Inhibitory and Noninhibitory Monoclonal Antibodies to Human

Harry V. Gelboin,* Inna Goldfarb, Kristopher W. Krausz, James Grogan,. Kenneth R. Korzekwa, Frank J. Gonzalez, and Magang Shou. Laboratory of Molecula...
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Chem. Res. Toxicol. 1996, 9, 1023-1030

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Inhibitory and Noninhibitory Monoclonal Antibodies to Human Cytochrome P450 2E1 Harry V. Gelboin,* Inna Goldfarb, Kristopher W. Krausz, James Grogan, Kenneth R. Korzekwa, Frank J. Gonzalez, and Magang Shou Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 37, Room 3E24, Bethesda, Maryland 20892 Received January 25, 1996X

A panel of 17 hybridomas producing (MAbs) against human cytochrome P450 2E1 (h2E1) was generated by immunizing mice with baculovirus-expressed h2E1. All 17 hybridoma clones gave positive ELISA or immunoblots with either baculovirus- or vaccinia virus-expressed h2E1. Two of the latter were further developed due to their desirable characteristics. MAb 1-73-18 was found to be a powerful inhibitor of P450 h2E1; however, it did not yield a positive immunoblot. MAb 2-106-12 was found to be noninhibitory but formed a strong positive immunoblot with P450 h2E1. These MAbs to h2E1 were highly specific and did not recognize six other human P450s as tested with ELISA or immunoblot analyses. The MAbs to baculovirus-expressed h2E1 also reacted with h2E1 expressed from a vaccinia virus vector system as well as with microsomal fractions of human and acetone-treated rat liver. MAb 1-73-18 inhibited h2E1 enzyme activity catalyzing the metabolism of phenanthrene by 85%, p-nitroanisole by 90%, 4-methylanisole by 60-80%, toluene by 90%, and chlorzoxazone by 90%. The inhibitory MAb 1-73-18 is uniquely useful for determining the contribution of h2E1 to the metabolism of h2E1 substrates in human liver containing multiple P450s. The quantitatively determined contribution of h2E1 to the metabolism of the above substrates ranged from 25% to 75%. Thus, h2E1 was responsible for the following percentages of the total metabolism in human liver: p-nitroanisole (35%), phenanthrene (23%), methylanisole to cresol (25%), methylanisole to methoxybenzyl alcohol (12%), toluene (40%), and chlorzoxazone (72%). The MAb 2-106-12 forming a strong immunoblot is useful for determining the amount of h2E1 protein in a tissue. Thus the utility of the inhibitory and immunoblot positive MAbs is complementary and can determine both the contribution of h2E1 to the metabolism of specific substrates and the amount of h2E1 protein in human tissue. The analyses of metabolism with the inhibitory MAb 1-73-18 can be generalized and applicable to all h2E1 substrates.

Introduction The inordinate value of MAbs1 for cytochrome P450 research has been reviewed (1) and demonstrated with human (2) and rat tissues (3, 4). Hybridoma cells producing MAbs are immortal, can be stored or grown indefinitely, and thus they are a continuous and reliable source of MAbs. The MAbs are chemically defined pure reagents binding to single epitopes on the protein antigen. In contrast, polyclonal antibodies are impure heterogeneous reagents binding multiple epitopes, exhibit serious problems with cross-reactivity, and are irreproducible with each preparation. The cytochrome P450 superfamily collectively performs the oxidative metabolism of a variety of xenobiotic chemicals of highly diverse structure including drugs, carcinogens, and environmental pollutants as well as endobiotics such as steroids and prostaglandins (5). Human 2E1 is an important member of the P450 superfamily. The h2E1 is especially active in the metabolism of low molecular weight chemicals such as benzene, toluene, and carcinogenic nitrosamines (6* Address correspondence to this author. Telephone (301) 496-6849; Fax (301) 496-8419. X Abstract published in Advance ACS Abstracts, August 1, 1996. 1 Abbreviations: MAbs, monoclonal antibodies; h2E1, human P450 2E1; BSA, N,O-bis(trimethylsilyl)acetamide; B[a]P 9,10-diol, benzo[a]pyrene trans-9,10-dihydrodiol; MtBSTFA, N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide; HPLC, high performance liquid chromatography; GC-MS, gas chromatography-mass spectrophotometry; ELISA, enzyme-linked immunoabsorbent assay; IB, immunoblot.

