Neuroscience Vol. 34, No.

0306-4522/90$3.00$0.00 Pergamon Press plc 0 1990IBRO

2, pp. 451463, 1990

Printed in Great Britain

REGIONAL DISTRIBUTION OF ETHANOL-INDUCIBLE CYTOCHROME P450 IIEl IN THE RAT CENTRAL NERVOUS SYSTEM T. HANssoN,*t N. TINDBERG,* M. INGELMAN-SUNDBERG*and C. K~HLER$ *Department of Physiological Chemistry, Karolinska Institute, S-104 01 Stockholm, Sweden fDepartment of Nemopharmacology, Astra Research Centre, S-151 85 Sodertalje, Sweden Abstract-A specific form of cytochrome P450, P450 IIEl, active in ethanol oxidation, is known to be induced about IO-fold in rat liver following ethanol treatment. This isozyme of P450 participates effectively in the metabolic activation of precarcinogens, such as N-dimethylnitrosamines, and of solvents such as carbon tetrachloride and benzene. In the present investigation, two different polyclonal antisera against P450 IIEl were used in order to map the regional distribution of this P450 form in the rat central nervous system. The presence of P450 IIEl in various brain regions was confirmed by Western blot analysis. The P450 IIEl-immunoreactivity was heterogeneously distributed between brain areas. Neuronal cell bodies and glial cells of presumed astroglial as well as oligodendroglial identity contained immunoreactivity. All fiber tracts harbored P450 IIEl-immunoreactive glial cells as did the ependymal lining of the ventricular wall as well as small and large vessels throughout the brain. P450 IIEl-immunoreactive glial cells were present in all areas of the neocortex, in the olfactory bulb, in the piriform cortex and in several different thalamic nuclei. In the cerebellum, P450 IIE-immunoreactivity was found in all cell layers and was exclusively localized to glial cells and their processes. Staining of blood vessels was prominent in the white matter where P450 IIEl-immunoreactive glial cells were seen to have end-feet on the vessels. A subgroup of pyramidal cells of the frontal cortex showed strong P450 IIEl-immunoreactivity, as did a component of the olfactory nerve which innervates the accessory bulb. In the hippocampal region, the pyramidal cells of all subfields were P450 IIEl-immunoreactive. Some polymorphic cells of the hilus and subfield CA stained intensely with the P450 IIEl antibodies. A high density of P450 IIElimmunoreactiviy was detected throughout the striatal complex. The immunoreactivity was localized to neuronal cell bodies as well as the neurophil. Fibers of the nigrostriatal system were strongly P450 IIEl-immunoreactive. Mechanical lesions of this pathway showed an accumulation of P450 IIElimmunoreactivity proximal to the lesion relative to the striatum and a depletion in the reticular part of the substantia nigra, suggesting that the antigen may be transported from the striatum to the substantia nigra. In the brain stem a high density of P450 IIE-immunoreactive neurons was detected in the substantia nigra, the pontine nucleus, lateral superior olive and the nucleus of the trigeminal nerve and facial nucleus. A great number of large- to medium-sized immunoreactive neurons were situated in the central gray and in the reticular formation. The present study shows that ethanol-inducible hepatic P450 IIEl is constitutively expressed in the rat brain. The distribution of this P450 form in certain populations of nerve cells might have implications for the regioselective toxicity of substrates for P450 IIEl.

The P450-dependent monooxygenase system is a multigene family of hemoproteins involved in detoxification and activation of endogenous compounds (e.g. steroids, fatty acids, prostaglandins and biogenic amines), as well as of exogenous substances (e.g.

drugs, chemical carcinogens, mutagens and other environmental chemicals). The multiple forms of P450 have different but overlapping substrate specificities. The cytochromes P450 are most abundant

tTo whom correspondence should be addressed at Department of Metabolism, Astra Research Centre, S-15185 Sodertalie, Sweden. Abbreviatiok: ABC, avidin-biotin complex; DAB, 3,3’diaminobenzidine: GFAP. alial fibrillarv acidic urotein: IR, immunoreactive immun&eactivity; PBS, phosphate: buffered saline; PMSF, phenylmethylsulfonylfluoride; P450, cytochrome P450; P450 IIEI, the ethanolinducible form of cytochrome P450, the product of the CYPZEI gene; SN, substantia nigra.

