Stable expression of human cytochrome P450 3A4 in V79 cells and its application for metabolic profiling of ergot derivatives

ejp ELSEVIER

environmental toxicology and pharmacology

European Journal of Pharmacology Environmental Toxicology and Pharmacology Section 293 (1995) 183-190

Stable expression of human cytochrome P450 3A4 in V79 cells and its application for metabolic profiling of ergot derivatives Reimund Rauschenbach, Hille Gieschen, Manfred Husemann, Birgit Salomon, Michael Hildebrand * Research Laboratories, Schering AG, D-13342 Berlin, Germany Received 28 December 1994; revised 6 March 1995; accepted 21 March 1995

Abstract

Expression of human cytochrome P450 (CYP) in heterologous cells is a means of specifically studying the role of these enzymes in drug metabolism. The complete cDNA encoding CYP3A4 (PCN1) was inserted into an expression vector containing the strong myeloproliferative sarcoma virus promoter in combination with the enhancer of the cytomegalovirus and stably expressed in V79 Chinese hamster cells. The presence of genomically integrated CYP3A4 cDNA cell clones was confirmed by polymerase chain reaction analysis. Transcription was detected by reverse transcribed polymerase chain reaction analysis. Functional expression could be demonstrated by conversion of testosterone to the specific 6fl-hydroxylated product. In recombinant V79 cells expressing CYP3A4 about 6% of the substrate was converted to 6/3-hydroxytestosterone. The metabolism of two dopaminergic ergot derivatives was investigated in live recombinant V79 cells. Both lisuride and terguride were monodeethylated. Keywords: Heterologous expression; Cytochrome P450 3A4; V79 Chinese hamster cell; Testosterone; Ergot derivative

1. Introduction

Microsomal cytochromes P450 are important enzymes to metabolize xenobiotics and serve generally as monooxygenases. This enzyme family comprises a large number of members with different substrate specificities and catalytic properties. For drug research and development P450 enzymes are of interest for several reasons. They predominantly mediate phase I metabolic reactions of drugs in man and in animal species used for pharmacological and toxicological characterization of new chemical entities. The insight into biotransformation helps to understand the fate of a compound in the biosystem. Furthermore, it plays an important role for the extrapolation of pharmacodynamic and toxicological effects observed in animals to man. This can only be scientifically justified, if at least qualitatively similar pharmacokinetic and metabolic profiles were ob-

Corresponding author. At: Institute of Pharmacokinetics, Schering AG, D-13342 Berlin, Germany. Tel.: +49-(0)30-4682731; Fax: +49(0)30-4681527.

0926-6917/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 9 2 6 - 6 9 1 7 ( 9 5 ) 0 0 0 1 6 - X

tained. For drug therapy the assignment of metabolic pathways to P450 enzymes is a prerequisite to predict interactions of different drugs taken concomitantly. If two compounds are substrates for a special P450 enzyme, a reduced clearance and elevated systemic levels (accompanied by unwanted effects) might result from comedicative use. Several drugs are able to reversibly or irreversibly inhibit P450 enzymes and the knowledge of such properties might be helpful for therapeutic dose regimens (Murray, 1992). Several strategies are used to investigate metabolism and to attribute specific reaction pathways to P450 enzymes using either intact biosystems, isolated organs (liver), cells (hepatocytes) or sub-cellular fractions (microsomes) (Hildebrand et al., 1994). The isolation and purification of individual enzymes has been another approach. Biotechnology, however, has offered an even more promising alternative by the stable expression of these enzymes in heterologous systems, like yeast, bacteria or mammalian cells. Thus, donor dependent variability of enzyme contents, time consuming isolation procedures and limited practical usability of enzymes can be avoided. As mentioned before different expression systems were estab-

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lished for P450 enzymes, also including the mitochondrial enzymes (Husemann and Petri, 1994), and all of them have different pros and cons (Doehmer and Greim, 1993). V79 cells, fibroblasts from Chinese hamsters, are an accepted toxicological model and were considered to exhibit no endogenous P450 contents (Glatt et al., 1987; Kiefer and Wiebel, 1989), which was one of the reasons for the introduction of this system to express several P450 enzymes from rats and humans (Doehmer and Oesch, 1991). These cell lines were used for several studies on metabolic degradation and/or toxicological effects (Dogra et al., 1990; Schmalix et al., 1993; Gieschen et al., 1994). Therefore V79 cells were selected to stably express a number of other P450 enzymes with special emphasis on its use for metabolic profiling. The CYP3A-family seems to be of high importance for biodegradation of drugs both in animals and in man. CYP3A represents approx. 30% of human liver total P450 contents and is also present in the intestinal mucosa. The list of its substrates includes a wide variety of drugs, belonging to very different therapeutical classes, and also endobiotics, like steroids (Shimada et al., 1994). Due to its importance, human CYP3A4 was expressed in V79 cell lines and applied to screen substrate properties of new and established drugs. The present paper describes the functional expression of the enzyme and the screening of two dopaminergic ergot derivatives.