14). It also metabolizes drugs such as chlorzoxazone (15, 16), acetaminophen (17), and commonly ingested caffeine (18). MAbs are uniquely precise and stable reagents (19) which detect and measure the amount of an individual P450 protein and when inhibitory can measure its contribution to the metabolism of diverse substrates in the presence of multiple P450s in different tissues of different individuals. Purified P450s from human tissue have been difficult to obtain in the amounts required for the immunization of the mice for hybridoma production, subsequent cloning, screening, and the enzymatic assays required for the measurement of P450 enzyme activity and its inhibition. We have obtained sufficient amounts of human P450 2E1 by cloning human P450 2E1 cDNA and expressing it into enzymatically active P450 2E1 with either the vaccinia virus (20) or baculovirus (21) expression systems. In the experiments presented here we used as immunogen h2E1 produced and purified from a baculovirus expression system. MAbs that recognize specific forms of P450 can be used for detection and quantitation of the distinct P450s. MAbs that inhibit the enzymatic activity of a specific P450 can be used to determine the quantitative contribution of the individual P450 to the metabolism of any substrate in a tissue preparation containing multiple P450s. This study reports the development of MAbs against human P450 2E1. One of the MAbs obtained, MAb 2-106-12, formed an immunoblot highly specific for h2E1 but not inhibitory to h2E1 enzyme activity. An-

S0893-228x(96)00015-X This article not subject to U.S. Copyright. Published 1996 by the American Chemical Society

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other, MAb designated 1-73-18, inhibited h2E1 enzyme activity toward the five different h2E1 substrates examined: phenanthrene, p-nitroanisole, 4-methylanisole, toluene, and chlorzoxazone. This MAb was used to measure the quantitative role of human 2E1 to the human liver metabolism of the five substrates.

Materials and Methods Chemicals. Chemicals used in this study were obtained from the following sources: p-nitroanisole, 4-nitrophenol, toluene, p-cresol, 4-methylanisole, and 4-methoxybenzyl alcohol from Aldrich, (Milwaukee,WI); deuterated (2,3,5,6-D) nitrophenol and benzyl alcohol (D7) from Cambridge Isotope Laboratories (Andover, MA); chlorzoxazone from Sigma (St. Louis, MO). NADPH from Boehringer Mannheim (Indianapolis, IN); 6-hydroxychlorzoxazone from Salford Ultrafine Chemicals and Research Ltd. (Manchester, U.K.); phenanthrene and benzo[a]pyrene-9,10 diol from the National Cancer Institute Chemical Carcinogen Repository; and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MtBSTFA), and N,O-bis(trimethylsilyl)acetamide (BSA) from Regis Technologies, Inc. (Morton Grove, IL). Preparation, Production, and Purification of MAbs. P450 cDNA incorporated into vaccinia virus vectors were used to express human P450s for enzyme specificity measurements (20). Spondoptera frugipedra (Sf9) cells were infected with a recombinant baculovirus containing human P450 2E1 cDNA. The h2E1 was expressed with this vector system, was purified from the cell membrane fractions using hydrophobic interaction and hydroxylapatite chromatography as described (21), and was used as immunogen. Two female Balb/c mice were inoculated intraperitoneally with the purified recombinant h2E1 protein. Mouse #1 received 30 µg of this immunogen with complete Freund’s adjuvant and two booster injections with incomplete Freund’s adjuvant on the 7th and 14th days. The splenocytes of this mouse were taken for fusion with nonsecreting myeloma cells P3/NSI/1-Ag4-1 (NS-1) on the 17th day. Mouse #2 received an initial inoculation of 35 µg of immunogen with complete Freund’s adjuvant and two booster injections with incomplete Freund’s adjuvant at 3 months. Fusion was performed 6 months after the first inoculation. This mouse received 10 µg of immunogen in PBS on each of the three days preceding the fusion. Fusion of the spleen cells with the mouse nonsecreting myeloma cells was performed as described previously (2, 3), with some modifications: the fused cells were plated in microtiter wells at a density of 1.5 × 104 cells per well, and HAT medium was supplemented with 10% of Hybridoma Cloning Factor from IGEN, Inc. Microtiter wells were examined daily for hybridoma growth starting with the 7th day after fusion until no new hybridomas were observed. Spent media of hybridomas approaching confluence were tested with ELISA using baculovirusexpressed h2E1 as antigen. Samples showing a positive response were tested for cross-reactivity with wild type baculoexpressed antigen. Those showing at least a 4-fold greater signal with baculovirus-expressed h2E1 compared to the wild type antigen were considered specific for P450 h2E1. Hybridomas producing specific immunoglobulins (Ig) were subcloned by limiting dilution. MAbs excreted from cloned hybridomas were further tested for immunoprecipitation and for their ability to inhibit h2E1-catalyzed metabolism of phenanthrene and p-nitroanisole. Isotyping of MAbs was carried out using alkaline phosphatase conjugated, affinity purified anti-mouse IgG1, IgG2a, IgG2b, and IgM from Zymed Laboratories Inc. (San Francisco, CA). Large scale production of MAbs was achieved by growing cloned hybridomas in culture. Immunoglobulins (Ig) from hybridoma culture supernatants were precipitated with 50% saturated ammonium sulfate, dialyzed in PBS, aliquoted, and stored at -20 °C. Additional amounts of MAb 1-73-18 and MAb 2-106-12 were prepared in mouse intraperitoneal ascites fluid. Hybridomas producing the desired MAbs were subcloned 3 times, expanded, and injected into pristine-primed BALB/c mice for production of MAbs in ascites fluid. Within two weeks,

