Human Hepatocyte Model Research Articles

Dietary polyphenols protect against N-nitrosamines and benzo(a)pyrene-induced DNA damage (strand breaks and oxidized purines/pyrimidines) in HepG2 human hepatoma cells

Dietary polyphenols have been reported to have a variety of biological actions, including anticarcinogenic and antioxidant activities. In the present study we investigated the protective effect of dietary polyphenols against N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR) and benzo(a)pyrene (BaP)-induced DNA damage (strand breaks and oxidized purines/pyrimidines) in HepG2 cells. Human hepatocellular carcinoma (HepG2) cells, which retain many specialized liver functions and drug metabolizing enzyme activities, were used as in vitro model for human hepatocytes. NDMA, NPYR and BaP were employed to induce DNA damage. DNA damage (strand breaks, oxidized pyrimidines and oxidized purines) was evaluated by the alkaline single cell gel electrophoresis or comet assay. None of the polyphenols concentrations tested in presence or absence of Fpg (formamidopyrimidine-DNA glycosylase), or Endo III (Endonuclease III) caused DNA damage per se. Increasing concentrations of BaP (25-100 microM) induced a significant increase of DNA strand breaks, Fpg and Endo III sensitive sites in a dose dependent manner. Myricetin and quercetin decreased DNA strand breaks and oxidized pyrimidines induced by NDMA, but not oxidized purines. However, both flavonoids reduced oxidized pyrimidines and purines induced by NPYR. DNA strand breaks induced by NPYR were prevented by quercetin, but not by myricetin. BaP-induced DNA strand breaks and oxidized pyrimidines were strongly reduced by myricetin and quercetin, respectively. While oxidized purines induced by BaP were reduced by quercetin, myricetin had no protective effect. (+)-Catechin and (-)-epicatechin reduced DNA strand breaks, oxidized pyrimidines and oxidized purines induced by NDMA. DNA strand breaks, and oxidized purines induced by NPYR were also prevented by (+)-catechin and (-)-epicatechin, while the maximum reduction of oxidized pyrimidines was found by (+)-catechin and (-)-epicatechin at 10 microM. (+)-Catechin and (-)-epicatechin decreased also DNA strand breaks and oxidized pyrimidines but not oxidized purines induced by BaP. Our results clearly indicate that polyphenols protect human derived cells against DNA strand breaks and oxidative DNA damage effects of NDMA, NPYR or BaP, three carcinogenic compounds which occur in the environment.

Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes

Drug biotransformation is one of the most important parameters of preclinical screening tests for the registration of new drug candidates. Conventional existing tests rely on nonhuman models which deliver an incomplete metabolic profile of drugs due to the lack of proper CYP450 expression as seen in human liver in vivo. In order to overcome this limitation, we used an organotypical model of human primary hepatocytes for the biotransformation of the drug diazepam with special reference to metabolites in both the cell matrix phase and supernatant and its interaction of three inducers (phenobarbital, dexamethasone, aroclor 1254) in different time responses (1, 2, 4, 8, 24 h). Phenobarbital showed the strongest inducing effect in generating desmethyldiazepam and induced up to a 150 fold increase in oxazepam-content which correlates with the increased availability of the precursor metabolites (temazepam and desmethyldiazepam). Aroclor 1254 and dexamethasone had the strongest inducing effect on temazepam and the second strongest on oxazepam. The strong and overlapping inductive role of phenobarbital strengthens the participation of CYP2B6 and CYP3A in diazepam N-demethylation and CYP3A in temazepam formation. Aroclor 1254 preferentially generated temazepam due to the interaction with CYP3A and potentially CYP2C19. In parallel we represented these data in the form of a mathematical model with two compartments explaining the dynamics of diazepam metabolism with the effect of these other inducers in human primary hepatocytes. The model consists of ten differential equations, with one for each concentration c i,j ( i = diazepam, temazepam, desmethyldiazepam, oxazepam, other metabolites) and one for each compartment ( j = cell matrix phase, supernatant), respectively. The parameters p k ( k = 1, 2, 3, 4, 13) are rate constants describing the biotransformation of diazepam and its metabolites and the other parameters ( k = 5, 6, 7, 8, 9, 10, 11, 12, 14, 15) explain the concentration changes in the two compartments.

