Journal of Hepatology 1994; 21: 95-102 Prh~ted hi Denmark. All rights reserved Munksgaard. Copenhagen

Copyright © Journal of Hepatology 1994

Journal of Hepatology ISSN 0168-8278

Protective effect of S-adenosyl-l-methionine on fiver damage induced by biliary obstruction in rats: a histological, ultrastructural and biochemical

approach

Pablo Muriel, Oscar R. Suarez, Patricia Gonzalez and Leonor Zufiiga

Centro de Investigacion y de Estudios A vanzados del L P.N., Departamento de Farmacologia y Toxicologio, Mexico

(Received 23 March 1993)

In human and experimental CCl4-1iver damage, S-adenosyl-l-methionine-synthetase and/or the intrahepatic content of S- adenosyl-l-methionine, are diminished and in human cirrhosis phospholipid methyltransferase is markedly reduced. There- fore the aim of this study was to investigate the effect of S-adenosyl-l-methionine administration on liver damage induced by 15-day bile duct ligation. Liver damage was analyzed by histological, ultrastructural and biochemical techniques. Biliary obstruction produced an increase in collagen content, dilation of the bile canaliculi and disorganization of mito- chondria. These effects were not observed in the bile-duct-ligated group receiving S-adenosyl-l-methionine. Biochemical results showed that bile duct ligatidn increased serum bilirubins, and alkaline phosphatase and ~-glutamyl transpeptidase activities. These effects were prevented significantly by S-adenosyl-l-methionine. On the other hand, glycogen content in the liver was depleted while lipid peroxidation was increased by biliary obstruction, S-adenosyl-l-methionine administra- tion prevented these effects. In the bile-duct-ligated group, hepatocyte and erythrocyte plasma membrane N a + / K + and Ca2+-ATPases were lower than in the control group (p<0.05). Administration of S-adenosyl-l-methionine preserved ATPase activities. The exogenous S-adenosyl-l-methionine supply is probably responsible for restoring transmethylation lost in liver diseases. © Journal of Hepatology.

Key words." ATPase; Bile duct ligation; Cirrhosis; Jaundice; Liver injury; Transmethylation

Bile-duct ligation in the rat is a useful tool for studying cholestasis and cirrhosis (1), it is also a good model for the evaluation of hepatoprotective drugs.

On the other hand, S-adenosyl-l-methionine (SAM) is the methyl donor in most transmethylation reactions and is utilized by methyltransferases in the formation of many biological compounds including plasma membrane lipids (2, 3). Transmethylation is an important means of altering the biological activity of a wide variety of compounds. In human and experimental liver damage SAM-synthetase and/or the intrahepatic SAM content are diminished and in human cirrhosis phospholipid methyltranspherase is markedly reduced (4-7). In addition, we have found that SAM administration may prevent liver damage and pre-

serves hepatocyte (8) and erythrocyte (9) plasma mem- brane Na+/K ÷ and Ca2+-ATPase activities in CCl4-cir- rhotic rats. Moreover, Corrales et al. (10) found that SAM treatment attenuated the fibrogenic effect of carbon tetrachloride and caused a decrease in the number of rats that developed cirrhosis.

However, since CCl4-induced cirrhosis is due to lipo- peroxidative processes, while human cirrhosis is not, it seems important to study the effect of SAM on liver dam- age which is not induced by free radicals, like bile-duct ligation in the rat. Therefore the aim of this study was to investigate the effect of SAM administration on liver damage induced by biliary obstruction determined by his- tological, ultrastructural and biochemical approaches.

Correspondence to: Dr Pablo Muriel, Centro de lnvestigacion y de Estudios Avanzados del I.P.N., Departemento de Farmacologia y Toxicolog- ia, Apdo. Postal 14-740, Mexico 07000, D.F., Mexico.

96 P. MURIEL et al.

Materials and Methods

Mater&Is

S-adenosyl-l-methionine (SAM, S A M Y R RM) was given as the pTtoluene sulphonate salt, containing 75% and 25% of the [ - ] and [+] isomer, respectively, and kindly pro- vided by Bio-Research Laboratories. The other reagents were of the best quality commercially available.

