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Inhibition of CO2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation
Biochimica et Biophysica Acta 883 (1986) 91-97 Elsevier
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BBA 22405
Inhibition of C O 2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation Elisa D i c k e r a n d A r t h u r I. C e d e r b a u m * Department of Biochemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, N Y 10029 (U.S.A.)
(Received February 21st, 1986)
Key words: CO 2 production; Aminopyrine; Methanol; Cyanamide; Aldehyde dehydrogenase; Formaldehyde oxidation; (Mitochondria)
Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the Iow-K a mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaidehyde inhibited the production of 14CO2 from t4C-labeled aminopyrine by 30-40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO 2 production, indicating that the inhibition of CO 2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H202 generation. Crotonaldehyde inhibited CO 2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO 2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards themetabolism of exogenously added formaldehyde. Introduction The production of 14CO2 after administration of 14C-labeled aminopyrine is being used as a liver function test [1,2] and being applied for the diagnosis of liver disease, including alcoholic liver disease [3,4]. The production of CO 2 from aminopyrine is complex and requires several different steps. The initial step involves the oxidation of aminopyrine to formaldehyde plus monomethylaminopyrine, as catalyzed by the NADPH-dependent mixed-function oxidase system. For-
* To whom correspondence should be addressed.
maldehyde is oxidized to formate, and formate then is oxidized to CO 2, primarily by folate-dependent reactions [5,6]. Several enzymes can oxidize formaldehyde to formate, including a specific glutathione-dependent formaldehyde dehydrogenase found in the cytosol [7-9], the low-K m mitochondrial aldehyde dehydrogenase [10-12], and peroxisomal catalase [13]. The quantitative roles of these enzymes in contributing towards the overall oxidation of formaldehyde is not clear, although the catalase system is believed to play a minor role in view of the low rates of H202 generation by normal liver cell preparations. Lowering of glutathione levels in hepatocytes [14], or in viva [15], resulted in a decrease in CO 2
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
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production from aminopyrine, suggesting the importance of the glutathione formaldehyde dehydrogenase in oxidizing the formaldehyde which arises from the oxidation of aminopyrine. However, agents that deplete glutathione, such as diethyl maleate or phorone, were recently shown to also inhibit the activity of the low-K m mitochondrial aldehyde dehydrogenase [16]. Cyanamide, a potent inhibitor of the low-K m mitochondrial aldehyde dehydrogenase [17,18], was recently shown to completely inhibit formaldehyde oxidation by intact mitochondria as well as the mitochondrial aldehyde dehydrogenase, but to have no effect on glutathione-dependent formaldehyde dehydrogenase activity [19]. In isolated hepatocytes, concentrations of cyanamide which block the oxidation of acetaldehyde by about 90% produced 30-50% inhibition of formaldehyde metabolism [19]. These results suggested that formaldehyde was metabolized in part by cyanamidesensitive (mitochondrial aldehyde dehydrogenase) and in part by cyanamide-insensitive (formaldehyde dehydrogenase) reactions. In the current report, the effect of cyanamide on the oxidation of aminopyrine to CO 2 was studied in order to evaluate which enzyme system(s) play important roles in oxidizing the formaldehyde which arises from the N-demethylation of aminopyrine. Similar experiments were carried out using methanol as a substrate, since its oxidation also results in the production of formaldehyde. Materials and Methods Male Sprague-Dawley rats weighing about 200-250 g received phenobarbital (1 m g / m l ) in the drinking water for 7 days to induce the microsomal mixed-function oxidase system [20]. Hepatocytes were prepared as previously described [19] and suspended in a medium consisting of Krebs-Ringer-bicarbonate buffer supplemented with 10 mM phosphate buffer (pH 7.4) plus 1.25% fatty acid-free bovine serum albumin saturated with a mixture of 95% 02/5% CO 2. Viability of the cells was greater than 90%. The metabolism of aminopyrine was determined in incubations containing the Krebs buffer, about 10-15 mg liver cell protein, 0.2 mM
methionine and 1 mM amino [dimethyl-14C1pyrine (final concentration of 0.083 ktCi per /Lmol) in a total volume of 3.0 ml. Methionine was added to promote the oxidation of formate to CO 2 [21,22]. Reactions were carried out at 37°C in 25 ml polycarbonate Erlenmeyer flasks containing hanging plastic center well cups. Reactions were terminated by injecting HCI (0.1 M final concentration) into the flask. Hyamine hydroxide was injected into the center well cup and CO 2 was collected in the center well for the next 60 rain. The flasks were opened and the cups were placed in scintillation vials containing Econofluor and counted in a scintillation counter with automatic quench correction. The material remaining in the flasks was deproteinized by the addition of 0.5 ml 7.5% ZnSO 4 plus 0.5 ml 0.4 M N a O H [23]. After centrifugation, separate aliquots were utilized for the determination of formaldehyde by a dimedone procedure and for the assay of formate by a formate dehydrogenase procedure. Assays were conducted as previously described [24]. In some experiments, [14C]formaldehyde (present at final concentrations of 0.2 or 1.0 mM) was used in place of aminopyrine. Pyrazole, 3 mM final concentration, was included to prevent reduction of formaldehyde by alcohol dehydrogenase. The same buffer plus methionine as described for the aminopyrine experiments was utilized. Methanol oxidation was studied in a similar manner as was aminopyrine, except that 3 mM pyrazole was present and [14]methanol was added to a final concentration of 5 mM with a specific activity of 0.5/~Ci per >mol. In some experiments, 2 mM glyoxylic acid was added to stimulate peroxisomal H202 generation and, hence, methanol oxidation. Amino[dimethyl-14C]pyrine (73.8 mCi per mmol), [14C]formaldehyde (10 mCi per mmol), hyamine hydroxide, Econofluor and Aquasol were from New England Nuclear (Boston, MA). [14C]Methanol (58 mCi per mmol) was from Amersham, Arlington Heights, IL. Formate dehydrogenase, dimedone, cyanamide, crotonaldehyde and fatty acid-free bovine serum albumin were from Sigma Chemical Co. (St. Louis, MO). All results are expressed as means __+S.D. Statistical significance was calculated by Students' t test.
