Acta pharmacol. et toxicol. 1977, 41,263-272.

From the Department of Physiology, University of Kuopio, SF-70101 Kuopio 10, Finland

Inducible Aldehyde Dehydrogenases in the Hepatic Cytosol of the Rat BY Riitta Torronen, Unto Nousiainen and Marios Marselos (Received November 23, 1976; Accepted February 14, 1977)

Abstract: Rats of the Wistar/Af/Han/MoW(Han 67) strain have previously been shown to respond in a variable way to phenobarbital treatment, as far as the induction of aldehyde dehydrogenase activity is concerned (MARSELOS 1976). This biochemical property is genetically determined and concerns the high-Km aldehyde dehydrogenase of the hepatic cytosol. In this study, administration of phenobarbital (1 mg/ml of drinking water, for 1 week) produces a uniform induction of aldehyde dehydrogenase in all rats, when measured with micromolar substrate concentration. The inducible low-Km enzyme of the cytosol is not genetically determined like the high-Km enzyme, and shows a wide specificity for aliphatic as well as for aromatic aldehydes. Despite the inducibility of the cytosolic enzymes, no alterations are found in the mitochondrial aldehyde dehydrogenase activities after phenobarbital treatment. The oxidation of D-glucuronolactone takes place only in the cytosol, and seems to be dependent on the low-Km aldehyde dehydrogenase. This is consistent with NMR studies, which showed that a very minimal amount of D-glucuronolactone is in aldehyde form under the measurement conditions usually applied. Further, the oxidation of D-glucuronolactone is also enhanced by phenobarbital in all rats without a genetic predisposition, and its dose-response curve is very similar to that of the low-Km aldehyde dehydrogenase.

Key-words: Liver - aldehyde dehydrogenase - D-glucuronolactone phenobarbital - rat.

- induction -

The induction olf a cytosolic aldehyde dehydrogenase (EC 1.2.1.3) was first described in the liver of mice by REDMOND & COHEN(1971). In rats, the inducibility of this enzyme has been found to be determined by genetic factors, so that it may vary significantly even with in the same animal strain (DEITRICH 1971; DEITRICH et al. 1972 & 1975; MARSELOS 1976). Kinetic studies on this inducible cytosdic aldehyde dehydrogenase have shown a relatively poor affinity for aldehydes, with a Km value in the millimolar

264

R. TURRUNEN, U. NOUSIAINEN AND M. MARSELOS

range (DEITRICH et al. 1972; KOIVULA & KOIVUSALO 1975a & b; ERIKSSON et al. 1975). Apart from this high-Km enzyme, the cytosol also contains another aldehyde dehydrogenase with a very low Km value, which has been conventionally considered as an artefactual result of mitochondria1 leakage (6. KOWULA 1975). Many investigators have suggested that D-glucuronolactoae dehydrogenase (EC 1.1.1.70) is an unspecific aldehyde dehydrogenase (SADAHROet al. 1966; AARTS& HrNNEN-BOUWMANS 1972; TONKES1973), consistent with the observation that in aqueous solution part of D-glucuronolactone is in a free aldehyde form (SADAHIRO et aE. 1966). In fact, chromatographic studies have shown that the two enzyme activities cannot be separated even after a considerable degree of purification (TOKES 1973; KOIVULA 1975). There is, however, much controversy about results concerning these enzymes. The aldehyde dehydrogenase oxidizing D-glucuronolactone has been found in one study to possess a micromolar Km value for acetaldehyde (TONKES & MARSH1973), while a millimolar value has been reported by KOIV~LA & KOIWSALO(1975a & b). Although D-glucuronolactone dehydrogenase is inducible and has a high apparent Km value for its substrate (HXNNINEN 1968; TONKES 1973), there are many differences between the activities of this enzyme and the inducible high-Km aldehyde dehydrogenase (DEITRICH 1971; MARSELQS1976). Unlike the cytosdic aldehyde dehydrogenase, Dglucuronolactone dehydrogenase is induced in all animals with no clear-cut genetic predisposition (MARSELDS 1976), and is extremely sensitive to disulfiram inhibition (MARSELDS 8 TORR~NEN 1976). The objectives of this study were 1) to find out if the low-Km enzymes of the cytosol and the mitochondria are inducible by phenobarbital, and 2) to define the amount of D-glucuronolactone which is in a free aIdehyde form under usual measurement conditions. Materials and methods Chemicals. Acetaldehyde, anisaldehyde, formaldehyde, phenobarbital and pyrazole were obtained from Merck AG (Darmstadt, W. Germany). Rotenone, EGTA (ethyleneglycol-bis-(3aminoethylether-N-N'-tetra-aceticacid) and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, U.S.A.). NAD was obtained from Boehringer & Sohne (Mannheim, W. Germany), D-glucuronolactone and propionaldehyde from Fluka AG (Buchs, Switzerland). All other reagents were of the best commercially available grade.

