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Co-localization of P450 enzymes in the rat substantia nigra with tyrosine hydroxylase
Pergamon
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Neuroscience Vol. 86, No. 2, pp. 511–519, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00649-0
CO-LOCALIZATION OF P450 ENZYMES IN THE RAT SUBSTANTIA NIGRA WITH TYROSINE HYDROXYLASE P. M. WATTS,* A. G. RIEDL,* D. C. DOUEK,† R. J. EDWARDS,‡ A. R. BOOBIS,‡ P. JENNER*¶ and C. D. MARSDEN§ *Neurodegenerative Disease Research Centre, Pharmacology Group, Biomedical Sciences Division, King’s College, London SW3 6LX, U.K. †MRC Clinical Sciences Centre and ‡Section on Clinical Pharmacology, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, U.K. §University Department of Clinical Neurology, Institute of Neurology, The National Hospital for Neurology and Neurosurgery, London WC1N 3BG, U.K. Abstract––Susceptibility to develop Parkinson’s disease has been linked to abnormalities of P450 enzyme function. Multiple P450 enzymes are expressed in brain but the relationship of these to Parkinson’s disease is unknown. We have investigated the expression of P450 enzymes in the rat substantia nigra and their co-localization in tyrosine hydroxylase-positive neurons and astrocytes. Immunohistochemistry was performed using anti-peptide antisera against the following P450 enzymes: CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, CYP2C13/2C6, CYP2D1, CYP2D4, CYP2E1, CYP3A1, CYP3A2 and NADPHP450 oxidoreductase. Immunoreactivity in nigral cells was found only for CYP2E1 and CYP2C13/2C6. CYP2E1 immunoreactivity was localized to many midbrain nuclei including the substantia nigra pars compacta but not the substantia nigra pars reticulata while immunoreactivity to CYP2C13/2C6 was found in the substantia nigra pars compacta, substantia nigra pars reticulata and many other midbrain nuclei. Sections of rat midbrain double labelled for either CYP2E1 or CYP2C13/2C6 and tyrosine hydroxylase or glial fibrillary acidic protein were examined for co-localization by confocal laser scanning microscopy. CYP2E1 and CYP2C13/2C6 immunoreactivity was found in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta but not in glial cells. CYP2C13/2C6, but not CYP2E1, was also found in non-glial, non-tyrosine hydroxylase-expressing cells in the substantia nigra pars reticulata. Isoniazid induction increased CYP2E1 fluorescence signal intensity from nigral dopaminergic neurons. At least two P450 enzymes are found in nigral dopamine containing cells and one, namely CYP2E1, is selectively localized to this cell population. CYP2E1 is a potent generator of free radicals which may contribute to nigral pathology in Parkinson’s disease. The expression of CYP2E1 in dopaminergic neurons in substantia nigra raises the possibility of a causal association with Parkinson’s disease. 1998 IBRO. Published by Elsevier Science Ltd. Key words: Parkinson’s disease, cytochrome P450, CYP2E1, CYP2C13/2C6, confocal microscopy, immunohistochemistry.
The cause of nigral dopaminergic cell degeneration in Parkinson’s disease (PD) is unknown but evidence exists which suggests that free radicals and oxidative stress may be involved.31 The trigger for the progressive nigral cell loss is also unknown but may involve exposure to environmental57 or endogenous neurotoxins coupled to a genetic susceptibility.18 There is controversial evidence suggesting a link between the expression of certain forms of P450 and ¶To whom correspondence should be addressed. Abbreviations: FITC, fluorescein isothiocyanate; FMO, flavin monooxygenase; GFAP, glial fibrillary acidic protein; isoniazid, isonicotinic acid hydrazide; LRSC, lissamine rhodamine sulphonyl chloride; MPTP, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine; NDS, normal donkey serum; NGS, normal goat serum; PBS, phosphatebuffered saline pH 7.4; PD, Parkinson’s disease; SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata; TH, tyrosine hydroxylase; VTA, ventral tegmental area.
susceptibility to developing PD. In particular, mutations in the CYP2D6 gene which result in failure of CYP2D6 expression, may be over-represented in some PD populations, typically conferring a two- to five-fold disease risk.2,9,36,53,59 In addition, allelic variants of human CYP1A1 exist with the homozygous CYPVal1A1 mutation conferring a six-fold relative risk to develop Parkinson’s disease compared to the wild type.56 Several P450 enzymes are found in monkey, human and rat brain. High levels of aromatase messenger RNA is present in the hypothalamus and amygdala of male Japanese monkeys,64 whilst CYP1A1,25 CYP1A2,25 CYP2E1,25 CYP2D6,26 CYP3A25 and NADPH-P450 oxidoreductase47 have been detected in various regions of the human brain. In the rat brain a range of P450 enzymes are reported to be present, including CYP1A1,38 CYP1A1/2,8,10,32,35,62 CYP2B1/2,6,7,33,38,62
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CYP2C7,61 CYP2C11,3,61 CYP2C12,27 CYP2C13,27 CYP2D1,40,60 CYP2D4,30 CYP2E1,5,7,8,15,29,54,58,65 CYP3A9,61 CYP4A3,55 CYP4A855,61 and NADPHP450 oxidoreductase.4,28,39,47 However, discrepancies exist regarding their presence and precise distribution within brain. For example, CYP1A1,8,35 CYP2D1,40 CYP2D4,30 CYP2E129,54 and NADPH-P450 oxidoreductase28 have been reported to be present in the rat substantia nigra. We believe that the discrepancies result from the specificity of the antibodies and antisera employed and that this has arisen from differing techniques of antibody production. Most studies have employed antisera raised against P450 enzymes from rat hepatic microsomes where even minor contamination of the immunizing P450 enzyme with other proteins will result in the production of cross-reactive antibodies. Even immunization with a pure P450 enzyme preparation results in antibodies which cross-react with similar or identical epitopes on closely-related P450 enzymes. To overcome this problem we have developed a series of highly specific anti-peptide antisera raised against unique peptide sequences of specific P450 enzymes. Using such antisera and peroxidase immunohistochemistry, we recently described the distribution of CYP1A1, CYP2B1/2, CYP2D1, CYP2E1, CYP3A1 and NADPH-P450 oxidoreductase in rat basal ganglia.48 These studies demonstrated selective localization of CYP1A1 restricted to the globus pallidus with CYP2E1 expressed in neuronal cells in the substantia nigra, paraventricular hypothalamic nucleus and striatal vasculature. CYP2E1 is also expressed in the cerebellum,29 hippocampus,29 cerebral cortices,54 caudate–putamen54 and globus pallidus.54 We now extend these findings by focusing on the expression of a wide range of P450 enzymes in the rat nigra using anti-peptide antisera directed against unique C-terminal or internal amino acid sequences. A panel of anti-P450 antisera targeted against each of the major xenobiotic-metabolizing P450 enzymes expressed in the rat were used. These recognized CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, CYP2C13/2C6, CYP2D1, CYP2D4, CYP2E1, CYP3A1, CYP3A2 and NADPH-P450 oxidoreductase. The localization of those P450 enzymes that stained nigral cells, as assessed by peroxidase immunohistochemistry, was investigated further by double staining with anti-P450 antisera and anti-tyrosine hydroxylase (TH) or anti-glial fibrillary acidic protein (GFAP) antibodies. Co-localization was detected by confocal laser scanning microscopy which allowed determination of P450 enzyme expression in dopaminergic neurons and astrocytes in the substantia nigra. Since CYP2E1 expression in brain was low, it was necessary to induce the enzyme and rats were treated with a classical hepatic CYP2E1 inducer, isonicotinic acid hydrazide (isoniazid) once daily for five days.43
EXPERIMENTAL PROCEDURES
Materials All chemicals were obtained from Sigma (Sigma Chemical Co. Ltd, Dorset, U.K.) unless otherwise stated. Animals Adult male adult Wistar rats (n=24; 150–200 g, Bantin and Kingman, Hull, U.K.) were housed six to a cage and given free access to pelleted food (Special Diet Services, Witham, U.K.) and water. All animal experiments were carried out in strict accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines. Preparation of antibodies Polyclonal rabbit anti-peptide antibodies directed against CYP1A1,24,48 CYP1A2,23 CYP2B1/2,48,51 CYP2D1,48 CYP2E1,24,48 CYP3A1,19,48 CYP3A219 and NADPH-P450 oxidoreductase48 were used. Antibodies directed against the C-termini of CYP2C12, CYP2C13/2C6 and CYP2D4 were produced by immunizing rabbits with the peptides PheIle-Pro-Val, Arg-Phe-Ile-Pro-Leu and Ala-Ser-Pro-Arg, respectively, by methods described previously.22,24 The specificity of antibody binding to the target P450 enzymes was confirmed by immunoblotting using hepatic microsomal fractions from untreated rats and rats treated with appropriate P450 enzyme inducing chemicals.19,22,24,51 Isonicotinic acid hydrazide treatment Male Wistar rats (n=6; 150–200 g) were treated with isonicotinic acid hydrazide (isoniazid; 200 mg/kg, i.p.) once daily for five days to induce CYP2E1. Control rats (n=6; 150–200 g) were injected with 0.9% saline. Animals were killed 3 h following the final treatment with isoniazid or saline by terminal anaesthesia with pentobarbitone (100 mg/ kg; Rhone Merieux Ltd, Harlow, U.K.) followed by transcardial perfusion with 0.1 M phosphate-buffered saline, pH 7.4 (PBS) at 4C (200 ml) and perfuse-fixed with 4% paraformaldehyde in PBS (200 ml). Tissue processing Brains and livers (used as positive controls to assess the reactivity of each antiserum) were removed and postfixed in 4% paraformaldehyde in 0.1 M PBS at 4C for 48 h. Tissues were then cryoprotected in 30% sucrose in 0.1 M PBS for a further 48 h and snap frozen in isopentane on dry ice. All tissue was subsequently stored at 70C. Peroxidase immunohistochemistry Contiguous coronal sections (30 µm) of midbrain were cut using a cryostat to include the entire substantia nigra according to the atlas of Paxinos and Watson.44 Immunostaining was performed on free-floating sections. Sections were washed twice in 0.1 M PBS before endogenous peroxidase activity was blocked by incubating sections in 70% methanol in water containing 0.3% hydrogen peroxide for 30 min. After two further washes in 0.1 M PBS, sections were incubated in 20% normal goat serum (NGS) in 0.1 M PBS for 1 h and then washed twice in 0.1 M PBS containing 1% NGS and 0.05% Triton X-100. Sections of midbrain and liver were incubated with antisera targeted against the C-terminal peptides for CYP1A1, CYP1A2, CYP2B1/2B2, CYP2C12, CYP2C13/2C6, CYP2D1, CYP2D4, CYP2E1, CYP3A1, CYP3A2 or NADPH-P450 oxidoreductase. Controls consisted of alternate sections of midbrain incubated with non-immune rabbit serum. Antisera or non-immune serum were diluted 1:1000 in 1% NGS in 0.1 M PBS and incubated overnight (18 h) at room temperature on a shaking platform. Immunostaining was performed according to the manufacturers instructions using a Vectastain ABC kit (Vector Laboratories, Peterborough, U.K.).