Preservation of hepatic phenotype in lentiviral-transduced primary human hepatocytes

Lentiviral vectors effectively transduce both dividing and non-dividing cells and stably integrate into the genome of the host cell. In this study, we evaluated the usefulness of a lentiviral system for genetic modulation of primary human hepatocyte cultures. Infection with GFP-expressing lentivectors shows that Huh7 and HepG2 cell lines, as well as primary cultures of human hepatocytes, are efficiently transduced by lentiviral vectors. Real-time RT-PCR analyses demonstrate that infection with lentivectors does not alter hepatic hallmarks such as the expression of the nuclear receptors CAR, PXR, RXRα, or HNF4α, or expression of the secretory protein, albumin. Additionally, infected hepatocytes retain the capacity for CYP3A4 induction in response to treatment with phenobarbital, a uniquely sensitive indicator of hepatic differentiation status. Lentivectors may be used for both over-expression and knockdown analyses in primary hepatocytes, as demonstrated in this study by >200-fold CAR over-expression and knockdown of CAR to less than 40% of endogenous levels, with corresponding effects on CYP2B6 expression. In summary, lentiviral vectors provide a novel methodology by which primary human hepatocytes may be stably genetically manipulated, with minimal effects on the differentiated hepatic phenotype. These approaches offer considerable advantage over current methodologies, providing a valuable alternative for use in pharmacological and toxicological investigations involving primary human hepatocyte models and potentially for cell-based therapeutics to treat hepatic dysfunction in vivo.

Evaluation of active hexose correlated compound hepatic metabolism and potential for drug interactions with chemotherapy agents.

Active hexose correlated compound (AHCC), a Basidiomycotina extract, is a well-tolerated nutritional supplement with no reported adverse effects. It has demonstrated potential antitumor activity and immune modulator activity. However, there is no current information regarding its metabolism and the potential for drug-drug interactions for AHCC in combination with chemotherapy. The objective of this study was to characterize AHCC hepatic metabolism, specifically involving the potential for drug interactions with selected chemotherapy agents. High-throughput cytochrome P-450 (CYP450) metabolism inhibition experiments were conducted in vitro evaluating CYP450 3A4, 2C8, 2C9, and 2D6 followed by an evaluation of AHCC as a substrate of these same isoenzymes. An ex vivo model of cryopreserved human hepatocytes was used to evaluate the CYP450 metabolism induction potential of AHCC for CYP450 3A4, 2C8/2C9, and 2D6. No inhibition of CYP450 activity was observed in presence of AHCC; however, AHCC was a substrate of CYP450 2D6. The CYP450 induction metabolism assays indicate that AHCC is an inducer of CYP450 2D6. AHCC does have the potential for drug-drug interactions involving CYP450 2D6, such as doxorubicin or ondansetron; however, the overall data suggest that AHCC would be safe to administer with most other chemotherapy agents that are not metabolized via the CYP450 2D6 pathway.

Effect of the St. John's Wort Constituent Hyperforin on Docetaxel Metabolism by Human Hepatocyte Cultures