Gelboin et al. the ascites fluid was removed, aliquoted and stored at -20 °C. The concentration of Ig of MAb 1-73-18 in ascites fluid was 0.52.0 mg/mL. The production of ascites fluid is simple and rapid, and yields large amounts of MAbs. The MAbs stored as ascites fluid generally are very stable, and ascites fluid has become a universal method for production and storage of MAbs. ELISA (Enzyme-Linked Immunosorbent Assay). Indirect ELISA was used for testing the reactivity of hybridoma supernatants or purified antibody preparation and to study their binding activity. The assay was performed by published methods (22) using alkaline phosphatase-conjugated goat F(ab′)2 fragment to mouse IgG (H+L) from Cappel Research Products, and to mouse IgG (γ chain specific) or to mouse IgM (µ chain specific) from Jackson Immuno-Research Laboratories. The antigens for coating polystyrene flat bottom immunoassay plates were as follows: (a) partially purified lysate of Sf9 cells infected with either wild type baculovirus or expressing P450 h2E1 (1-2 pmol/well); (b) lysates of Hep G2 cells infected with wild type or recombinant vaccinia virus expressing P450 h2E1 (5 pmol/ well); and (c) microsomal fractions from noninduced and induced rat liver prepared by routinely used methods (23). Immunoblot Assay. Proteins from the Sf9 cells, mammalian TK- embryoblasts, Hep G2 cells infected with wild type and recombinant baculovirus or vaccinia viruses, respectively, and liver microsomes, from humans or from untreated or acetone- or dexamethasone-treated rats, were separated by electrophoresis on SDS gels, transferred onto nitrocellulose filters, and probed with MAbs from cell culture or ascites fluid (2). MAb binding was detected using alkaline phosphataseconjugated goat F(ab′)2 anti-mouse IgG (H+L), and to mouse IgG (γ chain specific) or to mouse IgM (µ chain specific). Preparation of Human and Rat Liver Microsomes. Human liver specimens were obtained from four healthy human organ donors from the Cooperative Human Tissue Network of the Eastern Division (Philadelphia, PA). Liver microsomes from humans and acetone-induced rats were prepared as reported elsewhere (23). Cytochrome P450 content (24) and protein concentration (25) were determined according to procedures previously described. Preparation of Vaccinia Virus Expressed P450s. cDNA’s coding for different cytochrome P450 isozymes were constructed into vaccinia virus vectors (20). TK- embryoblasts or Hep G2 cells infected with recombinant vaccinia viruses were used as expression systems for the following P450s: human 1A2, 2B6, 2C8, 2C9, 2E1, 3A3, 3A4, and 3A5; mouse 1A1 and 1A2; and rat 2B1/2. The cells were harvested 24-48 h after infection. The membrane fractions of Hep G2 cells were used as a source of individual P450s for metabolic studies after measurement of P450 content by spectral analysis (24). Cell lysates from infected TK- embryoblasts or Hep G2 cells were used in ELISA and IB. MAb Inhibition of P450 h2E1-Catalyzed Metabolism. MAbs at protein concentration ranging from 10 to 500 µg were preincubated with 10-50 pmol of vaccinia-expressed h2E1 human liver microsomes or acetone-induced rat liver microsomes in 100 µL of 50 mM potassium phosphate buffer (pH 7.4) at 37 °C for 5 min, and the mixture was diluted with potassium phosphate buffer to 0.97 mL. Substrate and NADPH (1 mM) were added to a final volume of 1.0 mL to initiate the reaction. The concentrations of the substrates were as follows: 150 µM for p-nitroanisole, 200 µM for phenanthrene, and 500 µM each for chlorzoxazone, toluene, and 4-methylanisole. After 20 min, 4 volumes of dichloromethane (DCM) were added to stop the reaction. MAbs 1-68-11 (IgM class against rat 2C11) or Hy Hel (IgG against hen egg white lysozyme) at an amount of protein equivalent to the MAb 1-73-18 were used as a control. Internal standards for metabolite quantitation were B[a]P 9,10diol (D4) for the metabolism of phenanthrene and chlorzoxazone, deuterated nitrophenol for the metabolism of p-nitroanisole, and benzyl alcohol (D7) for the metabolism of toluene and 4-methylanisole. Extracts of products were analyzed by either reverse phase high performance liquid chromatography (HPLC) or gas