P450 enzymes in rat substantia nigra
Fig. 1. (a) Photomicrograph of rat substantia nigra stained for CYP2E1 using peroxidase immunohistochemistry and counterstained with Cresyl Violet. CYP2E1 immunoreactivity demonstrated throughout the midbrain including neurons of the SNpc and VTA. Scale bar=250 µm. (b) Photomicrograph of rat substantia nigra stained for CYP2C13/2C6 using peroxidase immunohistochemistry and counterstained with Cresyl Violet. CYP2C13/2C6-immunoreactive neurons present throughout the midbrain including the SNpc, SNpr and VTA. CYP2C13/2C6-positive neurons occasionally found in SNpr (arrow head). Scale bar=250 µm.
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P450 enzymes in rat substantia nigra Immunoreactive products were visualized using 3,3 diaminobenzidine (0.05% in Tris buffer) and 0.01% hydrogen peroxide. Sections were mounted onto slides and some were counterstained with Cresyl Violet to enhance visualization of cellular architecture before being dehydrated in 70%, 96% and 100% ethanol and cleared in xylene. For each of the P450 enzymes studied, sections of midbrain from at least six animals were examined at multiple levels throughout the substantia nigra. Sections of liver were similarly processed and served as positive controls for those P450 enzymes expressed in normal rat liver. Co-localization studies Sections of midbrain at the level of the exit of the third nerve (5.6mm from Bregma) were incubated in 0.1 M PBS containing 10% NGS and 10% normal donkey serum (NDS) for 1 h and then washed twice in 0.1 M PBS containing 0.05% Triton X-100, 0.5% NGS and 0.5% NDS. Sections were then incubated in the primary antisera which consisted of rabbit anti-CYP2E1 or rabbit antiCYP2C13/2C6 diluted 1/1000 and mouse monoclonal antiGFAP diluted 1/200 (0.1 µg/ml, Boehringer Mannheim Biochemica, Germany) or sheep polyclonal anti-TH diluted 1/250 (0.2 µg/ml, Pel Freez, AK, U.S.A.) made up in PBS with 0.5% NGS and 0.5% NDS, and incubated overnight (18 h) on a shaking platform. Tissues were washed twice in PBS with 0.5% NGS and 0.5% NDS and incubated in second layer antisera which consisted of biotinylated goat anti-rabbit polyclonal IgG diluted 1/200 (7 µg/ml, Vector Laboratories, Peterborough, U.K.) as a probe for the anti-P450 first layer and either affinity-purified lissamine rhodamine sulphonyl chloride (LRSC)-conjugated donkey anti-mouse F(ab )2 diluted 1/200 (7.5 µg/ml, Jackson Immunoresearch, PA, U.S.A., probing for anti-GFAP first layer) or affinity-purified LRSC-conjugated donkey antisheep F(ab )2 diluted 1/200 (7.5 µg/ml, Jackson Immunoresearch, PA, U.S.A., probing for anti-TH first layer), for 1 h in the dark. After two washes in PBS all sections were incubated in streptavidin-conjugated fluorescein isothiocyanate (FITC) diluted 1/50 (4 µg/ml, Caltag Labs, CA, U.S.A.) in PBS for 1 h in the dark and washed twice again in PBS. Sections were wet mounted in Vectashield (Vector Laboratories, Peterborough, U.K.) and stored at 4C in the dark until examined. Confocal microscopy and fluorophore detection A Leica IR confocal laser scanning microscope was used to image fluorescence staining. The argon/krypton laser produced excitation wavelengths of 488, 568 and 650 nm and four photomultipliers detected emission signals. Double staining with FITC and LRSC was scanned and detected simultaneously using narrow band filters to prevent crosstalk between signals. Eight optical sections for each tissue section were taken over a thickness of 15 µm and were summed using a maximal projection algorithm. Red (LRSC) and green (FITC) channels were overlayed or
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separated using Photoshop 3.0 software on a Macintosh PC computer. Background shadow was reduced equally for each channel by 30%. Sections taken from rats treated with isoniazid and saline and appropriate controls were stained for CYP2E1 and TH or GFAP as above and the level of CYP2E1 expressed quantified by measuring FITC signal intensity. This signal intensity, expressed on an arbitrary grey scale, was calculated from averages obtained from six cells from each of six isoniazid-treated or saline-treated animals, cells being selected from the medial aspect of the substantia nigra pars compacta (SNpc) at the level of the exit of the third nerve. TH fluorescence signal intensity was similarly quantified. Statistical analysis Differences between mean signal intensities for TH- and CYP2E1-expressing cells from isoniazid-treated and salinetreated animals were compared using Mann–Whitney U-test, where P<0.05 was considered significant. RESULTS
Peroxidase immunohistochemistry Liver sections, used as positive controls, produced immunoreactive centrilobular staining using antisera targeted against CYP1A2, CYP2B1/2, CYP2E1, CYP2C12, CYP2C13/2C6, CYP3A1, CYP3A2 and NADPH-P450 oxidoreductase. Antisera against CYP2D1 produced an intense uniform panlobular staining. No immunoreactivity to antisera against CYP1A1 or CYP2D4 was observed in liver sections. However, antisera against CYP1A1 stained the globus pallidus intensely, while antisera against CYP2D4 weakly stained white matter tracts in naive rat brain (data not shown). No immunoreactivity to CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, CYP2D1, CYP2D4, CYP3A1, CYP3A2 or NADPHP450 oxidoreductase was observed in the substantia nigra. Only CYP2E1 and CYP2C13/2C6 antisera produced staining of nigral cells. Staining was observed at all nigral levels and in all sections examined, from at least six animals. CYP2E1 immunoreactivity was detected throughout the midbrain including the SNpc and ventral tegmental area (VTA) in many cells with neuronal morphology (Fig. 1a). No staining was seen outside the nigra nor was there any reproducible signal in the substantia nigra pars reticulata (SNpr). CYP2C13/2C6 was detected in cells of neuronal morphology throughout the midbrain but was most
Fig. 2. Confocal immunofluorescence images of rat substantia nigra stained for TH or GFAP and CYP2E1 or CYP2C13/2C6. Scale bar=30 µm. (a) CYP2E1 expression in the SNpc of a naive animal (left), showing faint green fluorescence, TH expressing neurons (centre), showing strong red fluorescence and composite demonstrating co-localization of signals (right). (b) CYP2E1 expression in the SNpc of an isoniazid-treated animal (left) demonstrating enhanced CYP2E1 signal intensity, TH (centre) and composite demonstrating co-localization (right). (c) CYP2E1 expression in the SNpc of an isoniazidtreated animal (left), GFAP (centre), composite showing no co-localization (right). (d) CYP2C13/2C6immunoreactive neurons are present in the SNpc, SNpr and dorsal to the nigra (left), TH (centre) and composite demonstrating co-localization of signals in nigral neurons (right). Occasional CYP2C13/2C6positive, TH-negative cells were seen in the SNpr (arrow). (e) CYP2C13/2C6 in the SNpc and SNpr (left), GFAP (centre) and composite showing no co-localization (right). Occasional CYP2C13/2C6-positive, TH-negative cells were seen in the SNpr (arrow).
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Table 1. Fluorescence signal intensities for CYP2E1 and tyrosine hydroxylase in isoniazid-treated and saline-treated rats, meanS.D. Signal 2E1 TH
Saline
Isoniazid
71.56.2 216.28.8
165.425* 185.230
*P<0.05 (Mann–Whitney U-test) Each value represents the mean fluorescence signal intensities (expressed in relative units on a grey scale) taken from at least six neurons in the medial SNpc, n=6 animals in each group.
intense in cells of the SNpc and VTA (Fig. 1b). Occasional cells of neuronal morphology were stained for CYP2C13/2C6 in the SNpr. Isoniazid treatment resulted in more extensive hepatic centrilobular CYP2E1 staining but in brain there was no difference in neuronal CYP2E1 immunoreactivity in the substantia nigra compared with saline-treated animals. Control sections of midbrain incubated with non-immune serum showed only background staining, characterized by an even brown wash over the whole section. Confocal microscopy CYP2E1 signal was found to co-localise with TH in the SNpc in all sections examined but did not co-localise with GFAP. In normal animals CYP2E1 was expressed at low levels in a minority of dopaminergic neurons, the most intense signal being found in the medial SNpc and VTA. The CYP2E1 signal was virtually masked by the strong expression of TH, but co-localization of CYP2E1 and TH definitely occurred (Fig. 2a). Following treatment with the CYP2E1 inducer isoniazid, the majority of nigral dopaminergic neurons were found to express CYP2E1 (Fig. 2b) and signal intensity increased 2.3-fold, but had no effect on TH signal (Table 1). Some vascular endothelial cells stained for CYP2E1 after isoniazid treatment as we have previously shown in the striatum.48 Isoniazid treatment had no effect on the GFAP signal nor did it result in any CYP2E1 expression in astrocytes in the substantia nigra (Fig. 2c). CYP2C13/2C6 signal was found to co-localise with virtually all nigral TH-positive neurons in the SNpc (Fig. 2d). In addition, cells with neuronal morphology were observed expressing CYP2C13/2C6 in the SNpr but these were negative for both TH and GFAP. No CYP2C13/2C6 signal was detected in any of the GFAP-positive cells (Fig. 2e). Some GFAPpositive astrocytes were seen making intimate contact with CYP2C13/2C6-positive cells in the SNpr (Fig. 2e). Far fewer astrocytes were present in the SNpc and the majority of these were associated with capillaries and not neurons.
