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Catalase mediates acetaldehyde formation in the striatum of free-moving rats
NeuroToxicology 28 (2007) 1245–1248
Short communication
Catalase mediates acetaldehyde formation in the striatum of free-moving rats Mostofa Jamal *, Kiyoshi Ameno, Ikuo Uekita, Mitsuru Kumihashi, Weihuan Wang, Iwao Ijiri Department of Forensic Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita, Kagawa 761-0793, Japan Received 22 December 2006; accepted 2 May 2007 Available online 13 May 2007
Abstract Using brain microdialysis, we measured both ethanol (EtOH) and acetaldehyde (AcH) levels in the striatum of free-moving rats following the inhibition of EtOH oxidation pathways. Rats received intraperitoneal EtOH (1 g/kg) alone or in combination with 4-methylpyrazole (MP, 82 mg/ kg, an alcohol dehydrogenase inhibitor), and/or catalase inhibitor sodium azide (AZ, 10 mg/kg) or 3-amino-1,2,4-triazole (AT, 1 g/kg), and/or cyanamide (CY, 50 mg/kg, an aldehyde dehydrogenase inhibitor). Results revealed that both EtOH and AcH concentrations reached a plateau at 30 min after a dose of EtOH, and then gradually decreased for 4 h. AcH was identified in the CY + EtOH, CY + AT/AZ + EtOH, and CY + 4MP + EtOH groups. The CY + EtOH-induced peak AcH level was 195.2 19.4 mM, and this level was significantly higher than the values in other groups studied. The catalase or ADH inhibitor in combination with CY lowered considerably the AcH concentration in the brain. The EtOH level reached a maximum of 25.9 2.3 mM in the CY + 4-MP + EtOH group, and this level was markedly higher than in the EtOH group. No significant difference in brain EtOH levels was seen in any of the other groups examined. The findings strongly support the assumption that the enzyme catalase plays a significant role in AcH formation directly in the rat brain. # 2007 Elsevier Inc. All rights reserved. Keywords: Ethanol; Acetaldehyde; Catalase; In vivo microdialysis
1. Introduction The metabolism of ethanol (EtOH) in the brain is chiefly mediated by catalase. The substance derived from this metabolism, acetaldehyde (AcH), is involved in many of the changes of behavior caused by EtOH (Quertemont et al., 2005), but the presence of AcH in the brain continues to be a matter of controversy. There are two possible hypotheses with respect to AcH accumulation in the brain: (1) the peripheral high level of AcH escapes the liver and reaches the brain through the bloodbrain barrier (BBB) and (2) the production of AcH in the brain occurs via the local oxidation of EtOH by catalase. Catalase is an important metabolic route for the conversion of EtOH to AcH in the brain. Researchers had proved that the catalasemediated EtOH oxidation contributes to several behavioral effects of EtOH (Sanchis-Segura et al., 1999; Quertemont et al.,
* Corresponding author. Tel.: +81 87 891 2140; fax: +81 87 891 2141. E-mail address: [email protected] (M. Jamal). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.05.002
2003), but the question has been raised about how much AcH could produce behavioral changes after EtOH administration. EtOH is metabolized to AcH in the liver primarily by alcohol dehydrogenase (ADH). Other enzymes such as CYP2E1 and catalase may also influence EtOH oxidation (Ramchandani et al., 2001). Aldehyde dehydrogenase (ALDH) further catalyzes the oxidation of AcH to acetate. ADH is a major culprit in producing AcH via EtOH metabolism, but this enzyme is not present in the brain, in the isoenzyme that can metabolize EtOH (Galter et al., 2003). CYP2E1, another EtOH oxidative enzyme, is principally found in the liver. Recently it has been shown that CYP2E1 activity in the rat brain is low (Yadav et al., 2006). Catalase inhibitor sodium azide (AZ) or 3amino-1,2,4-triazole (AT) and ADH inhibitor 4-methypyrazole (4-MP) at high concentrations can prevent CYP2E1 activity by 60–70% (Quertemont et al., 2005; Salmela et al., 2001; Chow et al., 1992; Koop, 1990). AT and AZ are two potent catalase inhibitors and have been reported to inhibit catalase activity by 60–80% in the rat brain (Sinet et al., 1980; Canales, 2004; Zimatkin et al., 2006). Cyanamide (CY), an effective ALDH
