A. Callaerts

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.

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.

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.

Effect of the aging process on the gender and phenobarbital dependent expression of glutathione S-transferase subunits in brown Norway rat liver

The effect of age, gender and phenobarbital treatment on the hepatic cytosolic glutathione S-transferase subunit composition was studied in Brown Norway rats. Affinity chromatography followed by reversed phase HPLC was used in order to separate the various glutathione S-transferase subunits. Corresponding steady-state mRNA levels were measured by Northern Blot analysis using cDNA clones hybridizing to mRNA encoding glutathione S-transferase subunits 1/2, 3/4 and 7, respectively. In all the age groups studied (15, 25, 53, 99, 112 and 136 weeks) the total amount of glutathione S-transferase protein was in untreated rats significantly higher in males (132 micrograms/mg cytosolic protein) than in females (91 micrograms/mg cytosolic protein) and significant gender dependent differences in the subunit composition were demonstrated. Aging seemed to be of minor importance in untreated as well as in phenobarbital treated rats. Under control conditions, the subunit composition of male rats between 15 and 136 weeks old consisted of 28, 12, 11 and 49% of subunits 1, 2, 3 and 4 respectively and of female animals of the same age groups of 38, 26, 7 and 30%, respectively. In all the age groups studied phenobarbital administration (45 mg/kg body weight, i.p., once a day for 7 days) doubled total glutathione S-transferase protein in both genders and affected the subunit composition in a significant way, emphasizing the already existing differences between genders. Subunits 1, 2 and 3, especially, were increased in male rats in comparison to females resulting in the observation that levels of glutathione S-transferase subunits studied became higher in males than in their female counterparts. The HPLC results were confirmed by steady-state mRNA analysis. In untreated rats, higher levels of mRNA encoding glutathione S-transferase subunits 1/2 and 3/4 were present in male than in female livers. Phenobarbital treatment increased mRNA levels in both genders. Subunit 7 was never detected. These effects were demonstrated in both young and old rats.

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco–toxicology ☆

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Spectrophotometric measurement of flavin-containing monooxygenase activity in freshly isolated rat hepatocytes and their cultures

The determination of the mixed function flavin-containing monooxygenase activity in rat liver and in hepatocytes and their cultures by spectrophotometric measurement of the oxygenation of methimazole is complicated by an inhibition caused by some of the reagents used during this method. Optimal conditions were determined for measuring this enzyme activity in microsomal preparations of rat liver and its hepatocytes. Optimal flavin-containing monooxygenase activities were obtained for measurements performed in a 0.25 M N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-EDTA buffer at pH 8.7 and at a methimazole concentration of 2 mM. Data are also presented which show that no interferences caused by either cytochrome P450-dependent enzymes or by the reduction of methimazole disulfide by glutathione have to be taken into account when determining methimazole oxygenation. Finally, the above assay was also used to study flavin-containing monooxygenase activity in primary monolayer cultures of hepatocytes for 6 days.

Effect of phenobarbital on 7-Ethoxycoumarin-O-deethylase and microsomal epoxide hydrase activities in collagen gel cultures of rat hepatocytes.

Ethoxycoumarin-O-deethylase (ECOD) and microsomal epoxide hydrase (mEH) activities have been studied in collagen gel cultures of rat hepatocytes under control conditions and after exposure to phénobarbital. The hepatocytes were either sandwiched between two layers of extracellular matrix (type I collagen) or directly mixed with the same type of collagen gel. In preliminary experiments it was first tested whether it was possible to measure ECOD activity directly in intact hepatocytes that were embedded in a collagen gel matrix. Probably by a delay of substrate penetration through the collagen gel, the latter had to be removed by a collagenase digestion before ECOD activity measurements took place. When cultured in control medium, the activity of both phase I enzymes studied decreased as a function of culture time in sandwich as well as in immobilization cultures. After 4 days, however, a steady-state situation was reached and this level of enzymatic activity was observed for at least 2 wk. When the cells were exposed to 3.2 mm phenobarbital, an increased ECOD activity remained detectable for at least 14 days. In sandwich cultures a two- to threefold increase of ECOD activity was observed, whereas in immobilization cultures, increases of only 30 to 46% of the values observed in the control medium were measured. After exposure of the collagen gel cultures to 3.2 mm phenobarbital, the mEH activity was increased to the same extent (factor 2 to 2.5) in both culture models.

