ARCHIVES

Vol.

OF BIOCHEMISTRY

299, No. 1, November

AND

BIOPHYSICS

15, pp. 1633171,

1992

Catalytic Properties of the Human Cytochrome P450 2El Produced by cDNA Expression in Mammalian Cells Chris J. Patten,*,’ Hiroyuki Ishizaki,* Toshifumi Aoyama,? Maojung Lee,* Shu M. Ning,* Wen Huang,* Frank J. Gonzalez,? and C. S. Yang*,2 *Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855; and fLaboratory Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Received

June

5, 1992, and in revised

form

July

24, 1992

A full-length cDNA encoding human cytochrome P450 2El was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2El and comigrated with P450 2El from human liver microsomes. P450 2El expressed in Hep G2 cells, a human cell line which contains both cytochrome b, and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2El substrates: iV-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent K,,, and V,,, values for NDMA demethylation were 22 PM and 173 pmol/min/mg microsomal protein, respectively. P450 2El expressed in TK 143 cells, which do not contain bg, displayed K, microsomal and V,,, values of 3 1 PM and 34 pmol/min/mg protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the V,,, 2.2-fold and decreased the Km to 22 KM. Addition of b, to Hep G2 microsomes resulted in a 1.6-fold increase in V,,,,,, but showed no effect on the K,. P450 2E 1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 /.LM and V,,,,, of 2 13 pmol/min/mg microsomal protein. Addition of b, lowered the K, to 27 MM, but had no effect These results demonstrate conclusively that on V,,,. P450 2E 1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5. ‘~8 1992 Academic Press, Inc.

Cytochromes P450 constitute a multigene family of btype heme proteins which function in oxidizing a wide ’ Conducted in partial fulfillment of the requirement for a Ph.D. in the Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New JerseyGraduate School of Biomedical Sciences. ’ To whom correspondence should be addressed. Fax: (908) 932.5767. 0003-9861/92 $5.00 Copyright ~z‘ 1992 by Academic Press, All rights OS reproduction in any form

of

range of structurally unrelated compounds. Substrates of P45Os include xenobiotics such as drugs, carcinogens, and pesticides, and endobiotics such as steroids, fatty acids, and prostaglandins. P45Os function in both beneficial detoxification pathways in the hydroxylation of drugs to polar excretable metabolit,es and detrimental bioactivation pathways whereby procarcinogens are activated to their ultimate carcinogenic forms (1). Cytochrome P450 2El is responsible for the bioactivation of numerous potentially harmful compounds such as benzene, halogenated hydrocarbons, and nitrosamines (2). This enzyme species has been purified and characterized from the liver of rats, rabbits, and humans (3-5). Purified P450 2El reconstituted with NADPH:P450 oxidoreductase and phospholipid exhibits high activity toward a host of compounds that includes some nitrosamines, aniline, p-nitrophenol, ether, benzene, phenol, chloroform, carbon tetrachloride, and acetone (6). For certain substrates such as ether (7), p-nitrophenol (S), chloroform (9), phenol (10) and NDMA3 (11,12), the addition of cytochrome b5 to the reconstituted system was shown to enhance the enzyme activity. In the case of ether and NDMA, the b5 effect was greater at low substrate concentrations, leading to a decrease in the apparent K,,, values. Using purified rat enzymes, two studies showed that, the addition of b5 decreased the K, for NDMA demethylation 6- to g-fold (11, 12). In two other studies using purified human P450 2E1, the addition of b5 caused a roughly 4.5-fold decrease in the K,,, for NDMA demeth-

3 Abbreviations used: Hep G2-v2E1, Hep G2 cells infected with the 2El recombinant vaccinia virus; TK-143.v2E1, TK’143 cells infected with the 2El recombinant virus; TK 143-v2El-vOR, TK-143 cells infected simultaneously with both P450 2El and NADPH:P450 oxidoreductase recombinant viruses; NDMA, N-nitrosodimethylamine; NMBzA, N-nitrosomethylbenzylamine; b,, cytochrome b,; BUdR, 5. bromodeoxyuridine; PBS, phosphate-buffered saline; pfu, plaque forming units. 163

Inc. reserved.

164

PATTEN

ylation (5, 13). In all four sets of experiments b5 caused only a moderate increase in the V,,, (10 to 27%). In liver microsomes, NDMA is activated to its carcinogenic form by a high-affinity NDMA demethylase displaying an apparent K, of between 15 and 25 PM (13,14). Studies employing inhibitory antibodies (14,15) and P450 2El inducers (16) have implicated P450 2El as the enzyme responsible for the low K,,, form of NDMA demethylase. However, the reported K,,, values for NDMA demethylation using purified rat and human P450 2El were much higher, ranging from 300 to 560 PM in the presence of b5 (5,11-13). This discrepancy could be due to several factors such as the presence of glycerol, a competitive inhibitor for P450 2El (17), in the reconstituted system, damage to P450 2El during purification, the method of reconstitution (18), or a specific conformation requirement which cannot be achieved under the artificial conditions inherent in the reconstituted system. One may also question whether P450 2El is really responsible for the low Km form of NDMA demethylase. The present study deals with the catalytic properties of the human P450 2El expressed in mammalian cell lines by the vaccinia virus expression system. This system offers several advantages: first, the expressed P450 can be studied essentially free from the interference of other P45Os; second, the protein is expressed in its native, membrane-bound conformation; and finally, enzyme activities can be determined in the absence of glycerol. The results demonstrate that human P450 2El is expressed in the microsomal fraction in a catalytically active conformation capable of metabolizing NDMA and other P450 2El substrates. The use of cell lines which lack endogenous cytochrome b5 demonstrates the involvement of this hemoprotein in P450 2El catalysis. MATERIALS

