Both Cytochromes P450 2E1 and 3A Are Involved in the O

Equipe de recherche EA-948, Faculté de Médecine, UBO, 22 Avenue ... Juan Carlos Ledesma , Marta Miquel , María Pascual , Consuelo Guerri , Carlos M...
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Chem. Res. Toxicol. 1997, 10, 1205-1212

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Both Cytochromes P450 2E1 and 3A Are Involved in the O-Hydroxylation of p-Nitrophenol, a Catalytic Activity Known To Be Specific for P450 2E1 Alain Zerilli, Damrong Ratanasavanh, Danie`le Lucas, Thierry Goasduff, Yvonne Dre´ano, Christophe Menard, Daniel Picart, and Franc¸ ois Berthou* Equipe de recherche EA-948, Faculte´ de Me´ decine, UBO, 22 Avenue Camille Desmoulins, B.P. 815-29285-BREST Ce´ dex, France Received April 1, 1997X

4-Nitrophenol 2-hydroxylation activity was previously shown to be mainly catalyzed by P450 2E1 in animal species and humans. As this chemical compound is widely used as an in vitro probe for P450 2E1, this study was carried out to test its catalytic specificity. First, experiments were carried out on liver microsomes and hepatocyte cultures of rat treated with different inducers. Liver microsomes from pyrazole- and dexamethasone-treated rats hydroxylated p-nitrophenol with a metabolic rate increased by 2.5- and 2.7-fold vs control. Dexamethasone treatment increased the hepatic content of P450 3A but not that of P450 2E1. Two specific inhibitors of P450 3A catalytic activities, namely, ketoconazole and troleandomycin (TAO), inhibited up to 50% of 4-nitrophenol hydroxylation in dexamethasone-treated rats but not in controls. Hepatocyte cultures from dexamethasone-treated rats transformed p-nitrophenol into 4-nitrocatechol 7.8 times more than controls. This catalytic activity was inhibited by TAO. Similarly, hepatocyte cultures from pyrazole-treated rats hydroxylated p-nitrophenol with a metabolic ratio increased by about 8-fold vs control. This reaction was inhibited by diethyl dithiocarbamate and dimethyl sulfoxide, both inhibitors of P450 2E1. Second, the capability of human P450s other than P450 2E1 to catalyze the formation of 4-nitrocatechol was examined in a panel of 13 human liver microsomes. Diethyl dithiocarbamate and ketoconazole reduced 4-nitrophenol hydroxylase activity by 77% ((11) and 13% ((16), respectively. Furthermore, the residual activity following diethyl dithiocarbamate inhibition was significantly correlated with seven P450 3A4 catalytic activities. Finally, the use of human cell lines genetically engineered for expression of human P450s demonstrated that P450 2E1 and 3A4 hydroxylated 4-nitrophenol with turnovers of 19.5 and 1.65 min-1, respectively. In conclusion, P450 3A may make a significant contribution to 4-nitrophenol hydroxylase activity in man and rat.

Introduction Cytochromes P4501 comprise a superfamily of hemethiolate isozymes of major importance in the oxidation of drugs, environmental pollutants, dietary chemicals, carcinogens, and endogenous compounds. The various P450 isozymes exhibit distinct but overlapping patterns of substrate specificities. Such specific catalytic activities allow to characterize P450 isoforms in different media including microsomal preparations and cell cultures or even in vivo if the nontoxic substrate can be considered as a specific probe. In recent years, particular interest has focused on the ethanol-inducible P450 2E1. Indeed, this P450 isoform is of clinical relevance because it is involved in the oxidative metabolism of xenobiotics such as ethanol, aliphatic alcohols, low molecular weight toxins and carcinogens (1, 2), and endobiotics such as ketone bodies. Furthermore, it can also produce reactive oxygen species from molecular oxygen (3). Given the important role of * To whom correspondence should be addressed. Fax: (33) 2 98 01 66 03. E-mail: [email protected] or http://www.univbrest.fr/Recherche/Laboratoire/EA948. X Abstract published in Advance ACS Abstracts, September 15, 1997. 1 Abbreviations: DEDTC, diethyl dithiocarbamate; DTT, dithiothreitol; P450, cytochrome P450 (heme-thiolate protein, by the enzyme Commission, EC 1.14.14.1.); b5, cytochrome b5 (EC 4.4.2 group); DEX, dexamethasone; CHZ, chlorzoxazone; NIF, nifedipine oxidation; 4-NP, 4-nitrophenol; TAO, troleandomycin; SDS, sodium dodecyl sulfate.

