Acetaldehyde as Well as Ethanol Is Metabolized by Human CYP2E1

  1. Satoru Kunitoh1,
  2. Susumu Imaoka2,
  3. Toyoko Hiroi2,
  4. Yoshiyasu Yabusaki3,
  5. Takeyuki Monna1 and
  6. Yoshihiko Funae2
  1. 1Department of Public Health (S.K., T.M.) and 2Laboratory of Chemistry (S.I., T.H., Y.F.), Osaka City University Medical School, 1–4-54 Asahi-machi, Abeno-ku, Osaka, 545 Japan, and 3Biotechnology Laboratory (Y.Y), Sumitomo Chemical Co., Ltd., Hyogo, 665 Japan
     
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    Abstract

    Acetaldehyde was oxidized by rat and human hepatic microsomes in the presence of NADPH. We designated this NADPH-dependent oxidation system MAOS (microsomal acetaldehyde-oxidizing system), to distinguish it from the NAD-dependent acetaldehyde oxidation system of acetaldehyde dehydrogenase in mitochondria and cytosol. This activity was increased 2.3-fold by giving rats ethanol. Judging from theV max/Kmvalues, the metabolic capacity of rat hepatic microsomes for MAOS activity was increased 24-fold by ethanol. The acetaldehyde oxidation activity of eight forms of purified rat cytochrome P450 was investigated in a reconstituted system. CYP2E1 had the highest level, followed by CYP1A2 and 4A2. Immunoinhibition studies showed that an anti-CYP2E1 antibody inhibited 90% of the MAOS activity in rats given ethanol. NADPH-dependent acetate formation was 12% or 33.6% of the NAD-dependent acetate formation in liver homogenates of control rats and those treated with ethanol, respectively. We investigated human MAOS activity further. Among the 10 forms of human cytochrome P450 expressed in yeast, CYP2E1 had especially high acetaldehyde oxidation activity. The correlation of MAOS activity with the levels of immunoreactive CYP2E1 in individual human microsomes was highly significant (r 2 = 0.88, P < .01). These results indicate that hepatic CYP2E1 mainly contributes to MAOS in rats and humans, the pathway of which may play an alternative role against acetaldehyde in the liver after alcohol consumption together with acetaldehyde dehydrogenase in the metabolism of acetaldehyde.

    Ethanol is almost totally broken down by oxidative metabolism in vivo. Acetaldehyde, a reactive and toxic metabolite of ethanol, could affect drinking behavior and susceptibility to alcoholism. In addition, it is significant in the pathogenesis of liver damage (Lieber, 1990, 1991;Peters and Ward, 1988; Ingelman-Sandberg et al., 1993). Therefore, it is important to know how acetaldehyde is metabolizedin vivo. So far, the NAD-dependent ALDH system, located in mitochondria or cytosol, is the principle metabolic pathway in which acetaldehyde is converted to acetate (Koivula and Koivusalo, 1975;Tottmar et al., 1973; Tsutsumi et al., 1988).Weiner (1987) has shown by incubating rat liver slices with acetaldehyde that a low Km mitochondrial ALDH (ALDH2) might be responsible for 60% of the metabolism of acetaldehyde, whereas high Km cytosolic ALDH (ALDH1) metabolized an additional 20%, and the remaining 20% was caused by an undetermined system. The role of ALDH2 in humans has been examined by analyzing the genetic polymorphism of ALDH2 (Crabb et al., 1989; Singh et al., 1989; Enomoto et al., 1991; Harada et al., 1981). It has not been clarified whether enzymes other than ALDH are induced by ethanol and metabolize acetaldehyde.

    The oxidation of the aldehyde to the corresponding carboxylic acid in metabolic studies on tetrahydrocannabinol, a major constituent of marijuana, is catalyzed in mice by a hepatic microsomal aldehyde oxygenase involving P450 (Watanabe et al., 1991). Teleliuset al. (1991) have found that acetaldehyde is metabolized by CYP2E1 in starved and acetone-treated rats by measuring the disappearance of acetaldehyde, although acetate was not measured directly. These findings suggest that enzymes other than ALDH in rat and human hepatic microsomes contribute to the metabolism of acetaldehyde. In the past decade, much has been learned about the multiple forms of P450 in experimental animals and humans (Funae and Imaoka 1993; Guengerich and Shimada 1991). Among them, CYP2E1 is induced by alcohol intake, and it has high ethanol oxidation activity (Koop et al., 1984). The CYP2E1 induced by long-term alcohol intake is thought to play an important role in the MEOS (Lieber and DeCarli, 1970), which is apparently a different metabolic pathway of acetaldehyde production from that of the alcohol dehydrogenase system (Lieber, 1990, 1991). Recently, we found (Kunitoh et al., 1993; Asai et al., 1996) that CYP1A2 in addition to CYP2E1 is the major contributor to MEOS.