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inhibitor, can also block catalase activity in the rat brain (Sanchis-Segura et al., 1999). However, results of the previous investigations support the idea that no single catalase inhibitor can produce almost complete inhibition of catalase activity. In light of this evidence, several studies have been conducted using catalase inhibitors to examine the catalase-mediated EtOH effects in the brain (Sanchis-Segura et al., 1999; Quertemont et al., 2003). This is the first study demonstrating the manipulation of in vivo brain catalase activity in the brain of freely moving rat administrated 4-MP, AZ or AT, and CY following EtOH intake and the presumable production of AcH in the presence of these specific inhibitors of EtOH metabolism. Various effects on the brain of catalase-mediated AcH have been noted, but in most studies the concentration of AcH resulting from such manipulations has not been measured (Sanchis-Segura et al., 1999; Quertemont et al., 2003). On the other hand, a number of studies have shown in vitro brain AcH production through the action of enzymes of EtOH metabolism, but the majority of these works were conducted in brain homogenates (Gill et al., 1992; Aragon et al., 1992; Zimatkin et al., 2006). It is, therefore, essential to measure in vivo AcH following EtOH intake using specific inhibitors of EtOH metabolism in the brain of free-moving rats. 2. Materials and methods 2.1. Animal and drugs Male Wistar rats (9–10 weeks old, 250–300 g body weight) were used throughout the study. Animals were divided into the following experimental groups: (a) EtOH (1 g/kg), (b) CY (50 mg/kg) + EtOH, (c) CY + MP (82 mg/kg) + EtOH, (d) CY + AZ (10 mg/kg) + EtOH, (e) CY + AT (1 g/kg) + EtOH, (f) AT + EtOH (g) 4-MP + EtOH, (h) AZ + EtOH, and (i) CY alone. The animals received an i.p. injection of EtOH (20%, v/ v) 30 min, 1 h, and 5 h after a dose of AZ, CY or 4-MP, and AT, respectively. 4-MP, AT, and AZ were purchased from Sigma Chemical Gmbh, Germany, and CY was purchased from Wako Pure Chemical Industries, Japan.
as follows: column, injector and detector temperatures 90, 110 and 200 8C, respectively. The separation column was a SupelcowaxTM wide bore capillary column (60 m length, 0.53 mm i.d., 2 mm film thickness, Supelco, Bellefonte, PA, USA). Nitrogen was used as carrier gas at a flow rate of 20 ml/ min. Using this method, no artifactual AcH was detectable in the dialysate. In vitro probe recovery of EtOH and AcH was 72.2 3.6 and 52.6 5.9%, respectively, and the values were corrected. 2.3. Statistics The statistical software StatView (J-4.5, Berkeley, California, USA) was used to determine the significance of the differences. Statistical analysis of data used Student t-test. All data are expressed as the means S.D. 3. Results Fig. 1 shows the chromatograms of (A) a pure sample of EtOH (40 mM) and AcH (250 mM) and (B) a dialysate sample from a rat that received CY + EtOH (1 g/kg) i.p. The retention times were about 1.64 min for AcH and 2.55 min for EtOH in both the pure sample and the dialysate. Fig. 2 shows the time course changes of (A) EtOH and (B) AcH levels in the striatum. AcH was detected in the CY + EtOH, CY + AZ + EtOH, CY + AT + EtOH, and CY + 4-MP + EtOH groups. The AcH level reached a maximum of 195.2 19.4 mM at 30 min after EtOH injection followed by a gradual decrease for 4 h in the CY + EtOH group. The catalase or ADH inhibitor in combination with CY reduced the peak AcH levels to 129.3 12.1, 135.6 18.2, and 76.8 7.3 mM in the CY + AZ + EtOH, CY + AT + EtOH, and CY + 4-MP + EtOH groups, respectively. The CY + EtOHinduced peak value was considerably higher than in the other
