Role of cytochrome P4502E1 in alcoholic liver disease pathogenesis

Alcohol,Vol. 10, pp. 459-464, 1993

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Role of Cytochrome P4502E1 in Alcoholic Liver Disease Pathogenesis M. MORIMOTO,* A.-L. HAGBJORK,~ A. A. NANJI,~ M. INGELMAN-SUNDBERG,~ K. O. L I N D R O S , § P . C. F U , * E. A L B A N O ¶ A N D S. W . F R E N C H .1

*Department o f Pathology, Harbor-UCLA Medical Center, Torrance, CA 90509 t Department o f Physiological Chemistry, Karolinska Institute, Stockholm, Sweden ~Department o f Pathology, New England Deaconess Hospital and Harvard Medical School, Boston, MA §Research Laboratories o f the State Alcohol Monopoly (Alko), Helsinki, Finland ¶Department o f Experimental Medicine and Oncology, University o f Torino, Torino, Italy MORIMOTO, M., A.-L. HAGBJ(~RK, A. A. NANJI, M. INGELMAN-SUNDBERG, K. O. LINDROS, P. C. FU, E. ALBANO AND S. W. FRENCH. Role of cytochrome P4502EI in alcoholicliver diseasepathogenesis. ALCOHOL 10(6) 459--464, 1993.-The intragastric tube feeding model is ideal for the study of the role of dietary factors and the effect of drugs on experimental alcoholic liver disease (ALD), since the model allows us to study the effect of a single variable in the diet on the pathology of liver where the blood alcohol level (BAL) is maintained over 150 mge/e. By varying the dietary fatty acid composition we showed that the pathology was worsened by increasing linoleic acid or polyunsaturated fatty acids (PUFAs) in the diet where cytochrome P4502EI (CYP2EI) was increased posttranslationaUy by high BAL. Concomitant with the increase in CYP2EI there was evidence for an increase in lipid peroxidation (LP) by microsomes. Protein adducts of the products of LP were increased in the blood. Isoniazid (INH) enhanced this process and the pathology of ALD when INH was fed at therapeutic levels with ethanol. Preliminary studies show that diallyl sulfide, which inhibits and destroys liver CYP2E1 selectively, also modified the pathologic effects of ethanol. Thus we postulate that CYP2E1 induction plays a central role in the pathogenesis of ALD. ALD model Lipid peroxidation Cytochrome P4502EI

PUFA

Fatty acid

THE intragastric tube feeding model (23) of alcoholic liver disease (ALD) lends itself well to experiments where strict control of diet and alcohol intake and drug manipulation are required. Such is the case in experimental ALD. Using this approach it was established that high BAL and a high fat-low carbohydrate diet which contained polyunsaturated fatty acids (PUFAs) were required in order to induce A L D pathology in the rat (11,18,19,23). These observations suggested that CYP2E1 induction and its metabolism of ethanol might be involved in the generation of free radicals with damage to the liver and fibrosis resulting from lipid peroxidation (LP) (1,9,10,12-15,21). Complicating this hypothesis is the recent revelation that the five to six day cyclic swing in BAL when ethanol is infused at a fixed dose (22) can be explained by an autoadaptive mechanism where CYP2EI levels are increased at high BAL and decrease when BAL falls to low levels, regulated by a two-step induction of CYP2EI (2-4,20). So, is there a cyclic increase and decrease in LP during the swing up and down of the levels of BAL? Theoretically, interference in this induction of CYP2EI by BAL should also reduce the level of LP and also

Linoleic acid

Protein adducts

Diallyl sulfide

liver damage if the mechanism of A L D is through oxidative stress which results from increased free radical production. To address these questions we designed an experiment where CYP2EI levels are reduced by feeding a substrate of CYP2E 1 which reduces its levels by competitive inhibition and suicide-inhibitory action of DASO2, which is a metabolite of diallyl sulfide (DAS) (6,8). In pilot studies we found that DAS was effective in reducing the levels of microsomal CYP2EI and LP. When fed acutely the compound has been shown to protect the liver from the toxic effects of CC14 (8), including LP and histopathologically assessed damage to the liver. Details of our experiment using the intragastric tube feeding of ethanol and DAS, which were reported in part in an abstract (17), are the subject of this report. MATERIALS AND METHODS

