Search

The use of replication-defective adenoviruses to deliver transgenes into hepatocytes seems to be a promising approach to human liver gene therapy. However, the effects that the adenovirus-mediated expression of a foreign gene could have on the expression of other hepatic characteristic genes have not yet been properly examined. We have investigated this problem by using human hepatocytes infected with a recombinant E-1 defective adenovirus that carried a modified lacZ gene. The analysis of the biochemical functionality of transduced cells showed that the use of adenovirus: (1) was a very efficient way to introduce a foreign gene into human hepatocytes (80% transduced cells after 1 h contact, at an MOI of 15; approximately 100% transduced cells at an MOI of 20); (2) allowed the expression of the transgene to levels that enabled cells effectively to use lactose as an energy source; (3) does not affect urea synthesis, plasma protein synthesis and xenobiotic biotransformation activities (1A2, 2A1, 2B6, 3A3/5). Glycolysis was moderately increased (approx- imately 20%), while gluconeogenesis decreased (approximately 20%) in transduced hepatocyte; moreover, (4) the expression of inducible genes (acute-phase plasma proteins, CYPs) was not impaired in transduced human hepatocytes upon stimulation with IL-6 or methylcholantrene. The results of this research support the idea that efficient expression of transgenes can be achieved in human hepatocytes by means of adenoviral transduction, without altering these characteristic hepatic biochemical functions. [ABSTRACT FROM AUTHOR]

This paper reports cytotoxic effects and changes in the P450 system after exposing rat hepatocytes to four polyphenol-rich products widely used in Cuban traditional medicine (Mangifera indica L. (MSBE), Thalassia testudinum (Tt), Erythroxylum minutifolium and confusum extracts). Effects of mangiferin, the main polyphenol in MSBE, were also evaluated. Cytotoxicity was assayed by the MTT test after exposure of cells to the products (50-1000 microg/mL) for 24 or 72 h. The results showed that 500 microg/mL MSBE was moderately cytotoxic after 72 h, while mangiferin was not. Marked reductions in cell viability were produced by Erythroxylum extracts at concentrations > or = 200 microg/mL, whereas only moderate effects were induced by 1000 microg/mL Tt. Seven specific P450 activities were evaluated after 48 h exposure of cells to the products. MSBE reduced phenacetin O-deethylation (POD; CYP1A2) activity in a concentration-dependent manner (IC(50)=190 microg/mL). No decreases were observed in other activities. In contrast, mangiferin produced reductions in five P450 activities: IC(50) values of 132, 194, >200, 151 and 137 microg/ml for POD (CYP1A2), midazolam 1'-hydroxylation (M1OH; CYP3A1), diclofenac 4'-hydroxylation (D4OH; CYP2C6), S-mephenytoin 4'-hydroxylation (SM4OH), and chlorzoxazone 6-hydroxyaltion (C6OH; CYP2E1), respectively. E. minutifolium, E. confusum and Tt extracts produced small reductions in SM4OH and C6OH activities, but no significant changes were noted in the other P450 activities. On the other hand, all the products increased the benzyloxyresorufin O-debenzylation (BROD; CYP2B1) activity, with MSBE, mangiferin or E. minutifolium showing the highest effects (about 2-fold over control). Our results showed in vitro effects of these natural products on P450 systems, possibly leading to potential metabolic-based interactions.

The BC2 cell line derived from the human hepatocarcinoma, HGB, undergoes a spontaneous sharp differentiation process in culture as it becomes confluent, remains stably differentiated for several weeks, and may return to proliferation thereafter under appropriate density conditions. The relevance of the line as an hepatic model has been evaluated. Cells synthesize a large number of plasma proteins, and rates of glycogen and urea synthesis increase with time of confluency and become sensitive to insulin, reflecting the process of differentiation. Differentiated BC2 cells express the most relevant cytochrome P-450 (CYP) isozyme activities (CYP1A1/2, 2A6, 2B6, 2C9, 2E1, and 3A4) and conjugating enzymes (glutathione S-transferase and UDP-glucuronyltransferase) and also respond to model inducers. Methylcholanthrene induced an increase in CYP1A1/2 enzyme activity (eightfold), phenobarbital induced CYP2B6 activity (1.7-fold), and dexamethasone induced CYP3A4 activity (fivefold). In parallel, expression of the most relevant liver-enriched transcription factors, HNF-4, HNF-1, C/EBP-alpha and C/EBP-beta mRNAs, was significantly increased in differentiated cultures. This increase was largest in HNF-1 and HNF-4, which supports the idea that a redifferentiation process towards the hepatic phenotype takes place. BC2 is an hepatic cell line that is able to express most hepatic functions, especially the drug-biotransformation function, far more efficiently than any previously described human hepatoma cell line.