2.3. Construction of the CYP3A4 expression plasmid

2. Materials and methods

2.4. Cell culture

2.1. Cell lines and plasmids

The parental subline V79MZ cells was kindly supplied by J. Doehmer, Technical University, Munich, and used as recipient cell line for transfection with expression plasmids. The expression vector pMPSV-CMV-HE was received from H. Hauser, Gesellschaft fiir Biotechnologische Forschung, Braunschweig, Germany, and used for expression of CYP3A4 in V79 cells.

V79 cells were propagated in Dulbecco Vogt's modified Eagle's medium, DMEM (Seromed, Berlin, Germany), supplemented with 5% ( w / v ) fetal calf serum (Gibco, Eggenstein, Germany), 1 mM sodium pyruvate (Seromed, Berlin, Germany) and 2 mM L-glutamine (Gibco, Eggenstein, Germany) at 37°C under an atmosphere of 5% ( v / v ) CO 2 in air at 95% humidity. Hygromycin B resistant cell lines were grown in identical media containing 4 0 0 / z g / m i hygromycin B (Boehringer, Mannheim, Germany).

2.2. Cloning of CYP3A4 cDNA

2.5. Transfection of plasmids

A human liver cDNA library constructed in h g t l l (Clontech, Palo Alto, USA) was screened with a 32p_ labelled synthetic oligonucleotide corresponding to residues 168-193 of the published CYP3A4 cDNA sequence (Beaune et al., 1986; Gonzalez et al., 1988). Recombinant phages from positive plaques were purified by successive replating and rehybridization. Extracted ADNA was isolated and further characterized by restriction enzyme analysis or after prior amplification by polymerase chain reaction (PCR) with primers complementary to the lac5 gene of h g t l l flanking the EcoRI site. A positive but incomplete 1.4 kb cDNA insert was digested with KpnI and PvuI and subcloned into the pSelect-1 vector (Promega, Madison, WI, USA).

Transfection was carried out by a modified method described by Chen ancl Okayama (1988). V79 cells were seeded at a density of 1 × 10 6 cells/94-mm dish and cultured for about 20 h. Plasmids pMPSV-CMV-CYP3A4 (10 /zg) and p S K / H M R 2 7 2 (1 /xg) were diluted in 0.5 ml 50 mM N,N-bis[2-hydroxy-ethyl]-2-amino-ethanesulfonic acid (Calbiochem-Novabiochem, Bad Soden/Ts., Germany), 280 mM NaC1, 1.5 mM Na2HPO 4, pH 6.95. The DNA was precipitated by addition of 0.5 ml 250 mM CaCI: and incubated at room temperature for 20 min. The medium was removed and the mixture was added directly to the cells and left at room temperature for 30 min before medium was added. After 4 h incubation at 37°C, cells were treated with 25% ( v / v ) dimethyl sulfoxide in 30 mM

The 5' half of the coding region of CYP3A4 cDNA was amplified from the recloned insert with Taq polymerase and digested with PstI. The 5' PCR primer was designed to introduce a Kozak sequence and a Pstl site (Kozak, 1983). The resulting 1.0 kb P s t I / P s t I fragment was inserted into the pMPSV-CMV-HE vector partially cleaved with PstI (Wirth et al., 1991) yielding pMPSV-CMV3A4(5'). The missing 3' portion of CYP3A4 cDNA was amplified from reverse transcribed human liver total RNA (Clontech, Palo Alto, CA, USA) and digested with PstI and EcoRI. The resulting 0.5 kb fragment was inserted into pMPSV-CMV-HE vector cut with EcoRI and partially cleaved with PstI. Both recloned PCR fragments were completely sequenced using AmpliTaq polymerase (Applied Biosystems, Walterstadt, Germany). The I kb 5' portion of CYP3A4 cDNA was removed from pMPSV-CMV-3A4(5') with Pstl and ligated to the plasmid containing the 0.5 kb 3' fragment partially digested with PstI yielding pMPSV-CMV-CYP3A4. In the final construct the CYP3A4 gene was under the transcriptional control of the long terminal repeat promoter of the myeloproliferative sarcoma virus (MPSV-LTR) and the enhancer derived from the cytomegalovirus (CMV). The vector also contained a SV40 splice junction and a SV40 poly A tract for stabilisation of the mRNA.