St. John's wort is a commonly used herbal medication that increases cytochrome P450 3A (CYP3A) activity. Because docetaxel is inactivated by CYP3A, we studied the effects of the St. John's wort constituent hyperforin on docetaxel metabolism in a human hepatocyte model. Hepatocytes, isolated from three donor livers, were exposed to hyperforin (0.1, 0.5, or 1.5 micromol/L) or rifampin (10 micromol/L) for 48 hours. After 48 hours, hyperforin- or rifampin-containing medium was replaced with medium containing 100 micromol/L docetaxel. After 1 hour, docetaxel metabolism was characterized by liquid chromatography-tandem mass spectrometry. Subsequent incubations characterized the specific cytochrome P450s that produced the docetaxel metabolites observed in hepatocyte incubations. Rifampin induced docetaxel metabolism 6.8- to 32-fold above docetaxel metabolism in control cultures. Hyperforin induced docetaxel metabolism in all three hepatocyte preparations. Hyperforin induction was dose-dependent and, at maximum, was 2.6- to 7-fold greater than that in controls. Docetaxel metabolites identified in rifampin- and hyperforin-treated hepatocyte preparations included the previously described tert-butyl-hydroxylated metabolite and two previously unidentified metabolites involving hydroxylation on the baccatin ring. CYP3A4 produced the tert-butyl-hydroxylated metabolite and the two ring-hydroxylated metabolites. CYP2C8 produced one of the newly described ring-hydroxylated metabolites. Exposure to the St. John's wort constituent hyperforin induces docetaxel metabolism in vitro. This implies that subtherapeutic docetaxel concentrations may result when docetaxel is administered to patients using St. John's wort on a chronic basis. The results also show induction of previously undescribed metabolic pathways for docetaxel, one of which may be analogous to the known 6-alpha-hydroxylation of paclitaxel by CYP2C8.

750. AAV-2 Vectors and Protease-Activated Receptors: A Potential Interface between Coagulation and Gene Transfer

Phase I clinical trials demonstrated that AAV based on serotype 2 transduce human skeletal muscle and hepatocytes in vivo. AAV-2 is safe and well tolerated at doses tested, and an ongoing liver-directed study resulted in therapeutic FIX levels for hemophilia B. From these studies it is important to determine the minimum safe dose required for therapeutic efficacy. To this end, factors that increase or decrease expression by even modest amounts may become critical in the development of this novel therapeutic. We have uncovered evidence for an unexpected role of specific blood proteases in insuring optimal AAV-2 but not for AAV-5 or AAV-8 vectors. The use of specific inhibitors to thrombin or FXa (common anticoagulant drugs) prior to vector injection in mice resulted in low transgene expression (Blood 102:794a). Proteases trigger cellular responses at least in part through protease-activated receptors (PAR). Recent data show that PAR activation by thrombin enhances αVβ5-dependent (co-receptor for AAV-2) cellular function. Using PAR-1 and-2 knockout mice we sought to determine whether PAR or β5 integrin is required for AAV-2 transduction. Experiments were carried out in adult male C57Bl/6 mice homozygous (−/−) or heterozygous (+/−) for the null alleles of PAR-1, PAR-2 or β5 and compared to wild-type littermate as controls (+/+). A rAAV-2 encoding F.IX gene under the control of liver-specific promoter (hAAT/ApoE) was injected by tail vein at dose of 1 × 1012 vg/kg and circulating FIX levels were monitored for at least 10 weeks following vector injection. Injection of β5 deficient mice (n=6) resulted in FIX levels of 514 ng/ml which were lower than 3,837 ± 1,2 ng/ml among 12 controls (p < .0005). These results suggest that β5 co-receptor properties on AAV-2 transduction correlate well with previous in vitro observations (Nat Med 5:78). To test whether PARs have any role independently of β5, we further injected 19 mice of PAR-1 (−/−) or PAR-1 (+/−) genotype, and F.IX levels were 1,330 ± 376 and 1,782 ± 280 ng/ml, respectively. Among controls (n=10) levels of F.IX were 4,017 ± 1521 ng/ml. These differences were statistically significant when controls were compared to PAR-1 (−/−) or PAR-1 (+/−) at p < .005 and p <.05, respectively. Similarly, among 20 animals of PAR-2 mice (−/−) or (+/−) genotypes, FIX levels were 717 ± 222 ng/ml and 1,197 ± 648 ng/ml, respectively. Injection of controls (n=11) resulted in 3,540 ± 332 ng/ml. Next we injected AAV-2 vector in wild-type animals (n=4/group) with 10 μM/kg of specific activation peptides of PAR-1, PAR-2, or both PAR-1/-2 and compared with injection of scramble peptide as control (LSIGRL). Following simultaneous activation of PAR-1/-2 by SFLLRN, FIX levels were higher when compared to controls (2,654 vs. 1,248 ng/ml; p < 0.005). HepG2 cells, as a model for human hepatocytes, were transduced with AAV-2 following PAR-1/-2 activation and again F.IX secreted in the conditioned media was ~2-fold higher compared to control peptide. Together these data suggest a novel role of PARs and coagulation on gene transfer by viral vectors. PARs modulation may serve as an alternative strategy to improve viral vectors-cell interaction.