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P450 2E1 in xenobiotic metabolism and toxicity, there has been considerable interest in the research of model substrates. The availability of such probes is essential for the further investigation of P450 2E1 regulation. Among the compounds specifically metabolized by P450 2E1, two substrates have attracted attention as specific probes: chlorzoxazone (4) and 4-nitrophenol (4-NP) (5). The use of chlorzoxazone as a specific probe for P450 2E1 has been widely debated. Although rat P450 3A1/3A2 (6) and human P450 1A1 (7), 1A2 (8), and 3A4 (9) were demonstrated to be able to hydroxylate chlorzoxazone, it can be considered, however, that chlorzoxazone 6-hydroxylation can be used in vivo and in vitro as a tool to screen P450 2E1 activity in humans (10, 11). Concerning 4-nitrophenol, its 2-hydroxylation to form 4-nitrocatechol is known to be highly inducible by ethanol in laboratory animals such as rat (12) and rabbit (5). The predominant contribution of P450 2E1 to 4-NP hydroxylation is now generally accepted in mammalians including man (13). However, as suggested by different studies (13, 14), the role of other P450 isoforms to 4-NP hydroxylation cannot be totally precluded. In the present study, the specificity of p-nitrophenol as a substrate of P450 2E1 was reevaluated. This study was carried out using genetically expressed human P450, chemical inhibitions and immunoinhibitions in a panel of human liver microsomes, in vivo treatment of rats with pyrazole (inducer of P450 2E1) and dexamethasone © 1997 American Chemical Society

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(inducer of P450 3A), and induction or inhibition of rat hepatocyte cultures. These experiments suggest that 4-nitrophenol is not a specific substrate of P450 2E1 and, more importantly, can be metabolized by P450 3A isoforms in rat and man.

Materials and Methods Chemicals. All reagents were of analytical grade. Chlorzoxazone (5-chloro-2(3H)-benzoxazolone), p-nitrophenol, 4-nitrocatechol, troleandomycin (TAO), ketoconazole, diethyl dithiocarbamate (DEDTC), pyrazole, and dexamethasone (DEX) were from Sigma-Aldrich (L’Isle d’Abeau, France). The 6-hydroxylated metabolite of chlorzoxazone was synthesized according to Peter et al. (4). Rat Treatment and Preparation of Microsomes. Male Wistar rats (180-200 g; from IFA-Credo, L’Arbresle, France) were maintained on a 12-h light/dark cycle in a temperaturecontrolled environment. They had free access to water and standard food (M-25 Biscuit Extralabo, from Pie`trement, Provins, France). Pyrazole was injected intraperitoneally at 80 mg/kg/day for 4 days. Dexamethasone was injected ip in corn oil at 50 mg/ kg/day for 4 days. Animals were starved for 12 h before sacrifice. Control rats received vehicle. Liver microsomes were prepared as described previously (15) and stored at -80 °C until use. Rat Hepatocyte Cultures. Hepatocytes were isolated from male Wistar rats treated by pyrazole or dexamethasone according to the protocol described above. The whole liver was perfused by collagenase, and cells were seeded at a density of 2.5 × 106/28 cm2 Petri dish in 4 mL of nutrient medium (16). The culture medium consisted of a mixture (3/1, v/v) of minimum essential medium and medium 199, added with 10 µg/mL bovine insulin, 0.2% bovine serum albumin, and 10% fetal calf serum. After 4-h attachment, a culture medium containing 50, 100, or 200 µM 4-nitrophenol and, for inhibition experiments, 25 µM TAO or 30 or 50 µM DEDTC or dimethyl sulfoxide was used. After 4 or 20 h, the medium was removed and hydrolyzed overnight at 37 °C, pH 5.2, in 10% acetate buffer (2 M, v/v) with 1% (v/v) crude extract of Helix pomatia (Sigma, France). Hydrolysis was stopped by addition of 25% (v/v) perchloric acid (0.6 N) and 0.5 g of ammonium sulfate. The medium culture was extracted twice with 4 volumes of diethyl ether. The organic phases were evaporated under a nitrogen stream at 40 °C, redissolved in the HPLC mobile phase, and analyzed by HPLC electrochemical detection. Human Liver Microsomes. Human liver microsomes were from a microsome bank set up in the laboratory from many years (15, 17). The specific forms of P450 involved in different monooxygenation reactions have been previously characterized, especially P450 3A activities (17). These activities included nifedipine oxidation, tamoxifen and toremifene N-demethylations, testosterone 6β-hydroxylation, methadone (18) and buprenorphine (19) N-dealkylations, and erythromycin N-demethylation, all known to be specific for human P450 3A4. Monooxygenase Activities. Catalytic activities known to be specific to P450 3A isoforms (nifedipine oxidation) and P450 2E1 (chlorzoxazone 6-hydroxylation and 4-nitrophenol hydroxylation) were measured according to procedures previously described for microsomal preparations. The 6-hydroxylation of chlorzoxazone activity was measured in microsomal preparations according to a method previously described (11). The 4-NP hydroxylase activity was determined using HPLC, according to a previously described method (20). Absorbance was measured at 250 nm. Calibration curves were performed by injecting quantities of 4-NP ranging from 25 to 400 ng added to microsomal reaction medium not supplemented with NADPH and 4-NP. As a highly sensitive and specific assay for the determination of 4-nitrocatechol formed during the incubation of 4-NP with hepatocyte cultures was required, electrochemical detection was used. A Bas LC-4A electrochemical detection system (Bioanalytical Systems, West Lafayette, IN) with a cell equipped with