A new non-woven polyetherurethane support suitable to host cultured hepatocytes has been developed. Prior to its use in bioreactors and artificial liver devices, the biocompatibility of this new material was investigated. The experiments have shown that the survival and functionality of hepatocytes entrapped in the non-woven polymer were longer than that of monolayer cultured hepatocytes, under serum-free culture conditions. Hepatic specific metabolic functions, namely, synthesis of urea and synthesis and secretion of plasma proteins, were well maintained by hepatocytes entrapped in non-woven polyetherurethane sheets. Cells also retained the expression of biotransformation activities of 7-ethoxycoumarin-O-deethylase as well as CYP2A1, CYP2B1 and CYP3A1. The results presented in this paper point to non-woven polyetherurethane sheets as a suitable biocompatible support for functional, three-dimensional hepatocyte cultures., (Copyright 2000 Kluwer Academic Publishers)

The acceptable daily intake (ADI) for food additives is commonly derived from the NOAEL (no-observed-adverse-effect level) in long-term animal in vivo studies. To derive an ADI a safety or uncertainty factor (commonly 100) is applied to the NOAEL in the most sensitive test species. The 100-fold safety factor is considered to be the product of both species and inter-individual differences in toxicokinetics and toxicodynamics. Although in vitro data have previously been considered during the risk assessment of food additives, they have generally had no direct influence on the calculation of ADI values. In this review 18 food additives are evaluated for the availability of in vitro toxicity data which might be used for the derivation of a specific data-derived uncertainty factor. For the majority of the food additives reviewed, additional in vitro tests have been conducted which supplement and support the short- and long-term in vivo toxicity studies. However, it was recognized that these in vitro studies could not be used in isolation to derive an ADI; only when sufficient in vivo mechanistic data are available can such information be used in a regulatory context. Additional short-term studies are proposed for the food additives which, if conducted, would provide data that could then be used for the calculation of data-derived uncertainty factors.

Cytochrome P450 (CYP) activity is very low or even absent in human hepatomas, a phenomenon that is accompanied by low levels of some liver transcription factors, notably C/EBP alpha. To investigate a possible link between this transcription factor and hepatic CYP expression, we have stably transfected HepG2 cells with a C/EBP alpha vector containing a Zn-inducible metallothionein promoter. Expression of functional C/EBP alpha up to liver levels concomitantly increased the mRNAs of several members of the CYP2 family (2B6, 2C9 and 2D6), suggesting that this transcription factor may play a relevant role in controlling the hepatic expression of CYP enzymes.

The stability and inducibility of several P450 activities (namely, P450 1A1; 2A1, 2B1/2, 2C11, and 3A1) were studied in rat hepatocytes co-cultured with the MS epithelial cell line derived from monkey kidney. The results revealed that these monooxygenase activities were systematically higher in co-cultures than in conventional hepatocyte cultures. Pure cultures showed a rapid loss of monooxygenase activities, which were undetectable after 5 days. In contrast, all isozymes assayed were measurable in co-cultured hepatocytes on Day 7 (about 15 to 40% of the initial activities of Day 0 of culture). The beneficial effects of the co-culture system seemed to be more selective for certain cytochrome P450 isoforms, with P450 1A1 and 3A1 being the best stabilized isozymes after 1 wk. A clear response to inducers was observed in co-cultures, each isozyme showing a different induction pattern. 3-Methylcholanthrene produced a strong increase in P450 1A1 (7-ethoxyresorufin O-deethylase) activity and a low increase in P450 2A1 (testosterone 7 alpha-hydroxylation), whereas no changes were observed in the other activities. Phenobarbital treatment resulted in increases in P450 2B1/2 (7-pentoxyresorufin O-depentylase and 16 alpha- and 16 beta-hydroxylation of testosterone) activities, while minor effects were observed on P450 3A1 (testosterone 6 beta-hydroxylation) activity. Dexamethasone markedly increased P450 3A1 (testosterone 6 beta- and 15 beta-hydroxylation) activity and, to a lesser extent, P450 2B1/2 (16 beta-hydroxylation).