2.2. Microdialysis and head-space GC The microdialysis procedure and head-space GC (PerkinElmer, Norwalk, CT, USA) conditions were as previously described (Jamal et al., 2003a). In short, a microdialysis probe (8 mm long, Eicom, Japan) with a 3 mm long active dialysis membrane was implanted stereotaxically into the striatum (coordinates: 0.2 mm anterior to bregma, 3.0 mm lateral to midline and 3.0 mm ventral to dura) 24 h after surgery and the experiments were began. Ringer’s solution (147 mM NaCl, 4 mM KCl, 2.25 mM CaCl2) was perfused through the dialysis tube at a constant flow rate of 1 ml/min. The dialysates were collected at 15, 30, 45, 60, 90, 120, 180, and 240 min for 10 min each after EtOH injection into a vial containing 50 ml 0.002% tbutanol as an internal standard in 0.6N perchloric acid and then analyzed by head-space GC. Chromatographic conditions were
Fig. 1. The chromatographs of EtOH and AcH obtained from (A) a pure sample of EtOH and AcH and (B) a dialysate sample of rat. EtOH, ethanol; AcH, acetaldehyde; IS, internal standard.
M. Jamal et al. / NeuroToxicology 28 (2007) 1245–1248
Fig. 2. The time course effects of enzyme inhibitors on EtOH metabolic process. Data represents mean S.D. (n = 5). zP < 0.05, for the differences between EtOH and CY + 4-MP + EtOH. *P < 0.05, for the differences between CY + EtOH and CY + 4-MP + EtOH, yP < 0.05, for the differences between CY + EtOH and CY + AZ/AT + EtOH, and # P < 0.05, for the differences between CY + 4-MP + EtOH and CY + AZ/AT + EtOH. CY, cyanamide; EtOH, ethanol; AcH, acetaldehyde; AZ, sodium azide; AT, 3-amino-1,2,4triazole; 4-MP, 4-methylpyrazole.
groups examined. Treatment with the inhibitors did not modify significantly the levels of EtOH in the brain while no detectable AcH was observed. An exception was that treatment with CY + 4-MP + EtOH reached a peak EtOH level of 25.9 2.3 mM, and this level was significantly higher than that reached in the EtOH group. No significant difference in EtOH levels was found in the AZ + EtOH, AT + EtOH, or 4MP + EtOH groups, as compared with the EtOH group (data not shown). Neither EtOH nor AcH was detected in the CY group (data not shown). 4. Discussion The aim of this study was to evaluate the contribution of catalase to the accumulation of AcH in the brain of free-moving rats. Toward that end, we investigated the role of different oxidative mechanisms of EtOH metabolism to clarify the catalase-mediated EtOH oxidation in the brain. The results of the present study revealed that the administration of EtOH (1 g/kg) alone or in combination with catalase inhibitor AZ, or AT and ADH inhibitor 4-MP did not produce a detectable level of AcH in the brain. There are two possible reasons for this finding: either ALDH in the BBB prevented peripheral AcH from penetrating to the brain, or the rapid oxidation process of brain EtOH may have led to the insignificant production of AcH. Thus, these findings clarify that AcH does not penetrate the brain very well. AcH is formed mainly in the liver after EtOH consumption. High AcH concentration in the periphery is able to reach the brain through the BBB. As shown in our previous studies, AcH in the brain is approximately four-fold lower than that observed in the liver (Jamal et al., 2003b; Jamal et al., 2005). The present data demonstrated that the administration of CY + EtOH produced a high level of AcH (195.2 19.4 mM), whereas treatment with catalase inhibitor AZ or AT combined with CY followed by EtOH exhibited a significantly low level of AcH
