Gen. Pharmac. Vol. 16, No. 1, pp. 31-35, 1985 0306-3623/85 $3.00+0.00 Printed in Great Britain Pergamon Press Ltd

INFLUENCE OF ETHANOL UPON THE INTRODUCTION OF TYROSINE AMINOTRANSFERASE IN LIVER,

UPON THE NAD CONTENT IN LIVER AND BRAIN, AND UPON THE ACTIVITY OF

GLUTAMATE OXALATE AMINOTRANSFERASE AND GLUTAMATE PYRUVATE AMINOTRANSFERASE

IN THE SERUM OF RATS

HANS KROGER, REGINA GR~TZ and HELGA GRAHN Robert Koch-Institute, Nordufer 20, D-1000 Berlin 65, West Germany (Tel: (030)4503-230)

(Received 25 July 1984)

Abstract--1. The influence of ethanol, tryptophan, nicotinamide and methionine upon the activity of glutamate oxalate aminotransferase and glutamate pyruvate aminotransferase in serum as well as the induction of tyrosine aminotransferase in the liver, and of NAD ÷ + NADH in liver and brain are described.

2. After oral application of 6 g/kg ethanol, the activities of the examined enzymes and the concentrations of NAD ÷ + NADH in the brain as well as in the liver did not change over a period of 8 hr.

3. Administration of L-methionine lead--as is the case with DL-tryptophan--to a decrease of the NAD ÷ + NADH-concentration in the brain.

4. A simultaneous application of nicotinamide, OL-tryptophan, L-methionine of 1-methylnicotinamide together with ethanol caused a significant increase of the tyrosine aminotransferase induction in adrenalectomized animals.

5. Ethanol reduced the activity of the ADPR transferase in the nuclei of rat liver cells.

INTRODUCTION

Epidemiological investigations have clearly shown that there is a relationship between ethanol uptake and liver damage (Egh6je and Juhl, 1973). The causes of such liver damage are still unknown. Acute toxic doses of ethanol result in an enhanced induction of the t ryptophan oxygenase (M~rland, 1974) in liver. The application of ethanol over an extended longer period, however, led to a decrease of this enzyme 0Vl~dand, 1973; Badaway and Evans, 1973). The inducibility of the tyrosine aminotransferase (TAT) was also diminished in the liver of animals chroni- cally treated with ethanol (M~rland, 1974).

In the course of our investigations on the influence of different factors upon the metabolism of the rat liver, we measured the inducibility of T A T under the influence of ethanol under acute conditions. We also determined the influence of ethanol on the serum concentration of glutamate oxalate aminotransferase (GOT) and glutamate pyruvate aminotransferase (GPT) and N A D + + N A D H contents in liver and brain. Moreover , the activity of the A D P R transfer- ase in liver was analyzed.

MATERIALS AND METHODS

Animals

Rats of Wistar strain (Zentrale Versuchstierzucht des Bundesgesundheitsamtes, Berlin) were used. The adrenal- ectomized animals were administered 0.15 M NaCI over a period of 5 days after the operation. They then were used. Ethanol diluted to 50% with 0.15 M NaCI, was applied

orally. All the other substances were administered intra- peritoneaUy 4 hr before and together with ethanol. The animals were killed 8 hr after the ethanol administration.

Materials

Ethanol, nicotinamide (NA), OL-tryptophan (Merck, Darmstadt); 1-methyl-nicotinamide (Sigma, St Louis, MO); L-methionine (Schwarz/Mann, Orangebourg, N J); GOT and GPT test kits (Boehringer, Mannheim).

Tyrosine aminotransferase

The activity of the enzyme in liver was measured as described before (Kr6ger and Greuer, 1965). The protein content was determined according to the Biuret method (Gornall et al., 1949); it is given as #mol p-hydroxyphenyl pyruvate/hr × mg protein.

Glutamate oxalate aminotransferase and glutamate pyruvate amino t r ansf eras e

These enzymes were determined using the test kit from Boehringer (Mannheim) (Bergmeyer, 1974). Values are given as mU/ml.

N A D + + N A D H content

The NAD + + NADH-content was determined according to Nisselbaum and Green (1969). Values are given as /zmol/g liver and brain.

Cell nuclei

Liver cell nuclei were isolated as described by Blobel and Potter (1966).

ADPR transferase activity

The enzyme activity was determined according to Kidwell and Burdette (1974).

G.P. 16/l~ 31

32 HANS KR()GER et al.

Table 1. Influence of ethanol and 0.15 M NaCI on the activity of GOT and GPT in the serum, that of TAT and NAD + + N A D H content in the liver and the brain. For further details see Methods

Number GOT GPT TAT NAD + + N A D H Type of Treatment of (mU/ml) (mU/ml) (#mol/hr/mg protein) (/lmol/g) animals with animals serum serum liver liver brain

Adrenalectomized ethanol 4 104 + 22.0 12 -+ 5.0 0.64 -+ 0.29 0.79 _+ 0.00 0.24 -+ 0.02 (6 g/kg p.o.)