Animals Thirty-six male Wistar rats weighing 230-250 g were purchased from Charles River (Hollister, CA). The permanent intragastric eannula was implanted under thiopental sodium

Requests for reprints should be addressed to S. W. French, M.D., Department of Pathology, Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, CA 90509. 459

460

MORIMOTO ET AL. Tarrytown, NY). BAL was determined by Therapeutic Drug System (TDX) test (Abbott Laboratories, North Chicago, IL). Total P450 level was determined spectrophotometrically. The amounts of CYP2EI in the liver microsomes was determined by Western blot analysis (16). N A D P H oxidation and NADPH-dependent lipid peroxidation were measured by previously described methods (10,12).

TABLE 1 ALCOHOL DOSAGESWHICH CAUSE THE DEATH OF DIALLYL SULFIDE (DAS)-TREATEDRATS

Treatment Group

N

State

FeedingPeriod (day)

CO + DAS + E

4 6 6

Dead Alive Alive

15.5 ± 2.4* 14 14

CO + E

Ethanol Fed (g/kg body weight/day) 12.7 ± 1.0*t 10.3 ± 0.9* 12.9 ± 0.4*

Histology Liver samples were taken from five lobes of each rat at the sacrifice. The pathology score was quantified according to the criteria previously described (12) using sections stained with hematoxylin and eosin (H&E).

CO = corn oil, E = ethanol. *M ± SD. tp < 0.05 when compared with CO + DAS + E (alive) group and no significant difference compared with the CO + E group. One serum we could obtain from the dead rat showed high BAL (753 mg/dl). DAS caused the death to rats fed with the almost same amount of ethanol as the CO + E group.

Statistical Analysis The means + SD were calculated on all data. Comparison of the groups was done using the paired t test. The correlation coefficient was determined using the Spearman rank-order correlation method.

(2 mg/kg body weight, IP) and ketamine hydrochloride (6 mg/kg body weight, IM) anesthesia when a baseline liver biopsy was performed for morphology.

RESULTS

Diets The diets were prepared fresh as described previously (12) and contained corn oil (CO) contributing to 50% of liquid diet calories and 30-400/o of total calories. Rats were separated into six dietary groups of six each as follows: #1) ethanol (CO + E), #2) isocaloric dextrose pair-fed with #1 (CO + D), #3) ethanol and DAS (Sigma, St. Louis) (200 mg/kg body weight) (CO + DAS + E), #4) isocaioric dextrose and DAS pair-fed with #3 (CO + DAS + D), #5) ethanol (more than #3) and DAS, and #6) isocaloric dextrose and DAS pair-fed with #5. The last two groups (#5 and #6) were designed to determine the tolerance limit volume of ethanol in DAS-fed rats. The experimental liquid diet (1 kcal/ml) was fed continuously through a cannula at the rate of 180 ml/kg body weight/day to achieve adequate weight gain. An appropriate volume of 807o (w/v) ethanol solution was infused to keep a high BAL. The amounts of liquid diet and ethanol solution were adjusted according to the body weights determined weekly. BAL was monitored using a saliva alcohol test (Enzymatics, Inc., Hotsham, PA).

