Cell lines: a tool for in vitro drug metabolism studies.

Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, their widespread use is greatly hindered by the scarcity of suitable human liver samples. Moreover, the well-known in vitro phenotypic instability of hepatocytes, the irregular availability of fresh human liver for cell harvesting purposes, and the high batch-to-batch functional variability of hepatocyte preparations obtained from different human liver donors, seriously complicate their use in routine testing. To overcome these limitations, different cell line models have been proposed for drug metabolism screening. Human liver-derived cell lines would be ideal models for this purpose given their availability, unlimited life-span, stable phenotype, and the fact that they are easy to handle. However, the human hepatoma cells currently used (i.e. HepG2, Mz-Hep-1) show negligible levels of drug-metabolizing and do not constitute a real alternative to primary hepatocytes. Different strategies have been proposed to generate metabolically competent immortalized hepatocytes (transformation of human hepatocytes with plasmids encoding immortalizing genes, hepatocyte-like cells derived from stem cells, cell lines generated from transgenic animals, hepatocyte/hepatoma hydrid cells). Moreover, recombinant models heterologously expressing P450 enzymes in different host cells have been developed and successfully used in drug metabolism testing. In addition, new strategies have recently been explored to upregulate the expression of drug-metabolizing enzymes in cell lines of a human origin (i.e. transfection with expression vectors encoding key hepatic transcription factors). Among metabolic-based drug-drug interactions, P450 inhibition seems to be the most important. A major application of recombinant models expressing a single P450 is the screening of potential enzyme inhibitors. Therefore, pharmaceutical companies increasingly make use of cell lines to speed up the selection of new drugs with favourable pharmacokinetic and metabolic properties.

Strategies to in vitro assessment of major human CYP enzyme activities by using liquid chromatography tandem mass spectrometry.

At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Determining the role of CYP enzymes in the metabolism of a compound and evaluating the effect of NCEs on human CYP activities are key issues in pharmaceutical development as they may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. Reliable methods for determining enzyme activities are needed to characterize an individual CYP enzyme and to obtain a tool for the evaluation of its role in drug metabolism in humans. Different liquid chromatography tandem mass spectrometry methodologies have been developed for the fast and routine analysis of major in vivo and in vitro CYPs enzyme activities. The high sensitivity and selectivity of mass spectrometry allow traditional assays to be minimized, thus saving time, efforts and money. Therefore this technology has become the method of choice for the fast assessment of CYP enzyme activities in early drug discovery development. Our intention herein is to review the most recent approaches that have been developed to quickly assess CYPs activities using in vitro models and liquid chromatography coupled with mass spectrometry, as well as their application in early drug discovery.

Interactions of polyphenols with the P450 system: possible implications on human therapeutics.

Polyphenols are a family of natural compounds with many biological properties. This review focuses on their potential interaction on the cytochrome P450 system. Effects of phenolic acids, anthocyanins, stilbenes, catechins and other flavonoids on the drug metabolising function are revised. Their daily intake and presence in herbal medicines justify the study of potential drug-interaction to prevent undesirable clinical consequences.

Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes.

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.

Potential hepatoprotective effects of new Cuban natural products in rat hepatocytes culture.

The protective effects of five Cuban natural products (Mangifera indica L. (MSBE), Erythroxylum minutifolium, Erythroxylum confusum, Thalassia testudinum and Dictyota pinnatifida extracts and mangiferin) on the oxidative damage induced by model toxicants in rat hepatocyte cultures were studied. Cells were pre-incubated with the natural products (5-200 microg/mL) for 24 h. Then hepatotoxins (tert-butyl hydroperoxide, ethanol, carbon tetrachloride and lipopolysaccharide) were individually added and post-incubated for another 24 h. After treatments, cell viability was determined using the MTT assay. Mangiferin and MSBE exhibited the highest cytoprotective potential (EC50 between 50 and 125 microg/mL), followed by T. testudinum and Erythroxylum extracts, whereas no significant protective effects was produced by Dictyota extract treatment. Antioxidant properties of the natural products against lipid peroxidation and GSH depletion induced by tert-butyl hydroperoxide were then investigated. The results show that at 36 h pre-treatment of cells with mangiferin or MSBE, concentrations of T. testudinum and Erythroxylum extracts ranging from 25 to 100 microg/mL significantly inhibited lipid peroxidation induced by tert-butyl hydroperoxide (100 and 250 microM) and increased the GSH levels reduced by the toxicant. D. pinnatifida inhibited lipid peroxidation, but did not preserve GSH levels. In conclusion, MSBE, E. minutifolium, E. confusum and T. testudinum extracts and mangiferin showed hepatoprotective activity against induced damage in all the experimental series, where mangiferin and the extracts of MSBE and T. testudinum were the best candidates to inhibit "in vitro" damage to rat hepatocytes. This hepatoprotective effect found could be associated with the antioxidant properties observed for the products.