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(129.3 12.1 and 135.6 18.2, respectively) in the brain. The inhibition of a central source of AcH production represents an important limit for the accumulation of AcH in the brain. Several studies have reported that the ALDH inhibitor CY produced a high blood level of AcH after EtOH consumption in rats (Jamal et al., 2003b; Kinoshita et al., 1996). In light of these previous findings, our data indicated that CY + EtOH-induced high levels of brain AcH could be the result of accumulation from both the peripheral and catalase sources. On the other hand, the catalase inhibitors AZ and AT produced a low accumulation of brain AcH. It has been shown that treatment with catalase inhibitor decreased catalase activity in the rodent brain (Sinet et al., 1980; Canales, 2004; Zimatkin et al., 2006). Moreover, the inhibition of brain catalase activity was accompanied by a significant reduction in ethanol-induced behavior (Sanchis-Segura et al., 2005). It is, however, conceivable that catalase inhibitor-induced low levels of brain AcH might be the result of peripheral contribution. Combined treatment of 4-MP and CY followed by EtOH (1 g/kg) produced a significantly lower level of AcH (76.8 7.3 mM) in the brain, as compared with other groups studied. In particular, 4-MP can block the bulk of peripheral AcH production by inhibiting hepatic ADH activity. Thus, the existence of a lower level of brain AcH in this group is due presumably to the local production of brain AcH generated from EtOH oxidation by catalase. The results of the present context support the hypothesis that both catalase and the high level of peripherally formed AcH are strongly involved in the accumulation of AcH in the brain. The enzymes that metabolize EtOH to AcH in the central nervous system (such as catalase), as well as those that oxidize AcH to acetic acid (such as ALDH), would be major regulators of AcH accumulation in the brain. Thus, the data obtained from this study demonstrated that the contribution of catalase to ethanol oxidation in the rat brain was of great significance. 5. Conclusion We have confirmed the role of catalase in the process of brain EtOH oxidation. On the other hand, the study demonstrated that some other enzymes such as CYP2E1 and ADH may also be involved in EtOH metabolism, but these enzymes may have little effect on the process of brain AcH accumulation. However, the present results strongly support the conclusion that the enzyme catalase made a remarkable contribution to the production of AcH in the rat brain. Acknowledgement This study was funded by a Grant-in-Aid for Scientific Research (c) (No. 18590637) from the Ministry of Education, Science and Culture, Japan. References Aragon CM, Rogan F, Amit Z. Ethanol metabolism in rat brain homogenates by a catalase H2O2 system. Biochem Pharmacol 1992;44:93–8.
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Canales JJ. Catalase-independent early-gene expression in rat brain following acute ethanol exposure. Brain Res 2004;1016:96–101. Chow HH, Hutchaleelaha A, Mayersohn M. Inhibitory effect of 4-methylpyrazole on antipyrine clearance in rats. Life Sci 1992;50:661–6. Galter D, Carmine A, Buervenich S, Duester G, Olson L. Distribution of class I, III and IValcohol dehydrogenase mRNAs in the adult rat, mouse and human brain. Eur J Biochem 2003;270:1316–26. Gill K, Menez JF, Lucas D, Deitrich RA. Enzymatic production of acetaldehyde from ethanol in rat brain tissue. Alcohol Clin Exp Res 1992;16:910–5. Jamal M, Ameno K, Wang W, Kumihashi M, Ameno S, Ikuo U, et al. Inhibition of acetaldehyde metabolism decreases acetylcholine release in medial frontal cortex of freely moving rats. Brain Res 2005;1039:90–6. Jamal M, Ameno K, Kumihashi M, Ameno S, Kubota T, Wang W, et al. Microdialysis for the determination of acetaldehyde and ethanol concentration in the striatum of freely moving rats. J Chromatogr B 2003a;798:155–8. Jamal M, Ameno K, Ameno S, Okada N, Ijiri I. In vivo study of salsolinol produced by a high concentration of acetaldehyde in the striatum and nucleus accumbens of free-moving rats. Alcohol Clin Exp Res 2003b;27(Suppl.): 79S–84S. Kinoshita H, Ijiri I, Ameno S, Fuke C, Fujisawa Y, Ameno K. Inhibitory mechanism of intestinal ethanol absorption induced by high acetaldehyde concentrations: effect of intestinal blood flow and substance specificity. Alcohol Clin Exp Res 1996;20:510–3.
