Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 234, No. 1, October, pp. 187-196, 1984

Inhibition of the Oxidation of Acetaldehyde and Formaldehyde by Hepatocytes and Mitochondria by Crotonaldehyde’ ELISA DICKER

AND

ARTHUR I. CEDERBAUM2

Department of Biochemistry and Alcohol Research Center, Mount Sinai School of Medicine of the City University of New York, 1 Gustave Levy Place, New York, New York 10029 Received April

16, 1984

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 PM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than lo-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-K, mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme. 8 1~ Academic press, I,,~. The crotonol-crotonaldehyde system has been shown to be an effective aid in studying the mechanisms responsible for the effects of ethanol on hepatic functions (1). Crotonol reacted with liver alcohol dehydrogenase at about the same rate as ethanol, and both alcohols altered the cytosolic redox state to a similar degree; however, only ethanol produced reduction of the mitochondrial redox state (1, 2). 1 These studies were supported by USPHS Grants AA-03312 and AA-03608, and Research Career Development Award 5K02-AA-00003 (AIC) from the National Institute on Alcohol Abuse and Alcoholism. ‘To whom correspondence should be addressed.

This difference in effects on the mitochondrial redox state was related to the fact that, relative to acetaldehyde, crotonaldehyde was a poor substrate for mitochondrial aldehyde dehydrogenase (2). Siew et al. (3) partially purified two aldehyde dehydrogenases from rat liver mitochondria; one corresponded to the lowKm matrix aldehyde dehydrogenase, while the other corresponded to the high-K, outer-membrane or intermembrane-space aldehyde dehydrogenase. The former enzyme oxidized crotonaldehyde at a rate of about 10% of that found with acetaldehyde, while the latter enzyme oxidized crotonaldehyde at a rate of about 45% of

187

0003-9861/84 $3.00 Copyright All rights

0 1984 by Academic Press. Inc. of reproduction in any form reserved.

188

DICKER AND CEDERBAUM

that of acetaldehyde (3). We found that intact rat liver mitochondria oxidized crotonaldehyde at a rate of about 10% of that of acetaldehyde, whereas disrupted mitochondria, in the presence of NAD+, oxidized crotonaldehyde at about 25% of the rate found with acetaldehyde (2). The ability of acetaldehyde to affect the rate of glucose production from various precursors (4, 5) could not be duplicated by crotonaldehyde (2). This suggested that the effects of acetaldehyde required metabolism by the low-K, mitochondrial aldehyde dehydrogenase, rather than a direct action of acetaldehyde (2, 5). During the course of these experiments, it was noted that acetaldehyde had no effect on glucose production when crotonaldehyde was also present in the reaction system. Cyanamide, a potent inhibitor of the low-K, aldehyde dehydrogenase (68), also prevented the effects of acetaldehyde on glucose production by preventing the oxidation of acetaldehyde (5). This suggested the possibility that crotonaldehyde, similar to cyanamide, might prevent the effects of acetaldehyde by inhibiting the oxidation of acetaldehyde. Therefore, studies were carried out to determine the effect of crotonaldehyde on the oxidation of acetaldehyde by hepatocytes, intact mitochondria, and disrupted mitochondrial fractions (the latter taken as a reflection of aldehyde dehydrogenase activity when NAD+ was added to the system). Formaldehyde was included in this study because of recent interest in the toxicity of this compound; the fact that it is a product of cytochrome P-450catalyzed N-demethylation reactions; and the presence of two enzyme systems, the mitochondrial, and a specific glutathionedependent, cytosolic formaldehyde dehydrogenase, which play a role in formaldehyde oxidation (9-12). MATERIALS

AND METHODS

Hepatocytes were isolated from 24-h-fasted male Sprague-Dawley rats (250-350 g) as previously described (2, 5). The hepatocytes were suspended in Krebs-Ringer-bicarbonate-lo mM phosphate buffer, pH 7.4, supplemented with 1.50% fatty acid-free bovine serum albumin. The buffer was saturated