Zerilli et al. a glassy carbon working electrode was set at +0.7 V (21) against a Ag/AgCl reference electrode and a detection range of 10 nA. HPLC analysis was performed on a Beckman Ultrasphere ODS column (25 × 0.46 cm) with a mobile phase consisting of acetonitrile/water containing 0.5% (v/v) H3PO4 (25/75, v/v). Calibration curves were constructed in the range 2.5-15 ng of 4-nitrocatechol. Nifedipine oxidation was assayed according to Guengerich et al. (22) with 200 µM nifedipine. Kinetic studies of 4-NP hydroxylation were performed by incubation of 0.2 mg of liver microsomal proteins from DEXtreated rats in 50 mM potassium phosphate buffer (pH 6.8), 5 mM MgCl2, 1 mM ascorbic acid with variable concentrations of 4-NP ranging from 5 to 300 µM. Values of v vs S at various substrate concentrations were fit using nonlinear least-squares regression. Chemical Inhibitions of 4-NP Activity. 1. Inhibition of P450 3A. For the inhibition study of 4-NP metabolism, two types of inhibitors, both specific to P450 3A isoforms, were used: reversible inhibitors such as ketoconazole (23, 24) and mechanism-based inhibitors such as TAO (23). As this latter compound, a suicide inhibitor, required NADPH-dependent complexation for inactivation, 50 µM TAO was preincubated in the presence of 1 mM NADPH and 0.2 mg of microsomal protein at 37 °C for 20 min in a volume of 0.1 mL. Then the incubation medium was diluted 5-fold in 0.1 M potassium phosphate buffer (pH 6.8) containing 0.2 mM 4-NP and 1 mM NADPH. After 20-min incubation at 37 °C, 4-nitrocatechol was analyzed by HPLC as described above. The two inhibitors (ketoconazole was added at 2 µM) were added in methanol solution. Control reactions were performed by adding to the reaction medium the same amount of methanol, not exceeding 2% (v/v), that was needed for the addition of inhibitor. 2. Inhibition of P450 2E1. The effect of diethyl dithiocarbamate on 4-nitrocatechol formation was determined at a 4-NP concentration of 0.2 mM in a panel of 13 human liver microsomes. DEDTC, known to be a specific inhibitor of P450 2E1 catalytic activities (2, 9), was preincubated at 0.3 mM with microsomal proteins and NADPH for 10 min prior to the addition of 0.2 mM 4-NP. Metabolic rates were compared with corresponding controls. Metabolism of 4-Nitrophenol by Heterologously Expressed P450s. Human P450 2E1 (M106k), 3A4 (M107r), and 2A6 (M104r) were purchased from Gentest (Gentest Corp., Woburn, MA). Human B-lymphoblastoid cell lines transfected with these human P450 cDNAs plus NADPH-P450 reductase expressed highly specific catalytic P450 reductase activities and contained cytochrome b5 (50 pmol/mg of protein). Human P450 3A4 (P207) and 3A5 (P235) expressed in baculovirus insect cells were also used. These preparations did not contain cytochrome b5. Another human P450 3A4 (P202), coexpressed in baculovirus insect cell with cytochrome b5 (500 pmol/mg of protein), was tested for the 4-nitrophenol hydroxylation reaction. Purified rabbit cytochrome b5 (Oxford Biomedical Research, MI) was added to the incubation mixture in the molar ratio 2/1 for b5/ P450. Microsomal proteins, 0.2 mg, of these transfected cells were incubated with 0.2 mM 4-NP and 1 mM NADPH for 20 min at 37 °C. The hydroxylated metabolite was analyzed as described above. Immunoinhibitions of 4-NP Activity. For immunoinhibition studies, polyclonal rabbit anti-rat P450 3A2 serum (Gentest, MA) and polyclonal rabbit anti-rat P450 2E1 (gift from Dr. Song, Bethesda, MD) were incubated with 0.1 mg of microsomal proteins for 30 min at room temperature prior to NADPH and 4-NP additions. Antibody P450 3A2 and P450 2E1 were incubated at concentrations of 10 µL of immune serum/40 pmol of P450 (i.e., 2.5 mg of IgG/nmol of P450) and 7.5 mg of Ig/nmol of P450, respectively. Normal rabbit nonimmune serum was incubated in the same conditions as control. These antibodies did not cross-react with other P450 families other than P450 2E1 (25) and the P450 3A family (according to Gentest specifications). Immunoquantification of P450 2E1 and 3A. P450 3A isoforms were immunoquantified by means of a polyclonal anti-