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Table 1. ELISA and Immunoblot Analysis of Binding of MAbs against Baculo-Expressed h2E1 with Vaccinia-Expressed h2E1 and Rat 2E1 from Microsomes of Acetone-Induced Rat Liver ELISAa MAbs 1-10-3 1-18-27 1-42-4 1-48-1 1-83-1 1-93-5 1-393-1 1-36-1 1-53-1 1-67-3 1-72-6 1-73-18 1-88-11 1-156-3 2-24-7 2-106-12 2-155-45

Ig class G G G G G G G M M M M M M M G G G

bacuo-h2E1, 1 pmol 1.89 1.66 1.95 1.44 1.61 1.94 1.41 0.95 1.22 0.85 0.81 1.28 1.57 0.51 1.99 1.87 1.99

vaccinia-h2E1, 5 pmol 0.62 0.31 0.74 0.20 0.20 0.74 0.07 0.27 0.27 0.15 0.14 0.43 0.51 0.10 0.97 0.18 0.26

immunoblotb RLM,c 5 pmol 0.17 0.04 0.52 0.14 0.12 0.76 0.05 0.61 0.41 0.23 0.26 0.10 0.42 0.34 0.17 0.16 0.21

baculo-h2E1, 1 pmol (+) + +++ +++ + +++ ++ + +++ ++ (+) +++ ++ ++ +++

vaccinia-h2E1, 1 pmol (+) + +++ +++ n.d. +++ + + +++ ++ (+) +++ + ++ +++

RLM, 3 pmol nte nt nt +++ +++ + +++ ++ + +++ ++ (+) ++ +++

inhibitiond + -

a Antigen-binding activity was determined at 405 nm. b Qualitative estimates of antigen-binding activity. c Acetone-induced rat liver microsomes. d Metabolism of phenanthrene and p-nitroanisole was carried out respectively for screening inhibitory MAbs as listed above. e Not tested.

chromatography-mass spectrophotometry (GC-MS) as described below. High Performance Liquid Chromatography. The HPLC was performed on a Hewlett-Packard Model HP1050 liquid chromatograph equipped with an HP Model 1050 autosampler, a ternary solvent delivery system, and a multiple-wavelength detector, controlled by a Hewlett Packard HPLC2D Chemstation software and a Compaq Prolinea 4/66 personnel computer. The separation of phenanthrene metabolites was analyzed as described in Figure 4 legend. Chlorzoxazone and its 6-hydroxy metabolite were separated on a 20/20 ODS column (4.6 mm i.d. × 200 mm, TLC, Springfield, VA), eluted with a gradient of 10% acetonitrile in water containing 0.5% H3PO4 (pH ) 3) to 80% acetonitrile at a flow rate of 1 mL/min. Retention times of the 6-hydroxy metabolite and chlorzoxazone were 9.0 and 13.2 min, respectively. Gas Chromatography-Mass Spectrophotometry (GCMS). GC-MS analysis was performed on a Hewlett-Packard 5890 instrument with a 5971 mass selective detector and a HP Vectra QS/20 PC computer using an HP-G1030A MS ChemStation (DOS series) software. A Heliflex (Deerfield, IL) AT-1 silica capillary column (30 m × 0.25 mm × 1 µm film) was used. Metabolites of p-nitroanisole and toluene with MtBSTFA (metabolites of 4-methylanisole with BSA) were derivatized at 60 °C overnight to butyldimethylsilyl (trimethylsily) products. Samples were injected into the column via an autosampler and eluted with a carrier gas of helium under 6 psi. Ionization was by electron impact (70 eV). Temperature program was gradient from 50 to 150 °C at 30 °C/min and then from 150 to 280 °C at 10 °C/min.

Results A single fusion of the isolated spleen cells from mouse #1 with myeloma cells resulted in the formation of 443 hybridoma clones. Fourteen of these produced MAbs that bound to h2E1 as measured by ELISA. Seven of these were of the IgG class and seven of the IgM class. Of 1459 hybrids obtained from the fusion of spleen cells of the second mouse with myeloma cells, only three produced MAbs of the IgG class specific for h2E1. Table 1 shows the combined data from the two experiments. The MAb producing hybridomas were subcloned 1-3 times, grown in the flasks for the production of large amounts of MAbs which were concentrated and used for cross-reactivity studies as measured by ELISA and immunoblotting and for inhibitory activity toward h2E1-catalyzed metabolism.