with a mixture of 95% 02-5s COs. Cell viability was routinely better than 90%. Protein was determined by the method of Lowry et al (13). The oxidation of acetaldehyde or formaldehyde by hepatocytes was assayed at 37°C in 25-ml polycarbonate Erlenmeyer flasks containing the above buffer and about 5-10 mg liver cell protein in a final volume of 3 ml. The cells were incubated with 3 mM pyrazole for 5 min prior to initiating the reaction by the addition of acetaldehyde or formaldehyde. The pyrazole was included to prevent reduction of the aldehyde to the alcohol via alcohol dehydrogenase. For the formaldehyde experiments, 0.2 mM methionine was also included in the reaction mixture. The appropriate concentrations of crotonaldehyde were added immediately before the addition of the aldehyde substrate. Reactions with formaldehyde were terminated after 5 min by the addition of 1 ml trichloroacetic acid (TCA) to a final concentration of 4.5% w/v. The samples were centrifuged, and the remaining formaldehyde was determined in aliquots of the supernatant by the method of Nash (14). Zero-time controls contained the acid added before the cells. The uptake of formaldehyde was calculated by subtracting the remaining formaldehyde from the initial zero-time value (15). Reactions with acetaldehyde were terminated at varying time intervals (1 to 10 min) by the addition of HCl to a final concentration of 0.25 N. The flasks were sealed with tight rubber serum stoppers and incubated at 60°C for 20 to 30 min. The remaining acetaldehyde was detected by head-space gas chromatography (16). Zero-time controls contained the HCl added before the cells, and the differences between the remaining acetaldehyde and the initial zero-time values was used to calculate the uptake of acetaldehyde. The accumulation of acetaldehyde during the metabolism of ethanol was determined in a reaction system containing the Krebs buffer, 10 mM ethanol, and about 20 mg liver cell protein in a final volume of 3 ml. In some cases, 5 mM pyruvate was added to increase the rate of ethanol oxidation. Crotonaldehyde was added immediately prior to initiating the reaction with ethanol. Reactions were terminated after 5, 10, and 15 min by the addition of HCI, and the concentration of acetaldehyde was determined as described above. All values were corrected for zero-time controls. Rat liver mitochondria were prepared as previously described (17). The oxidation of acetaldehyde or formaldehyde by intact mitochondria was assayed at 37°C in a reaction system containing 0.25 M mannitol, 3.3 mM MgC12, 10 mM potassium phosphate, pH ‘7.4, 3.3 mM ADP, and 3 to 5 mg mitochondrial protein in a final volume of 3 ml. Reactions were initiated by the addition of acetaldehyde or formaldehyde (final substrate concentrations of either 0.2 or 1 mM), and were terminated after 3 to 5 min by

CROTONALDEHYDE

INHIBITION

OF OXIDATION TABLE

189

OF ALDEHYDES

I

OXIDATION OF CROTONALDEHYDE AND ACETALDEHYDE, AND THE EFFECF OF CROTONALDEHYDE ON ACETALDEHYDE OXIDATION BY MITOCHONDRIAL ALDEHYDE DEHYDROGENASE’ Rate of aldehyde Concentration of crotonaldehyde bM) 0 0.01 0.02 0.05 0.10 0.20 0.5 1.0

Crotonaldehyde

1.07 2.02 2.20 2.83 3.91 4.96 4.15

rf: 0.65 + 0.27 + 0.28 + 0.72 f 1.01 k 1.62 f 0.24

oxidation

Effect of crotonaldehyde (%I

Acetaldehyde 24.14 16.69 13.59 8.27 7.78 7.27 6.31 6.46

& + + f + + + +

(nmol min-’

2.44 3.40 1.78 1.09 1.46 1.28 0.91 1.58

-31 -44* -66;’ -68** -7Of’ -74** -73**

mg mitochondrial

Net acetaldehyde 24.14 15.62 11.57 6.07 4.95 3.36 1.35 2.31

+ f f i 5 f * f

2.49 3.59 1.85 0.83 0.74 0.27 0.71 1.82

protein-‘) Effect of crotonaldehyde 6) -35 -52* -75** -79** -86** -94** -g(-J**

a The oxidation of varying concentrations of crotonaldehyde (0.01 to 1 mM), and of 0.2 mM acetaldehyde in the absence and presence of crotonaldehyde, by disrupted mitochondria was assayed as described under Materials and Methods. The “Net” column was calculated from the differences between the rates in the presence of acetaldehyde minus the rates given by crotonaldehyde alone. Results are from three experiments. l P < 0.05. ** P < 0.005.