DISCUSSION
This is the first comprehensive investigation of the expression of multiple P450 enzymes in the substantia nigra and also the first to use exclusively anti-peptide antisera. The antisera used were highly specific, as a result of using short synthetic peptide sequences unique to each P450 enzyme.24 CYP2E1 was previously reported in neurons in the rat substantia nigra but diffuse staining was also observed in the SNpr and in many other brain regions including cells identified as glia.29 Others found CYP2E1 in the rat nigra after induction with ethanol, but there was no attempt to define its localization or the cell type in which it was expressed.54 We have confirmed and extended these findings by the observation in the present studies that CYP2E1 is selectively localized in dopaminergic nigral neurons. We previously failed to find the more extensive distribution of CYP2E1,48 previously reported by others,29,54 and in the present study we were unable to demonstrate CYP2E1 expression in nigral astrocytes even after administration of isoniazid. In vitro, CYP2E1 expression in primary cultures of rat cortical astrocytes has been co-localized with GFAP using confocal microscopy,37 but this does not appear to occur in vivo in the substantia nigra. The localization of CYP2E1 to the rat SNpc is supported by the finding of mRNA for CYP2E1 in human substantia nigra.25 The inducibility of CYP2E1 in the SNpc by isoniazid is evidence that the enzyme is present in the substantia nigra in a functional form. We have also demonstrated previously that isoniazid induces CYP2E1 in striatal vasculature in the rat.48 Alternatively CYP2E1 may have a role in neurodegeneration or in the detoxication of environmental neurotoxins similar to the metabolism of 1-methyl-4phenyl-1,2,3,6 tetrahydropyridine (MPTP) carried out by CYP2D6 and other P450 enzymes.17 Electron leakage from P450 redox reactions in the nigra might be expected to induce oxidative stress under certain conditions. Some P450 enzymes, including CYP2E1, are particularly susceptible to electron leakage and the extent of this appears to be dependent on the affinity of substrate for the enzyme. Such an increase in formation of reactive oxygen species from P450 enzymes in nigral dopaminergic neurons is a potential cause of the oxidative stress which has been detected in substantia nigra in Parkinson’s disease.20 The hepatic P450 enzyme responsible for vitamin D 25-hydroxylation and CYP1A2, both oxidise dopamine to an aminochrome in vitro, this reaction being supported by an electron donor other than NADPH-P450 oxidoreductase.52 CYP2E1 could also participate in such reactions and might be responsible for dopamine degradation. Oxidized metabolites might include hydrogen peroxide and neurotoxin 6-hydroxydopamine. This might explain the previously reported covalent binding of dopamine or a
P450 enzymes in rat substantia nigra
metabolite to rat brain microsomal protein after co-incubation in vitro, this reaction being dependent on NADPH and oxygen.49 Alternatively, CYP2E1 activity in the nigra might be of importance in the activation of pro-neurotoxins in a manner analogous to the activation of MPTP by monoamine oxidase B. CYP2E1 is capable of oxidizing alcohols to aldehydes. Acetaldehyde condenses with dopamine to form potentially neurotoxic isoquinolines,34 which have been detected in normal human and rat brain as well as in tissue from patients with PD.41 There is also increasing evidence that exposure to n-hexane as a component of petroleum spirit and of other solvents is linked to the development of a parkinsonian syndrome.45 n-Hexane is neurotoxic only following activation to 2,5-hexandione and this reaction can in part be catalysed by CYP2E1.1 With the low levels of n-hexane in the atmosphere from the incomplete combustion of petrol and the presence of CYP2E1 in the nigra, neurotoxic 2,5-hexanedione may be formed. The significance of the widespread neuronal distribution of CYP2C13/2C6 in the brain and the intense immunostaining of this enzyme observed in the SNpc dopaminergic neurons is unclear. CYP2C13 participates in the 6-hydroxylation of testosterone50 and it may therefore be important for the degradation of testosterone or the synthesis of other hormones. Indeed, high levels of testosterone are present in human substantia nigra.12 CYP2C13 expression in nigral dopaminergic neurons might therefore be expected to be functionally important possibly by reducing the aromatase interconversion of androgen to oestrogen46 which would be expected to affect the firing patterns of these cells16 and their susceptibility to MPTP.21 In contrast to previous reports we found no evidence of CYP2D140 nor CYP2D430 expression in the rat nigra. Our anti-peptide antibody against CYP2D4 failed to stain liver but did stain white matter tracts. Wyss et al.63 have also observed CYP2D4 immunoreactivity in white matter tracts.
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Norris et al.40 used a polyclonal antibody raised against purified CYP2D1 and this may cross-react with all CYP2D forms. Thus, it is still possible that rat nigra contains CYP2D2, CYP2D3 or CYP2D5. Therefore expression of these P450 enzymes requires further investigation. The presence of CYP1A1/235 and NADPH-P450 oxidoreductase28 in the rat nigra have previously been reported using polyclonal antisera prepared against purified rat liver proteins. We could not confirm the presence of these P450 enzymes using our anti-peptide antisera. The absence of NADPH-P450 oxidoreductase in nigra, despite the presence of CYP2E1 and CYP2C13/2C6 reported has a corollary in mouse heart14 and rabbit lung endothelial cells42 where CYP1A1 is present in the absence of the reductase. As P450 enzyme function is dependent on an electron donor, other electron donors may be acting in this role in nigral dopaminergic neurons. Flavin mononoxygenases (FMOs) have been detected in rat brainstem11 and FMO3 and FMO4 have been detected in the human substantia nigra.13
CONCLUSION
The expression of CYP2E1 in nigral dopaminergic neurons in the rat raises important questions about its physiological function at this site and if expressed in humans, it could be involved in mechanisms of cell death including Parkinson’s disease. The presence of CYP2E1 has been reported in the human brain, although there are no reports of it being found in the substantia nigra. CYP2E1 expression may vary between individuals, as a result of polymorphisms in CYP2E1 promoters. Clearly such polymorphic variation could be a risk factor for the development of PD. Acknowledgements—This work was supported by the Medical Research Council, the Wellcome Trust, the Parkinson’s Disease Society and the National Parkinson Foundation, Miami, U.S.A.
REFERENCES
1. 2. 3. 4. 5. 6. 7.
Abou-Donia M., Hu Z., Lapadula D. M. and Gupta R. P. (1991) Mechanisms of joint neurotoxicity of n-hexane, methyl isobutylketone and O-ethyl-O-4-nitrophenyl phenylphosphophonothioate in hens. J. Pharmac. exp. Ther. 257, 282–289. Akhmedova S. N., Pushnova E. A., Yakimovsky A. F., Avtonomov V. V. and Schwartz E. I. (1995) Frequency of a specific cytochrome P4502D6B (CYP2D6B) mutant allele in clinically differentiated groups of patients with Parkinson’s disease. Biochem. molec. Med. 54, 88–90. Alkayed N. J., Narayanan J., Gebremedhin D., Medhora M., Roman R. J. and Harder D. R. (1996) Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. Stroke 27, 971–979. Anandatheerthavarada H. K., Boyd M. R. and Ravindranath V. (1992) Characterization of a phenobarbital-inducible cytochrome P-450, NADPH-cytochrome P-450 reductase and reconstituted cytochrome P-450 mono-oxygenase system from rat brain: evidence for constitutive presence in rat and human brain. Biochem. J. 288, 483–488. Anandatheerthavarada H. K., Shankar S. K., Bhamre S., Boyd M. R., Song B. J. and Ravindranath V. (1993) Induction of brain cytochrome P-450IIE1 by chronic ethanol treatment. Brain Res. 601, 279–285. Anandatheerthavarada H. K., Shankar S. K. and Ravindranath V. (1990) Rat brain cytochromes P-450: catalytic, immunochemical properties and inducibility of multiple forms. Brain Res. 536, 339–343. Anandatheerthavarada H. K., Williams J. F. and Wecker L. (1993) The chronic administration of nicotine induces cytochrome P-450 in rat brain. J. Neurochem. 60, 1941–1944.