Determination of major human cytochrome P450s activities in 96-well plates using liquid chromatography tandem mass spectrometry.

At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Evaluation of the effect of NCEs on human CYP450 enzyme activities is a key issue in pharmaceutical development as it may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. A liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method has been developed for the fast and routine analysis of major human CYP450s enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in primary hepatocyte cell cultures. The high sensitivity and selectivity of mass spectrometry has allowed traditional assays to be minimized, thus enabling the use of 96-well plate format which markedly reduced the number of hepatocytes needed for each cytochrome CYP450 activity measurement, a fact that is particularly critical concerning human hepatocytes.

Effects of Mangifera indica L. aqueous extract (Vimang) on primary culture of rat hepatocytes.

Vimang is an aqueous extract from stem bark of Mangifera indica L. (Mango) with pharmacological properties. It is a mixture of polyphenols (as main components), terpenoids, steroids, fatty acids and microelements. In the present work we studied the cytotoxic effects of Vimang on rat hepatocytes, possible interactions of the extract with drug-metabolizing enzymes and its effects on GSH levels and lipid peroxidation. No cytotoxic effects were observed after 24 h exposure to Vimang of up to 1000 microg/mL, while a moderate cytotoxicity was observed after 48 and 72 h of exposure at higher concentrations (500 and 1000 microg/mL). The effect of the extract (50-400 microg/mL) on several P450 isozymes was evaluated. Exposure of hepatocytes to Vimang at concentrations of up to 100 microg/mL produced a significant reduction (60%) in 7-methoxyresorufin-O-demethylase (MROD; CYP1A2) activity, an increase (50%) in 7-penthoxyresorufin-O-depentylase (PROD; CYP2B1) activity, while no significant effect was observed with other isozymes. To our knowledge, this is the first report regarding the modulation of the activity of the P450 system by an extract of Mangifera indica L. The antioxidant properties of Vimang were also evaluated in t-butyl-hydroperoxide-treated hepatocytes. A 36-h pre-treatment of cells with Vimang (25-200 microg/mL) strongly inhibited the decrease of GSH levels and lipid peroxidation induced by t-butyl-hydroperoxide dose- and time-dependently.

In vitro ADME medium/high-throughput screening in drug preclinical development.

The study of the ADME features of the huge number of new chemical entities (NCEs) produced mainly by combinatorial chemistry has become a bottleneck in the drug development process. In response the pharmaceutical industry is involved in the development of new medium/high-throughput screening capabilities. The aim of this paper is to review some of the available in vitro ADME systems adapted to screening requirements together with the technological approaches which can be linked to medium/high-throughput molecular screening.

[Risk management project: reactive or proactive approach?]. / Progetto Risk Management: metodologia reattiva o proattiva?

Risk management in healthcare refers to the process of developing strategies aimed at preventing and controlling the risk of occurrence of errors and harmful events. The final objective is primarily that of increasing patient safety and secondarily, that of reducing the financial burden of adverse events. The implementation of a risk management system is therefore of vital strategic importance. Nevertheless, a fundamental question that needs to be answered in the operational phase is: should a proactive or reactive approach to risk management be taken? In our view, proactive risk management has many advantages over a reactive approach and is therefore preferable. The reactive approach should be taken exclusively to obtain information regarding risk and errors, in the preliminary, as well as monitoring and follow-up phases of the project.

Human hepatocytes as a tool for studying toxicity and drug metabolism.

Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolising enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment.

Human hepatocytes in primary culture: the choice to investigate drug metabolism in man.