AND

METHODS

Chemicals. N-Nitrosodi[‘?]methylamine (49 mCi/mmole) and Nnitrosomethyl[‘4C]benzylamine (45 mCi/mmol) were custom synthesized by SRI International (Menlo Park, CA). Acetaminophen, glutathione, phenol, catechol, hydroquinone, and benzenetriol were purchased from Sigma Chemical Co. (St. Louis, MO). Tissue culture reagents were purchased from GIBCO BRL (Gaithersburg, MD). All other reagents were the highest grades available commercially. Wild-type vaccinia virus (strain WR) Virus, cell lines, andplasmids. and TK-143 cells were obtained from B. Moss (National Institutes of Health, Bethesda, MD). Hep G2 and CV-1 cells were obtained from ATCC (Rockville, MD). Cells were grown in Dulbecco’s modified essential medium containing 10% fetal bovine serum. For Hep G2 cells, nonessential amino acids were included in the medium. TK-143 cell medium also contained 25 pg/ml of BUdR. Expression vector pSCl1 was provided by B. Moss. A full-length cDNA for human P450 2E1 was inserted into pSCl1 by blunt end ligation. All procedures for the vaccinia Vaccinia virus expression system. virus expression system are based on the method of Chakrabarti et al. (19) with modifications by Gonzalez et al. (20). In brief, 6-cm dishes of confluent CV-1 cells were preinfected with wild-type vaccinia virus (6 X lo5 pfu). One hour later, the cells were transfected with a mixture of wild-type vaccinia DNA, calf thymus DNA, and pSCll/PEl expression vector. The cells were then cultured for several days during which time

ET AL. the recombinant virus is formed in uivo by insertion of the expression vector into homologous sites (thymidine kinase locus) of the wild-type vaccinia genome. After this time period, the cells were harvested and used to infect TK-143 cells which had been cultured with BUdR in the growth medium. The resulting recombinant virus was selected from wildtype vaccinia virus and spontaneous mutants based on two characteristics: (i) the ability to grow in the presence of BUdR, and (ii) the ability to express /!-galactosidase. The latter characteristic was detected by the addition of X-gal to the agar overlay, which results in blue color formation at the site of a recombinant virus plaque. Infection of cells and microsomepreparation. Flasks of confluent Hep G2 or TK-143 cells were infected with the recombinant vaccinia virus at a multiplicity of infection (pfu/cell) of 25. After about 18 h of infection, the cells were harvested, washed once in PBS, and lysed by brief sonication. The cell suspension was centrifuged for 30 min at 10,OOOgand the pellet was discarded. The supernatant was centrifuged at 100,OOOg for 90 min to pellet the microsomal fraction. The resulting microsomal pellets were resuspended in 0.35 M sucrose. In some cases, the microsomal pellets were washed once in PBS and subjected to a second high-speed centrifugation. Microsomes from infected Hep G2 cells were designated Hep G2-v2E1, and microsomes from infected TK-143 cells were termed TK-143-v2El. Coinfection of TK-143 cells with the P450 2El and NADPH:P450 oxidoreductase recombinant viruses was accomplished by previously described methods (21). The titers of the two viruses were adjusted to give a 50:50 mixture, and the multiplicity of infection was kept at 25. Microsomes from coinfected cells were designated TK-143. vBEl-vOR. NIlMA metabolism. NDMA demethylation was measured by the method of Yang et al. (22). The incubation mixture contained 50 mM Tris-HCl (pH 7.0 at 37”C), 10 mM MgCl,, 150 mM KCl, an NADPH generating system (0.4 mM NADP, 10 mM glucose 6-phosphate, and 0.4 unit/ml glucose&phosphate dehydrogenase), [‘?]NDMA, and the desired amount of cell microsomes. Except where indicated, the final NDMA concentration was 30 pM (0.049 +Ci). The reaction was initiated by the addition of the NADPH generating system, and terminated with 60 ~1 of Na acetate. After 30 ~1 of HCHO and 60 ~1 of dimedome were added, the mixture was incubated for 30 min at 50°C. The formaldemethane derivative was extracted into hexane and mixed with Betaflour for counting. p-Nitrophenol metabolism. p-Nitrophenol hydroxylase activity was determined by the method of Reinke et al. (23) with minor modifications. The reaction mixture contained 80 mM potassium phosphate (pH 7.0), an NADPH generating system, p-nitrophenol, and cell microsomes. The reaction was terminated by the addition of 31 ~1 perchloric acid and centrifuged to remove protein. The supernatant (90 111)was mixed with 9 ~1 of 10 N NaOH, and the absorbance at 546 nm was measured in a Beckman microcuvette. 4-Nitrocatechol formation was quantitated using an extinction coefficient of 10.28 mM-l cm-‘. Phenol metabolism. Incubations contained 80 mM potassium phosphate (pH 7.0), an NADPH generating system, phenol, and microsomes in a final volume of 200 ~1. The reaction was terminated by the addition of 1 ml ice-cold ethyl acetate. Phase separation was facilitated by centrifugation, and 850 ~1 of the upper phase was removed and dried under reduced pressure with sodium sulfate. The residue was resuspended in 200 ~1 of distilled water, 100 ~1 of which was injected onto a Waters HPLC system equipped with a Supelco C-18 column (25 cm). Metabolites were eluted from the column at a flow rate of 2 ml/min with a gradient consisting of aqueous solution A (5% methanol and 200 ~1 formic acid per liter) and B (60% methanol, 10% acetonitrile, 200 ~1 formic acid per liter). Initial conditions were 100% A and 0% B; at the start of the run, B was increased 8% per minute for 10 min; at 10 min, B was increased 10% per minute for 2 min. Column eluants were monitored amperometrically using a Waters electrochemical detector set at +0.5 V. Typical product retention times were benzenetriol, 3.5 min, hydroquinone, 5.0 min, and catechol, 8.0 min. Products were quantified by comparisons with known amounts of products that had been subjected to extraction and HPLC procedures identical to those of the samples.