    Here, we investigated NADPH-dependent acetaldehyde metabolism in rat and human hepatic microsomes (MAOS) by directly measuring acetate levels. We clarified which forms of rat and human P450s contribute to this oxidation system.

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    Materials and Methods

    Chemicals.

    Acetaldehyde was purchased from Merck-Schuchardt (München, Germany). [14C]Acetaldehyde (specific activity, 4.5 mCi/mmol) and [14C]acetate (specific activity, 52 mCi/mmol) were obtained from Du Pont/NEN Research Products (Boston, MA). The anion-exchange column (Bond Elute) was purchased from Uniflex (Tokyo, Japan). DLPC was obtained from the Sigma Chemical Co. (St. Louis, MO). NADPH and NAD+ were obtained from the Oriental Yeast Co.(Tokyo, Japan). Other reagents and organic solvents were obtained from Wako Pure Chemical Industries (Tokyo, Japan).

    Rat P450s.

    Male Sprague-Dawley rats at 4 weeks of age were purchased from Clea Japan Inc. (Tokyo, Japan). Twenty rats were pair-fed with liquid diets containing either ethanol or isocaloric carbohydrates according to DeCarli and Lieber (1967). 3-MC (40 mg/kg, dissolved in corn oil) was given intraperitoneally 2 times every other day or PB (80 mg/kg, dissolved in saline) was given intraperitoneally daily for 4 days, respectively. Hepatic microsomes were prepared as described (Funae and Imaoka 1985) and frozen at −80°C until used. P450s were purified from hepatic microsomes by ion-exchange high-performance liquid chromatography followed by 7 to 15% polyethylene glycol fractionation and octylamino-Sepharose chromatography as described (Funae and Imaoka 1987; Yasukochi and Masters 1976). NADPH-P450 reductase and cytochrome b5 were purified as reported (Funae and Imaoka 1985, 1987). The specific activity of the purified reductase was 38 U/mg of protein. The specific content of purified cytochrome b5 was 28 nmol/mg of protein.

    Human P450s and human microsomes.

    Microsomes were prepared from yeast cells expressing human P450 1A1, 1A2, 2A6, 2B6, 2C9, 2C18, 2D6, 2E1 and 3A4 as described elsewhere (Imaoka et al., 1996). Human samples (Nos. 1–9) were obtained from Japanese patients with hepatocellular carcinoma. Tissue was selected from areas of the liver which were visually free of tumor, frozen as soon as possible and stored −80°C until use. Human sample 10 was also obtained from organ donor who died in an accident. Informed consent was obtained from the patients or their relatives. The tissues were used for research purposes with the full approval of the Institutional Committee for Research Subjects. Hepatic microsomes were prepared as described for those of the rat (Funae and Imaoka, 1985).

    Assay of acetaldehyde oxidation activity.

