Inhibition of oxidative stress and apoptosis enables extended maintenance of integrity and function of isolated hepatocytes in suspension

Aims: Isolated hepatocytes in suspension may offer an alternative culture system for bioartificial liver devices. However, maintenance of isolated hepatocyte suspensions in conventional media leads to rapid loss of cell integrity. The aim of this study was to develop a modified medium to better maintain hepatocyte integrity. Methods: Isolated rat hepatocytes were prepared by collagenase digestion. Hepatocytes were purified in a Percoll gradient, suspended in bicarbonate buffered isotonic saline supplemented with d‐α‐tocopherol succinate and glucose and medium changed at 24 h (modified medium). The properties of cells treated this way were compared with those prepared by collagenase digestion and suspension in bicarbonate buffered isotonic saline (basic medium). Both media were maintained at 30°C for 48 h on an orbital shaker. Markers for oxidative stress, apoptosis and metabolic function were measured enzymatically. Cell morphology was assessed by electron microscopy. Results: When compared to collagenase‐isolated hepatocytes maintained in basic medium, hepatocytes purified by Percoll (Amersham Biosciences, Castle Hill, Australia) and maintained in modified medium demonstrated significantly increased glutathione (GSH) and GSH : glutathione disulphide (GSSH) ratios, decreased lipid peroxidation product formation, decreased caspase‐3 protease activity, reduced uptake of trypan blue and loss of lactate dehydrogenase (LDH) and increase preservation of cellular adenosine triphosphate concentration ([ATP]), urea synthesis, ammonia removal and glycogen content. Cell morphology was substantially preserved following 48 h of maintenance in the modified medium. Conclusions: The use of Percoll and modified medium reduces cell injury and apoptosis and greatly improves maintenance of cell function and morphology. The modifications reported here and the use of isolated hepatocyte suspensions in bioartificial liver devices are worthy of further investigation.     

Protective effects of a hibernation-inducer on hepatocyte injury induced by hypothermic preservation

Background/Purpose

For hepatocyte-based cell therapy to be realistic, the method chosen for cryopreservation or hypothermic preservation is critical. The aim of the present study was to clarify whether D-Ala2-Leu5-enkephalin (DADLE), a hibernation inducer, has protective effects on hepatocytes with regard to hypothermic preservation injury.

Methods

A suspension of rat hepatocytes was stored at 4?°C for 24?h with or without DADLE. Their viability was measured by the trypan blue dye exclusion method, and alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels in the preservation solution were measured. After 24?h of cold storage, viable hepatocytes were cultured at 37?°C for another 24?h. Then albumin production and lidocaine clearance were measured.

Results

DADLE significantly improved the survival rate of hepatocytes. The levels of ALT and LDH in the preservation solution with DADLE were significantly lower than those in the preservation solution without DADLE. The treated viable hepatocytes maintained both albumin synthesis and lidocaine clearance.

Conclusions

DADLE appears to have protective effects on hepatocytes with regard to hypothermic preservation injury in vitro. This hibernation-inducer is useful in prolonged hypothermic preservation for hepatocyte-based therapy.     

Relationship between cellular ATP content and cellular functions of primary cultured rat hepatocytes in hypoxia

The importance of oxygen in maintaining the functional integrity of hepatocytes has been well established in a variety of experimental models, such as in vivo , perfused liver and isolated hepatocytes. However, one of the shortcomings of these systems is their short life span. Therefore, we have examined the effects of long-term hypoxia on cellular adenine nucleotide content and cellular functions, such as albumin production, urea production and DNA synthesis, in adult rat hepatocytes in primary culture. Hepatocytes were cultured at a density of 11 × 10⁴ and 5 × 10⁴ cells/0.18 mL per cm² for the study of albumin and urea production and DNA synthesis, respectively, at various oxygen tensions (20, 12, 8 and 5%) for 24 h. Cellular ATP content in cultured hepatocytes in hypoxia gradually declined, corresponding to the decrease in oxygen tension, and the cellular ATP level at 5% oxygen was approximately 20% of that at 20% oxygen. Albumin production also decreased in parallel with the decrease in cellular ATP content in cultured hepatocytes in hypoxia. However, even when cellular ATP content gradually declined corresponding with the decrease in oxygen tension in cultured hepatocytes in hypoxia, such as at 8 or 5% oxygen, urea production remained at a high level; in contrast, DNA synthesis was completely suppressed. These results suggest that the cellular ATP content decreases in cultured hepatocytes during long-term hypoxia in relation to oxygen tension and that the relationship between decreased ATP levels and liver function in cultured hepatocytes during hypoxia differs for albumin production, urea production and DNA synthesis.     