Intact ethanol 8 86 -+ 12.4 12 _+ 1.9 0.61 -+ 0.22 0.71 _+ 0.10 0.22 _+ 0.05 (6 g/kg p.o.)

Adrenalectomized 0.15 M NaCl 4 83 _ 6.4 9 _+ 0.6 0.55 + 0.07 0.83 ± 0.10 0.22 ± 0.02 (10 ml/kg i.p.)

Intact 0.15 M NaCl 4 8 6 + 11.4 9-+0.8 0.51 +0 .07 0.75-+0.09 0.21 -+0.02 (10 mlg/kg i.p.)

RESULTS

Influence of ethanol Ethanol administered orally to both adrenal-

ectomized and intact animals cause only minor changes in the activities of the enzymes tested (Table 1). The same holds for the NAD + + NADH content in liver and brain.

Influence of ethanol and nicotinamide Nicotinamide is known to increase the NAD con-

centration. We found the increase to be higher in adrenalectomized animals than in intact ones (Table 2). Moreover, Table 2 shows that in adrenal- ectomized animals the simultaneous administration of ethanol and nicotinamide causes significantly higher increase of the TAT activity than nicotinamide alone (see also Krrger and Gr/itz, 1978).

Influence of ethanol and OL-tryptophan As can be seen from Table 3, DL-tryptophan raises

the activity of TAT (see also Krrger et al., 1968; Krrger and Gr/itz, 1979). Ethanol enhances this effect. This combination was bound to be very toxic for adrenalectomized animals. After administration of DL-tryptophan to intact animals, there was a slight increase of the GOT and GPT activities in serum. Moreover, the nicotinamide adenine dinucleotide (NAD) content in the brain is reduced under influence of tryptophan.

Influence of ethanol and L-methionine Simultaneous administration of ethanol and

L-methionine increases the TAT activity of the liver. The NAD content in brain, however, decreases under the influence of L-methionine alone (Table 4).

Influence of ethanol, nicotinamide, and L-methionine In normal animals, the enzymes GOT and GPT in

serum are slightly increased under this treatment. With this combination the induction of the TAT in adrenalectomized animals is significantly enhanced by the administration of ethanol (Table 5).

Influence of ethanol, DL-tryptophan, and L-methionine The TAT activity rises significantly in the adrenal-

ectomized animals (Table 6), if ethanol, OL-trytophan and L-methionine are administered in combination. Here, too, the NAD concentration in brain is dimin- ished in comparison to normal animals (Table 1).

Influence of ethanol and l-methyl-nicotinamide It is remarkable that the GOT and GPT activities

proved to be relatively high under treatment with 1-methyl-nicotinamide (Table 7). Also, in the brain of all animals and in the liver of intact animals, a lower NAD content than in untreated animals was found (compare Table 1). The combination of ethanol and l-methyl-nicotinamide is toxic for the adrenal- ectomized animals.

Table 2. Influence of ethanol and nicotinamide (NA) on the activity of GOT and GPT in the serum, that of TAT and N A D + + N A D H content in the liver and the brain, For further details see Methods

Number GOT GPT TAT NAD + + N A D H Type of Treatment of (mU/ml) (mU/ml) (/~mol/hr/mg protein) ~mol /g ) animals with animals serum serum liver liver brain

Adrenalectomized ethanol 4 108 + 48. I 16 + 7.1 3.86 + 0.74 3.67 4- 0.67 0,38 + 0.06

Intact

Adrenalectomized

Intact

(6 g/kg p.o.) + NA (500 mg/kg i.p.)

ethanol 4 113 _+ 51.9 15 _+ 2.8 (6 g/kg p.o.) + NA (500 mg/kg i.p.)

NA 4 88 + 12.3 l l + 1.0 (500 mg/kg i.p.)

lqA 4 106_+09.1 12_+1.2 (500 mg/kg i.p.)

4 .30+ 1.66 2.51 +0.61 0 .38+0 .14

1.83 __+ 0.48 3.45 + 0.70 0.46 -t- 0.02

3.39 __ 0.65 1.83 __+ 0.60 0.24 ____ 0.08

Effect of ethanol upon the liver metabolism 33

Table 3. Influence of ethanol and DL-tryptophan on the activity of GOT and GPT in the serum, that of TAT and NAD + + NADH content in the liver and the brain. For further details see Methods

Number GOT GPT TAT NAD + + NADH Type of Treatment of (mU/ml) (mU/ml) (~mol/hr/mg protein) (pmol/g) animals with animals serum serum liver liver brain

Adrenalectomized ethanol (6 g/kg p.o.) DL-tryptophan (300 mg/kg i.p.)

