Antoine Vercruysse

Phase I and phase II xenobiotic biotransformation in cultures and co-cultures of adult rat hepatocytes

The aim of this study was to measure the activity of phase I and II key enzymes in the biotransformation of xenobiotics and their inducibility by phenobarbital (2 mM) in two currently used in vitro models, namely adult rat hepatocytes, conventionally cultured or co-cultured with rat epithelial cells derived from primitive biliary duct cells. For phase I, the cytochrome P450 content and the enzymic activities of 7-ethoxycoumarin O-deethylase and aldrin epoxidase have been determined, for phase II glutathione S-transferase activity was measured. In conventional cultures, all phase I parameters investigated declined continuously as a function of culture time. Two mM phenobarbital had inducing effects on 7-ethoxycoumarin O-deethylase and glutathione S-transferases but not on aldrin epoxidase. In co-cultures, after an initial decrease, a steady state situation developed for all the parameters measured, lasting for at least 10 days. The cytochrome P450 content, the 7-ethoxycoumarin O-deethylase, aldrin epoxidase and glutathione S-transferase activities were maintained from 3 to 4 days on at 25, 100, 15 and 50%, respectively, of their corresponding value obtained for freshly isolated hepatocytes. After phenobarbital treatment, the parameters mentioned were significantly increased with the exception of the aldrin epoxidase activity of which the inducibility was nearly completely lost.

Maintenance and induction in co-cultured rat hepatocytes of components of the cytochrome P450-mediated mono-oxygenase

Hepatocytes grown in culture rapidly lose many of the cytochromes P450 (CYP) responsible for metabolizing foreign compounds. Among the proteins most readily lost are members of the CYP2B subfamily. We have investigated, by RNase protection assays, the ability of rat hepatocytes, cultured conventionally or co-cultured with rat liver epithelial cells, to maintain the expression of genes encoding members of the CYP2B subfamily, and the inducibility of this expression by phenobarbital. After 4 days of conventional hepatocyte culture CYP2B mRNAs were undetectable, but remained inducible by phenobarbital. In co-cultured hepatocytes the abundance of the mRNAs remained relatively constant from 4-14 days. After 7 days of co-culture the concentration of the mRNAs was increased 12-15-fold by phenobarbital. RNase protection assays with probes capable of distinguishing between CYP2B1 and 2B2 mRNAs demonstrated that the ratios of the abundance and inducibility of the two mRNAs were the same in co-culture as in vivo. Co-cultured hepatocytes also maintained the expression of genes coding for two other components of the cytochrome P450-mediated mono-oxygenase, namely cytochrome P450 reductase and cytochrome b5.

Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNA replication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes

In this study, we investigated the combined effects of EGF and collagen I gel on the phenotype of cultured rat hepatocytes and we focussed our investigations on the regulation of xenobiotic-mediated induction of CYP, cell cycle progression and activation of capases 8 and 3. We found that EGF, added to basal culture medium or phenobarbital (3.2 mM) containing medium, provoked a moderate decrease of CYP1A1 and CYP2B1/2 activities. However, EGF did not exert any inhibitory effect on 3-methylcholantrene (5 microM) and beta-naphtoflavone (25 microM) induction of CYP1A1 activities. In collagen gel sandwich cultures, hepatocytes remained arrested in mid-G1 phase of the cell cycle, even in the presence of EGF. In conventional primary cultures, caspases 8 and 3 were activated at 3 and 5 days after plating respectively. In collagen gel sandwich cultures, we found that neither collagen I nor EGF prevented activation of caspase 8 while collagen I gel inhibited activation of caspase 3, preventing spontaneous apoptosis of cultured rat hepatocytes. In contrast, EGF transiently increased caspase 3 activity at day 1 after plating. Altogether, our data demonstrate that collagen I gel triggers intracellular signals which strongly affect cultured hepatocyte phenotype, leading to a cell cycle arrest in G1 phase and long-term survival through the inhibition of caspase 3 activation and that EGF-free medium improves survival and liver-specific gene expression in hepatocytes maintained in collagen I gel sandwich cultures.