In vitro analysis of cryopreserved alginate–poly‐l‐lysine–alginate‐microencapsulated human hepatocytes

Background: The availability of well‐characterized human hepatocytes that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after microencapsulated cryopreservation and investigated whether these cryopreserved microencapsulated hepatocytes can be used for clinical applications. Methods: Adult hepatocytes of 18 separate donors were isolated with a two‐step extracorporeal collagenase perfusion technique. After pre‐incubation at 4 °C for 12–24 h in HepatoZYME‐SFM, hepatocytes were microencapsulated using alginate–poly‐l ‐lysine–alginate microcapsules. The microencapsulated hepatocytes were transferred to a complete medium containing 10% dimethyl sulphoxide. They were immediately placed into an isopropanol progressive freezing container at ?80 °C overnight and immersed in liquid nitrogen the next day. During the post‐thawing culture period, albumin secretion, urea synthesis, cell cycle, mRNA and protein levels, as well as the morphology and pathology structure of pre‐incubation before microencapsulated cryopreservation (PMC) groups were analysed. Results: Compared with the immediate cryopreservation (IC) groups, we found significant improvement in the mRNA and protein levels in the attached cells, and higher secretion of albumin and urea levels after thawing. In the attached cultured human cryopreserved/thawed hepatocytes from the PMC group, albumin production was not significantly different from those of the direct culture groups on days 2, 3 and 4. The preserved morphology in the PMC group compared with the IC group was obvious. Conclusions: The results of the present study suggested recovery of the functional and morphological integrity of human hepatocytes after pre‐incubation at 4 °C for 12–24 h before microencapsulated cryopreservation. These studies offer the possibility for clinical applications in pharmacotoxicology, bioartificial liver and cell therapy in humans.     

Animal model for liver cell banking from non-heart beating donors after prolonged ischaemia time

BACKGROUND AND AIM: Although there is a growing interest on the use of non-heart beating donors to enlarge the liver donor pool, livers with prolonged warm ischaemia time are not currently considered for organ transplantation. We hypothesised that these organs may represent a source of hepatocytes for cell transplantation and/or use in bioartificial liver devices. Thus, we investigated if prolonged ischaemia could influence the recovery and viability of functional hepatocytes dissociated from rat livers. METHODS: Hepatocytes were isolated from the liver within 15 min after death (t=15 min) and after 4, 8 and 12h of ischaemia. Cells were either maintained in culture or cryopreserved. In all products, we evaluated cell recovery and viability, hepatocyte markers and cellular functions, including albumin and urea production. RESULTS: The number of cells per gram of tissue was similar at 15 min, 4 and 8h, while it was significantly decreased at 12h. About 0.2 x 10(6) viable cells expressing hepatocyte markers and producing albumin and urea were isolated up to 8h of ischaemia per gram of tissue. CONCLUSIONS: Recovery of viable and functional hepatocytes seems possible after prolonged ischaemia time. These data warrant the evaluation of hepatocyte isolation from human livers of non-heart beating donors.     