Different types of hepatic tissue, including whole or split livers from organ donors or waste liver from therapeutic liver resections, are used to prepare human hepatocyte cultures. Characteristics of liver samples from different origins (gender, age, healthy/pathological status, xenobiotic treatment) as sources of human hepatocytes are key factors which notably determine viability and functionality of hepatocytes. The characterisation of the CYP system can be assessed in terms of activity (using specific substrates/inhibitors), protein (antibody analysis) and molecular biology-based mRNA amplification techniques (PCR technology and DNA microarrays). It could reasonably be considered that human hepatocytes reflect the heterogeneity of CYP expression in human liver and is a suitable model for drug metabolism studies. Several key issues need to be addressed at the early stages of drug development to better select drug candidates (metabolic profile and rate, identification of CYPs involved, drug-drug interactions due to enzyme induction/inhibition). The metabolic stability and metabolite profile of new chemicals can be easily investigated by incubating the drugs with fully competent metabolic models like hepatocyte suspensions or 24 h-cultured hepatocytes. CYP inhibitory effects are usually screened in recombinant CYP enzymes or microsomes, however, the actual concentration of substrate and inhibitor available to the CYP enzyme depends on processes missing in subcellular models (transport mechanisms, cytosolic enzymes, binding to intracellular proteins). Since intact cells more closely reflect the environment to which drugs are exposed in the liver, cultured hepatocytes constitute a more predictive model for drug-drug interactions. Screening of CYP inducers cannot be done in microsomes as it requires a cellular system fully capable of expressing CYP genes. Primary hepatocytes are still the unique in vitro model for global examination of inductive potential of drugs (monitored as increases in mRNA content or activity).

Effect of xenobiotics on monooxygenase activities in cultured human hepatocytes.

The activity of human cytochrome P450 monooxygenases, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase can be increased by 3-methylcholanthrene, phenobarbital and ethanol in human hepatocytes maintained in primary culture. Total cytochrome P450 content increased two-fold after 48 hr of incubation with methylcholanthrene or phenobarbital and 1.5-fold after incubation with ethanol. The three chemicals elicited different effects on cytochrome P450 dependent activities. Addition of 3-methylcholanthrene caused a time- and concentration-dependent increase in both monooxygenase activities, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase, while phenobarbital and ethanol increased 7-ethoxycoumarin O-deethylase activity but had no effect on aryl hydrocarbon hydroxylase. Dexamethasone per se had little or no effect on either monooxygenase activities, but potentiated the effect of the three chemicals on 7-ethoxycoumarin O-deethylase.

The immunosuppressant drug FK506 prevents Fas-induced apoptosis in human hepatocytes.

FK506 is a potent immunosuppressive drug used for the prevention of graft rejection in organ transplantation. Experimental and clinical studies have shown correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell death after ischemia-reperfusion injury in the rat liver. Fas-mediated apoptosis is very likely involved in allograft rejection and experimental evidence has shown a decrease of FasR expression in mouse hepatocytes produced by the drugs. On the basis of these findings we have investigated the protective effect of FK506 in comparison with cyclosporine A (CsA) on Fas-induced apoptosis, by analysing the activation of downstream effector caspases in human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies against FasR, which resulted in a significant activation of caspase-3 after 12 h. Prevention of the downstream activation of the caspase cascade and apoptosis was observed when hepatocytes were pre-treated for 3 h with immunosuppressant drugs. A significant reduction (ca. 30-40%) of caspase-3 activation by 5 microM FK506 and CsA was observed. Along with less activation of caspase-3 a decrease of apoptotic DNA fragmentation was found. In addition, FK506 significantly reduced not only caspase-8 but also caspase-9 activation, to a similar extent as CsA, thus suggesting a protective effect at the mitochondrial level of this drug, as has already been reported for CsA. These effects of FK506 help to explain its strong anti-rejection properties and suggest promising benefits of pharmacological preconditioning on ischemia-reperfusion injury following liver transplantation.

Rat hepatocytes cultured on a monkey kidney cell line: Expression of biotransformation and hepatic metabolic activities.