Koop DR. Inhibition of ethanol-inducible cytochrome P450IIE1 by 3-amino1,2,4-triazole. Chem Res Toxicol 1990;3:377–83. Quertemont E, Tambour S, Tirelli E. The role of acetaldehyde in the neurobehavioral effects of ethanol: a comprehensive review of animal studies. Prog Neurobiol 2005;75:247–74. Quertemont E, Escarabajal MD, De Witte P. Role of catalase in ethanol-induced conditioned taste aversion: a study with 3-amino-1,2,4-triazole. Drug Alcohol Depend 2003;70:77–83. Ramchandani VA, Bosron WF, Li TK. Research advances in ethanol metabolism. Pathol Biol 2001;49:676–82. Salmela KS, Tsyrlov IB, Lieber CS. Azide inhibits human cytochrome P4502E1, 1A2, and 3A4. Alcohol Clin Exp Res 2001;25:253–60. Sanchis-Segura C, Miquel M, Correa M, Aragon CM. Cyanamide reduces brain catalase and ethanol-induced locomotor activity: is there a functional link? Psychopharmacol 1999;144:83–9. Sanchis-Segura C, Correa M, Miquel M, Aragon CM. Catalase inhibition in the Arcuate nucleus blocks ethanol effects on the locomotor activity of rats. Neurosci Lett 2005;376:66–70. Sinet PM, Heikkila RE, Cohen G. Hydrogen peroxide production by rat brain in vivo. J Neurochem 1980;34:1421–8. Zimatkin SM, Pronko SP, Vasiliou V, Gonzalez FJ, Deitrich RA. Enzymatic mechanisms of ethanol oxidation in the brain. Alcohol Clin Exp Res 2006;30:1500–5.
Short communication
Catalase mediates acetaldehyde formation in the striatum of free-moving rats Mostofa Jamal *, Kiyoshi Ameno, Ikuo Uekita, Mitsuru Kumihashi, Weihuan Wang, Iwao Ijiri Department of Forensic Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita, Kagawa 761-0793, Japan Received 22 December 2006; accepted 2 May 2007 Available online 13 May 2007
Abstract Using brain microdialysis, we measured both ethanol (EtOH) and acetaldehyde (AcH) levels in the striatum of free-moving rats following the inhibition of EtOH oxidation pathways. Rats received intraperitoneal EtOH (1 g/kg) alone or in combination with 4-methylpyrazole (MP, 82 mg/ kg, an alcohol dehydrogenase inhibitor), and/or catalase inhibitor sodium azide (AZ, 10 mg/kg) or 3-amino-1,2,4-triazole (AT, 1 g/kg), and/or cyanamide (CY, 50 mg/kg, an aldehyde dehydrogenase inhibitor). Results revealed that both EtOH and AcH concentrations reached a plateau at 30 min after a dose of EtOH, and then gradually decreased for 4 h. AcH was identified in the CY + EtOH, CY + AT/AZ + EtOH, and CY + 4MP + EtOH groups. The CY + EtOH-induced peak AcH level was 195.2 19.4 mM, and this level was significantly higher than the values in other groups studied. The catalase or ADH inhibitor in combination with CY lowered considerably the AcH concentration in the brain. The EtOH level reached a maximum of 25.9 2.3 mM in the CY + 4-MP + EtOH group, and this level was markedly higher than in the EtOH group. No significant difference in brain EtOH levels was seen in any of the other groups examined. The findings strongly support the assumption that the enzyme catalase plays a significant role in AcH formation directly in the rat brain. # 2007 Elsevier Inc. All rights reserved. Keywords: Ethanol; Acetaldehyde; Catalase; In vivo microdialysis
1. Introduction The metabolism of ethanol (EtOH) in the brain is chiefly mediated by catalase. The substance derived from this metabolism, acetaldehyde (AcH), is involved in many of the changes of behavior caused by EtOH (Quertemont et al., 2005), but the presence of AcH in the brain continues to be a matter of controversy. There are two possible hypotheses with respect to AcH accumulation in the brain: (1) the peripheral high level of AcH escapes the liver and reaches the brain through the bloodbrain barrier (BBB) and (2) the production of AcH in the brain occurs via the local oxidation of EtOH by catalase. Catalase is an important metabolic route for the conversion of EtOH to AcH in the brain. Researchers had proved that the catalasemediated EtOH oxidation contributes to several behavioral effects of EtOH (Sanchis-Segura et al., 1999; Quertemont et al.,