    [14C]Acetaldehyde was diluted with H2O (1 ml) and passed through Bond Elute to remove contaminating [14C]acetate. The flow-through fraction was collected and used in the assay. This procedure reduced the assay background to 100 to 200 cpm. The amount of [14C]acetate produced by P450 was assayed by the modified method of Von Korff (1969) andWickramasinghe (1986). The reaction mixture (total volume, 100 μl) containing microsomes (200 μg) and [14C]acetaldehyde (300 μM) was incubated for 15 min at 37°C in 0.1 M Tris-acetate buffer (pH 7.2) in tightly capped tubes. The rate of acetate formation in a reconstituted system with purified P450 was measured as follows. Purified P450 (30 pmol), NADPH-P450 reductase (0.3 U), cytochrome b5 (30 pmol), DLPC (5 μg) and [14C]acetaldehyde (300 μM) were incubated in 0.1 M Tris-acetate buffer (pH 7.2). The reaction was started by adding 10 mM NADPH (20 μl) and stopped by adding excess nonradioactive acetaldehyde (20 mM). The reaction mixture (0.1 ml) was applied to a Bond Elute anion-exchange column (1 ml) and washed with H2O (2 ml). The acetate produced by P450 was eluted with 1 N HCl (2 ml) and quantified by a liquid scintillation counter (Beckman Instruments Inc., Fullerton, CA). For comparison of NAD- or NADPH-dependent acetaldehyde metabolism, the fraction of homogenates in rat livers was used instead of that of microsomes. Reaction mixture (total volume, 100 μl) containing homogenates (500 μg) and [14C]acetaldehyde (300 μM) were incubated for 15 min at 37°C in 0.1 M Tris-acetate buffer (pH 7.2). The reaction was started by the addition of 10 mM NADPH (20 μl) or 1.7 mM NAD+ (5.6 μl) [according to Sugata et al.(1988)] and stopped by adding excess nonradioactive acetaldehyde (20 mM). When [14C]acetaldehyde was incubated with P450 without NADPH or NAD+, the counts were at background level. The standard curve for acetate was linear between 0 and 100 μM. The detection limit was 0.1 μM (130 cpm vs. 107 cpm background). The percent recovery of the added authentic acetate was from 95% to 105%. The acetate formation was linear up to 30 min in the presence of hepatic microsomes and purified P450. Catalytic activities of all enzyme preparations were assayed under conditions in which the metabolism was proportional to the P450 concentrations and incubation period. The amounts of NADPH-P450 reductase and DLPC were enough for the purified P450 to have optimal activity, and the NADPH concentration was saturating. Statistical analysis was performed by means of the Student’s t test.

    Immunochemical study.

    The antibody against purified rat P450s was raised in female Japanease white rabbits obtained from Biotech (Saga, Japan) and the immunoglobulin G (IgG) fraction was prepared by ammonium sulfate precipitation and affinity chromatography with protein A Sepharose as described (Imaoka et al., 1987). The purified P450s used in immunization migrated as a single band on SDS-PAGE, and their specific contents were 13 to 17 nmol/mg. The antibody was characterizied as described (Imaoka et al., 1987, 1989). Proteins were separated by SDS-PAGE with a 7.5% acrylamide gel. Immunoblotting and inhibition studies proceeded as described (Imaoka et al., 1987). Immunoblots were quantified by densitometry of immunostained bands with purified rat and human P450 as a standard (Imaoka et al., 1996). The intensity of the band was linear from 0.2 to 1.0 pmol of P450 applied to SDS-PAGE.

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    Results

    Acetate formation activities of rat hepatic microsomes and purified P450.

    The turnover rate of acetate formed by microsomes of control rats and of those given ethanol, 3-MC or PB are shown in table1. The rate of acetate formation by hepatic microsomes from rats given ethanol was higher than that by corresponding control rats by 130%. Acetate formation by microsomes of PB-treated rats was lower than that of controls by 70% (P < .01). Acetate formation by 3-MC-treated rats was not statistically different from that of control rats (table 1). We studied the acetate formation activities of purified rat P450s in a reconstituted system (table 2). CYP2E1 formed acetate at the highest rate among the eight forms of P450. CYP1A2 and 4A2 also had high rates of acetate formation activity, whereas that by CYP2C11 and 3A2, the major forms in male rats, was low.

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    Table 1

    Acetate formation activity of rat hepatic microsomes

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    Table 2

    Acetaldehyde oxidation activity of purified rat P450s

    Apparent kinetic parameters (Km andVmax) of rat hepatic microsomes for MAOS activity.

    The apparent Km andV max values of rat hepatic microsomes for MAOS activity were determined from Lineweaver-Burk plots at acetaldehyde concentrations ranging from 50 μM to 5 mM (table3). The Km value of microsomes treated with ethanol was 35 μM, which was lower than that of the control. The V max value of microsomes given ethanol was 5.01 nmol/min/mg, which was 3-fold higher than that of the control. Judging from theV max/Kmvalues, the metabolic capacity of hepatic microsomes for MAOS activity was increased 24-fold by ethanol.

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    Table 3

    V max and K m of rat hepatic microsomes for MAOS activity

    Effect of antibody against CYP2E1 on MAOS activity of rat hepatic microsomes.