Intact ethanol (6 g/kg p.o.) + DL-tryptophan (300 mg/kg i.p.)

Adrenalectomized VL-tryptophan (300 mg/kg i.p.)

Intact DL-tryptophan (300 mg/kg i.p.)

1 94 I! 4.03 1.30 0.10

3 of the employed animals died

4 119±12.8 19±2.8 3.15±1.86 0.89±0.10 0.16±0.02

3 103± 11.7 12±3.1 0.75±0.03 i.46±0.10 0.10+0.05

4 140±26.7 23±2.5 i.39±0,46 0.96±0.08 0.15±0.02

Table 4. Influence of ethanol and L-methionine on the activity of GOT and GPT in the serum, that of TAT and NAD + + NADH content in the liver and the brain. For further details see Methods

Number GOT GPT TAT NAD + + NADH Type of Treatment of (mU/ml) (mU/ml) (pmol/hr/mg protein) ~mol/g) animals with animals serum serum liver liver brain

Adrenalectomized ethanol 4 84± 12.3 9± !.3 0.88±0.12 0.89 ± 0.19 0.17±0.06

Intact

Adrenalectomized

Intact

(6 g/kg p.o.) + L-methionine (300 mg/kg i.p.)

ethanol 4 108 ± 21.5 14 ± 2.0 (6 8/kg p.o.) + L-methionine (300 mg/kg i.p.)

L-methionine 4 il4+7.1 i1±0.6 (300 mg/kg i.p.)

L-methionine 4 106 ± 42.2 23 + 14.6 (300 mg/kg i.p.)

2.49-t- 1 . 37 0.77±0.10 0.19±0.09

0.33+0.05 0.72+0.11 0.19 +0.09

0.84+0.29 0.74 + 0.06 0.13 +0.04

Influence of ethanol upon the ADPR transferase activ- ity

Two hours after the administration of ethanol, the ADPR tranferase activity is distinctly lower than in the controls (see Table 8). Tn this connection, the effect o f some other substances was tested, too.

DISCUSSION

Earlier investigations with tyrosine amino- transferase have shown that tryptophan, nicotin- amide, and especially the combination of tryptophan and L-methionine caused an induction of this enzyme (Kr6ger and Grfitz, 1978; Kr6ger et al., 1968; Kr6ger

Table 5. Influence of ethanol, nicotinamide (NA), and L-methionine on the activity of GOT and Girl ̀ in the serum, that of TAT and NAD + + NADH content in the liver and the brain. For further details see Methods

Number GOT GPT TAT NAD + + NADH Type of Treatment of (mU/ml) (mU/ml) (pmol/hr/mg protein) (pmol/g) animals with animals serum serum fiver liver brain

Adrenalectomized ethanol 4 95 + 35.3 12 + 4.8 3.14 + 0.36 2.71 + 1.02 0.22 + 0.03 (6 g/kg p.o.) + NA (500 mg/kg i.p.) + L-methionine (300 mg/k 8 i.p.)

Intact ethanol 4 148+ 17.6 21 +3.7 6.47+2.64 1.55+0.36 0.26+0.10 (6 g/kg p.o.) + NA (500 mg/kg i.p.) + L-methionine (300 mg/kg i.p.)

Adrenalectomized NA 4 109 5:7.2 14 + 3.2 1.17 + 0.22 2.58 + 0.36 0.28 + 0.18 (500 mg/kg i.p.) + L-methionine (300 mg/kg i.p.)

Intact NA 4 115+ 16.6 19 + 3.4 4.74+i.77 3.32+1.27 0.24+0.11 (500 mg/kg i.p.) + L-methionine (300 mg/kg i.p.)

34 HANS KROGER et al.

Table 6. Influence of ethanol, DL-tryptophan, and L-methionine on the activity of GOT and GPT in the serum, that of TAT and NAD + + NADH content in the liver and the brain. For further details see Methods

Number GOT GPT Type of Treatment animals with

Adrenalectomized ethanol (6 g/kg p.o.) + OL-tryptophan (300 mg/kg i.p.) + L-methionine (300 mg/kg i.p.)

Intact ethanol (6 g/kg p.o.) + DL-tryptophan (300 mg/kg i.p.) + L-methionine (300 mg/kg i.p.)

Adrenalectomized DL-tryptophan (300 mg/kg i.p.) + L-methionine (300 mg/kg i.p.)

