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Alcohol & Alcoholism, VoL 28, SIB, pp. 69-75, 1993
Elsevier Science Ltd
Medical Council on Alcoholism
Printed in Great Britain. All rights reserved
0735-D414/93 $6.00 + 0.00
Pergamon
IMMUNOHISTOCHEMICAL DEMONSTRATION OF ETHANOL-INDUCIBLE
P450 2EI IN RAT BRAIN
TETSURO SOHDA, MASANORI SHIMIZU, SEIICHIRO KAMIMURA and MAKOTO OKUMURA
First Department of Internal Medicine, School of Medicine, Fukuoka University, Nanakuma, Johnan-ku, Fukuoka 814-01, Japan
Abstract -The microsomal ethanol-oxidizing system (MEOS) is a P450-dependent pathway for
ethanol oxidation in hepatic microsomes, The bulk of MEOS activity is catalyzed by P450 2EI (an
ethanol-inducible isozyme of P450) in animal livers treated with ethanoL Rat brains also metabolize
certain drugs, and it is theorized that a mechanism for drug metabolism exists within the brain which
acts on P450, We compared immunohistochemical localization of P450 2EI between ethanol-treated
rats and pair-fed control rats. In control rats, immunoreactive P450 2EI was detected in minute amounts
in both basal ganglia and cerebellar cortices. After ethanol treatment, the content of P450 2E I increased
in the basal ganglia, and the enzyme was also induced in the cerebellar cortices, substantia nigra and
hippocampus. We speculate that the rat brain metabolizes ethanol by P450 2EI.
We examined the distribution of P450 2El
in various parts of the brain using
immunohistochemical staining techniques, both
before and after long-tenn ethanol administration.
This research is an important step towards
elucidating the mechanism of ethanol metabolism
in rat brains.
INTRODUCTION
It is well established that ethanol has a toxic effect
on the liver. The microsomal ethanol-oxidizing
system (MEOS), the term used to describe the
P450-dependent pathway of ethanol oxidation in
hepatic microsomes, contributes to hepatocyte
injury from chronic alcohol intake. The bulk of
MEOS activity is catalyzed by P450 2El (an
ethanol-inducible isozyme of P450) in animals
treated with ethanol.
Recently, the intralobular distribution of P450
2El in rat and human livers using an
immunohistochemical staining procedure was
described (Tsutsumi et al., 1989). P450 2Eldependent enzyme activities have also been
detected in extrahepatic tissues such as lung,
kidney, pancreas and alimentary tract as well as in
the liver (Shimizu et al., 1989, 1990; Sohda et al.,
1992). However, not much is known about the
effect of ethanol on P450 2El content and
associated activities in the brain.
In contrast, recent reports indicate that P450
lAI and P450 2B 1 can be found in brain areas
which metabolize morphine (Fishman et al., 1976)
and convert estradiol and estrone into
catecholestrogens in rats (Fishman and Norton,
1975). These results indicate that a mechanism for
drug metabolism, which acts on P450, exists
within the brain (Kapitulnik et al., 1987).
MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats (140-150 g) were
fed a nutritionally adequate liquid diet for 21 days
in which ethanol provided 36% of the total caloric
intake (Lieber and DeCarli, 1989). Pair-fed
littennates consumed the same diet except that an
isocaloric amount of carbohydrate was substituted
for ethanol. Rats were fed with liquid diets until
sacrificed.
Polyclonal antibodies
Three rabbit polyclonal antibodies against P450
isozymes (P450 2El antibody and P450 lAI
antibody from Oxygene Co., U.S.A., P450 2Bl
from Oxford Co., U.S.A.) were used: (1) P450
2EI antibody for ethanol inducible P450; (2) P450
lAI antibody for 3-methylcholanthrene; and (3)
P450 2B 1 antibody for phenobarbital.
69
70
Fig. 1. Distribution of P450 2EI in rat brain basal ganglion.
A: Nerve cells of the caudate putamen from an ethanol-fed rat exhibited a dark staining reaction, indicating
specific binding of the anti-P450 2EI antibody. B: Only a weak reaction was obtained in the corresponding
cells from this pair-fed control rat. C: Negative control was carried out with immunoadsorbed antiserum. x16.
P450 2EI IN RAT BRAIN
Preparation of tissue samples and microsomes
After decapitation, the brains were rapidly
removed. Tissue blocks were immediately cut,
embedded in Tissue-Tek medium, and snap-frozen
in liquid nitrogen. The remaining tissue was used
to isolate microsomes as in the method described
by Ardies et al. (1987). Protein concentration was
determined by the method of Lowry et al. (1951).
Microsomal P450 content was calculated
according to the method of Omura and Sato from
the CO-reduced difference spectrum based on an
extinction coefficient of 91 mM-i cm- i (Omura
and Sato, 1964).