* Corresponding author. Tel.: +81 87 891 2140; fax: +81 87 891 2141. E-mail address: [email protected] (M. Jamal). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.05.002
2003), but the question has been raised about how much AcH could produce behavioral changes after EtOH administration. EtOH is metabolized to AcH in the liver primarily by alcohol dehydrogenase (ADH). Other enzymes such as CYP2E1 and catalase may also influence EtOH oxidation (Ramchandani et al., 2001). Aldehyde dehydrogenase (ALDH) further catalyzes the oxidation of AcH to acetate. ADH is a major culprit in producing AcH via EtOH metabolism, but this enzyme is not present in the brain, in the isoenzyme that can metabolize EtOH (Galter et al., 2003). CYP2E1, another EtOH oxidative enzyme, is principally found in the liver. Recently it has been shown that CYP2E1 activity in the rat brain is low (Yadav et al., 2006). Catalase inhibitor sodium azide (AZ) or 3amino-1,2,4-triazole (AT) and ADH inhibitor 4-methypyrazole (4-MP) at high concentrations can prevent CYP2E1 activity by 60–70% (Quertemont et al., 2005; Salmela et al., 2001; Chow et al., 1992; Koop, 1990). AT and AZ are two potent catalase inhibitors and have been reported to inhibit catalase activity by 60–80% in the rat brain (Sinet et al., 1980; Canales, 2004; Zimatkin et al., 2006). Cyanamide (CY), an effective ALDH
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inhibitor, can also block catalase activity in the rat brain (Sanchis-Segura et al., 1999). However, results of the previous investigations support the idea that no single catalase inhibitor can produce almost complete inhibition of catalase activity. In light of this evidence, several studies have been conducted using catalase inhibitors to examine the catalase-mediated EtOH effects in the brain (Sanchis-Segura et al., 1999; Quertemont et al., 2003). This is the first study demonstrating the manipulation of in vivo brain catalase activity in the brain of freely moving rat administrated 4-MP, AZ or AT, and CY following EtOH intake and the presumable production of AcH in the presence of these specific inhibitors of EtOH metabolism. Various effects on the brain of catalase-mediated AcH have been noted, but in most studies the concentration of AcH resulting from such manipulations has not been measured (Sanchis-Segura et al., 1999; Quertemont et al., 2003). On the other hand, a number of studies have shown in vitro brain AcH production through the action of enzymes of EtOH metabolism, but the majority of these works were conducted in brain homogenates (Gill et al., 1992; Aragon et al., 1992; Zimatkin et al., 2006). It is, therefore, essential to measure in vivo AcH following EtOH intake using specific inhibitors of EtOH metabolism in the brain of free-moving rats. 2. Materials and methods 2.1. Animal and drugs Male Wistar rats (9–10 weeks old, 250–300 g body weight) were used throughout the study. Animals were divided into the following experimental groups: (a) EtOH (1 g/kg), (b) CY (50 mg/kg) + EtOH, (c) CY + MP (82 mg/kg) + EtOH, (d) CY + AZ (10 mg/kg) + EtOH, (e) CY + AT (1 g/kg) + EtOH, (f) AT + EtOH (g) 4-MP + EtOH, (h) AZ + EtOH, and (i) CY alone. The animals received an i.p. injection of EtOH (20%, v/ v) 30 min, 1 h, and 5 h after a dose of AZ, CY or 4-MP, and AT, respectively. 4-MP, AT, and AZ were purchased from Sigma Chemical Gmbh, Germany, and CY was purchased from Wako Pure Chemical Industries, Japan.