VACCINIA

VIRUS-EXPRESSED

The incubation conditions were the Metabolism of acetaminophen. same as those described for phenol metabolism except that 10 mM glutathione was included. The formation of the benzoquinonimine was determined as a glutathione conjugate by the HPLC method of Buckpitt (24) with some modifications. A Supelco C-18 column (15 cm) and an isocratic mobile phase consisting of 10% methanol and 0.1% trifluoroacetic acid in water were used. The flow rate was 1.5 ml/min and the product was eluted at around 8 min. The conjugate was detected by uv absorption with a wavelength setting of 254 nm, and electrochemical detection with a setting of +0.7 V. Acetaminophen was used as a standard to quantitate the conjugate in both detection systems. In the uv range the two compounds have been shown to have similar extinction coeffcients (25). For electrochemical detection the conjugate peak area was multiplied by a factor of 1.7 to correct for the differences in the responses of these two compounds.

Metabolism of NMBzA. The oxidation of [14C]NMBzA to [‘?]benzaldehyde was measured by a radiometric HPLC procedure. The incubation conditions were the same as those described for NDMA metabolism, with the exception that the final volume was increased to 150 ~1. The reaction was terminated by the addition of 15 ~1 of a mixture of 16% zinc sulfate and 66 mM semicarbazide followed by 15 ~1 of saturated barium hydroxide. The mixture was centrifuged for 20 min at 4OOOg, and 100 ~1 of the supernatant was injected onto a HPLC equipped with a C-18 Supelco column (25 cm). An isocratic mobile phase consisting of 45% acetonitrile and 3% acetic acid in water (pH 4.2) was used at a flow rate of 2 ml/min. Benzaldehyde was elut,ed at approximately 3 min and the parent compound eluted at approximately 4 min. Peaks were quantitated using a Waters radiomatic detector (Series A-100). Benzaldehyde can be reduced to benzalcohol and oxidized to benzoic acid under the current incubation conditions. These three products were not clearly separated in the HPLC and were quantitated together as one peak. Other methods

Rat liver cytochrome bg? P450 2E1, and NADPH: P45O oxidoreductase were purified by previously described methods (11). The specific contents of the final preparations of cytochrome b, and P450 2El were 40 and 11 nmol/mg, respectively. Cytochrome b, was incorporated into cell microsomes essentially by the method of Cinti and 0201s (26). In the control experiments, microsomes were incubated similarly in the absence of b,. Determination of b, content in cell microsomes using NADPH and NADH was by the method described by Estabrook et al. (27). Manganese b5 was prepared by the method of 0~01s and Strittmatter (28). Trypsin b5 was prepared essentially by the method of Reid et al. (29), with the exception that a Sepharose 2’,5’ADP column was included as an additional purification step (30). For the reconstituted system, glycerol was first removed from P450 2El and NADPH:P450 oxidoreductase by dialysis against 2 liters of a buffer consisting of 0.5 M sucrose and 50 mM potassium phosphate (pH 7.25). The standard reconstituted system consisted of 0.016 nmol P450 2E1, 210 units reductase, 0.032 nmol cytochrome b,, 11 pg dilauroyl phosphatidylcholine, and the other components described for measuring NDMA demethylation. P450 determination was by the method of Omura and Sato (31) using a buffer consisting of 25% glycerol, 50 mM potassium phosphate (pH 7.4), and 0.5% E911 detergent. SDS-gel electrophoresis was by the met,hod of Laemmli (32). Immunoblot analysis utilized previously described methods (33). For P450 2El immunodetection a rabbit anti-rat polyclonal antibody was used (33). Immunodetection of cytochrome b, utilized a guinea pig anti-hamster polyclonal antibody (34). Kinetic parameters, Km and V,,,,, were determined by nonlinear regression analysis using the KaleidaGraph comput,er program from Abelbeck Software.

HUMAN

Expression of P450 2El and spectral properties. Immunoblot analysis of microsomes from Hep G2

and TK-143 cells infected with the P450 2El recombinant

2El

165

vaccinia virus showed a polypeptide band (M, 52,500) which comigrated with P450 2El from human liver microsomes (Fig. 1). Based on densitometric scans of the immunoblots, expression levels were approximately two times higher in Hep G2 microsomes than in TK- 143. A protein band representing P450 2El was not detected in microsomes from uninfected control cells or in cells infected with the wild-type vaccinia virus (Fig. 1). An immunoblot analysis for P450 2El synthesis using a multiplicity of infection of 25 and infection times of 8, l&30, and 48 h showed that an 18-h infection time produced the highest P450 2El protein levels (results not shown). Infection times of between 17 and 19 h were hence used routinely throughout this study. Detergent-solubilized microsomes from Hep G2 cells infected with the P450 2El recombinant virus showed a characteristic CO-ferrous P450 spectrum with an absorption maximum at around 453 nm (spectrum not shown). The red-shifted nature of the spectrum is consistent with the absorption spectrum of purified rat and human P450 2El (5, 11). The specific content of the expressed protein was calculated to be 36 pmol/mg microsomal protein. The CO-reduced minus oxidized difference spectrum of uninfected control microsomes did not show a peak of absorbance in the 450-nm region. Catalytic activities. P450 2El expressed in Hep G2 cells, a cell line which contains sufficient amounts of NADPH:P450 oxidoreductase to support P450-dependent activities (35), was able to catalyze the oxidation of NDMA, p-nitrophenol, phenol, and acetaminophen (Table IA). With phenol as the substrate, hydroquinone was the major product formed, and the formation of catechol and benzenetriol were also detectable. With control Hep G2 microsomes, little H14CH0, hydroquinone, and acetaminophen conjugate were formed (< 1 pmol/min/mg). Catechol, benzenetriol, and p-nitrocatechol were below the limits of detection (1.3, 0.4, and 500 pmol, respectively).

ABCDEFG FIG.