Animals. Adult male albino rats of the WistarJAf/Han/MoI/(Han 67) strain were used. The animals have been selectively developed into two genetically different substrains, according to the induction (RR, reactors) or not (IT,non-reactors) of the soluble high-Km et al. 1975; MARSELOS& PIETIKAINEN1975). In aldehyde dehydrogenase (MARSELOS

INDUCIBLE ALDEHYDE DEHYDROGENASES

265

these experiments, we used animals of the second and third filial generation, and in all cases the response to phenobarbital was of the rr- or RR-type, as predicted. All rats had a free access to commercial chow (Hankkija Ltd., Finland) and to tapwater, unless otherwise specified. For the induction experiments, phenobarbital was administered with the drinking water (1 mg/ml) for 7 days, or as indicated. Preparation of the tissues. The rats were stunned by a blow on the head, and were bled by cutting the cervical vessels. The excised livers were homogenized in 3 vol. of ice-cold KCI (0.15 M). In the experiments for the cytosolic enzymes, the hoinogenate was centrifuged at 10,000 x g for 20 min. An equal volume of calcium chloride (0.024 M, in 0.25 M sucrose) was added to the supernatant, and the suspension was stirred and left to stand in ice. After 10 min., it was centrifuged again at 10.000 X g for 20 rnin., in order to obtain the cytosolic fraction (LITERST et al. 1976). In the experiments for the comparison of the mitochondria1 and cytosolic enzymes, the respective fractions were separated by differential centrifugation as described by CHAPPEL& HANSFORD (1972). The mitochondria were washed three times with HEPES buffer (5 mM, pH 7.6) containing sucrose (0.25 M) and EGTA (1 mM). The mitochondrial pellet was solubilized in 1 ml of Na-deoxycholate (1 % in 0.15 M-KCI). Enzyme activities. All measurements were carried out in a Perkin-Elmer spectrophotometer (model 402), by following the formation of reduced NAD (extinction coefficient 6.22 X 109). Aldehyde dehydrogmase was measured in Na-pyrophosphate buffer (0.1 M, pH 8.0) at 25", with NAD as coenzyme (0.5 mM). Pyrazole (0.1 mM) was added to inhibit alcohol dehydrogenase, and rotenone (1 mM in methanol, 0.2 % of the final kolume) was added to mitochondria1 samples, in order to inhibit the enzyme NADH oxidase. Acetaldehyde (5 and 0.05 mM), propionaldehyde (5 and 0.05 mM), anisaldehyde (0.5 and 0.05 mM), benzaldehyde (0.05 mM), p-carboxybenzaldehyde (0.05 mM), formaldehyde (0.5 mM), glyoxal (0.05 mM) and D,L-glyceraldehyde (0.05 mM) were used as substrates. D-glucuronolactone dehydrogenase was measured in exactly the same conditions as above, with D-glucuronolactone (50 mM) as the substrate. Protein determination was carried out by the method of LOWRYe f al. (1951),and bovine serum albumin was used as standard. Statistical analysis of the results was performed with Student's t-test, and P values less than 0.025 were considered significant. Nuclear magnetic resonance studies ('H-NMR). The presence of D-glucuronaldehyde in a D-glucuronolactone solution was estimated by a Perkin-Elmer NMR spectrometer (model R 12 B) from a sample of D-glucuronolactone (50 mM) dissolved in heavy water. Furthermore, a JEOL PMX 60 NMR spectrometer was used for the detection of the aldehyde group in buffered solutions of D-glucuronolactone (5, 50 and 500 mM, in pyrophosphate buffer, 0.1 M, pH 8.0).

Results

Studies with IH-NMR spectrometry failed to show the existence of an aldehyde group in the solutions of D-glucuronolactone, whether made up in

266

R. TtiRRiSNEN, U. NOUSIAINEN AND M. MARSELOS

heavy water or in pyrophosphate buffer. The lack of a spectral peak typical of the aldehyde group indicates that less than 3 % of the D-glucuronolactone is converted into the aldehyde form according to the measurement conditions. This amount is below the sensitivity of the instruments used, so that its exact percentage is impossible to define. Induction of the high-Km aldehyde dehydrogenase by phenobarbital could be detected in the RR animals by using the substrates acetaldehyde, anisaldehyde, benzaldehyde, D-glucuronolactone and propionaldehyde. When the rr-substrain was used, an almost 2-fold increase could be detected in the enzyme activity of the cytosol, measured with D-glucuronolactone or with micromolar concentrations of acetaldehyde, anisaldehyde and propionaldehyde. The latter substrates did not give any statistically significant difference between control and phenobarbital-treated rr rats, when used in higher concentrations reflecting the total cytosolic activity. Finally, p-carboxybenzaldehyde, D,L-glyceraldehyde, glyolxal and formaldehyde could not be used as the substrate either for the induced low-Km enzyme (rr rats), or for the induced high-Km enzyme (RR rats) (table 1). In isolated mitochondria from rr rats, no increase of aldehyde dehydrogenase activity was found with millimolar or micromolar acetaldehyde con-