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NaH2PO 4, 150 mM NaC1, pH 7.5, for 1 min and then washed twice with DMEM. Medium containing 400 ~ g / m l hygromycin B was added after 2 days of incubation at 37°C. Resistant colonies appearing after 12-14 days were picked with cotton buds and grown in mass culture for further studies. The pSK/HMR272 plasmid serving as a selection marker was constructed by insertion of a 2.8 kb BamHI/HindlII fragment containing the hygromycin B phosphotransferase gene including the thymidine kinase promoter and the thymidine kinase terminator of the herpes simplex virus from plasmid pHMR272 (Bernard et al., 1985) into the pBluescript II vector (Stratagene, La Jolla, CA, USA).

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analysis was carried out with the RNA preparation as a template to exclude DNA contamination. Transcription of CYP3A4 cDNA into mRNA was detected by reverse transcribed polymerase chain reaction (RT-PCR). The first strand cDNA synthesis was performed after priming with random hexamers. Amplification was performed as described for chromosomal DNA. Both primers corresponded to residues within the CYP3A4 cD~NA (5' primer: 554-580, 3' primer: 1141-1166) resulting in a 0.6 kb fragment after amplification. Amplification with glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers served as control for the integrity of the RNA resulting in a 0.4 bp amplified fragment corresponding to bases 324-717 of the GAPDH gene (Tokunaga et al., 1987).

2.6. Preparation of chromosomal DNA and PCR analysis 2.8. Enzyme assay and analysis of testosterone The presence of genomically integrated CYP3A4 cDNA in recombinant cells was confirmed by PCR analysis. Chromosomal DNA was isolated according to a modified method described by Sambrook et al. (.1989) by incubation of about 1 x 10 6 cells with proteinase K in 200 /zl digestion buffer (100 mM NaCI, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 0.5% ( w / v ) sodium dodecyl sulfate, 100 /zg/ml proteinase K) at 50°C for 16 h. The viscosity of the lysate was reduced by drawing five times through a hypodermic needle to shear the chromosomal DNA into smaller fragments. Samples were extracted twice with 1 vol. of phenol/chloroform/isoamyl alcohol (25:24:1, v / v / v ) . DNA was recovered by ethanol precipitation and resuspended in 10 mM Tris-HC1, pH 8.0, 1 mM EDTA, at 37°C overnight. Amplification was carried out with 35 cycles using 1 /xg chromosomal DNA as template. The 5' primer corresponded to residues 913-937 within the CYP3A4 cDNA and the 3' primer to a sequence within the proximal vector region. The amplified product had a length of 0.6 kb.

2.7. Preparation of total RNA and RT-PCR analysis Preparation of total RNA was performed according to Chomczynski and Sacchi (1987) using a total RNA isolation kit (Promega, Madison, WI, USA). A control PCR

A

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Enzymatic activity of CYP3A4 in live cells was determined by conversion of testosterone to 6fl-hydroxytestosterone. Cells were seeded at a density of 1 x 10 6 cells/94 mm petri dish in 10 ml of DMEM medium and grown for 16 h at 37°C. Medium was replaced by 10 ml of fresh medium containing 50 /xM testosterone. After further growth for 24 h, products from cell culture supernatants were pre-purified on Bond Elut C18 reverse phase cartridges (Varian, Harbor City, CA, USA) and then analysed by reverse phase HPLC (Hypersil ODS, 125 X 4.6 mm, 5 /zm, Bischoff Analysentechnik und -gedite, Leonberg, Germany) with an isocratic solvent system (methanol:water, 45:55, v / v ) at ambient temperature, applying a flow rate of 1.0 ml/min and UV detection at 254 nm. Protein concentration of cytosol free pellets from cells used in enzyme assays was determined according to Lowry et al. (1951).

2.9. Enzyme assay and analysis of ergot deriuatives The metabolism studies were performed in live cell cultures. Cells were seeded at a density of 2 X 10 6 cells/75 cm 2 flask and incubated for 24 h in 10 ml medium. After medium exchange 0.3 ~ M of 14C-labelled substrate, lisuride or terguride (Fig. 1), was added and incubated for

B

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Fig. 1. Structuresof the ergot derivativeslisuride (A) and terguride(B). The compoundswere 14C-labelledat the indicated( * ) positions.

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48 h. Supernatant was removed and 2 ml were extracted with 4 ml ice-cold acetone/methanol (3:1, v / v ) . After centrifugation, decanting and removal of the liquid phase by N 2 the residues were dissolved in the mobile phase and analyzed by HPLC. Metabolites were separated on a Spherisorb ODS II ( 1 2 5 × 4 . 6 mm, 5 /zm, M&WChromatographietechnik, Berlin, Germany) as stationary phase at ambient temperature. The mobile phase consisted of methanol, H20 and 0.067 M aqueous Na2HPO 4 (55:25:25, v / v / v ) and was used at a flow rate of 1.0 ml/min. The HPLC-chromatograms were recorded both by fluorescence (extinction 282 nm, emission 370 nm) and by on-line radioactivity detection (RAMONA LS-4, Raytest, Germany).

