0145-6008/96/2001-0047A$03.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 20, No. 1 February 1996

Effect of Vitamin E Deficiency on Inhibition of Liver Regeneration by Long-Term Administration of Alcohol

Takashi Tanaka, Yasutaka Goto, Miki Imano, Hirohide Asai, Takao Yamada, Shuji Kawai, Satoru Kunitoh, Kyoko Kondo, Terumi Yamashita, Takeyuki Monna, Shuhei Nishiguchi, Tetsuo Kuroki, and Shuzo Otani

The effects of vitamin E (VE) deficiency on liver regeneration sup- pressed by long-term administration of alcohol were studied. Male rats were divided into two groups: the alcohol group and the control group. In addition, each group was subdivided into two groups ac- cording to the presence or not of VE. Altogether, four groups were provided: a group maintained on the VE-deficient alcohol diet (group EA), a group maintained on the VE-deficient control diet (group EC), a group maintained on the ordinary alcohol diet (group A), and a group maintained on the ordinary control diet (group C). After pair- feeding for 6 weeks, partial hepatectomy was performed to deter- mine the ornithine decarboxylase (ODC) activity, polyamine levels, lipid peroxide levels, and DNA synthesis. DNA synthesis at 24 hr after partial hepatectomy was suppressed significantly in the alcohol ad- ministration group, regardless of the presence or not of VE. DNA synthesis at 48 hr after partial hepatectomy tended to show low values in group EA, compared with group A. As for the hepatic ODC activity, group EA showed the lowest value at 4 hr after partial hep- atectomy. Of polyamines, the putrescine level In group EA at 4 hr after partial hepatectomy was significantly low, compared with the other three groups. The levels of spermidine and spermine de- creased by long-term administration of alcohol, but the effect of VE deficiency was not found. The lipid peroxide level increased signifi- cantly in the VE-deficient diet administration group, but the effect of alcohol administration was not found. These results suggested that the decrease in putrescine after ODC suppression by VE deficiency in addition to the decrease in spermidine and spermine caused by long-term alcohol administration were concerned with suppression of DNA synthesis later.

Key Words: Alcohol, Vitamin E, Liver Regeneration, Lipid Peroxide.

ANY OF the patients with alcoholic liver diseases are M accompanied by the nutrition disorder caused by the deficiency of protein and various vitamins. Particularly, vitamin E (VE) has an action to catch an active oxygen produced in the metabolism of alcohol and deter the chain reaction of peroxidation. It has been reported that a defi- ciency of VE is concerned with the progression of alcoholic liver disorder in a long-term alcohol administration model in rats.' On the other hand, in the relationship between alcohol and liver regeneration, it has been reported that long-term alcohol administration suppresses the liver re-

From the Department of Public Health (T.T., YG., MI., HA. , Ta.Y, Sh.R, Sa.R, RK, Te.Y, T.M.), Third Department of Internal Medicine (S.N., TIC), and Second Department of Biochemistry (S.O.), Osaka City University Medical School, Osaka, Japan.

Received for publication August 18, 1995; accepted November 16, 1995 Reprint requests: Takashi Tanaka, M.D., Department of Public Health,

Osaka City University Medical School, 1-4-54, Asahi-machi, Abeno-ku, Osaka, 545 Japan.

Copyright 0 1996 by The Research Society on Alcoholism.

Alcohol Clin Exp Res, Vol20, No 1, 1996: pp 47A-50A

generation via the polyamine metabolism in the process of liver regeneration after partial hepatectomy in rats.273

In the present study, we used VE-deficient diets concur- rently and studied the effect of VE deficiency on the sup- pression of liver regeneration by long-term alcohol admin- istration measuring the DNA synthesis and polyamine metabolism as indicators of liver regeneration.

MATERIALS AND METHODS

Experimental Design

Six-week-old male Wistar rats were purchased from Clea Japan, Inc. (Tokyo, Japan) and were divided into four groups: control group main- tained with a control liquid diet (group C), alcohol group maintained with an alcohol iiquid diet (group A), group EC maintained on a VE-deficient control liquid diet with VE removed from the control diet, and group EA maintained on a VE-deficient alcohol liquid diet with VE removed from the alcohol diet. All of the liquid diets were purchased from the Oriental Yeast Co. (Tokyo, Japan) and, with the alcohol liquid diet, 36% of the total calories were provided by ethanol. After pair-feeding for 6 weeks, the animals in the group given alcohol (groups A and EA) were starved for 16 hr to eliminate the most recent effects of alcohol. The animals in groups C and EC were starved at the same time. Approximately 70% of the liver was then resected (partial hepatectomy) by the method of Higgins and Anderson4 under light ether anesthesia. As for the diet after partial hepatectomy, the control diet was given to all the groups to eliminate the influence of acute alcohol administration (i.e., the control diet given to group A and the VE-deficient control diet to group EA). At the time of operation, VE levels, lipid peroxide levels, and polyamine levels were measured in the resected liver. Ornithine decarboxylase (ODC) activity and polyamine levels were assayed in the regenerating liver at 4 and 24 hr after surgery. DNA synthesis in the regenerating liver was measured at 24 and 48 hr after surgery.

