THE JOURNAL OF BIOLOGICAL CHEMISTRY Vnl. 256. No. 2, Issue of January 25, pp. 1044-1052, 1981 Printed in U.S.A.

Regulation of Three Forms of Cytochrome P-450 and Epoxide Hydrolase in Rat Liver Microsomes EFFECTS OF AGE, SEX, AND INDUCTION*

(Received for publication, June 4, 1980, and in revised form, September 17, 1980)

Paul E. Thomas, Linda M. Reik, Dene E. Ryan, and Wayne Levin From the Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc., Nutley, New Jersey 07110

A procedure for the preparation of monospecific an- tibody directed against rat liver microsomal cyto- chrome P-450. is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450,, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-sol- ubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activ- ity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and ( d ) remove only the homologous antigen from deter- gent-solubilized microsomes when covalently bound to a solid support. With a radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The lev- els of these cytochrome P-450 isozymes vary independ- ently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450., P-4501,, and P- 450, in the metabolism of xenobiotics by rat liver mi- crosomes. A large proportion of benzo(a)pyrene metab- olism and testosterone 16a-hydroxylation in micro- somes from untreated rats is not catalyzed by cyto- chromes P-450,, P-450b, and P-450,.

Epoxide hydrolase, another microsomal enzyme in- volved in the metabolism of xenobiotics, was also quan- titated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The induc- tion of epoxide hydrolase varies independently of the induction of cytochromes P-450,, P-450b, and P-450,.

The liver microsomal monooxygenase system exhibits un- usually broad substrate specificity; it metabolizes endogenous substrates such as steroids, bile acids, and fatty acids and also innumerable exogenous substances such as drugs, pesticides, and carcinogens, etc. (1). The metabolism of so many struc- turally dissimilar substrates is not typical of other enzyme systems. Multiple forms of the terminal oxidase, cytochrome P-450,’ have been the favored hypothesis to explain the ver-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The term cytochrome P-450 is used to designate any or all forms of liver microsomal cytochrome P-450. In rats, cytochrome P-450,, is the major hemoprotein induced by phenobarbital; cytochrome P-450, is the predominant hemoprotein inducible by 3-methylcholanthrene, and cytochrome P-450. is a minor form induced by both compounds

satility of the liver microsomal enzyme system and differences in metabolism depending on the age, sex, strain, and treatment of an animal. This hypothesis has been substantiated by the recent isolation of several distinct forms of cytochrome P-450 from rabbits, mice, and rats (5).

Although several forms of purified cytochrome P-450 have been well characterized, there is little understanding of their regulation in tissues because of the lack of specific assays. Spectral properties, metabolic activity, or mobility on SDS- PAGE’ are not sufficiently specific characteristics to permit quantitation of one form of cytochrome P-450 in the presence of a mixture of isozymes (6). However, antibodies monospecific for a single form of cytochrome P-450 can provide the neces- sary specificity to distinguish individual forms of cytochrome P-450. Recently, we have demonstrated the potential use of a radial immunodiffusion assay with monospecific antibodies to quantitate cytochromes P-450b and P-450, in rat liver micro- somes (7).

In this study, we describe the preparation of antibody monospecific for cytochrome P-450,. Monospecific antibodies to cytochromes P-450,, P-4501,, P-450,, and epoxide hydrolase are used as probes to examine the regulation and quantitation of these enzymes in rat liver microsomes.

EXPERIMENTAL PROCEDURES

Chemicals-PB was obtained from Merck & Co., Inc. and MC and sodium cholate were purchased from Sigma Chemical Co. Trans- stilbene oxide and y-chlordane were purchased from Aldrich Chemical Co., Inc. and Velsicol Chemical Corp., respectively. Pregnenolone- l6a-carbonitrile was a gift from G. D. Searle and Co., and Aroclor 1254 (Lot KC-12-638) was a gift of Monsanto Co. SKF-525A (2- diethylaminoethyl 2,2-diphenylvalerate hydrochloride) was obtained from Smith Kline and French Co., and isosafrole was from Eastman Kodak Co. Corn oil was purchased from Matheson, Coleman, and Bell. Agarose (Sea Kem medium electroendosmosis and low elec- troendosmosis) was purchased from Marine Colloids Inc. Emulgen 911 was obtained from Kao-Atlas Ltd., Japan. [7,8-”Hloctene 1,2- oxide was a &t of Dr. D. M. Jerina. [7-’H]testosterone, [4-14C]- testosterone, and [N-methyl-14C]benzphetamine hydrochloride were purchased from New England Nuclear. Morphine (free base) was purchased from Mallinckrodt Chemical Works.

Xenobiotic Treatment of Rats and Preparation of Hepatic Micro- somes-Long Evans rats from Blue Spruce Farms, Altamont, NY were maintained on 12-h light cycles every 24 h in wire bottom cages x on corn cob bedding with water and Ralston Purina Rodent Chow 5001 ad libitum. Rats were either immature (50 to 60 g) or sexually

(2). Cytochrome P-450, is also known as cytochrome P-448 (3) and cytochrome P,-450 (4).

a The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; PB, sodium phenobarbital; MC, 3-methylcholanthrene; SKF-525A, 2-diethylaminoethyl 2.2-diphen- ylvalerate hydrochloride.

1044

Regulation of Cytochrome P-450 and Epoxide Hydrolase 1045

mature adults (150 to 190 g). The following xenobiotics were admin- istered by intraperitoneal injection on four consecutive days and the rats were killed by decapitation 24 h after the last injection: PB (75 mg/kg) in water, MC (25 mg/kg) in corn oil, pregnenolone-l6a- carbonitrile (25 mg/kg) in corn oil, trans-stilbene oxide (300 mg/kg) in corn oil, y-chlordane (50 mg/kg) in corn oil, SKF-525A (75 mg/kg) in water, and isosafrole (150 mg/kg) in corn oil. Aroclor 1254 (300 mg/kg), dissolved in corn oil, was given as a single intraperitoneal injection 4 days before death. Morphine was administered continu- ously by implanting a pellet containing 75 mg of morphine (free base) 96 and 48 h before sacrifice (8).

Total microsomal cytochrome P-450 was determined by the method of Omura and Sato (9) in 0.1 M potassium phosphate buffer (pH 7.4) with 20% glycerol. Cytochrome P-450 in liver microsomes from rats treated with SKF-525A was determined as previously described (10). Protein was determined by the method of Lowry et al. (11) using bovine serum albumin as standard.

