Vol. 150, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 15, 1988 Pages 925-930

IMMUNOCHEMICAL DETECTION OF CYTOCBROME P450c-M/F

AND NADPH-CYTOCHBOME P450 REDUCTASE IN RAT LIVER AND KIDNEY

Osamu Sugita+, Kazuo Nagashima*. Shigeru Sassa+

and Attallah Kappas +

The Rockefeller University, New York, N.P., and

Department of Pathology, Hokkaido University, Sapporo, Japan*

Received November 24, 1987

SUMMARY: The localization and distribution of NADPH- cytochrome P450 reductase and cytochrome P450c,M/F were investigated immunohietochemically in the liver and the kidney of untreated rats employing both an unlabelled antibody peroxidase-antiperoxidase method and a peroxidase labelled primary antibody technique. In both immunohisto- chemical procedures, the reductase and P450c-M,F were detected in hepatocytes throughout the liver. In con- trast. the reductase and P450s-r,F in the ~~~~~~~~~~~~~~_p:,fr detectable in the proximal tu u e cells.

The oxidative metabolism of a variety of chemicals. steroids,

vitamins, fatty acids, and prostaglandins (1) is catalyzed by the

monooxygenase system. which consists of NADPH-cytochrome P450

reductase and P450. A number of isozymes of cytochrome P450 have

been identified (2-4). while no isozymes of the reductase are

known (5). Histochemical techniques based on the use of specific

antibodies offer a useful means for the study of the specific

localization of these membrane-bound components in the mono-

oxygenase system. In this study we examined. by immunohisto-

chemical techniques, the distribution of cytochrome P450c-M/F and

of cytochrome P450 reductase in the liver and the kidney of

untreated rats.

0006-291X/88 $1.50

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Copyright 0 1988 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

Vol. 150, No. 3, 1988 BlOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS ABD HETHODS

Puificadnn nf NAPPHz~rLnrhrnne L4%2 radn~L.aaa and rrurhrnme : P450 reductase was purified from the liver of male rats

;~:a~~e%awley. -2OOg body weight. Charles River Breeding Labs) treated with phenobarbital according to the method of Yasukochi and Masters (6). with the following modifications. Microsomes were solubilixed using 5CmM Tris (pli 7.7)/lmM dithiothreitol/ lX(w/v) Emulgen 913(Kao-Atlas, Tokyo)/2OX(v/v) glycerol at a protein concentration of approximately 4.5mg/ml. The purified P450 reductase had a specific activity of 35.7 units/mg of protein when assayed with low ionic strength buffer (7) and had a molecular weight of approximately 78.000 daltons on sodium dodecylsulfate- polyacrylamide gel electrophoresis. Cytochrome P45OC_M/F was purified from the liver of untreated male rats by cholate solubilization. polyethylene glycol(PEG) precipitation, diethyl- aminoethylcellulose(DEAE) anion exchange chromatography and hydroxyapatite chromatography according to our method (8). The purified preparation (Mr ~48.500 daltons) was electrophoretically homogeneous and contained w 15nmol P450/mg protein.

PreparaLinn nf anGPndica: Antibodies against rat liver NADPH- cytochrome P450 reductase were raised in adult male New Zealand rabbits according to Masters et al.(g). An IgG fraction was purified from rabbit serum(9). Antibodies against P450 prepared by injecting 20 ug of the homogeneously purifie

F- l‘q ;;;ge

with an equal volume of Freund’s complete adjuvant into the backs of 3 Rhode Island Red hens. The same amount of the antigen with Freund’s incomplete adjuvant was injected 2 weeks after the first injection. After an increase in the specific immune titer in sera was confirmed, eggs were collected and an IgP fraction was isolated using PEG cuts as described previously (10). An antibody against P45OC_M,F was specific for P45OC,

W’ and did not react

with other cytochrome P45O’s (8) or P4 reductasetdata not shown).