The present study deals with the expression of cytochrome P-450 and two monooxygenase activities (aryl hydrocarbon hydroxylation and 7-ethoxycoumarin O-deethylation) by hepatocytes cultured on feeder layers of MS cells, in an attempt to investigate the influence of culture medium composition on phase I activities of hepatocytes. When compared with culture medium I (serum-free, hormone-supplemented Ham's F12), 4-day MS hepatocyte cultures supplemented with nicotinamide (medium II) allowed better retention by hepatocytes of cytochrome P-450 and phase I activities (aryl hydrocarbon hydroxylation and 7-ethoxycoumarin O-deethylation). A moderate additive effect was found after increasing branched-chain amino acid composition (medium III). Increasing the concentration of all amino acids, galactose and pyruvate (medium IV) resulted in higher levels of cytochrome P-450, aryl hydrocarbon hydroxylation and 7-ethoxycoumarin O-deethylation which, after 8 days in culture, were at 33, 49 and 43% of day 0 values, respectively. A further increase of branched-chain amino acid concentration produced only a moderate effect (medium V). Under these culture conditions, hepatocytes also expressed basic metabolic functions. After 10 days, MS hepatocyte cultures retained 43, 71 and 65% of ureogenesis, triglyceride secretion and albumin production measured at day 0. In contrast, pure hepatocytes cultured under the same conditions expressed these three hepatic functions only until the fifth or sixth day of culture.

Prolonged expression of biotransformation activities of rat hepatocytes co-cultured with established cell lines.

Co-culture of hepatocytes with fibroblastic cells (C3H/10T1 2 and 3T3) allowed the expression of phase I and II biotransformation enzymes for a prolonged time in culture. After 7 days in co-culture, cytochrome P-450 and monooxygenase activities were still 20-30% of the initial value, while in pure hepatocyte cultures they were undetectable. NADPH-cytochrome c reductase and conjugation activities remained at nearly initial levels for at least 7 days in co-cultured hepatocytes. Finally, in co-cultures a clear and prolonged induction of monooxygenase activities by 3-methylcholanthrene and phenobarbital was observed.

Inhibition of monooxygenase activities in human hepatocytes by interferons.

The effects of human recombinant interferons alpha and gamma on monooxygenase activities in cultured human hepatocytes have been investigated. Dose-response and time course studies showed that interferons reduced 7-ethoxyresorufin O-deethylase activity of human hepatocytes after a 12-hr incubation with 300 U/ml interferons alpha and gamma (52% and 38% decrease, respectively). A reduction in 7-ethoxyresorufin O-deethylase activity was also observed in HepG2 cells, although in these cells maximal inhibition was observed after 24 hr of treatment with 1000 U/ml (a 41% and 28% decrease with interferon alpha and gamma, respectively). A decrease in activity was also observed in 7-pentoxyresorufin O-depentylase, 7-ethoxycoumarin O-deethylase and testosterone 2alpha- and 6beta-hydroxylase. It is noteworthy that the marked increase in 7-ethoxyresorufin O-deethylase activity detected in human hepatocytes after incubation with 2 mum-3-methylcholanthrene (10-fold over non-treated cells) was reduced by 40% in the presence of interferons (300 U/ml), thus indicating that the inducibility of monooxygenases could be altered by interferon treatment. The inhibitory effect of interferons on 7-ethoxyresorufin O-deethylase was transient and HepG2 cells recovered their normal activity 24 hr after interferon removal from culture medium. This study provides the first direct evidence that interferons down-regulate the level of monooxygenases in human hepatic cells and prevent, in part, their induction by xenobiotics.

Functionality of cultured human hepatocytes from elective samples, cadaveric grafts and hepatectomies.

The major possible sources of human liver for hepatocyte isolation are elective liver biopsies, cadaveric liver grafts and therapeutic liver resections. The suitability in terms of metabolic-competent hepatocyte cultures and risk/benefit of these resources has been comparatively studied. To this end, viability of isolated hepatocytes, yield of isolation procedure, hepatocyte survival during culture and CYP activities were the parameters analysed. The best results were found in hepatocytes prepared from elective biopsies, whereas a marked reduction in viability and functional competence was seen in hepatocytes from hepatectomy samples. Metabolic differences were observed in total CYP oxidative metabolism (7-ethoxycoumarin O-deethylation, total testosterone hydroxylation), as well as in CYP3A4, CYP2C9 or CYP2C19 activities (testosterone oxidations at 6beta-, 16beta- and 17-positions, respectively). Vascular control during the hepatectomy procedure influenced hepatocyte functionality: higher CYP activities were found in hepatocytes isolated from samples obtained under non-ischemic conditions or continuous vascular clamping than in those obtained under intermittent vascular clamping. In addition to cellular functionality, other criteria such as sample availability or ethical aspects should be considered. Elective biopsies have low, but not absent, surgical risk. However, the better functionality and the higher accessibility of elective liver samples in comparison to the other groups suggest this source of liver tissue as the most appropriate for cell harvesting purposes.