as follows: column, injector and detector temperatures 90, 110 and 200 8C, respectively. The separation column was a SupelcowaxTM wide bore capillary column (60 m length, 0.53 mm i.d., 2 mm film thickness, Supelco, Bellefonte, PA, USA). Nitrogen was used as carrier gas at a flow rate of 20 ml/ min. Using this method, no artifactual AcH was detectable in the dialysate. In vitro probe recovery of EtOH and AcH was 72.2 3.6 and 52.6 5.9%, respectively, and the values were corrected. 2.3. Statistics The statistical software StatView (J-4.5, Berkeley, California, USA) was used to determine the significance of the differences. Statistical analysis of data used Student t-test. All data are expressed as the means S.D. 3. Results Fig. 1 shows the chromatograms of (A) a pure sample of EtOH (40 mM) and AcH (250 mM) and (B) a dialysate sample from a rat that received CY + EtOH (1 g/kg) i.p. The retention times were about 1.64 min for AcH and 2.55 min for EtOH in both the pure sample and the dialysate. Fig. 2 shows the time course changes of (A) EtOH and (B) AcH levels in the striatum. AcH was detected in the CY + EtOH, CY + AZ + EtOH, CY + AT + EtOH, and CY + 4-MP + EtOH groups. The AcH level reached a maximum of 195.2 19.4 mM at 30 min after EtOH injection followed by a gradual decrease for 4 h in the CY + EtOH group. The catalase or ADH inhibitor in combination with CY reduced the peak AcH levels to 129.3 12.1, 135.6 18.2, and 76.8 7.3 mM in the CY + AZ + EtOH, CY + AT + EtOH, and CY + 4-MP + EtOH groups, respectively. The CY + EtOHinduced peak value was considerably higher than in the other
2.2. Microdialysis and head-space GC The microdialysis procedure and head-space GC (PerkinElmer, Norwalk, CT, USA) conditions were as previously described (Jamal et al., 2003a). In short, a microdialysis probe (8 mm long, Eicom, Japan) with a 3 mm long active dialysis membrane was implanted stereotaxically into the striatum (coordinates: 0.2 mm anterior to bregma, 3.0 mm lateral to midline and 3.0 mm ventral to dura) 24 h after surgery and the experiments were began. Ringer’s solution (147 mM NaCl, 4 mM KCl, 2.25 mM CaCl2) was perfused through the dialysis tube at a constant flow rate of 1 ml/min. The dialysates were collected at 15, 30, 45, 60, 90, 120, 180, and 240 min for 10 min each after EtOH injection into a vial containing 50 ml 0.002% tbutanol as an internal standard in 0.6N perchloric acid and then analyzed by head-space GC. Chromatographic conditions were
Fig. 1. The chromatographs of EtOH and AcH obtained from (A) a pure sample of EtOH and AcH and (B) a dialysate sample of rat. EtOH, ethanol; AcH, acetaldehyde; IS, internal standard.
M. Jamal et al. / NeuroToxicology 28 (2007) 1245–1248
Fig. 2. The time course effects of enzyme inhibitors on EtOH metabolic process. Data represents mean S.D. (n = 5). zP < 0.05, for the differences between EtOH and CY + 4-MP + EtOH. *P < 0.05, for the differences between CY + EtOH and CY + 4-MP + EtOH, yP < 0.05, for the differences between CY + EtOH and CY + AZ/AT + EtOH, and # P < 0.05, for the differences between CY + 4-MP + EtOH and CY + AZ/AT + EtOH. CY, cyanamide; EtOH, ethanol; AcH, acetaldehyde; AZ, sodium azide; AT, 3-amino-1,2,4triazole; 4-MP, 4-methylpyrazole.
