mice. Oral ZNC-2381, administered at doses of 10, 30 and 100 mg/kg 1 h before inducing hepatic injury with anti-Fas ab, dose-dependently inhibited the increase in serum alanine aminotransferase (s-ALT) activity 8 h after injection of anti-Fas ab. Increases in DNA fragmentation (nucleosome assay) and caspase-3 activity in the liver 2 h after injection of anti-Fas ab were also inhibited by ZNC-2381 in a dose-dependent manner. As shown by histopathological examination, ZNC-2381 dose-dependently inhibited the appearance of TUNEL-positive apoptotic cells in the liver. Moreover, in studies in vitro, ZNC-2381 (1– 100 µmol/l) concentration-dependently inhibited increases in DNA fragmentation and caspase-3 activity caused by anti-Fas ab in isolated mouse hepatocytes. N- Acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-cho), a caspase-3-specific inhibitor, inhibited hepatocellular apoptosis caused by anti-Fas ab both in vivo and in vitro, as well as the increase in s-ALT activity in vivo. These results demonstrate that orally administered ZNC-2381 inhibits hepatocellular apoptosis induced by anti-Fas ab and presents the progression of hepatic injury. We propose that the mechanism of action of ZNC-2381 may involve blockade of the signal transduction pathway (caspase-3) of apoptosis mediated by anti-Fas ab.

Modulation of Blood Glucose by Melatonin: A Direct Action on Melatonin Receptors in Mouse Hepatocytes

Melatonin receptors were studied in isolated mouse hepatocytes using the 2[¹²⁵I]iodomelatonin binding assay. The binding of 2[¹²⁵I]iodomelatonin to hepatocytes isolated from the mouse using collagenase was stable, saturable, reversible and of high affinity. The equilibrium dissociation constant (K_d) obtained from saturation studies was 10.0 ± 0.4 pmol/l (n = 16), which was comparable to the K_d obtained from kinetics studies (6.9 ± 1.2 pmol/l, n = 3), and the maximum number of binding sites (B_max) was 2.9 ± 0.4 fmol/mg protein (n = 16). The relative order of potency of indoles in competing for 2[¹²⁵I]iodomelatonin binding was 2-iodomelatonin > 2-phenylmelatonin > 6-chloromelatonin > melatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that the binding was mediated by the ML₁ receptor subtype. The linear Rosenthal plots, the close proximity of the Hill coefficient to unity and the monophasic competition curves suggest that a single class of 2[¹²⁵I]iodomelatonin binding sites is present in the mouse hepatocytes. Guanosine 5′-O-(3-thiotriphosphate) dose-dependently inhibited 2[¹²⁵I]iodomelatonin by lowering the affinity of binding, while no inhibitory effects of adenosine nucleotides were observed, suggesting that the binding sites are G-protein linked. Western immunoblotting was used to identify the melatonin receptor subtype in mouse hepatocytes using anti-Mel_1a and anti-Mel_1b. Hepatocyte membrane extract reacted with anti-Mel_1b but not anti-Mel_1a giving a peptide-blockable band of 36 kD, supporting the hypothesis that the melatonin receptors in mouse hepatocytes are of the Mel_1b subtype. Melatonin injection and a high plasma glucose level affected 2[¹²⁵I]iodomelatonin binding in the whole mouse liver homogenates. Plasma glucose was elevated by mid-light intraperitoneal injection of melatonin (4 and 40 mg/kg body weight) in a dose-dependent manner with maximum elevation achieved 1 h after injection. 2[¹²⁵I]Iodomelatonin binding at this time showed increased K_d with no changes in B_max. When the plasma glucose returned to normal within 2 h, the binding remained lowered with increased K_d but no changes in B_max. Elevation of plasma glucose by 2-deoxyglucose injection (500 mg/kg), on the other hand, decreased the binding by decreasing the B_max without affecting the K_d. Suppression of plasma glucose by insulin injection (3 IU/kg) did not change the binding. Thus, melatonin may act directly on the liver to elevate the plasma glucose level, and changes in plasma glucose level itself may in turn affect hepatic melatonin binding.