518 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
P. M. Watts et al. Anandatheerthavarada H. K., Williams J. F. and Wecker L. (1993) Differential effect of chronic nicotine administration on brain cytochrome P4501A1/2 and P4502E1. Biochem. biophys. Res. Commun. 194, 312–318. Armstrong M., Daly A. K., Cholerton S., Bateman D. N. and Idle J. R. (1992) Mutant debrisoquine hydroxylation genes in Parkinson’s disease. Lancet 339, 1017–1018. Bhagwat S. V., Boyd M. R. and Ravindranath V. (1995) Brain mitochondrial cytochromes P450: xenobiotic metabolism, presence of multiple forms and their selective inducibility. Arch. Biochem. Biophys. 320, 73–83. Bhamre S., Bhagwat S. V., Shankar S. K., Williams D. E. and Ravindranath V. (1993) Cerebral flavin-containing monooxygenase-mediated metabolism of antidepressants in brain: immunochemical properties and immunocytochemical localization. J. Pharmac. exp. Ther. 267, 555–559. Bixo M., Backstrom T., Winblad B. and Andersson A. (1995) Estradiol and testosterone in specific regions of the human female brain in different endocrine states. J. Steroid Biochem. molec. Biol. 55, 297–303. Bootman M. A., Phillips I. R. and Shephard E. A. (1996) Flavin-containing monooxygenase expression in the human brain. XIth International Symposium on Microsomes and Drug Oxidations, p. 23. Brittebo E. B. (1994) Metabolic activation of the food mutagen Trp-P-1 in endothelial cells of heart and kidney in cytochrome P450-induced mice. Carcinogenesis 15, 667–672. Cammer W., Downing M., Clarke W. and Schenkman J. B. (1991) Immunocytochemical staining of the RLM6 form of cytochrome P-450 in oligodendrocytes and myelin of rat brain. J. Histochem. Cytochem. 39, 1089–1094. Chiodo L. A. and Caggiula A. R. (1983) Substantia nigra dopamine neurons: alterations in basal discharge rates and autoreceptor sensitivity induced by estrogen. Neuropharmacology 22, 593–599. Coleman T., Ellis S. W., Martin I. J., Lennard M. S. and Tucker G. T. (1996) 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) is N-demethylated by cytochromes P450 2D6, 1A2 and 3A4-implications for susceptibility to Parkinson’s disease. J. Pharmac. exp. Ther. 277, 685–690. Daly A. K., Cholerton S., Armstrong M. and Idle J. R. (1994) Genotyping for polymorphisms in xenobiotic metabolism as a predictor of disease susceptibility. Environ. Health Perspect. 102, 55–61. Debri K., Boobis A. R., Davies D. S. and Edwards R. J. (1995) Distribution and induction of CYP3A1 and CYP3A2 in rat liver and extra-hepatic tissues. Biochem. Pharmac. 50, 2047–2056. Dexter D. T., Holley A. E., Flitter W. D., Slater T. F., Wells F. R., Daniel S. E., Lees A. J., Jenner P. and Marsden C. D. (1994) Increased levels of lipid hydroperoxides in the Parkinsonian substantia nigra: an HPLC and ESR study. Movement Disorders 9, 92–97. Dluzen D. E. (1996) Effects of testosterone upon MPTP-induced neurotoxicity of the nigrostriatal dopaminergic system of C57/B1 mice. Brain Res. 715, 113–118. Edwards R. J., Murray B. P. and Boobis A. R. (1991) Anti-peptide antibodies in studies of cytochromes P450 IA. Meth. Enzym. 206, 220–233. Edwards R. J., Murray B. P., Singleton A. M., Murray S., Davies D. S. and Boobis A. R. (1993) Identification of the epitope of an anti-peptide antibody which binds to CYP1A2 in many species including man. Biochem. Pharmac. 46, 213–220. Edwards R. J., Singleton A. M., Murray B. P., Davies D. S. and Boobis A. R. (1995) Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes. Biochem. Pharmac. 49, 39–47. Farin F. M. and Omiecinski C. J. (1993) Regiospecific expression of cytochrome P-450s and microsomal epoxide hydrolase in human brain tissue. J. Toxicol. environ. Health 40, 317–335. Gilham D. E., Cairns W., Paine M. J. I., Modi S., Poulsom R., Roberts G. C. K. and Wolf C. R. (1997) Metabolism of MPTP by cytochrome P4502D6 and the demonstration of 2D6 mRNA in human foetal and adult brain by in situ hybridization. Xenobiotica 27, 111–125. Hagihara K., Shiosaka S., Lee Y., Kato J., Hatano O., Takakusu A., Emi Y., Omura T. and Tohyama M. (1990) Presence of sex difference of cytochrome P-450 in rat preoptic area and hypothalamus with reference to coexistence with oxytocin. Brain Res. 515, 69–78. Haglund L., Kohler C., Haaparanta T., Goldstein M. and Gustafsson J. A. (1984) Presence of NADPH-cytochrome P450 reductase in central catecholaminergic neurones. Nature 307, 259–262. Hansson T., Tindberg N., Ingelman-Sundberg M. and Kohler C. (1990) Regional distribution of ethanol-inducible cytochrome P450 IIE1 in the rat central nervous system. Neuroscience 34, 451–463. Hedlund E., Wyss A., Kainu T., Backlund M., Kohler C., Pelto-Huikko M., Gustafsson J. A. and Warner M. (1996) Cytochrome P4502D4 in the brain: specific neuronal regulation by clozapine and toluene. Molec. Pharmac. 50, 342–350. Jenner P., Dexter D. T., Sian J., Schapira A. H. V. and Marsden C. D. (1992) Oxidative stress as a cause of nigral cell death in Parkinson’s disease and incidental Lewy Body disease. Ann. Neurol. 32, S82–S87. Kapitulnik J., Gelboin H. V., Guengerich F. P. and Jacobowitz D. M. (1987) Immunohistochemical localization of cytochrome P-450 in rat brain. Neuroscience 20, 829–833. Kempermann G., Knoth R., Gebicke-Haerter P. J., Stolz B. J. and Volk B. (1994) Cytochrome P450 in rat astrocytes in vivo and in vitro: intracellular localization and induction by phenytoin. J. Neurosci. Res. 39, 576–588. Kennedy N. P. and Tipton K. F. (1990) Ethanol metabolism and alcoholic liver disease. Essays Biochem. 25, 137–195. Kohler C., Eriksson L. G., Hansson T., Warner M. and Gustafsson J. A. (1988) Immunohistochemical localization of cytochrome P-450 in the rat brain. Neurosci. Lett. 84, 109–114. Kurth M. C. and Kurth J. H. (1993) Variant cytochrome P450 CYP2D6 allelic frequencies in Parkinson’s disease. Am. J. med. Genetics 48, 166–168. Montoliu C., Sancho-Tello M., Azorin I., Burgal M., Valles S., Renau-Piqueras J. and Guerri C. (1995) Ethanol increases cytochrome P4502E1 and induces oxidative stress in astrocytes. J. Neurochem. 65, 2561–2570. Naslund B. M. A., Glaumann H., Warner M., Gustafsson J. A. and Hansson T. (1988) Cytochrome P-450 b and c in the rat brain and pituitary gland. Molec. Pharmac. 33, 31–37. Norris P. J., Hardwick J. P. and Emson P. C. (1994) Localization of NADPH cytochrome P450 oxidoreductase in rat brain by immunohistochemistry and in situ hybridization and a comparison with the distribution of neuronal NADPH-diaphorase staining. Neuroscience 61, 331–350.
P450 enzymes in rat substantia nigra 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.
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Norris P. J., Hardwick J. P. and Emson P. C. (1996) Regional distribution of cytochrome P450 2D1 in the rat central nervous system. J. comp. Neurol. 366, 244–258. Ohta S., Kohno M., Makino Y., Tachikawa O. and Hirobe M. (1987) Tetrahydroisoquinoline and 1-methyltetrahydroisoquinoline are present in the human brain: relation to Parkinson’s disease. Biomed. Res. 8, 453–456. Overby L. H., Nishio S., Weir A., Carver G. T., Plopper C. G. and Philpot R. M. (1992) Distribution of cytochrome P450 1A1 and NADPH-cytochrome P450 reductase in lungs of rabbits treated with 2,3,7,8-tetrachlorodibenzo-pdioxin: ultrastructural immunolocalization and in situ hybridization. Molec. Pharmac. 41, 1039–1046. Park K. S., Sohn D. H., Veech R. L. and Song B. J. (1993) Translational activation of ethanol-inducible cytochrome P450 (CYP2E1) by isoniazid. Eur. J. Pharmac. 248, 7–14. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. 2nd edn. Academic, London. Pezzoli G., Antonini A., Barbieri S., Canesi M., Perbellini L., Zecchinelli A., Mariani C. B., Bonetti A. and Leenders K. L. (1995) n-Hexane induced parkinsonism: pathogenic hypotheses. Movement Disorders 10, 279–282. Pfaff D. W. and Schwartz-Giblin S. (1988). Cellular mechanisms of female reproductive behaviour. In The Physiology of Reproduction (eds Knobil E. and Neill J. D.). pp. 1487–1568, Raven, New York. Ravindranath V., Anandatheerthavarada H. K. and Shankar S. K. (1990) NADPH cytochrome P-450 reductase in rat, mouse and human brain. Biochem. Pharmac. 39, 1013–1018. Riedl A. G., Watts P. M., Edwards R. J., Boobis A. R., Jenner P. and Marsden C. D. (1996) Selective localization of P450 enzymes and NADPH-P450 oxidoreductase in rat basal ganglia using anti-peptide antisera. Brain Res. 743, 324–328. Sasame H. A., Ames M. M. and Nelson S. D. (1977) Cytochrome P450 and NADPH cytochrome c reductase in rat brain formation of reactive catechol metabolites. Biochem. biophys. Res. Commun. 78, 919–926. Schenkman J. B. (1992) Steroid metabolism by constitutive cytochromes P450. J. Steroid Biochem. molec. Biol. 43, 1023–1030. Schulz T. G., Stahlmann R., Edwards R. J., Debri K., Davies D. S. and Neubert D. (1995) Enoxacin is an inducer of CYP1A2 in rat liver. Biochem. Pharmac. 50, 1517–1520. Segura-Aguilar J. (1996) Peroxidase activity of liver microsomal vitamin D 25-hydroxylase and cytochrome P4501A2 catalyses 25-hydroxylation of vitamin D3 and oxidation of dopamine to aminochrome. Biochem. molec. Med. 58, 122–129. Smith C. A. D., Gough A. C., Leigh P. N., Summers B. A., Harding A. E., Maranganore D. M., Sturman S. G., Schapira A. H. V., Williams A. C., Spurr N. K. and Wolf C. R. (1992) Debrisoquine hydroxylase gene polymorphism and susceptibility to Parkinson’s disease. Lancet 339, 1375–1377. Sohda T., Shimizu M., Kamimura S. and Okumura M. (1993) Immunohistochemical demonstration of ethanolinducible P450 2E1 in rat brain. Alcohol Alcohol. 28, 69–75. Stromstedt M., Warner M. and Gustafsson J. A. (1994) Cytochrome P450s of the 4A subfamily in the brain. J. Neurochem. 63, 671–676. Takakubo F., Yamamoto M., Ogawa N., Yamashita Y., Mizuno Y. and Kondon I. (1996) Genetic association between cytochrome P450IA1 gene and susceptibility to Parkinson’s disease. J. neural Transm. 103, 843–849. Tanner C. M. (1989) The role of environmental toxins in the etiology of Parkinson’s disease. Trends Neurosci. 12, 49–54. Tindberg N. and Ingelman-Sundberg M. (1996) Expression, catalytic activity, and inducibility of cytochrome P450 2E1 (CYP2E1) in the rat central nervous system. J. Neurochem. 67, 2066–2073. Tsuneoka Y., Matsuo Y., Iwahashi K., Takeuchi H. and Ichikawa Y. (1993) A novel cytochrome P-450IID6 mutant gene associated with Parkinson’s disease. J. Biochem. 114, 263–266. Tyndale R. F., Sunahara R., Inaba T., Kalow W., Gonzalez F. J. and Niznik H. B. (1991) Neuronal cytochrome P450IID1 (debrisoquine/sparteine-type): potent inhibition of activity by ()cocaine and nucleotide sequence identity to human hepatic P450 gene CYP 2D6. Molec. Pharmac. 40, 63–68. Warner M. and Gustafsson J. A. (1994) Effect of ethanol on cytochrome P450 in the rat brain. Proc. natn. Acad. Sci. U.S.A. 91, 1019–1023. Warner M., Kohler C., Hansson T. and Gustafsson J. A. (1988) Regional distribution of cytochrome P-450 in the rat brain: spectral quantitation and contribution of P-450b,e and P-450c,d. J. Neurochem. 50, 1057–1065. Wyss A., Gustafsson J. A. and Warner M. (1995) Cytochromes P450 of the 2D subfamily in rat brain. Molec. Pharmac. 47, 1148–1155. Yamada-Mouri N., Hirata S., Hayashi M. and Kato J. (1995) Analysis of the expression and the first exon of aromatase mRNA in monkey brain. J. Steroid Biochem. molec. Biol. 55, 17–23. Yoo M., Ryu H. M., Shin S. W., Yun C. H., Lee S. C., Ji Y. M. and You K. H. (1997) Identification of cytochrome P450 2E1 in rat brain. Biochem. biophys. Res. Commun. 231, 254–256. (Accepted 1 December 1997)
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Neuroscience Vol. 86, No. 2, pp. 511–519, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00649-0
CO-LOCALIZATION OF P450 ENZYMES IN THE RAT SUBSTANTIA NIGRA WITH TYROSINE HYDROXYLASE P. M. WATTS,* A. G. RIEDL,* D. C. DOUEK,† R. J. EDWARDS,‡ A. R. BOOBIS,‡ P. JENNER*¶ and C. D. MARSDEN§ *Neurodegenerative Disease Research Centre, Pharmacology Group, Biomedical Sciences Division, King’s College, London SW3 6LX, U.K. †MRC Clinical Sciences Centre and ‡Section on Clinical Pharmacology, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, U.K. §University Department of Clinical Neurology, Institute of Neurology, The National Hospital for Neurology and Neurosurgery, London WC1N 3BG, U.K. Abstract––Susceptibility to develop Parkinson’s disease has been linked to abnormalities of P450 enzyme function. Multiple P450 enzymes are expressed in brain but the relationship of these to Parkinson’s disease is unknown. We have investigated the expression of P450 enzymes in the rat substantia nigra and their co-localization in tyrosine hydroxylase-positive neurons and astrocytes. Immunohistochemistry was performed using anti-peptide antisera against the following P450 enzymes: CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, CYP2C13/2C6, CYP2D1, CYP2D4, CYP2E1, CYP3A1, CYP3A2 and NADPHP450 oxidoreductase. Immunoreactivity in nigral cells was found only for CYP2E1 and CYP2C13/2C6. CYP2E1 immunoreactivity was localized to many midbrain nuclei including the substantia nigra pars compacta but not the substantia nigra pars reticulata while immunoreactivity to CYP2C13/2C6 was found in the substantia nigra pars compacta, substantia nigra pars reticulata and many other midbrain nuclei. Sections of rat midbrain double labelled for either CYP2E1 or CYP2C13/2C6 and tyrosine hydroxylase or glial fibrillary acidic protein were examined for co-localization by confocal laser scanning microscopy. CYP2E1 and CYP2C13/2C6 immunoreactivity was found in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta but not in glial cells. CYP2C13/2C6, but not CYP2E1, was also found in non-glial, non-tyrosine hydroxylase-expressing cells in the substantia nigra pars reticulata. Isoniazid induction increased CYP2E1 fluorescence signal intensity from nigral dopaminergic neurons. At least two P450 enzymes are found in nigral dopamine containing cells and one, namely CYP2E1, is selectively localized to this cell population. CYP2E1 is a potent generator of free radicals which may contribute to nigral pathology in Parkinson’s disease. The expression of CYP2E1 in dopaminergic neurons in substantia nigra raises the possibility of a causal association with Parkinson’s disease. 1998 IBRO. Published by Elsevier Science Ltd. Key words: Parkinson’s disease, cytochrome P450, CYP2E1, CYP2C13/2C6, confocal microscopy, immunohistochemistry.