p-Nitrophenol: Substrate for P450 2E1 and 3A Enzymes rat P450 3A2 (isolated from phenobarbital-treated rat; supplied by Gentest) antiserum from goat as previously described (17). This antibody did not distinguish between the members of the P450 3A family. So, the blot quantified by means of imageprocessing scan analysis (Vilber-Lourmat, France) indicated the amount of total P450 3A in microsomal preparations. P450 2E1 was immunoquantified using polyclonal anti-rat P450 2E1 antibody (Amersham, U.K.) by means of chemiluminescence detection (ECL Western blot kit, Amersham). Northern Blot Analysis. Total RNA was extracted from 500 mg of rat liver by the acid guanidium thiocyanate phenol chloroform method (26). The final RNA pellet was washed with 80% ethanol, vacuum-dried, and dissolved in 400 µL of diethyl pyrocarbonate-treated water. The concentration of RNA was estimated by absorbance at 260 nm, and the purity was determined by the 260/280 nm ratio, which ranged from 1.6 to 1.8. Digoxigenin-Labeled DNA Probe Preparation. The DNA probes were obtained from RT-PCR products of hepatic RNA isolated from rat treated by dexamethasone (3A1) or control rat (2E1). Briefly 1 µg of total RNA, after denaturation at 75 °C for 5 min, was reverse transcripted at 42 °C for 50 min using 200 ng of random hexamers and 200 units of reverse transcriptase (Superscript II, Gibco Brl), 50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 1 mM dNTP, 50 mM DTT in a total volume of 10 µL. Amplification mixture consisted of 5 µL of cDNA template, 1 µM each of primers [3A1, ATC CGA TAT GGA GAT CAC sense primer and GAA GAA GTC CTT GTC TGC antisense primer (27); 2E1, GAG GCG CAA TTC CTG GTG GAG GAGC sense primer and GTC ATA GTT TAA GGG ATA ACA antisense primer (28)], 0.2 mM dNTP, 0.5 unit of Taq polymerase (Perkin Elmer, CT), 5 µL of 10× buffer [100 mM Tris HCl (pH 8.3), 500 mM KCl], and 1.5 mM MgCl2 in a total volume of 50 µL. PCR was performed in a personal thermocycler (Biometra, Kontron, France) programmed for an initial denaturation of 3 min at 95 °C followed by 30 cycles of amplification. For P450 3A1 amplification, each cycle was composed of 30 s at 95 °C, 1 min at 55 °C, and 1 min at 72 °C; for P450 2E1 amplification, each cycle was composed of 30 s at 95 °C, 1 min at 50 °C, and 2 min at 72 °C. A final extension was conducted at 72 °C for 5 min. The total PCR reaction was subjected to electrophoresis on a 1.5% agarose gel, and the PCR products (579 bp for 3A1 and 1086 bp for 2E1), visualized by ethidium bromide, were extracted from the gel using a centrifugation filter (Spin X, Costar, MA) and ethanol precipitated. The pellet was washed in 70% ethanol, dried, and redissolved in H2O. An aliquot of the purified PCR products was used to synthesize the digoxigenin-labeled cDNA probe by PCR under the same conditions as for the first PCR but using a mixture containing 0.2 mM each of dNTP (dATP, dCTP, and dGTP), 0.19 mM dTTP, 0.01 mM digoxigenin-11-dUTP (Boehringer, Mannheim, Germany) in a total volume of 100 µL. Nothern Hybridization. An aliquot of total RNA samples (10-20 µg) was subjected to denaturing electrophoresis on agarose-formaldehyde gels and transferred to nitrocellulose filters. The filters were prehybridized in a solution of 2% blocking reagent (Boehringer), 5 × SSC, 50% formamide, 50 mM sodium phosphate (pH 7.0), 7% SDS, 0.1% laurylsarcosine at 55 °C for 2 h. The prehybridization solution was replaced with hybridization solution containing the heat-denatured digoxigenin-labeled probe, and hybridization was conducted at the same temperature for 18-20 h. The filters were washed twice with 2 × SSC, 0.1% SDS for 5 min at room temperature and twice with 0.1 × SSC, 0.1% SDS for 15 min at 68 °C. The probe detection was performed by using the anti-digoxigenin alkaline phosphatase antibody and a chemiluminescent substrate (CSPD) as described by the manufacturer (Boehringer).

Results Kinetic Studies. Rates of 4-NP hydroxylation by liver microsomes of DEX-treated rat were fit to both Michaelis-Menten (v vs S) and Eadie-Hofstee plots (v vs v/S),

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Figure 1. Steady-state kinetics of 4-NP hydroxylation by liver microsomes from DEX-treated rats: plots of (A) v (expressed in nmol of 4-nitrocatechol formed per min per mg of microsomal protein) vs S (substrate concentration) and (B) v vs v/S. (C) The same results were fitted to a plot according to the Hill equation: [log (Vm - v)/v] vs log [S]. S50 and Vm were estimated as 55 µM and 5.1 nmol/(min‚mg of protein).

using nonlinear regression (Figure 1). Curves showed significant departures from linearity in the EadieHofstee representation, which cannot be explained by the contribution of two distinct enzymes. The sigmoidal nature of the v vs S plot was fit using the equation:

v)