the addition of TCA3 (formaldehyde) or HCl (acetaldehyde). Analyses and calculations were carried out as described above for the hepatocytes. Oxygen consumption was assayed at 30°C using a Clark oxygen electrode and a Yellow Springs oxygen monitor. The reaction system was the same as described for the oxidation of the aldehydes, except for the omission of ADP. Substrates utilized were either 10 mM succinate or 10 mM glutamate plus 3 mM malate. State 3 respiration was initiated by the addition of 0.25 mM ADP. Aldehyde dehydrogenase activity was .determined in a reaction mixture containing 50 mM sodium pyrophosphate, pH 9,1 mM NAD+, 0.01 mM rotenone, and about 2 mg mitochondrial protein in a final volume of 3 ml (2, 7). The mitochondria were disrupted with sodium deoxycholate (final concentration, 0.25%). Reactions were initiated by the addition of acetaldehyde (or, in some cases, crotonaldehyde), and the absorbance change at 340 nm was determined. The activity of the glutathione-dependent formaldehyde dehydrogenase was determined using a 100,OOOg soluble supernatant fraction prepared by differential centrifugation in 0.25 M sucrose, 0.01 M Tris-HCl, pH 7.4, 0.001 M EDTA. The soluble supernatant fraction was dialyzed overnight against a 200 mM sodium pyrophosphate buffer, pH 8, to 3 Abbreviation

used: TCA, trichloroacetic

acid.

remove glutathione. The oxidation of formaldehyde was assayed, in the absence or presence of 1 mM glutathione, in a reaction mixture containing 100 mM pyrophosphate buffer, pH 8, 0.67 mM NAD+, 3 mM pyrazole, and about 10 mg of supernatant protein in a final volume of 3 ml. Reactions were initiated by the addition of formaldehyde (0.1 to 2 mM), and the production of NADH was recorded at 340 nm. The glutathione-dependent formaldehyde dehydrogenase activity was taken as the net difference in the rates in the absence and presence of glutathione. All values represent the means * SEM. Statistical analysis was performed by Student’s t test. RESULTS

Oxidation of acetaldehgde lq disrupted mitochondm’al fractions. As a measure of mitochondrial aldehyde dehydrogenase activity, the oxidation of acetaldehyde by disrupted mitochondria in the presence of excess NAD+ was determined. At an acetaldehyde concentration of 0.2 ITIM, the rate of production of NADH was about 24 nmol min-’ mg mitochondrial protein-l (Table I). In contrast, crotonaldehyde, over a substrate range varying from 0.01 to 1 mM, was oxidized at a rate that was only 10 to 20% of that found with acetaldehyde

190

DICKER AND CEDERBAUM

0

2

1 C~r~~~ntr.atlon

of

Acetaldehyde

3 I mMI

FIG. 1. The effect of 0.1 mM crotonaldehyde on the oxidation of varying concentrations of acetaldehyde by mitochondrial aldehyde dehydrogenase. Assays were carried out using deoxycholate-disrupted mitochondria as described under Materials and Methods. Results are from four experiments. The percentage inhibition by 0.1 mM crotonaldehyde was 80, 69, 52, 38, and 27% at acetaldehyde concentrations of 0.2, 0.5, 1, 2, and 3 mM, respectively. 0, Absence of crotonaldehyde; 0, 0.1 mM crotonaldehyde.

(Table I). However, crotonaldehyde was an effective inhibitor of the oxidation of acetaldehyde; significant inhibition was

observed at a crotonaldehyde concentration of 0.01 mM, and maximum inhibition was found at a crotonaldehyde concentration of about 0.2 mM (Table I). To determine the kinetics of inhibition by crotonaldehyde, the concentration of acetaldehyde was varied over the range 0.2-3 mM, in the absence and presence of 0.1 mM crotonaldehyde. Crotonaldehyde was a more effective inhibitor of the oxidation of low concentrations than of high concentrations of acetaldehyde (Fig. 1). Regression analysis of a Hanes-Woolf plot (s/z, vs. V) of the data of Fig. 1 revealed that the K, and V,,,,, for acetaldehyde were 0.022 mM and 32 nmol min-’ mg protein-‘, respectively, in the absence of crotonaldehyde, and 0.54 mM and 31 nmol min-l mg protein-’ in the presence of crotonaldehyde. Thus, crotonaldehyde was a competitive inhibitor of the oxidation of acetaldehyde. The Ki for crotonaldehyde was about 5 PM. Oxidation of acetaldehyde and fmldehyde by intact mitochondria. Intact rat liver mitochondria oxidized acetaldehyde and formaldehyde at similar rates (Table II). The rate of oxidation of the two aldehydes did not increase when the concentration of aldehyde was elevated five-

TABLE II EFFECT OF CROTONALDEHYDE ON THE OXIDATION OF ACETALDEHYDE AND FORMALDEHYDE BY INTACT RAT LIVER MITOCHONDRIA~ Rate of oxidation

of aldehyde

(nmol min-’

mg mitochondrial

Acetaldehyde Concentration of crotonaldehyde (mM) 0

0.1 0.2 0.5 1.0

Formaldehyde

0.2 mM

18.40 3.32 3.96 2.76 1.64

f f f + f

1.0 mM Effect (%)

Rate 2.20 1.00 1.44 1.04 1.04

-82’ -78’ -95’ -910

protein-‘)

Effect (%)

Rate 18.80 12.76 10.00 8.84 8.28

f f + f f

1.92 2.24 0.36 2.60 1.84

1.0 mM

0.2mM

-32” -47” -53.’ -66..