RESULTS

P450

1. Immunoblot analysis of human P450 2El in cell lines and liver microsomes. Lane A, acetone induced rat liver microsomes (0.6 pg); lane B, human liver microsomes (2.8 fig); lane C, TK 143.v2El microsomes (90 pg); lane D, Hep G2-v2EI microsomes (90 pg); lane E, control TK- 143 microsomes (90 pg); lane F, control Hep G2 microsomes (90 fig); and lane G, purified rat liver P450 2El incorporated into control Hep G2 microsomes.

PATTEN TABLE Catalytic

Conditions A. Hep Hep Hep Hep Hep

G2+2El G2-v2El G2-v2El G2-v2El G2-v2El

B. TK-143.v2El-vOR TK-143~v2El-vOR TK-143 + purified reductase

Activity

I

NDMA NDMA p-Nitrophenol Acetaminophen Phenol

NDMA Acetaminophen NDMA

2.5

220

of Expressed

Substrate

ET AL.

Human

Product

2El 200

Activity (pmol/min/mg)

H’%HO H’“CH0 p-Nitrocatechol GSH-conjugate Hydroquinone Catechol Benzenetriol

Cl.0 98 680 184 390 7.0

HWHO GSH-conjugate HWHO

20 17 Cl.0

n :E

0.7

Note. Incubations were carried out with microsomes prepared from Hep G2 (A) or TK-143 cells (B). For NDMA metabolism, 25 pg of Hep G2 microsomes and 35 bg of TK-143 microsomes were used. For phenol, p-nitrophenol, and acetaminophen metabolism 125 pg of both Hep G2 and TK-143 microsomes was used, respectively. The final substrate concentrations were NDMA (30 PM), phenol (500 PM), acetaminophen (640 PM), andp-nitrophenol (100 PM). Incubations were carried out at 37°C for the following time periods: NDMA demethylation, 20 min; metabolism of phenol, p-nitrophenol, and acetaminophen, 45 min. Other assay conditions are described under Materials and Methods.

TK-I43 cells contain lesser amounts of NADPH:P450 oxidoreductase and required additional reductase to display maximal P450 activity (36). This was accomplished either by adding purified reductase to the incubation mixture or by coinfection of a human NADPH:P450 oxidoreductase recombinant virus. Table IB demonstrates the ability of TK-143 microsomes with expressed P450 2El and NADPH:P450 oxidoreductase to metabolize NDMA and acetaminophen. The observed activity in TK-143 microsomes is shown to be several-fold less than the activity of Hep G2 microsomes. This is most likely due to both the lower levels of protein expression in TK-143 cells vs Hep G2 cells (Fig. 1, lanes C and D), and the lack of cytochrome b5 in TK-143 cells (see Table IIIA). The pH optimum for the demethylation of NDMA by Hep G2-v2El microsomes was 6.7 (Fig. 2). This value agrees with the pH optimum reported for purified rat P450 2El (12) and for acetone-induced rat liver microsomes (unpublished result). Human liver microsomes, however, showed optimum activity at a pH of about 7.1 (Fig. 2). A pH optimum of greater than 7.0 was observed in one other human liver microsome sample. Incorporation of cytochrome b, into cell microsomes. The cytochrome b6 content of cell microsomes

was determined by immunoblot and spectral analysis. The immunoblot in Fig. 3 demonstrates detectable levels of b, in Hep G2 microsomes but not in TK-143 microsomes. In subsequent experiments, purified rat liver b, was incorporated into TK-143 microsomes by incubating b5 and microsomes in a small volume at 37°C followed by the

160

P 0+ ;

E”

‘E ;E

? 0

160

a

=

140

=

I”

120

0.5 6.4

6.6

6.6

7.0

7.2

7.4

I 7.6

PH FIG. 2. pH profile of NDMA demethylation by microsomes prepared from Hep G2-v2E1 cells and human liver. The assay was carried out with the procedure described under Materials and Methods with the exception that the standard NDMA demethylase buffer (Tris-HCl, KCl, and MgCl,) was replaced with 80 mM potassium phosphate buffer adjusted to the following pH values: 6.4, 6.6, 6.7, 6.8, 7.0, 7.2, and 7.6.

addition of more buffer and by a high-speed centrifugation to remove unbound bs. This procedure resulted in a large increase of microsomal b5 (lanes D through F of Fig. 3). Cytochrome bS can be enzymatically reduced by either NADH or NADPH through the NADH:cytochrome b5 oxidoreductase or NADPH:P450 oxidoreductase pathways, respectively (37, 38). The addition of either coenzyme to Hep G2 microsomes produced a typical b5 spectrum with a peak at 423 nm and trough at 409 nm (spectrum not shown) which further verifies the presence

A I3 C‘D

E

F G

H

I

J

FIG.

3. Immunoblot analysis of cytochrome b, in microsomes from and cell lines. Lanes A, B, and C, 200 pg each of control Hep G‘2, CV-1, and TK -143 microsomes, respectively. Lanes D, E, and F contain liver

13.3 pg each of b5 enriched TK-143 microsomes. Incorporation was carried out essentially by the method described in Table II. The final h, concentrations in the incorporation mixtures were as follows: lane D, 0.8 PM; lane E, 8.0 PM; and lane F, 23 FM. Lanes G and H contain 6 gg each of acetone-induced rat liver microsomes and human liver microsomes, respectively. Lanes I and ,J, 0.5 pg each of purified rat and rabbit liver h5.