The cause of nigral dopaminergic cell degeneration in Parkinson’s disease (PD) is unknown but evidence exists which suggests that free radicals and oxidative stress may be involved.31 The trigger for the progressive nigral cell loss is also unknown but may involve exposure to environmental57 or endogenous neurotoxins coupled to a genetic susceptibility.18 There is controversial evidence suggesting a link between the expression of certain forms of P450 and ¶To whom correspondence should be addressed. Abbreviations: FITC, fluorescein isothiocyanate; FMO, flavin monooxygenase; GFAP, glial fibrillary acidic protein; isoniazid, isonicotinic acid hydrazide; LRSC, lissamine rhodamine sulphonyl chloride; MPTP, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine; NDS, normal donkey serum; NGS, normal goat serum; PBS, phosphatebuffered saline pH 7.4; PD, Parkinson’s disease; SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata; TH, tyrosine hydroxylase; VTA, ventral tegmental area.
susceptibility to developing PD. In particular, mutations in the CYP2D6 gene which result in failure of CYP2D6 expression, may be over-represented in some PD populations, typically conferring a two- to five-fold disease risk.2,9,36,53,59 In addition, allelic variants of human CYP1A1 exist with the homozygous CYPVal1A1 mutation conferring a six-fold relative risk to develop Parkinson’s disease compared to the wild type.56 Several P450 enzymes are found in monkey, human and rat brain. High levels of aromatase messenger RNA is present in the hypothalamus and amygdala of male Japanese monkeys,64 whilst CYP1A1,25 CYP1A2,25 CYP2E1,25 CYP2D6,26 CYP3A25 and NADPH-P450 oxidoreductase47 have been detected in various regions of the human brain. In the rat brain a range of P450 enzymes are reported to be present, including CYP1A1,38 CYP1A1/2,8,10,32,35,62 CYP2B1/2,6,7,33,38,62
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CYP2C7,61 CYP2C11,3,61 CYP2C12,27 CYP2C13,27 CYP2D1,40,60 CYP2D4,30 CYP2E1,5,7,8,15,29,54,58,65 CYP3A9,61 CYP4A3,55 CYP4A855,61 and NADPHP450 oxidoreductase.4,28,39,47 However, discrepancies exist regarding their presence and precise distribution within brain. For example, CYP1A1,8,35 CYP2D1,40 CYP2D4,30 CYP2E129,54 and NADPH-P450 oxidoreductase28 have been reported to be present in the rat substantia nigra. We believe that the discrepancies result from the specificity of the antibodies and antisera employed and that this has arisen from differing techniques of antibody production. Most studies have employed antisera raised against P450 enzymes from rat hepatic microsomes where even minor contamination of the immunizing P450 enzyme with other proteins will result in the production of cross-reactive antibodies. Even immunization with a pure P450 enzyme preparation results in antibodies which cross-react with similar or identical epitopes on closely-related P450 enzymes. To overcome this problem we have developed a series of highly specific anti-peptide antisera raised against unique peptide sequences of specific P450 enzymes. Using such antisera and peroxidase immunohistochemistry, we recently described the distribution of CYP1A1, CYP2B1/2, CYP2D1, CYP2E1, CYP3A1 and NADPH-P450 oxidoreductase in rat basal ganglia.48 These studies demonstrated selective localization of CYP1A1 restricted to the globus pallidus with CYP2E1 expressed in neuronal cells in the substantia nigra, paraventricular hypothalamic nucleus and striatal vasculature. CYP2E1 is also expressed in the cerebellum,29 hippocampus,29 cerebral cortices,54 caudate–putamen54 and globus pallidus.54 We now extend these findings by focusing on the expression of a wide range of P450 enzymes in the rat nigra using anti-peptide antisera directed against unique C-terminal or internal amino acid sequences. A panel of anti-P450 antisera targeted against each of the major xenobiotic-metabolizing P450 enzymes expressed in the rat were used. These recognized CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, CYP2C13/2C6, CYP2D1, CYP2D4, CYP2E1, CYP3A1, CYP3A2 and NADPH-P450 oxidoreductase. The localization of those P450 enzymes that stained nigral cells, as assessed by peroxidase immunohistochemistry, was investigated further by double staining with anti-P450 antisera and anti-tyrosine hydroxylase (TH) or anti-glial fibrillary acidic protein (GFAP) antibodies. Co-localization was detected by confocal laser scanning microscopy which allowed determination of P450 enzyme expression in dopaminergic neurons and astrocytes in the substantia nigra. Since CYP2E1 expression in brain was low, it was necessary to induce the enzyme and rats were treated with a classical hepatic CYP2E1 inducer, isonicotinic acid hydrazide (isoniazid) once daily for five days.43
EXPERIMENTAL PROCEDURES
Materials All chemicals were obtained from Sigma (Sigma Chemical Co. Ltd, Dorset, U.K.) unless otherwise stated. Animals Adult male adult Wistar rats (n=24; 150–200 g, Bantin and Kingman, Hull, U.K.) were housed six to a cage and given free access to pelleted food (Special Diet Services, Witham, U.K.) and water. All animal experiments were carried out in strict accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines. Preparation of antibodies Polyclonal rabbit anti-peptide antibodies directed against CYP1A1,24,48 CYP1A2,23 CYP2B1/2,48,51 CYP2D1,48 CYP2E1,24,48 CYP3A1,19,48 CYP3A219 and NADPH-P450 oxidoreductase48 were used. Antibodies directed against the C-termini of CYP2C12, CYP2C13/2C6 and CYP2D4 were produced by immunizing rabbits with the peptides PheIle-Pro-Val, Arg-Phe-Ile-Pro-Leu and Ala-Ser-Pro-Arg, respectively, by methods described previously.22,24 The specificity of antibody binding to the target P450 enzymes was confirmed by immunoblotting using hepatic microsomal fractions from untreated rats and rats treated with appropriate P450 enzyme inducing chemicals.19,22,24,51 Isonicotinic acid hydrazide treatment Male Wistar rats (n=6; 150–200 g) were treated with isonicotinic acid hydrazide (isoniazid; 200 mg/kg, i.p.) once daily for five days to induce CYP2E1. Control rats (n=6; 150–200 g) were injected with 0.9% saline. Animals were killed 3 h following the final treatment with isoniazid or saline by terminal anaesthesia with pentobarbitone (100 mg/ kg; Rhone Merieux Ltd, Harlow, U.K.) followed by transcardial perfusion with 0.1 M phosphate-buffered saline, pH 7.4 (PBS) at 4C (200 ml) and perfuse-fixed with 4% paraformaldehyde in PBS (200 ml). Tissue processing Brains and livers (used as positive controls to assess the reactivity of each antiserum) were removed and postfixed in 4% paraformaldehyde in 0.1 M PBS at 4C for 48 h. Tissues were then cryoprotected in 30% sucrose in 0.1 M PBS for a further 48 h and snap frozen in isopentane on dry ice. All tissue was subsequently stored at 70C. Peroxidase immunohistochemistry Contiguous coronal sections (30 µm) of midbrain were cut using a cryostat to include the entire substantia nigra according to the atlas of Paxinos and Watson.44 Immunostaining was performed on free-floating sections. Sections were washed twice in 0.1 M PBS before endogenous peroxidase activity was blocked by incubating sections in 70% methanol in water containing 0.3% hydrogen peroxide for 30 min. After two further washes in 0.1 M PBS, sections were incubated in 20% normal goat serum (NGS) in 0.1 M PBS for 1 h and then washed twice in 0.1 M PBS containing 1% NGS and 0.05% Triton X-100. Sections of midbrain and liver were incubated with antisera targeted against the C-terminal peptides for CYP1A1, CYP1A2, CYP2B1/2B2, CYP2C12, CYP2C13/2C6, CYP2D1, CYP2D4, CYP2E1, CYP3A1, CYP3A2 or NADPH-P450 oxidoreductase. Controls consisted of alternate sections of midbrain incubated with non-immune rabbit serum. Antisera or non-immune serum were diluted 1:1000 in 1% NGS in 0.1 M PBS and incubated overnight (18 h) at room temperature on a shaking platform. Immunostaining was performed according to the manufacturers instructions using a Vectastain ABC kit (Vector Laboratories, Peterborough, U.K.).