In vitro effect of methanol on folate-deficient rat hepatocytes

Methanol is primarily metabolized by oxidation to formaldehyde and then to formic acid. These processes are accompanied by formation of superoxide anion and hydrogen peroxide. This paper reports the in vitro antioxidant effect of vitamin E on isolated hepatocytes of folic acid deficient rats rendered so as to emulate a human hepatocyte model. These hepatocytes were treated with 320 μmol of methanol per million cells and incubated for 30 min. The microsomal fraction of these hepatocytes showed a decreased level of superoxide dismutase (SOD), with increase in lipid peroxidation (LPO) shown by increase in recorded levels of malondialdehyde (MDA). Catalase activity was shown to be increased. Levels of reduced glutathione (GSH) were decreased and the activity of glutathione peroxidase (GSH-Px) and of glutathione reductase (GSSG-R) were not altered. The hepatocytes of folate deficient rats pretreated with vitamin E, when subjected to methanol treatment, showed no significant change in SOD levels and a significant decrease in MDA levels. The catalase activity in this group of animals showed a highly significant decrease. These animals had normal levels of GSH, while a significant fall in GSH-Px and GSSG-R levels were observed. These results suggest that Vitamin E exerts a protective effect on hepatocytes by acting as a free radical scavenger, proving its usefulness in treating methanol toxicity.

Synergetic signaling for apoptosis in vitro by ethanol and acetaminophen

In vitro , ethanol in combination with acetaminophen induces hepatocyte apoptosis resembling immune-mediated fulminant hepatic failure in human beings. Intracellular pathways originating at the mitochondria are linked to apoptosis. I studied ethanol-induced apoptosis and hepatocytotoxicity after using an in vitro model of normal human primary hepatocytes that were exposed to 5 or 10 mM acetaminophen, 40 or 100 mM ethanol, 40 mM ethanol + 5 mM acetaminophen, or 40 mM ethanol + 10 mM acetaminophen, or nonexposed (control; plain medium). Transmission electron microscopy was performed at different time points after exposure to the various treatments. Apoptosis, as assessed by transmission electron microscopy, was increased in a time-dependent manner after exposure to ethanol + acetaminophen. In the ethanol + acetaminophen model, mitochondrial injury was associated with apoptosis of hepatocytes. Ultrastructural damage and induction of apoptosis were seen in response to N-acetyl-benzoquinone-imine plus ethanol, supporting the suggestion that the damage was due to the active metabolite of acetaminophen. The modulation of mitochondrial damage in vitro may have implications for the development of new therapeutic strategies to prevent apoptosis.

Effect of selenium supplementation on the activities of glutathione metabolizing enzymes in human hepatoma Hep G2 cell line

Cell culture is an important tool for studying injury to cells exposed to oxidative stress. The human hepatoblastoma derived Hep G2 cells retain their morphology and most of their function in culture and are therefore widely used as an in vitro model of human hepatocytes. Conventional cell culture media are deficient in selenium, which is essential for activation of glutathione peroxidase (GPx), a key enzyme in the defense against oxidative stress. Supplementation of the culture media with 1 μM sodium selenite increased the activities of total GPx by threefold and the selenium-dependent GPx by fourfold as compared to cells cultured in control media. The non-selenium-dependent GPx activity was unchanged. The activities of the other glutathione (GSH)-related enzymes were practically unchanged despite a tendency toward elevation. The activities of oxidized glutathione (GSSG) reductase and catalase increased by 22.4 and 27.4%, respectively. These relatively small increases did not carry statistical significance. Supplementation of tissue culture media with selenium may prove important, particularly for cell protection against oxidative stress.