VACCINIA

VIRUS-EXPRESSED

of endogenous b5 in Hep G2 cells. The specific contents of b5 in cell microsomes are shown in Table II. Based on these results, the reducible b5 content in Hep G2 microsomes was estimated to be 5 to 11 times less than the amount in human liver microsomes. The b, content of 16 individual human liver microsome samples was found to range from 0.28 to 0.66 nmol/mg protein (unpublished results). A b, spectrum was not detected in TK-143 microsomes, which is in agreement with the immunoblot in Fig. 3. Table II also demonstrates that enrichment of both Hep G2 and TK-143 microsomes with purified rat liver bs shows a corresponding increase in the amount of NADPH (or NADH) reducible b,. The observation that exogenously incorporated cytochrome b5 can be enzymatically reduced is a strong indication that the b5 is incorporated in its native membrane-bound conformation (39). The effects of Effects of b5 on NDMA demethylation. exogenously added b5 on NDMA demethylation by TK-143-v2El-vOR microsomes are shown in Table III. With increasing amounts of b5 incorporated, the rate of NDMA demethylation increased (experiment A). At the highest level of b5 incorporation (2.1 nmol/mg), a 4.7-fold stimulation in NDMA demethylase activity was observed. In experiment B, b5 was added directly to the incubation mixture without separating the unbound b5 by centrifugation. Under these conditions, NDMA demethylation was increased 2.6-fold. Substitution of rat b5 with purified rabbit b5 had a similar stimulatory effect on NDMA demethylation. However, incorporation of trypsin-solubilized rat b5 had no effect on enzymatic activity. This result is consistent with previous studies showing that the hy-

TABLE

II

Cytochrome b, Incorporation into Cell Microsomes Conditions

Reducing agent

Hep G2 microsomes Hep G2 microsomes TK TK

+ b5

14%vOR microsomes 143.vOR microsomes

+ b,s

NADPH NADH NADPH NADH NADPH NADPH

Reducible b5 (nmol/md 0.04 0.06 0.68 0.91 ND” 0.41

Note. Where indicated (+b5), microsomes from Hep G2 or W-143 cells were fortified with rat liver b5 by incubating microsome with b, at 37°C for 40 min. For Hep G2 microsomes the incubation consisted of 3.6 mg of microsomes and 28 pM b5 (1.8 nmol bb/mg protein) in a final volume of 270 ~1. For TK-143 microsomes the incubation consisted of 6 GM b,. Residual b, which did not bind to microsomes was removed by diluting the mixture to 10 ml with cold PBS followed by centrifugation at 100,OOOgfor 60 min. The washing procedure was repeated, and the final microsomal pellet resuspended in 0.25 M sucrose. Cytochrome b5 content was assayed spectrophotometrically as described under Materials and Methods. a Not detectable.

HUMAN

167

P450 2El TABLE

III

The Effect of Cytochrome b5 on NDMA Demethylase Activity Bound b, (nmol/mg)

Conditions A. TK-143-vZEl-vOR TK 143~v2El-vOR, TK 143.v2El-vOR, TK -143.v2El-vOR, B. TK-143.v2El-vOR TK-143.v2El-vOR, 37°C preincubation TK-143.v2El-vOR, 0°C preincubation TK 143.v2El-vOR, 37°C preincubation TK- 143.v2El-vOR, 37°C preincubation

Activity (pmol/min/mg) 14

fb, (0.8 PM) +b, (8.0 PM) +b, (23 PM)

0.14

49

0.8

58 66

2.1

12 +b, (rat) 31 +b, (rat) 24

+b, (rabbit) 32

+ trypsin

b, 11

Note. In experiment A, increasing amounts of rat liver b5 (final concentration shown in parentheses) were incubated with TK-143.v2E1vOR microsomes by the method described in Table II. The bl, incorporation of these microsomes is demonstrated in Fig. 3 (lanes C, D, E, and F) and quantitated by the dithionite reduced vs oxidized difference spectrum. In experiment B, microsomes and b, (2 pM or 0.8 nmol/mg microsomal protein) were mixed together in a small volume (-20 ~1) and incubated for 13 min at either 37 or 0°C. Then, the other components of the NDMA reaction were added, and NDMA demethylase activity was assayed.

drophobic tail of b5 is necessary for membrane insertion (40). It also suggests that the observed stimulation is not the result of a nonspecific protein or heme effect. The result in experiment B, demonstrating that preincubating b5 and microsomes at 0°C resulted in less stimulation of NDMA metabolism than at 37”C, further suggests that the stimulation is dependent on membrane bound bg. Previous studies have demonstrated that greater amounts of bs are incorporated into microsomes at 37°C than at 0°C (40, 26). The results in Table III clearly demonstrate that b5 can increase P450 2El-dependent metabolism of NDMA and that the stimulation requires insertion of b, into the microsomal membrane where the hemoprotein can interact with P450 2El or NADPH:P450 oxidoreductase or both. Kinetic parameters of NDMA and NMBzA metabolism. The substrate dependence of NDMA demethylation with expressed human P450 2El is shown in Fig. 4 and Table IV. Simple Michaelis-Menten kinetics were observed. Hep G2-v2El microsomes showed a low K,,, value that is within the range reported for rat (14) and human liver microsomes [Table IV and Ref. (13)]. Addition of b, to the microsomes increased the V,,, of the reaction 1.6.fold, but had no effect on the K, of NDMA demethylation. The K, for NDMA demethylation with TK-143-v2El microsomes was slightly higher; incorporation of b, into these microsomes lowered the K,,, to a

168

PATTEN

250

Km value was decreased by the addition of b5. Substitution

1

200 ;150 2 5 100 :a. 50 0 0

20

40

60

[NDMA]

80

100

120

PM

using Hep FIG. 4. Substrate dependence of NDMA demethylation G2-v2El micrasomes in the presence and absence of exogenously added cytochrome b,. The assay method is described in the note to Table IV and under Materials and Methods. The K,,, values were 22 and 20 pM, respectively, in the presence (0) and absence (0) of b,. The V,,,,, values were 278 and 158 pmol/min/mg in the presence and absence of b,, respectively.