3. Results

3.1. Cloning of CYP3A4 cDNA from a human liver cDNA library About 9 × 105 plaques of a human liver cDNA library were screened using a synthetic oligonucleotide derived from a specific 5' region of the CYP3A4 cDNA sequence which had minimal homology to the 89% identical CYP3A5 sequence (Aoyama et al., 1989). Six positive plaques were identified. Five of the six clones contained detectable inserts of 1.0-1.8 kb calculated length. Further restriction analysis revealed that only the inserts from two clones of about 1.2 kb and 1.4 kb length corresponded to the CYP3A4 cDNA (data not shown). The inserts of the other clones did not correspond to any of the expected pattern and were not further considered. Terminal sequencing of the 1.4 kb insert demonstrated that it contained the complete 5' region of the CYP3A4 cDNA including the 5' untranslated region. Restriction analysis of the 3' terminal

MPSV-LTR SV40 splice jun

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pMPSV-CMV-CYP3A4 5075bp pBR328 ori

CYP3A4 (hPCNI)

CMVenh

SV40 polyA

Fig. 2. Structure of plasmid pMPSV-CMV-CYP3A4 for expression of human CYP3A4.

1 2 3 4 5 6 7 8

0.6 kb

Fig. 3. PCR analysis of the genomic DNA of V79h3A4 cells. Total genomic DNA was isolated from hygromycin B-resistant V79 cells and subjected to PCR using two synthetic primers one of which hybridized internal of the CYP3A4 cDNA whereas the other was complementary to the pMPSV-CMV vector proximal to the insert amplifying a 0.6 kb fragment. Lane 1, pMPSV-CMV-CYP3A4 plasmid (positive control); lane 2, V79 cells (negative control); lanes 3-8, V79h3A4 cell clones.

portion showed that about 150 bp of the coding region were missing. 3.2. Construction of recombinant cell lines expressing CYP3A 4 The strategy for construction of an expression plasmid aimed at the complementation of the missing 3' portion and at the optimization of the 5' terminal sequence for optimal expression in animal cells. The complete cDNA was obtained by assembling a 1.0 kb 5' PCR fragment amplified from a purified human liver Agtll cDNA clone and a 0.5 kb 3' RT-PCR fragment prepared from human liver total RNA. Complete sequencing revealed that no mistakes were generated during amplification and that the CYP3A4 form, termed PCN1 (Gonzalez et al., 1988), was obtained. Expression of full length cDNA was put under the control of the strong MPSV-LTR promoter and the CMV enhancer contained in the vector pMPSV-CMV-HE yielding the expression plasmid pMPSV-CMV-CYP3A4 (Fig. 2). The plasmid pMPSV-CMV-CYP3A4 was cotransfected with the pSK/HMR272 selection plasmid into V79 cells by the calcium phosphate procedure (Chen and Okayama, 1988). Hygromycin B resistant colonies appeared 12 days after transfection. 13 clones were further analyzed for cDNA integration, transcription and functional expression of CYP3A4. 3.3. Characterization of recombinant cell lines The presence of the genomically integrated CYP3A4 cDNA in hygromycin B resistant cells was confirmed by PCR. Total genomic DNA was subjected to PCR using a 5'

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R. Raus~enbach et aL / E u r J. PharmacoL Environ. ToxicoL ~ a r m a c o L S e c ~ n 2 ~ ( 1 9 ~ 1 ~ - 1 ~

1

3 2

5 4

7 6

9 8

11 13 15 10 12 14

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Fig. 4. PCR analysis of reverse transcribed total" RNA from V79h3A4 cells. Total RNA was isolated from V79h3A4 cells and reverse transcribed using random hexamer primers. The generated single strand cDNA was subjected to PCR using two CYP3A4 cDNA primers amplifying a 0.6 kb region of the CYP3A4. RT-PCR with GAPDH specific primers served as control for the integrity of the used RNA (0.4 kb). Lane 1, pMPSV-CMV-CYP3A4 plasmid DNA (positive control); lane 2, V79 cells using CYP3A4 primers (negative control); lane 3, V79 cells using GAPDH-specific primers; lanes 4, 6, 8, 10, 12, 14, V79h3A4 cell clones using CYP3A4 internal primers; lanes 5, 7, 9, 11, 13, 15, V79h3A4 cell clones using GAPDH-specific primers.