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Results
Effect of cyanamide on the oxidation of formaldehyde Initial experiments were conducted to evaluate the effectiveness of cyanamide as an inhibitor of formaldehyde oxidation by hepatocytes from phenobarbital-treated rats. The addition of [14C]formaldehyde (0.2 mM final concentration) to the hepatocytes resulted in the production of t4CO2 and [t4C]formate. Cyanamide inhibited the production of both products of formaldehyde metabolism (Table I). When the concentration of formaldehyde was raised to 1 raM, formation of products (mainly formate) increased, and cyanamide was again inhibitory. Since cyanamide is a potent inhibitor of the low-K m mitochondrial aldehyde dehydrogenase, but does not affect formaldehyde dehydrogenase [19], the partial sensitivity of formaldehyde oxidation to cyanamide suggests that both enzyme pathways contribute towards the overall metabolism of added formaldehyde~ Cyanamide was therefore utilized to evaluate the metabolism of formaldehyde generated from the oxidation of aminopyrine.
Effect of cyanamide on the oxidation of aminopyrine A time-course for the oxidation of aminopyrine by isolated hepatocytes is shown in Table II. Under these conditions, CO 2 was the major product detected, with small amounts of formaldehyde also being produced. Essentially no formate was
found, which is probably due to the presence of excess methionine added to the reaction system. In the presence of cyanamide, the rate of CO 2 production was reduced about 40% at all time periods (Table II). Coupled to this inhibition of CO 2 production by cyanamide there was a corresponding increase in formaldehyde production; the net decrease in CO 2 production was, in fact, exactly equivalent to the net increase in formaldehyde production, such that total production of CO 2 plus formaldehyde was not altered by cyanamide (Table II). The fact that cyanamide changed the distribution of products, but not the total yield of products, suggests that cyanamide did not alter the oxidation of aminopyrine at the level of cytochrome P-450. Recent work by Thurman and co-workers has implicated a critical role for the availability of NADPH as a limiting factor in regulating drug metabolism by intact cells [25,26]. We had previously shown that adding several metabolic substrates could increase the overall oxidation of aminopyrine, presumably by increasing the generation of NADPH [24]. Results in Table III show that the addition of either pyruvate or xylitol resulted in an increase in CO2 and formaldehyde production from aminopyrine; total product formation was increased about 35% by the metabolic substrates. The effects of cyanamide were identical in the absence and presence of the metabolic substrates. Under all conditions, cyanamide decreased the production of CO 2 from aminopyrine,
TABLE I EFFECT OF CYANAMIDE
ON THE OXIDATION
OF FORMALDEHYDE
T h e o x i d a t i o n of f o r m a l d e h y d e w a s a s s a y e d in the a b s e n c e o r p r e s e n c e o f 0.10 m M c y a n a m i d e . Results are f r o m three e x p e r i m e n t s . Concentration of formaldehyde (mM)
0.2
1.0
Reaction product
CO 2 formate CO 2 + formate CO 2 formate CO 2 + formate
* P < 0.05; ** P < 0.02; *** P < 0.001.
Rate of product formation (nmol/min per mg hepatocyte protein) - Cyanamide
+ Cyanamide
0.94 + 0,11 0.45 ___0,05 1.38 + 0.15 1.17+0,02 2.19 _+0.53 3.36 _+0.55
0.67 + 0.04 0.24 4- 0.03 0.91 -+ 0.07 0.58_+0.03 0.62 _+0.07 1.19 _+0.06
Effect of c y a n a m i d e (%)
- 29 - 47 - 34 -50 - 72 - 65
* ** * *** ** **
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T A B L E II EFFECT OF CYANAMIDE
ON THE OXIDATION OF AMINOPYRINE
The o x i d a t i o n of a m i n o p y r i n e to C O 2 plus f o r m a l d e h y d e was assayed in the absence or presence of 0.10 m M c y a n a m i d e , Results are from four experiments. R e a c t i o n time (rain)
Reaction product
5 10 15 5 10 15 5 10 15
CO 2
formaldehyde
CO 2 + formaldehyde
R a t e of p r o d u c t f o r m a t i o n ( n m o l / m i n per mg h e p a t o c y t e protein)
Effect of cyanamide
- Cyanamide
+ Cyanamide
( %)
4.17_+0.46 6.86-+ 1.32 12.00 ± 2.69 1.23 -+ 0.12 1.42 _+0.08 1.94_+0.21 5.40 _+0.31 8.28_+ 1.07 13.94 _+ 1.47
2.54_+0.44 3.84_+0.40 7.99 -+ 2.06 2.58 -+ 0.65 4.37 _+ 1.29 6.29_+ 1.65 5.12 _+0.52 8.21 _+ 1.07 14.28 _+ 1.79
- 39 -44 - 33 + 110 + 208 + 224 - 5 - 1 + 2
* * * * * *
* P < 0.05
whereas the production of formaldehyde was increased (Table III). The increase in formaldehyde was equivalent to the decrease in CO 2 production, as the sum of CO 2 plus formaldehyde production was not altered by cyanamide (Table III).