P450 enzymes in rat substantia nigra
Fig. 1. (a) Photomicrograph of rat substantia nigra stained for CYP2E1 using peroxidase immunohistochemistry and counterstained with Cresyl Violet. CYP2E1 immunoreactivity demonstrated throughout the midbrain including neurons of the SNpc and VTA. Scale bar=250 µm. (b) Photomicrograph of rat substantia nigra stained for CYP2C13/2C6 using peroxidase immunohistochemistry and counterstained with Cresyl Violet. CYP2C13/2C6-immunoreactive neurons present throughout the midbrain including the SNpc, SNpr and VTA. CYP2C13/2C6-positive neurons occasionally found in SNpr (arrow head). Scale bar=250 µm.
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P450 enzymes in rat substantia nigra Immunoreactive products were visualized using 3,3 diaminobenzidine (0.05% in Tris buffer) and 0.01% hydrogen peroxide. Sections were mounted onto slides and some were counterstained with Cresyl Violet to enhance visualization of cellular architecture before being dehydrated in 70%, 96% and 100% ethanol and cleared in xylene. For each of the P450 enzymes studied, sections of midbrain from at least six animals were examined at multiple levels throughout the substantia nigra. Sections of liver were similarly processed and served as positive controls for those P450 enzymes expressed in normal rat liver. Co-localization studies Sections of midbrain at the level of the exit of the third nerve (5.6mm from Bregma) were incubated in 0.1 M PBS containing 10% NGS and 10% normal donkey serum (NDS) for 1 h and then washed twice in 0.1 M PBS containing 0.05% Triton X-100, 0.5% NGS and 0.5% NDS. Sections were then incubated in the primary antisera which consisted of rabbit anti-CYP2E1 or rabbit antiCYP2C13/2C6 diluted 1/1000 and mouse monoclonal antiGFAP diluted 1/200 (0.1 µg/ml, Boehringer Mannheim Biochemica, Germany) or sheep polyclonal anti-TH diluted 1/250 (0.2 µg/ml, Pel Freez, AK, U.S.A.) made up in PBS with 0.5% NGS and 0.5% NDS, and incubated overnight (18 h) on a shaking platform. Tissues were washed twice in PBS with 0.5% NGS and 0.5% NDS and incubated in second layer antisera which consisted of biotinylated goat anti-rabbit polyclonal IgG diluted 1/200 (7 µg/ml, Vector Laboratories, Peterborough, U.K.) as a probe for the anti-P450 first layer and either affinity-purified lissamine rhodamine sulphonyl chloride (LRSC)-conjugated donkey anti-mouse F(ab )2 diluted 1/200 (7.5 µg/ml, Jackson Immunoresearch, PA, U.S.A., probing for anti-GFAP first layer) or affinity-purified LRSC-conjugated donkey antisheep F(ab )2 diluted 1/200 (7.5 µg/ml, Jackson Immunoresearch, PA, U.S.A., probing for anti-TH first layer), for 1 h in the dark. After two washes in PBS all sections were incubated in streptavidin-conjugated fluorescein isothiocyanate (FITC) diluted 1/50 (4 µg/ml, Caltag Labs, CA, U.S.A.) in PBS for 1 h in the dark and washed twice again in PBS. Sections were wet mounted in Vectashield (Vector Laboratories, Peterborough, U.K.) and stored at 4C in the dark until examined. Confocal microscopy and fluorophore detection A Leica IR confocal laser scanning microscope was used to image fluorescence staining. The argon/krypton laser produced excitation wavelengths of 488, 568 and 650 nm and four photomultipliers detected emission signals. Double staining with FITC and LRSC was scanned and detected simultaneously using narrow band filters to prevent crosstalk between signals. Eight optical sections for each tissue section were taken over a thickness of 15 µm and were summed using a maximal projection algorithm. Red (LRSC) and green (FITC) channels were overlayed or
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separated using Photoshop 3.0 software on a Macintosh PC computer. Background shadow was reduced equally for each channel by 30%. Sections taken from rats treated with isoniazid and saline and appropriate controls were stained for CYP2E1 and TH or GFAP as above and the level of CYP2E1 expressed quantified by measuring FITC signal intensity. This signal intensity, expressed on an arbitrary grey scale, was calculated from averages obtained from six cells from each of six isoniazid-treated or saline-treated animals, cells being selected from the medial aspect of the substantia nigra pars compacta (SNpc) at the level of the exit of the third nerve. TH fluorescence signal intensity was similarly quantified. Statistical analysis Differences between mean signal intensities for TH- and CYP2E1-expressing cells from isoniazid-treated and salinetreated animals were compared using Mann–Whitney U-test, where P<0.05 was considered significant. RESULTS
Peroxidase immunohistochemistry Liver sections, used as positive controls, produced immunoreactive centrilobular staining using antisera targeted against CYP1A2, CYP2B1/2, CYP2E1, CYP2C12, CYP2C13/2C6, CYP3A1, CYP3A2 and NADPH-P450 oxidoreductase. Antisera against CYP2D1 produced an intense uniform panlobular staining. No immunoreactivity to antisera against CYP1A1 or CYP2D4 was observed in liver sections. However, antisera against CYP1A1 stained the globus pallidus intensely, while antisera against CYP2D4 weakly stained white matter tracts in naive rat brain (data not shown). No immunoreactivity to CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, CYP2D1, CYP2D4, CYP3A1, CYP3A2 or NADPHP450 oxidoreductase was observed in the substantia nigra. Only CYP2E1 and CYP2C13/2C6 antisera produced staining of nigral cells. Staining was observed at all nigral levels and in all sections examined, from at least six animals. CYP2E1 immunoreactivity was detected throughout the midbrain including the SNpc and ventral tegmental area (VTA) in many cells with neuronal morphology (Fig. 1a). No staining was seen outside the nigra nor was there any reproducible signal in the substantia nigra pars reticulata (SNpr). CYP2C13/2C6 was detected in cells of neuronal morphology throughout the midbrain but was most
Fig. 2. Confocal immunofluorescence images of rat substantia nigra stained for TH or GFAP and CYP2E1 or CYP2C13/2C6. Scale bar=30 µm. (a) CYP2E1 expression in the SNpc of a naive animal (left), showing faint green fluorescence, TH expressing neurons (centre), showing strong red fluorescence and composite demonstrating co-localization of signals (right). (b) CYP2E1 expression in the SNpc of an isoniazid-treated animal (left) demonstrating enhanced CYP2E1 signal intensity, TH (centre) and composite demonstrating co-localization (right). (c) CYP2E1 expression in the SNpc of an isoniazidtreated animal (left), GFAP (centre), composite showing no co-localization (right). (d) CYP2C13/2C6immunoreactive neurons are present in the SNpc, SNpr and dorsal to the nigra (left), TH (centre) and composite demonstrating co-localization of signals in nigral neurons (right). Occasional CYP2C13/2C6positive, TH-negative cells were seen in the SNpr (arrow). (e) CYP2C13/2C6 in the SNpc and SNpr (left), GFAP (centre) and composite showing no co-localization (right). Occasional CYP2C13/2C6-positive, TH-negative cells were seen in the SNpr (arrow).
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Table 1. Fluorescence signal intensities for CYP2E1 and tyrosine hydroxylase in isoniazid-treated and saline-treated rats, meanS.D. Signal 2E1 TH
Saline
Isoniazid
71.56.2 216.28.8
165.425* 185.230
*P<0.05 (Mann–Whitney U-test) Each value represents the mean fluorescence signal intensities (expressed in relative units on a grey scale) taken from at least six neurons in the medial SNpc, n=6 animals in each group.
intense in cells of the SNpc and VTA (Fig. 1b). Occasional cells of neuronal morphology were stained for CYP2C13/2C6 in the SNpr. Isoniazid treatment resulted in more extensive hepatic centrilobular CYP2E1 staining but in brain there was no difference in neuronal CYP2E1 immunoreactivity in the substantia nigra compared with saline-treated animals. Control sections of midbrain incubated with non-immune serum showed only background staining, characterized by an even brown wash over the whole section. Confocal microscopy CYP2E1 signal was found to co-localise with TH in the SNpc in all sections examined but did not co-localise with GFAP. In normal animals CYP2E1 was expressed at low levels in a minority of dopaminergic neurons, the most intense signal being found in the medial SNpc and VTA. The CYP2E1 signal was virtually masked by the strong expression of TH, but co-localization of CYP2E1 and TH definitely occurred (Fig. 2a). Following treatment with the CYP2E1 inducer isoniazid, the majority of nigral dopaminergic neurons were found to express CYP2E1 (Fig. 2b) and signal intensity increased 2.3-fold, but had no effect on TH signal (Table 1). Some vascular endothelial cells stained for CYP2E1 after isoniazid treatment as we have previously shown in the striatum.48 Isoniazid treatment had no effect on the GFAP signal nor did it result in any CYP2E1 expression in astrocytes in the substantia nigra (Fig. 2c). CYP2C13/2C6 signal was found to co-localise with virtually all nigral TH-positive neurons in the SNpc (Fig. 2d). In addition, cells with neuronal morphology were observed expressing CYP2C13/2C6 in the SNpr but these were negative for both TH and GFAP. No CYP2C13/2C6 signal was detected in any of the GFAP-positive cells (Fig. 2e). Some GFAPpositive astrocytes were seen making intimate contact with CYP2C13/2C6-positive cells in the SNpr (Fig. 2e). Far fewer astrocytes were present in the SNpc and the majority of these were associated with capillaries and not neurons.