value comparable to that of Hep G2-v2El. The b5 effect on the I’,,,,, with TK-143 microsomes, producing a 2.2fold increase, was greater than the effect seen with Hep G2 microsomes. Subjecting Hep G2-v2El microsomes to several freeze-thaw cycles, a condition to which the samples were subjected in some of our earlier studies, increased the K, for NDMA demethylation. However, the

TABLE Kinetic

ET AL.

of ferrous b5 with manganese bg, a form of b5 which cannot be enzymatically reduced by either the NADH or the NADPH pathway (41), was shown to decrease the V,,, and increase the Km for NDMA metabolism by TK-143v2El microsomes (Table IV). This provides evidence that the changes in K,,, and V,,, produced by b5 are dependent on the electron-transferring properties of b5 and not just on the physical presence of the hemoprotein. Purified rat P450 2El and purified NADPH:P450 oxidoreductase were incorporated into control Hep G2 microsomes by applying the same techniques used for incorporating b5. Evidence for the incorporation is provided by the immunoblot in Fig. 1, lane G. Purified P450 2El reconstituted in this manner shows a Km for NDMA demethylation that was three times higher than that with expressed P450 2El in Hep G2 cells (Table IV). The b5 effect on the kinetics of NDMA demethylation with purified P450 2El bound to microsomes was shown to mimic the effects seen with expressed P450 2E1, i.e., the V,,,, was increased, whereas the K,,, was not significantly affected. On the other hand, b5 showed a very large effect in reducing the Km when P450 2El and NADPHP450 oxidoreductase preparations (with glycerol removed from the buffer by dialysis) were reconstituted in the presence of phospholipid (Table IV). This effect is similar to our previous results with the reconstituted system in the presence of glycerol (11). In the presence of b,, however, the observed Km is several-fold lower than that observed previously (11).

IV

Parameters of NDMA Demethylation Catalyzed by Expressed Human 2E1 in the Presence and Absence of Cytochrome b5 V,,, bmol/min/mg)

Km (FM) Conditions HepGB-v2El TK-143-v2El-vOR HepG2-v2El frozerib TK-143-v2El-vOR HepG2 + purified 2Eld t reductase Reconstituted systeme (without glycerol) Human liver microsomes

Without

added b,

21.7 + 1.5 31.0 * 2.9 33.5 33.7

With added bs 22.3 + 0.6 19.0 * 3.5” 23.3 55.8’

With added bs

b5 Effect on V,,,

173 k 16 34+- 5 163 34

277 zk 14 75% 8 200 21’

1.6 2.2 1.2 0.6

Without

added b,

66

59

134

245

2.4

2462 16.3

49

2050 6000

2300

1.1

Note. Incubation conditions are the same as those described in the note to Table I and under Materials and Methods for NDMA metabolism. Rat liver b, was incorporated into cell microsomes according to the method described in (B) of Table III; i.e., high-speed centrifugation to remove unbound b, was not carried out. NDMA concentrations were 10, 20, 30, 60, 100, 500, 1000, 4000, and 20,000 pM. Correlation coefficients were between 0.992 and 0.999. ’ P < 0.01 in comparison to those without added b,; n = 3 with both Hep G2 and TK 143 microsomes. b Cell microsomes were subjected to four freeze-thaw cycles. ’ Instead of Fe-b,, Mn-b, was used. d Purified rat P450 2El and NADPH:P450 reductase were incorporated into control Hep G2 microsomes by the method described for b:, incorporation. Unbound P450 2El and reductase were removed by high speed centrifugation. ’ Standard reconstituted system using purified components. Details are described under Materials and Methods.

VACCINIA

VIRUS-EXPRESSED

The substrate dependence of NMBzA oxidation by Hep G2-v2El microsomes is presented in Fig. 5. The results show that P450 2El can metabolize NMBzA with apparent K, and V,, values that are comparable to those of NDMA metabolism, indicating a high specificity toward this substrate by human P450 2El. Upon the addition of changed, whereas the bg, the V,,, was not significantly K,,, was slightly decreased.

HUMAN

169

P450 2El

250 (

iv--

---

200

DISCUSSION In the present study we describe the expression of human P450 2El in mammalian cells using the vaccinia virus expression system. Evidence which substantiates the authenticity of the expressed P450 2El is as follows: First, a typical P450 spectrum was observed. Second, immunoblot experiments showed that the expressed protein was translocated to the endoplasmic reticulum membrane and had the same mobility on SDS-polyacrylamide gels as P450 2El from human liver microsomes. Finally, expressed P450 2El was shown to possess full catalytic activity toward substrates that are known to be mainly metabolized by P450 2El in the liver. Control Hep G2 and TK-143 microsomes did not show significant activity toward any of the substrates tested, nor was there any indication of P450 2El from the immunoblots, thus demonstrating the lack of endogenous P450 2El in these cell lines. Human P450 2El expressed in Hep G2 cells demonstrated a K,,, for NDMA demethylation that was well within the range recently reported for liver microsomes (13, 14). This result provides direct evidence that 2El is the low K,,, NDMA demethylase isozyme. The vaccinia virus system is appropriate for this experiment because it allows for the insertion of the P450 molecule into mammalian endoplasmic reticulum where it can assume its native conformation. Thus, artifacts present in the reconstituted system which might distort kinetic parameters can be eliminated. Of further importance is the observation that expressed P450 2El showed an apparent low Km (high affinity) for the metabolism of NMBzA, a well-known esophageal carcinogen. The activation of NMBzA can occur through two pathways: debenzylation, which leads to the formation of benzaldehyde and a methylating species, and demethylation, which leads to the formation of formaldehyde and a benzylating species. With the present assay system, 14C-labeled benzaldehyde was quantitated together with benzalcohol and benzoic acid as one peak in HPLC. Although these three compounds are the products of the debenzylation reaction, some of the products detected could be originated from the benzylating species generated in the demethylation pathway. Therefore, the observed apparent Km could comprise kinetic parameters from both the debenzylation and the demethylation reactions. Because the present method is much more sensitive than