Cross-Immunoreactivity of MAbs in ELISA. Table 1 shows the Ig class of 17 MAbs and their binding activity, measured by ELISA, to baculovirus- and vaccinia virus-expressed human P450 2E1 and liver microsomes from acetone-treated rats. The concentration of immunogloblins used in each series of experiments was 10.0, 1.0, and 0.1 µg/mL, and the results with 10.0 µg/ mL are shown. Four MAbs of the IgG class (1-10-3, 1-424, 1-93-5, 2-24-7) and two of the IgM class (1-73-18, 1-8811) reacted with vaccinia virus-expressed h2E1. Five of the 17 MAbs tested (1-42-4, 1-93-5, 1-36-1, 1-53-1, 1-8811) cross-reacted with liver microsomes from acetonetreated rats. The remaining 11 MAbs did not cross-react with liver microsomes from acetone-treated and control rats at a 100 µg/mL concentration of immunoglobulin (data not shown). Cross-Immunoreactivity of MAbs Measured by the Immunoblot Assay. Table 1 also shows a qualitative estimation of antigen-binding activity of the selected MAbs with an immunoblot (IB) assay. Nine MAbs of the IgG class and six of the IgM class bound the baculovirusexpressed h2E1 with variable intensity. Only two, 1-42-4 (IgG) and 1-73-18 (IgM), did not bind to baculovirusexpressed h2E1. Fourteen out of 15 MAbs reacting with baculovirus-expressed 2E1 also reacted with vaccinia virus-expressed h2E1 antigen with an identical intensity. Thirteen MAbs that bound to baculovirus-expressed h2E1 by immunoblot were tested with liver microsomes from acetone-treated rats. A qualitative estimation of their immunoprecipitation is shown in Table 1. Only two of the thirteen MAbs, 2-24-7 (IgG) and 1-156-3 (IgM), did not recognize P450 2E1 in rat liver microsomes. No cross-reactivity was found between the MAbs detecting h2E1 and six other baculovirus or vaccinia virus-expressed human P450s. These MAbs to h2E1 did not cross-react with baculovirus-expressed h3A4 or with vaccinia virus-expressed human 1A2, 2C8, 2C9, 3A3, 3A4, and 3A5. Data for MAb 1-73-18 are shown as a typical example of a number of MAbs that reacted with the h2E1 but not with the heterologous expressed P450s tested (Table 2). MAb 2-106-12 to h2E1 binds by IB to 0.25 pmol of baculovirus-expressed h2E1 and 0.1-1.0 pmol of vaccinia

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Table 2. ELISA Analyses of Cross-Reactivity of MAb 1-73-18 to Baculo-Expressed h2E1 with Heterologous Baculo- and Vaccinia-Expressed P450s P450s

MAb 1-73-18a

*bv wild bvh2E1

0.046 1.179b

vv wild vvh2E1 vvh1A2 vvh2C8 vvh2C9 vvh3A3 vvh3A4

0.147 0.402 0.119 0.112 0.130 0.147 0.134

P450s

MAb 1-73-18a

bv Expressed bvh3A4

0.033

vv Expressed vvh3A5 vvrh2B1 vvrh3A1 vvrh4A1 vvrh4A4 vvmh1A1 vvmh1A2

0.124 0.036 0.047 0.038 0.035 0.049 0.035

a Assay: ELISA using 5 pmol per well of expressed P450s from vaccinia (vv) and 1 pmol per well from baculovirus (bv). MAb concentration is 1 µg/mL. b Optical density at 405 nm. bv: baculoexpressed virus. vv: vaccinia-expressed virus. h, r, m: human, rat, and mouse, respectively.

Figure 1. Immunoblot analysis of cross-reactivity between MAb 2-106-12 and vaccinia virus-expressed human isoforms of P450. Samples (1 pmol) were applied to the wells in the following order: (A) h1A2; (B) h2B6; (C) h2C8; (D) h2E1; (E) h2C9; (F) h3A3; (G) h3A5; (H) molecular weight standard; (I) vaccinia virus, wild type.

Figure 2. Immunoblot analysis of cross-reactivity between MAb 2-106-12 and human, mouse, and rat vaccinia-expressed isoforms of P450. Samples were applied to the wells in the following order: (A) rat 2A1, 3.0 pmol; (B) mouse 1A1, 1.0 pmol; (C) mouse 1A2, 1.0 pmol; (D) vaccinia virus-expressed h2E1, 1 pmol; (E) baculovirus-expressed h2E1, 0.25 pmol; (F) h3A4, 2.0 pmol; (G) rat 2B1, 2.0 pmol; (H) h2B6, 1.5 pmol.

Figure 3. Immunoblot analysis of human and rat liver microsomes with MAb 2-106-12. Samples were applied to the wells in the following order: (A) untreated rat liver microsomes, 3.5 µg; (B) dexamethasone-treated rat liver microsomes, 4.0 µg; (C) acetone-treated rat liver microsomes, 4 µg; (D) molecular weight standard; (E, F, G) human liver microsomes “M”, 6, 2, and 0.2 µg; (H) baculo-expressed h2E1, 1.0 pmol; (I, J) human liver microsomes “K”, 20.0 and 5.0 µg.

virus-expressed h2E1. This MAb did not cross-react with other vaccinia virus-expressed human P450s as shown in Figures 1 and 2. These were 1A2, 2B6, 2C8, 2C9, 3A3, 3A4, and 3A5, mouse 1A1 and 1A2, and rat 2A1 and 2B1. Figure 3 shows the positive binding of MAb 2-106-12 with liver microsomes from acetone and dexamethasonetreated and untreated rats. The microsomal fraction of the two human livers also showed strong bands at the level of 54 kDa when tested with MAb 2-106-12.