in the liver but also present in many extrahepatic tissues. The P450 genes are under complex and distinct control, by both exogenous and endogenous compounds, e.g. hormones.26~27 Although measurable levels of microsomal P450 and related monooxygenase activities have been detected within the rat brain,*’ only sparse information is available regarding the brain P450 and the capabilities of the different cell types to bioactivate and detoxicate lipid soluble compounds. The diversity of neuronal phenotypes in the central nervous system raises the question of selective expression of different forms of P450 with respect to cell classes, circuits and systems. Recently, we have shown that the rat brain contains various forms of cytochrome P450, which, however, are present at very low levels when expressed on a per gram wt tissue basis.25.37Nevertheless, immunohistochemical studies have demon-

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strated the presence of P45Os in various populations of glial and neuronal cells throughout the rat neuraxis, indicating that individual cells may contain significant amounts of these enzymes.s~‘s~37 A specific ethanol-inducible form of cytochrome P450 (P450 IIEI) has in recent years been shown to be responsible for the metabolic activation of a large number of toxicologically relevant compounds to cytotoxic and/or carcinogenic metabolites. These agents include acetoaminophen,’ ethanol>** ~-nitrosodimethylamine33 and various solvents.‘Z~‘s~3’ Furthe~ore, P450 IIEI has an apparently high rate of oxidase activity causing the formation of reactive oxygen species being able to initiate lipid peroxidation.2,34 This form of P450 has also been implicated in the metabolism of acetone and acetol.” The presence of P450 IIEI in the brain might thus be of physiological and toxicological importance. It was therefore considered of interest to investigate the distribution of P450 IIEl in various regions of the rat brain and we here present a first immunohistochemical mapping of this form of cytochrome P450 in the rat brain. EXPERIIMENTAL PROCEDURES Animals

Male Sprague-Dawley rats (weight 200g; Alab AB, Stockholm, Sweden) were kept at normal housing conditions and fed ad lib&m with tap water and Brood Stock Feed-R3 (Ewos AB, Slidertllje, Sweden) before use. In four rats, mechanical lesions of the nigrostriatal pathways were performed using a sharp duraknife. The lesioned rats were killed seven days later. Two rats received an intracerebroventricular injection of colchicine (Sigma Chemical Company, St. Louis, MO) (12Opg in 20~1) into the right lateral ventricle. The rats were killed 24 h after injection. Anti~ud~e.sand control e.~per~me~t~ Cytochrome P450 HE1 was purified from rat liver microsomes as described earlier.” Two antisera (A and B) were raised in rabbits against two different batches of protein purified on two different occasions. Characterizations of the antisera have been published elsewhere.‘,‘0,j4The specificity of the antisera is characterized by: (i) the recognition of only one band in Western blot of liver microsomes from control, benzene-, phenobarbital-, ~-naphtoflavone-, imidazole-, ethanol-, acetone-, Me,SG- or isoniazid-treated rats; (ii) the absence of cross reactivity in Western blot, according to the induction response and electrophoretic mobility, with any of rat liver microsomal cytochromes P450 IAI, IA2, IIAl, IIBI, IIB2, IIC3, IIC7, IIClI or IIIA (Refs 2, 3 and unpublished observations); and (iii) by the failure to recognize any type of P450 present in the periportal region of the liver lobule.’ The two antisera gave quali~tively similar patterns of IR when tested on tissue sections from rat brain. The IR was not seen under control conditions in the absence of primary antiserum nor following incubations with antiserum preadsorbed with highly purified antigen (10m6M) (Fig. IB). All rats were deeply anesthetized by pento~rbi~l (Mebumal, ACO. Stockholm; 60 mg/kg, i.p.), tracheotomized and provided with artificial respiration. They were perfused through the ascending aorta with $0 ml of saline followed by 500 ml of a fixative containing lysine: periodate: paraformaldehyde prepared as described by McLean and