VmSn (Sn50 + Sn)

where S50 is the substrate concentration showing halfmaximal velocity, namely, 55 µM, n is a measure of cooperativity, and Vm is the maximal velocity of 5.1 nmol/ (min‚mg). The n value obtained from the plot of [log(Vm - v)/v] vs log S (Hill plot) through linear regression was 1.80. So, the 4-NP hydroxylation by DEX-treated liver microsomes showed substrate cooperativity, as judged by the Hill plot. In Vivo Treatment of Rats. Rats were treated with two families of inducers: pyrazole known to induce P450 2E1 and dexamethasone known to induce P450 3A isoforms. Figure 2 shows that, as expected, pyrazole treatment increased both 4-NP hydroxylation activity and P450 2E1 content in liver by about 2.5-fold vs control. Oppositely, the findings of DEX treatment were unexpected. P450 2E1 content was not modified, while the 4-NP hydroxylation activity increased by about 2.7-fold vs control. Pyrazole and dexamethasone were assessed to induce P450 2E1 and 3A activities, respectively. Chlorzoxazone 6-hydroxylation increased by 2.1-fold vs control by pyrazole treatment but was not significantly modified by dexamethasone treatment. PCN treatment was shown to increase 4-NP hydroxylation by 1.3-fold vs control (data not shown). As DEX treatment did not increase P450 2E1 expression, assessed by Western and Northern blots (Figure 2), this step of the work suggests that P450 3A isoforms could be involved in the 4-NP hydroxylation. As expected, dexamethasone treatment increased nifedipine oxidation by 4.5-fold vs control and liver P450 3A

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Figure 4. Effect of two chemical inhibitors (TAO, open bar, and ketoconazole, hatched bar), on 4-nitrophenol hydroxylation activity of liver microsomes from untreated rat (UT) and dexamethasone (DEX)-treated rats. Data are from three untreated rats and four DEX-treated animals. Figure 2. Effect of pyrazole and dexamethasone treatment on 4-nitrophenol hydroxylation (open bar), chlorzoxazone 6-hydroxylation (solid bar), and P450 2E1 content (hatched bar) in rat liver microsomes (A) and P450 2E1 mRNA levels (B). Results are expressed as means ( standard deviation of four rats.

Figure 5. Metabolic rates of 4-nitrophenol hydroxylation by rat hepatocyte cultures. 50, 100, or 200 µM 4-NP was incubated for 4 h with 2.5 × 106 hepatocytes of untreated (UT) or dexamethasone (DEX)-pretreated rat or DEX-pretreated rat in the presence of 25 µM TAO. Results are from three plates of two rats. Figure 3. Effect of pyrazole and dexamethasone treatment on nifedipine oxidation NIF (open bar), P450 3A content (hatched bar), and P450 3A mRNA (solid bar) levels in rat liver microsomes. Data are expressed as relative units vs untreated rats; data from three animals.

content and P450 3A1 mRNA by 2.3-fold (Figure 3). Pyrazole treatment decreased the catalytic activity specific for P450 3A. The assertion that P450 3A isoforms could be involved in the 4-NP hydroxylation was confirmed by inhibition experiments. Two chemical inhibitors were incubated at concentrations close to their “windows of selectivity”, i.e., 2 µM for ketoconazole and 50 µM for TAO. The preincubation of TAO with NADPH inhibited 46.2 ( 6.5% (n ) 4 rats) of 4-NP hydroxylation activity in DEX-treated rats but only 8.8 ( 3% (n ) 3 rats) in untreated rats (Figure 4). The same inhibition profile was observed with incubation of 2 µM ketoconazole. In order to confirm these results, complementary experiments were performed by using rat hepatocyte cultures. Rat Hepatocyte Cultures. The 4-nitrocatechol metabolite was glucuronoconjugated by hepatocyte cultures. So, analysis needed a previous enzymatic hydrolysis (data not shown). In order to increase the specificity of detection, it was measured by an electrochemical detector with an oxidation potential of 0.7 V. Rat hepatocyte cultures from DEX-treated rat hydroxylated 4-NP with a metabolic rate increased by 7.8 ( 1.2-fold vs untreated rat (Figure 5), whatever the substrate concentration. This catalytic activity was inhibited by about 70.9 ( 7.1% vs control by TAO (25

Table 1. Metabolic Rates of 4-Nitrophenol Hydroxylation by Rat Hepatocyte Culturesa 4-nitrophenol concentration (µM) rat treatment

100

200

untreated pyrazole-treated: without inhibitor +DEDTC (30 µM) +DEDTC (50 µM) +DMSO (30 mM) +DMSO (70 mM)

0.11 ( 0.01 0.60 ( 0.03

0.15 ( 0.01 1.23 ( 0.08

0.15 ( 0.02 0.12 ( 0.01 0.23 ( 0.01 0.16 ( 0.01

0.29 ( 0.03 0.21 ( 0.01 0.45 ( 0.09 0.39 ( 0.05

a Substrate, at the concentration of 100 or 200 µM, was incubated for 4 h with 2.5 × 106 hepatocytes of untreated and pyrazole-treated rat. For the inhibition experiments, diethyl dithiocarbamate (DEDTC) or dimethyl sulfoxide (DMSO) was added to the culture medium. Results from four plates from two rats (means ( SD) are expressed as nmol/(mL‚4 h) of 4-nitrocatechol formed.