Effect (%)

Rate 18.28 3.60 1.83 1.93 2.78

f f + + f

0.79 1.11 1.23 1.04 1.09

-8O* -90’ -89* -8~5~

’ The oxidation of acetaldehyde or formaldehyde (0.2 or 1 mM final substrate concentrations) of the indicated concentrations of crotonaldehyde, as described under Materials and Methods. crotonaldehyde. Reeults are from four (acetaldehyde) or six (formaldehyde) experiments. l P < 0.001. ** P < 0.02.

Effect (%)

Rate 22.98 8.95 5.24 3.94 4.78

k + * k f

2.05 2.76 1.70 1.86 1.36

-61* -77’ -88* -79’

was assayed in the presence Effect refers to the effect of

CROTONALDEHYDE

INHIBITION

OF OXIDATION

OF ALDEHYDES

191

TABLE III fold (0.2 to 1 mM, Table II), suggesting metabolism by the low-K, mitochondrial EFFECT OF CROTONALDEHYDE ON MITOCHONDRIAL aldehyde dehydrogenase. The addition of OXYGEN CONSUMPTION” crotonaldehyde resulted in a marked inhibition of the oxidation of acetaldehyde Rate of oxygen consumption and formaldehyde; significant inhibition (natoms oxygen min-’ mg mitochondriai protein-‘) was observed at a crotonaldehyde concentration of 0.1 mM (Table II). CrotonaldeGlutamate hyde inhibited the oxidation of 0.2 and 1 Succinate + malate Concentration mM formaldehyde to similar extents (Taof ble II). However, the inhibition by croton- crotonaldehyde State State State State aldehyde of acetaldehyde oxidation was 4 4 3 3 (mM) greater at 0.2 than at 1 InM acetaldehyde, i.e., increasing the concentration of acet0 23.3 122.0 23.3 118.6 aldehyde relieved the inhibition by cro0.25 30.4 114.9 23.4 114.9 tonaldehyde. The ineffectiveness of form0.5 29.7 114.9 22.4 110.6 1.5 21.7 25.4 97.6 33.9 aldehyde, as opposed to the ability of the same concentration of acetaldehyde to ‘Oxygen uptake in the presence of ADP (State 3) relieve the inhibition produced by crotonaldehyde, probably reflects the fact that, and after utilization of ADP (State 4) was assayed relative to acetaldehyde, formaldehyde is as described under Materials and Methods. Results are from two experiments. a poor substrate for the low-K, aldehyde dehydrogenase (15). To determine if the effect of crotonaldehyde on the oxidation of acetaldehyde presence of pyrazole (to prevent reduction and formaldehyde was relatively specific of the aldehydes to the alcohols via alcohol for the oxidation of aldehydes or reflected dehydrogenase), hepatocytes oxidized 0.2 an inhibitory action on the mitochondrial or 1 mM acetaldehyde at rates of about respiratory chain (which would prevent 10 nmol min-’ mg cellular protein-’ (Table reoxidation of the NADH produced in the IV). Crotonaldehyde inhibited acetaldealdehyde dehydrogenase reaction), the ef- hyde oxidation by the hepatocytes, with fect of crotonaldehyde on the oxidation of the extent of inhibition being greater at other mitochondrial substrates was stud- 0.2 mM than at 1 mM acetaldehyde (Table ied. Table III shows that crotonaldehyde, IV). The results on the sensitivity of acat concentrations (0.25 or 0.5 mM) that etaldehyde oxidation by hepatocytes to inhibited the oxidation of acetaldehyde crotonaldehyde were similar to the results and formaldehyde by >80%, had no effect on the sensitivity of acetaldehyde oxidaon the State 4 and State 3 rates of oxi- tion by mitochondria and disrupted midation of either succinate or glutamate. tochondria to crotonaldehyde, which probCrotonaldehyde (0.25 mM) had no ef- ably reflects the fact that, in hepatofect on the oxidation of P-hydroxybutyrate, cytes, most of the acetaldehyde is oxidized an NAD+-dependent substrate (data not by the low-K, aldehyde dehydrogenase shown). Some inhibition of State 3 respi- (19-22). Figure 2 shows an acetaldehyde subration was noted at high concentrations of crotonaldehyde, which probably reflects strate concentration curve in the absence direct toxic effects by high levels of alde- and presence of 0.2 or 0.5 mM crotonalhydes on the respiratory chain (17, 18). dehyde. Crotonaldehyde was more effecThus, at low concentrations, crotonaldetive in inhibiting the oxidation of lower hyde does not appear to be an inhibitor than of higher concentrations of acetalor an uncoupler of the mitochondrial re- dehyde (Fig. 2). Regression analysis of a spiratory chain. Hanes-Woolf plot of the data in Fig. 2 indicated that Km values for acetaldehyde Oxidation qf acetaldehgde and fmaldehyde by isolated hepatocytes. In the were 17, 210, and 370 PM, while V,,,