primer hybridizing internal of the CYP3A4 cDNA and a 3' primer homologous to the proximal vector sequence, respectively. The expected 0.6 kb amplification fragment was detected in genomic DNA from five recombinant V79 cell clones (Fig. 3). An amplification product with the identical length was obtained from pMPSV-CMV-CYP3A4 DNA used as a positive control. Identity of the products

A2s4 A

TES

was proved by restriction enzyme analysis (data not shown). No PCR products were obtained from chromosomal DNA of parent cells. Transcription of the CYP3A4 cDNA was demonstrated by RT-PCR analysis. First strand cDNA obtained from total RNA was subjected to PCR with two CYP3A4specific primers. Transcription was detected in all five

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Fig. 5. HPLC analysis of a lipophilic extract from the recombinant V79h3A4 cell clone (A) and parent cell line V79 (B) after incubation with 50 /zM testosterone. TES = testosterone; 6/3-OH-TES = 6fl-hydroxytestosterone; AND = androstenedione.

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B

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14

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Fig. 6. Metabolic pattern of lisuride after incubation with non-transfected V79 cells (A) and V79h3A4 cells (B). LIS = lisuride, MDL = monodeethyllisuride.

clones by occurrence of a 0.6 kb amplification product which was not detected in non-transfected cells (Fig. 4). The occurrence of a 0.4 kb fragment from control RT-PCR reactions with GAPDH-specific primers demonstrated that the cDNA was intact. PCR reactions using total RNA without prior reverse transcription as template did not result in amplification products proving the absence of contaminating DNA (data not shown). 3.4. Functional expression o f CYP3A4 in V79h3A4 cells

Functional expression of CYP3A4 in recombinant V79 cell clones was demonstrated by the appearance of a 6fl-hydroxylated product after conversion of 50 /zM testosterone, which did not occur in parental V79 cells (Fig. 5). Furthermore, to a minor extent, other unidentified degradation products were present. The specific enzyme activity determined from 24 h incubation of substrate and recombinant V79h3A4 was about 25 pmol min-1 mg-1 protein. Both CYP3A4 expressing V79 cells and nontransfected V79 cells additionally converted testosterone to androstenedione. 3.5. Metabolism o f ergot derivatiues in V79h3A4 cells

Human CYP3A4 expressing V79 cells were further investigated by determining their metabolic activity towards two ergot derivatives, lisuride and terguride, as unknown substrates. Lisuride was monodeethylated by human CYP3A4 (Fig. 6). The other peaks observed in the chromatograms were also detectable with non-transfected V79 cells. This reflected that additional, probably non-P450 metabolic activ-

ity was present. Due to its total amount and the interest in 3A4 mediated reactions, which could unequivocally be attributed to monodeethylation the other minor products were not identified. In case of terguride the same reaction was observed. The N-monodeethylated metabolites of both substrates were identified by cochromatography of the synthetic reference compound. Metabolic pattern obtained by fluorescence and radioactivity detection were similar. Two unidentified degradation products observed in V79h3A4 cells were detected with parental cells as well.

4. Discussion Cytochrome P450 3A4 is the most abundant P450 enzyme in human liver and able to metabolize a great variety of compounds including steroids, antibiotics, carcinogens and drugs (Waxman et al., 1988; Bork et al., 1989; Combalbert et al., 1989; Guengerich, 1990; Kerlan et al., 1992). Because of its broad substrate and catalytic specificity heterologous expression of this specific P450 enzyme is important for investigating the metabolism of clinically important drugs. V79 Chinese hamster cells genetically engineered for expression of distinct cytochrome P450 enzymes and their use in metabolic and mutagenicity studies were reported for rat CYP2B1 (Doehmer et al., 1988), rat CYP1A1 (Dogra et al., 1990), human CYP1A1 (Schmalix et al., 1993), rat CYP1A2 (W/51fel et al., 1991), human CYP1A2 (W/51fel et al., 1992) and human CYP2E1 (Doehmer, personal communication, 1994). Among mammalian cell lines V79 cells are particularly suitable for the stable expression of any cytochrome P450 enzyme because of the