Adult Rat Hepatocytes in Primary Culture. VII. Proliferative and Functional Properties of Cells from Ethanol-Intoxicated Animals: Evidence for a Reversible Albumin 'Production Defect'

Properties of long-term cultures of hepatocytes isolated from nutritionally maintained adult rats treated in vivo for 8–12 days with ethanol to achieve blood levels of 200–220 mg ethanol/dl (experimental group) and from isocalorically maintained mock-treated (control) rats were compared. Two kinds of treatment protocols were used: intragastric intubation or inhalation chambers. Similar results were observed with cultured cells obtained from either protocol. Hepatocytes in ‘monolayers’ from both animal groups showed unimpaired proliferative responses with respect to (a) cell multiplication and DNA synthesis rates during an asynchronous 12-day growth cycle and (b) proliferogenic peptides [insulin, glucagon, and epidermal growth factor (EGF)] that reinitiate DNA synthesis parasynchronously during late stationary phase (days 12–13). Quantitative differences in [³H]leucine uptake into total proteins (cellular plus ‘secreted’) were not detected during the growth cycle. However, during stationary phase (days 8–12), 25%-75% less [³H]leucine-labeled albumin was released into culture fluids by hepatocytes from experimental livers than by hepatocytes from control livers. More pronounced differences were seen when albumin in the same culture fluids was measured by radioimmunoassay in contrast to measurements of [³H]leucine-labeled albumin in these fluids. The defect was reversible since it was not seen in stationary-phase hepatocyte cultures prepared from rats withdrawn more than 7 days from ethanol treatment whose blood alcohol levels had fallen to 0 mg ethanol/dl. These findings are consistent with the hypothesis that, with respect to albumin production by hepatocytes, chronic ethanol exposure induces a specific but reversible ‘defect’. Because this defect is not seen in short-term hepatocyte cultures, long-term cultures should facilitate attempts to understand the molecular and cellular causes of this functional impairment.     

Primary porcine hepatocytes with portal vein serum cultured on microcarriers or in spheroidal aggregates

AIM:To develop a culture mode providing durable biomaterials with high yields and activities used in bioartificial liver.METHODS:Hepatocytes were isolated from a whole pig liver by Seglen s method of orthotopic perfusion with collagenase. In culture on microcarriers, primary porcine hepatocytes were inoculated at a concentration of 5center dot10(7)/mL into the static culture systems containing 2g/L Cytodex-3, then supplemented with 100mL/L fetal calf serum (FCS) or 100mL/L porcine portal vein serum (PPVS) respectively. In spheroidal aggregate culture hepatocytes were inoculated into 100mL siliconized flasks at a concentration of 5.0center dot10(6)/mL.RESULTS:In culture on microcarriers hepatocytes tended to aggregate on Cytodex-3 obviously after being inoculated. Typical multi-cellular aggregated spheroids could be found in the two systems 24h-48h after hepatocytes were cultured. The morphological charact-eristics and synthetic functions were maintained for 5wk in FCS culture system and 8wk in PPVS culture system. In spheroidal aggregate culture about 80%-90% isolated hepatocytes became aggregated spheroids 24h after cultured in suspension and mean diameter of the spheroids was 100&mgr;m. The relationship among the hepatocytes resembled that in the liver in vivo. Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytes cultured on monolayers.CONCLUSION:As high-yields and high-activity modes of culture on microcarriers or in spheroidal aggregate culture with portal vein serum are promising to provide biomaterials for bioartificial liver (BAL) efficiently.     

Subzero nonfreezing storage of rat hepatocytes using UW solution and 1,4-butanediol. II- functional testing on rewarming and gene expression of urea cycle enzymes

In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 °C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 °C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0°C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group. Also, we have investigated the enzyme activities and the relative expression at messenger RNAs levels of two of the Urea cycle (UC) enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), during 60 min of rewarming. Results were compared with the ammonium removal efficiency of the three groups.In conclusion: These data indicated that hepatocytes preserved under cold or subzero conditions up to 120 h followed by 60 min of rewarming, maintain UC enzymes at levels similar to freshly isolated hepatocytes, allowing their use in bioartificial liver devices.     