Intact OL-tryptophan (300 mg/kg i.p.) + L-methionine (300 mg/kg i.p.)

of (mU/ml) (mU/ml) animals serum serum

4 109 _+ 17.9 12 _+ 3.0

TAT NAD + + NADH (/~ mol/hr/mg protein) (~ mol/g)

liver liver brain

3.65_+0.68 1.06_+0.19 0.14_+0.08

4 126-+14.7 18_+4.8 4.36-+0.99 0.81_+0.15 0.13-+0.03

4 83-+16.4 9-+0.5 0.66-+0.12 1.01+0.19 0.17+.03

4 117_+37.3 18+__1.9 2 .56__+1 .59 0.83__+0.17 0.14-+0.05

Table 7. Influence of ethanol and l-methyl nicotinamide (I-MNA) on the activity of GOT and GPT in the serum, that of TAT and NAD ÷ + NADH content in the liver and the brain. For further details see Methods

Number G O T GPT TAT NAD + + NADH Type of Treatment of (mU/ml) (mU/ml) (llmol/hr/mg protein) (/tmol/g) animals with animals serum serum liver liver brain

2.68 1.05 0.18 Adrenalectomized ethanol 2 145 14 (6 g/kg p.o.) + I -MNA 2 of the 4 employed animals died (500 mg/kg i.p.)

Intact ethanol 4 134 _+ 22.7 19 _+ 2.9 1.04 -+ 0.86 (6 g/kg p.o.) + 1-MNA (500 mg/kg i.p.)

I-MNA (500 mg/kg i.p.)

I-MNA (500 mg/kg i.p.)

Adrenalectomized

Intact

0.85_+0.08 0.15+0.03

4 96_+ 12.7 II _+ 1.7 0.47_+0.03 0.75_+0.07 0.21 +0.14

4 161-+41.8 27-+9.1 1.14_+0.40 0.43_+0.34 0.14_+0.04

a n d Gr/ i tz , 1979). F r o m these s tud ie s we c o n c l u d e d t h a t N A D is i nvo l ved in the r e g u l a t i o n o f th is e n z y m e . Poss ib le , the p o l y - A D P R a n d the A D P - r i bose de r ived f r o m t he se were effective here, s ince a n i n v o l v e m e n t o f t hese s u b s t a n c e s in t he r e g u l a t i o n a n d d i f f e ren t i a t ion , respec t ive ly , h a s been a s s u m e d (C ap - l an a n d R o s e n b e r g , 1975; R a s t l a n d Swet ly , 1978). U n d e r the in f luence o f e t h a n o l , t he T A T i n d u c t i o n c a u s e d by D L - t r y p t o p h a n was e n h a n c e d especia l ly in t he a d r e n a l e c t o m i z e d a n i m a l s .

Table 8. Influence of ethanol and other substances upon the ADPR transferase activity in the liver cell nuclei. The animals were killed 2hr after administration of the

substances

cpm/mg DNA

NaCI (0.15 M; 10 ml/kg i.p.) 7538 Ethanol (6 g/kg p.o.) 5616 Ethanol (6 g/kg p.o.)

+ L-tryptophan (300 mg/kg i.p.) 6945 + L-methionine (300 mg/kg i.p.)

L-tryptophan (300 mg/kg i.p.) 6275 + L-methionine (300 mg/kg i.p.)

Ethanol (6 g/kg p.o.) 6297 + Nicotinamide (500 mg/kg i.p.)

Nicotinamide (500 mg/kg i.p.) 7000

T h i s effect was even in tens i f ied by L - m e t h i o n i n e . T h e N A D + + N A D H c o n t e n t was h a r d l y a l t e red u n d e r these a c u t e c o n d i t i o n s . A d i f fe ren t i a t ed d e t e r m i n a t i o n s h o w e d t h a t e t h a n o l ch ron i ca l l y a d m i n i s t e r e d ove r a pe r i od o f 14 d a y s c a u s e d a dec r ea se o f N A D + a n d a n inc rease o f N A D H ( P u n j a n i et al., 1979).

T h i s effect as well as a r e d u c t i o n o f the t r y p t o p h a n - o x y g e n a s e ac t iv i ty c o u l d be p r e v e n t e d by a s i m u l t a n e o u s a d m i n i s t r a t i o n o f p y r a z i n a m i d e ( P u n - j an i et al., 1979). W h e n a n a l y z i n g t he A D P R t r ans fe r - ase, we f o u n d t he ac t iv i ty o f th is e n z y m e to b e c o m e dec r ea sed by e t h a n o l . W e bel ieve t h a t the effect o f the e t h a n o l is d u e to an in t e r f e rence wi th the N A D - a d e n o r i b o s y l a t i o n m e t a b o l i s m . In a d r e n a l - e c t o m i z e d a n i m a l s t he r e was a p r o n o u n c e d effect. T h i s m i g h t be d u e to the fac t t h a t t he co r t i co s t e ro id s h a v e a n in f luence o n t he A D P R t r ans f e r a se .

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