µM). These results confirm that 4-NP hydroxylation was catalyzed by P450 3A isoforms inducible by dexamethasone. Rat hepatocyte cultures from pyrazole-treated rat hydroxylated 4-NP with a metabolic rate increased by 5.3- and 8.1-fold vs untreated rat with 0.1 and 0.2 mM substrate concentration (Table 1). This catalytic activity was inhibited by 30 and 50 µM DEDTC or dimethyl sulfoxide so efficiently that it decreased to the level of control hepatocytes. Metabolism of 4-NP by Human Liver Microsomes. The formation of 4-nitrocatechol exhibited simple Michaelis-Menten kinetics in human liver microsomes when initial rate conditions were ensured (20-min reaction, 0.2 mg of protein and 0.2 mM 4-NP). The apparent Km was about 40 µM (data not shown) and was in agreement with

p-Nitrophenol: Substrate for P450 2E1 and 3A Enzymes

Figure 6. Inhibitory effect of (A) chemical compounds (diethyl dithiocarbamate or DEDTC, gray bar, and ketoconazole, black bar) and (B) anti-rat P450 2E1 and 3A2 antibodies on 4-nitrophenol 2-hydroxylation activity. Experiments were carried out on a panel of 13 human liver microsomes (A) and one sample Br016 (B). 4-NP was incubated at 0.2 mM concentration with 2 µM ketoconazole or 0.3 mM DEDTC preincubated 10 min in the presence of 1 mM NADPH. Polyclonal anti-rat P450 2E1 and 3A2 were preincubated with microsomal proteins at 7.5 mg of Ig/nmol of P450 or 10 µL of immune serum/40 pmol of P450 (i.e., 2.5 mg of IgG/nmol of P450). Each value represents the average of duplicate experiments.

previous findings (13). As expected, the rate of formation of 4-nitrocatechol [2.38 ( 1.55 nmol/(min‚mg), n ) 15] was correlated significantly with liver immunoquantified 2E1 levels (r ) 0.74, n ) 15), chlorzoxazone 6-hydroxylation activity (r ) 0.90, n ) 15), and N-nitrosodimethylamine N-demethylase activity (r ) 0.88, n ) 10). P450 2E1 inhibition by preincubation of 13 liver microsomes with 300 µM DEDTC was 77.3 ( 11% (Figure 6A). These data indicate, as expected, that P450 2E1 participates in 4-NP hydroxylation, but the lack of complete inhibition of this reaction suggests that additional P450s may also be involved. The latter is supported by finding that ketoconazole, a specific inhibitor of P450 3A, inhibited 12.6 ( 15.9% of control activity (Figure 6A). This means that DEDTC plus ketoconazole inhibited 85.8% ( 10.9% of total 4-NP activity in 13 human liver microsomes. The combination of chemical inhibitors and specific polyclonal antibodies (Figure 6B) confirms that P450 2E1 is the major enzyme involved in the 2-hydroxylation of p-nitrophenol but that P450 3A4 is also involved. Ketoconazole and polyclonal anti-rat P450 3A4 inhibited 23% and 10% of total activity of the Br016 sample, respectively. The combination of DEDTC and anti-P450 3A4 was able to inhibit 89.5% of total activity. Ketoconazole combined with anti-P450 2E1 inhibited 77.5% of total activity, while they inhibited alone 23.7% and 68% of total activity (Figure 6B) of the Br016 sample. The combination of two antibodies (anti-P450 2E1 and antiP450 3A), added at 7.5 mg of Ig/nmol of P450 and 2.5

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Figure 7. Correlation between the rate of p-nitrophenol 2-hydroxylation at 0.2 mM (following preincubation with DEDTC, 0.3 mM) and nifedipine oxidation (A) and tamoxifen N-demethylation (B) activities in a panel of 13 human liver microsomes. Coefficient correlation, r, was calculated by the leastsquares regression method.

mg of IgG/nmol pf P450, respectively, inhibited 92% of total activity. Futhermore, the 4-NP residual activity after DEDTC inhibition was significantly correlated with seven catalytic activities supported by P450 3A, namely, nifedipine oxidation, tamoxifen N-demethylation (Figure 7), testosterone 6β-hydroxylation (r ) 0.87), methadone N-demethylation (r ) 0.74), buprenorphine N-dealkylation (r ) 0.74), toremifene N-demethylation (r ) 0.81), erythromycin N-demethylation (r ) 0.87), and P450 3A immunoquantified (r ) 0.70). The regression lines of Figure 7 failed to intercept the y axis representing the residual 4-nitrophenol hydroxylation activity, suggesting that P450s other than P450 2E1 and 3A could be implicated in the metabolism of 4-NP. This assertion was in agreement with Figure 6B which showed that the combination of P450 2E1 and 3A chemical inhibitors or two polyclonal antibodies did not inhibit completely the 4-NP activity. Metabolism of 4-NP by Individual P450s. Incubations of 4-NP with microsomal preparations of human β-lymphoblastoid cells genetically engineered for stable expression of human P450 2E1, 3A4, 3A5, and 2A6 demonstrated that 4-nitrophenol was hydroxylated by many P450 isoforms, namely, 2E1 and 3A4 but also 2A6 (Table 2). P450 2E1 catalytic activity was very sensitive to the presence of cytochrome b5, that, added in the ratio 2/1 for b5/P450, increased 2-fold the 4-nitrophenol 2-hydroxylase activity. Conversely, the effect of cytochrome b5 on the ability of P450 3A4 to metabolize p-nitrophenol was somewhat complex. Coexpression of cytochrome b5 with P450 3A4 provided a superior turnover number when compared to reconstitued systems where addition

