ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 193, No. 2, April 1, pp. 551-559, 1979

The Effect of Pargyline on the Metabolism of Ethanol Acetaldehyde by Isolated Rat Liver Cells1 ARTHUR Department

of Biochemistry,

I. CEDERBAUM

AND

ELISA

DICKER

Mount Sinai School of Medicine of the City University New York, New York 10029

Received

March 8, 1978; revised

September

and

of New York,

1, 1978

The effect of pargyline on the uptake of acetaldehyde (in the presence of pyrazole) by isolated rat liver cells was studied after incubating the liver cells for 0, 10,30,45, and 60 min with 0.40, 1.30, and 2.6 InM pargyline. Without any incubation period, pargyline had no effect on acetaldehyde uptake. With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. This suggests the possibility that pargyline is metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action. Pargyline was also a more effective inhibitor of the uptake of lower concentrations of acetaldehyde, e.g., 0.167 mM, than of higher concentrations (1.0 mM) of acetaldehyde, especially after short incubation periods or when pyrazole was omitted from the reaction medium. After a 20- to 30-min incubation period, pargyline inhibited the control rate of ethanol oxidation by the liver cells, as well as the accelerated rate of ethanol oxidation found in the presence of pyruvate or an uncoupling agent. Pargyline had no effect on hepatic oxygen consumption. During ethanol oxidation, a time-dependent release of acetaldehyde into the medium was observed. Pyruvate, by increasing the rate of ethanol oxidation, increased the output of acetaldehyde five- to tenfold. Pargyline increased the output of acetaldehyde two- to threefold, despite decreasing the rate of ethanol metabolism by the liver cells. These data indicate that pargyline inhibits the low K, aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. Although most of the acetaldehyde generated during the oxidation of ethanol is removed by the liver cells in an effective manner, changes in the activity of aldehyde dehydrogenase or the rate of acetaldehyde generation significantly alter the hepatic output of acetaldehyde.

Acetaldehyde, the initial metabolite produced during the oxidation of ethanol, is rapidly metabolized in the liver. Several NAD+-dependent aldehyde dehydrogenases exist in the cytosolic, microsomal, and mitochondrial fractions of the liver cell, which are capable of oxidizing acetaldehyde (l-5). Recent reports have indicated that most of the acetaldehyde produced during ethanol oxidation is metabolized by a mitochondrial enzyme with a high affinity for acetaldehyde (2, 3, S-10). Other NAD+-dependent enzymes, with a low affinity for acetaldehyde ’ These studies were supported by a Research Scientist Career Development Award (5K02-AA 00003-03) from the National Institute on Alcohol Abuse and Alcoholism and Mount Sinai Alcohol Research Center Grant AA 03508-01

(K, of about 1 mM), exist in the cytosol, endoplasmic reticulum, and mitochondrial matrix and outer membrane fractions (1, 2, 4, 11). The detailed studies of Parrilla et al. (10) showed that most of the acetaldehyde is oxidized within the mitochondria when the concentration of acetaldehyde is below 0.4 mM. Above this concentration, cytosolic oxidation of acetaldehyde assumes a significant role in acetaldehyde oxidation. The classic inhibitor of aldehyde dehydrogenase is tetraethylthiuram disulfide (disulfiram); its administration results in elevated acetaldehyde blood levels after ethanol ingestion (12,13). However, disulfiram lacks specificity (14), is inhibitory toward other enzyme systems as well, e.g., NAD+dependent mitochondrial oxygen consump551