Abnormal fatty acid turnover in the phospholipids of the red blood cell membranes of cystic fibrosis patients (in vitro study).

Summary: A study was carried out in order to investigate whether the abnormal in vitro turnover of fatty acids in the phospholipids of the red blood cell membranes of cystic fibrosis patients is intrinsic to the membrane, or whether it is induced by extrinsic serum factors. Red blood cells of cystic fibrosis patients and healthy subjects were labeled in vitro with [14C]linoleic acid, bound to albumin. The labeled cells were reincubated in autologous and homologous serum. The radioactivity present in the serum lipids and in the major phospholipid fractions of the red cell membranes was measured.Conclusions of this study are: 1) not all of the cystic fibrosis patients examined individually show an abnormal in vitro turnover of the red cell fatty acids, although they all presented abnormal fatty acid patterns for the red blood cell phospholipids, the platelet phospholipids, and the plasma lipids. 2) The in vitro abnormal fatty acid turnover occurs only in the incubations where red cells of cystic fibrosis patients are involved (in homologous and autologous serum) and not where red cells of healthy subjects are incubated in serum of cystic fibrosis patients. Consequently, the abnormal turnover is intrinsic to the red cells and is not induced by extrinsic serum factors.

Inhibition of gluconeogenesis by sodium valproate and its metabolites in isolated rat hepatocytes

The effects of sodium valproate (VP) and the sodium salts of its metabolites (2-en-VP, 4-en-VP, 4-OH-VP, 5-OH-VP and 2-propylglutaric acid) on gluconeogenesis have been examined using isolated rat hepatocytes. VP and its metabolites have a concentration-dependent inhibitory effect on gluconeogenesis from lactate: with increasing drug concentration, the lag in the onset of gluconeogenesis increases and the rate of glucose synthesis decreases. The toxic effect on glucose synthesis from lactate is very dependent on the metabolite used: the inhibitory effect is highest for VP and 4-en-VP, followed by 5-OH-VP, 4-OH-VP, 2-en-VP and finally by 2-propylglutaric acid. Thus, delta-dehydrogenation and omega-oxidation products in particular have a toxic effect on gluconeogenesis in isolated rat hepatocytes. Induction of the omega-oxidation pathway by enzymes inducers such as phenobarbitone may play a role in the eventual hepatotoxicity of VP. With glycerol the gluconeogenic substrate, the inhibitory effect of VP is also present, although to a smaller extent than with lactate. Glucagon abolishes the inhibitory action of VP.

Hepatocyte Cultures in Drug Metabolism and Toxicological Research and Testing

When a new chemical entity is tested for its safety for humans and their environment, in vivo experiments on living animals are usually conducted. However, in the early preclinical stage of drug development, in vitro techniques, and more specifically hepatocyte-based in vitro models, are currently being applied. A major problem, however, related to the use of hepatocytes and their cultures is their limited viability, which is associated with the loss of phase I and phase II biotransformation capacity. Therefore, in order to keep the hepatocytes functional for a longer period, the in vivo microenvironment is mimicked in vitro as closely as possible through the addition of soluble medium components, coculture with helper cells, or culture on an extracellular matrix (sandwich culture). We discuss the advantages and disadvantages of current short- and long-term hepatocyte culture systems are discussed as well as their proper use in toxicological research and testing.