Purification of Antigens-Cytochromes P-450;,, P-450t,, and P-450, were purified to apparent homogeneity from Aroclor 1254-, PB-, or MC-treated rats as described (2). PB-treated rats were used for the isolation of hepatic microsomal epoxide hydrolase to apparent ho- mogeneity (12). Microsomal NADPH-cytochrome P-450 reductase (NADPH-cytochrome c reductase) was purified from the livers of PB-treated rats to a specific activity of 40,000 units/mg of protein using the assay conditions of Phillips and Langdon (13) a t 22°C. The reductase was purified by the procedure of Dignam and Strobe1 (14) through and including the DEAE-Sephadex A-25 chromatography step, and purification was completed by affinity chromatography on 2',5"ADP-Sepharose 4B as described by Yasukochi and Masters (15).

Preparation of Monospecific Antibodies to Liuer Microsomal Epoxide Hydrolase, NADPH-Cytochrome P-450 Reductase, Cyto- chrome P-45&, and Cytochrome P-450,"Antibody against epoxide hydrolase was produced in sheep and has been previously character- ized (16). Antibodies against cytochromes P-450t, and P-450, were raised in rabbits as previously described (17) except the antigens were administered at 30 to 40 intradermal sites instead of 3 sites. Purified immunoglobulin G, used in the present study, was isolated (17) from a pool of high titer antisera from one sheep or a t least six rabbits bled monthly for 6 months or more. Anti-NADPH-cytochrome P-450 reductase was produced in sheep as described for cytochrome P-450 (17).

Monospecificity of antibodies to cytochromes P-45o1, and P-450, was ensured by the immunoabsorption technique recently described (7). Briefly, anti-P-450, was absorbed with solid phase crude cyto- chrome P-450 from PB-treated rats, and anti-P-45Ot, was absorbed with crude cytochrome P-450 from MC-treated rats.

Preparation of Monospecific Antibody to Cytochrome P-450a- Antibody to purified cytochrome P-450, was produced in sheep as described (17). Despite the high purity of cytochrome P-450, from Aroclor 1254-treated rats (2, 18), the antibody produced against this protein showed trace recognition of at least one other microsomal protein on Ouchterlony double diffusion plates. The following proce- dure was used to obtain monospecific antibody to cytochrome P-450,. The contaminating antigen was obtained by chromatographing liver microsomes from Aroclor 1254-treated rats which had been solubilized with detergent on a Whatman DE-52 column a t room temperature using the conditions originally described by Warner et al. (19). The column fractions were screened for cytochrome P-450. and the con- taminating antigen using the Ouchterlony double diffusion technique with anti-P-450. and purified cytochrome P-459 as standard. The fust major peak of protein eluting from the column contained the contaminating antigen but little or no cytochrome P-450,,. This frac- tion was dialyzed and bound to Sepharose 4B using cyanogen bromide (7). Antiserum to cytochrome P-450,, in 10 mM potassium phosphate, pH 7.4, 0.2 M KCI, and 0.2 mM EDTA, was passed through a column of covalently bound contaminating antigen, and fractions were col- lected (7). The eluting fractions which were devoid of antibody to the contaminating protein were pooled, and the IgG fraction was purified as described (17).

Immunochemical Procedures-The Ouchterlony double diffusion plates contain the following reagents (expressed as final concentra- tions) which are combined before the pH is adjusted to pH 7.4 with NaOH: 1.0 M glycine, 10 mM potassium phosphate, 0.2 mM EDTA, 86 mM NaCI, and 15 mM sodium azide. Sea Kem medium electroendos- mosis agarose (0.9% final concentration) is added to this solution which is heated to boiling with a reflux condenser attached to the flask until no trace of agarose can be detected. Emulgen 911 is added to a final concentration of 0.2%. Petri dishes (21.5 cm2 area) are filled

with 3 ml of hot agarose solution and allowed to cool. Wells of 4-mm diameter and with 8-mm center to center spacing were punched in solidified agarose and loaded with 12 pl of an appropriate sample. Immunodiffusion was performed at room temperature in a humid atmosphere on a level surface for 2 days or more.

Immunoaffinity purification of cytochrome P-450,, from rat liver microsomes was performed as previously described for cytochromes P-450h and P-450, (7).

Radial immunodiffusion for quantitation of cytochrome P-450 and epoxide hydrolase was performed as previously described (7) with the following modifications. The radial immunodiffusion plates were pre- pared as described above for Ouchterlony double diffusion analysis with the addition of 0.5% sodium cholate and 5%) glycerol (final concentration) to the gel solution. Low electroendosmosis agarose (Sea Kem LE) was substituted for medium electroendosmosis agarose (Sea Kem ME). Liver microsomes were solubilized as described (7) and four different concentrations (in duplicate) of liver microsomes and purified cytochrome P-450 standards were used for each immu- nodiffusion assay. Radial immunodiffusion precipitin rings (ring di- ameter squared minus well diameter squared) varied linearly with dilutions of either standards or solubilized microsomes. All determi- nations on liver microsomes were performed a t least twice, and certain microsomal preparations were included in each experiment to verify the reproducibility of the results.

Enzyme Assays-The metabolism of benzo(a)pyrene (20). [N- methyl-'4C]benzphetamine (17), and [4-"C]testosterone in the 68, 7a, and 16a positions (21) was measured as previously described. In some experiments, 7a- hydroxylation of testosterone was measured by the release of tritiated water from [7-.'H]testosterone. Tritiated testoster- one, nominally labeled in the 7-position, was diluted to 69 mCi/mmol with unlabeled testosterone. The substrate was purified by thin layer chromatography with 6 8 methanol in toluene as solvent. The UV spot corresponding to testosterone was scraped from the plate and the gel was extracted with acetone. The sample was centrifuged to remove traces of silica gel, and the acetone was removed and evapo- rated to dryness. Testosterone was dissolved in methanol and ad- justed to a final concentration of 6.25 IIIM based on its absorbance at 242 nm ( E = 16,040 M ' cm"). Since the distribution of tritium between the 701- and 70-positions is not precisely known and varies among different lots of [7-:'H]testosterone, absolute turnover numbers cannot be obtained with this assay. However, in a double label experiment with [4-14C]testosterone. and subsequent separation of testosterone metabolites (21), it was determined that a 20% release of tritium occurred at the 7a-position with our sample of tritiated substrate. Although tritium is present in both the 7a- and 7[l-position of [7-:'H]testosterone, previous studies with rat liver microsomes have failed to detect 7P-hydroxytestosterone as a metabolite (21, 22). The assay method for quantitation of tritiated water was performed ex- actly as described for zoxazolamine (23). All enzyme assays were under conditions of constant product formation with respect to time and protein. In the reconstituted system, saturating concentrations of lipid (dilauroylphosphatidylcholine) and NADPH-cytochrome P-450 reductase were used, with limiting amounts of cytochrome P-450 (2, 17). Dilauroylphosphatidylcholine was prepared in 0.02 M Tris-HC1 (pH 8.0) containing 1 mM EDTA and sonicated immediately before use.