000%9861/79/040551-09$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tion (15) or dopamine-P-hydroxylase (14), and interferes with the metabolism of several drugs (16). Pargyline, a monoamine oxidase inhibitor, is also known to elevate blood acetaldehyde levels in mice and rats metabolizing ethanol (1’7, 18). Pargyline in vivo, but not in vitro, was shown to inhibit aldehyde dehydrogenase activity in mouse liver homogenates (18). In rats, pargyline inhibited the high-affinity mitochondrial aldehyde dehydrogenase, as determined with propionaldehyde and formaldehyde as substrates (19,20). In these reports, aldehyde dehydrogenase activities were measured in disrupted cellular fractions in the presence of externally added NAD+. In the present article the effect of pargyline in vitro on the oxidation of acetaldehyde and ethanol by intact liver cells was evaluated. Pargyline, only after an incubation period with the liver cells, was found to inhibit the metabolism of acetaldehyde. This inhibition was associated with an accumulation of acetaldehyde during ethanol metabolism, as well as a decrease in the rate of ethanol oxidation. METHODS Isolated rat liver cells were prepared from fed, male Sprague-Dawley rats, weighing about 250 to 350 g, as previously described (21). The cells were washed twice and suspended in Hanks’ buffer-l0 rnM sodium phosphate, pH 7.4-2.5% fatty acid-free bovine serum albumin. Trypan blue exclusion by the isolated liver cells was routinely >90%, whereas leakage of lactic dehydrogenase was 110%. Protein was determined by the method of Lowry et al. (22). Oxygen consumption was assayed for 3 to 5 min at 37°C using a Clark oxygen eIectrode and Yellow Springs oxygen monitor. The oxidation of acetaldehyde by isolated liver cells was assayed using procedures similar to those previously described for acetaldehyde oxidation by isolated mitochondria (23). Plastic 25ml Erlenmeyer flasks containing Hanks’ phosphate-albumin buffer and liver cell protein concentrations varying from 5 to 20 mg of protein were kept on ice prior to the addition of acetaldehyde. Where indicated, the liver cells were incubated with pargyline for the indicated time period at 37°C and then placed in an ice bucket. Pyrazole was added to a final concentration of 3 mM and the reaction was initiated by the addition of acetaldehyde (final volume of 3.0 ml and final acetaldehyde concentrations as indicated under Results). The flasks were immediately closed with serum caps

AND DICKER containing center-well cups and transferred to the 3’7°C water bath. The reaction was terminated after 5 min by the addition of 1 ml of trichloroacetie acid (final concentration of 4.5%) through the serum cap. The flasks were removed and 0.3 ml of 15 mM semicarbazide HCl in 180 mM potassium phosphate, pH 7.4, was injected into the center well. After an overnight diffusion period at room temperature, aliquots of the center well were diluted with H20 to 3.0 ml and the absorbance at 224 nm was determined. A millimolar extinction coefficient of 9.41 was utilized to calculate the remaining concentration of acetaldehyde. Zero-time controls contained the acid added before the acetaldehyde. Control experiments also included liver cells incubated with pargyline in the absence of acetaldehyde. Incubations were carried out in triplicate. The oxidation of ethanol was carried out, under air, in stoppered 25ml plastic Erlenmeyer flasks at 37°C. The reaction mixture consisted of Hanks’-phosphatealbumin buffer, 20 to 30 mg of liver cell protein and ethanol at a final concentration of 12.5 mM in a final reaction volume of 3.0 ml. The liver cells were incubated with the indicated concentrations of pargyline (or buffer in the case of no pargyline) for 20 to 30 min. The appropriate addition, e.g., pyruvate, glutamine or FCCP* was then added and the reaction was initiated by the addition of ethanol. The reaction was terminated after 60 min by the addition of trichloroacetic acid (final concentration of 4.5%). The remaining ethanol was determined in aliquots of the supernatant obtained from a IO-min centrifugation at 12,OOOg, using 1 mM NAD+, 10 units of yeast alcohol dehydrogenase, and a buffer consisting of 75 mM sodium pyrophosphate, 25 mM semicarbazide HCl, and 10 mM glycine, pH 9.6. Zero-time controls contained the acid added before the ethanol. Experiments involving the accumulation of acetaldehyde during ethanol oxidation were carried out as described above except that after the addition of ethanol, the flasks were closed with serum caps containing center-well cups. After 60, 120, or 180 min, the reaction was terminated with acid through the serum cap, semicarbazide was injected into the center well, and after an overnight diffusion period the concentration of acetaldehyde was determined as described above. Source of materials. Pargyline was obtained from Saber Laboratories, Morton Grove, Illinois. Pyrazole was ohtained from Pfaltz and Bauer, Inc., Flushing, New York. Collagenase (CLS Type II) was from Worthington Biochemicals, Freehold, New Jersey. FCCP, antimycin, and yeast alcohol dehydrogenase were from Boehringer Mannheim Biochemicals, Indianapolis, Indiana. Rotenone, oligomycin, hyaluronidase, NAD+, and fatty acid-free bovine serum * Abbreviation used: FCCP, carbonyl p-trifluoromethoxyphenylhydraaone.

cyanide

553

EFFECTOFPARGYLINEONACETALDEHYDEANDETHANOLMETABOLISM albumin3 were from Sigma Chemical Co., St. Louis, Missouri. Statistics. All values refer to means 2 standard errors of the mean. Statistical analysis was performed by Student’s t test. Results were considered significant when p < 0.05. In most casesp values were considerably lower. RESULTS

