Effects of ethanol treatment and castration on liver alcohol dehydrogenase activity

Alcohol, Vol. 2, pp. 39--41, 1985. c. AnkhoInternationalInc. Printed in the U.S.A.

0741-8329/85$3.00 ~- .00

Effects of Ethanol Treatment and Castration on Liver Alcohol Dehydrogenase Activity R A C H E L B. G I L L I O N , K A T H R Y N E. C R O W , 1 R I C H A R D D. B A T T A N D M I C H A E L J. H A R D M A N

D e p a r t m e n t o f Chemistry, Biochemistry and Biophysics, M a s s e y University, Palmerston North, N e w Zealand

GILLION, R. B., K. E. CROW, R. D. BATT AND M. J. HARDMAN. Effects of ethanol treatment and castration on liver alcohol dehydrogenase activity. ALCOHOL 2(1) 39--41, 1985.--Induction of alcohol dehydrogenase (ADH) activity by chronic ethanol treatment and castration has previously been reported to occur in Sprague-Dawley rats. In the present study, no induction was found following chronic ethanol treatment and only a low level of induction was found with castration. However the activity of ADH was high in control animals compared with those used in other studies. The activity of ADH in control animals was not decreased by testosterone administration, which has been shown to reverse induction of the enzyme produced by chronic ethanol treatment or castration in other studies. It is concluded that the male Sprague-Dawley rat is not necessarily a suitable animal for the study of ADH induction by chronic ethanol treatment and that further unknown factors must be identified before the regulation of ADH activity in vivo is fully understood. Alcohol dehydrogenase Sprague-Dawley rats Testosterone administration

Chronic ethanol treatment

IT has been concluded by Cicero et aL that the male Sprague-Dawley rat is a suitable animal in which to study the effects of chronic ethanol administration on liver alcohol dehydrogenase (ADH) activity [2]. This conclusion was based on findings of consistent, significant induction of the enzyme with both chronic ethanol treatment and castration [1,2]. The present study began with the aim of reproducing these results to further our research on factors regulating the rate of ethanol metabolism.

Castration

weighed and then homogenised in 3 volumes of 0.05 M Hepes buffer pH 8.4 [3]. The activity of ADH was measured in the supernatant fraction after centrifugation of the homogenates at 34,000 g for 45 minutes. The assay method has been described previously [3]. Protein concentrations in the supernatant fractions were determined by the Lowry method [5]. RESULTS

Chronic Ethanol Administration Rats which received 5% ethanol as their sole fluid drank similar volumes to those taken by control animals which received water. Both ethanol drinking and control animals ate similar quantities of food and showed similar growth curves (Fig. i). The average amount of ethanol consumed was 7 g per kg per day. The concentration of ethanol in the solution provided for the ethanol-drinking animals was critical. If rats were given 10% ethanol, they drank only sufficient fluid to give about the same ethanol intake, 7 g per kg per day. Therefore their fluid intake was halved, the amount of food eaten decreased, and the animals did not thrive. At the end of 14 days, ethanol-consuming and control rats were killed and liver ADH activity was determined. Table 1 shows that there were no significant differences in enzyme activity between controls and ethanol-consuming animals. This was true whether results were expressed as activity per gram wet weight of liver, per liver, or per mg protein.

METHOD The animals used in the present study were male Sprague-Dawley rats, with an initial weight of 100 g. The animals were housed in a temperature-regulated room (24°C) with artificial light providing a 12 hr light/dark cycle. They were provided with a dry pellet diet ad lib. For experiments on chronic ethanol treatment, the rats were provided with 5% ethanol (weight/volume) as the sole drinking fluid. Control animals received water. Castration was carried out under ether anesthesia and control animals were given a sham operation. Testosterone was administered by a subcutaneous injection of 0.5 mg of testosterone propionate per day. The control animals received an injection of vehicle only (50 ~1 of corn oil). At the end of each experimental period, the rats were killed by cervical dislocation, the livers rapidly removed,

~Requests for reprints should be addressed to K. E. Crow.