Critical evaluation of 7-ethoxycoumarin O-deethylase activity measurement in intact isolated rat hepatocytes

In the determination of 7-ethoxycoumarin O-deethylase activity in intact isolated rat hepatocytes various factors influence the assay, including: the decay of 7-ethoxycoumarin fluorescence which is temperature and pH dependent; the measured fluorescence which has to be adjusted for the inner filter effect; glucose addition to the medium which influences the activity; all organic solvents which inhibit the enzymic activity, with dimethylformamide provoking the smallest effect (partial competitive inhibition); the enzymic reaction which is inhibited by the product of reaction; and the presence of bovine serum albumin in the medium which affects the enzymic activity. Biphasic kinetics are observed for the O-deethylation of ethoxycoumarin in intact isolated rat hepatocytes. For the high-affinity component, Km and Vmax values are 5 microM and 43 pmol/min X 10(6) cells and for the low-affinity component are 414 microM and 915 pmol/min X 10(6) cells. Addition of the substrate in dimethylformamide or omitting bovine serum albumin from the medium cause important changes in these kinetic parameters.

Effect of phenobarbital on the expression of glutathione S-transferase isoenzymes in cultured rat hepatocytes

Cultured adult rat hepatocytes were treated daily with 3.2 mM phenobarbital (PB) in order to study its effect on the expression of cytosolic glutathione S‐transferase isoenzymes. Glutathione S‐transferase (GST) activities, using 1‐chloro‐2,4‐dinitrobenzene and 1,2‐dichloro‐4‐nitrobenzene as substrates, were increased when PB was present in the culture medium. After purification and separation of GST on glutathione Sepharose 6 B and reversed‐phase HPLC, respectively, it was observed in vitro that PB caused an increase in the relative amounts of subunits 1, 3 and 7 compared to subunits 2 and 4. Using Northern blot technique, elevated levels of GST subunit 1/2 and 7 mRNA were measured, after addition of PB to the cultures.

Glutathione dependent detoxication in adult rat hepatocytes under various culture conditions

In order to obtain more information concerning the effects of culture and medium conditions on the glutathione dependent detoxication system in hepatocyte cultures, glutathione reductase (GR) and glutathione peroxidase (GPx) activities were studied in both pure cultures of adult rat hepatocytes and their co-cultures with rat epithelial cells. Cells were isolated either with an oxygen saturated Krebs Henseleit buffer (KHB) or with a non-gassed Hepes buffer. As medium conditions, additions of 10% fetal calf serum (FCS), 25 mM nicotinamide, 0.1 μM selenium and 2% dimethylsulphoxide, respectively, to the culture medium were examined. It was found that co-cultures of rat hepatocytes can cope better with oxidative stress than pure cultures do. This conclusion was reached from the following observations. When oxygenated KHB was used as isolating buffer, GR and GPx activities increased during the first days of pure culture and then slowly decreased. This was observed for all the medium conditions studied and no significant differences between the different media could be observed. For co-cultures, however, after some initial variations GR and GPx activities reached stabilized levels which were not only significantly lower than those observed for pure cultures, but were also maintained throughout the whole culture period. Supplementation of the medium had no effect on these findings with the exception of high GPx activities when Se was added to the co-culture medium. When Hepes buffer with a low oxygen content was used in cell isolation, pure cultures showed significantly lower GR and GPx activities than those first mentioned. Both approached the values measured for cocultures. The shape of the enzymatic activity curve as a function of culture time remained essentially unchanged. In co-cultures no significant differences could be observed in GPx activities when both perfusion techniques were involved, with the exception of the measurements done during the first 2 days of co-culture. Using the non-gassed Hepes buffer instead of the oxygenated KHB had no statistical effect on the GSH content of the hepatocytes in either of the two culture systems.

High-performance liquid chromatography of benzophenone derivatives for the determination of benzodiazepines in clinical emergencies

A screening procedure for fifteen 1,4-benzodiazepines in biological material (urine), suitable for clinical toxicology, is described. The benzodiazepines and/or their metabolites are hydrolysed to their benzophenones; after alkalinization, the benzophenones are extracted with chloroform. The extract is evaporated to dryness and the dried residue is dissolved in methanol and analysed by high-performance liquid chromatography on a reversed-phase column at 254 nm with methanol-water (1:1) as eluent.