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