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Table 2. p-Nitrophenol 2-Hydroxylation Activity of Human Heterologously Expressed P450s

P450

specific catalytic activity (min-1)c

4-NP activity (min-1)

cytochrome b5 content (pmol/mg)

2E1 + OR 2E1 + OR + b5a 3A5 + OR 3A4 + OR 3A4 + OR + b5a 3A4 + OR + b5b 2A6 + OR

9.3* nd 3.6** 4.4** nd 173** 20.8***

9.2 19.5 0.1 1.45 1.65 5.8 2.7

50 360 50 50 160 500 50

a Rabbit cytochrome b added (ratio P450/b ) 0.5). b Cyto5 5 chrome coexpressed with P450 in insect cells (Gentest). c According to the supplier. *4-NP hydroxylase, **testosterone 6β-hydroxylase, ***coumarin 7-hydroxylase. Activities are expressed as turnover number [min-1 or pmol/(min‚pmol of P450)]. OR: NADPH-P450 oxidoreductase coexpressed. Each value represents the average of duplicates. Catalytic activities were not detectable in control microsomal preparations of cells not genetically modified.

of purified cytochrome b5 had no significant effect on catalytic activity.

Discussion The current view is that the chemistry used by the different P450 isoforms is relatively uniform and that catalytic selectivity is predominantly a function of how this protein binds a potential substrate and positions atoms toward the reactive center provided by the heme prosthetic group. P450 isoforms are generally characterized by a specific substrate that they metabolize regioand stereospecifically. But this specificity is generally rather broad. Although 4-NP has been suggested to be an in vitro probe to test P450 2E1 activity in mammalians (5, 13), its specificity has not been thoroughly evaluated. The present study by enzyme induction and chemical inhibition techniques showed the involvement of two P450 families, namely, P450 2E1 and 3A isoforms, in the hydroxylation of p-nitrophenol. At a substate concentration of 200 µM, P450 3A isoforms were found to be minor contributors in control rat liver microsomes. In contrast, as in DEX-treated rats the 4-nitrophenol hydroxylase activity increased 2.7-fold vs control rats, P450 3A isoforms were clearly involved in this reaction. The same pattern of induction was observed with rat hepatocyte cultures. The increase of this hydroxylation activity did not involve P450 2E1 because the hepatic content of this P450 isoform was not modified in rat liver following DEX treatment. Futhermore, the part of 4-NP hydroxylation supported by P450 3A in DEX rat liver and hepatocytes was inhibited by TAO and ketoconazole, specific inhibitors (24, 29, 30). Oppositely, this reaction was not significantly inhibited by the same compounds in untreated rat liver microsomes. Two members of the steroid-inducible P450 subfamily, exhibiting 89% amino acid sequence homology, have been described (31): 3A1 and 3A2. P450 3A12 was not detectable in hepatic microsomes from untreated rats. Following dexamethasone treatment, the level of this P450 isoform reached about 30% of the total microsomal content. P450 3A2, a constitutive isoform in untreated rats, was clearly less inducible than P450 3A1 by dexa2 P450 3A1 is an allelic variant of P450 3A23; see: Mahnke, A., Strotkamp, D., Roos, P. H., Hanstein, W. G., Chabot, G. G., and Ne´f, P. (1997) Expression and inducibility of cytochrome P450 3A9 (CYP3A9) and other members of the CYP3A subfamily in rat liver. Arch. Biochem. Biophys. 337, 62-68.