Ethanol-induced apoptosis in vitro

Objectives: The aim is to study the apoptotic process in a human hepatocyte model for ethanol (EtOH)-induced apoptosis. Design and methods: Normal human primary hepatocytes (HPH) and Hep G2 cells were exposed to increasing EtOH. 6000 cells/sample were analyzed by transmission electron microscopy. Results: Apoptotic cells were observed (mmol/L EtOH): 40: 6 ± 0.5%, 60: 13 ± 2% ( p < 0.05), 80: 26 ± 1% (p < 0.001) (vs. control). Two consecutive doses of 80 mmol/L for 24 h each additionally increased apoptosis 55 ± 3% ( p < 0.0001 vs. control and p < 0.001 vs. single dose). In response to this exposure, there is a stronger apoptotic activity in HPH when compared to Hep G2 ( p < 0.05). Conclusions: In vitro, EtOH-induced apoptosis is regulated by dose level and the frequency of exposure.

Maintained function of primary human hepatocytes by cellular interactions in coculture: implications for liver support systems.

The application of primary hepatocytes in hybrid artificial liver systems has been hampered by the gradual loss of differentiated morphology and function in vitro. Therefore, we have established a coculture model of autologous human hepatocytes and biliary epithelial cells (BEC) in collagen gel in the presence of hepatotrophic growth factors. Furthermore, we examined the effect of hepatocyte cell perfusion in a women multicompartment capillary membrane system. Normal hepatocytes isolated from human liver produced albumin for more than 2 weeks in serum-free media, and were further stimulated by conditioned medium. When cocultured with BEC, albumin secretion was greatly enhanced, suggesting that cellular interactions promote tissue-specific differentiation. When perfused in bioreactors, albumin levels were maintained at steady state for longer than 2 weeks. These data indicate that differentiation of primary hum hepatocytes can be maintained by coculture interactions and three-dimensional hybrid organ devices, providing appropriate growth factors and matrix for tissue regeneration.

Maintained function of primary human hepatocytes by cellular interactions in coculture: implications for liver support systems

Abstract The application of primary hepatocytes in hybrid artificial liver systems has been hampered by the gradual loss of differentiated morphology and function in vitro. Therefore, we have established a coculture model of autologous human hepatocytes and biliary epithelial cells (BEC) in collagen gel in the presence of hepatotrophic growth factors. Furthermore, we examined the effect of hepatocyte cell perfusion in a woven multicompartment capillary membrane system. Normal hepatocytes isolated from human liver produced albumin for more than 2 weeks in serum-free media, and were further stimulated by conditioned medium. When cocultured with BEC, albumin secretion was greatly enhanced, suggesting that cellular interactions promote tissue-specific differentiation. When perfused in bioreactors, albumin levels were maintained at steady state for longer than 2 weeks. These data indicate that differentiation of primary hum hepatocytes can be maintained by coculture interactions and three-dimensional hybrid organ devices, providing appropriate growth factors and matrix for tissue regeneration.

Establishment and application of an experimental model of human fetal hepatocytes for investigation of the protective effects of silybin and polyporus umbellalus polysaccharides.