DISCUSSION
This is the first comprehensive investigation of the expression of multiple P450 enzymes in the substantia nigra and also the first to use exclusively anti-peptide antisera. The antisera used were highly specific, as a result of using short synthetic peptide sequences unique to each P450 enzyme.24 CYP2E1 was previously reported in neurons in the rat substantia nigra but diffuse staining was also observed in the SNpr and in many other brain regions including cells identified as glia.29 Others found CYP2E1 in the rat nigra after induction with ethanol, but there was no attempt to define its localization or the cell type in which it was expressed.54 We have confirmed and extended these findings by the observation in the present studies that CYP2E1 is selectively localized in dopaminergic nigral neurons. We previously failed to find the more extensive distribution of CYP2E1,48 previously reported by others,29,54 and in the present study we were unable to demonstrate CYP2E1 expression in nigral astrocytes even after administration of isoniazid. In vitro, CYP2E1 expression in primary cultures of rat cortical astrocytes has been co-localized with GFAP using confocal microscopy,37 but this does not appear to occur in vivo in the substantia nigra. The localization of CYP2E1 to the rat SNpc is supported by the finding of mRNA for CYP2E1 in human substantia nigra.25 The inducibility of CYP2E1 in the SNpc by isoniazid is evidence that the enzyme is present in the substantia nigra in a functional form. We have also demonstrated previously that isoniazid induces CYP2E1 in striatal vasculature in the rat.48 Alternatively CYP2E1 may have a role in neurodegeneration or in the detoxication of environmental neurotoxins similar to the metabolism of 1-methyl-4phenyl-1,2,3,6 tetrahydropyridine (MPTP) carried out by CYP2D6 and other P450 enzymes.17 Electron leakage from P450 redox reactions in the nigra might be expected to induce oxidative stress under certain conditions. Some P450 enzymes, including CYP2E1, are particularly susceptible to electron leakage and the extent of this appears to be dependent on the affinity of substrate for the enzyme. Such an increase in formation of reactive oxygen species from P450 enzymes in nigral dopaminergic neurons is a potential cause of the oxidative stress which has been detected in substantia nigra in Parkinson’s disease.20 The hepatic P450 enzyme responsible for vitamin D 25-hydroxylation and CYP1A2, both oxidise dopamine to an aminochrome in vitro, this reaction being supported by an electron donor other than NADPH-P450 oxidoreductase.52 CYP2E1 could also participate in such reactions and might be responsible for dopamine degradation. Oxidized metabolites might include hydrogen peroxide and neurotoxin 6-hydroxydopamine. This might explain the previously reported covalent binding of dopamine or a
P450 enzymes in rat substantia nigra
metabolite to rat brain microsomal protein after co-incubation in vitro, this reaction being dependent on NADPH and oxygen.49 Alternatively, CYP2E1 activity in the nigra might be of importance in the activation of pro-neurotoxins in a manner analogous to the activation of MPTP by monoamine oxidase B. CYP2E1 is capable of oxidizing alcohols to aldehydes. Acetaldehyde condenses with dopamine to form potentially neurotoxic isoquinolines,34 which have been detected in normal human and rat brain as well as in tissue from patients with PD.41 There is also increasing evidence that exposure to n-hexane as a component of petroleum spirit and of other solvents is linked to the development of a parkinsonian syndrome.45 n-Hexane is neurotoxic only following activation to 2,5-hexandione and this reaction can in part be catalysed by CYP2E1.1 With the low levels of n-hexane in the atmosphere from the incomplete combustion of petrol and the presence of CYP2E1 in the nigra, neurotoxic 2,5-hexanedione may be formed. The significance of the widespread neuronal distribution of CYP2C13/2C6 in the brain and the intense immunostaining of this enzyme observed in the SNpc dopaminergic neurons is unclear. CYP2C13 participates in the 6-hydroxylation of testosterone50 and it may therefore be important for the degradation of testosterone or the synthesis of other hormones. Indeed, high levels of testosterone are present in human substantia nigra.12 CYP2C13 expression in nigral dopaminergic neurons might therefore be expected to be functionally important possibly by reducing the aromatase interconversion of androgen to oestrogen46 which would be expected to affect the firing patterns of these cells16 and their susceptibility to MPTP.21 In contrast to previous reports we found no evidence of CYP2D140 nor CYP2D430 expression in the rat nigra. Our anti-peptide antibody against CYP2D4 failed to stain liver but did stain white matter tracts. Wyss et al.63 have also observed CYP2D4 immunoreactivity in white matter tracts.
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Norris et al.40 used a polyclonal antibody raised against purified CYP2D1 and this may cross-react with all CYP2D forms. Thus, it is still possible that rat nigra contains CYP2D2, CYP2D3 or CYP2D5. Therefore expression of these P450 enzymes requires further investigation. The presence of CYP1A1/235 and NADPH-P450 oxidoreductase28 in the rat nigra have previously been reported using polyclonal antisera prepared against purified rat liver proteins. We could not confirm the presence of these P450 enzymes using our anti-peptide antisera. The absence of NADPH-P450 oxidoreductase in nigra, despite the presence of CYP2E1 and CYP2C13/2C6 reported has a corollary in mouse heart14 and rabbit lung endothelial cells42 where CYP1A1 is present in the absence of the reductase. As P450 enzyme function is dependent on an electron donor, other electron donors may be acting in this role in nigral dopaminergic neurons. Flavin mononoxygenases (FMOs) have been detected in rat brainstem11 and FMO3 and FMO4 have been detected in the human substantia nigra.13
CONCLUSION
The expression of CYP2E1 in nigral dopaminergic neurons in the rat raises important questions about its physiological function at this site and if expressed in humans, it could be involved in mechanisms of cell death including Parkinson’s disease. The presence of CYP2E1 has been reported in the human brain, although there are no reports of it being found in the substantia nigra. CYP2E1 expression may vary between individuals, as a result of polymorphisms in CYP2E1 promoters. Clearly such polymorphic variation could be a risk factor for the development of PD. Acknowledgements—This work was supported by the Medical Research Council, the Wellcome Trust, the Parkinson’s Disease Society and the National Parkinson Foundation, Miami, U.S.A.
REFERENCES
1. 2. 3. 4. 5. 6. 7.
Abou-Donia M., Hu Z., Lapadula D. M. and Gupta R. P. (1991) Mechanisms of joint neurotoxicity of n-hexane, methyl isobutylketone and O-ethyl-O-4-nitrophenyl phenylphosphophonothioate in hens. J. Pharmac. exp. Ther. 257, 282–289. Akhmedova S. N., Pushnova E. A., Yakimovsky A. F., Avtonomov V. V. and Schwartz E. I. (1995) Frequency of a specific cytochrome P4502D6B (CYP2D6B) mutant allele in clinically differentiated groups of patients with Parkinson’s disease. Biochem. molec. Med. 54, 88–90. Alkayed N. J., Narayanan J., Gebremedhin D., Medhora M., Roman R. J. and Harder D. R. (1996) Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. Stroke 27, 971–979. Anandatheerthavarada H. K., Boyd M. R. and Ravindranath V. (1992) Characterization of a phenobarbital-inducible cytochrome P-450, NADPH-cytochrome P-450 reductase and reconstituted cytochrome P-450 mono-oxygenase system from rat brain: evidence for constitutive presence in rat and human brain. Biochem. J. 288, 483–488. Anandatheerthavarada H. K., Shankar S. K., Bhamre S., Boyd M. R., Song B. J. and Ravindranath V. (1993) Induction of brain cytochrome P-450IIE1 by chronic ethanol treatment. Brain Res. 601, 279–285. Anandatheerthavarada H. K., Shankar S. K. and Ravindranath V. (1990) Rat brain cytochromes P-450: catalytic, immunochemical properties and inducibility of multiple forms. Brain Res. 536, 339–343. Anandatheerthavarada H. K., Williams J. F. and Wecker L. (1993) The chronic administration of nicotine induces cytochrome P-450 in rat brain. J. Neurochem. 60, 1941–1944.