Immunohistochemical staining
Frozen 5-llm sections were cut in a cryostat,
mounted on albumin-coated slides and air-dried
for 30 min. Following fixation with cold acetone
for 20 min, these sections were again air-dried and
washed in a phosphate-buffer saline solution
(PBS) with a pH of 7.4. Sections were initially
treated for 30 min with 0.03% hydrogen peroxide
in methanol to eliminate endogenous peroxidase
activity. The same sections were then washed with
71
PBS and overlaid with a 1: 10 dilution of normal
goat serum for 10 min to block nonspecific protein
binding. Subsequently, polyclonal antibodies at a
final dilution of 1:200 in PBS were placed on the
sections overnight at 4°C. After washes in PBS,
the sections were incubated for 30 min with
biotinylated goat anti-rabbit IgG, washed again in
PBS and incubated with streptavidin-biotinylated
horseradish peroxidase. After further washes in
PBS, peroxidase activity was developed in 10 roM
Tris-HCI buffer, pH 7.4, containing 3,3'diaminobenzidine and 0.05% hydrogen peroxide
for 5 min. All sections were subsequently
counterstained with hematoxylin. In each section,
immunoreaction intensity was ranked blindly
according to the following scale: +2, + and -.
RESULTS
Effects of ethanol treatment on rat brain
microsomal P450
The effects of ethanol administration on total
microsomal P450 content are shown in Table 1.
The aggregate content of total P450 in brain
microsomes was increased approximately two-fold
Fig. 2. Distribution of P450 2EI in the basal ganglion of an ethanol-fed rat, at higher magnification.
Nerve cells of the caudate putamen exhibited positive staining for P450 2EI, while nerve fibers of the caudate
putamen were negative. x 100.
72
T. SOHDA et al.
A
Fig. 3. Immunohistochemical staining of sections of the cerebellar cortex.
Positive staining is seen in the small cortical cells and small granular cells, whereas negative staining is observed in Purkinje's
cells (~) from an ethanol-fed rat (A) and a pair-fed control rat (B) using anti-P450 2El antibodies. x 40.
as compared with that in the control rats, and
the difference between this group and the controls
was statistically significant (P < 0.05, Student's ttest).
Immunohistochemistry
In the immunohistochemical staining method
used, P450 2EI could be detected only in the
nerve cells of the caudate putamen, globus
pallidus and cerebellar cortices from the control
rats. Higher levels of P450 2EI were found in the
nerve cells of the caudate putamen and the globus
pallidus of the chronically ethanol-fed rats than in
the controls. In the cerebellar cortices,
P4502E1 IN RAT BRAIN
73
Fig. 4. Distribution of the other isozymes of P450 in the basal ganglion of two rat brain specimens.
In both cases, a weakly positive reaction was observed in the nerve cells of the caudate putamen from an
ethanol-fed rat using anti-P450 IAI antibody (A) and anti-P450 2BI (B) antibody. x 16.
hippocampus and substantia nigra were also
stained with antibodies against P450 2EI after
ethanol consumption.
Purkinje's cells were easily degenerated by
ethanol, hypoxia, arsenic intoxication and ageing.
No P450 2EI was detected in the Purkinje's cells
of the cerebellar cortices of either the ethanoltreated or the control rat groups. In the caudate
putamen and globus pallidus, P450 IAI and P450
2B I staining intensities were less noticeable.
These results can be found in Table 2.
DISCUSSION
This is the first immunohistochemical
demonstration and localization study of P450 2EI
in rat brains. Immunoreactive P450 2EI was found
to be localized in the ba~al ganglia (caudate
putamen, globus pallidus), and cerebellar cortices
in control rat brains. After ethanol treatment,
levels of P450 2EI increased in the basal ganglia,
and the enzyme was also found in the cerebellar
cortices, substantia nigra and hippocampus.
74
T. SOHDA et al.
Table 1. Effect of ethanol treatment on rat brain microsomal
P450 content
Rat
Treatment
Total P450*
EI
CI
E2
C2
E3
C3
Ethanol
Control
Ethanol
Control
Ethanol
Control
0.0749
0.0232
0.1567
0.0546
0.1175
0.0720
Ethanol
Control
0.1167 ± 0.0409t
0.0499 ± 0.0247
*In nmoles P450 per mg microsomal protein.
tMean ± SE. Significantly increased (P < 0.05) over the
controls.
The total P450 content in the microsomes of
ethanol-treated rat brains was definitely higher
than that of the control brains (Table 1). Increased
total P450 content in ethanol-treated rats may be
due to an increase of P450 2El, because the
immunohistological distribution of other major
isozymes of P450 (lAl and 2Bl) was unchanged
by ethanol treatment. This result indicates that
P450 2E1 could be induced in certain specific
areas of the brain following ethanol treatment.
Moreover, evidence of P450 1AI, a 3methylcholanthrene-inducible P450 enzyme and
P450 2BI, a phenobarbital-inducible P450 enzyme
is reported in rat brain areas, by Kapitulnik et ai.