To establish a new experimental model system of human fetal hepatocytes to study the mechanisms underlying the protective effect of silybin and polyporus umbellalus polysaccharides (PSP) on the cellular ultrastructure. Human fetal hepatocytes were obtained from the liver of a human fetus that resulted from a medically necessary induced labor; the mother provided informed consent for sampling, experimental use and publication of findings. The hepatocytes were cultured and then pretreated with silybin or PSP or without either (control), after which the treated cells were exposed to CCl4 for 4 h. Changes in cellular ultrastructure were observed by scanning electron microscopy and transmission electron microscopy, and changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST) and superoxide dismutase (SOD) were assayed. Levels of ALT and AST were significantly decreased, and level of SOD was elevated in the two pretreatment groups following CCl4 exposure, as compared to the control group. The cellular integrity and ultrastructure were well preserved in the two pretreatment groups but were seriously damaged in the control group. The CCl4-induced hepatotoxic cell model system of human fetal hepatocytes is an effective tool for studying the hepatoprotective effect of drugs and may be applicable for studies to screen medicines for treatment of hepatitis.

Differences in hypolipidaemic effects of two statins on Hep G2 cells or human hepatocytes in primary culture.

1. The objective of this study was to compare in cultured human hepatocytes or Hep G2 cells, changes in the fate of unesterified low density lipoprotein (LDL)-cholesterol induced by crilvastatin, a new cholesterol lowering drug and a reference statin, simvastatin. 2. The experiments were carried out for 20 h, each well contained 4.2 x 10(5)/cm2 Hep G2 cells or 0.5 x 10(5)/Cm2 human hepatocytes, 130 microM ursodeoxycholate, 0.68 microCi or 1.59 microCi unesterified human [14C]-LDL-cholesterol, crilvastatin or simvastatin at 0 or 50 microM (both cell types) or 300 microM (Hep-G2 cells). Incubation with the two drugs resulted in increased amounts of unesterified [14C]-LDL-cholesterol taken by the two cell types, compared to control. 3. Crilvastatin 50 microM led to significantly higher quantities of [14C]-glyco-tauro-conjugated bile salts, compared to simvastatin. Statins reduced the apo B100 level secreted by the two cell types (simvastatin) or human hepatocytes (crilvastatin). Crilvastatin enhanced both the level of apo A1 secreted by the Hep G2 cells and the level of APF, a high density lipoprotein (HDL) and biliary apoprotein. 4. Crilvastatin not only acts by stimulating LDL-cholesterol uptake by hepatocytes, but also by enhancing the catabolism of LDL-cholesterol in bile salts and probably by stimulating HDL and/or bile component secretion. Such a mechanism was not previously described for HMG CoA reductase inhibitors. Our results on APF show that this apoprotein could be considered also as an indicator of changes in bile and/or HDL compartments. 5. The human hepatocyte model appeared to be a suitable and relevant model in the pharmacological-metabolic experiments carried out in this study. It led to more consistent data than those obtained with Hep G2 cells.

Primary cultured normal human hepatocytes for hepatitis B virus receptor studies

Background/Aims: We analyzed the hepatitis B virus envelope specificities (HBs, preS2 and preS1) involved in virus attachment to normal human hepatocytes, and we performed in vitro hepatitis B virus infection experiments without addition of dimethyl sulfoxide and polyethylene glycol, which may affect cell membrane integrity, in order to study further the early steps of the life cycle of the hepatitis B virus. Methods: Primary normal human hepatocytes were prepared from surgical biopsies by the two-step collagenase perfusion technique, and cultured in a fetal calf serum-free medium supplemented with 10 −6 M dexamethasone. Cell-binding assays, ligand blotting and immunohistochemistry experiments were carried out using our anti-idiotypic (Ab2) antibodies (Ab2s/preS1, Ab2s/preS2 and Ab2s/HBs). Results: Probing primary normal human hepatocytes, the 35-kDa major preS1-binding protein (preS1-BP35) we have previously identified in human hepatoma HepG2 cells was recognized in blotting, whereas both HBs- and preS1-specificities of the hepatitis B virus envelope interacted strongly with normal human hepatocyte cell membrane in cell-binding assays and immunohistochemistry experiments. Hepatitis B virus infectivity studies confirmed a great inter-experimental variability depending on donors and liver perfusion, and demonstrated a great intra-experimental variability depending on the serum-derived hepatitis B virus isolate used for the inoculation. In our culture conditions, only increased detection of the RC and CCC DNA forms of hepatitis B virus in cells and of hepatitis B virus surface antigens in medium was observed 4 to 8 days after exposure of cells to hepatitis B virus. Conclusion: These findings support a potential role for preS1-BP35 as a receptor protein for hepatitis B virus. In our hands, limitation(s) in the hepatitis B virus life cycle may occur at some step after virion binding, and likely result from complex regulation of reverse transcription of the RNA and translation of core protein by extrahepatic host factors or/and by the virus itself. However, the normal human hepatocyte model developed here is available for studying the initial steps in hepatitis B virus entry into cells.