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absence of P450-mediated metabolic activation of xenobiotics (Glatt et al., 1987; Kiefer and Wiebel, 1989). As compared to other expression systems the V79 cell line was selected for CYP3A4 expression because of its mammalian origin and its special biological features not contained in other systems. Recombinant V79 cells expressing a selected human P450 can be used to specifically study the substrate and catalytic activity of the enzyme towards potential drugs. A recombinant V79 cell line which functionally expressed the chromosomally integrated human CYP3A4 cDNA was established. The MPSV promoter in combination with the CMV enhancer was capable of sufficiently promoting the expression of CYP3A4 cDNA in this cell line as demonstrated by conversion of testosterone to 6fl-hydroxytestosterone. The conversion of testosterone to androstenedione by CYP3A4 expressing V79 cells as well as parent V79 cells indicated an endogenous reductase activity. Stable and transient expression of CYP3A4 in different cell lines had been established by many laboratories. These expression systems include COS cells (Gonzalez et al., 1988), HepG2 cells (Waxman et al., 1991), human B lymphoblastoid cell lines (Crespi et al., 1991), insect cells by baculovirus (Buters et al., 1994), V79 and CHO cells (Tynes, 1992), yeast (Brian et al., 1990; Renaud et al., 1990) and E. coli (Gillam et al., 1993). However, comparisons are difficult because different substrates had been studied and because different cell fractions and different specific values have been used for evaluating the enzymatic activity. Human CYP3A4 is involved in many N-dealkylation processes as N-demethylation of diltiazem, tamoxifen, erythromycin and N-deethylation of lidocain, amiodarone and other compounds (Coutts et al., 1994). As the investigated ergot derivatives lisuride and terguride were known to be N-dealkylated in vivo and in other in vitro systems (Toda and Oshino, 1981; Huempel et al., 1984,1989; Frrster, 1987), human CYP3A4 was screened for being responsible for this reaction. V79 cells expressing functional human CYP3A4 could be successfully applied for these studies. With CYP3A4 metabolic activity was limited to single N-dealkylation, although a second identical reaction to a dideethlylated metabolite is known to occur in vivo (Huempel et al., 1984,1989; Frrster, 1987). Another ergot derivative CQA 206-291 was also shown to be monodeethylated at the sulfonamide moiety by CYP3A4 in human microsomes and in CYP3A4 expressing COS1 cells. The consecutive dideethylated conversion product of this substrate was not formed either (Ball et al., 1992). Human CYP1A1, expressed in genetically engineered V79 cells, but not CYP1A2, was identified to be responsible for the monodeethylation of both substrates, whereas rat CYP1A1 is only capable to degrade lisuride (Gieschen et al., 1994). Evaluating these results it can be concluded that a combination of P450 enzymes seems to be responsible for this

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reaction. As all investigations performed up to now only revealed single dealkylation by cytochromes P450, it still remains to be clarified, which enzymes catalyse the in vivo relevant dideethylation. In conclusion, V79 cells expressing P450 enzymes can contribute to a profound insight and understanding of complex biodegradation processes.

Acknowledgements We thank D. Henschel, Da. Schmidt and D. Klews for their excellent technical assistance and De. Schmidt for synthesis of oligonucleotides. Furthermore, we thank Dr. J. Doehmer (Institut fiir Toxikologie und Umwelthygiene, University of Munich, Germany) for providing parental V79 cells. The work was part of a research project sponsored by the Schering AG, Berlin, Germany.

References Aoyama, T., S. Yamano, D.J. Waxman, D.P. Lapenson, U.A. Meyer, V. Fischer, R. Tyndale, T. Inaba, W. Kalow, H.V. Gelboin and F.J. Gonzalez, 1989, Cytochrome P-450 hPCN3, a novel cytochrome P-450 IliA gene product that is differentially expressed in adult human liver, J. Biol. Chem. 264, 10388. Ball, S.E., G. Maurer, M. Zollinger, M. Ladona and A.E.M. Vickers, 1992, Characterization of the cytochrome P-450 gene family responsible for the N-dealkylation of the ergot alkaloid CQA 206-291 in humans, Drug Metab. Dispos. 20, 56. Beaune, P.H., D.R. Umbenhauer, R.W. Bork, R.S. Lloyd and F.P. Guengerich, 1986, Isolation and sequence determination of a cDNA clone related to human liver cytochrome P-450 nifedipine oxidase, Proc. Natl. Acad. Sci. USA 83, 8064. Bernard, H.-U., G. Kr/immer and W.G. R6wekamp, 1985, Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture, Exp. Cell Res. 158, 237. Bork, R.W., T. Muto, P.H. Beaune, P.K. Srivastana, R.S. Lloyd and F.P. Guengerich, 1989, Characterization of mRNA species related to human liver cytochrome P-450 nifedipine oxidase and the regulation of catalytic activity, J. Biol. Chem. 264, 910. Brian, W.R., M.-A. Sari, M. Iwasaki, T. Shimada, L.S. Kaminsky and F.P. Guengerich, 1990, Catalytic activities of human liver cytochrome P-450 IIiA4 expressed in Saccharomyces cerevisiae, Biochemistry 29, 11280. Buters, J.T.M., K.R. Korzekwa, K.L. Kunze, Y. Omata, J.P. Hardwick and F.J. Gonzalez, 1994, cDNA-directed expression of human cytochrome P450 CYP3A4 using baculovirus, Drug Metab. Dispos. 22, 688. Chen, C.A. and H. Okayama, 1988, Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA, Biotechniques 6, 632. Chomczynski, P. and N. Sacchi, 1987, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162, 156. Combalbert, J., I. Fabre, G. Fabre, I. Dalet, J. Derancourt, J.P. Cano and P. Maurel, 1989, Metabolism of cyclosporin A, IV: purification and identification of the rifampicin-inducible human liver cytochrome P450 (cyclosporin A oxidase) as a product of P4501IiA subfamily, Drug Metab. Dispos. 17, 197. Crespi, C.L, F.J. Gonzalez, D.T. Steimel, T.R. Turner, H.V. Gelboin,