When inhibition of metabolism by antibodies was studied, all assay tubes were brought to the same final concentration of IgG with control IgC. Microsomes were preincubated with IgG for 10 min at 22°C in 0.25 ml of phosphate-buffered saline (pH 7.4) before addition of cofactors and substrate. In the reconstituted system, cytochrome P-450 was first exposed to antibody for 5 min at 22"C, NADPH- cytochrome P-450 reductase and lipid were added, followed by an additional 5-min incubation a t 22°C. After this preincubation, the tubes were returned to ice and all necessary cofactors and buffer were added. Reactions were initiated by the addition of substrate and incubated a t 37°C.

The metabolism of [7,8-"H]octene 1.2-oxide (2 Ci/mol) by micro- somal epoxide hydrolase was determined as previously described (24).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis- SDS-PAGE was performed in 7.5% acrylamide gels by the method of Laemmli (25). A slab gel apparatus (Hoefer Scientific Instruments) was used to form a slab gel (0.75 mm X 10 cm X 15 cm). The protein in gels was fixed in a solution of 25% isopropanol and 10% acetic acid (1 h), stained in the same solution containing 0.05% Coomassie brilliant blue R250 (1 h), and destained in 108 isopropanol and 10% acetic acid (14 h).

1046 Regulation of Cytochrome P-450 and Epoxide Hydrolase

RESULTS

Characterization of Monospecific Antibody Against Cytochrome P-450.

Although cytochrome P-450, from Aroclor 1254-treated rats is highly purified (2, 18), anti-P-450, recognizes a t least one other microsomal protein. It is known that antibodies can be produced to contaminating protein present at no more than 1% of the total protein concentration if the contaminant is highly antigenic (26). Microsomal proteins differ greatly in antigenicity (27), and antibodies should not be assumed to be monospecific based solely on the apparent punty of the anti- gen of immunization. The antibody titer to the contaminant was very low relative to the titer against cytochrome P-450.. The lack of monospecificity of anti-P-450. was only apparent when double immunodiffusion analysis was performed under highly sensitive conditions as described under “Experimental Procedures.” A suitable immunoabsorbent for making anti-P- 450. monospecific was obtained by fractionating liver micro- somes from Aroclor 1254-treated rats on a DEAE-cellulose column at room temperature (19). The protein fraction which did not bind to DEAE-cellulose was used as the immunoab- sorbent described under “Experimental Procedures.”

An Ouchterlony double diffusion analysis of anti-P-450, with several antigens is shown in Fig. 1. Monospecific anti-P- 450. reacts with microsomes from control, PB-, or MC-treated rats to give a single well defined immunodiffusion band which shows immunochemical identity with purified cytochrome P- 450.. The formation of a single immunodiffusion band when anti-P-450. reacts with a complex mixture of antigens (liver microsomes) suggests that the antibody is reacting with only one protein and is therefore, monospecific. Fig. 1 provides additional evidence that anti-P-450. is monospecific by show- ing that two other forms of rat liver cytochrome P-450, P-4501, and P-450,. do not cross-react with anti-P-450,. In a radial immunodiffusion assay, cytochromes P-4501, and P-450,. do not cross-react with anti-P-450, a t a concentration 64 times greater than the minimum detectable concentration of cyto- chrome P-450,. These results indicate that cytochrome P-450, is immunochemically distinct from cytochromes P-4501, and P-450, and provide additional evidence for the specificity of anti-P-450..

FIG. 1. Ouchterlony immunodiffusion analysis of the speci- ficity of antibody to cytochrome P-450.. The central two wells were filled with IgC (25 rng/rnl) against cytochrome 1’-450,,. Cyto- chromes I’-450., (2.2 p ~ ) , P-450,. (5.0 p ~ ) , and P-450,. (5.0 p ~ ) purified from Aroclor 1254-treated rats were in the surrounding wells as indicated. Liver microsomes solubilized with detergent (10 p~ cyto- chrome 1’-450) were from untreated (control) rats or rats treated with MC or I’B as labeled. All wells in the agarose gel were 4 mm diameter except for wells containing microsomes from PB and control rats which were 5 mm in diameter.

“9

A B C . D “4

FIG. 2. SDS-PAGE of immunoaffinity purified cytochrome P-450.. Well A contains 7 pg of liver microsomal protein from MC- treated rats. Well B contains 0.3 pg of authentic cytochrome P-450,,, and well C contains an equivalent amount of immunoaffinity purified protein. Well D contains 0.5 pg of sheep IgG.

FIG. 3. Double immunodiffusion analysis of rat liver micro- somes from Aroclor 1254-treated rats using antibodies against five microsomal enzymes. Liver microsomes ( L i t w Micro.) were solubilized with detergents (5) and placed in the wells at a final concentration of 10 p~ cytochrome P-450. Additional NADPH-cyto- chrome P-450 reductase was added to the microsomal preparation because this enzyme is normally present in insufficient amount to form a well defined immunoprecipitin band. Immunoglobulin C prep- arations, monospecific for the following microsomal enzymes, were placed in the indicated wells: three distinct forms of cytochrome P-450 (Anti-P-450,. Anti-P-450*, and Anti-P-450,.), epoxide hydro- lase (Anti- E H ) . and NADPH-cytochrome P-450 reductase (Anti- Reduc.).