Interleukin-6 (IL-6 or BSF-2/IFN beta 2) is a component of normal human skin. IL-6 was immunologically detected in basal keratinocytes, endothelial cells and in a number of mononucleated cells and fibroblasts in normal skin and sudoriparous ducts. In psoriasis, intense labelling of the cytoplasm in the vicinity of keratinocyte membranes was detected in all epidermal layers and other skin appendages. The fact that this interleukin acts synergistically with respect to IL-1 and Tumour Necrosis Factor (TNF) strengthens the hypothesis whereby IL-6 may contribute via its receptor action to EGF function in modulating cell hyper-proliferation in psoriasis.

We have developed new co-cultures of continuous cell lines 3T3 (clone A31) and C3H/10T1/2 (clone 8) with hepatocytes as an alternative to co-cultures with noncontinuous epithelial cells. In this biological system we studied in detail the expression of the hepatic biotransformation system. After 7 d in culture, total cytochrome P-450 content and the monooxygenase activities aryl hydrocarbon hydroxylase and 7-ethoxycoumarin o-deethylase still maintained about 30% of their initial value, whereas in pure cultured hepatocytes these activities were undetectable. A significant response to induction by methylcholanthrene and phenobarbital of monooxygenase activities was observed in co-cultures for 7 d. NADPH-cytochrome c reductase activity remained unchanged for at least 7 d in co-cultured hepatocytes, whereas in pure cultures this activity was reduced to about 75% of the initial value after only 24 h. Finally, the activity of the conjugating enzymes UDP-Gt and GSH-t was maintained at nearly the initial levels during the complete period of study. The easy handling of continuous cell lines and the maintenance of the biotransformation system of hepatocytes in co-culture make this approach simpler and easier to standardize.

High yields of human hepatocytes (up to 23 X 10(6) viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham's F12 containing 0.2% bovine serum albumin, 10(-8) M insulin, and 10(-8) M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250 +/- 177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50 +/- 0.17 nmol glucose.mg-1.min-1) similar to that reported for human liver. Insulin at 10(-8) M activated glycolysis (X1.40) and glycogenesis (X1.34), and glucagon at 10(-9) M stimulated gluconeogenesis (X1.35) and glycogenolysis (X2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, haptoglobin, alpha 2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10(-9) M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol.mg-1 cell protein.min-1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol.mg-1). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.

Recombinant human IL-6 (rhIL-6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL-6 stimulated human hepatocytes with [35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C-reactive protein, haptoglobin, alpha 1-antichymotrypsin and fibrinogen were strongly induced (26-, 23-, 8.6-, 4.6- and 3.8-fold increases, respectively). Moderate increases were found for alpha 1-antitrypsin (2.7-fold) and alpha 1-acid glycoprotein (2.7-fold). RhIL-6 had no effect on alpha 2-macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, alpha 1-antichymotrypsin, alpha 1-antitrypsin and alpha 1-acid glycoprotein, dexamethasone enhanced the action of rhIL-6. We conclude that rhIL-6 controls the acute phase response in human liver cells.

The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (-2 degrees C/min down to -30 degrees C and then quick freezing to -196 degrees C) and a fast freezing rate (FFR) (direct freezing of tubes to -196 degrees C: -39 degrees C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37 degrees C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes.