42] Use of human hepatocytes to study P450 gene induction

The human hepatocyte model system is useful for investigations of interspecies and interindividual differences in P450-related activities, including induction of gene expression in response to xenobiotic exposure and the molecular events controlling gene expression through the transfection of reporter gene constructs. The CYP3A family is the major steroid-inducible cytochrome P450s in rats, rabbits, and humans. No single animal model faithfully predicts the responses observed in human hepatocyte preparations. Hepatocytes can be cryopreserved and reestablished into culture, and the frozen/thawed cells still respond to P450-inducing compounds with enhanced gene transcription and drug-metabolizing capacity. It is crucial to gain a complete understanding of the genetic structure, regulation, and functional activity of human cytochrome P450s because of the relative importance of cytochrome P450s in the biotransformation of a variety of exogenous and endogenous compounds.

Role of cellular ribose-5-phosphate content in the regulation of 5-phosphoribosyl-1-pyrophosphate and de novo purine synthesis in a human hepatoma cell line

5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo purine synthesis. However, the role of ribose-5-phosphate (R5P), the precursor for PRPP, in the regulation of PRPP and de novo purine synthesis has not yet been clarified conclusively. This study was designed to clarify interrelationships between R5P content, PRPP availability, and the rate of de novo purine synthesis in the cultured human hepatoma cell line (HepG 2 ), a plausible model for normal human hepatocytes. Increasing glucose concentration in the culture media from 0 to 10 mmol/L resulted in a 2.9-fold elevation of cellular R5P content (from 107 ± 31 to 311 ± 57 nmol/g protein), associated with a correlated increase of 7.14-fold in cellular PRPP availability (from 4.76 ± 3.4 to 34 ± 8.4 pmol/mg protein/min) and of 149-fold in the rate of de novo purine synthesis (from 55 to 8,204 dpm/mg protein/h). Plotting the rate of de novo purine synthesis versus R5P content indicates that at a wide range of R5P content, including that prevailing in hepatocytes under physiological conditions, the rate of purine synthesis depends on R5P content. A similar dependence was also demonstrated for PRPP availability. The rate of de novo purine synthesis exhibited a sigmoidal dependence on PRPP availability. The demonstration in human hepatocytes of dependence of the rate of purine synthesis on R5P content has implications concerning the pathogenesis of purine overproduction associated with several inborn and acquired conditions in man.