groups examined. Treatment with the inhibitors did not modify significantly the levels of EtOH in the brain while no detectable AcH was observed. An exception was that treatment with CY + 4-MP + EtOH reached a peak EtOH level of 25.9 2.3 mM, and this level was significantly higher than that reached in the EtOH group. No significant difference in EtOH levels was found in the AZ + EtOH, AT + EtOH, or 4MP + EtOH groups, as compared with the EtOH group (data not shown). Neither EtOH nor AcH was detected in the CY group (data not shown). 4. Discussion The aim of this study was to evaluate the contribution of catalase to the accumulation of AcH in the brain of free-moving rats. Toward that end, we investigated the role of different oxidative mechanisms of EtOH metabolism to clarify the catalase-mediated EtOH oxidation in the brain. The results of the present study revealed that the administration of EtOH (1 g/kg) alone or in combination with catalase inhibitor AZ, or AT and ADH inhibitor 4-MP did not produce a detectable level of AcH in the brain. There are two possible reasons for this finding: either ALDH in the BBB prevented peripheral AcH from penetrating to the brain, or the rapid oxidation process of brain EtOH may have led to the insignificant production of AcH. Thus, these findings clarify that AcH does not penetrate the brain very well. AcH is formed mainly in the liver after EtOH consumption. High AcH concentration in the periphery is able to reach the brain through the BBB. As shown in our previous studies, AcH in the brain is approximately four-fold lower than that observed in the liver (Jamal et al., 2003b; Jamal et al., 2005). The present data demonstrated that the administration of CY + EtOH produced a high level of AcH (195.2 19.4 mM), whereas treatment with catalase inhibitor AZ or AT combined with CY followed by EtOH exhibited a significantly low level of AcH
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(129.3 12.1 and 135.6 18.2, respectively) in the brain. The inhibition of a central source of AcH production represents an important limit for the accumulation of AcH in the brain. Several studies have reported that the ALDH inhibitor CY produced a high blood level of AcH after EtOH consumption in rats (Jamal et al., 2003b; Kinoshita et al., 1996). In light of these previous findings, our data indicated that CY + EtOH-induced high levels of brain AcH could be the result of accumulation from both the peripheral and catalase sources. On the other hand, the catalase inhibitors AZ and AT produced a low accumulation of brain AcH. It has been shown that treatment with catalase inhibitor decreased catalase activity in the rodent brain (Sinet et al., 1980; Canales, 2004; Zimatkin et al., 2006). Moreover, the inhibition of brain catalase activity was accompanied by a significant reduction in ethanol-induced behavior (Sanchis-Segura et al., 2005). It is, however, conceivable that catalase inhibitor-induced low levels of brain AcH might be the result of peripheral contribution. Combined treatment of 4-MP and CY followed by EtOH (1 g/kg) produced a significantly lower level of AcH (76.8 7.3 mM) in the brain, as compared with other groups studied. In particular, 4-MP can block the bulk of peripheral AcH production by inhibiting hepatic ADH activity. Thus, the existence of a lower level of brain AcH in this group is due presumably to the local production of brain AcH generated from EtOH oxidation by catalase. The results of the present context support the hypothesis that both catalase and the high level of peripherally formed AcH are strongly involved in the accumulation of AcH in the brain. The enzymes that metabolize EtOH to AcH in the central nervous system (such as catalase), as well as those that oxidize AcH to acetic acid (such as ALDH), would be major regulators of AcH accumulation in the brain. Thus, the data obtained from this study demonstrated that the contribution of catalase to ethanol oxidation in the rat brain was of great significance. 5. Conclusion We have confirmed the role of catalase in the process of brain EtOH oxidation. On the other hand, the study demonstrated that some other enzymes such as CYP2E1 and ADH may also be involved in EtOH metabolism, but these enzymes may have little effect on the process of brain AcH accumulation. However, the present results strongly support the conclusion that the enzyme catalase made a remarkable contribution to the production of AcH in the rat brain. Acknowledgement This study was funded by a Grant-in-Aid for Scientific Research (c) (No. 18590637) from the Ministry of Education, Science and Culture, Japan. References Aragon CM, Rogan F, Amit Z. Ethanol metabolism in rat brain homogenates by a catalase H2O2 system. Biochem Pharmacol 1992;44:93–8.
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