Assay of DNA Synthesis [3H]Thymidine (10 gCi/lOO g body weight) was injected into rats

intraperitoneally 1 hr before they were killed at 24 or 48 hr after partial hepatectomy. One hour later, the remaining liver was removed and ho- mogenized with 4 volumes of cold distilled water, and then 1 ml of the homogenate was mixed with an equal amount of 10% trichloroacetic acid (TCA). The mixture was centrifuged at 800 X g for 10 min. The precipitate was washed three times, suspended in 5% TCA, and heated at 90°C for 15 min to solubilize the DNA. Radioactivity of the acid-insoluble fraction was counted with a liquid scintillation counter.

Assay of ODC Activity

The liver was homogenized in 3 volumes of 50 mM Tris-HC1 buffer (pH 7.5) containing 0.1 mM EDTA and 1 mM dithiothreitol. The homogenate was centrifuged at 30,000 X g for 30 min, and the supernatant was assayed for ODC-activity by the measurement of the 14C0, produced from DL-[~- ''C]ornithine by the method of Murakami et a1.'

47A

48A TANAKA ET AL

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. A .A

EC 15000 EC

El EA la EA

20000 - **

10000 **p < 0.01 *p c 0.05

*p < 0.05

5000

0 O h r 4hr 24 hr 24 hr 48 hr

Fig. 1. Effect of VE deficiency on lipid peroxide levels in rat liver 0, 4, and 24 hr after partial hepatectomy. Results are shown as means 2 SD. n = 8 for each treatment.

Fig. 2. Effect of VE deficiency on DNA synthesis in rat liver 24 and 48 hr after partial hepatectomy. Results are shown as means 2 SD. n = 8 for each treatment.

Estimation of Intracellular Polyamine Levels

The liver was homogenized in 3 volumes of the same buffer used in ODC assays and then mixed with an equal amount of 10% TCA and centrifuged at 10,000 X g for 30 min. Five-microliter portions of the supernatant were analyzed for polyamines with a Shimadzu LC3A HPLC apparatus (Kyoto, Japan) with a cation-exchange column (ISC-05). Poly- amines separated by the column were reacted with o-phthalaldehyde, and fluorescence was measured.

Other Analyses

Intrahepatic VE (a-tocopherol) levels were measured by the method of Abe et a1.6 Lipid peroxide levels were measured by the method of Masugi and Nakamura.’

Statistical Analysis

by Student’s t test. Data were expressed as the means 2 SD. Statistical analysis was done

RESULTS

After pair-feeding for 6 weeks, mean body weights of the four groups were not significantly different, and the main- tenance condition was good. Intrahepatic VE levels were 43.7 t 11.0 pg/g liver in group C, 38.1 ? 6.0 pg/g liver in group A, 4.7 t 0.8 pg/g liver in group EC, and 3.0 ? 0.2 pg/g liver in group EA. It was significantly low in two groups maintained on the VE-deficient diets, and it showed a significantly lower value in group EA than in group EC.

Effect of VE Deficiency on Lipid Peroxide Levels in Liver Intrahepatic lipid peroxide levels tended to increase after

partial hepatectomy in all the four groups. The levels in groups EC and EA showed a significantly higher value at 0 and 4 hr after partial hepatectomy than those in groups C and A. The effect of long-term alcohol administration was not found at 0 and 4 hr after partial hepatectomy, but at 24 hr after partial hepatectomy the lipid peroxide levels in groups EA and A were lower than those in groups EC and C, respectively (Fig. 1).

Effect of VE Deficiency on DNA Synthesis in the Liver At 24 hr after surgery, the rate of [3H]thymidine incor-

poration into DNA fraction as an indicator for DNA syn- thesis was significantly lower in groups A and EA, com- pared with groups C and EC, respectively. Thus, a significant suppression by long-term alcohol administration was observed, but no effect of VE deficiency was found. At 48 hr after partial hepatectomy, no effect of long-term alcohol administration was found, but group EA tended to show low value, compared with group A (Fig. 2). DNA synthesis in group EA was suppressed most severely on the whole during liver regeneration.

Effect of VE Deficiency on ODC Activity in the Liver ODC activity, a rate-limiting enzyme in polyamine me-

tabolism, at 4 hr after partial hepatectomy, tended to show low values by stage in order of group C, group A, group EC, and group EA, and the difference between groups C and EA was statistically significant. At 24 hr after partial hep- atectomy, ODC activity tended to show high values with long-term alcohol administration and low values with VE deficiency (Fig. 3).