DICKER

AND

CEDERBAUM

TABLE EFFECT OF CROTONALDEHYDE

Concentration of crotonaldehyde ma

IV

ON THE OXIDATION

OF ACETALDEHYDE

Rate of oxidation

of acetaldehyde protein-‘)

BY ISOLATED

(nmol min-’

0.2 mM Acetaldehyde

0 0.05 0.10 0.20 0.50 1.0

9.55 7.65 6.03 4.09 3.22 2.16

+ f iz f + f

0.50 0.66 0.93 0.33 0.42 0.22

mg liver cell

1 mM Acetaldehyde

Effect (%I

Rate

HEPATOCYTES’

-20 -37* -57***

-@3*** -7.y.

Effect (%)

Rate 11.62 11.20 10.68 8.65 8.41 6.84

+ + f + + f

0.80 0.68 1.00 0.29 0.79 0.94

a The effect of crotonaldehyde on the oxidation of 0.2 or 1 mM acetaldehyde by isolated assayed as described under Materials and Methods. Results are from five experiments

-4 -8 -26** -2a* -41** hepatocytes

was

* P < 0.05. ** P < 0.01. ***p < 0.001.

values were 9.7, 10, and 9.2 nmol min-’ liver cell protein-‘, in the absence and presence of either 0.2 or 0.5 mM croton-

5 0i g O 8

25 Concentration

.5 of

.75 Acetaldehyde

1 I mM I

FIG. 2. The effect of 0.2 and 0.5 mu crotonaldehyde on the oxidation of varying concentrations of acetaldehyde by isolated hepatocytes. Results are from four experiments. The present inhibition by 0.2 mM crotonaldehyde was 56, 46, 29, and 11%. and by 0.5 mM crotonaldehyde was 80, 57, 44, and 30%, at acetaldehyde concentrations of 0.1, 0.2, 0.5, and 1 mM, respectively. 0, Absence of crotonaldehyde; 0, 0.2 mM crotonaldehyde; 0, 0.5 mM crotonaldehyde.

aldehyde, respectively. Thus, crotonaldehyde was a competitive inhibitor of the oxidation of acetaldehyde by intact hepatocytes. The Ki value for crotonaldehyde was about 20 PM. At concentrations of 0.2 or 1 mM, formaldehyde was oxidized at rates of 5 and 18 nmol min-’ mg cell protein-‘, respectively (Table V). Crotonaldehyde produced some inhibition of the oxidation of formaldehyde; however, this inhibition did not exceed about 40% and was lower than the inhibition found with intact mitochondria (Table V, to be compared with Table II). Formaldehyde, besides being a substrate for the low-K, mitochondrial aldehyde dehydrogenase, can also be oxidized by a cytosolic, glutathione-dependent formaldehyde dehydrogenase (9-12). Table V shows that cyanamide, a potent inhibitor of the low-K, aldehyde dehydrogenase (6-ES),produced about 25 to 35% inhibition of formaldehyde oxidation. This extent of inhibition was similar to the inhibition produced by crotonaldehyde, and suggested the possibility that both cyanamide and crotonaldehyde are inhibiting that portion of formaldehyde oxidation which occurs via the mitochondrial aldehyde dehydrogenase, with little effect on that

CROTONALDEHYDE

INHIBITION

OF OXIDATION

TABLE

193

OF ALDEHYDES

V

EFFECT OF CROTONALDEHYDE ON THE OXIDATION OF FORMALDEHYDE BY ISOLATED RAT LIVER CELLS Oxidation

Concentration of formaldehyde (mM) 0.2

1.0

Concentration of crotonaldehyde bM)

(-)