1

0 0

100

200 [NMBzA]

i---

i.--

300 PM

400

_

I

500

FIG. 5. Substrate dependence of NMBzA metabolism using Hep GZv2El microsomes with and without exogenous cytochrome b,. The assay method is described under Materials and Methods. The amount ofprotein used in the incubation was 50 pg. The incubation time was 15 min. The substrate concentrations used were 20, 50, 100, 200, and 400 pM. The K,,, values in the presence (0) and absence (0) of bs were 27 and 47 FM, respectively. The V,., values in the presence and absence of b5 were 239 and 213 pmol/min/mg, respectively.

that used previously (42), metabolism of low concentrations of NMBzA could be studied and the K,,, value observed was lower than previously estimated. Strittmatter and colleagues have demonstrated that a large excess of amphipathic b5 can be bound to microsomal membranes in vitro (39). Several criteria, such as the ability to undergo rapid reduction by NADH:b5 reductase (43) and the resistance to intermembrane transfer (44), demonstrated that the extra-bound b5 possessed all the properties of endogenous b5. In the present study we have used previously described methods to insert amphipathic b5 into cell microsomes. The results show that the extrabound bf, can be reduced enzymatically and can accelerate 2El-dependent NDMA demethylation. The method we have chosen to incorporate b5 into cell microsomes is viewed as an alternative approach to coexpressing b, as a recombinant vaccinia virus (21). However, in view of the above mentioned studies, it is suggested that the extrabound b5 is incorporated in its native conformation and that the b5 effects observed with cell microsomes are reflective of how endogenous b5 effects 2El-dependent NDMA demethylation in liver microsomes. The effects of cytochrome b5 exogenously bound to cell microsomes on the kinetic parameters of NDMA demethylation are somewhat different from those observed with the reconstituted system in which b:, drastically decreased the Km. With Hep G2 microsomes, the bound b, did not decrease the Km, but slightly increased the V,,,. With TK-143 microsomes, which were shown not to contain endogenous bg, the added b5 decreased the K,,, and increased the V,,, more than twofold. It is possible that in Hep G2 microsomes, the levels of endogenous b5 are enough to maintain the low Km form of P450 2El. The

170

PATTEN

observed decrease in the K,,, of the TK-143 microsomes suggests that b5 either directly affects the reversible binding of NDMA to the active site of P450 2El or through its electron-transferring properties affects one of the rate constants which contribute to the Km value. Our studies using manganese b5 suggest that the electron-transferring capacity of b5 is important in affecting the K,,, values. This interpretation, together with the observation of the increase in the V,,,,, by br,, is consistent with the previously demonstrated role of b, in accelerating the flow of the second electron to P450 and improving the coupling between NADPH oxidation and product hydroxylation. The K,,, value for P450 2El-dependent NDMA demethylation and the influence of b, on this parameter appear to be greatly affected by the membrane structure and lipid environment surrounding this enzyme system. In rat and human liver microsomes (13, 14), Hep G2v2E1, and b, reconstituted TK-143-v2El microsomes, an apparent K, value of around 20 PM was observed (Table IV). When purified P450 2El was incorporated into the microsomal membrane of Hep G2 cells, it displayed a K,,, of 66 PM, and this value was not affected by the presence of exogenous b5 (Table IV). Similarly, exogenously incorporated b5 had only a small effect on the Km and increased the V,,, by eightfold using control TK-143 microsomes (which had no endogenous b5) enriched with purified P450 2El and NADPH:P450 oxidoreductase (results not shown). However, b5 produced a drastic effect in decreasing the K,,, in a nonmembrane reconstituted NDMA demethylase system. The microsomal membrane seems to present an environment which allows efficient interaction for catalysis between P450 2El and NADPH: P450 oxidoreductase, whereas in the nonmembrane reconstituted system such interaction is effected more effectively by b,. The K,,, is believed to be determined not only by the reversible binding of substrates to the active site of P450 2El but also by the subsequent steps in the catalysis (45). It appears that although similar activities can be demonstrated in different types of microsomes and reconstituted systems, the kinetic parameters are more sensitive to the membrane environment and are affected by the interactions among P450 2E1, NADPH:P450 oxidoreductase, and b5 in the membrane or reconstituted system. This concept is consistent with the finding that the kinetic parameter changed slightly when Hep G2-v2El microsomes were repeatedly frozen and thawed. The observation that the pH optimal for NDMA demethylation in Hep G2-v2El microsomes was slightly lower than the optimum in human liver microsomes (pH 6.7 vs 7.1) is also probably due to a difference in the membrane environment due to sample collection and storage, although other interpretations are also possible. In conclusion, the present work demonstrates that heterologously expressed P450 2El can catalyze many established P450 BEl-dependent reactions. Low K,,, activity was demonstrated for the activation of NDMA and

ET AL.

NMBzA. Cytochrome b5 was shown to affect the metabolism of both nitrosamines. ACKNOWLEDGMENTS This work was supported by NIH Grant ES 03938 and NIEHS Center Grant ES 05022. We thank Dr. Shanthi Paranawithana for helpful discussions and Dr. John Y. L. Chiang for supplying the cytochrome b5 antibody. We also thank Dorothy Wang for computer assistance and Dr. J. S. Yoo for determining the bs content of several human microsome samples.

REFERENCES 1. Guengerich, 2. Guengerich,

F. P. (1991) J. Biol. Chem. 266, 10019-10022. F. P., Kim, D. H., and Iwasaki, M. (1991) Chem. Res. Tel-id. 4, 168-179. 3. Ryan, D. E., Ramanathan, L., Iida, S., Thomas, P. E., Haniu, M., Shively, J. E., Lieber, C. S., and Levin, W. (1985) J. Biol. Chem.