Figure 4. MAb 1-73-18 inhibition of phenanthrene metabolism. MAb 1-73-18 was preincubated with 2E1 (15 pmol), HLM (50 pmol), or acetone-induced RLM (50 pmol), respectively, in 50 mM KPi for 5 min at 37 °C. 200 µM phenanthrene and 1 mM NADPH were added in 1 mL as a final volume and incubated for 20 min. MAb 1-68-11 against rat 2C11 was used as control. Reaction was terminated with 4 volumes of dichloromethane, and B[a]P 9,10-diol (1.5 µM) was added as an internal standard. Extracts were analyzed by reverse phase HPLC. % control was obtained by the ratio of phenanthrene-9,10-diol formed in the presence and absence of MAb 1-73-18.

MAb Inhibition of 2E1-Catalyzed Metabolism. The inhibitory effect of the 17 MAbs listed in Table 1 was examined by measuring the effect of each MAb on the vaccinia virus-expressed h2E1-catalyzed metabolism of two known substrates of h2E1, phenanthrene and pnitroanisole. Of the 17 MAbs tested, only MAb 1-73-18 exhibited inhibitory activity. Figure 4 shows that MAb 1-73-18 inhibits h2E1-catalyzed phenanthrene metabolism by 90%. MAb 1-73-18 inhibits phenanthrene metabolism by human microsomes by 25%, which indicates that 25% of phenanthrene metabolism in human liver is catalyzed by P450 2E1 and 75% by P450s other than h2E1. The amount of actual metabolism in human liver catalyzed by h2E1 is likely greater by about 10% than that indicated since the purified 2E1 was inhibited by 90% rather than 100%. This experiment clearly demonstrates the utility of inhibitory MAbs for determining the role of a specific P450 in a tissue containing multiple P450s. Phenanthrene metabolism in rat liver microsomes, however, is not inhibited by the MAb 1-73-18, indicating that its entire metabolism in rat liver microsomes is catalyzed by P450s that do not contain an epitope recognized by MAb 1-73-18. These P450s may be other than 2E1 or a rat 2E1 lacking the MAb 1-73-18 epitope. Figure 5 shows the effect of MAb 1-73-18 on the expressed h2E1-catalyzed metabolism of p-nitroanisole. The metabolism of this substrate by purified h2E1 is inhibited 90% by MAb 1-73-18. Figure 5 also shows that human liver microsomes are inhibited by about 35-45% by the MAb 1-73-18, which indicates that h2E1 is responsible for 35-45% of the p-nitroanisole metabolism in human liver. The remaining 60-65% of the p-nitroanisole metabolism is catalyzed by P450s which are insensitive to inhibition by MAb 1-73-18. Thus, the analysis of p-nitroanisole metabolism with MAb 1-73-18 determines the contribution of 2E1 to its total metabolism. The metabolism of p-nitroanisole by rat liver microsomes is completely insensitive to MAb 1-73-18 inhibition, indicating that its metabolism in rat microsomes is catalyzed by a P450 containing an epitope not recognized by MAb 1-73-18. Figure 6 shows the effect of MAb 1-73-18 on toluene metabolism by the expressed P450 2E1. The

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Figure 5. MAb 1-73-18 of p-nitroanisole metabolism. MAb 1-73-18 was preincubated with 2E1 (15 pmol), HLM (50 pmol), or acetone-induced RLM (20 pmol), respectively, in 50 µM KPi for 5 min at 37 °C. 500 µM p-nitroanisole and 1 mM NADPH were added in 1 mL as a final volume and incubated for 20 min. MAb 1-68-11 against rat 2C9 was used as control. Reaction was terminated with 4 volumes of dichloromethane, and deuterated nitrophenol (D4, 5 µM) was added as an internal standard. Extracts were derivatized with BSA and analyzed by GC-MS. % control was obtained by the ratio of nitrophenol formed (nmol/ min, nmol of P450) in the presence to that in the absence of MAb 1-73-18.

Figure 7. MAb 1-73-18 inhibition of 4-methylanisole metabolism. MAb 1-73-18 was preincubated with 2E1 (30 pmol), HLM (50 pmol), or acetone-induced RLM (100 pmol), respectively, in 50 mM KPi for 5 min at 37 °C. 500 µM toluene and 1 mM NADPH were added in 1 mL as a final volume and incubated for 20 min. MAb 1-68-11 against rat 2C11 was used as control. Reaction was terminated with 4 volumes of dichloromethane, and deuterated benzyl alcohol (D7, 50 µM) was used as an internal standard. Extracts were derivatized with BSA and analyzed by GC-MS. % control was obtained by the ratio of p-cresol formed in the presence to that in the absence of MAb 1-73-18.