Nakane.‘” The brains were removed from the scull and postfixed for 4 h. after which they were transferred to phosphate-buffered saline (PBS) containing 20% (w/v) sucrose. Sections (30pm thick) were cut on a freezing microtome, collected in wells containing PBS. Sagittal and horizontal sections, respectively, were incubated floating free in antiP450 IIEI antibodies diluted f : 5000-l: 15,000 in PBS containing 0.2% Triton X-100 and 1% normal goat serum (Dako Patts, Copenhagen, Denmark) for six days. The antigen-antibody complex was made visible by the avidin-biotin complex (ABC) method of Hsu et al.’ using the commercial ABC Kit (Vector Laboratories, Burligame, CA) with 3’,3’-diamino~nzjdine (DAB; Sigma Chemical Co., St. Louis, MO) as the chromogen, In separate experiments, the DAB-reacted sections were incubated with a rnonoclonal antibody against glial tibrillary acidic protein (GFAP) (Chemicon, U.S.A.; diluted I : 12,000) which was visualized using 4-chloro-napthol (Sigma) as a chromogen. These sections were coverslipped in gIycerol:PBS (3:l) without previous defatting or dehydration. Control experiments included the preadso~tion of the antiserum with purified antigen for 24 h at 4°C or omission of the primary antiserum. No staining of the tissue sections occurred in either of these control experiments. Subcellular ,fractionation The animals were decapitated and the brains quickly removed, then washed in ice-cold 0.15 M KCI. Dissection of the hippocampus and ventral striate took place at once, after which the material was kept on ice in four volumes of homogenization buffer: IO mM potassium phosphate buffer containing 1.14% (w/v) KCI, pH 7.4, freshly made 0. I mM phenylmethylsulfonylfluoride (PMSF) and 200 1(M butylated hydroxytoluene. The homogenate was centrifuged at 3OOg for 10 min. The resulting supernatant was subsequently centrifuged at lO,~g for 20 min. giving a mitochondrial pellet, which was resuspended in 50mM sodium/potassium phosphate buffer, pH 7.4, and a microsomal supernatant. A microsomal pellet was obtained by centrifugation at 100,000g for 60 min. Both microsomal and mitochondrial fractions held protein concentrations of approximately IS mg/ml, and were stored at N, at -80°C. Western ~mu~ob~ottin~ Sodium dod~ylsuphate-polyacrylamide gel electrophoresis was performed using the discontinuous system described by Laemmli” and a Bio-Rad mini Protean II apparatus. Slab gels of an acrylamide concentration of 8.5% were used and 20 pg of protein in each well was applied. The amount of P450 IIEI in microsomes and mitochondrial fractions was determined by Western btot, using the same two antisera that were used for the immunohistochemical analysis. The secondary swine anti-rabbit antibody from Dakopatts a/s, Denmark, was coupled to alkaline phosphatase. The developing agents NBT and BCIP from BioRad, were used in an 80 mM carbonate buffer containing 10 mM MgCl,, pH 9.8. In some experiments, the brain fractions were chromatographcd on ~~chloro-amphetamine Sepharose, as described by Warner et a1.37prior to Western blot analysis. RESULTS

General covtments

The two different P450 IIEI antisera used in the present study gave a qualitatively similar staining pattern (not shown). Both antisera gave positive signals in Western immunoblotting with microsomal and mitochondrial fractions obtained from rat brain. The antisera recognized a protein from the

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Fig. I. A. Photomicrograph showing the distribution of P450 IIEl-IR in sagittal sections from male rat brain. B. Preadsorption of the antisera with the highly purified liver P450 IIEl blocked the immunostaining in the brain tissues. Photomicrographs are from computer digitized pictures.