Effect of VE Deficiency on Polyamine Levels in Liver Concentration of intrahepatic putrescine, the polyamine

synthesized directly by ODC, showed the lowest value in group EA at 4 hr after partial hepatectomy, which was statistically significant, compared with Groups C or EC. At 24 hr after surgery, the putrescine levels in the four groups were not significantly different (Fig. 4). Concentration of intrahepatic spermidine, the second polyamine to be syn- thesized, showed significant changes at 24 hr after partial hepatectomy. Intrahepatic spermidine levels decreased in groups A and EA, compared with groups C and EC. But, this decrease was caused by the effect of long-term alcohol administration, and the effect of VE deficiency was not found (Fig. 5) . Concentration of intrahepatic spermine, the third polyamine to be synthesized, showed significant

VITAMIN E DEFICIENCY IN LIVER REGENERATION

250 -

200 -

150 -

100 -

50 -

0-

49A

8000 s k

3 8 g 4000

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# 6000

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3 2000 u 0 n

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Fig. 3. Effect of VE deficiency on ODC activity in rat liver 4 and 24 hr after partial hepatectomy. Results are shown as means 'c SD. n = 8 for each treatment. prot., protein.

(nmol/g l iver)

40

10

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4hr 24 hr Fig. 4. Effect of VE deficiency on intracellular putrescine levels 4 and 24 hr

after partial hepatectomy in rats. Results are shown as means f SD. n = 8 for each treatment.

(nmoVg l iver)

350 1 300 .A

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250

200

150

100

50

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Fig. 5. Effect of VE deficiency on intracellular spermidine levels 0, 4, and 24 hr after partial hepatectomy in rats. Results are shown as means 2 SD. n = 8 for each treatment.

changes before partial hepatectomy. Intrahepatic spermine levels decreased in groups A and EA, compared with groups C and EC; but, this change was also caused by the effect of long-term alcohol administration, and the effect of VE Deficiency was not found (Fig. 6).

(nmoVg l iver)

O h r 4hr 24 hr

Fig. 6. Effect of VE deficiency on intracellular spermine levels 0, 4, and 24 hr after partial hepatectomy in rats. Results are shown as means 'c SD. n = 8 for each treatment.

DISCUSSION

There are various antioxidation systems in the living body, and these systems protect the living body from vari- ous disorders by active oxygens. VE can be mentioned as one of the substances that have the antioxidation effect.8 When alcohol is administered, VE in the living body catches an active oxygen produced and prevents the chain reaction of peroxidation. Experimentally, in a long-term alcohol administration model using rats, it has been re- ported that VE deficiency is concerned with the progres- sion of alcoholic liver disorder.'

In the relation of alcohol and liver regeneration, it has been reported that long-term alcohol administration sup- presses liver regeneration via the polyamine metabolism in the process of liver regeneration after partial hepatectomy in rats.273 In the present study, we used VE-deficient liquid diet in combination with long-term alcohol administration and studied its effect on liver regeneration after partial hepatectomy in rats.

According to the results of DNA synthesis, an indicator for liver regeneration, the suppressive effect was the most marked in the group maintained on the VE-deficient alco- hol diet (group EA) (Fig. 2). When the polyamine metab- olism was then studied, in group EA, in addition to the decrease of intrahepatic spermidine and spermine levels (figs. 4 and 5) , intrahepatic putrescine level was decreased by the inhibition of ODC induction (Figs. 3 and 4).

A similar phenomenon has been also seen on the acute alcohol administration model in rats. We have reported that the inhibition of ODC induction by acute alcohol administration and concomitant decrease in the intrahe- patic putrescine level at 4 hr after partial hepatectomy lead to suppression of DNA synthesis at 24 hr after partial hepatectomy .'

Therefore, for the process of liver regeneration to pro- ceed smoothly, two conditions are needed from the aspect of polyamine metabolism. First, it is required that spermi- dine and spermine, of which absolute quantity is large, exist in abundance. It is required that the total polyamine level

50A TANAKA ET AL

in hepatocyte be maintained sufficiently. Second, it is re- quired that putrescine be synthesized sufficiently in hepa- tocyte with the induction of ODC. When these two condi- tions exist together, the early stage of liver regeneration process seems to advance smoothly. In the case of the experimental model in the present study, it is speculated that spermidine and spermine decreased by long-term al- cohol administration (the first condition) and that not enough ODC induction occurred because of VE deficiency, resulting in insufficient synthesis of putrescine (the second condition).

A close relationship between VE and liver regeneration has been suggested. Ueda" has observed a marked in- crease in lipid peroxide content in the regenerating liver of rats maintained on a VE-deficient diet, suggesting that VE plays a leading role in the defense system and that lipid peroxide is concerned largely with liver regeneration. In the present study, the amount of VE that is a defense factor showed the lowest value in group EA, which may be con- cerned with the suppression of DNA synthesis later.

In conclusion, long-term administration of alcohol sup- pressed the process of liver regeneration after partial hep- atectomy, and VE deficiency suppressed it more severely. As the mechanism of this phenomenon, it is possible that VE deficiency-in addition to the decrease of intrahepatic spermidine and spermine levels with long-term alcohol ad- ministration-suppresses the ODC induction and that the

concomitant decrease of intrahepatic putrescine level is concerned with the inhibition of DNA synthesis later.

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