(1987). In the present study, P450 2El was
detected in the same areas. These results also
suggest that the brain may metabolize ethanol in
the same pathway as morphine.
Several reports indicate that total P450 content
of the rat brain was relatively low compared with
that in the liver (Tsutsumi et ai., 1989). However,
we speculated that P450 may be highly
concentrated in specific areas of the brain.
It is known that Purkinje's cells in the cerebellar
cortices are easily damaged by ethanol
intoxication leading to ataxia. It has been indicated
that P450 2El correlates with tissue injury and
carcinogenesis due to the cytotoxicity of
acetaldehyde (Sorrell and Tuma, 1985), lipid
peroxidation (Castillo et ai., 1992) and
metabolism of procarcinogens (Garro et ai., 1981;
Seitz et al., 1981; Farinati et al., 1985). In our
study, P450 2E1 was not found in Purkinje's cells
of the cerebellar cortices, while this enzyme was
found to be localized in the small cortical cells and
Table 2. Distribution of P450 2E 1 in various parts of the rat
brain
Cereberal cortex
Caudate putamen
Globus pallidus
Hippocampus
Substantia nigra
Cerebellar cortex
Medulla oblongata
EI
E2
E3
CI
C2
C3
+2
+
+
+2
+2
+
+
+2
+2
+
+
+
+
+
+
+
+
+
+
+2
+
+
+
+
Frozen rat brain sections were stained immunochemically with
anti-P450 2EI antibodies. +2, + and - denote intense,
slight, and no specific immunostaining, respectively.
small granular cells of the cerebellar cortices. We
propose that it is the small cortical cells and the
small granular cells of the cerebellar cortices, not
Purkinje's cells, which are affected by ethanol.
Acknowledgements - Thanks to Tim Cornish of the
Department of Communication Studies, Simon Fraser
University, Canada, for English language revision.
REFERENCES
Ardies, C. M. et al. (1987) Purification of NADPH:
cytochrome c (cytochrome P-450) reductase from hamster
liver microsomes by detergent extraction and affinity
chromatography. Analytical Biochemistry 162, 39-46.
Castillo, T. et al. (1992) Role of cytochrome P-450 2EI in
ethanol-, carbon tetrachloride- and iron-dependent
microsomal lipid peroxidation. Hepatology 16, 992-996.
Farinati, F. et al. (1985) Effect of chronic ethanol consumption
on activation of nitrosopyrrolidine to a mutagen by rat upper
alimentary tract, lung and hepatic tissue. Drug Metabolism
Disposition 13, 210-214.
Fishman, J. and Norton, B. (1975) Catechol estrogen formation
in the central nervous system of the rat. Endocrinology 96,
1054-1059.
Fishman, J., Hahn, E. F. and Norton, B. I. (1976) Ndemethylation of morphine in rat brain is localized in sites
with high opiate receptor content. Nature 261, 64-65.
Garro, A. J., Seitz, H. K. and Lieber, C. S. (1981)
Enhancement of dimethyl-nitrosamine metabolism and
activation to mutagen following chronic ethanol
consumption. Cancer Research 41, 120-124.
Kapitulnik, J. et at. (1987) Immunohistochemical localization
of cytochrome P450 in rat brain. Neuroscience 20, 829-833.
Lieber, C. S. and DeCarli, L. M. (1989) Liquid diet technique
of ethanol administration: 1989 update. Alcohol and
Alcoholism 24, 197-211.
Lowry, O. H. et al. (1951) Protein measurement with the Folin
phenol reagent. Journal of Biological Chemistry 193,
265-275.
Omura, T. and Sato, R. (1964) The carbon monoxide-binding
P450 2El IN RAT BRAIN
pigment of liver microsomes. Journal of Biological
Chemistry 239, 2370-2378.
Seitz, H. K., Garro, A. 1. and Lieber, C. S. (1981) Enhanced
pulmonary and intestinal activation of procarcinogens and
mutagens after chronic ethanol consumption in the rat.
European Journal of Clinical Investigation 11, 33-38.
Shimizu, M. et al. (1989) Extrahepatic localization of ethanolinducible P450IIEI (Abstract). Alcoholism: Clinical and
Experimental Research 13, 325.
Shimizu, M. et al. (1990) Immunohistochemical localization of
ethanol-inducible P450IIEI in the rat alimentary tract.
Gastroenterology 99, 1044-1053.
75
Sorrell, M. F. and Tuma, D. J. (1985) Hypothesis - Alcoholic
liver injury and the covalent binding of acetaldehyde.
Alcoholism: Clinical and Experimental Research 9,
306-309.
Sohda, T., Shimizu, M. and Okumura, M. (1992) Distribution
of pancreas of ethanol-inducible P450IIE1 in rats fed ethanol
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Tsutsumi, M. et al. (1989) The intralobular distribution of
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