39

40

GILLION, CROW, BATT AND HARDMAN

200-

,

TABLE 1 LIVER ALCOHOL DEHYDROGENASE ACTIVITY IN ETHANOL-DRINKING* AND CONTROLt RATS

control

o etl~nol 180

ADH Activity:~

o~ -.c-

160

~

140

L-

> <

12C

0

2

/-,

6

8

10

12

14

o- moles per rain per g liver

# moles per rain per liver

n moles per rain per mg protein

Experiment 1 Controls Ethanol fed

4.0 ± 0.3 4.4 ± 0.3

36 : 2 37 ± 3

35 ± 2 36 ± 3

Experiment 2 Controls Ethanol fed

4.6 ± 0.3 4.0 ± 0.4

39 _ 3 33 _+ 3

38 ± 2 33 ± 3

Doy FIG. 1. Weight gains for ethanol-drinking and control rats. Ethanol-drinking animals were provided with 5% (w/v) ethanol as their sole fluid, control animals received water. Rats were selected so that the average initial weights for the two groups differed by less than 2 g. Results are means±SEM for l0 animals in each group.

*5% ethanol for 14 days. tWater for 14 days. :]:Results are expressed as means_SEM, with 10 rats in each group.

TABLE 2 THE EFFECT OF CASTRATION ON ADH ACTIVITY

TABLE 3 THE EFFECT OF TESTOSTERONE ADMINISTRATION ON ADH ACTIVITY

ADH Activity:[:

ADH Activity*

Experiment 1" Sham-operated controls Castrates Experiment 2t Sham-operated controls Castrates

~t moles per rain per g liver

~ moles per rain per liver

n moles per rain per mg protein

3.9 ± 0.2

41 +_ 2

33 ± 2

4.5 ± 0.5

41 ± 2

40 ± 4

5.0 ~ 0.3

53 ± 3

41 ± 3

6.0 ~ 0.3

53 ± 2

49 ± 2

Treatment Testosterone Corn-oil controls

~t moles per min per g liver

p, moles per rain per liver

n moles per rain per mg protein

4.9 __. 0.4 4.7 +_ 0.2

32 -+ 2 32 ± 1

45 ± 3 44 ± 2

*Results expressed as means ± SEM, n=8.

*n=9, t n = 10. SResults are expressed as means _+ SEM.

Castration C a s t r a t e d and s h a m - o p e r a t e d a n i m a l s were m a i n t a i n e d on s t a n d a r d l a b o r a t o r y diet a n d w a t e r ad lib for 16 ( E x p e r i m e n t I) o r 17 ( E x p e r i m e n t 2) d a y s following surgery. Weight g a i n s w e r e m o n i t o r e d , with c a s t r a t e d a n i m a l s s h o w i n g signific a n t l y l o w e r a v e r a g e gains te.g., E x p e r i m e n t 1, day 16, controls 2 3 1 ± 8 ; c a s t r a t e s 207_+41. O n day 16 or 17 the a n i m a l s w e r e killed a n d liver A D H activity w a s m e a s u r e d . C a s t r a t e d a n i m a l s s h o w e d slightly higher A D H activity t h a n c o n t r o l s w h e n the activity w a s c a l c u l a t e d as t t m o l e s o f e t h a n o l o x i d i s e d p e r m i n u t e p e r g wet weight o f liver o r p e r mg p r o t e i n (Table 2). T h e i n c r e a s e was not statistically signific a n t in E x p e r i m e n t 1, but was significant (,o<0.05) in E x p e r i m e n t 2. H o w e v e r the A D H activity p e r total liver w a s iden-

tical in c o n t r o l and c a s t r a t e d a n i m a l s in b o t h e x p e r i m e n t s (Table 2). T h i s w a s b e c a u s e the c a s t r a t e d a n i m a l s had smaller livers. T h e p e r c e n t a g e d e c r e a s e in liver size in cast r a t e d a n i m a l s w a s the same as the p e r c e n t a g e d e c r e a s e in b o d y weight, so that liver w e i g h t / b o d y weight ratios rem a i n e d t h e same as for c o n t r o l s .

Testosterone Administratitm Following a d m i n i s t r a t i o n of t e s t o s t e r o n e for 6 d a y s , A D H activity w a s m e a s u r e d in livers from t r e a t e d and c o n t r o l animals. T a b l e 3 s h o w s that t h e r e was n o difference in A D H activity b e t w e e n the t w o groups.