Cyanamide

0 0.05

5.07 k 0.22 4.71 f 0.21

0.10 0.20 0.50 1.00

3.77 3.46 3.40 2.97

0.26 0.23 0.31 0.27

0

18.32 + 0.80

0.05 0.10 0.20 0.50 1.00

15.04 12.35 11.90 11.69 11.09

+ f f f +

(+) Cyanamide Effect @)

Rate

+ + + +

of formaldehyde (nmol min-’ mg liver cell protein-‘)

0.49 0.54 0.80 0.67 0.89

-7 -26** -32; -338 -41* -

-1f.J’** -32* -358 -36* -39*

Effect @)

Rate 3.75 f 0.17 3.87 f 0.23 3.64 3.59 3.18 3.02

+ + + +

0.26 0.26 0.19 0.29

12.50 + 0.83 11.11 12.15 11.68 11.12 10.69

f f f + +

0.73 0.64 0.35 0.99 1.05

-3 -4 -15*** -1g*** -11 -3 -7 -11 -14

“The effect of crotonaldehyde on the oxidation of 0.2 or 1mM formaldehyde by isolated hepatocytes, in the absence and presence of 0.1 mM cyanamide, was assayed as described under Materials and Methods. Results are from five experiments. * P < 0.001.

** P < 0.005. *** 0.05 > P < 0.10.

portion of formaldehyde oxidation which occurs via the cytosolic formaldehyde dehydrogenase. Consistent with this possibility were the observations that crotonaldehyde did not significantly inhibit the oxidation of formaldehyde in the presence of cyanamide (Table V), i.e., once the lowK, mitochondrial pathway was inhibited by cyanamide, crotonaldehyde was no longer inhibitory. Cyanamide was previously shown to be without any effect on formaldehyde oxidation by the glutathionedependent formaldehyde dehydrogenase. Crotonaldehyde was found not to be a substrate for the formaldehyde dehydrogenase, nor did it affect the oxidation of formaldehyde at concentrations which nearly completely blocked mitochondrial oxidation of formaldehyde. The rate of glutathione-dependent oxidation of 0.2 mM formaldehyde by the 100,000~ soluble supernatant fraction was 6.7 nmol min-’ mg protein-’ in the absence of crotonaldehyde,

and 6.3 nmol min-’ mg protein-’ in the presence of 1 mM crotonaldehyde. A similar lack of effect by crotonaldehyde was noted at formaldehyde concentrations ranging from 0.1 to 2 InM (data not shown).

Eflect of crotonaldehyde cm the accumulaticm of acetaldehyde. The above experiments demonstrated that crotonaldehyde inhibited the oxidation of acetaldehyde added to hepatocytes or mitochondria. Experiments were carried out to determine if crotonaldehyde would also block the metabolism of acetaldehyde generated during the metabolism of ethanol. In the absence of crotonaldehyde, there was little or no accumulation of acetaldehyde over the 15-min time course of ethanol oxidation (Table VI; a short time course was utilized in these experiments to lower the removal of crotonaldehyde via oxidation). There was considerable accumulation of acetaldehyde when crotonaldehyde was present (Table VI).

194

DICKER

AND TABLE

CEDERBAUM VI

EFFECT OF CROTONALDEHYDE ON THE ACCUMULATION OF ACETALDEHYDE DURING THE OXIDATION OF ETHANOL BY ISOLATED HEPATOCYTES’ Accumulation Concentration of crotonaldehyde (mM)

Rate

0

0.22 + 0.04

0.2 0.4 0.6 1.0

1.10 2.16 2.37 3.09

of acetaldehyde

(nmol min-’

mg liver cell protein-‘)

(-) Pyruvate

+ + + +

0.19 0.22 0.25 0.50

(+) Pyruvate Effect (%) +400 +881 +997 +1304

Rate 3.11 + 0.84 4.73 5.08 5.52 6.09

+ 0.59 5~ 0.64 f 0.66 f 0.55

Effect 6) +52 +63 +77 +96

‘The accumulation of acetaldehyde, in the absence and presence of 5 mM pyruvate, during the oxidation of 10 mM ethanol was assayed as described under Materials and Methods. Effect refers to the effect of crotonaldehyde. Results are from three experiments,