Supplementation of Ham's F12 culture medium with essential amino acids (EAA) up to the rat plasma levels increased the rates of synthesis of albumin and transferrin by cultured rat hepatocytes by 1.3 and 1.7, respectively. Fifty percent of this increase could be attributed to three of the EAA: the branched-chain amino acids (BCAA: Leu Ile and Val). Non-branched-chain essential amino acids (non-BC-EAA) stimulated only 25% of the increase produced by the whole EAA mixture. When each EAA was tested individually, none of them caused an appreciable increase in albumin and transferrin in culture medium. When the concentrations of all EAA were raised to rat postprandial portal levels, albumin and transferrin synthesis rates reached a maximum, increasing by 3.2 and 3.5, respectively. Supplementation with BCAA at postprandial portal concentrations increased albumin and transferrin synthesis rates by 2.2 and 2.0, respectively, and had no noteworthy effect on the synthesis of cellular proteins. Non-BC-EAA at their postprandial portal concentrations increased albumin and transferrin synthesis rates by 1.7 and 1.9, respectively. Supplementation with alanine to reach a nitrogen content equal to that of the modified EAA-enriched medium had no stimulatory effect. Our results show that EAA have a specific effect on the synthesis of plasma proteins by cultured hepatocytes, and that BCAA at physiologic concentrations account for the major part of this stimulatory effect. Consequently, EAA and particularly BCAA concentration should be elevated in serum-free nutrient media to sustain maximum plasma protein synthesis.

The properties of the reaction of fluorescamine with proteins are the basis for the development of a sensitive, general and simple method for the assay of proteolytic activities. More importantly, the assay measures the initial step(s) of proteolytic attack, making it specially suitable for the examination of the controlling factors that regulate proteolytic degradation and/or the detection of 'specific' proteinases. The method allows the simple determination of the general parameters of enzyme action, V and Km, using proteins, i.e. the physiological substrates of the proteinases. The more appropriate proteins to be used as substrates are the N-amino-terminally blocked ones. Many proteins fulfill this requirement. If the particular protein whose degradation has to be studied lacks this modification, three different approaches can be used to study its degradation: (a) the accumulation of N-amino termini in excess over that of the intact substrate; (b) a gel filtration/continuous method and (c) the chemical blockage of its amino groups. The particular advantages of each of these approaches are discussed.

The three monokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interleukin-6 (IL-6) modulate acute phase plasma protein synthesis in adult human hepatocytes. Only IL-6 stimulates the synthesis of the full spectrum of acute phase proteins as seen in inflammatory states in humans, i.e. synthesis and secretion of C-reactive protein, serum amyloid A, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin and haptoglobin are increased while albumin, transferrin and fibronectin are decreased. IL-1 beta as well as TNF alpha, although having a moderate effect on the positive acute phase proteins and inhibiting the synthesis of fibrinogen, albumin and transferrin, fail to induce serum amyloid A and C-reactive protein. These data suggest that IL-6 plays the key role in the regulation of acute phase protein synthesis in human hepatocytes.

The plasma half-life of recombinant human interleukin-6 (rhIL-6) was determined in rats by measuring the disappearance of the biological activity as well as of the radioactivity of 125I-rhIL-6 from the circulation. The kinetics of clearance were biphasic. It consisted of a rapid initial disappearance corresponding to a half-life of 3 min, and of a second slow one corresponding to a half-life of about 55 min. By cellulose-acetate electrophoresis it was shown that rhIL-6 binds to a plasma protein resulting in a complex migrating in the beta-gamma region; 20 min after intravenous injection, about 80% of the 125I-rhIL-6 that had disappeared from the circulation was found in the liver. 125I-rhIL-6 was exclusively localized on the surface of parenchymal cells suggesting the existence of an interleukin-6 receptor on the hepatocytes.

This study examines the factors involved in the rapid glycolysis and glycogenolysis that occur during the first stages of hepatocyte culture: a) Shortly after seeding glycolysis, estimated as lactate released to culture medium, increased 10 times in comparison to that reported in vivo. By 8 to 9 h of culture, hepatocytes were nearly glycogen-depleted even in the presence of insulin. b) 6-Phosphofructo-2-kinase remained 100% active during this period. The proportion of the initial active phosphorylase (87%) decreased to 57% by 7 h of culture. c) Fructose 2,6-bisphosphate content was initially similar to that found in liver of fed animals, decreased after seeding and increased thereafter up to four times the initial concentration. In spite of changes in the concentration of this activator, the glycolytic rate remained high and constant. d) ADP and AMP increased sharply after cell plating, reaching values 1.7 and 3.5 times higher. The rise in AMP levels may be involved in the activation of glycolysis and glycogenolysis, because this metabolite is known to act as an allosteric activator of phosphofructokinase and glycogen phosphorylase. This metabolic situation resembles that of cells under hypoxia.