Immunoaffinity chromatography provides the most critical test of the specificity of anti-P-450. (Fig. 2). Liver microsomes from MC-treated rats (well A) were solubilized with detergent and slowly passed through a column of anti-P-450, covalently

Regulation of Cytochrome P-450 and Epoxide Hydrolase 1047

bound to Sepharose. Proteins recognized by the antibody were eluted with KSCN, as described previously for other forms of cytochrome P-450 (7), and were electrophoresed by SDS- PAGE as shown in Fig. 2. One major polypeptide eluted from the column (well C) and this protein-staining band is identical in mobility with highly purified cytochrome P-450, (well B). Immunoaffinity purified cytochrome P-450, (well C) contains a small amount of a polypeptide with a molecular weight larger than cytochrome P-450,. This contaminating poly- peptide is identical in molecular weight with the protein stripped by KSCN from a control IgG Sepharose immunoaf- finity column and is identified as the heavy chain of IgG by its mobility. Other investigators have also reported on the lability of the covalent bond formed between protein and CNBr-activated Sepharose (28, 29).

By the above criteria, antibodies to cytochromes P-450b and P-450,. are also monospecific (7) as well as antibody to micro- somal epoxide hydrolase (16, 30). These antibodies together with a fifth antibody, anti-NADPH-cytochrome P-450 reduc- tase, are compared in the immunodiffusion plate in Fig. 3. Each antibody reacts with its homologous antigen in liver microsomes from Aroclor 1254-treated rats giving a single immunodiffusion band. There is no detectable immunochem- ical relatedness among the five microsomal proteins as evi- denced by the lack of interaction of the precipitin bands.

"A ' I 1 1 -

Ant1-P450c -G-G

"0"" """"_"""____~ - '. Anti - P 4 5 0 b

3 i? z 0 V 6ol \ \

\ \ - ' \ Anti -P450a

0 , I I - - - - - - - - _ - A -

0 2 4 6 8 IO 12 14 16 rng ANTIBODY/nrnole CYTOCHROME P450

Anta- P 4 5 0 0

-&--A" ""-0""- """_1,-= --n 100 ""-

Antl -P450b

c Benzo[a]pyrene hydroxylase

Specificity of Antibody Inhibition of Catalytic Activity

Results of Ouchterlony double diffusion analysis and im- munoaffinity chromatography have demonstrated a lack of immunological cross-reaction among cytochromes P-450,, P- 450h, and P-450,. Monospecificity of anti-P-450,, anti-P-450h, and anti-P-450, was further tested by the ability to inhibit enzymatic activity of the homologous and not the heterolo- gous antigens. A cross-reaction of antibodies with heterolo- gous antigens could result in inhibition of catalytic activity without immunoprecipitation. Cytochromes P-450,, P-450b, and P-450, are efficient catalysts of the 7a-hydroxylation of testosterone, the N-demethylation of benzphetamine, and the metabolism of benzo(a)pyrene, respectively (2). The inhibi- tory properties of the three monospecific antibodies toward the catalytic activity of cytochromes P-450,, P-450,,, and P- 450, are presented in Fig. 4, A to C, respectively. In Fig. 4A, anti-P-450, specifically inhibits 7a-hydroxylation of testoster- one catalyzed by cytochrome P-450,, while the heterologous antibodies, anti-P-450h and anti-P-450,, have no effect on this reaction. Anti-P-450,,, but not anti-P-450, or anti-P-450,. (Fig. 4B), effectively inhibits benzphetamine N-demethylation cat- alyzed by cytochrome P-450h. Fig. 4C shows that anti-P-450,. completely inhibits the metabolism of benzo(a)pyrene by cytochrome P-450,, but this reaction is insensitive to the

O t

Benzphetarnme N-demethylation

\ Antl -P450b

o 1 I I I I I I I 1

0" " -~ - - - - - "" 0- """"""""""o

0 2 4 6 8 IO 12 14 16 rng ANTIBODY/nrnole CYTOCHROME P 4 5 0

FIG. 4. The effects of three monospecific antibodies on the catalytic activity of purified cytochromes P-450 in the recon- stituted system. The amount of each antibody is expressed in milligrams of antibody per nmol of cytochrome P-450 to normalize for different cytochrome P-450 concentrations among the three as- says. The 7a-hydroxylation of testosterone (A) , benzphetamine N - demethylation ( B ) , and benzo(a)pyrene metabolism ( C ) was cata- lyzed by cytochromes P-450., P-450,,, and P-450,., respectively. The assays for catalytic activity are described under "Experimental Pro- cedures."

rng ANTIBODY/nrnole CYTOCHROME P 4 5 0

1048 Regulation of Cytochrome P-450 and Epoxide Hydrolase

presence of anti-P-4501, and anti-P-450,. These results show the marked specificity of the three monospecific antibodies for their respective antigens.

TABLE I Immunochemical quantitation of three forms of hepatic

microsomal cytochrome P-450 from immature male rats treated with different inducers

Total cytochrome P-450 was estimated spectrally as described under "Experimental Procedures." Each determination was made on the liver microsomes from a pool of eight rat livers. Numbers in parentheses give the amount of cytochrome P-450 as specific content in nanomoles of cytochrome P-450 per mg of protein. Unknown cytochrome P-450 is derived from the arithmetic difference between 100'%, and the sum of cytochromes P-454, 1'-4501,, and P-450..

I'er cent of total cvtochrome 1'-450

nmlJ//mfi pn~le ln

Untreated (con- 0.77

Corn oil 0.86 Phenobarbital 2.0 3-Methylcholan- 1.9

Aroclor 1254 3.5 y-Chlordane 1.9 Trans-stilbene 1.6

Pregnenolone 16a- 1.1

2.3

trol)

threne

oxide

carbonitrile SKF-525A Momhine 0.86

6 (0.05)

6 (0.05) 7 (0.14)

11 (0.21)

7 (0.25) 4 (0.08) 4 (0.06)

5 (0.06)

4 (0.09) 6 (0.05)

2 (0.02)

2 (0.02) 57 (1.14)

1 (0.02)

36 ( 1.26) 46 (0.87) 47 (0.75)

3 (0.03)

35 (0.81) 2 (0.02)

3 (0.03)

4 (0.03) 2 (0.04)

77 (1.46)

39 (1.37) 3 (0.06) 2 (0.03)

2 (0.02)

4 (0.09) 4 (0.03)

89

88 34 11

18 47 47

90

57 88

PL50( FL5Oc

P150( PL50a

P L 5 0 b v - " - PL50t: EH

"

A B C U E F G H I FIG. 5 (left). SDS-PAGE of liver microsomes from rats

treated with various xenobiotics. Samples contain 7 pg of micro- somal protein from corn oil-treated rats (well B) , rats treated with morphine (well C ) , y-chlordane (well D), SKF-525A (well E ) , trans- stilbene oxide (well F), and pregnenolone 16a-carbonitrile (well G ) . Microsomes from untreated rats are in well H. Wells A and Z contain a mixture of purified cytochromes P-450,. P-45Oh. and P-450,. (0.25 pg each) in ascending order, respectively.