DISCUSSION In c o n t r a s t with o t h e r r e c e n t s t u d i e s [2, ! I, 12], the activity o f liver A D H in the p r e s e n t s t u d y was not i n d u c e d by c h r o n i c e t h a n o l t r e a t m e n t . Also, t h e small i n c r e a s e in A D H activity with c a s t r a t i o n (15-21%, T a b l e 2) was less t h a n t h a t o b s e r v e d in o t h e r s t u d i e s [ 1 , 2 , 8] (35-70%, see T a b l e 4) u s i n g the s a m e strain o f rats and t h e s a m e A D H assay. H o w e v e r ,

EFFECTS OF ETHANOL AND CASTRATION ON ADH TABLE 4 ADH ACTIVITY IN OTHER STUDIES ADH activity* Reference No. 1 2 2 8 12 This studyt This study?

Controls 1.02 1.4 1.6 2.7 1.03 4.2 4.45

--- 0.02 --- 0.1 ±0.1 ±0.1 ± 0.08 ± 0.4 ± 0.3

Chronic Ethanol

Castrates

-2.15 --- 0.1 --1.58 ± 0.07 4.3 ± 0.3 --

1.38 --- 0.05 -2.3 _ 0 . 1 4.6 ± 0 . 1 --5.25 ± 0.4

*/zMoles per min per g wet weight liver. *Averages of results from Experiments i and 2 are shown.

Table 4 shows that liver A D H activity in control animals in the present study was as high as, or e v e n higher than, the activity found after induction (by chronic ethanol treatment or by castration) in other studies [1, 2, 8, 12]. This suggests that in o u r animals A D H activity was already ' i n d u c e d . ' Such induction could be due to dietary factors, o r perhaps to e n v i r o n m e n t a l stress [9]. W h a t e v e r the explanation o f the high control activities in the present study, it appears that e x p e r i m e n t a l induction o f A D H may be more likely if the initial activity o f the e n z y m e is low. This conclusion is supported by the results o f a n o t h e r study [4] in which it was

41 shown that animals with low initial rates o f ethanol metabolism s h o w e d the greatest increase in rate following chronic ethanol treatment. O t h e r studies have shown that ethanol or castrationinduced increases in A D H activity can be r e v e r s e d by administration of testosterone [1, 2, 8, 11]. H o w e v e r , testosterone treatment did not d e c r e a s e A D H activity in the present study. That testosterone status is not the only regulator o f A D H activity is also suggested by o t h e r e x p e r i m e n t s [2] in which changes in serum testosterone levels did not always result in changes in liver A D H activity. A n u m b e r o f o t h e r h o r m o n e effects on A D H activity have been reported recently [6, 7, 10] and the overall hormonal control picture for the e n z y m e may p r o v e to be very complex. The results of this study show that the activity o f liver A D H in Sprague-Dawley rats is not always increased by chronic ethanol treatment. T h u s this strain is not necessarily more suitable than any o t h e r for the study of the effects o f chronic ethanol treatment on A D H activity. Until all the factors influencing A D H activity are clearly identified, the answer to the question 'is A D H induced by chronic ethanol t r e a t m e n t ' must remain ' s o m e t i m e s . '

ACKNOWLEDGEMENTS We would like to thank Mr. J. E. Ormsby for carrying out the surgical procedures, and Ms. K. M. Stowell for helpful discussions. This work was supported by the Medical Research Council of New Zealand.

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7. Mezey, E. and J. J. Potter. Effects of thyroidectomy and triiodothyronine administration on rat liver alcohol dehydrogenase. Gastroenterology 80: 566-574, 1981. 8. Mezey, E., J. J. Potter, S. M. Harmon and P. D. Tsitouras. Effects of castration and testosterone administration on rat liver alcohol dehydrogenase activity. Biochem Pharmacol 29: 3175-3180, 1980. 9. Mezey, E., J. J. Potter and R. Kvetnansky. Effect of stress by repeated immobilisation on hepatic alcohol dehydrogenase activity and ethanol metabolism. Biochem Pharmacol 2g: 657-663, 1979. 10. Mezey, E., J. J. Potter and P. D. Tsitouras. Liver alcohol dehydrogenase activity in the female rat: Effects of ovariectomy and estradiol administration. Life Sci 29: 1171-1176, 1981. 11. Rachamin, G., J. A. Macdonald, S. Wahid, J. J. Clapp, J. M. Khanna and Y. Israel. Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Biochem J 186: 483--490, 1980. 12. Wahid, S., J. M. Khanna, F. J. Carmichael, K. O. Lindros, G. Rachamin and Y. Israel. Alcohol-induced redox changes in the liver of the spontaneously hypertensive rat. Biochem Pharmacol 30: 1277-1282, 1981.