5 0-GIUAI

AC

AC

5 mM

L

5 0 mM

OOSmM

3

2

-

I

I!4 AC

0.05 mM

C

C

Pb

C

Pb

C

Pb

Pb

C

Pb

Fig. I . Oxidation of acetaldehyde (Ac) and D-glucuronolactone (D-GIUAL) by the cytosolic and mitochondria1 fractions of the liver of rr animals. Each substrate has been used in the indicated concentration, and measurenients have been carried out as described in Materials and Methods. Five control ( C ) and five phenobarbital-treated animals (Pb) are quoted (mean rt: S.D.). No measurable activity could be detected in the mitochondria with D-glucuronolactone as the substrate. The asterisk denotes P less than 0.025.

~

p-Carboxybenzaldehyde D,L-GI yceraldehyde Glyoxal Formaldehyde

Propionaldehyde

Benzaldehyde D-Glucuronolactone

Anisaldehyde

Acetaldehyde

Substrate

~~

~

~~

0.05 0.5

0.05 0.05

0.05 50 5 0.05

0.05

5 0.05 0.5

Concentration (mM)

t 0.1 (89 %) f 0.2 (116 %)

5.8 f 0.4 (93 ‘7%) 1.5 k 0.1 (107 %)

6.3 t 0.7 1.4 f 0.2 0.4 f 0.1 1.8 f 0.4 0.3 2.0

2.9 f 0.4 (116 %) 0.7 f 0.1 (164 %)* 2.8 f 0.1 (115 %) 1 h k 0.6 (541 %)* 3.2 t 1.2 (95 96) 1.3 f 0.2 (156 %)* 6.4 P 1.0 (110 %) 1.7 f 0.3 (179 %)*

~

2.5 f 0.5 0.4 f 0.1 2.4 P 0.7 0.3 f 0.1 3.4 f 0.2 0.9 f 0.1 5.8 k 1.2 1.0 f 0.1

~

Pb

rr Control

~

~

6.5 f 0.3 1.3 f 0.1 0.4 k 0.1 2.2 ? 0.3

0.8 P 0.1 7.3 k 1.7 1.2 P 0.3

f 0.5 k 0.1 3.0 f 0.5 0.5 f 0.1 4.3 f 0.3

Control 2.7 0.3

~

~~

Pb

0.5 2.5

f 0.1 (134 %) t 0.3 (116 76)

6.1 ? 0.2 (94 %) 1.5 P 0.1 (115 76)

26.3 P 5.1 (984 %)* 1 1 f 0.1 (327%)* 9.3 f 0.4 (305 %)* 8.9 f 1.7 (1896 %)* 30.3 f. 2.4 (874 %)* 2 1 k 0.3 (259 %)* 63.8 f 4.3 (874 %)* 4 5 t 0.4 (375 %)*

RR

Cytosolic aldehyde dehydrogenase activity measured with different substrates, in rats of the rr and RR substrains. Activities after phenobarbital (Pb) treatment are compared to those of the untreated control animals, and are quoted in parentheses as percentage change. The asterisks denote values of P less than 0.025,with regard to the respective controls. The data presented are the means ( f S.D.) of five animals (nmol NADH/rnin./mg protein).

Table 1

3

268

R. TeRRONEN, U. NOUSIAINEN AND M. MARSELOS

centration. In addition, no apparent activity could be shown in the mitochondria with D-glucuronolactme as the substrate. Despite the lack of induction of the mitochondria1 enzymes, the cytosol isolated from the same animals again had increased activities after phenobarbital treatment, when measured with D-glucuronolactone or with a micromolar acetaldehyde concentration (fig. 1). No induction of the mitochondrial enzymes could be detected when RR rats were used (results not presented). The pattern of induction of the low-Km aldehyde dehydrogenase and the D-glucuronolactone dehydrogenase was followed by monitoring the time response of the increase of the two enzyme activities after phenobarbital administration. Both substrates gave a similar rise of activity during the first four days of treatment, and the two activities almost reached the maximum of their induction after the fifth day (fig. 2).

I

2

3

5

6

days

Fig. 2. The induction of the cytosolic oxidation of D-glucuronolactone (50 mM, of acetaldehyde (0.05 mM, *---+). Each point represents the mean (k S.D.)of at least three animals of the rr substrain. Measurements have been done from controls (0 time), or from rats which had consumed 0.1 % phenobarbital solution for 1 to 5 days as indicated. Even after only one day of phenobarbital intake. there was a statistically significant difference from the controls (P 0.02.5).

M) and