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R. Rauschenbach et al. /Eur. J. Pharmacol. End,iron. Toxicol. Pharmacol. Section 293 (1995) 183-190

B.W. Penman, and R. Langenbach, 1991, A metabolically competent human cell line expressing five cDNAs encoding procarcinogenactivating enzymes: application to mutagenicity testing, Chem. Res. Toxicol. 4, 566. Coutts, R.T., P. Su and G.B. Baker, 1994, Involvement of CYP2D6, CYP3A4, and other cytochrome P-450 isozymes in N-dealkylation reactions, J. Pharmacol. Toxicol. Methods 31, 177. Doehmer, J., S. Dogra, T. Friedberg, S. Monier, M. Adesnik, H. Glatt and F. Oesch, 1988, Stable expression of rat cytochrome P-4501IB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin Bj, Proc. Natl. Acad. Sci. USA 85, 5769. Doehmer, J. and H. Greim, 1993, Cytochromes P450 in genetically engineered cell cultures: The gene technological approach, in: Cytochrome P450. Handbook of Experimental Pharmacology, Vol. 105, eds. J.B. Schenkman and H. Greim (Springer-Verlag, Berlin) p. 415. Doehmer, J. and F. Oesch, 1991, V79 Chinese hamster cells genetically engineered for stable expression of cytochromes P450, in: Methods in Enzymology, eds. E.E. Johnson and M.R. Waterman (Academic Press, San Diego, CA), vol. 206, p. 117. Dogra, S., J. Doehmer, H. Glatt, H. M61ders, P. Siegert, T. Friedberg, A. Seidel and F. Oesch, 1990, Stable expression of rat cytochrome P-450IA1 cDNA in V79 Chinese hamster cells and their use in mutagenicity testing, Mol. Pharmacol. 37, 608. F6rster, K., 1987, Untersuchungen zur Biotransformation von Tergurid: Vergleich zwischen dem Modell der isoliert perfundierten Rattenleber und dem Ganztierversuch an Ratte, Hund und Affe, PhD-thesis, Ludwig-Maximilians- Universit~it, Munich. Gieschen, H., M. Hildebrand and B. Salomon, 1994, Metabolism of two dopaminergic ergot derivatives in genetically engineered V79 cells expressing CYP450 enzymes, in: Cytochrome P450 Biochemistry, Biophysics and Molecular Biology, ed. M.C. Lechner (John Libbey Eurotext, Paris) p. 467. Gillam, E.M.J., T. Baba, B.-R. Kim, S. Ohmori and F.P. Guengerich, 1993, Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme, Arch. Biochem. Biophys. 305, 123. Glatt, H., I. Gemperlein, G. Turchi, H. Heinritz, J. Doehmer and F. Oesch, 1987, Search for cell culture systems with diverse xenobioticmetabolizing activities and their use in toxicological studies, Mol. Toxicol. 1, 313. Gonzalez, F.J., B.J. Schmid, M. Umeno, O.W. McBride, J.P. Hardwick, U.A. Meyer, H.V. Gelboin and J.R. Idle, 1988, Human P450PCN1: sequence, chromosome localization, and direct evidence through cDNA expression that P450PCN1 is nifedipine oxidase, DNA 7, 79. Guengerich, F.P., 1990, Metabolism of 17ot-ethynylestradiol in humans, Life Sci. 47, 1981. Hildebrand, M., M. Huempel, H. Gieschen and C. Kraus, 1994, Xenobiotic metabolism -From intact biosystem to single enzymes, in: Assessment of the use of single cytochrome P450 enzymes in drug research, eds. M. Hildebrand and M. Waterman (Springer-Verlag, Berlin) p. 1. Huempel, M., W. Krause, G.-A. Hoyer, H. Wendt and G. Pommerenke, 1984, The pharmacokinetics and biotransformation of 14C-lisuride hydrogen maleate in rhesus monkey and man, Eur. J. Drug Metab. Pharmacokin. 9, 347. Huempel, M., D. Sostarek, H. Gieschen and C. Labitzky, 1989, Studies on the biotransformation of lonazolac, bromerguride, lisuride and terguride in laboratory animals and their hepatocytes, Xenobiotica 19, 361. Husemann, M. and T. Petri, 1994, Mammalian cell lines stably expressing bovine adrenal ll/3-hydroxylase cDNA, in: Cytochrome P450 Biochemistry, Biophysics and Molecular Biology, ed. M.C. Lechner (John Libbey Eurotext, Paris) p. 709.