for crotonaldehyde binding to the low-K, aldehyde dehydrogenase. The Km for acetaldehyde oxidation by intact mitochondria is generally below 10 PM (19-21), whereas that for formaldehyde oxidation is about 0.19 to 0.38 mM (15,25). The ability of increasing concentrations of acetaldehyde, but not the same concentrations of formaldehyde, to protect against the inhibition by crotonaldehyde (Table II) probably is due to acetaldehyde being a preferred substrate relative to formaldehyde. Recently, we have shown that acetaldehyde is an effective inhibitor of the oxidation of formaldehyde, whereas formaldehyde is a poor inhibitor of the DISCUSSION oxidation of acetaldehyde (15). As a consequence, acetaldehyde, but not formalResults in the current report confirm dehyde (at the concentrations utilized), that crotonaldehyde is a poor substrate can relieve the inhibition produced by for oxidation by the mitochondrial alde- crotonaldehyde. hyde dehydrogenase at pH 7.4 (1, 2). AlCrotonaldehyde inhibits the oxidation though a poor substrate for oxidation, of acetaldehyde by disrupted mitochoncrotonaldehyde effectively inhibits the ox- drial fractions, by intact mitochondria, idation of acetaldehyde and formaldehyde. and by intact hepatocytes. The inhibition Siew et al. (3) reported an apparent Km by crotonaldehyde is identical when comvalue for crotonaldehyde oxidation by the paring disrupted mitochondria with intact partially purified low-K, aldehyde dehy- mitochondria, e.g., 0.1 InM crotonaldehyde drogenase of about 32 PM. Crotonaldehyde produces 79% inhibition with disrupted is a competitive inhibitor of acetaldehyde mitochondria and 82% inhibition with inoxidation, with an apparent Ki of about 5 tact mitochondria (at 0.2 mM acetaldehyde to 20 PM, which is similar to the K,,, value substrate concentration). However, the

The accumulation of acetaldehyde which occurred in the presence of crotonaldehyde was about the same as that previously found in the presence of cyanamide (5). A significant increase in the accumulation of acetaldehyde occurred when pyruvate was added to the hepatocytes (Table VI). This probably reflects the ability of pyruvate to increase the rate of ethanol oxidation and, thus, of acetaldehyde production (23,24). Crotonaldehyde produced an additional increase in this already elevated acetaldehyde accumulation system (Table VI).

CROTONALDEHYDE

INHIBITION

inhibition with hepatocytes (3’7%) is less. This could reflect the oxidation and, hence, removal of crotonaldehyde by the cytosolic aldehyde dehydrogenase. We have previously shown that, unlike studies with mitochondria, where the rate of crotonaldehyde oxidation is only 10 to 15% that of acetaldehyde, the rate of crotonaldehyde oxidation by intact hepatocytes is 30 to 50% of that of acetaldehyde (2). Most of this crotonaldehyde oxidation by hepatocytes is apparently mediated by the cytosolic aldehyde dehydrogenase and not mitochondrial aldehyde dehydrogenase, since (a) cyanamide was a poor inhibitor of crotonaldehyde oxidation; and (b) the oxidation of crotonaldehyde was associated with an increase in the lactate/ pyruvate ratio, whereas the @hydroxybutyrate/acetoacetate ratio was not altered (2). Thus, crotonaldehydes causes reduction of the cytosolic, but not the mitochondrial, NAD/NADH redox ratio. In view of the cytosolic oxidation of crotonaldehyde, more crotonaldehyde is required to produce effective inhibition of acetaldehyde oxidation by hepatocytes than in studies with mitochondria. Very low levels of acetaldehyde are detected during the metabolism of ethanol, attesting to the rapid, efficient removal of acetaldehyde under normal conditions. Crotonaldehyde increases the accumulation of acetaldehyde more than lo-fold, suggesting that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, inhibits the oxidation of acetaldehyde generated by the metabolism of ethanol. It is also possible that crotonaldehyde may increase the accumulation of acetaldehyde by increasing the rate of ethanol oxidation (the rate of acetaldehyde generation) via reoxidation of the NADH produced in the alcohol dehydrogenase reaction (crotonaldehyde + NADH or enzyme-NADH complex - crotonol + NAD+ + enzyme). Indeed, pyruvate, which increases the rate of ethanol oxidation, also increases the accumulation of acetaldehyde ((23, 24), Table VI). Crotonaldehyde produced an increase in the accumulation of acetaldehyde even in the presence of pyruvate, indicating that impairment of

