Yves Vandenberghe

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.

Influence of medium composition and culture conditions on glutathione S-transferase activity in adult rat hepatocytes during culture.

SummaryGlutathione S-transferase (GST) activity was measured in adult rat hepatocytes during either pure culture or coculture with another rat liver cell type in various media. Addition of nicotinamide, selenium, or dimethylsulfoxide, deprivation of cyst(e)ie and the use of two complex media were tested. Whatever the conditions used, after a constant decrease during the first 24 h, GST remained active over the whole culture period (1–2 wk). However, various patterns were observed: GST activity either remained relatively stable to approximately 50% of the initial value or showed a moderate or strong increase. The highest values were found in pure hepatocyte cultures maintained in the presence of nicotinamide or dimethylsulfoxide. Similar changes were observed using 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene as substrates for GST. Addition of 10−4M indomethacin resulted in 37 to 60% inhibition of enzyme activity. Thus, these results demonstrate that GST remained expressed during culture but its levels markedly varied depending on the medium composition and type and age of culture.

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.

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.

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.

Effects of dimethylsulphoxide on phase I and II biotransformation in cultured rat hepatocytes.

Addition of 2% dimethylsulphoxide (DMSO) to the medium has been described to retain biochemical and morphological differentiation of cultured rat hepatocytes. The maintenance of differentiated hepatocytes for a long time with stable phase I and II biotransformation capacities provides an important model for pharmacological and toxicological studies. This work has been undertaken to study the effects of DMSO on phase I and II parameters in cultured hepatocytes. For phase I cytochrome P-450 content, 7-ethoxycoumarin O-deethylase and aldrin epoxidase activities and for phase II glutathione S-transferase (GST) activity and its isoenzyme profile have been measured. In control cells cytochrome P-450 content and the enzymic activities measured declined as a function of culture time. Addition of DMSO partially prevented the loss of all parameters measured. The changes in GST activity were related to changes in its isoenzyme pattern. In control cells subunits 1 and 2 decreased, 3 and 4 increased and de novo expression of 7 was observed. When DMSO was added, the subunit profile better resembled that observed in vivo and the expression of subunit 7 was suppressed. In conclusion DMSO protects cultured hepatocytes from dedifferentiation and stabilizes the cells.

Transcriptional- and post-transcriptional-dependent regulation of glutathione S-transferase expression in rat hepatocytes as a function of culture conditions

Transcriptional activity of the glutathione S‐transferase (GST) α (subunits 1 and 2), μ (subunits 3 and 4) and π (subunit 7) gene families has been analyzed using the nuclear ‘run‐on’ technique on adult rat hepatocytes maintained for 4 days in conventional culture and for 4 and 12 days in co‐culture with rat liver epithelial cells. Several medium conditions are included in this study, namely with or without fetal calf serum and with nicotinamide or dimethylsulphoxide. Hepatocytes co‐cultured for 4 days maintain approximately 30–70% of the α gene family transcriptional activity, whatever the medium conditions, when compared to freshly isolated hepatocytes. A marked decrease is observed after 12 days of co‐culture or when hepatocytes are maintained in conventional culture. The transcriptional activity of the μ gene family is maintained at 40–160% when hepatocytes are cultured with or without fetal calf serum, and is inducible by nicotinamide (approximately 4‐fold) and dimethylsulphoxide (approximately 2‐fold) in conventional culture and/or in co‐culture. In contrast to freshly isolated hepatocytes, GST π gene transcriptional activity is observed in conventional and co‐cultured hepatocytes, irrespective of the medium conditions. Dimethylsulphoxide treatment however, represses the expression of GST 7 in vitro. These results demonstrate that the expression of GST α, μ and π genes in conventional and co‐cultured rat hepatocytes is controlled primarily at the level of transcription. It cannot be excluded, however, that dimethylsulphoxide stabilizes the GST mRNA levels in vitro.

Regulation of glutathione S-transferase gene expression by phenobarbital in cultured adult rat hepatocytes

Previous studies, by using Northern blotting analyses, showed that phenobarbital (PB) affects the steady‐state mRNA levels of glutathione S‐transferase (GST) subunits , and 7 in both conventional cultures of adult rat hepatocytes and co‐cultures, with rat liver epithelial cells [Vandenberghe et al., 1989, FEBS Lett. 251, 59–64; Morel et al., 1989, FEBS Lett. 258, 99–102]. To determine whether PB acts at the transcriptional level, nuclear ‘run‐on’ experiments using cDNA probes hybridizing to GST subunits , and 7 mRNA were performed on purified nuclei isolated from control and PB treated hepatocytes seeded under conventional and co‐culture conditions. Data from this study demonstrate that the increase in steady‐state mRNA levels observed in both conventional culture and co‐culture after 4 days PB exposure results from an increased transcriptional activity or the GST genes. However, a substantial increase in steady‐state mRNA levels in the absence of a commensurate increase in transcriptional activity at 12 days of co‐culture, indicates that the barbiturate has also a stabilizing effect in vitro on the GST mRNAs.

Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes

mRNA levels of glutathione S‐transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.