Aminopyrine oxidation in the presence of crotonaldehyde Acetaldehyde was shown to be a competitive
inhibitor of formaldehyde oxidation by the low-K m mitochondrial aldehyde dehydrogenase, but to have no effect on the glutathione-dependent formaldehyde dehydrogenase activity [19]. When acetaldehyde was added to the hepatocytes, there was a slight decrease in CO 2 production from aminopyrine, which was accompanied by an increase in formaldehyde accumulation. The decreases in CO2 was equivalent to the gain in
T A B L E 1II EFFECT OF CYANAMIDE STRATES
ON THE OXIDATION
OF AMINOPYRINE
IN T H E P R E S E N C E
OF METABOLIC
SUB-
The o x i d a t i o n of a m i n o p y r i n e to C O 2 plus f o r m a l d e h y d e was assayed in the absence or presence of 0.10 m M c y a n a m i d e . W h e r e indicated, either 5 m M p y r u v a t e or 5 m M xylitol was a d d e d to the h e p a t o c y t e suspensions. Results are from three experiments. Addition
Reaction product
R a t e of p r o d u c t f o r m a t i o n ( n m o l / m i n per mg h e p a t o c y t e protein)
Effect of cyanamide
- Cyanamide
+ Cyanamide
(%)
None
CO z formaldehyde CO a + formaldehyde CO 2 formaldehyde CO 2 + formaldehyde CO 2 formaldehyde CO z + formaldehyde
0.65 _+0.13 0.14 _+0.01 0.79 + 0.19 0.82_+0.04 0.22 -+ 0.07 1.05 -+ 0.06 0.78 + 0.13 0.29 _+0.05 1.08 + 0.13
0.39 + 0.04 0.41 + 0.13 0.80 _+0.13 0.51 + 0 . 0 8 0.62 + 0.15 1.12 + 0,17 0.40 _+0.07 0.65 _+0.09 1.05 + 0.16
- 40 + 193 + 1 - 38 + 182 + 7 - 49 + 124 - 3
Pyruvate
Xylitol
* P < 0.05; ** P < 0.02; *** P < 0,001.
* *** ** *** * **
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TABLE IV E F F E C T OF A C E T A L D E H Y D E A N D C R O T O N A L D E H Y D E ON A M I N O P Y R I N E O X I D A T I O N The oxidation of aminopyrine was determined in the absence or presence of either 0.5 m M acetaldehyde or 1 mM crotonaldehyde. Results are from three or four experiments. Addition
Rate of aminopyrine oxidation ( n m o l / m i n per mg hepatocyte protein)
None Acetaldehyde Crotonaldehyde
CO 2
effect (%)
formaldehyde
effect ( %)
CO 2 + formaldehyde
effect (%)
0.755:0.13 0.60 + 0.09 0.47 + 0.11
- 20 - 37 *
0.09+0.01 0.21 5:0.04 0.30 5:0.04
+ 133 * + 233 **
0.845:0.13 0.81 5:0.13 0.77 5:0.11
- 4 - 8
• P <0.05; ** P < 0.02.
formaldehyde (Table IV). Although in agreement with the cyanamide results, the effects of acetaldehyde were small, which probably reflect the rapid rates of acetaldehyde oxidation by the hepatocytes so that effective inhibitory concentrations did not last for a sufficient period. Pyrazole was not added to block the reduction of acetaldehyde to ethanol, since pyrazole itself blocked aminopyrine oxidation. Therefore, we evaluated the effects of crotonaldehyde, which was also shown to inhibit formaldehyde oxidation by mitochondrial aldehyde dehydrogenase but not by formaldehyde dehydrogenase [27]. Crotonaldehyde is oxidized by hepatocytes at rates that are about one-quarter that found with acetaldehyde [27]; therefore, effective inhibitory concentrations of this aldehyde would persist for a longer period
than with acetaldehyde. Crotonaldehyde inhibited CO 2 production from aminopyrine by 37%, whereas formaldehyde production was doubled (Table IV). As was found with cyanamide, the decrease in CO 2 was equal to the increase in formaldehyde, suggesting a change in the distribution of products rather than an inhibition of aminopyrine oxidation. Effect of crotonaldehyde on methanol oxidation Similar experiments were conducted to evaluate the enzymes contributing towards the oxidation of formaldehyde which arises from the oxidation of methanol. In rat liver, methanol is oxidized primarily by catalase, and not alcohol dehydrogenase [28, 29]. Since rates of H202 generation are limiting for catalase-dependent oxidations [13],
TABLE V E F F E C T OF C R O T O N A L D E H Y D E ON M E T H A N O L O X I D A T I O N The oxidation of methanol to CO 2, formaldehyde or formate was assayed in the absence or presence of 1 mM crotonaldehyde. Where indicated, 2 mM glyoxylic acid was also present. Results are from three or four experiments. Addition
None
Glyoxylate
Product
CO 2 formaldehyde formate total CO 2 formaldehyde formate total
* P <0.05 ** P < 0 . 0 1
?.ate of product formation ( n m o l / m i n per mg hepatocyte protein)
Effect of
- Crotonaldehyde
+ Crotonaldehyde
crotonaldehyde (%)
0.875:0.19 0.19 5:0.06 0.06 + 0.02 1.12 + 0.11 1.88 5:0.21 0.22 4- 0.06 0.14 5:0.03 2.24 4- 0.16
0.49_+0.12 0.63 _+0.20 0.02 5:0.01 1.14 5:0.10 0.95 5:0.19 0.89 5:0.10 0.08 + 0.01 1.92 ___0.14
-44 + 232 - 67 +2 - 49 + 230 - 43 - 14
* *
* **