brain preparations with identical mobility on sodium dodecyl sulphatepolyacrylamide gel electrophoresis as liver P450 IIEl (Fig. 2). The incubation of brain sections with anti-P450 IIEl antisera resulted in staining of a large number of cells in most areas of the brain. Neuronal cell bodies and glial cells of presumed astroglial as well as oligodendroglial identity were found to contain immunoreactivity (IR). In some cases IR could also be visualized in major fiber tracts (see below). In general, P450 IIEl-like IR could be detected throughout the rat neuraxis. The IR was heterogeneously distributed between brain areas with the highest density of staining in the basal ganglia (caudatus, putamen), nucleus accumbens and olfactory tubercle (Fig. 1A). All fiber tracts harbored P450 IIEl-IR cells, as did the ependymal lining of the ventricular walls (Fig. 3C). Olfactory bulb, cortical areas and hippocampal region P450 IIEl-IR glial cells were present in all layers of the olfactory bulb with particularly high densities in the external plexiform layer (Fig. 3B). Scattered glial cells were also detected in the glomerular layer and among fibers of the olfactory nerve. Some bundles of olfactory nerve fibers harbored P450 IIEl-IR while others were not stained. In several cases entire

glomeruli contained intense IR, suggesting the presence of P450 IIEl-IR in terminals of the olfactory nerve. The mitral and tufted cells were not stained. A component of the olfactory nerve which innervates the accessory olfactory bulb showed strong IR (Fig. 3A). No staining of neurons was detected in any part of the olfactory bulb. All areas of the neocortex harbored P450 IIEl-IR glial cells and blood vessels. In addition, a subgroup of pyramidal cells in layers 4 and 5 showed strong staining of their somata as well as distinct cytoplasmic staining of both apical and basal dendrites (Fig. 3D). In the hippocampus, the pyramidal cells of all subfields (CA1 through CA4) and in the subiculum were P450 IIEl-IR (Fig. 4A). Shorter and longer dendritic processes of these cells contained IR. In addition, some polymorphic cells of the hilus and CA3 stained intensely with the anti-P450 IIEl antibodies (Fig. 4B and C). Moderate staining of the glial cells were detected in the molecular layer of the dentate gyrus (Fig. 4B). Weak IR was detected in neurons of all layers of the entorhinal area. The piriform cortex was rich in P450 IIEl-IR glial cells, especially in layer I where the large astrocytes were stained. A large number of blood vessels but relatively few neurons were stained in this area.

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proximal to the lesion relative to the striatum and a depletion in the reticular part of the substantia nigra (SN), suggesting that the antigen may be transported from the striatum to the SN (Fig. 6A and C). However, a slight decrease of IR in the accumbens and medial part of the caudate suggests that some transport of P450 IIEl occurs in the nigrostriatal directions as well. A number of large glial cells around the site of the lesion were surrounded by an immunoreactive halo (Fig. 6BD), seemingly representing staining of numerous fine glial cell processes. The possibility that the immunoreactivity in the SN emanated from the striatal cell bodies led us to administer colchicine in order to determine whether this treatment might affect the distribution pattern of IR in the striatonigral system. Tissue sections from animals treated with colchicine showed no increases in staining densities in comparison with those from untreated rats. Thus, no depletion of staining was seen in the fibers or in the SN.

S/A

C

VS

HC

L

Fig. 2. Western blot analysis of P450 lIEI in subcellular fractions of the rat brain, A. Analysis of hippocampal mitochondrial preparations from starved and acetonetreated rats (S/A, lane 1) and from control rats (C, lane 2). B. Analysis of mitochondriai control preparations from ventral striate (VS), hippocampus (HC) and liver microsomes (L) previously fractionated on pchloroamphetamine-Sepharose according to Warner et a/.’

Tha~amus

was detected in several different P450 IIEl-IR thalamic nuclei. The highest density of IR was found in the anteroventral, ventrolateral, the anterior pretectal areas and in the reticular thalamic nucleus (Fig. 5D). In the reticular nculeus IR neurons predominated while P450 IIEl-IR glial cells and blood vessels were more common in the other nuclei. P450 IIEl-IR glial cells and neuronal cells were also found in the superior colliculus.