FIG. 6 (right). SDS-PAGE of rat liver microsomes comparing

Quantitation of Liver Microsomal Cytochrome P-450 and Epoxide Hydrolase

Effect of Xenobiotic Treatment-Xenobiotic treatment of animals markedly affects hepatic drug-metabolizing capacity, but these effects could not previously be correlated with levels of specific forms of cytochrome P-450. Monospecific antibod- ies against cytochromes P-450., P-4501,, and P-450,. can be used in radial immunodiffusion assays to quantitate the re- spective antigen in microsomal preparations. Electrophoreti- cally homogeneous forms of cytochrome P-450 (2) were used as standards in the assay. The levels of the three forms of cytochrome P-450 in liver microsomes from rats treated with different compounds are shown in Table I. Corn oil was used to administer some of the inducers, and the data shows that the levels of cytochrome P-450 are not significantly altered by corn oil. PB induces the specific content of total liver micro- somal cytochrome P-450 approximately 2.5-fold and selec- tively induces cytochrome P-4501, more than 50-fold. y-Chlor- dane, trans-stilbene oxide, and SKF-525A have similar but less marked effects than PB on cytochrome P-4501, levels per mg of protein. MC treatment results in a dramatic increase in cytochrome P-450,. whether measured as a percentage of the total cytochrome P-450 or as nanomoles of cytochrome P-450,. per mg of protein. Aroclor 1254, a polychlorinated biphenyl mixture, preferentially induces cytochromes P-45O1, and P- 450, and also increases the specific content of cytochrome P- 450. from 0.05 to 0.25 nmol/mg of protein. PB and MC also induce cytochrome P-450, but the increase is considerably less than increases in cytochromes P-4501, and P-450,.. Pregneno- lone 16a-carbonitrile has no effect on levels of these three forms of cytochrome P-450 although total cytochrome P-450

"" .. . ... - .... " _"... m i e ... -

Female

age, sex, and xenobiotic treatment. The microsome samples (5 pg of protein) were prepared from control rats (CONT) or rats treated with PB, MC, or Aroclor 1254 (AR). In each pair of gel samples (underlined), microsomes from immature rats are shown on the left and microsomes from adult rats are shown on the right. Only imma- ture male rats were used for Aroclor 1254 treatment. The standard sample ( S ) is a mixture of purified P-450.. epoxide hydrolase ( E H ) , cytochrome P-450,,, and cytochrome P-450, (0.5 pg each) in ascending order, respectively.

Regulation of Cytochrome P-450 and Epoxide Hydrolase 1049

is increased. Morphine treatment had no significant effect on rat liver microsomal cytochrome P-450.

Interestingly, unknown forms of cytochrome P-450, z.e. im- munochemically distinct from cytochromes P-450., P-450b, and P-450,, predominate in untreated, corn oil-, pregnenolone 16a-carbonitrile-, and morphine-treated rats. The present re- sults (Table I) do not address unknown forms which may be selectively induced by any of the xenobiotics used in this study, since not all forms of rat liver cytochrome P-450 have been purified and characterized.

The changes in levels of cytochromes P-450,, P-450t,, and P-450, after treatment with various compounds (Table I) are supported by the elect.rophoretic profile of these microsomal preparations in SDS-PAGE. Profiles of the protein-staining bands obtained from these microsomes appear in Figs. 5 and 6. Fig. 5 shows that y-chlordane, trans-stilbene oxide, and SKF-525A induce a protein(s) with the same molecular weight as cytochrome P-450b. Microsomes from morphine- and corn oil-treated rats do not differ in electrophoretic pattern from the profile of microsomes from untreated rats which supports the results in Table I. The only inducers which increase levels of cytochrome P-450, are MC and Aroclor 1254 (Fig. 6). As shown in Table I, only three inducers increase the specific content of cytochrome P-450, more than 2-fold (PB, MC, and Aroclor 1254) and this selective increase is supported by the results of SDS-PAGE (Fig. 6).

Epoxide hydrolase, a microsomal enzyme functioning in sequence with the cytochrome P-450-dependent monooxygen- ase system in the metabolism of alkene and arene oxides (31), can also be assayed by radial immunodiffusion in liver micro- somes from rats treated with various xenobiotics. Epoxide hydrolase concentration in microsomes was determined by a radial immunodiffusion assay using monospecific antibody to microsomal epoxide hydrolase, and enzymatic activity was determined by the rate of hydration of octene 1,Z-oxide (Table 11). Epoxide hydrolase represents approximately 2% of the total hepatic microsomal protein from untreated immature male rats when measured by radial immunodiffusion. Mor- phine and corn oil have little or no effect on levels or catalytic activity of epoxide hydrolase. Interestingly SKF-525A, which is a good inducer of cytochrome P-450, caused no significant change in epoxide hydrolase levels. The other xenobiotic treatments yield significant increases in both enzyme concen-

TABLE I1 Comparison of the immunoquantitation of expoxide hydrolase with

octene 1,2-oxide hydrolase activity in liver microsomes of rats treated with a variety of xenobiotics

Details of the epoxide hydrolase catalytic activity assay and the radial immunodiffusion assay are given under “Experimental Proce- dures.” Each determination was made on the liver microsomes from a pool of eight rat livers.

Epoxide hydrolase Rat pretreatment (concentra-

Concentra- Activity tion x 100)

Activity/

tion

Untreated Corn oil Pregnenolone l6a-carboni-

trile Trans-stilbene oxide SKF-525A y-Chlordane Morphine Phenobarbital 3-Methylcholanthrene Aroclor-1254 Isosafrole

protein

0.018 0.019 0.027

0.056 0.021 0.048 0.019 0.038 0.031 0.043 0.031

mg/mg r~mol/min/mg protein

21 22 34

72 27 64 27 51 41 60 38

12 12 13

13 13 13 14 13 13 14 12

TABLE I11 Age and sex differences in the levels of three forms of cytochrome P-450 in hepatic microsomes from rats treated with different

inducers Total cytochrome P-450 was estimated spectrally as described

under “Experimental Procedures.” Each determination was made on the liver microsomes from a pool of two to six rat livers. Numbers in parentheses give the data expressed as specific content in nanomoles of cytochrome P-450 per mg of protein. Unknown cytochrome P-450 is derived from the arithmetic difference between 100% and the sum of cytochromes P-450,, P-4501,, and P-450,.