96 methanol oxidation was studied in the absence and presence of glyoxylic acid. The latter is a substrate for peroxisomal glyoxylase and stimulates HzO 2 generation. Methanol was oxidized primarily to CO 2, with small amounts of formaldehyde also accumulating (Table V). The addition of glyoxylic acid doubled the rate of methanol oxidation. When cyanamide was added, in the absence or presence of glyoxylic acid, production of CO 2, formaldehyde and formate from methanol were all diminished by more than 70%, i.e., cyanamide inhibited total methanol oxidation rather than changing the distribution of the products. In view of this effect of cyanamide, this inhibitor could not be utilized to assess pathways of metabolism of formaldehyde derived from methanol oxidation. Therefore, we employed crotonaldehyde to block the mitochondrial but not the formaldehyde dehydrogenase system. As shown in Table V, crotonaldehyde decreased the production of CO 2 from methanol by about 45% in the absence or presence of glyoxylate. Accompanying this decrease in CO 2 was an accumulation of formaldehyde which was equivalent to the loss in CO 2 production (Table V). Discussion
Cyanamide is a potent inhibitor of the low-K m mitochondrial aldehyde dehydrogenase and was previously shown to nearly completely inhibit the oxidation of formaldehyde by this enzyme system [19]. Since cyanamide did not affect the activity of formaldehyde dehydrogenase, this inhibitor appears to be a valuable agent in assessing the enzyme pathways responsible for the oxidation of formaldehyde to formate. Initial experiments indicate that in the hepatocytes from phenobarbitaltreated rats, in analogy to results found with hepatocytes from uninduced rats, cyanamide caused partial, but not complete inhibition of the oxidation of formaldehyde to formate plus CO 2. This partial sensitivity to cyanamide suggests that formaldehyde is being oxidized by cyanamide-sensitive (mitochondrial aldehyde dehydrogenase) and -insensitive (formaldehyde dehydrogenase) pathways. The greater sensitivity of 1 mM formaldehyde oxidation to inhibition by cyanamide, c o m -
pared to 0.2 mM formaldehyde, reflects a greater contributory role of the mitochondrial enzyme at higher concentrations of formaldehyde, which approximates the K,, values for formaldehyde by the two enzyme pathways (unpublished data). The oxidation of aminopyrine to CO 2 was depressed about 40% by cyanamide, which is similar to the inhibition of CO 2 production from 0.2 mM formaldehyde. If cyanamide was blocking CO 2 production by inhibiting the oxidation of formaldehyde to formate, then cyanamide should promote the accumulation of formaldehyde, and the amount of formaldehyde which accumulates should be equivalent to the decrease in the amount of CO 2. This proved to be the case under a variety of reaction time periods and in the absence or presence of metabolic substrates. Cyanamide changed the distribution of products but did not alter the yield of products, consistent with an inhibition of the metabolism of the formaldehyde arising from the metabolism of aminopyrine. Essentially, identical results are found with crotonaldehyde which, similar to cyanamide, inhibits mitochondrial aldehyde dehydrogenase-dependent oxidation of formaldehyde but does not affect formaldehyde dehydrogenase activity [27]. With regard to methanol oxidation, crotonaldehyde also produced some inhibition of CO 2 production, that was equivalent to the increase in formaldehyde accumulation. Cyanamide could not be utilized in the studies with methanol since it blocked the production of all products rather than changing the distribution of products. Cyanamide itself is not the ultimate inhibitor of mitochondrial aldehyde dehydrogenase, but rather is converted to the actual inhibitor [17,30]. Recent studies have shown that catalase is the cyanamide-activating enzyme [31-33], and that during this activation, the peroxidatic activity of catalase was inhibited [34]. Hence, inhibition of methanol oxidation by cyanamide reflects this inhibition of the peroxidatic activity of catalase towards alcohols. The results with cyanamide and crotonaldehyde suggest that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde generated via the mixed-function oxidase system or via methanol oxidation, just as both enzyme systems contribute towards the metabo-
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lism of exogenously added formaldehyde. The usefulness of the aminopyrine breath test will depend not only on the quantity and integrity of the mixed-function oxidase system, but also on the activity of the formaldehyde oxidizing enzymes, the glutathione-requiring formaldehyde dehydrogenase and the low-K m mitochondrial aldehyde dehydrogenase.
Acknowledgements These studies were supported by USPHS Grant AA-03312 from the National Institute on Alcohol Abuse and Alcoholism. We thank Ms. Roslyn C. King for typing the manuscript.