Rate of Alcohol Utilization All of the rats fed with DAS and a high volume of ethanol were dead within 20 days. Data of two rats which died due to bleeding from the operation scar were excluded. The others died at 15.5 + 2.4 days, probably due to alcohol intoxication because they showed signs of alcohol intoxication prior to death and one serum sample we obtained soon after death showed a high BAL of 753 mg/dl. We also excluded data from one rat (CO + DAS + E) since it was morbid prior to sacrifice because of bleeding near the implanted tube at the stomach. The amount of ethanol which caused the death of DAS-treated rats was compared with amounts of the other two groups of ethanol-fed rats at the time of the second week (Table 1). The body weight gains were less in DAS-fed groups (Table 2). The fact that the ethanol volume required to keep high BAL was less in DAS-fed rats largely explains this result. The amount of ethanol required to maintain high BAL at one month was less in CO + DAS + E compared to CO + E, though there was not a statistically significant difference in BAL between two groups (Table 3). These data suggest that DAS destroys the adaptation mechanism to ethanol metabolism in rats.

Biochemical Analysis Alanine aminotransferase (ALT) activity was measured by an automatic analyzer (Technicon axon system, Technicon,

TABLE 2 BODY WEIGHT GAIN (BWG) AFTER ONE MONTH OF FEEDING EXPERIMENTALDIETS

Treatment Group

N

CO CO CO CO

6 6 6 5

+ + + +

D E DAS + D DAS + E

BWG (g) 53.7 59.6 26.8 24.1

+ ± ± ±

20.3 17.9 20.7 23.0

Rate of BWGin Last Two Weeks* (g/day) 3.0 3.4 1.8 2.2

+ ± ± +

1.7 0.6 0.9 0.7

AverageCalories of D or E (kcal/kg/day) 78.5 78.5 61.6 61.3

+ + ± ±

2.3 2.3 2.6 2.8

Means + SDs. D = dextrose, E = ethanol, CO = corn oil, DAS = diailyl sulfide. The body weight gains were less in DAS-fed groups (CO + DAS + D, CO + DAS + E) because the ethanol volume required to keep high BAL was less in DAS-fed rats. *Calculated using the body weight in the growing phase.

6 6 6 5

13.7 + 0.8§ -11.7 ± 1.1§

224 + 80 -262 ± 183

BAL (mg/dl) 0.74 1.53 0.40 1.02

± + + ±

0.11" 0.22* 0.16" 0.15"

P-450 (nmol/mg) 43.8 563.5 3.4 110.3

+ 15.5" + 84.2* ± 3.8* ± 47.9*

CYP2EI (pmol/mg) 9.1 15.3 7.6 15.8

+ + ± +

1.9t 3.9t 3.3t 1.7t

NADPH-ox (nmol/ mg • min) 0.060 0.246 0.002 0.023

+ + ± ±

0.032~ 0.207~ 0.005~ 0.012~

Lipid Peroxidation (nmol/mg • min)

40 59 35 51

+ 7 ± 26 ± 14 ± 51

ALT (U/L)

10.9 16.7 14.4 19.5

+ ± ± ±

1.5§ 1.9§ 1.4§ 0.9§

Liver (g)

0¶ 3.7 ± 0.5¶ 0¶ 2.2 ± 0.41

Liver Fat

0.8 7.0 0.8 4.0

+ + ± ±

1.2¶ 0.9¶ 1.0¶ 0.7¶

Pathology Score

M + SD. CO = corn oil, D = dextrose, E = ethanol. DAS reduced P450, CYP2EI, lipid peroxidation, and pathology score. *p < 0.05 when compared in all combinations, tP < 0.05. ~p < 0.05 when compared in all combinations except CO + D with CO + E (p = 0.08). §p < 0.01. ¶p <0.01 when compared in all combinations except CO + D w i t h C O + DAS + D.

D E DAS + D DAS + E

CO CO DO CO

+ + + +

N

Treatment Group

Ethanol Fed (g/kg/d)

TABLE 3 EFFECT OF DIALLYL SULFIDE (DAS) ON VARIOUS PARAMETERS IN THE RAT LIVER

462

MORIMOTO ET AL.

FIG. 1. Protoporphyrin pigments were seen in the bile ductules (arrow) in a CO + DAS + D rat liver after one month of feeding. H&E, x 156. CV = central vein.