Per cent of total cytochrome P-450 Total Un- p-450 P-450, P-450h P-450, known

P-450 -

Immature male Control Phenobarbital 3-Methylcholan-

threne

Adult male Control Phenobarbital 3-Methylcholan-

threne

Immature female Control Phenobarbital 3-Methylcholan-

threne

Adult female Control Phenobarbital 3-Methylcholan-

threne

nmoNmg protein

0.75 7 (0.05) 4 (0.03) 4 (0.03) 1.96 7 (0.14) 57 (1.12) 2 (0.04) 1.87 11 (0.21) 1 (0.02) 77 (1.44)

1.11 3 (0.03) 2 (0.02) 1 (0.01) 2.51 1 (0.03) 43 (1.08) 1 (0.03) 1.93 3 (0.06) 1 (0.02) 68 (1.31)

0.72 8 (0.06) 3 (0.02) 4 (0.03) 2.10 8 (0.17) 62 (1.30) 3 (0.06) 1.66 14 (0.23) 1 (0.02) 79 (1.31)

~

85 34 11

94 55 28

85 27 6

92 47 11

tration and catalytic activity of epoxide hydrolase, with the greatest induction by trans-stilbene oxide. The ratio of cata- lytic activity to enzyme concentration is essentially constant despite wide variation in epoxide hydrolase levels after treat- ment with these xenobiotics.

Quantitation of Liver Microsomal Cytochrome P-450

Age and Sex Differences-Hepatic drug-metabolizing ca- pacity is sensitive to age and sex differences as well as xeno- biotic pretreatment. Table I11 summarizes the results of radial immunodiffusion assays comparing sex and age differences in levels of three forms of cytochrome P-450 in microsomes from untreated, PB-, and MC-treated rats. Treatment of rats with P B or MC gives rise to a significant increase in the specific content of total cytochrome P-450 (nanomoles/mg of protein). PB or MC administration also significantly increases the specific content of cytochrome P-450, in female and immature male rats but not in adult males. MC is a better inducer of cytochrome P-450, than PB (Tables I and 111) which is con- sistent with previous observations that MC is a better inducer of the 7a-hydroxylation of testosterone than is PB (32). Cy- tochrome P-450, is lower in untreated, PB-, or MC-treated adult male rats compared to immature male rats, but this age difference is not seen in the female rat (Tables I11 and IV). The induction of cytochrome P-450 by either PB or MC is minimal, however, compared to the induction of cytochromes P-450b and P-450,. The induction of cytochrome P-45% by PB is similar in immature and adult male rats but significantly different in immature and adult female rats (Table IV). A similar pattern was observed in the induction of cytochrome

1050 Regulation of Cytochrome P-450 and Epoxide Hydrolase

TABLE IV Quantitation of cytochromes P-4.50, and P-4.506 in the livers of rats

treated with phenobarbital: effects of age and sex All values are reported as the mean of four individual rats f

standard deviation. P-450t,, adult female uersus immature female ( p < 0.01); P-450h, adult female uersus adult male ( p < 0.02); P-450., adult male uersus immature male ( p < 0.01); P-450,, adult male versus adult female ( p < 0.01).

Per cent of total cytochrome P-450

P-450” P-4501, Sex and age Total P-450

nmol/mgprotein Male

Adult Immature 2.35 f 0.34 10.6 +- 1.9 58.3 f 3.9

2.6 f 0.12 2.6 f 0.2 56.7 f 5.1

Female

Adult Immature 2.0 -t 0.14 13.2 f 0.9 60.0 -t 6.0

2.13 k 0.15 10.7 f 1.5 47.4 f 3.3

P-450, by MC but statistical analyses were not performed. The SDS-PAGE profie of these liver microsomal prepara-

tions is shown in Fig. 6. To facilitate comparisons between age groups, the first well in each pair contains microsomes from immature rats and the second sample is from the adult. All major changes in the three forms of cytochrome P-450 shown in Table I11 can be supported by changes in the staining intensity of bands with the same mobility as the corresponding standards. The most prominent increases in protein-staining bands are observed when PB or MC is used to induce cyto- chrome P-4501, or P-450,, respectively. Treatment with PB or MC gives detectable increases in cytochrome P-450, in all animals except the adult male rat. The SDS-PAGE profiie in Fig. 6 illustrates the decrease in cytochrome P-450, in the untreated adult male compared to the immature male rat. The lack of a significant age difference in cytochrome P-450, in female rats (Tables 111 and IV) can also be seen in the SDS- PAGE patterns. PB and Aroclor 1254 induce other protein bands with mobilities different than those attributable to the three forms of cytochrome P-450 identified here (Fig. 6). One of these bands in the cytochrome P-450 region (Mr = 45,000 to 60,000) is epoxide hydrolase (M, = 49,000) which is induced by PB and Aroclor 1254.

While the SDS-PAGE profies support the immunoquanti- tation data in Tables I to 111, this technique cannot be substi- tuted for immunoquantitation. Caution must be taken when interpreting the electrophoretic profiles of microsomal prep- arations since microsomal proteins other than cytochrome P- 450 are known to have minimum molecular weights in the 45,000 to 60,000 region (cf Ref. 2), and it is not known whether any protein-staining band is comprised of more than one protein.

DISCUSSION

Results of immunoaffinity chromatography, Ouchterlony double diffusion analysis, and selective inhibition of catalytic activity by only the homologous antibody support the concept that antibodies prepared against cytochromes P-450,, P-4501,, and P-450, are monospecific for their respective antigens. Consequently, these antibodies are uniquely suited to provide the necessary specificity to quantitate a single form of cyto- chrome P-450 in the presence of a mixture of forms. The possibility that another form(s) of cytochrome P-450 exists in rat liver microsomes which shows immunochemical related- ness with cytochromes P-450,, P-4501,, and P-450, cannot be eliminated. We have previously shown the potential of the high specificity of anti-P-450b and anti-P-450, to assay for the corresponding antigens in rat liver microsomes (7). These

studies have been extended to include immunoquantitation of cytochrome P-450, in rat liver microsomes and the effects of age, sex, and xenobiotic treatment on the levels of these three cytochrome P-450 isozymes.