Basal forebrain including the striatal complex

Mesencephalon and medulla

In the diagonal band of Broca a subpopulation of large- and medium-sized neurons were stained with the anti-P450 IIEl antibodies. These cells appear to be part of a larger system of neurons that can be followed into the substantia innominata and into the ventral striatum. Their size and distribution suggests that they may be part of the basal forebrain cholinergic system. In the lateral septum, a string of IR neurons was detected in the dorsolateral part. Neuronal cell bodies were present in the bed nucleus of the stria terminalis. A high density of P450 IIEl-IR was detected throughout the striatal complex (caudate nucleus, globus pallidum, nucleus accumbens and olfactory tubercle) (Fig. 1). The ~mmunoreactivity was localized to neuronal cell bodies (Fig. 5A and B) as well as the neurophil. In the caudate nucleus, islands of high staining density were observed, creating a patchy appearance within the structure (Fig. 5A). The neurophi1 staining in the globus pallidum was higher than that in the rest of the striatum. Fibers of the striatonigral system were strongly P450 IIEI-IR, indicating that the antigen was transported to or from the striatum. Indeed, mechanical lesions of this pathway showed an accumulation of P450 IIEl-IR

In the ventral part of the rostra1 mesen~phalon, a high density of P450 IIEl-IR was detected in the SN. In the reticular part this was diffusely distributed, while in the compact part a small population of neuronal cell bodies was immunostained (Fig. 5C). Scattered IR cells were detected in the reticular formation. Strongly P450 IIEl-IR neurons were present in the pontine nucleus, lateral superior olive, the nucleus of the trigeminal nerve and the facial nucleus (Fig. 7A--C). In addition, a large number of large- to medium-sized neurons were situated in the central gray and in the reticular formation. Dense IR was present in the nucleus of the spinal trigeminal nerve with scattered IR neurons in the reticular nucleus of the medulla. Cerebellum

The cerebellum was found to be rich in P450 IIEl-IR structures (Fig. 8A-D). The IR was localized exclusively to glial cells and their processes. Prominent staining was found in the radial processes of the Bergmann glial cells and in glial cells which formed a fine reticulated pattern of the white matter. The granular cell layer harbored scattered immunostained

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Fig 3. A. A component of the olfactory nerve which innervates the accessory bulb containing strong P45450 IIEI-IR. 3. High densities of P450 IIEl-IR glial cells present in the external plexiform layer of the olfactory bulb. C. Strong P450 IIEI-IR cells in the ependymal lining of the ventricular (third) walls. D. A subgroup of pyramidal cells in layers 4 and 5 showed strong staining of their somata with distinct cytoplasmatic staining of both apical and basal dendrites. Note that the nucleus lacks IR. fc, frontal cortex; 3V, third ventricle. Arrows in D mark apical and basal dendrites. Magnifications: A, x 31; B, x 500; C, x312; D, x200.

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Fig. 4

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Fig. 5. Photomicrographs showing P450 IIEI-IR cells in the striatum (A and B), substantia nigra (C) and reticular nucleus of the thalamus (D). A. In the caudate nucleus, islands of high IR density created a patchy appearance. The IR in the striatal complex was localized to neuronal cell bodies as well as in the neurophil. B. P450 IIEI-IR neurons in the striatum. C. A population of P450 IIEl-IR neuronal cell bodies in the compact part of the substantia nigra. D. Strong P450 IIEI-IR neurons in the reticular thalamic nucleus. STRI, striatal complex; gp, globus pallidus; SNc, substantia nigra compacta. Open arrowheads in A mark islands of high IR density. Arrowheads in B indicate IR neuronal cell bodies. Magnifications: A, x 31; B, x200; C and D, x 125.

ghal cells. Staining of blood vessels was found throughout the cerebellum. This was most prominent in the white matter, where frequently P450 IIEI-IR glial cells were seen to have end-feet on the vessels (Fig. 8C). Blood vessels Small and large blood vessels throughout the brain were P450 IIEl-IR (Fig. 9). The IR appears to be mainly located in the endothelial cells.