    To obtain evidence that CYP2E1 contributes to MAOS, we performed inhibition studies with anti-CYP2E1 antibody with hepatic microsomes of rats treated with ethanol and control rats (fig.1). Anti-CYP1A2 antibody was used for comparison, because this P450 metabolized acetaldehyde in a reconstituted system. Antibody against CYP2E1 inhibited the MAOS activity by 56 and 90% when antibody (0.5 mg IgG/mg protein) was added to microsomes of control and ethanol-treated rats, respectively. Induced CYP2E1 was a major contributor to the MAOS. Inhibition was not detected by giving anti-CYP1A2 antibody (0.5 mg IgG/mg protein) to hepatic microsomes of control and ethanol-treated rats. These results indicated that contribution of CYP1A2 to the MAOS was low, although it contributes to MEOS (Kunitoh et al., 1993). CYP2E1 was a main contributor to MAOS, especially in the ethanol-treated rats.

    Figure 1
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    Figure 1

    Effect of antibody against CYP2E1 and 1A2 on MAOS of rat hepatic microsomes. Various amounts of IgG were incubated with microsomes (200 μg) for 10 min at room temperature. The remaining components were added, and the reaction was initiated. The mixture was incubated for 15 min at 37°C in 0.1 M Tris-acetate buffer, pH 7.2. MAOS metabolites were analyzed as described under “Methods.” (A) Effect of anti-CYP2E1 and 1A2 antibodies on microsomes from control. (B) Effect of anti-CYP2E1 and 1A2 antibodies on microsomes from rats treated with ethanol. Residual activity is expressed as a percentage of the value measured with control rabbit IgG instead of anti-CYP2E1 and 1A2 IgG. The filled and open circles indicate the residual activity in the presence of anti-CYP2E1 and 1A2 antibodies, respectively.

    MAOS activities of human hepatic microsomes.

    We investigated whether CYP2E1 was a major contributor to human MAOS by human P450s expressed in yeasts. The MAOS activities of 10 expressed human P450s was determined (table 4). Among these P450s, CYP2E1 had especially high acetaldehyde oxidation activity and CYP1A2, 2C18 and 3A4 had moderate activity. Control cells that did not express P450 had negligible activity. To assess contribution of CYP2E1 to MAOS activity in human hepatic microsomes, MAOS activity of human hepatic microsomes was investigated. Human hepatic microsomal samples were prepared from liver tissues of 10 patients. The case histories of the patients are shown in table 5, along with the CYP2E1 contents and MAOS activities. The MAOS activity of human hepatic microsomes was between 0.19 and 1.32 nmol/min/mg of protein. The variation was related to the P450 contents in the human liver samples. To obtain evidence that CYP2E1 contributes to human MAOS, we performed inhibition studies with anti-CYP2E1 antibody with human hepatic microsomes of sample 10 (fig. 2). Anti-CYP2E1 antibody inhibited the MAOS activity by 55%, which suggested that CYP2E1 played a major role in human MAOS. To obtain further evidence for the role of human CYP2E1 in MAOS, the rates of MAOS activity in individual human microsomes were compared with the levels of CYP2E1 determined by means of immunoblotting with anti-CYP2E1 antibody (fig. 3). The specificity of antibodies was investigated by Western blotting the P450s expressed in B-lymphoblastoid cells (data not shown). This anti-CYP2E1 antibody reacts only with the corresponding human P450, and human hepatic microsomes gave a single stained band (Imaoka et al., 1996). The contents of CYP2E1 are shown in table 5. MAOS activity significantly correlated with the levels of immunoreactive CYP2E1 (r 2 = 0.88, P < .01). These results indicated that CYP2E1 mainly contributes to acetaldehyde oxidation in human hepatic microsomes.

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    Table 4

    Acetaldehyde oxidation activity of human P450 expressed in yeast

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    Table 5

    Case histories, P450 2E1 contents and the MAOS activity of human hepatic microsomes5-a

    Figure 2
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    Figure 2

    Correlation of MAOS activity in human hepatic microsomes with the levels of CYP2E1. The reaction proceeded as described in the legend to figure 1. The levels of CYP2E1 were assayed by quantitative immunoblotting.  

    Figure 3
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    Figure 3

    Effect of antibody against CYP2E1 and 1A2 on MAOS of hepatic microsomes from human sample 10. The reaction proceeded as described in the legend to figure 1.  

    NAD- or NADPH-dependent acetaldehyde metabolism in rat liver homogenate.