β-glucuronidase latency in isolated murine hepatocytes

The physiological function of microsomal β-glucuronidase is unclear. Substrates may be either glucuronides produced in the lumen of endoplasmic reticulum (ER) or those taken up by hepatocytes. In the latter case, efficient inward transport of glucuronides at the plasma membrane and the ER membrane would be required. Therefore, the potential role of β-glucuronidase in ER was investigated. Isolated mouse hepatocytes and mouse and rat liver microsomal vesicles were used in the experiments. Selective permeabilization of the plasma membrane of isolated hepatocytes with saponin or digitonin resulted in an almost 4-fold elevation in the rate of p-nitrophenol glucuronide hydrolysis, while the permeabilization of plasma membrane plus ER membrane by Triton X-100 caused a further 2-fold elevation. In microsomal vesicles, the p-nitrophenol glucuronide or phenolphthalein glucuronide β-glucuronidase activity showed about 50% latency as revealed by alamethicin or Triton X-100 treatment. A light-scattering study indicated that the microsomes are relatively impermeable to both glucuronides and to glucuronate. On the basis of our results, the role of liver microsomal β-glucuronidase in the deconjugation of glucuronides taken up by the liver seems unlikely. Hydrolysis of the glucuronides produced in the ER lumen may play a role in substrate supply for ascorbate synthesis or in “proofreading” of glucuronidation.

TNF-alpha regulates transforming growth factor-alpha expression in regenerating murine liver and isolated hepatocytes.

TNF-alpha is a pleotropic proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes, primarily through its ability to induce the expression of inflammatory and cytotoxic mediators. TNF-alpha is also involved in cell growth accompanying the healing process in multiple organ systems and influences liver repair following hepatotoxic damage or regeneration following partial hepatectomy. In this respect, TNF-alpha is a known mitogen for hepatocytes. In this paper we describe a novel role for TNF-alpha in the modulation of expression of TGF-alpha, the latter being a complete hepatocyte mitogen. TNF-alpha directly up-regulates TGF-alpha mRNA by up to 7-fold in isolated mouse hepatocytes, whereas neutralization of TNF-alpha significantly decreased liver mRNA and protein expression of TGF-alpha following chemical-induced hepatotoxicity. That TNF-alpha directly stimulated TGF-alpha was suggested by the inability of either anti-IL-6 Abs or cycloheximide to inhibit TNF-alpha-induced TGF-alpha expression in hepatocytes. However, in the presence of anti-TGF-alpha neutralizing Abs, the mitogenic activity of TNF-alpha is abrogated. Using cells transfected with the TGF-alpha promoter, and an RNA polymerase inhibitor, it was shown that TNF-alpha modulates TGF-alpha expression through both pre- and posttranscriptional events. Taken together, these data suggest that TNF-alpha participates in liver repair and regeneration, in part, by directly inducing the expression of TGF-alpha.

Regulation of Glucuronidation by Glutathione Redox State through the Alteration of UDP-Glucose Supply Originating from Glycogen Metabolism

The effect of altered redox state of glutathione was investigated onp-nitrophenol glucuronidation in isolated mouse hepatocytes. Decrease of GSH/GSSG ratio provoked by various agents caused increased glucuronidation which was accompanied by stimulated glycogenolysis and elevated UDP-glucose content. The stimulation of glycogenolysis and glucuronidation by glutathione consumption could be prevented by the reduction of oxidized glutathione with dithiothreitol and by the glycogenolysis inhibitor fructose. In permeabilized hepatocytes glycogen metabolism, bypassed by the addition of UDP-glucose, stimulated glucuronidation which was insensitive to glutathione depletion. In liver microsomes either UDP-glucuronosyltransferase activity or UDP-glucuronic acid transport was not influenced by GSH/GSSG ratio. These results suggest that alteration of the GSH/GSSG ratio regulates glucuronidation by affecting enzymes of the glycogen metabolism via the modification of UDP-glucuronate supply.

Role of Metabolic Intermediates in Lipopolysaccharide/Cytokine-Mediated Production of Nitric Oxide in Isolated Mouse Hepatocytes

Induction of nitric oxide synthase by bacterial endotoxinin vivocan be mimicked by treating cultured hepatocytes with a combination of lipopolysaccharide and cytokines (LPS/cytokines), but the role of LPS/cytokine-induced nitric oxide in hepatocyte glucose metabolism is ambiguous. In this study, intermediary metabolite effects on LPS/cytokine-induced hepatocyte nitric oxide synthesis were examined. Pyruvate, lactate, oxaloacetate, and fumarate all showed some inhibitory effects on hepatocyte nitric oxide synthesis. However, these metabolic intermediates could not improve the mitochondrial respiration of LPS/cytokine-treated hepatocytes. Phosphoenolpyruvate carboxykinase activity (or flux) relating factors, glucocorticoids and cAMP, also blocked LPS/cytokine-induced nitric oxide synthesis. Insulin was much less potent than cAMP and glucocorticoids, and phorbol ester did not show any effect on hepatocyte nitric oxide synthesis. These results suggest that LPS/cytokine-induced nitric oxide synthesis is related, at least partly, to phosphoenolpyruvate carboxykinase activity (or flux) in hepatocytes.