Although changes in levels of different forms of cytochrome P-450 have been postulated to explain differences in hepatic drug metabolism with age and sex (33, 34), no specific assay has been available to test this proposal. The results presented here demonstrate age and sex differences in levels of three forms of cytochrome P-450 in control as well as PB- or MC- treated rats.

Inducers of drug metabolism have been divided into differ- ent classes based on the enzymatic and spectral properties of hepatic microsomes isolated after pretreatment of rats with xenobiotics. PB and MC are representatives of the two classes (1) which have been studied extensively, while pregnenolone 16a-carbonitrile is known to possess unique inducing proper- ties and belongs in a separate class (35-37). Those studies did not address specifically which forms of cytochrome P-450 were induced.

Aroclor 1254 treatment increases the specific content of all three forms of cytochrome P-450 and, therefore, possesses inducing properties similar to a mixture of PB and MC. Three xenobiotics used in the present study, y-chlordane, trans- stilbene oxide, and SKF-525A, selectively increase the level of cytochrome P-450b, and can be considered “PB-like” inducers. There are no structural similarities among these inducers of cytochrome P-450b which might be responsible for the specific increase in this hemoprotein. Using a variety of enzymatic assays and other indirect tests on microsomes, both y-chlor- dane and SKF-525A have been characterized as “PB-like” inducers (38-41). These three inducers have no effect on the levels of cytochrome P-450, and very little effect on the levels of cytochrome P-450,. MC and Aroclor 1254 are the only xenobiotics tested in this study which have a marked effect on cytochrome P-450, levels.

Trans-stilbene oxide was originally characterized as a selec- tive inducer of epoxide hydrolase (42). Recent studies have shown that trans-stilbene oxide treatment increases micro- somal monooxygenase activities as well as cytochrome P-450, but these studies have not indicated whether it belongs in the “MC-” or “PB-like” class or a separate class of inducer (43- 45). However, since not all forms of rat liver cytochrome P- 450 have been purified and characterized, the present results do not address unknown forms which may be selectively induced by these xenobiotics.

Pregnenolone 16a-carbonitrile treatment of rats increases the specific content of total cytochrome P-450 but has no effect on levels of cytochromes P-450,, P-450h, and P-450,. This xenobiotic belongs to a third class of inducers (35, 36) which induces a form of cytochrome P-450 recently purified and characterized (37). This purified form of cytochrome P- 450 was shown to be distinct from the major forms of cyto- chrome P-450 induced by either PB or MC by several criteria including immunochemical properties (37).

The monospecific antibodies to cytochromes P-450., P-450h, and P-450, have been used to determine the contribution of these isozymes to the overall catalytic activity of microsomal preparations. The results of such a study on benzo(a)pyrene metabolism are presented in Table V. Anti-P-450b and anti-P- 450, do not significantly inhibit benzo(a)pyrene-metabolizing activity in microsomes from MC-treated rats. The addition of anti-P-450, results in a 43%, 23%, and 88% inhibition of benzo(a)pyrene metabolism to fluorescent phenolic metabo- lites in microsomes from control, PB-, and MC-treated rats, respectively. Other forms of cytochrome P-450 not yet char- acterized must, therefore, be involved in benzo(a)pyrene me-

Regulation of Cytochrome P-450 and Epoxide Hydrolase 1051

TABLE V Immune complex inhibition of benzo(a)pyrene metabolism catalyzed by liver microsomes from immature male rats

Benzo(a)pyrene metabolism to fluorescent phenols and inhibition of this reaction by antibodies are described under “Experimental Procedures.” Antibody concentration is expressed in milligrams of IgG per nmol of cytochrome P-450. Catalytic activity is expressed as nanomoles of fluorescent products formed (equivalent to nanomoles of 3-OH benzo(a)pyrene) per min per nmol of cytochrome P-450.

Metabolism of benzo(a)p.wene Treatment of rats and antibody

Concentration Per cent of Con- trol Activity

- nmol/min/nmol

Untreated (control) 0.25 100 4 mg anti-P-450,/nmol 0.19 76 15 mg anti-P-450,/nmol 0.15 57

Phenobarbital treated 0.32 100 4 mg anti-P-450Jnmol 0.26 80 15 mg anti-P-45OC/nmol 0.25 77

3-Methylcholanthrene treated 1.58 100 4 mg anti-P-450, /nmol 0.38 24 15 mg anti-€”450,/nmol 0.19 12 4 mg anti-P-4501,/nmol 1.64 104 15 mg anti-P-4501Jnrnol 1.39 88 4 mg anti-P-450a/nmol 1.69 107 15 mg anti-P-450.Jnmol 1.44 91

tabolism by liver microsomes since inhibition by anti-P-450,. was not the same for all three microsomal preparations. Other evidence has shown that the high benzo(a)pyrene-metaboliz- ing activity in microsomes from pregnenolone-16a-carboni- trile-treated rats is insensitive to an antibody directed pri- marily against cytochrome P-450, (36).

From our data, cytochrome P-450,. represents at least 88% of the benzo(a)pyrene hydroxylase activity and approximately 77% of the total cytochrome P-450 in rats treated with MC. These data contrast with the speculation of Nebert and co- workers (46, 47). They propose that a hemoprotein with a molecular weight of 56,000 induced by MC in rat liver micro- somes has a CO maximum at or near 450 nm (termed P,-450) and is responsible for most of the benzo(a)pyrene metabolism. They also conclude that another form of cytochrome P-450 (P-448) induced by MC has a CO maximum a t 447 to 448 nm, is responsible for most of the acetanilide metabolism catalyzed by these microsomes, and has a minimum molecular weight of 54,000. Our data are not consistent with this nomenclature. Purified cytochrome P-450, (CO maximum at 447 nm) has a minimum molecular weight of 56,000 (2) and accounts for at least 88% of the benzo(a)pyrene metabolism in rat liver mi- crosomes from MC-treated rats. In addition, cytochrome P- 450,. has low catalytic activity for acetanilide (48). If a hemo- protein with a molecular weight of 54,000 is also induced by MC in rat liver microsomes, then at least three forms of cytochrome P-450 are induced by this hydrocarbon since cytochrome P-450, (Mr = 48,000) is also induced by MC (cf “Results”). The proposal by Nebert and co-workers (46, 47) provides clear evidence of the dangers involved in formulating such concepts from data obtained with microsomes which are known to contain a number of different forms of cytochrome P-450, at least some of which are under different regulatory control.