Toxicity of the antitumoral drug datelliptium in hepatic cells: Use of models in vitro for the prediction of toxicity in vivo.

Datelliptium is a DNA-intercalating agent derived from ellipticine. The drug has potent antitumoral activity in vitro and in vivo. The first clinical use of the drug revealed unexpected hepatotoxic effects in humans that had not been observed in animals. Using different hepatic models in vitro (rat hepatocytes in suspension and in culture, cultured human hepatocytes and rat and human hepatoma cell lines), the possibility of prediction in vitro of the hepatic toxicity of a drug has been investigated. Cytotoxic effects were evaluated by measuring the leakage of intracellular lactate dehydrogenase and the ability of cells to reduce MTT after exposure to concentrations of datelliptium ranging from 0.1 to 1000 mum. According to these endpoint parameters, the concentrations of the drug that produced 50% of maximal inhibitory effect (IC(50)) were in the range 100 to 195 mum in rat hepatocyte suspension and hepatocyte cultures after 2 hr of treatment, 7-9 mum in cultured rat and human hepatocytes after 23 hr of treatment, and about 200-320 mum in HepG2 and FaO cells after 23 hr of treatment. Metabolic parameters were generally more sensitive than cytotoxic endpoints for detecting toxic effects of datelliptium on hepatocytes in the two experimental models used. Metabolic effects on rat hepatocyte suspension and culture were evaluated respectively after 2 and 23 hr of exposure to the drug. Triglyceride secretion was the most sensitive parameter and the IC(10) values (concentration causing 10% of maximal inhibitory effect) were 0.03 and 0.9 mum in hepatocyte suspension and culture, respectively. Glycogen, albumin and cellular protein synthesis were similarly altered in both cellular systems and the IC(10) values were in the range 0.5-3.5 mum. Ureogenesis and gluconeogenesis were relatively insensitive parameters in cell suspensions (IC(10) values 16.4 and 10.3 mum, respectively) compared with those in hepatocyte culture (IC(10) values 3.6 and 3.1 mum, respectively). The concentrations of datelliptium reported in blood, and particularly in liver, are higher than the concentrations that produce impairment of cell metabolism in vitro. This may be an indicator of the toxicological risk of datelliptium and anticipates the hepatotoxicity observed in vivo.

Cytochrome P450 activities in pure and co-cultured rat hepatocytes. Effects of model inducers.

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).

Evaluation of the xenobiotic biotransformation capability of six rodent hepatoma cell lines in comparison with rat hepatocytes.

Phase I and II activities were examined in six rodent hepatoma cell lines and compared with those of cultured rat hepatocytes both in basal conditions and after exposure to 5 microM methylcholanthrene, 2 mM phenobarbital, and 15 microM beta-naphtoflavone. The metabolic profile of testosterone was also studied. The highest aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities were found in MH1C1 cells. Comparable values for 7-ethoxyresorufin O-deethylase activity, ranging from 21.6 to 42.9 pmol/mg x min, were observed in the hepatocytes and hepatoma cells, except the HTC cells. In contrast, only Fao cells showed 7-pentoxyresorufin O-depentylase activity at levels similar to those of hepatocytes (6.2 +/- 1.0 and 7.4 +/- 1.2 pmol/mg x min, respectively). Rat hepatocytes actively hydroxylated p-nitrophenol, but this activity was not measurable in hepatoma cells. Glutathione transferase activity was maintained in all the hepatoma cell lines at similar levels to those found in hepatocytes (684 +/- 56 nmol/mg x min). The seven hydroxylated metabolites of testosterone produced by cultured hepatocytes were negligible in hepatoma cells. Exposure of cells to inducers revealed that aryl hydrocarbon hydroxylase activity was mainly increased after treatment with 3-methylcholanthrene and beta-naphtoflavone, and the highest values were found in rat hepatocytes followed by MH1C1 and Fao cells. 3-Methylcholanthrene and naphtoflavone treatment also resulted in a marked increase in 7-ethoxyresorufin O-deethylase activity in hepatocytes as well as in H4IIC3, McA-Rh7777, MH1C1, and Fao cells.(ABSTRACT TRUNCATED AT 250 WORDS)