The role of lipoprotein lipase and apoprotein E in the recognition of chylomicrons and chylomicron remnants by cultured isolated mouse hepatocytes.

Lipoprotein lipase (LPL) has been proposed to play a role in the uptake of chylomicron remnants by hepatocytes by mediating the binding of these lipoproteins to cell-surface glycosaminoglycans and to the low-density-lipoprotein receptor-related protein (LRP). This proposal is based on studies that examined the binding of chylomicrons to HepG2 cells, fibroblasts and Chinese hamster ovary cells in culture, in the presence of large amounts of LPL [Beisiegel (1995) Curr. Opin. Lipidol. 6, 117-122]. We have investigated whether LPL attached to the surface of chylomicrons enhances the binding and uptake of these lipoproteins to isolated hepatocytes maintained in culture. Bovine milk LPL was bound to mouse chylomicrons, double-labelled in vivo with [3H]retinol (in retinyl esters) and with [14C]palmitic acid (in triacylglycerols), collected from the mesenteric lymph of normal mice and from mice lacking the apoprotein E (apo E) gene. Normal chylomicrons (containing apo E) and apo E-free chylomicrons, with or without bound LPL, were incubated with cultured hepatocytes isolated from mice lacking the apo E gene. At 0 degree C LPL did not enhance the binding of the normal or apo E-free chylomicrons by the hepatocytes. When incubations were performed at 37 degrees C the triacylglycerols of normal and apo E-free chylomicrons were hydrolysed by LPL and there was a significant uptake of [14C]fatty acids and [3H]retinol by the hepatocytes. The addition of heparin or lactoferrin, a known inhibitor of hepatic uptake of chylomicron remnants, to the incubation medium inhibited the uptake of [3H]retinol, present in the lipoprotein core, but not the uptake of the [14C]fatty acids. We conclude that: (1) LPL attached to chylomicrons in amounts sufficient to effectively hydrolyse their core triacylglycerols does not enhance the binding of these lipoproteins to the surface of isolated hepatocytes; (2) the recognition and uptake of chylomicrons by hepatocytes requires that these lipoproteins be first hydrolysed by LPL; and (3) the uptake of lipolysed chylomicrons (remnants) by hepatocytes does not require the mediation of apo E.

Comparative Short-Term Effects of Methyl Tertiary Butyl Ether and Unleaded Gasoline Vapor in Female B6C3F1 Mice

PS-6 unleaded gasoline (UG) and methyltert-butyl ether (MTBE), an UG additive, with long-term exposure at high concentrations increased liver tumors selectively in female mice. PS-6 UG is a liver tumor promoter inN-nitrosodiethylamine-initiated female mice and produces short-term effects potentially relevant to its tumor promoting ability. The new formulation of UG (91-01) and MTBE were evaluated for similar short-term effects in mouse liver. Mice were exposed to 7814 ppm MTBE, 2014 ppm 91-01 UG, or 2028 ppm PS-6 UG vapor for 3 or 21 days, 6 hr/day, 5 days/week. Relative liver weights increased and uterine weights decreased in MTBE-, 91-01 UG-, and PS-6 UG-exposed mice. Because the decrease in relative uterine weight is suggestive of hormonal modulation, we evaluated the effects of MTBE, 91-01 UG, and PS-6 UGin vivoon hepatic 17-β estradiol metabolismin vitro.Gavage treatment with either blend of UG and with MTBE increased estrogen metabolism in isolated mouse hepatocytes. Hepatic microsomal P450 activity was assessed by 7-pentoxyresorufin-O-dealkylase (PROD) and 7-ethoxyresorufin-O-de-ethylase (EROD) activities. Similar increases in P450 content and PROD and EROD activities were observed in all exposed mice as compared to controls. No hepatoxicity was observed in any treatment group. The hepatic labeling index, as measured by the incorporation of 5-bromo-2′-deoxyuridine, was increased in all exposed mice at 3 days but not 21 days, indicating that MTBE and 91-01 UG are also hepatic mitogens. These data demonstrate that a newer blend of UG and the UG additive MTBE elicit short-term effects similar to those of PS-6 UG. Given that these effects are potentially related to tumor promotion and the general lack of genotoxic activity, MTBE and 91-01 UG may exhibit tumor promoting activity similar to that seen with PS-6 UG. Since the liver is under multihormonal control, the increase in hepatic estrogen metabolism and uterine effects support a potential role for endocrine modulations in both MTBE- and UG-induced hepatocarcinogenesis.