Kremers et al. (49) have raised the possibility of the exist- ence of more than one form of rat liver microsomal cyto- chrome P-450 involved in the 16a-hydroxylation of steroids. Their data on the effects of sex and enzyme induction on microsomal metabolism of four different steroids at the 16a- position indicated that steroid 16a-hydroxylase may be het-

erogeneous. The results of our immunoquantitation and in- hibition studies with monospecific antibodies have established the role of more than one form of rat liver microsomal cyto- chrome P-450 in the 16a-hydroxylation of testosterone (Table VI). Purified cytochrome P-450h, but not cytochromes P-450,, and P-450,, efficiently catalyzes the 16a-hydroxylation of tes- tosterone. It is known that testosterone 16a-hydroxylase ac- tivity is very low in untreated immature male rats and that this metabolic activity increases markedly with age (32). Im- munoquantitation data (cf “Results”) indicate that cyto- chrome P-450,, does not increase with age in untreated male rats, suggesting an age-dependent development of another form of cytochrome P-450 involved in the 16a-hydroxylation of testosterone. The results of inhibition studies with anti-P- 450h in untreated and PB-treated rats confirm this observation (Table VI). Immature and adult male rats treated with PB have high rates of 16a-hydroxylation and a high proportion of total cytochrome P-450 as P-450h, and in these rats, 16n- hydroxylation was the most sensitive to anti-P-45Ok, inhibition. However, in the untreated adult male rat which has high 16a- hydroxylase activity and a low proportion of total cytochrome P-450 as P-450h, only 14% inhibition by anti-P-4501, was ob- served. These marked differences in anti-P-450h inhibition of 16a-hydroxylation establish the existence of more than one steroid 16a-hydroxylase in rat liver microsomes. Anti-P-4501, had no effect on the 7a- and GP-hydroxylation of testosterone in any of the treatment groups (data not shown) which con- firms our earlier data on the lack of 7a- and GB-hydroxylase activity of purified cytochrome P-4501, (2).

Many of the same compounds which induce various forms of cytochrome P-450 also induce expoxide hydrolase, but not to the same extent. Studies to date, which have examined the induction of epoxide hydrolase, have been limited to measure- ments of catalytic activity. Our results on immunoquantitation provide a firm foundation for using octene oxide hydration as a reliable index of the magnitude of epoxide hydrolase induc- tion by xenobiotics. This correlation is apparent by the rela- tively constant ratio of octene oxide hydration to absolute enzyme concentration. The ratio of catalytic activity of epox- ide hydrolase to enzyme concentration has recently been

TABLE VI Immune complex inhibition of testosterone hydroxylation catalyzed

by liver microsomes from immature and adult male rats Assay conditions for the hydroxylation of testosterone are given

under “Experimental Procedures.” The amount of antibody is ex- pressed in milligrams of IgG per nmol of microsomal cytochrome P- 450. Catalytic activity is expressed as nanomoles of 16a-hydroxytes- tosterone formed Der min per nmol of cvtochrome P-450.

Age, treatment, and antibody concentration

Immature Untreated 4 mg anti-P-450t,/nmol 15 mg anti-P-450t,/nmol

Phenobarbital treated 4 mg anti-P-450t,/nmol 15 mg anti-P-4501,/nmol

Adult Untreated 4 mg anti-P-450t,/nmol 15 mg anti-P-4501,/nrnol

Phenobarbital treated 4 mg anti-P-450h/nmol 15 mg anti-P-4501,/nmol

~

Testosterone I&-hydroxyl-

ation

nrnol/min/nmol

0.19 0.15 0.12

0.86 0.22 0.12

1.12 1.18 0.96

1.02 0.40 0.35

Per cent of control activity

”.

100 89 65

100 25 14

100 105 86

100 39 34

1052 Regulation of Cytochrome P-450 and Epoxide Hydrolase

shown to be in good agreement when rats were treated with 2-acetylaminofluorene or PB (27).

The agreement between catalytic activity of epoxide hydro- lase for octene oxide and absolute enzyme concentrat ion in rat l iver microsomes is not found with the cytochrome P-450- dependent monooxygenase system. The reasons for this dis- crepancy are readily apparent s ince ( a ) multiple forms of cy tochrome P-450 with overlapping substrate specificities can vary as a function of age, sex, and pretreatment with xeno- biotics; (6 ) the involvement of a second enzyme in these react ions (NADPH-cytochrome P-450 reductase) may be a rate-determining step for some b u t not all enzymatic reactions; a n d ( c ) the requi rement of lipid for this e lectron transport cha in can be influenced by a variety of dietary and drug t rea tments . We anticipate that the ability to quan t i t a t e indi- vidual forms of cytochrome P-450 should result in a better understanding of the complex nature of steroid and xenobiotic metabolism as well as carcinogen activation and inactivation.

Acknololedgments-We thank Dr. D. M. Jerina for the [7,8- 'Hloctene 1,2-oxide and Mrs. Ann Marie Williams for assistance in the preparation of this manuscript.

Noted Added in Proof-We have found that antibody directed against cytochrome P-450, cross-reacts with purified cytochrome P- 450,, to a small extent in the Ouchterlony test giving a reaction of apparent partial identity (Ryan, D. E., Thomas, P. E., and Levin, W. (1980) J . Biol. Chem. 255, 7941-7955). Nevertheless, in the radial immunodiffusion assay, where anti-P-450, is uniformly distributed throughout the agarose gel, cytochrome P-450,1 does not form a precipitin ring regardless of the concentration used in the assay. Subsequently, the reaction of cytochrome P-450d with anti-P-450, was removed by immunoabsorption, and the absorbed antibody was used in the radial immunodiffusion assay. The percentage of cytochrome P-450,. in microsomes did not change as a result of using the absorbed antibody for the radial immunodiffusion assay. From these results, we conclude that the quantitation of cytochrome P-450, is not influ- enced by the presence of cytochrome P-450,1 in microsomal prepara- tions.

2. 1.

3.

4.

5.

6.

7.

8.

9. 10.

11.

12.

13.

14.

15.

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