DISCUSSION

The present light microscopical study shows that a protein related to ethanol-inducible cytochrome P450 IIEl is constitutively expressed in both glial cells and neuronal cell bodies, fibers and terminals in many regions of the rat brain. In addition, a large number of small and large blood vessels containing P450 IIEl-IR are observed throughout the brain. The presence of authentic P450 IIEl in the rat brain is supported by the finding that (i) preadsorption of the

Fig. 4. A. The distribution of P450 IIEI-IR cells in the neocortex and the hippocampal region, The pyramidal cells of all subfields (CA1 through CA4) and in the subiculum were P450 IIEI-IR. B. Polymorphic cells of the hilus stained intensely with the anti-P450 HE1 antibodies. Moderate staining of glial cells was detected in the molecular layer of dentate gyrus. No IR was detected in the granule cell layer. C. Intensely stained polymorphic cells with shorter and longer dendritic processes as well as pyramidal cells in the CA3 subfield. Note the lack of IR in stratum lucidum. cx, neocortex; hc, hippocampal region; H, hilus; sub, subiculum; ml, molecular layer; g, granule layer; so, stratum oriens; sp, stratum pyramidal; sl, lucidum; sr, stratum radiatum. Magnifications: A, x 31; B and C, x 312.

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Fig. 6. Photomicrographs showing the distribution of P450 IIEI-IR in a horizontal tissue section following mechanical lesions of the nigrostriatal pathway. A and C. Mechanical lesion of this pathway caused a depletion of P450 IIEI-IR in the reticular part of the substantia nigra and an accumulation of P450 IIEl-IR proximal to the lesion. A number of large glial cells were present around the site of the lesion. These cells were surrounded by an immunoreactive halo (B and D). STRI, striatal complex; HPC, hippocampal region; gp, globus pallidus; SN, substantia nigra. Asterisks in A and C indicate the site of lesion. A is a photomicrograph from a computer-digitized picture. Magnifications: B, x 3 12; C, x 3 1; D, x 500.

antisera with the highly purified liver P450 IIEI completely blocked the immunostaining in the brain tissues sections, (ii) the antisera recognized a protein of identical mobility on sodium dodecyl sulphatepolyacrylamide gel electrophoresis as that of hepatic P450 IIEI when Western blot analysis was carried out on microsomal and mitochondrial fractions from rat brain and (iii) identical IR-patterns were observed with two different antisera directed against two different preparations of P450 IIEI. In most regions of the rat brain the pattern of P450 IIEl-IR was distinctly different from that obtained

previously with antibodies against P450 IIBl, IAI, IA2 and PB/PCN-E, respectively.5,‘5.37 P450 IIB 1-, IA2- and PB/PCN-E-IR is mainly associated with glial cells. The present study showed, however, that certain similarities exist between the staining pattern obtained with anti-P450 IAI and IIEI antibodies. Thus, several forebrafn areas which have been shown to harbor P450 IAl-IR neuronal cell bodies also contained P450 IIEl-IR cells. This was also true for certain populations of neurons in the brain stem and the medulla. The neurons in the striatal complex and SN, however, contain a high density of P450 IIE 1-IR

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Fig. 7. Photomicrographs of a sagittat section showing the distribution of P450 IIEI-IR in the brainstem. A. Neurons of facial nucleus, motor trigeminal nucleus and med superior olive nucleus contained strong P4.50 IIEI-IR. B and C. Strong P450 IIE-IR neurons in the facial nucleus at two different magnifications. 7, facial nucleus; MSO, med superior olive; MO& motor trigeminal nucleus. Magnifications: A, x 31; B, x 200; c, x 500.