    To clarify the role of MAOS in liver, MAOS should be compared with the NAD-dependent ALDH system. To compare the role of MAOS to that of the ALDH system in acetaldehyde metabolism, we examined the effect of NAD+ or NADPH on acetate formation in the rat liver homogenate (table 6). The levels of NADPH-dependent acetate formation were 12% and 33.6% of the NAD-dependent acetate formation in control rats and rats given ethanol, respectively, which indicated that NADPH-, but not NAD-dependent acetaldehyde oxidation was increased by ethanol.

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    Table 6

    Effect of NAD or NADPH on acetaldehyde metabolism in rat liver homogenates

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    Discussion

    We developed a direct assay of acetate formed from [14C]acetaldehyde by a NADPH-dependent oxidation system. The advantages of this procedure were new separation method of acetaldehyde and acetate by use of an anion-exchange column to detect acetate specifically. The contribution of P450s to the MAOS was studied by this method. We found that acetaldehyde was metabolized by MAOS, effectively by liver microsomes of rats given ethanol, and that CYP2E1 was a major contributor to MAOS. This is in agreement with the findings of Telelius et al. (1991). TheKm value of rat hepatic microsomes treated with ethanol for MAOS activity was 35 μM. ThisKm value was similar to that of ALDH1 (high Km ALDH), although it was higher than that of ALDH2 (low Km ALDH, 1–10 μM) (Singh et al., 1989; Enomoto et al., 1991). Judging from theV max/Kmvalues, the metabolic capacity of hepatic microsomes for MAOS activity was increased 24-fold by ethanol treatment. Hansson et al.(1990) reported that CYP2E1 was increased in the brain when ALDH was inhibited with disulfiram in ethanol-treated rats and the concentration of acetaldehyde in the blood increased to more than 50 μM. This might be of interest with regard to the findings of CYP2E1-dependent acetaldehyde metabolism.

    Although it is difficult to compare precisely the biological contribution of MAOS with that of the ALDH system in acetaldehyde metabolism, we examined the effect of NAD+ or NADPH on acetate formation in the rat liver homogenate. This was the first study of comparison between the role of MAOS and that of ALDH in vitro. The results indicate that NADPH- but not NAD-dependent acetaldehyde oxidation was increased by ethanol. Therefore, MAOS activity might be more important for acetaldehyde metabolism when MAOS is induced by ethanol.

    We investigated human MAOS activity with 10 human P450s expressed in yeast. Among them, CYP2E1 had the highest acetaldehyde oxidation activity as in the rat. These P450s are present in hepatic microsomes of adult human (Imaoka et al., 1996; Wrighton and Stevens 1992). Pretty high levels of CYP2E1 (about 7% of the total P450) were detected; and no marked sex, race or age-related differences in CYP2E1 content were found when interindividual variations in the levels of P450 enzymes were investigated in the hepatic microsomes of 30 Japanese and 30 Caucasian subjects (Shimada et al., 1994). In individual human microsomes, the level of immunoreactive CYP2E1 was significantly correlated with MAOS activity. Japanease individuals homozygous and heterozygous for ALDH2*2 exhibits a significantly high blood acetaldehyde level after low to moderate consumption of alcohol, which causes facial flushing, tachycardia and other unpleasant symptoms. Nevertheless, the amount of daily alcohol intake and total intake among Japanese individuals heterozygous for ALDH2*2 tends to increase (Enomoto et al., 1991). These results and our findings suggest that increase of alcohol intake is caused by induction of MAOS, especially CYP2E1 and MAOS plays an important role in acetaldehyde metabolism.

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    Acknowledgments

    We thank Dr. K. Furuichi and Miss A. Tominaga for their generous support throughout this study.

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    Footnotes

    • Send reprint requests to: Yoshihiko Funae, Ph.D., Labolatory of Chemistry, Osaka City University Medical School, 1–4-54 Asahi-machi, Abeno-ku, Osaka 545 Japan.

    • Abbreviations:
      P450
      cytochrome P450
      SDS-PAGE
      sodium dodecyl sulfate polyacrylamide gel electrophoresis
      3-MC
      3-methylcholanthrene
      PB
      phenobarbital
      MAOS
      microsomal acetaldehyde-oxidizing system
      MEOS
      microsomal ethanol-oxidizing system
      DLPC
      dilauroylphosphatidylcholine
      ALDH
      acetaldehyde dehydrogenase
      • Received July 2, 1996.
      • Accepted October 1, 1996.
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    1. JPET February 1, 1997 vol. 280 no. 2 527-532
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