3,4-Methylenedioxymethamphetamine (ecstasy)-induced hepatotoxicity: effect on cytosolic calcium signals in isolated hepatocytes

Abstract: Aims/Background: Hepatocellular damage has been reported as a consequence of 3,4-methylenedioxymethamphetamine (MDMA) intake. However, little is known about the cellular mechanisms involved. The present study was undertaken to evaluate the effects of MDMA on cell viability as well as free calcium levels ([Ca²⁺]_i) in short-term cultured hepatocytes. Reduced glutathione (GSH), adenosine-5′-triphosphate (ATP) and lipid peroxidation were investigated to evaluate the toxic effect of MDMA, in vitro, using freshly isolated rat hepatocytes. Methods: In order to measure cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i), rat hepatocytes were loaded with the Ca²⁺ indicator fura-2-acetoxymethylester (fura-2-AM). Results: A sustained rise of ([Ca²⁺]_i) after incubation with MDMA was the most noteworthy finding. In Ca²⁺-free medium, MDMA caused a reduced increase of ([Ca²⁺]_i). On the other hand, MDMA (0.1–5 mM) induced a concentration-dependent and time exposure-dependent GSH and ATP depletion. Although it did not reach statistical significance, GSH deficits were accompanied by a tendency to increase lipid peroxidation 3 h after MDMA incubation. Conclusions: The above data suggest that the marked rise of ([Ca²⁺]_i) and subsequent ATP and GSH depletion can lead to a rapid decrease in cell viability.     

Binding and action of insulin and glucagon in monolayer cultures and fresh suspensions of rat hepatocytes

Insulin and glucagon binding, and the subsequent stimulation of amino-acid transport, were investigated in adult-rat hepatocytes. Cells were used either in suspension shortly after isolation, or as monolayers after 20 h of culture in a serum-free medium. At 37°C, hepatocytes in monolayer cultures bound 2.5 times as much insulin and glucagon as did freshly isolated cells, owing to an increase in the total number of binding sites per cell. For both hormones, these differences could be accounted for mainly by a greater number of low-affinity binding sites in primary cultured hepatocytes compared with freshly isolated cells. Exposure of hepatocytes to insulin or glucagon for 2–3 h at 37°C in a medium free from amino acids increased the capacity (primary cultures) or induced the emergence (fresh suspensions) of a similar high-affinity component (K_m ～ 1 mM) of α-aminoisobutyric-acid (AIB) transport. Primary cultured hepatocytes were more sensitive to insulin (half-maximal effect occurred with insulin at ～0.3 nM) than freshly isolated cells (half-maximal effect ～0.7 nM) for the stimulation of AIB transport, whereas the dose-response curves were virtually indistinguishable for the glucagon stimulation of AIB transport in both preparations of cells (half-maximal effect occurred with glucagon at ～1.5 nM).These results indicate that, despite differences in the apparent insulin- and glucagon-binding capacities (which involved mainly a low affinity site), both freshly isolated and primary cultured (20-h monolayers) hepatocytes behave similarly in response to insulin and glucagon with regard to the stimulation of amino acid transport.     

Redistribution of canalicular organic anion transport activity in isolated and cultured rat hepatocytes

The hepatocanalicular transport of a large number of organic anions, such as bilirubin glucuronides and glutathione conjugates in the rat, is mediated by an adenosine triphosphate (ATP)-dependent transport system, which is termed canalicular multispecific organic anion transporter (cMOAT). This system is mainly defined by its deficiency in mutant TR⁻ rats. We have previously reported that in cultured hepatocytes the fluorescent organic anion glutathione-bimane (GS-B) accumulates in intracellular vesicles and that this transport is mediated by cMOAT. We now show that this intracellular accumulation of fluorescent organic anion is largely absent in freshly isolated hepatocytes but appears when cells are incubated in suspension at 37°C or cultured for periods of 1 to 24 hours. The appearance of intracellular cMOAT activity coincides with the disappearance of 70% of cMOAT activity from the plasma membrane as measured by the transport activity of the cells for the organic anion dinitrophenyl-glutathione (GS-DNP). Both the appearance of intracellular cMOAT and the disappearance of transport activity from the plasma membrane were completely inhibited at temperatures below 20°C. Residual cMOAT activity in 24-hour cultured hepatocytes could be further diminished by incubation of the cells with 1 μmol/L monensin or 10 mmol/L methylamine. We conclude that after disruption of the cell polarity by collagenase isolation of the hepatocytes, remnants of apical membrane containing cMOAT are rapidly endocytosed when the cells are kept at 37°C. Evidence suggests that at least part of the transporters may recycle back to the plasma membrane after endocytosis. These observations may be relevant for the understanding of regulation of canalicular transport.     