Biochemical Data Data at one month of treatment is shown in Table 3. DAS reduced the P450 level by half (CO + DAS + D, CO + DAS + E) compared to rats without DAS (CO + D, CO + E), although ethanol doubled it compared to controls (CO + D, CO + DAS + D). DAS reduced the CYP2E1 level dramatically in control rats (CO + DAS + D) and suppressed the induction of CYP2EI strongly in spite of high BAL (CO + DAS + E). DAS did not affect the NADPH

oxidation level, although ethanol increased it. LP was decreased by DAS treatment, and there was a strong positive correlation with the CYP2E1 level (R = 0.748, p < 0.001). In ALT activity, there was not a significant difference between groups, but it tended to be higher in ethanol-fed groups.

Histology The increase of liver weight was observed in CO + E, CO + DAS + D, and CO + DAS + E groups compared to

FIG. 2. Periportal fatty accumulation was seen in a CO + DAS + E rat liver after one month of feeding. H&E, x 156. CV = central vein.

P4502E1 AND ALD

463

CO + D. DAS induced a hepatomegaly which was more severe in combination with ethanol. There were no abnormal findings in CO + D. In CO + E, pericentral fatty accumulation was observed with the score of 3.7 + 0.5. In CO + DAS + D there was no fatty accumulation, but protoporphyrin pigment was observed in bile ductules (Fig. 1). In CO + DAS + E fatty change in the periportal areas (Fig. 2) and protoporphyrin pigment in bile ductules were observed. The mitosis of hepatocyte was seen frequently. The degrees of fatty change, spotty necrosis, and inflammation were more severe in CO + E than in CO + DAS + E, which contributed to the higher score in the former group compared to the latter group (Table 3). A strong positive correlation between the pathology score and CYP2E1 was found (R = 0.826, p < 0.005). LP was also correlated strongly with the pathology score (R = 0.477, p < 0.005). DISCUSSION CYP2E1, the ethanol-inducible form of cytochrome P450, is thought to play a key role in the pathogenesis of ALD, due to the preferential distribution in zone 3 (15,24) and the potential for free radical generation (14). We have previously reported that the induction of CYP2E1 by ethanol, which is enhanced by corn oil (21), is closely related to ALD, and that isoniazid (INH), the inducer of CYP2EI, aggravated the pathology of ALD in the intragastric rat feedingmodel. DAS, an essential oil of onion and garlic, is identified as an inhibitor of CYP2E1 (6,8). Some investigators focused on

a chemopreventive effect of cancer by DAS (7,25), although it has not been used in the investigation of chronic ALD. In this study we evaluated the role of CYP2EI in the experimental design where CYP2E1 is reduced by DAS. DAS destroyed the adaptation mechanism for ethanol in rats. This data suggests that the induction of CYP2E1 in microsomes is the main mechanism for pharmacological adaptation to alcohol ingestion. DAS reduced the CYP2E1 level effectively and suppressed LP by microsomes as measured in vitro. The pathology score was lower in CO + DAS + E than in CO + E in accordance with the reduced CYP2EI as well as LP. Further study is needed to evaluate the role of CYP2EI in the pathogenesis of ALD. DAS did not inhibit alcohol dehydrogenase activity in vitro until 1 mM concentration of DAS was used. The bleeding from the operation scar was observed in some DAS- and ethanol-fed rats but not in other groups. Ailium sativum (garlic) has been reported to reduce coagulation time and increase fibrinolytic activity. The active ingredient responsible for this is thought to be DAS (5). The high dose of ethanol was thought to enhance this effect of DAS. DAS induced the rapid turnover of berne of CYP2E1 and stimulated the synthesis of heme in the liver. This process is postulated to induce a protoporphyrin. We concluded that CYP2EI plays a key role in the pathogenesis of ALD because the inhibition of CYP2E1 induction prevented the production of lipid peroxide and the central fatty accumulation and reduced the pathology score despite of high BAL.

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