Figure 6. MAb 1-73-18 inhibition of toluene metabolism. MAb 1-73-18 was preincubated with 2E1 (25 pmol), HLM (50 pmol), or acetone-induced RLM (50 pmol), respectively, in 50 mM KPi for 5 min at 37 °C. 500 µM toluene and 1 mM NADPH were added in 1 mL as a final volume and incubated for 20 min. MAb 1-68-11 against rat 2C11 was used as control. This reaction was terminated with 4 volumes of dichloromethane, and deuterated benzyl alcohol (D7, 50 µM) was used as an internal standard. Extracts were derivatized with BSA and analyzed by GC-MS. % control was obtained by the ratio of benzyl alcohol formed (nmol/min, nmol of P450) in the presence to that in the absence of MAb 1-73-18.

Figure 8. MAb 1-73-18 inhibition of 4-methylanisole metabolism. Experimental details were the same as in Figure 7, except that % control was obtained by the ratio of benzyl alcohol formed in the presence to that in the absence of MAb 1-73-18.

inhibition by the 1-73-18 is 90%, which demonstrates the strong inhibitory effect of this MAb h2E1 activity. Rat liver microsomes are entirely unaffected by the inhibitory 1-73-18 monoclonal antibody. Toluene metabolism in human liver microsomes is inhibited by 40-50%, and thus h2E1 contributes 40-50% of the total metabolism of toluene in human liver microsomes. The remainder 50-60% is catalyzed by P450s not containing the epitope recognized by MAb 1-73-18. Figures 7 and 8 show the effect of inhibitory MAb 1-73-18 on 4-methylanisole metabolism to two different products. Methylanisole conversion to p-cresol is shown in Figure 7, and its conversion to 4-methoxybenzyl alcohol is shown in Figure 8. In the case of conversion of methylanisole to p-cresol, the MAb inhibited the reaction by 60%. Rat liver microsomal metabolism was unaffected. The human liver microsomes were inhibited by approximately 30%,

indicating that methylanisole conversion to p-cresol by 2E1 in human liver is approximately 30% and the remainder is catalyzed by other P450s. In the case of 4-methylanisole, conversion to methoxybenzyl alcohol by the expressed 2E1 was inhibited by 80%, whereas the rat microsomes were not inhibited. The human liver microsomes were only inhibited to a small degree, about 5-10%, indicating that the 2E1 contributes very little to the conversion of 4-methylanisole to methoxylbenzyl alcohol in human liver. In the case of methylanisole metabolism, the MAb indicates that different P450s are involved since its inhibitory effects are different for each metabolic analyses. Figure 9 shows the effect of MAb 1-73-18 on the metabolism of the muscle relaxant chlorzoxazone by expressed P450 h2E1 and by rat and human liver microsomes. The metabolism by the purified expressed 2E1 is inhibited to a very high extent 90% by MAb 1-73-18. Human microsomes are also inhibited extensively, by 70-80% by MAb 1-73-18, indicating that the 2E1 is responsible for 70-80% of the chlorzoxazone metabolism in human liver, whereas 20-30% of its metabolism is catalyzed by P450s other than 2E1 which do not contain the MAb 1-73-18 binding epitope. The rat liver microsomes are essentially unaffected by the MAb

1028 Chem. Res. Toxicol., Vol. 9, No. 6, 1996

Figure 9. MAb 1-73-18 inhibition of chlorzoxazone metabolism. MAb 1-73-18 was preincubated with 2E1 (30 pmol), HLM (50 pmol), or acetone-induced RLM (100 pmol), respectively, in 50 mM KPi for 5 min at 37 °C. 500 µM chlorzoxazone and 1 mM NADPH were added in 1 mL as a final volume and incubated for 20 min. MAb 1-68-11 (IgM for rat 2C11) was used as a control. Reaction was terminated with 4 volumes of dichloromethane, and B[a]P 9,10-diol (1.5 µM) was added as an internal standard. Extracts were analyzed by reverse phase HPLC. % control was obtained by the ratio of 6-OH-chlorzoxazone formed in the presence to that in the absence of MAb 1-7318. Table 3. Small Molecules Reported or Substrates for P450 2E1 acrylonitrile (6, 26) p-nitrophenol (27, 29, 32) 1,1,2,2-tetrafluoro-1(2,2,2-trifluoroethoxy)ethane (33) 1,1-dichloroethylene (32, 34) N,N-dimethylformamide (35) furan (36) ethylene dibromide (6) ethylene dichloride (6) vinyl chloride (6) vinyl bromide (6) viyl carbamate (6) ethyl carbamate (6) CCl4 (6) CHCl3 (6) CH3CCl3 (6)

trichloroethylene (6, 37) 1,2-dichloropropane (6) benzene (6, 12) styrene (6) 1,3-butadiene (38) p-nitroanisole (14) 1,1,2,3,3,3-hexafluoropropyl methyl ether (39) acetone (40) toluene (41) lauric acid (42) 1,1-dichloro-2,2,2trifluoroethane (43) acetaldehyde (44) carbon disulfide (45)