Synthesis of specific estradiol-induced protein by surviving rat uteri and cell-free uterine extracts

A cell-free system was developed for the synthesis of the specific ‘induced protein’ (IP) which is stimulated in the rat uterus within the first hour after treatment with estradiol-17β. Surviving uteri or uterine extracts were incubated with [³H]-labeled (estrogen-treated) or with [¹⁴C]- or [³S]-labeled (controls) leucine, glutamic acid or methionine, and the 150,000 g_av supernatant solution was analyzed by electrophoresis on cellulose acetate. IP was detected by determining the isotope incorporation ratio. The 12,000 g_max post-mitochondrial supernatant suspension from uteri of 10- or 20-day old rats, prepared 1 h after injection of estradiol-17β (4 or 5 μg/rat), was found to synthesize IP. Treatment of surviving uteri with 30 nM estradiol-17β for l h at 37°C also permitted the extraction of a post-mitochondrial supernatant suspension capable of synthesizing IP.The electrophoretic distribution of radiolabeled soluble proteins was different in the cell-free system and surviving uteri. However, both preparations showed a single protein band with increased incorporation of tritiated amino acid after estradiol treatment. This band had an electrophoretic mobility relative to bovine serum albumin of approximately 1.1, characteristic of IP. Appearance of this band was prevented by addition of antibodies to estradiol or of cordycepin during incubation of the uteri with estradiol. The induction of IP in surviving uteri followed by IP synthesis in a cell-free system provides a versatile model for the more detailed analysis of the regulation of specific protein synthesis by estradiol.     

Subzero nonfreezing storage of rat hepatocytes using modified University of Wisconsin solution (mUW) and 1,4-butanediol. I-effects on cellular metabolites during cold storage

Various cryopreservation techniques have been investigated to extend the storage of isolated hepatocytes; however, most have a reduced viability after rewarming due to ice crystal formation. Subzero nonfreezing conditions could theoretically reduce organ metabolism without damage due to ice crystal formation. In the present work we evaluated the viability and metabolic parameters of isolated rat hepatocytes preserved in subzero nonfreezing condition. Cell suspensions were maintained in modified University of Wisconsin (mUW) solution using 8% -1,4-butanediol as cryoprotectant, up to 120 h at -4°C. The time course evolution of hepatocytes viability were measured by LDH release and propidium iodide assay. The cellular concentrations of glutathione, ATP, glycogen and the lactate production during cold storage were also determined. Finally, results were compared with conventional hypothermic storage at 0 °C in mUW solution without cryoprotectant. After 5 days of subzero storage, we found an improvement in the ability of rat hepatocytes to maintain the metabolic resources in comparison with the cold preserved group.     

Effect of glycyrrhizin on lysis of hepatocyte membranes induced by anti-liver cell membrane antibody

Studies were made on why glycyrrhizin injection decreases the plasma aspartate aminotransferase (AST) and alanine aminotransferase activities in patients with chronic hepatitis.¹ For this, rat hepatocytes were isolated, and incubated with antibody raised against rat liver cell membranes, and the effect of glycyrrhizin on their release of transaminase was investigated. Isolated rat hepatocytes released AST on incubation with anti-liver cell antibody in the presence of complement. At this time, their endogenous phospholipase A₂ activity was increased. Cultured hepatocytes also released the transaminase in the presence of venom phospholipase A₂. Glycyrrhizin suppressed the release of transaminase in the presence of either anti-liver cell membrane antibody or phospholipase A₂. These results suggest that antibody treatment raised the phospholipase A₂ activity in liver cell membranes, resulting in release of transaminases, and that glycyrrhizin suppressed this increase in phospholipase A₂ activity and so inhibited the release of transaminase.     