while little or no staining

has so far been found for other P45Os. Since the anti P450 IIEI antiserum used

in the present study does not cross-react with P450 IA 1, it appears that a number of neurons in untreated rat brain contain multiple forms of P450. In cerebellum prominent staining was found with anti-P450 IIEl antisera in a large number of cerebellar structures, including Bergmann glial cells,

astrocytes and oligodendrocytes, but not in Purkinjie cells. Although all cell layers harbored P450 IIEI-IR glial cells, the white matter seemed to be particularly well equipped with those as well as with stained blood vessels with numerous attached glial cell endfeet. The distribution of P450 IIEl in cerebellum is similar to that seen previously for other types of p45o.w.37

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___-Fig.

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-.

_-_

_/--.i

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Fig. 9. Photomicrographs showing small and large blood vessels containing P450 IIEI-IR in the corpus callosum. A. The white matter contained a large number of P450 IIEl-IR glial cells in close contact with the vessels. B. A higher magnification of a blood vessel with attached glial cells. CC, corpus callosum; bv, blood vessel. magnifications: A, x 125; B, x 312.

A large number of different neuronal cell types expresssed immunodetectable amounts of P450 IIEl. The populations of immunoreactive neurons represented acetylchohne-containing motorneurons in medulla, dopaminergic neurons in SN, and presumed gluamatergic pyramidal cells in cortex and in hippocampus. The P450 IIEl-IR is thus not

associated with one single class of chemically identified cells. Thus the distribution of P450 IIEl-IR among different types of neurons would indicate that this form of P450 may participate in a common metabolic reaction rather than in a selective pathway of metabolism of a specific transmittor substance.

Fig. 8. Photomicrographs showing the distribution of P450 IIEI-IR in the cerebellum. A. The P450 IIEl-IR was localized to all cell layers of the cerebellum; molecular layer, granular layer and white matter. B and D. P450 IIEI-IR in Bergmann glial cells and their processes in th; molecular layer. Purkinje cells did not contain P450 IIEl-IR (C and D). Stained blood vessels were found throughout the cerebellum (A and C). C. The IR appeared mainly to’be located in the endothelial cells. Freque&y IR glial cells were seen to have endfeet on the vessels. ml, molecular layer; gl, granular layer; wm, white matter; pc, Purkinje cell; bv, blood vessel. Arrowheads in B and C indicate Bergmann glial cell processes. Arrow in C marks astrocyte endfoot on the vessel. Magnifications: A, x 125; B, x 312; C, x 500; D, x 1250.

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The high density of P450 IIEl-IR in basal ganglia, frontal cortex and hippocampus is of particular interest since these regions show morphological and biochemical changes following ethanol consumption in experimental animals.‘9,20.23,24~35 This might indicate a correlation between the presence of P450 IIEl, a form which metabolizes ethanol, and CNS effects within specific brain regions. Such a role for P450 IIEl in regional toxicity has been demonstrated in rat liver. Thus, a heterogeneous distribution of this P450 form and the region-specific expression and induction of the enzyme following ethanol consumption and/or solvent exposure has been implicated to explain the selective hepatotoxicity by substances known to be metabolized by this isozyme.’ Among the highest densities of P450 IIEl-IR were found in the caudate nucleus and pars reticulata of the SN. In the caudate nucleus, P450 IIEl-IR was found in neuronal cell bodies as well as in the neurophil. The observation that lesion of the nigrostriatal pathway significantly depleted the IR in the SN cells indicates that the P450 IIEl-IR cells in the SN are dependent on an intact nigrostriatal pathway. An accumulation of P450 IIEl-IR proximal to the lesion and a depletion of IR in the SN seem to indicate that the antigen is synthesized in striatal cell bodies and axonally transported to the terminals. Treatment of animals with colchicine, to block axonal transport, did not reveal additional staining in the striatal neurons, nor did it affect the intensity of staining of fibers or of terminals in the SN. Our data could indicate that P450 proteins are not transported by fast axonal transport from the cell body down to the terminal but by some other mechanism insensitive to colchicine. The absence of effects on IR in the cell bodies following colchicine treatment is in line with the data presented by Kapitulnik et af.,14 who showed strong staining of nerve fibers but minor staining of neuronal cell bodies in rats treated with colchicine and using antLP45OIA 1 antibodies. Recently. evidence was presented for the presence of a number of xenobiotic metabolizing enzymes including P450, nicotinamide adenine dinucleotide phosphate P450 reductase and glucuronosyl transferase in brain micro-vessel endothelial cells from rat brain.4 Our immunocytochemical data show