OF OXIDATION

OF ALDEHYDES

195

acetaldehyde oxidation plays a role in the increased accumulation of acetaldehyde during the oxidation of ethanol in the presence of crotonaldehyde. Formaldehyde, besides being a substrate for the low-K, mitochondrial aldehyde dehydrogenase, can also be oxidized by a glutathione-dependent, cytosolic formaldehyde dehydrogenase (9-12). Whereas crotonaldehyde is a potent inhibitor of mitochondrial oxidation of formaldehyde, it is without any effect on the activity of formaldehyde dehydrogenase. This is similar to results recently reported for cyanamide (15). Crotonaldehyde inhibits formaldehyde oxidation by intact hepatocytes, indicating that part of the formaldehyde oxidation occurs via the mitochondrial aldehyde dehydrogenase. In fact, the inhibition by crotonaldehyde is similar to the inhibition produced by cyanamide, and the inhibition by cyanamide and crotonaldehyde is not additive, suggesting inhibition of the same formaldehyde oxidizing enzyme system, i.e., the mitochondrial aldehyde dehydrogenase. Presumably, formaldehyde oxidation, which is insensitive to crotonaldehyde and cyanamide, reflects oxidation via formaldehyde dehydrogenase. In summary, crotonaldehyde has been shown to be a competitive inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, intact mitochondria, and intact hepatocytes. The fact that inhibition is competitive may be of value, since other commonly used inhibitors of aldehyde dehydrogenase (cyanamide, disulfiram) are irreversible inhibitors of the enzyme. REFERENCES 1. STUBBS, M., VEECH, R. L., AND KREBS, H. A. (1972) Biochem J. 126, 59-65. 2. CEDERBAUM, A. I., AND DICKER, E. (1982) Alwholism Clin. Exp. Res. 6. 100-109. 3. SIEW, C., DEITRICH, R. A., AND ERWIN, V. G. (1976) Arch Biochem Biophys. 176, 638-649. 4. CEDERBAUM, A. I., AND DICKER, E. (1979) Arch. Biochem. Biophys. 197,415-423. 5. CEDERBAUM, A. I., AND DICKER, E. (1981) Biochem Pharmacol 30, 3079-3088. 6. DEITRICH, R. A., TROXELL, P. A., WORTH, W. S.,

196

7. 8. 9. 10. 11. 12.

13.

14. 15. 16. 17.

DICKER

AND

AND ERWIN, V. G. (1976) B&hem. Pharmmd 25,2733-2737. MARCHNER, H., AND TOTTMAR, 0. (1978) Ada Pharmucol Toxicd 43,219-232. KITSON, T. M., AND CROW, K. E. (1979) B&hem. Phmm.acol. 23,2551-2556. STRITTMATTER, P., AND BALL, E. G. (1955) J. Biol Chem 213,445-461. GOODMAN, J. L., AND TEPHLY, T. R. (1971) Biochim Biophya Acta 252,489-505. UOTILA, L., AND KOIVUSALO, M. (1974) J. BioL Chim. 249, 7653-7663. JONES, D. P., THOR, H., ANDERSON, B., AND ORRENIUS, S. (1978) J. Bid Chew. 253, 60316037. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Bid Chem. 193, 265-273. NASH, T. C. (1953) Biockm J. R&416-422. DICKER, E., AND CEDERBAIJM, A. I. (1984) Arch. B&&em. Biophys 232,179-188. COHEN, G., AND CEDERBAUM, A. I. (1980) Arch Biock Biophys 199.438-447. CEDERBAUM, A. I., AND RUBIN, E. (1977) Biochem Pharmacol 26.1349-1353.

CEDERBAUM 18. CEDERBAUM, A. I., LIEBER, C. S., AND RUBIN, E. (1974) Arch Biochem. Biophys. 161,26-39. 19. PARRILLA, R., OHKAWA, K., LINDROS, K. O., ZIMMERMAN, U. J. P., KOBAYASHI, K., AND WILLIAMSON, J. R. (1974) J. Bid Chem 249,49264933. 26. TO~TMAR, O., PETTERSSON, H., AND KIESSLING, K. H. (1973) Biochem .I. 135, 577-586. 21. GRUNNET, N. (1973) Eur. J. Bioche-m 35, 236243. 22. LINDROS, K. O., PEKKANEN, L., AND KOIVULA, T. (1977) Actu Pharmacd Toxic& 40,134-144. 23. LINDROS, K. 0. (1975) in The Role of Acetaldehyde in the Actions of Ethanol (Lindros, K. O., and Eriksson, C. J. P., eds.), Vol. 23, pp. 67-81, Finnish Found. Alcohol Studies, Helsinki, Finland. 24. TOTTMAR, S. 0. C., AND MARCHNER, H. (1975) in The Role of Acetaldehyde in the Actions of Ethanol (Lindros, K. O., and Eriksson, C. J. P., eds.), Vol. 23, pp. 27-66, Finnish Found. Alcohol Studies, Helsinki, Finland. 25. CINTI, D. L., KEYES, S. R., LAMELIN, M. A., DENK, H., AND SCHENKMAN, J. B. (1976) J. Bid Chem 251, 1571-1577.