The control of the synthesis and secretion of plasminogen activator by rat sertoli cells in culture

Sertoli cells in primary cultures produce plasminogen activator activity, and release it into the medium at rates greatly influenced by a variety of factors, including cell density, the presence of hormones, incubation temperature and duration of culture. In Sertoli cells maintained in culture in the presence of dibutyryl cAMP, the amounts of plasminogen activator activity secreted per cell were maximal at cell densities up to 2.5 μg DNA/cm² (350 units/μg cell DNA), and declined to 40 units/μg cell DNA at a density of 22 μg DNA/cm². Concentrations of follicle-stimulating hormone (FSH) required to elicit half-maximal stimulation of the production of plasminogen activator activity were 0.37 μg/ml for oFSH-NIH S12 and 8 ng/ml for the more purified oFSH-S1528C2. The ED₅₀ for dibutyryl cAMP was found to be 0.08 mM. Addition of an inhibitor of phosphodiesterase (3-isobutyl-l-methylxanthine) enhanced the formation of plasminogen activator by cells cultured in the presence of FSH.Addition to the culture medium of testosterone, epidermal growth factor, insulin, human chorionic gonadotropin or prostaglandins (E₁, E₂ or F_1α) did not result in increased production of PA activity by Sertoli cells. Cells in culture for as long as 14 days remained responsive to FSH or dibutyryl cAMP. Increases of cellular levels of plasminogen activator became evident within 2–4 h after addition of either FSH or dibutyryl cAMP to the medium. The stimulation by FSH or dibutyryl cAMP of the production of plasminogen activator activity was shown to be dependent upon de novo synthesis of RNA and protein. Levels of enzyme activity released by Sertoli cells maintained in culture for 48 h at 37°C were approx. 50% higher than plasminogen activator released by cells cultured at 32°C. The control of the production of plasminogen activator activity by Sertoli cells was discussed in relation to the control of plasminogen activator production by granulose cells, and the possible role of plasminogen activator in gonadal functions.     

Metabolic studies on the micropinocytic process in endothelial cells.

Tufts of capillary endothelium isolated from rat epididymal fat ingest [¹⁴C]sucrose by the process of micropinocytosis. Ingestion of sucrose for periods in excess of 30 min was quantitated using the concentration of cellular DNA as a standard. Capillaries incubated in the metabolic inhibitors 2,4-dinitrophenol, NaN₃, or iodoacetate exhibit a dose-dependent reduction of cellular ATP. Micropinocytic ingestion of [¹⁴C]sucrose is not affected by concentrations of these inhibitors which markedly reduce or deplete ATP pools. Pure oxygen or nitrogen atmospheres also have no effect on micropinocytosis. Ingestion of [¹⁴C]sucrose is reduced, however, at temperatures lower than 37° or in the presence of Zn²⁺.     

Hybrid bioartificial liver support in cynomolgus monkeys with D-galactosamine-induced acute liver failure

AIM: To evaluate a hybrid bioartificial liver support system (HBALSS) in cynomolgus monkeys with acute liver failure.METHODS: To establish a model of acute liver failure, 0.3 g/kg of D-galactosamine was injected intravenously into cynomolgus monkeys. Chinese human liver cells were introduced into a perfusion bioreactor to carry out hybrid bioartificial liver support treatment. Forty-eight hours after the injection, one group of cynomolgus monkeys received HBALSS care, and a second experimental group received no treatment. Clinical manifestations of all animals, survival time, liver and kidney functions and serum biochemistry changes were recorded. Simultaneous detection of the number, viability and function of hepatocytes in the hybrid bioartificial liver were also performed.RESULTS: Forty-eight hours after the injection of D-galactosamine, serum biochemistry levels were significantly increased, whereas albumin levels and the Fischer index were significantly reduced compared to baseline (all Ps < 0.05). Of the ten monkeys in the HBALSS treatment group, five survived, with an average duration of survival of 128 ± 3 h. All cynomolgus monkeys in the control group died, with a duration of survival of 112 ± 2 h. Survival time was significantly longer with HBALSS treatment (P < 0.05). Moreover, the number, viability and function of hepatocytes were maintained at a high level with HBALSS.CONCLUSION: The novel hybrid bioartificial liver plays a significant role in liver support by significantly reducing serum biochemistry levels and extending animal survival time.     