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13 Chance, B. (1950) J. Biol. Chem. 182, 649-658 14 Zahlten, R.N. and Bhatt, H.S. (1983) in Biochemistry of Metabolic Processes (Lennon, D., Stratman, F.W. and Zahlten, R.W., eds.), pp. 429-440, Elsevier Science Publishers, Amsterdam 15 Bhatt, H.S. and Combes, B. (1985) Hepatology 5, 615-621 16 Dicker, E. and Cederbaum, A.I. (1985) Biochim. Biophys. Acta 843, 107-113. 17 Deitrich, R.A., Troxell, P.A., Worth, W.S. and Erwin, V.G. (1976) Biochem. Pharmacol. 25, 2733-2737 18 Marchner, H. and Tottmar, O. (1978) Acta Pharmacol. Toxicol. 43, 219-232 19 Dicker, E. and Cederbaum, A.I. (1984) Arch. Biochem. Biophys. 232, 179-188 20 Marshall, W.J. and McLean, A.E.M. (1969) BiOchem. Pharmacol. 18, 153-157 21 Krebs, H.A., Hems, R. and Tyler, B. (1976) Biochem. J. 158, 341-353 22 Waydhas, C., Weigl, K. and Sies, H. (1978) Eur. J. Biochem. 89, 143-150 23 Billings, R.E. and Tephly, T.R. (1979) Biochem. Pharmacol. 28, 2985-2991 24 Dicker, E. and Cederbaum, A.I. (1983) J. Pharmacol. Exp. Ther. 227, 687-693 25 Thurman, R.G. and Kauffman, F.C. (1980) Pharmacol. Rev. 31,229-251 26 Reinke, L.A., Kauffman, F.C. and Thurman, R.G. (1980) Biochem. Pharmacol. 29, 813-819 27 Dicker, E. and Cederbaum, A.I. (1984) Arch. Biochem. Biophys. 234, 187-196 28 Tephly, T.R. (1977) Fed. Proc. 36, 1627-1633 29 Makar, A.B. and Tephly, T.R. (1976) Nature 261,715-716 30 Kitson, T.M. and Crow, K.E. (1979) Biochem. Pharmacol. 28, 2551-2556 31 DeMaster, E.G., Shirota, F.N. and Nagasawa, H.T. (1984) Biochem. Biophys. Res. Commun. 122, 358-365 32 Svanas, G.W. and Weiner, H. (1985) Biochem. Pharmacol. 34, 1197-1204 33 DeMaster, E.G., Shirota, F.N. and Nagasawa, H.T. (1985) Alcohol 2, 117-121 34 Cederbaum, A.I. and Dicker, E. (1986) Toxicol. Lett. 29, 107-114
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BBA 22405
Inhibition of C O 2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation Elisa D i c k e r a n d A r t h u r I. C e d e r b a u m * Department of Biochemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, N Y 10029 (U.S.A.)
(Received February 21st, 1986)
Key words: CO 2 production; Aminopyrine; Methanol; Cyanamide; Aldehyde dehydrogenase; Formaldehyde oxidation; (Mitochondria)
Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the Iow-K a mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaidehyde inhibited the production of 14CO2 from t4C-labeled aminopyrine by 30-40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO 2 production, indicating that the inhibition of CO 2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H202 generation. Crotonaldehyde inhibited CO 2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO 2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards themetabolism of exogenously added formaldehyde. Introduction The production of 14CO2 after administration of 14C-labeled aminopyrine is being used as a liver function test [1,2] and being applied for the diagnosis of liver disease, including alcoholic liver disease [3,4]. The production of CO 2 from aminopyrine is complex and requires several different steps. The initial step involves the oxidation of aminopyrine to formaldehyde plus monomethylaminopyrine, as catalyzed by the NADPH-dependent mixed-function oxidase system. For-
* To whom correspondence should be addressed.
maldehyde is oxidized to formate, and formate then is oxidized to CO 2, primarily by folate-dependent reactions [5,6]. Several enzymes can oxidize formaldehyde to formate, including a specific glutathione-dependent formaldehyde dehydrogenase found in the cytosol [7-9], the low-K m mitochondrial aldehyde dehydrogenase [10-12], and peroxisomal catalase [13]. The quantitative roles of these enzymes in contributing towards the overall oxidation of formaldehyde is not clear, although the catalase system is believed to play a minor role in view of the low rates of H202 generation by normal liver cell preparations. Lowering of glutathione levels in hepatocytes [14], or in viva [15], resulted in a decrease in CO 2
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production from aminopyrine, suggesting the importance of the glutathione formaldehyde dehydrogenase in oxidizing the formaldehyde which arises from the oxidation of aminopyrine. However, agents that deplete glutathione, such as diethyl maleate or phorone, were recently shown to also inhibit the activity of the low-K m mitochondrial aldehyde dehydrogenase [16]. Cyanamide, a potent inhibitor of the low-K m mitochondrial aldehyde dehydrogenase [17,18], was recently shown to completely inhibit formaldehyde oxidation by intact mitochondria as well as the mitochondrial aldehyde dehydrogenase, but to have no effect on glutathione-dependent formaldehyde dehydrogenase activity [19]. In isolated hepatocytes, concentrations of cyanamide which block the oxidation of acetaldehyde by about 90% produced 30-50% inhibition of formaldehyde metabolism [19]. These results suggested that formaldehyde was metabolized in part by cyanamidesensitive (mitochondrial aldehyde dehydrogenase) and in part by cyanamide-insensitive (formaldehyde dehydrogenase) reactions. In the current report, the effect of cyanamide on the oxidation of aminopyrine to CO 2 was studied in order to evaluate which enzyme system(s) play important roles in oxidizing the formaldehyde which arises from the N-demethylation of aminopyrine. Similar experiments were carried out using methanol as a substrate, since its oxidation also results in the production of formaldehyde. Materials and Methods Male Sprague-Dawley rats weighing about 200-250 g received phenobarbital (1 m g / m l ) in the drinking water for 7 days to induce the microsomal mixed-function oxidase system [20]. Hepatocytes were prepared as previously described [19] and suspended in a medium consisting of Krebs-Ringer-bicarbonate buffer supplemented with 10 mM phosphate buffer (pH 7.4) plus 1.25% fatty acid-free bovine serum albumin saturated with a mixture of 95% 02/5% CO 2. Viability of the cells was greater than 90%. The metabolism of aminopyrine was determined in incubations containing the Krebs buffer, about 10-15 mg liver cell protein, 0.2 mM