518 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
P. M. Watts et al. Anandatheerthavarada H. K., Williams J. F. and Wecker L. (1993) Differential effect of chronic nicotine administration on brain cytochrome P4501A1/2 and P4502E1. Biochem. biophys. Res. Commun. 194, 312–318. Armstrong M., Daly A. K., Cholerton S., Bateman D. N. and Idle J. R. (1992) Mutant debrisoquine hydroxylation genes in Parkinson’s disease. Lancet 339, 1017–1018. Bhagwat S. V., Boyd M. R. and Ravindranath V. (1995) Brain mitochondrial cytochromes P450: xenobiotic metabolism, presence of multiple forms and their selective inducibility. Arch. Biochem. Biophys. 320, 73–83. Bhamre S., Bhagwat S. V., Shankar S. K., Williams D. E. and Ravindranath V. (1993) Cerebral flavin-containing monooxygenase-mediated metabolism of antidepressants in brain: immunochemical properties and immunocytochemical localization. J. Pharmac. exp. Ther. 267, 555–559. Bixo M., Backstrom T., Winblad B. and Andersson A. (1995) Estradiol and testosterone in specific regions of the human female brain in different endocrine states. J. Steroid Biochem. molec. Biol. 55, 297–303. Bootman M. A., Phillips I. R. and Shephard E. A. (1996) Flavin-containing monooxygenase expression in the human brain. XIth International Symposium on Microsomes and Drug Oxidations, p. 23. Brittebo E. B. (1994) Metabolic activation of the food mutagen Trp-P-1 in endothelial cells of heart and kidney in cytochrome P450-induced mice. Carcinogenesis 15, 667–672. Cammer W., Downing M., Clarke W. and Schenkman J. B. (1991) Immunocytochemical staining of the RLM6 form of cytochrome P-450 in oligodendrocytes and myelin of rat brain. J. Histochem. Cytochem. 39, 1089–1094. Chiodo L. A. and Caggiula A. R. (1983) Substantia nigra dopamine neurons: alterations in basal discharge rates and autoreceptor sensitivity induced by estrogen. Neuropharmacology 22, 593–599. Coleman T., Ellis S. W., Martin I. J., Lennard M. S. and Tucker G. T. (1996) 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) is N-demethylated by cytochromes P450 2D6, 1A2 and 3A4-implications for susceptibility to Parkinson’s disease. J. Pharmac. exp. Ther. 277, 685–690. Daly A. K., Cholerton S., Armstrong M. and Idle J. R. (1994) Genotyping for polymorphisms in xenobiotic metabolism as a predictor of disease susceptibility. Environ. Health Perspect. 102, 55–61. Debri K., Boobis A. R., Davies D. S. and Edwards R. J. (1995) Distribution and induction of CYP3A1 and CYP3A2 in rat liver and extra-hepatic tissues. Biochem. Pharmac. 50, 2047–2056. Dexter D. T., Holley A. E., Flitter W. D., Slater T. F., Wells F. R., Daniel S. E., Lees A. J., Jenner P. and Marsden C. D. (1994) Increased levels of lipid hydroperoxides in the Parkinsonian substantia nigra: an HPLC and ESR study. Movement Disorders 9, 92–97. Dluzen D. E. (1996) Effects of testosterone upon MPTP-induced neurotoxicity of the nigrostriatal dopaminergic system of C57/B1 mice. Brain Res. 715, 113–118. Edwards R. J., Murray B. P. and Boobis A. R. (1991) Anti-peptide antibodies in studies of cytochromes P450 IA. Meth. Enzym. 206, 220–233. Edwards R. J., Murray B. P., Singleton A. M., Murray S., Davies D. S. and Boobis A. R. (1993) Identification of the epitope of an anti-peptide antibody which binds to CYP1A2 in many species including man. Biochem. Pharmac. 46, 213–220. Edwards R. J., Singleton A. M., Murray B. P., Davies D. S. and Boobis A. R. (1995) Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes. Biochem. Pharmac. 49, 39–47. Farin F. M. and Omiecinski C. J. (1993) Regiospecific expression of cytochrome P-450s and microsomal epoxide hydrolase in human brain tissue. J. Toxicol. environ. Health 40, 317–335. Gilham D. E., Cairns W., Paine M. J. I., Modi S., Poulsom R., Roberts G. C. K. and Wolf C. R. (1997) Metabolism of MPTP by cytochrome P4502D6 and the demonstration of 2D6 mRNA in human foetal and adult brain by in situ hybridization. Xenobiotica 27, 111–125. Hagihara K., Shiosaka S., Lee Y., Kato J., Hatano O., Takakusu A., Emi Y., Omura T. and Tohyama M. (1990) Presence of sex difference of cytochrome P-450 in rat preoptic area and hypothalamus with reference to coexistence with oxytocin. Brain Res. 515, 69–78. Haglund L., Kohler C., Haaparanta T., Goldstein M. and Gustafsson J. A. (1984) Presence of NADPH-cytochrome P450 reductase in central catecholaminergic neurones. Nature 307, 259–262. Hansson T., Tindberg N., Ingelman-Sundberg M. and Kohler C. (1990) Regional distribution of ethanol-inducible cytochrome P450 IIE1 in the rat central nervous system. Neuroscience 34, 451–463. Hedlund E., Wyss A., Kainu T., Backlund M., Kohler C., Pelto-Huikko M., Gustafsson J. A. and Warner M. (1996) Cytochrome P4502D4 in the brain: specific neuronal regulation by clozapine and toluene. Molec. Pharmac. 50, 342–350. Jenner P., Dexter D. T., Sian J., Schapira A. H. V. and Marsden C. D. (1992) Oxidative stress as a cause of nigral cell death in Parkinson’s disease and incidental Lewy Body disease. Ann. Neurol. 32, S82–S87. Kapitulnik J., Gelboin H. V., Guengerich F. P. and Jacobowitz D. M. (1987) Immunohistochemical localization of cytochrome P-450 in rat brain. Neuroscience 20, 829–833. Kempermann G., Knoth R., Gebicke-Haerter P. J., Stolz B. J. and Volk B. (1994) Cytochrome P450 in rat astrocytes in vivo and in vitro: intracellular localization and induction by phenytoin. J. Neurosci. Res. 39, 576–588. Kennedy N. P. and Tipton K. F. (1990) Ethanol metabolism and alcoholic liver disease. Essays Biochem. 25, 137–195. Kohler C., Eriksson L. G., Hansson T., Warner M. and Gustafsson J. A. (1988) Immunohistochemical localization of cytochrome P-450 in the rat brain. Neurosci. Lett. 84, 109–114. Kurth M. C. and Kurth J. H. (1993) Variant cytochrome P450 CYP2D6 allelic frequencies in Parkinson’s disease. Am. J. med. Genetics 48, 166–168. Montoliu C., Sancho-Tello M., Azorin I., Burgal M., Valles S., Renau-Piqueras J. and Guerri C. (1995) Ethanol increases cytochrome P4502E1 and induces oxidative stress in astrocytes. J. Neurochem. 65, 2561–2570. Naslund B. M. A., Glaumann H., Warner M., Gustafsson J. A. and Hansson T. (1988) Cytochrome P-450 b and c in the rat brain and pituitary gland. Molec. Pharmac. 33, 31–37. Norris P. J., Hardwick J. P. and Emson P. C. (1994) Localization of NADPH cytochrome P450 oxidoreductase in rat brain by immunohistochemistry and in situ hybridization and a comparison with the distribution of neuronal NADPH-diaphorase staining. Neuroscience 61, 331–350.
P450 enzymes in rat substantia nigra 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.
519
Norris P. J., Hardwick J. P. and Emson P. C. (1996) Regional distribution of cytochrome P450 2D1 in the rat central nervous system. J. comp. Neurol. 366, 244–258. Ohta S., Kohno M., Makino Y., Tachikawa O. and Hirobe M. (1987) Tetrahydroisoquinoline and 1-methyltetrahydroisoquinoline are present in the human brain: relation to Parkinson’s disease. Biomed. Res. 8, 453–456. Overby L. H., Nishio S., Weir A., Carver G. T., Plopper C. G. and Philpot R. M. (1992) Distribution of cytochrome P450 1A1 and NADPH-cytochrome P450 reductase in lungs of rabbits treated with 2,3,7,8-tetrachlorodibenzo-pdioxin: ultrastructural immunolocalization and in situ hybridization. Molec. Pharmac. 41, 1039–1046. Park K. S., Sohn D. H., Veech R. L. and Song B. J. (1993) Translational activation of ethanol-inducible cytochrome P450 (CYP2E1) by isoniazid. Eur. J. Pharmac. 248, 7–14. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. 2nd edn. Academic, London. Pezzoli G., Antonini A., Barbieri S., Canesi M., Perbellini L., Zecchinelli A., Mariani C. B., Bonetti A. and Leenders K. L. (1995) n-Hexane induced parkinsonism: pathogenic hypotheses. Movement Disorders 10, 279–282. Pfaff D. W. and Schwartz-Giblin S. (1988). Cellular mechanisms of female reproductive behaviour. In The Physiology of Reproduction (eds Knobil E. and Neill J. D.). pp. 1487–1568, Raven, New York. Ravindranath V., Anandatheerthavarada H. K. and Shankar S. K. (1990) NADPH cytochrome P-450 reductase in rat, mouse and human brain. Biochem. Pharmac. 39, 1013–1018. Riedl A. G., Watts P. M., Edwards R. J., Boobis A. R., Jenner P. and Marsden C. D. (1996) Selective localization of P450 enzymes and NADPH-P450 oxidoreductase in rat basal ganglia using anti-peptide antisera. Brain Res. 743, 324–328. Sasame H. A., Ames M. M. and Nelson S. D. (1977) Cytochrome P450 and NADPH cytochrome c reductase in rat brain formation of reactive catechol metabolites. Biochem. biophys. Res. Commun. 78, 919–926. Schenkman J. B. (1992) Steroid metabolism by constitutive cytochromes P450. J. Steroid Biochem. molec. Biol. 43, 1023–1030. Schulz T. G., Stahlmann R., Edwards R. J., Debri K., Davies D. S. and Neubert D. (1995) Enoxacin is an inducer of CYP1A2 in rat liver. Biochem. Pharmac. 50, 1517–1520. Segura-Aguilar J. (1996) Peroxidase activity of liver microsomal vitamin D 25-hydroxylase and cytochrome P4501A2 catalyses 25-hydroxylation of vitamin D3 and oxidation of dopamine to aminochrome. Biochem. molec. Med. 58, 122–129. Smith C. A. D., Gough A. C., Leigh P. N., Summers B. A., Harding A. E., Maranganore D. M., Sturman S. G., Schapira A. H. V., Williams A. C., Spurr N. K. and Wolf C. R. (1992) Debrisoquine hydroxylase gene polymorphism and susceptibility to Parkinson’s disease. Lancet 339, 1375–1377. Sohda T., Shimizu M., Kamimura S. and Okumura M. (1993) Immunohistochemical demonstration of ethanolinducible P450 2E1 in rat brain. Alcohol Alcohol. 28, 69–75. Stromstedt M., Warner M. and Gustafsson J. A. (1994) Cytochrome P450s of the 4A subfamily in the brain. J. Neurochem. 63, 671–676. Takakubo F., Yamamoto M., Ogawa N., Yamashita Y., Mizuno Y. and Kondon I. (1996) Genetic association between cytochrome P450IA1 gene and susceptibility to Parkinson’s disease. J. neural Transm. 103, 843–849. Tanner C. M. (1989) The role of environmental toxins in the etiology of Parkinson’s disease. Trends Neurosci. 12, 49–54. Tindberg N. and Ingelman-Sundberg M. (1996) Expression, catalytic activity, and inducibility of cytochrome P450 2E1 (CYP2E1) in the rat central nervous system. J. Neurochem. 67, 2066–2073. Tsuneoka Y., Matsuo Y., Iwahashi K., Takeuchi H. and Ichikawa Y. (1993) A novel cytochrome P-450IID6 mutant gene associated with Parkinson’s disease. J. Biochem. 114, 263–266. Tyndale R. F., Sunahara R., Inaba T., Kalow W., Gonzalez F. J. and Niznik H. B. (1991) Neuronal cytochrome P450IID1 (debrisoquine/sparteine-type): potent inhibition of activity by ()cocaine and nucleotide sequence identity to human hepatic P450 gene CYP 2D6. Molec. Pharmac. 40, 63–68. Warner M. and Gustafsson J. A. (1994) Effect of ethanol on cytochrome P450 in the rat brain. Proc. natn. Acad. Sci. U.S.A. 91, 1019–1023. Warner M., Kohler C., Hansson T. and Gustafsson J. A. (1988) Regional distribution of cytochrome P-450 in the rat brain: spectral quantitation and contribution of P-450b,e and P-450c,d. J. Neurochem. 50, 1057–1065. Wyss A., Gustafsson J. A. and Warner M. (1995) Cytochromes P450 of the 2D subfamily in rat brain. Molec. Pharmac. 47, 1148–1155. Yamada-Mouri N., Hirata S., Hayashi M. and Kato J. (1995) Analysis of the expression and the first exon of aromatase mRNA in monkey brain. J. Steroid Biochem. molec. Biol. 55, 17–23. Yoo M., Ryu H. M., Shin S. W., Yun C. H., Lee S. C., Ji Y. M. and You K. H. (1997) Identification of cytochrome P450 2E1 in rat brain. Biochem. biophys. Res. Commun. 231, 254–256. (Accepted 1 December 1997)