Acetaldehyde Uptake by Isolated Rat Liver Cells The addition of acetaldehyde to a suspension of rat liver cells resulted in the rapid removal of acetaldehyde from the incubation medium. Acetaldehyde uptake was linear with time for at least 15 min and was proportional to the amount of liver cell protein up to at least 20 mg of protein per flask. To allow accurate measurements of acetaldehyde uptake at a constant reaction period of 5 min the amount of liver cell protein was varied from 5 (lowest acetaldehyde concentration) to 20 (highest acetaldehyde concentration) mg. Since acetaldehyde may be reduced to ethanol via alcohol dehydrogenase, acetaldehyde uptake was studied in the absence and presence of pyrazole, a specific inhibitor of alcohol dehydrogenase. In the absence of pyrazole, the rate of acetaldehyde uptake was dependent on the concentration of acetaldehyde in the range of 0.021 to 1.0 mM (Fig. 1). In the presence of 3 InM pyrazole (a concentration which depressed the oxidation of ethanol by about 90%), acetaldehyde uptake was dependent on the concentration of acetaldehyde in the range of 0.021 to 0.33 mM but then began to gradually level off (Fig. 1). mle was particularly effective in lowering the rate of acetaldehyde uptake at higher concentrations of acetaldehyde. The percentage decrease in acetaldehyde uptake produced by pyrazole was 3,0,12,14,27,35, and 42% at acetaldehyde concentrations of 0.021, 0.042, 0.083, 0.167, 0.33, 0.67, and 1.0 mM, respectively. The effect of pyrazole was statistically significant at the latter three concentrations of acetaldehyde. These data 3 Inhibition by pargyline of acetaldehyde and ethanol metabolism was observed only with thefatty acid-free bovine serum albumin (Sigma A 6663) and not fraction V albumin.

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suggest that at the lower concentrations of acetaldehyde, most of the acetaldehyde is metabolized by oxidative pathways that are insensitive to pyrazole, whereas at the higher concentrations, the increment in the rate of acetaldehyde uptake primarily reflects reduction of the acetaldehyde to ethanol, as cytosol enzymes, sensitive to pyrazole, become major factors. In the presence of pyrazole, acetaldehyde (0.167 to 1.0 mM) uptake was inhibited 65 to 75% (n = 4) by inhibitors of mitochondrial oxygen consumption such as rotenone (0.005 mM), antimycin (0.005 mM), or oligomycin (0.005 mM). The pyrazole-insensitive rate of acetaldehyde oxidation (10 to 13 mnoUn/mg of liver cell protein, corresponding to a rate of 2.1 to 2.7 ymol/min/g of liver wet wt) is in agreement with other reports where acetaldehyde uptake rates varying from 1.5 to 3.5 ~moYmin/g of liver wet wt have been recorded (9, 10, 24-27). The Effect of Pargyline on Acetaldehyde Uptake It has been reported that the direct addition of 0.5 or 1.0 mu pargyline to liver homogenates had no effect on acetaldehyde metabolism (18,20). In preliminary experiments, we observed that up to 2 mM pargyline had no effect on state 4 or state 3 oxidation of acetaldehyde by isolated rat liver mitoehondria (unpublished observations). Since administration of pargyline

554

CEDERBAUM

in vivo results in inhibition of acetaldehyde metabolism, pargyline may be metabolized in viva to the effective inhibitor or some in vivo factor may be required for the action of pargyline (18, 28). Coprine, another inhibitor of aldehyde dehydrogenase, inhibited the low K, enzyme in vivo but not in vitro, suggesting that an active metabolite of coprine which was formed in vivo was the effective inhibitory agent (29). In in vivo experiments, strong elevations of blood acetaldehyde levels in mice were found after only 15 min of pretreatment with pargyline (18). Therefore, liver cells were incubated with pargyline or buffer for 0, 10, 30, 45, or 60 min at 37°C before initiating the reaction with acetaldehyde (final concentrations of 0.167, 0.33, 0.67, and 1.0 mM). Without any incubation period, pargyline had no effect on acetaldehyde uptake with all four concentrations of acetaldehyde tested (Figs. I

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,

0 f67mM

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AND DICKER

2a-d). With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. For example, with 0.167 mM acetaldehyde (Fig. 2a), the extent of inhibition of acetaldehyde uptake increased from 0 to 7, 19, 31, and 50% (0.4 mM pargyline) or from 3 to 18,42,49, and 56% (1.3 mM pargyline) when the incubation period of the liver cells with pargyline was increased from 0 to 10,30,45, and 60 min, respectively. The inhibition of acetaldehyde uptake by pargyline was dependent on the concentration of pargyline as well as the time of incubation of the liver cells with pargyline. In addition, the inhibition of acetaldehyde uptake by pargyline, especially at “short” incubation periods, was dependent on the concentration of acetaldehyde as pargyline was a more effective inhibitor of the uptake of 0.167 and 0.33 InM acetaldehyde than of 1.0