methionine and 1 mM amino [dimethyl-14C1pyrine (final concentration of 0.083 ktCi per /Lmol) in a total volume of 3.0 ml. Methionine was added to promote the oxidation of formate to CO 2 [21,22]. Reactions were carried out at 37°C in 25 ml polycarbonate Erlenmeyer flasks containing hanging plastic center well cups. Reactions were terminated by injecting HCI (0.1 M final concentration) into the flask. Hyamine hydroxide was injected into the center well cup and CO 2 was collected in the center well for the next 60 rain. The flasks were opened and the cups were placed in scintillation vials containing Econofluor and counted in a scintillation counter with automatic quench correction. The material remaining in the flasks was deproteinized by the addition of 0.5 ml 7.5% ZnSO 4 plus 0.5 ml 0.4 M N a O H [23]. After centrifugation, separate aliquots were utilized for the determination of formaldehyde by a dimedone procedure and for the assay of formate by a formate dehydrogenase procedure. Assays were conducted as previously described [24]. In some experiments, [14C]formaldehyde (present at final concentrations of 0.2 or 1.0 mM) was used in place of aminopyrine. Pyrazole, 3 mM final concentration, was included to prevent reduction of formaldehyde by alcohol dehydrogenase. The same buffer plus methionine as described for the aminopyrine experiments was utilized. Methanol oxidation was studied in a similar manner as was aminopyrine, except that 3 mM pyrazole was present and [14]methanol was added to a final concentration of 5 mM with a specific activity of 0.5/~Ci per >mol. In some experiments, 2 mM glyoxylic acid was added to stimulate peroxisomal H202 generation and, hence, methanol oxidation. Amino[dimethyl-14C]pyrine (73.8 mCi per mmol), [14C]formaldehyde (10 mCi per mmol), hyamine hydroxide, Econofluor and Aquasol were from New England Nuclear (Boston, MA). [14C]Methanol (58 mCi per mmol) was from Amersham, Arlington Heights, IL. Formate dehydrogenase, dimedone, cyanamide, crotonaldehyde and fatty acid-free bovine serum albumin were from Sigma Chemical Co. (St. Louis, MO). All results are expressed as means __+S.D. Statistical significance was calculated by Students' t test.
93
Results
Effect of cyanamide on the oxidation of formaldehyde Initial experiments were conducted to evaluate the effectiveness of cyanamide as an inhibitor of formaldehyde oxidation by hepatocytes from phenobarbital-treated rats. The addition of [14C]formaldehyde (0.2 mM final concentration) to the hepatocytes resulted in the production of t4CO2 and [t4C]formate. Cyanamide inhibited the production of both products of formaldehyde metabolism (Table I). When the concentration of formaldehyde was raised to 1 raM, formation of products (mainly formate) increased, and cyanamide was again inhibitory. Since cyanamide is a potent inhibitor of the low-K m mitochondrial aldehyde dehydrogenase, but does not affect formaldehyde dehydrogenase [19], the partial sensitivity of formaldehyde oxidation to cyanamide suggests that both enzyme pathways contribute towards the overall metabolism of added formaldehyde~ Cyanamide was therefore utilized to evaluate the metabolism of formaldehyde generated from the oxidation of aminopyrine.
Effect of cyanamide on the oxidation of aminopyrine A time-course for the oxidation of aminopyrine by isolated hepatocytes is shown in Table II. Under these conditions, CO 2 was the major product detected, with small amounts of formaldehyde also being produced. Essentially no formate was
found, which is probably due to the presence of excess methionine added to the reaction system. In the presence of cyanamide, the rate of CO 2 production was reduced about 40% at all time periods (Table II). Coupled to this inhibition of CO 2 production by cyanamide there was a corresponding increase in formaldehyde production; the net decrease in CO 2 production was, in fact, exactly equivalent to the net increase in formaldehyde production, such that total production of CO 2 plus formaldehyde was not altered by cyanamide (Table II). The fact that cyanamide changed the distribution of products, but not the total yield of products, suggests that cyanamide did not alter the oxidation of aminopyrine at the level of cytochrome P-450. Recent work by Thurman and co-workers has implicated a critical role for the availability of NADPH as a limiting factor in regulating drug metabolism by intact cells [25,26]. We had previously shown that adding several metabolic substrates could increase the overall oxidation of aminopyrine, presumably by increasing the generation of NADPH [24]. Results in Table III show that the addition of either pyruvate or xylitol resulted in an increase in CO2 and formaldehyde production from aminopyrine; total product formation was increased about 35% by the metabolic substrates. The effects of cyanamide were identical in the absence and presence of the metabolic substrates. Under all conditions, cyanamide decreased the production of CO 2 from aminopyrine,
TABLE I EFFECT OF CYANAMIDE
ON THE OXIDATION
OF FORMALDEHYDE
T h e o x i d a t i o n of f o r m a l d e h y d e w a s a s s a y e d in the a b s e n c e o r p r e s e n c e o f 0.10 m M c y a n a m i d e . Results are f r o m three e x p e r i m e n t s . Concentration of formaldehyde (mM)
0.2
1.0
Reaction product
CO 2 formate CO 2 + formate CO 2 formate CO 2 + formate
* P < 0.05; ** P < 0.02; *** P < 0.001.