Pathway of cholesterol efflux from human hepatoma cells

Studies have been carried out with HepG2 cells, as a model for human hepatocytes, to explore a novel proposition that the liver contributes free cholesterol to the plasma lipoproteins which participate in the process of reverse cholesterol transport. Specifically, we compared efflux of cholesterol from HepG2 cells and human fibroblasts (a model for extrahepatic tissues) after labeling cells with [ 14C]cholesterol. Incubation of both types of cells with human serum resulted in the efflux of [ 14C]cholesterol and net cholesterol flux from the cells to the medium and its subsequent esterification. Rates of cholesterol efflux from HepG2 cells and fibroblasts were similar. Nondenaturing two-dimensional gel electrophoresis showed that about 10% of the cell-derived [ 14C]cholesterol moves rapidly through pre β 1-, pre β 3- and pre β 2-HDL particles into αHDL and LDL, although the majority moves directly to αHDL and LDL, with most of [ 14C]cholesterol and cholesterol mass accumulating in LDL. When cells were incubated with equivalent concentrations of isolated lipoproteins, HDL was much more effective in promoting [ 14C]cholesterol efflux than LDL, suggesting that unesterified cholesterol is initially transferred to HDL and then to LDL. Incubation with whole serum in contrast to isolated lipoproteins did not enhance cholesterol efflux despite a 3-fold higher esterification rate. We also investigated the identity of newly secreted lipoproteins following the labeling of HepG2 cells with [ 14C]cholesterol: 72% of labeled cholesterol was released as LDL, 20% was released as pre β 2-HDL and 8% as small αHDL particles. Novel apo A-I rich but [ 14C]cholesterol-deficient pre β 2-HDL particles were also secreted by HepG2 cells.

The transporter for the HMG-CoA reductase inhibitor pravastatin is not present in Hep G2 cells. Evidence for the nonidentity of the carrier for pravastatin and certain transport systems for BSP

The hydrophilic HMG-CoA reductase inhibitor pravastatin is not taken up via a carrier-mediated system into Hep G2 cells. Therefore, Hep G2 cells are not a good model for human hepatocytes with respect to elucidation of the effect of hydrophilic HMG-CoA reductase inhibitors. Sulfobromophthalein (BSP), on the other hand, is taken up into Hep G2 cells by carrier systems with K m and V max values almost identical to freshly isolated hepatocytes. These results indicate that the hepatocellular BSP transporting proteins expressed in Hep G2 cells (bilitranslocase and BSP/bilirubin binding protein) are not involved in the hepatocellular uptake of pravastatin. In contrast to the hepatocellular sodium-taurocholate cotransporter, which is not functioning in Hep G2 cells, we found a saturable transport of cholate with K m and V max values identical to those in cultured rat hepatocytes in the presence of sodium.

Comparison of sulfur amino acid utilization for GSH synthesis between HepG2 cells and cultured rat hepatocytes

HepG2 cells are widely used as a model of human hepatocytes for studies of drug metabolism and toxicity. However, GSH metabolism in HepG2 cells is poorly characterized. This report describes the utilization of sulfur amino acids for GSH synthesis in HepG2 cells. In contrast to primary cultures of rat hepatocytes, which rely mostly on methionine for GSH synthesis, HepG2 cells use cystine. Their inability to utilize methionine for GSH synthesis was not due to lack of methionine uptake or low cellular ATP levels, but rather to the lack of S-adenosyl-methionine synthetase activity. When HepG2 cells were cultured overnight in medium containing cystine as the only sulfur amino acid, addition of glutamate or acivicin had minimal to no effect on cell GSH; however, addition of threonine significantly depleted cell GSH. When cystine (0.18 mM) uptake was measured, glutamate (2.5 mM), which inhibited cystine uptake in cultured rat hepatocytes, had a minimal effect in HepG2 cells. Instead, threonine (20 mM) strongly inhibited the apparent uptake of cystine by HepG2 cells. Strong inhibition by threonine of apparent cystine uptake was actually due to inhibition of cysteine uptake, which resulted from GSH-cystine mixed disulfide exchange. Radio-HPLC confirmed this. After incubating cells with [ 35S]cystine (0.18 mM) for 10 min, the total counts inside the cell matched the counts in the uptake medium in the form of GSH-cysteine mixed disulfide. Finally, HepG2 cells took up cysteine by both Na +-dependent and -independent mechanisms. The former exhibited high affinity and low capacity, whereas the latter exhibited the opposite. At a physiologic concentration of cysteine (10 μM), 68% of cysteine uptake occured via the Na +-dependent system and 32% via system L 1.