In vitro biotransformation of 2-methylpropene (isobutene): Epoxide formation in mice liver

Until now, no data are available concerning the biotransformation and toxicity of 2-methylpropene (or isobutene), a gaseous alkene widely used in industry (rubber, fuel additives, plastic polymers, adhesives, anti-oxidants). In this work, the biotransformation of 2-methyl-propene (MP) has been studied, using total liver homogenates of mice, supplemented with a NADPH-generating system. In analogy to other olefins, 2-methylpropene is metabolized to its epoxide 2-methyl-1,2-epoxypropane (MEP), as proved by the identification by gas chromatography coupled with mass spectrometry. The epoxidation is cytochromeP-450 dependent, as shown by experiments in the absence of the NADPH-generating system and in the presence of various concentrations of metyrapone and SKF 525-A, two known inhibitors of the mono-oxygenases. A simple gas chromatographic headspace method has been developed for the quantitative determination of the epoxide formed. The formation of MEP is never linear in function of time and it reaches a maximum after 20 min. Thereafter is decreases continuously to undetectable levels. This observation can be explained by the immediate action of epoxide hydrolase and glutathione S-transferase, converting the epoxide to 2-methyl-1,2-propanediol and to the glutathione conjugate respectively. The involvement of both enzymes has been demonstrated by the addition of 3,3,3-trichloropropene oxide and indomethacin. These inhibitors of, respectively, epoxide hydrolase and glutathione S-transferase increase the epoxide formation in a significant way. The actual concentration of MEP is therefore not only dependent on its formation by cytochromeP-450 dependent mono-oxygenases, but also on its conversion by epoxide hydrolase and glutathione S-transferase, both very active in liver tissue.

Age- and gender-related changes in the hepatic metabolism of 2-methylpropene and relationship to epoxide metabolizing enzymes.

The effect of age and gender on the in vitro biotransformation of 2-methylpropene, an alkene metabolized to 2-methyl-1,2-epoxypropane, was studied. The epoxide concentration and the epoxide metabolizing enzymatic activities were investigated in male and female Brown Norway rats of different ages. Liver tissue of senescent rats was exposed to smaller 2-methyl-1,2-epoxypropane concentrations than that of young animals, although changes during ageing were rather modest. With advancing age a feminization of male glutathione S-transferase and cytosolic epoxide hydrolase activities was found, as well as a significant decline of the female microsomal epoxide hydrolase activity and an increase of the cytochrome P-450 content in the oldest female rats.

Observation of hepatotoxic effects of 2-n-pentylaminoacetamide (Milacemide) in rat liver by a combined in vivo/ in vitro approach

Milacemide or 2-n-pentylaminoacetamide hydrochloride, a new glycine derivative, was found to cause elevations of plasma transaminases in patients suffering from severe depression and Alzheimers disease. However, no signs of liver toxicity were observed during the course of earlier conducted subchronic and chronic in vivo studies in rodents and cynomolgus monkeys. In this study an in vivo/in vitro approach has been proposed to detect early alterations in key metabolic and functional liver capacities. Milacemide was administered by continuous i.v. infusion for 7 days to male Sprague-Dawley rats using subcutaneously implanted osmotic pumps. Doses were given of 0, 250 and 500 mg/kg per day. Body weight and food intake were recorded and at day 7 of exposure, Milacemide concentration, glucose, urea, triglycerides and cholesterol levels and alanine (ALT) and aspartate aminotransferase (AST) activities were measured in plasma. Non-esterified fatty acids were determined in serum. On day 8, after overnight fasting, hepatocytes were isolated. A portion of the cells derived from untreated animals (no osmotic pumps) were cultured in a primary monolayer and exposed in vitro to different Milacemide concentrations.The xenobiotic biotransformation capacity of the isolated hepatocytes was studied by measuring the cytochrome P450 content, ethoxycoumarin-O-deethylase (ECOD), pentoxyresorufin-O-deethylase (PROD), ethoxyresorufin-O-deethylase (EROD), aldrin epoxidase (AE), epoxide hydrolase (EH) and glutathione S-transferase (GST) enzyme activities. Triglycerides, cholesterol and phospholipid contents were measured on the isolated cells. At plasma concentrations of 43 and 130 μM Milacemide, the ALT activity was unchanged or significantly decreased, whereas the AST activity was increased in both cases. Other clinical chemistry parameters remained unchanged. Weight gain was significantly lower in rats treated with the high Milacemide dose. In addition, decreased food consumption was observed in all treated animals leading to significantly lower food efficiency factors for the rats treated with the high dose. Milacemide had a specific inhibitory effect on xenobiotic biotransformation: ECOD activity decreased to 60% of the control value for both Milacemide doses, PROD activity remained unaffected whereas EROD activity decreased to 65% of the control value. A decrease was also observed at the highest drug concentration for AE (to 41%), EH␣(to 65%), cytochrome P450 content (to 80%) and GST (to␣85%). At 500 mg Milacemide kg/day, hepatocyte triglycerides levels increased 3.1-fold while cholesterol and phospholipid levels remained unaffected. Electron and light microscopy on total liver and isolated hepatocytes indicated a concentration-dependent accumulation of lipid droplets, the occurrence of numerous vacuoles in the cytoplasm and other structural abnormalities. When the cultured hepatocytes of control animals (without osmotic pumps) were exposed to Milacemide, the appearance of vacuoles and myeloid bodies could be confirmed in vitro. The results of this study using an in vivo/in vitro approach clearly show potential hepatotoxic properties of Milacemide, an effect not observed in conventional toxicity studies.