Rate of product formation (nmol/min per mg hepatocyte protein) - Cyanamide
+ Cyanamide
0.94 + 0,11 0.45 ___0,05 1.38 + 0.15 1.17+0,02 2.19 _+0.53 3.36 _+0.55
0.67 + 0.04 0.24 4- 0.03 0.91 -+ 0.07 0.58_+0.03 0.62 _+0.07 1.19 _+0.06
Effect of c y a n a m i d e (%)
- 29 - 47 - 34 -50 - 72 - 65
* ** * *** ** **
94
T A B L E II EFFECT OF CYANAMIDE
ON THE OXIDATION OF AMINOPYRINE
The o x i d a t i o n of a m i n o p y r i n e to C O 2 plus f o r m a l d e h y d e was assayed in the absence or presence of 0.10 m M c y a n a m i d e , Results are from four experiments. R e a c t i o n time (rain)
Reaction product
5 10 15 5 10 15 5 10 15
CO 2
formaldehyde
CO 2 + formaldehyde
R a t e of p r o d u c t f o r m a t i o n ( n m o l / m i n per mg h e p a t o c y t e protein)
Effect of cyanamide
- Cyanamide
+ Cyanamide
( %)
4.17_+0.46 6.86-+ 1.32 12.00 ± 2.69 1.23 -+ 0.12 1.42 _+0.08 1.94_+0.21 5.40 _+0.31 8.28_+ 1.07 13.94 _+ 1.47
2.54_+0.44 3.84_+0.40 7.99 -+ 2.06 2.58 -+ 0.65 4.37 _+ 1.29 6.29_+ 1.65 5.12 _+0.52 8.21 _+ 1.07 14.28 _+ 1.79
- 39 -44 - 33 + 110 + 208 + 224 - 5 - 1 + 2
* * * * * *
* P < 0.05
whereas the production of formaldehyde was increased (Table III). The increase in formaldehyde was equivalent to the decrease in CO 2 production, as the sum of CO 2 plus formaldehyde production was not altered by cyanamide (Table III).
Aminopyrine oxidation in the presence of crotonaldehyde Acetaldehyde was shown to be a competitive
inhibitor of formaldehyde oxidation by the low-K m mitochondrial aldehyde dehydrogenase, but to have no effect on the glutathione-dependent formaldehyde dehydrogenase activity [19]. When acetaldehyde was added to the hepatocytes, there was a slight decrease in CO 2 production from aminopyrine, which was accompanied by an increase in formaldehyde accumulation. The decreases in CO2 was equivalent to the gain in
T A B L E 1II EFFECT OF CYANAMIDE STRATES
ON THE OXIDATION
OF AMINOPYRINE
IN T H E P R E S E N C E
OF METABOLIC
SUB-
The o x i d a t i o n of a m i n o p y r i n e to C O 2 plus f o r m a l d e h y d e was assayed in the absence or presence of 0.10 m M c y a n a m i d e . W h e r e indicated, either 5 m M p y r u v a t e or 5 m M xylitol was a d d e d to the h e p a t o c y t e suspensions. Results are from three experiments. Addition
Reaction product
R a t e of p r o d u c t f o r m a t i o n ( n m o l / m i n per mg h e p a t o c y t e protein)
Effect of cyanamide
- Cyanamide
+ Cyanamide
(%)
None
CO z formaldehyde CO a + formaldehyde CO 2 formaldehyde CO 2 + formaldehyde CO 2 formaldehyde CO z + formaldehyde
0.65 _+0.13 0.14 _+0.01 0.79 + 0.19 0.82_+0.04 0.22 -+ 0.07 1.05 -+ 0.06 0.78 + 0.13 0.29 _+0.05 1.08 + 0.13
0.39 + 0.04 0.41 + 0.13 0.80 _+0.13 0.51 + 0 . 0 8 0.62 + 0.15 1.12 + 0,17 0.40 _+0.07 0.65 _+0.09 1.05 + 0.16
- 40 + 193 + 1 - 38 + 182 + 7 - 49 + 124 - 3
Pyruvate
Xylitol
* P < 0.05; ** P < 0.02; *** P < 0,001.
* *** ** *** * **
95
TABLE IV E F F E C T OF A C E T A L D E H Y D E A N D C R O T O N A L D E H Y D E ON A M I N O P Y R I N E O X I D A T I O N The oxidation of aminopyrine was determined in the absence or presence of either 0.5 m M acetaldehyde or 1 mM crotonaldehyde. Results are from three or four experiments. Addition
Rate of aminopyrine oxidation ( n m o l / m i n per mg hepatocyte protein)
None Acetaldehyde Crotonaldehyde
CO 2
effect (%)
formaldehyde
effect ( %)
CO 2 + formaldehyde
effect (%)
0.755:0.13 0.60 + 0.09 0.47 + 0.11
- 20 - 37 *
0.09+0.01 0.21 5:0.04 0.30 5:0.04
+ 133 * + 233 **
0.845:0.13 0.81 5:0.13 0.77 5:0.11
- 4 - 8
• P <0.05; ** P < 0.02.