Kerlan, V., Y. Dreano, J.P. Bercovici, P.H. Beaune, H.H. Floch and F. Berthou, 1992, Nature of cytochromes P450 involved in the 2-/4-hydroxylation of estradiol in human liver microsomes, Biochem. Pharmacol. 44, 1745. Kiefer, F. and F.-J. Wiebel, 1989, Chinese hamster cells express cytochrome P-450 activity after simultaneous exposure to polycyclic aromatic hydrocarbons and aminophylline, Toxicol. Lett. 48, 265. Kozak, M., 1983, Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles, Microbiol. Rev. 47, 1. Lowry, O.H., N.J. Rosebrough, L.A. Farr and R.J. Randall, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265. Murray, M., 1992, P450-Enzymes; Inhibition mechanisms, genetic regulation and effects of liver disease, Clin. Pharmacokinet. Con. 23, 132. Renaud, J.-P., C. Cullin, D. Pompon, P. Beaune and D. Mansuy, 1990, Expression of human liver cytochrome P450 ILIA4 in yeast. A functional model for the hepatic enzyme, Eur. J. Biochem. 194, 889. Sambrook, J., E.F. Fritsch and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd edn. (Cold Spring Harbor Laboratory Press, New York, New York, USA). Schmalix, W.A., H. M~iser, F. Kiefer, R. Reen, F.J. Wiebel, F. Gonzalez, A. Seidel, H. Glatt, H. Greim and J. Doehmer, 1993, Stable expression of human cytochrome P450 1AI cDNA in V79 Chinese, hamster cells and metabolic activation of benzo[a]pyrene, Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. 248, 251. Shimada, T., H. Yamazaki, M. Mimura, Y. Inui and F.P. Guengerich, 1994, Interindividual variations in human liver cytochrome P450 enzymes involved in the oxidation of drags, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians, J. Pharmacol. Exp. Ther. 270, 414. Toda, T. and N. Oshino, 1981, Biotransformation of lisuride in the hemoglobin-free perfused rat liver and in the whole animal, Drug Metab. Dispos. 9, 108. Tokunaga, K., Y. Nakamura, K. Sakata, K. Fujimori, M. Ohkubo, K. Sawada and S. Sakiyama, 1987, Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancer, Cancer Res. 47, 5616. Tynes, R., 1992, Mammalian cell lines recombinant for phase 1 oxidations which are useful for investigative in vitro human drug metabolism, in: Proceedings of the 9th International Symposium on Microsomes and Drug Oxidations, Jerusalem, p. 175. Waxman, D.J., C. Attisano, F.P. Guengerich and D.P. Lapenson, 1988, Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6/3-hydroxylase cytochrome P-450 enzyme, Arch. Biochem. Biophys. 263, 424. Waxman, D.J., D.P. Lapenson, T. Aoyama, H.V. Gelboin, F.J. Gonzalez and K. Korzekawa, 1991, Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s, Arch. Biochem. Biophys. 290, 160. Wirth, M., L. Schumacher and H. Hauser, 1991, Construction of new expression vectors for mammalian cells using the immediate early enhancer of the human cytomegalovirus to increase expression from heterologous enhancer/promotors, in: Protein Glycosylation, Vol. 15, Cellular, Biotechnical and Analytical Aspects, ed. H.S. Conradt (VCH Publishers, Weinheim, Germany) p. 49. W61fel, C., K.L. Platt, S. Dogra, H. Glatt, F. W~ichter and J. Doehmer, 1991, Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17/3-estradiol and 2-aminofluorene in V79 Chinese hamster cells, Mol. Carcinogen 4, 489. W61fel, C., B. Heinrich-Hirsch, T. Schulz-Schalge, A. Seidel, H. Frank, U. Ramp, F. W~ichter, F.J. Wiebel, F. Gonzalez, H. Greim and J. Doehmer, 1992, Genetically engineered V79 Chinese hamster cells for stable expression of human cytochrome P450IA2, Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. 228, 95.