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I 2.6 OF or the possibility that pargyline and acetaldehyde compete for aldehyde dehydrogenase. Since pargyline inhibits the oxidation of low concentrations of acetaldehyde, i.e., concentrations which are likely to arise during the metabolism of ethanol, it is to be expected that pargyline would increase the levels of acetaldehyde which accumulate during the oxidation of ethanol. Indeed, pargyline doubled or tripled the levels of acetaldehyde released from the liver cells during ethanol oxidation. Associated with these increased levels of acetaldehyde is an inhibition of ethanol metabolism. Thus, in contrast to pyruvate, pargyline increased the output of acetaldehyde from the liver cells even though the rate of ethanol metabolism was decreased by pargyline. Pargyline was reported to have no effect

558

CEDERBAUM

on the activity of alcohol dehydrogenase (20). Pargyline did not affect hepatic oxygen consumption, suggesting that pargyline does not interfere with the reoxidation of reducing equivalents. It is therefore likely that the inability to remove acetaldehyde effectively in the presence of pargyline decreases the rate of ethanol oxidation. In the perfused liver, low concentrations of acetaldehyde retarded the rate of ethanol elimination (25). In view of the kinetic characteristics of alcohol dehydrogenase, which favor the backward direction, the inhibition of ethanol metabolism by pargyline probably reflects the reduction of the accumulated high levels of acetaldehyde back to ethanol. Ethanol oxidation is assayed as the disappearance of ethanol from the medium. In addition, the accumulation of high levels of acetaldehyde could interfere with the transport of reducing equivalents generated from the alcohol dehydrogenase reaction into the mitochondria via substrate shuttles (34). In vivo pargyline was shown to decrease the rate of blood ethanol clearance in mice and rats by 40 to 60% (18, 19). As shown in Fig. 3, identical results can be obtained with isolated rat liver cells. The administration of other inhibitors of aldehyde dehydrogenase, e.g., disulfiram (18, 35, 36), coprine (29), and n-butyraldoxime (37), is also associated with an accumulation of acetaldehyde and a decreased rate of ethanol oxidation. During the oxidation of ethanol by liver cells, detectable levels of acetaldehyde were found to accumulate in the medium. However, it is apparent that the liver cells effectively remove most of the acetaldehyde that is generated by the, oxidation of ethanol. For example, after a 60-min incubation period an ethanol oxidation rate of 375 nmollmg of liver cell protein is associated with an acetaldehyde accumulation of about 2 to 3 nmol/mg of liver cell protein (about 20 PM), i.e., less than 1% of the acetaldehyde produced from ethanol metabolism is released into the medium. It can be calculated that the rate of acetaldehyde metabolism by the liver cells exceeds the rate of acetaldehyde generation. In the presence of pyrazole, acetaldehyde is metabolized at a rate of about 10 nmol/min/mg of liver cell

AND DICKER

protein or 0.60 pmolih/mg of liver cell protein which is greater than the acetaldehyde generation rate (ethanol oxidation) of about 0.38 ymol/h/mg of liver cell protein. However, these data suggest that in isolated liver cells, the acetaldehyde oxidation rate is not far in excess over the acetaldehyde generation rate. In fact, when the acetaldehyde generation rate is accelerated, e.g., a doubling of the rate of ethanol metabolism in the presence of pyruvate, up to tenfold greater levels of acetaldehyde are released into the medium (Fig. 4). In perfusion studies, Lindros (24) reported that raising the concentration of pyruvate from 0.1 to 0.5 mM, increased acetaldehyde output sevenfold. These data support the concept that there appears to be a good balance between the formation and elimination rates of acetaldehyde, i.e., hepatic output of acetaldehyde is low under normal conditions (36). However, moderate changes in aldehyde dehydrogenase activity or acetaldehyde generation can significantly alter the hepatic output of acetaldehyde (8, 24, 33, 36, 38) (Figs. 4 and 5). Acetaldehyde uptake is inhibited up to 60% by pargyline (Fig. 2). This inhibition is associated with an increased release of acetaldehyde into the medium during ethanol metabolism (Fig. 5). Since pargyline is an effective inhibitor of acetaldehyde metabolism only after an incubation period with the liver cells, it appears that either pargyline is required to be metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action, The fact that pargyline inhibits ethanol metabolism, increases acetaldehyde accumulation, and inhibits the oxidation of low concentrations of acetaldehyde, i.e., concentrations which are likely to arise during ethanol oxidation, suggests that pargyline specifically inhibits the low K, aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. REFERENCES

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