formaldehyde (Table IV). Although in agreement with the cyanamide results, the effects of acetaldehyde were small, which probably reflect the rapid rates of acetaldehyde oxidation by the hepatocytes so that effective inhibitory concentrations did not last for a sufficient period. Pyrazole was not added to block the reduction of acetaldehyde to ethanol, since pyrazole itself blocked aminopyrine oxidation. Therefore, we evaluated the effects of crotonaldehyde, which was also shown to inhibit formaldehyde oxidation by mitochondrial aldehyde dehydrogenase but not by formaldehyde dehydrogenase [27]. Crotonaldehyde is oxidized by hepatocytes at rates that are about one-quarter that found with acetaldehyde [27]; therefore, effective inhibitory concentrations of this aldehyde would persist for a longer period
than with acetaldehyde. Crotonaldehyde inhibited CO 2 production from aminopyrine by 37%, whereas formaldehyde production was doubled (Table IV). As was found with cyanamide, the decrease in CO 2 was equal to the increase in formaldehyde, suggesting a change in the distribution of products rather than an inhibition of aminopyrine oxidation. Effect of crotonaldehyde on methanol oxidation Similar experiments were conducted to evaluate the enzymes contributing towards the oxidation of formaldehyde which arises from the oxidation of methanol. In rat liver, methanol is oxidized primarily by catalase, and not alcohol dehydrogenase [28, 29]. Since rates of H202 generation are limiting for catalase-dependent oxidations [13],
TABLE V E F F E C T OF C R O T O N A L D E H Y D E ON M E T H A N O L O X I D A T I O N The oxidation of methanol to CO 2, formaldehyde or formate was assayed in the absence or presence of 1 mM crotonaldehyde. Where indicated, 2 mM glyoxylic acid was also present. Results are from three or four experiments. Addition
None
Glyoxylate
Product
CO 2 formaldehyde formate total CO 2 formaldehyde formate total
* P <0.05 ** P < 0 . 0 1
?.ate of product formation ( n m o l / m i n per mg hepatocyte protein)
Effect of
- Crotonaldehyde
+ Crotonaldehyde
crotonaldehyde (%)
0.875:0.19 0.19 5:0.06 0.06 + 0.02 1.12 + 0.11 1.88 5:0.21 0.22 4- 0.06 0.14 5:0.03 2.24 4- 0.16
0.49_+0.12 0.63 _+0.20 0.02 5:0.01 1.14 5:0.10 0.95 5:0.19 0.89 5:0.10 0.08 + 0.01 1.92 ___0.14
-44 + 232 - 67 +2 - 49 + 230 - 43 - 14
* *
* **
96 methanol oxidation was studied in the absence and presence of glyoxylic acid. The latter is a substrate for peroxisomal glyoxylase and stimulates HzO 2 generation. Methanol was oxidized primarily to CO 2, with small amounts of formaldehyde also accumulating (Table V). The addition of glyoxylic acid doubled the rate of methanol oxidation. When cyanamide was added, in the absence or presence of glyoxylic acid, production of CO 2, formaldehyde and formate from methanol were all diminished by more than 70%, i.e., cyanamide inhibited total methanol oxidation rather than changing the distribution of the products. In view of this effect of cyanamide, this inhibitor could not be utilized to assess pathways of metabolism of formaldehyde derived from methanol oxidation. Therefore, we employed crotonaldehyde to block the mitochondrial but not the formaldehyde dehydrogenase system. As shown in Table V, crotonaldehyde decreased the production of CO 2 from methanol by about 45% in the absence or presence of glyoxylate. Accompanying this decrease in CO 2 was an accumulation of formaldehyde which was equivalent to the loss in CO 2 production (Table V). Discussion
Cyanamide is a potent inhibitor of the low-K m mitochondrial aldehyde dehydrogenase and was previously shown to nearly completely inhibit the oxidation of formaldehyde by this enzyme system [19]. Since cyanamide did not affect the activity of formaldehyde dehydrogenase, this inhibitor appears to be a valuable agent in assessing the enzyme pathways responsible for the oxidation of formaldehyde to formate. Initial experiments indicate that in the hepatocytes from phenobarbitaltreated rats, in analogy to results found with hepatocytes from uninduced rats, cyanamide caused partial, but not complete inhibition of the oxidation of formaldehyde to formate plus CO 2. This partial sensitivity to cyanamide suggests that formaldehyde is being oxidized by cyanamide-sensitive (mitochondrial aldehyde dehydrogenase) and -insensitive (formaldehyde dehydrogenase) pathways. The greater sensitivity of 1 mM formaldehyde oxidation to inhibition by cyanamide, c o m -
pared to 0.2 mM formaldehyde, reflects a greater contributory role of the mitochondrial enzyme at higher concentrations of formaldehyde, which approximates the K,, values for formaldehyde by the two enzyme pathways (unpublished data). The oxidation of aminopyrine to CO 2 was depressed about 40% by cyanamide, which is similar to the inhibition of CO 2 production from 0.2 mM formaldehyde. If cyanamide was blocking CO 2 production by inhibiting the oxidation of formaldehyde to formate, then cyanamide should promote the accumulation of formaldehyde, and the amount of formaldehyde which accumulates should be equivalent to the decrease in the amount of CO 2. This proved to be the case under a variety of reaction time periods and in the absence or presence of metabolic substrates. Cyanamide changed the distribution of products but did not alter the yield of products, consistent with an inhibition of the metabolism of the formaldehyde arising from the metabolism of aminopyrine. Essentially, identical results are found with crotonaldehyde which, similar to cyanamide, inhibits mitochondrial aldehyde dehydrogenase-dependent oxidation of formaldehyde but does not affect formaldehyde dehydrogenase activity [27]. With regard to methanol oxidation, crotonaldehyde also produced some inhibition of CO 2 production, that was equivalent to the increase in formaldehyde accumulation. Cyanamide could not be utilized in the studies with methanol since it blocked the production of all products rather than changing the distribution of products. Cyanamide itself is not the ultimate inhibitor of mitochondrial aldehyde dehydrogenase, but rather is converted to the actual inhibitor [17,30]. Recent studies have shown that catalase is the cyanamide-activating enzyme [31-33], and that during this activation, the peroxidatic activity of catalase was inhibited [34]. Hence, inhibition of methanol oxidation by cyanamide reflects this inhibition of the peroxidatic activity of catalase towards alcohols. The results with cyanamide and crotonaldehyde suggest that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde generated via the mixed-function oxidase system or via methanol oxidation, just as both enzyme systems contribute towards the metabo-
97
lism of exogenously added formaldehyde. The usefulness of the aminopyrine breath test will depend not only on the quantity and integrity of the mixed-function oxidase system, but also on the activity of the formaldehyde oxidizing enzymes, the glutathione-requiring formaldehyde dehydrogenase and the low-K m mitochondrial aldehyde dehydrogenase.
Acknowledgements These studies were supported by USPHS Grant AA-03312 from the National Institute on Alcohol Abuse and Alcoholism. We thank Ms. Roslyn C. King for typing the manuscript.
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