260,6385-6393. 4. Koop, D. R., Morgan, E. T., Tarr, G. E., and Coon, M. J. (1982) J. Biol. Chem. 257, 8472-8480. S. A., Thomas, P. E., Ryan, D. E., and Levin, W. (1987) Arch. Biochem. Biophys. 258,292-297. 6. Koop, D. R. (1992) FASEB J. 6, 724-730. I. Brady, J. F., Lee, M. J., Li, M., Ishizaki, H., and Yang, C. S. (1988)

5. Wrighton,

Mol. Pharmacol. 33,148-154. 8. Koop, D. R. (1986) Mol. Pharmacol. 29, 399-404. 9. Brady, 3. F., Li, D., Ishizaki, H., Lee, M. J., Ning, S. M., Xiao, F., and Yang, C. S. (1989) Toxicol. Appl. Pharmacol. 100,342-349. 10. Koop, D. R., Laethem, C. L., and Schnier, G. G. (1989) Toxicol. Appl. Pharmacol.

98, 278-288.

11. Patten, C. J., Ning, S. M., Lu, A. Y. H., and Yang, C. S. (1986) Arch. Biochem. Biophys. 251, 629-638. 12. Levin, W., Thomas, P. E., Oldfield, N., and Ryan, D. E. (1986) Arch. Biochem. Biophys. 248,158-165.

13. Ishizaki, H., Yoo, J. S. H., Guengerich, F. P., and Yang, C. S. (1991) FASEB J. [Abstract

No. 66441

14. Yoo, J. S. H., Ishizaki, H., and Yang, C. S. (1990) Carcinogenesis 11,2239-2243. 15. Yang, C. S., Koop, D. R., Wang, T., and Coon, M. J. (1985) Biochem. Biophys. Res. Commun. 128,1007-1013. 16. Tu, Y. Y., Peng, R., Chang, Z. F., and Yang, C. S. (1983) Chem. Biol. Interact.

44, 247-260.

17. Yoo, J. S. H., Cheung, R., Patten, C. J., Wade, D., and Yang, C. S. (1887) Cancer Res. 47, 3373-3377. 18. Gorsky, L. D., and Coon, M. J. (1986) Drug Metab. Dispos. 14,8996. 19. Chakrabarti, S., Brechling, K., and Moss, B. (1985) Mol. Cell. Biol. 5,3403-3409. 20. Gonzalez, F. J., Aoyama, T., and Gelboin, H. V. (1991) Meth. Enzymol. 206, 89-92. 21. Aoyama, T., Nagata, K., Yamazoe, Y., Kato, R., Matsunaga, E., Gelboin, H. V., and Gonzalaz, F. J. (1990) Proc. N&l. Acad. Sci. USA 87,5425-5429. 22. Yang, C. S., Patten, C. J., Ishizaki, H., and Yoo, J. S. H. (1991) Meth. Enzymol. 206, 595-603. 23. Reinke, L. A., and Moyer, M. J. (1985) Drug Metab. Dispos. 13,

548-552. 24. Buckpitt, A. R., Rollins, D. E., Nelson, S. D., Franklin, R. B., and Mitchell, J. R. (1977) Anal. Biochem. 83, 168-177. 25. Moldeus, P. (1978) Biochem. Pharmacol. 27, 2859-2863.

VACCINIA

VIRUS-EXPRESSED

26. Cinti, D., and Ozols, J. (1975) Biochim. Biophys. Acta. 410,32%44. 27. Estabrook, R. W., and Werringloer, J. (1978) Meth. Enzymol. 52, 212-220. P. (1964) J. Biol. Chem. 239, 101828. Ozois, J., and Strittmatter, 1023. 29. Reid, L. S., and Mauk, A. G. (1982) J. Am. Chem. Sot. 104, 841845. 30. Yasukochi, Y., and Masters, B. S. S. (1976) J. Biol. Chem. 251, 5337-5344, 31. Omura, T., and Sato, R. (1964) J. BioL Chem. 239, 2370-2378. 32. Laemmli, U. K. (1970) Nature 227, 680-685. 33. Yoo, J. S. H., Ning, S. M., Patten, C. J., and Yang, C. S. (1987) Cancer Res. 47, 992-998. 34. Chiang, J. Y. L., Fisher, C. W., Steggles, A., and Tang, P. M. (1985) Biochim. Biophys. Acta 830, 11-19. 35. Aoyama, T., Korzekwa, K., Nagata, K., Gillette, J., Gelboin, H. V., and Gonzalez, F. J. (1990) Endocrinology 126, 3101-3106. 36. Yamano, S., Aoyama, T., Mcbride, 0. W., Hardwick, J. P., Gelboin, H. V., and Gonzalez, F. J. (1989) Mol. Pharmacol. 35, 83-86.

HUMAN

171

P450 2El

37. Fisher, G. J., and Gaylor, J. L. (1982) J. Biol. Chem. 257, 7455. 38. Enoch, H. G., and Strittmatter, 8981.

7449-

P. (1979) J. Biol. Chem. 254,8976-

39. Strittmatter, P., Rogers, M. J., and Spatz, L. (1972) J. Biol. Chem. 247, 7188-7194. 40. Enomoto, K., and Sato, R. (1973) Biochem. Biophys. Res. Commun. 51,1-7. 41. Morgan, E. T., and Coon, M. J. (1984) Drug Metab. Dispos. 12, 358-364. 42. Yoo, J. S. H., Guengerich, 88, 1499%1504.

F. P., and Yang, C. S. (1988) Cancer Res.

43. Rogers, M. J., and Stittmatter, 900.

P. (1974) J. Biol. Chem. 249, 895-

44. Enoch, H. G., Fleming, P. J., and Strittmatter, Chem. 254, 6483-6488.

P. (1979) J. Biol.

45. Yang, C. S., Ishizaki, H., Lee, M., Wade, D., and Fadel, A. (1991) Chem. Res. Toxicol. 4, 408-413.