methasone (31, 32). Phenobarbital treatment of rats also induced 4-NP hydroxylation 1.8-fold vs control (data not shown). Data from DEX and phenobarbital induction experiments allowed to assert that the P450 3A1 isoform was the major P450 3A isoform involved in the 4-NP hydroxylation in addition to P450 2E1. This assertion was supported by immunoinhibition experiments (33). A polyclonal antibody P450 2E1 was able to inhibit up to 60% of 4-NP activity, while polyclonal antibody P450 3A2 inhibited 30% in DEX-treated liver microsomes (33). The flavonoid R-naphthoflavone (R-NF) slightly stimulated the 4-NP activity up to 106 ( 2% vs control (data not shown). The effects of R-NF, yielding either stimulation, no effect, or inhibition, depend on the reaction (34). Futhermore, the sigmoidal patterns of the v vs S plot were not observed with liver microsomal preparation from control rat (data not shown). The S50, equivalent to Km, was estimated to be 55 µM, i.e., close to the Km of 30 µM reported elsewhere for human liver microsomes (13). The sigmoidal plot was not due to the presence of two distinct enzymes, as shown by the Eadie-Hofstee plot. The substrate cooperativity was validated by the Hill plot. Such a mechanism has been described as characteristic of the human P450 3A4 isoform (34). The P450 1A family was previously shown to not be involved in 4-NP hydroxylation (35). Indeed, this hydroxylase activity decreased by 0.6-fold in liver microsomes from 3-methylcholanthrene- or β-naphthoflavonetreated rats vs control (36). Such a decrease of P450 2E1 catalytic activity by P450 1A inducers has been previously reported (35). As the combination of two chemical inhibitors (DEDTC and ketoconazole) or two polyclonal antibodies (anti-P450 2E1 and 3A) was not able to inhibit completely the 4-nitrophenol hydroxylation activity in human liver microsomes, the involvement of other P450s could not be totally precluded. This observation was in agreement with the findings of Figure 7 showing that when P450 3A4-dependent activities were equal to zero, there was still some 4-nitrophenol hydroxylation activity. As the extrapolation of findings from rat to man has often been questioned, the question of the specificity of P450 2E1 in 4-nitrophenol 2-hydroxylation in man was also raised. To evaluate the hypothesis that P450 3A4 could be involved in the catalytic activity, additional studies were conducted using selective inhibitors and correlation analyses in a panel of 13 human liver microsomes. A role for P450 3A4 in the formation of 4-nitrocatechol is indicated by several lines of evidence: (i) the lack of total inhibition by diethyl dithiocarbamate, a specific inhibitor of P450 2E1 (2, 9); (ii) the partial inhibition by ketoconazole, a specific inhibitor of P450 3A4 (24), and by antibody anti-P450 3A2; (iii) significant correlations between residual p-nitrophenol hydroxylation by P450 2E1-inactivated microsomes and immunoquantified P450 3A and its corresponding catalytic activities. In light of the data presented, it is apparent that P450 3A plays a relatively important role in the biotransformation of 4-NP in human liver microsomes. The inhibition studies suggest that the contribution of P450 3A can be estimated by 10-25% of total activity. Finally, such an assertion was strengthened by metabolic studies performed by using pure human P450 heterologously expressed in different systems. The highest catalytic activity was obtained with pure P450 2E1 reconstituted with b5. The turnover number of 19.5 min-1 is in agreement with the value of 24.3 min-1

p-Nitrophenol: Substrate for P450 2E1 and 3A Enzymes

reported by Chen et al. (37). Interestingly, cytochrome b5 markedly stimulated p-nitrophenol hydroxylation, confirming that b5 has a strong stimulatory effect on P450 2E1-mediated two-electron oxidations (37, 38). The ratio b5/P450 present in microsomes of lymphoblastoids genetically modified was 0.28 (Gentest communication) vs about 1.7 in human liver microsomes (39). In the presence of extra added b5, this ratio was 2, the optimal range being 1-2 (38). The effect of b5 on P450 3A4mediated metabolism was complex. Indeed, b5 did not have a significant stimulatory effect on the catalytic activity of the reconstituted system. Added cytochrome b5 did not couple with P450 3A4 expressed in human lymphoblastoid cells. However such a coupling has been demonstrated to be efficient when P450 3A4 was expressed in yeast (40), bacteria (41), or insect cells (42). Coexpression of NADPH-P450 reductase and b5 with P450 3A4 in yeast (43) or insect cells (Gentest) allowed to optimize catalytic activities. Such differences are most likely the result of employing heterologous systems having differing phospholipid or glutathione contents, compounds known to influence catalytic activities of P450 3A (44). The turnover numbers reported in Table 2 allow to extrapolate the relative contribution of human liver P450 2E1 and 3A4 to p-nitrophenol hydroxylase activity. On the basis of average hepatic contents of P450 2E1 and 3A, 120 and 250 pmol/mg of protein, respectively (45, 46), it can be estimated that the total catalytic activity should be 2.7 nmol/(min‚mg) with a contribution of 13% for P450 3A4. These rough calculations are in full agreement with the findings reported in this study: catalytic activity of 2.38 ((1.55) nmol/(min‚mg) and 13% inhibition by ketoconazole. The low catalytic activity of P450 2A6 and its low hepatic content allow to preclude a significant contribution of this P450. In summary, although p-nitrophenol hydroxylation was significantly increased by inducers of P450 2E1 such as pyrazole, pyridine, and acetone, it could be also used as a specific marker of the induction of P450 3A isoforms in rat since P450 3A isoforms are involved in this hydroxylation. In man, P450 3A4 may make a significant contribution to this catalytic activity, especially in individuals with high hepatic levels of this P450 isoform.

Acknowledgment. This work was financially supported by a grant from the European Community (Contract ERB BMH4-CT96-0184). Thanks are due to Ms. Nathalie Vaillant for careful typing of the manuscript.

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