Irnmwal d&-inn: Rats (Sprague-Dawley. male, - 200 g body weight) were perfused through the left ventricle with a fixative consisting of PX(w/v) paraformaldehyde in lysine-periodate solution (11). Tissue sections were pre ared at 4’C. rinsed overnight in 0.05M phosphate buffer (pH 7.4 P containing lOX(w/v) sucrose. Tissue sections were immediately frozen in n-hexane which had been chilled in dry ice-acetone and mounted in O.C.T. compound (Tissue-Tek, Miles). The frozen sections were first exposed to O.~%(V/V) H202 in 95% methanol for 30 min to block endogenous peroxidase activity. After pre-incubation with lO%(v/v) normal goat serum for the reductase. and 2.5X(w/v) bovine serum albumin (BSA) for P450. tissue sections were incubated for 16 h at 4’C with the appropriate antibody or preimmune antibody in dilutions ranging from 1:lOO to 1:3000, All dilutions were prepared in 20mM potassium phosphate buffer (pH 7.4) containing 0.15 M NaCl and 2.5 % BSA. When unlabelled primary antibodies were used, the sections were subsequently exposed to horseradish per- oxidase(HRPO)-labelled goat anti-IgG (Cappel) for the reductase. and rabbit anti-IgY (Cappel) for P450. diluted 1:lOO and finally to the peroxidase-antiperoxidase complex at a dilution of 1:lOO. Non-immunized rabbit serum and chicken IgY served as control. All sections were finally exposed to 3.3’-diaminobenxidine (0.3mg/ml) and R O2 (10mM potassium p osphate buffer. pH 7.4. containing 0.15 M NaCl), E

(0.05%. v/v) in phosphate-buffered saline

for visualieation of staining. For direct immunoperoxidase method.

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BRPO-labelled anti-P450C-M/Fr and RRPO-labelled IgY from untreated hen were prepared according to the method of Nakane et a1.(12).

RESULTS

In liver sections incubated with anti-P450CDM/F. positive

stains were seen homogeneously in hepatocytes throughout the liver

with some intensification in the centrilobular region (Fig. 1A).

In liver sections which were incubated with preimmune IgY, no

positive stains were seen (data not shown). Immunohistochemical

Figure 1. Immunohistoche,mical localization of cytochrome P450cM/P and NADPH-cytochrome P450 reductase in untreated rat liver (magnification 100 X1. (A) A section which had been exposed to chicken IgY anti-rat P-450

t&b&/F' (B) A section which had been exposed to rabbit anti-rat P450 reductase.

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Vol. 150, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

staining of NADPB-cytochrome P450 reductase was very similar to

that of cytochrome P450C-R,P throughout the liver (Fig. 1B).

Staining by both the unlebelled antibody-PAP method and the HRPO-

labelled primary antibodies gave comparable results, indicating

that the positive staining reflects the specificity of primary

antibodies. Although both P450C-M,F and the reductaae were stained

intensely in hepatocytes. they were not seen in cells lining the

portal vein, the hepatic artery and the bile ducts (Fig. 1A 8 1B).

In kidney sections incubated with anti-P450C-h,P. positive stains

were seen onlyinthe proximal tubule cells. but not in the glome-

ruli or in the distal tubule cells (Fig. 2A). Immunohistochemical

staining of the reductase in the kidney demonsttated similar

distribution of the reductase to that of P45OC-M/F(Fig* 2B)*

DISCUSSION

The results of this study demonstrate that P450C-M,F, the

major constitutive form of cytochrome P450 in the liver (8). can

be demonstrated immunohistochemically both in the liver and the

kidney. In the liver both P450C-M/F and the reductase are dis-

tributed uniformly. while in the kidney. they are present only in

the proximal tubules. Several immunohistochemical studies showed

the localization of various P450 isozymes in the liver (13-16).

Immunohistochemical staining of the reductase was also investi-

gated in several tissues including the liver (17). Haaparanta et

al. (5) reported the presence of both the reductase and cytochrome

P450 in the rat ventral prostate using immunohistochemical techni-

ques. To our knowledge, there have been no reports concerning the

simultaneous immunohistochemical examination of the reductase and

P450 in the liver and the kidney. Since cytochrome P450 and the

reductase function as components of the microsomal mixed function

oxidaee system, and there is evidence that they form a binary

complex (18) to perform the mixed function oxidation. they would

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Vol. 150, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 2. Immunohistochemical localization of cytochrome P450cM,P and NADPH-cytochrome P450 reductase in untreated rat kidney (magnification 100 X). (A) A section which had been exposed to chicken IgY anti-rat P45OG-,I,. (B) A section which had been exposed to rabbit IgG anti-rat P450 reductaae.

be expected to localize in close association with each other in

the endoplasmic reticulum. Our data demonstrate clearly that

these elements are present in very close proximity both in the

liver and the kidney.

In our earlier study (8). we demonstrated that cytochrome

P450 C-M/p purified from rat liver catalyzes estradiol 2- and 16K-

hydroxylation. Thus the immunohistochemical demonstration of

P450,,,/, in the kidney. together with the reductase. the essen-

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Vol. 150, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tial component in the mixed function oxidation system. suggests

that estradiol hydroxylations may also take place in the kidney.

ACK%OULKDGLLBBTS

This study was supported in part by grants from USPBS DK- 32890. ES-01055 and the Suntory Fund for Biomedical Research. The excellent technical assistance of Ms. Luba Garbaczewski is grate- fully acknowledged.

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