Ontogenesis of insulin processing in fetal rat hepatocytes

Summary We studied insulin processing and hepatic glycogenesis in cultured hepatocytes isolated from rat fetuses of 17, 19, and 21 days of gestation. Steady-state insulin binding increased by 250% between days 17 and 19, from 145±8 to 361±52 fmol/mg protein, and by an additional 40% (405±69 fmol/mg protein) by 21 days of gestation. At 37°C, ¹²⁵I-insulin was rapidly (t_1/2<5 min) internalized by hepatocytes at all three ages, reaching maximal levels (63–76% of the total cell-associated radioactivity) by 15 min. ¹²⁵I-labelled degradation products appeared rapidly (t_1/2<15 min) within the cells. Yet, the majority (68–77%) of the intracellular radioactivity consisted of intact ¹²⁵I-insulin, even after 4 h at 37°C. Hepatocytes pre-loaded with ¹²⁵I-insulin and then acid-stripped of surface-bound radioactivity, rapidly released both intact ¹²⁵I-insulin (retroendocytosis) and its radiolabelled degradation products. While intact insulin was initially released more rapidly (t_1/2<6 min), and reached a plateau after 15–30 min, the degradation products continued to accumulate in the medium for at least 4 h. Methylamine inhibited intracellular ¹²⁵I-insulin degradation at all three gestational ages and also blocked insulin-stimulated glycogenesis in 19- and 21-day hepatocytes, without altering basal glycogen synthesis. Insulin-stimulated glycogenesis was not induced in 17-day fetal rat hepatocytes in control or methylamine-treated cultures. We conclude that both degradative and retroendocytotic pathways for processing insulin are present in fetal rat hepatocytes by 17 days of gestation. Further, insulin-receptor processing was functionally related to the glycogenic action of insulin in responsive 19- and 21-day fetal rat hepatocytes     

Protective effects of polydatin against CCl4-induced injury to primarily cultured rat hepatocytes

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Resistance of three immortalized human hepatocyte cell lines to acetaminophen and N-acetyl-p-benzoquinoneimine toxicity

BACKGROUND/AIMS: Acetaminophen toxicity in hepatocytes is attributed to generation of the toxic metabolite N-acetyl-p-benzoquinoneimine, leading to depletion of intracellular glutathione, alteration of redox potential and ultimately, cellular necrosis. We aimed to determine the effect of acetaminophen and N-acetyl-p-benzoquinoneimine on three human hepatocyte cell lines HH25, HH29 and HHY41, and for comparison, on primary rat hepatocytes, a cell type that is relatively resistant to acetaminophen-induced toxicity. METHODS: We investigated the effect of incubation of rat hepatocytes and 3 hepatocyte cell lines with acetaminophen or N-acetyl-p-benzoquinoneimine on LDH release, glutathione status, mitochondrial function, CYP1A activity, albumin synthesis and DNA content. RESULTS: We demonstrated that HH25, HH29 and HHY41 are resistant to the toxic effects of acetaminophen under conditions that induce cytotoxicity in rat primary hepatocytes, as indicated by maintenance of glutathione levels and basal LDH release. Incubation with N-acetyl-p-benzoquinoneimine caused a dose-dependent cytotoxicity in rat hepatocytes. Under comparable conditions N-acetyl-p-benzoquinoneimine had no effect on any of the hepatocyte cell lines. Nevertheless, when culturing the cells for a further 48 h, a decrease in glutathione levels, albumin synthesis, CYP1A activity, DNA content and mitochondrial function was apparent. CONCLUSION: HH25, HH29 and HHY41 cells are highly resistant to acetaminophen and N-acetyl-p-benzoquinoneimine-induced toxicity. They tolerate a much higher concentration of both toxins for a longer period of time compared to rat primary hepatocytes. These results are of relevance in the use of these cell lines to investigate acetaminophen hepatotoxicity, and may be of importance in the choice of cells for use in bioartificial liver support systems.