the presence of P450 IIEl in brain vessel endothelial cells. Recent immunohistochemical experiments” presented evidence for the presence of P4502- and P4505-IR in capillary endothelial cells and in larger blood vessels of rabbit lung. Furthermore, a role for P450 in the vascular metabolism of the endogenous substrate arachidonic acid to vasoactive compounds has been proposed. 29,3’ Such a function of brain capillary endothelial P450 could have a profound implication for its role in regulation of brain microcirculation. It has been shown that hepatic P450 IIEl metabolizes acetone.“x’6 Since brain can use ketone bodies as substrates for energy metabolism during the neonatal period and after prolonged starvation.6,2” such compounds may well constitute endogenous substrates for P450 IIEl. CONCLUSION

In view of the well-known effects of ethanol on motor activity and memory, the observation that P450 IIEl-IR was found in basal ganglia and in several areas of the cortex, including the hippocampus and several nuclei in the medulla, inspires the study of the role of brain P450 IIEl in ethanol metabolism and/or in metabolism of endogenous compounds. The expression of P450 IIEl in the CNS is also of interest because of the toxicological importance of this P450 form.2,“,‘4 It might thus be suggested that metabolic activation of numerous different organic solvents, which are specific substrates for this isozyme, might take place in the P450 IIEl -containing neurons. Furthermore, the P450 IIEl -dependent metabolism of ethanol to acetaldehyde and the P450 IIEl-catalysed metabolic activation of certain drugs, might be connected with the development of specific neuronal cell damage. Further exploration of the distribution and biochemical characterization of P450 IIEl will be relevant to the understanding of the molecular mechanisms of ethanol toxicity in the CNS. Acknowledgemenrs~This work was supported by grants from 0. E. Edla Johanssons Stiftelse, The Swedish Medical Research Council and the National Board of Laboratory Animals.

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I. 2. 3. 4. 5. 6. 7.

Coon M. J., Koop D. R.. Reeve L. E. and Crump B. L. (1984) Alcohol metabolism and toxicity: role of cytochrome P450. Fundam. appl. Toxic. 4, 134-143. Ekstriim G. and Ingelman-Sundberg M. (1989) Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P450. Biochem. Pharmac. 38, 13 13-13 19. Eliasson E., Johansson I. and Ingehnan-Sundberg M. (1988) Ligand-dependent maintenance of ethanol-inducible cytochrome P450 in primary rat hepatocyte cell cultures. Biochem. hiophys. Res. Commun. 150, 436443. Ghersi-Egea J.-F., Minn A. and Siest G. (1988) A new aspect of the protective functions of the blood--brain barrier: activities of four drug-metabolizing enzymes in isolated rat brain microvessels. Life Sci. 42, 2515-2523. Hansson T., Kiihler C., Astrom A., Warner M. and Gustafsson J.-A. lmmunohistochemical evidence for the existence of multiple forms of cytochrome P450 and of NADPH P450 reductase in glial cells of the rat brain (to be submitted). Hawkins R. A., Williamsson D. H. and Krebs H. A. (1971) Ketone-body utilization by adult and suckling rat brain in 1Gt.0.Biochem. J. 122, 13Sl8. Hsu S. M., Raine L. and Fanger H. (1982) The use of avidinbiotin peroxidase complex (ABNC) in immunoperoxidase techniques. A comparison between ABC and unlabelled antibody (PAP) procedures. J. Histochem. 29, 577-580.

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