Table 4 Carcinogens Reported or Substrates for P450 2E1

Drugs Reported or Substrates for P450 2E1

N-nitrosodimethylamine (7, 8) N-nitrosodiethylamine (46, 47) N-nitrosonornicotine(NNN) (7) N-nitrosobis(2-oxopropyl)amine (48)

chlorzoxazone (16, 49) acetaminophen (17, 28, 51) enflurane (51, 52) sevoflurane, isoflurane and methoxyflurane (11) eicosatetraenoic acid (53) caffeine (18, 54) ethoxycoumarin (31)

1-73-18, indicating an absence of the 2E1 epitope for MAb 1-73-18 in rat liver microsomes.

Discussion Human cytochrome P450 h2E1 is the major and a most important P450 engaged in the metabolism of a variety of environmentally important small molecules, some of which are carcinogenic in humans, as well as drugs of therapeutic value (6, 15, 26-31). Table 3 is a partial list of small molecule substrates for P450 2E1. Many of these compounds are found in the environment and workplace. Table 4 includes several commonly used drugs, e.g., chlorzoxazone (15, 16), acetaminophen (17), and caffeine (18), as well as the carcinogens N-nitrosodimethylamine

Gelboin et al.

and nitrosonornicotine (8). In this study, we have isolated MAbs which are highly specific for P450 h2E1. This was accomplished by the use of the baculovirus expression system for producing the highly purified human P450 2E1 in sufficient amounts for successful immunization of the mice. In this system, no other P450s are found in the immunogen preparation other than h2E1. One of the MAbs studied, 2-106-12, yielded a strong IB with P450 h2E1 with very high specificity. The MAb 2-106-12 can thus be used to determine the amount of 2E1 protein in human tissues, and it would be an important tool for determining the individual and tissue distribution and phenotypic polymorphisms of the 2E1 protein in humans. In a previous study, we reported (2) the isolation of MAbs to human 3A4 which bind or inhibit the metabolic activity of h3A4, which was demonstrated with six different substrates: testosterone, diazepam, nitroanisole, phenanthrene, cyclosporin, and taxol. The P450 3A4 enzyme is considered the major contributor to the metabolism of a very large variety of clinically used drugs. It metabolizes a wide range of large molecules. In constrast, 2E1 metabolizes mostly small molecules and including some larger molecular size drugs. This paper describes the production of a monoclonal antibody (MAb 1-73-18) which has a very high inhibitory activity toward human 2E1-catalyzed metabolism. This property enables the measurement of the precise contribution of 2E1 to the metabolism of a variety of substrates in a tissue which ordinarily has a large mixture of different P450s. In this study, MAb 1-73-18 was found to inhibit the h2E1catalyzed metabolism of p-nitroanisole, 4-methylanisole, phenanthrene, toluene, and the drug chlorzoxazone. In the case of toluene, chlorzoxazone, p-nitroanisole, and phenanthrene, the inhibition of the expressed human 2E1 is about 90%. In the case of 4-methylanisole, the inhibition was less, and this may be a reflection of the failure of the antibody to efficiently occupy the active site of the enzyme. The MAb inhibitory to P450 h2E1 reported in this paper complements to a large extent the action of the MAb inhibitory to P450 h3A4 reported previously (2). The use of expression systems for production of single cytochrome P450s of both animal and human origin has led to major advances for determining their substrate and product specificity. Thus, each P450 can be characterized for its substrate and product specificity. The knowledge of the specificity of the P450 enzyme, however, does not necessarily determine a quantitative role for an individual P450 in metabolism of a given substrate in the tissue. This is especially true when more than one P450 engages the metabolism of a single substrate. Furthermore, the amount of a particular P450 present in the liver or other tissues would also govern its quantitative role in metabolism. Since the amount of a specific P450 in a tissue is generally unknown and is usually variable, the binding Mab can be used independently to determine the amount of an individual P450 present. The inhibitory MAb specific for a single P450 inhibits the reaction to an extent which defines the contribution of the single P450 in the presence of multiple P450s. A MAb able to measure the amount of a specific P450 protein can elucidate polymorphisms that are not under control of structural genes but rather can arise from changes in regulatory mechanisms, or in regulatory genes that result in unique P450 phenotypes. Thus, MAbs to P450 are highly complementary to cDNA expression for the analyses of P450 content and function and will play a general and unique role in defining P450-catalyzed reactions.

Monoclonal Antibodies to Human Cytochrome P450 2E1

Acknowledgment. Thanks to Michelle Hudson for her valuable clerical help in the preparation of the manuscript.

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