Methods

Obstructive jaundice was induced in male Wistar rats weighing around 200 g by double ligation and division of the common bile duct. The animals were sacrificed after 15 days of biliary obstruction. Control animals were sham operated. S-adenosyl-l-methionine 20 mg/kg i.m. was given three times daily throughout the experiment period.

Animals were sacrificed under light ether anesthesia, blood was collected by heart puncture, and the liver was rapidly removed. Serum was obtained for the following determinations: alkaline phosphatase (11) and ),-glutamyl transpeptidase (12) activity and for bilirubin content (Kit Merck-M6xico).

Small liver sections fixed in Bouin's were used for Tri- chromic staining for histological examination under light microscopy. Liver pieces were also separated for glycogen (13). Malondialdehyde was determined in liver homogen- ates according to Ohkawa et al. (14).

For ultrastructural analysis, liver blocks approximately 0.5 to 0.7 mm 3 were fixed with glutaraldehyde 2.5% in phosphate-buffer 0.1 M (pH=7.2) for 1 h, I% OsO4 was added to continue fixing (30 min at 27°C and 30 min at 4°C). After dehydration with increasing concentrations of ethyl alcohol, the blocks were embedded in epoxy resin. Ultrathin sections were cut and then examined with a Jeol-2000 EX electron microscope at an accelerating volt- age of 80 KV.

Hepatocyte plasma membranes were isolated according to the method described by Pohl et al. (15). The mem- branes were frozen in liquid nitrogen and were resus- pended for further use in 20 mM imidazole (pH 7.8). Pro- tein determinations were performed according to the method described by Bradford (16).

Preparation of erythrocyte membranes was performed according to the procedure described by Rose & Oklander (17). Recovery of red blood cell membrane proteins from 3 g of packed red blood cells was 5 to 6 mg.

The activities of hepatocyte Na+/K + and high affinity CaZ+-ATPases were determined in liver plasma mem- branes according to the methods described by Ames (18) and Lotersztajn & Pecker (19), respectively. The activities of erythrocyte Na+/K + and Ca2+-ATPases were deter-

mined in plasma membranes according to Lee & Au (20) and Ames (18).

For statistical analysis, the ANOVA with Tukey (21) test was used to compare the groups. A difference was considered significant when p<0.05.

Results

Liver damage induced by 15 days of biliary obstruction (bile-duct-ligated rats) is accompanied by an increase in collagen content around the central vein (Fig. 1B). In con- trast, liver sections from bile-duct-ligated rats that re- ceived S-adenosyl-l-methionine revealed less collagen con- tent (Fig. IC) and resembled control (Fig. IA). Administration of SAM per se produced no histological alterations (Fig. ID).

In bile-duct-ligated rats electron microscopy studies re- vealed (Fig. 2B) heavily dilated bile canaliculi (filled ar- rows), disorganized mitochondria with a loss of cristae and internal structure (arrow heads) and diminished rough endoplasmic reticulum (small filled arrows). In ad- dition, collagen fibers were more frequently observed in bile-duct-ligated rats (Fig. 2D) than in sham-operated rats. Ligated rats that received SAM showed no alter- ations (Fig. 2C).

Fig. 3 shows several-fold increases in serum content of total (panel A), conjugated (panel B) and unconjugated (panel C) bilirubins. In addition, as shown in this figure, SAM treatment partially (p<0.05), but significantly pre- vented the increase in all bilirubins.

Serum alkaline phosphatase activity increased signifi- cantly after bile-duct ligation (Fig. 4 upper panel). Treat- ment with SAM completely prevented the elevation in serum alkaline phosphatase activity.

Fig. 4, lower panel, shows y-glutamyl traspeptidase serum activity. As shown in this figure, biliary obstruction produced a more than seven-fold increase in serum y-glut- amyl-transpeptidase activity and SAM treatment partly but significantly prevented this increase.

Liver glycogen content decreased about 10 times after biliary obstruction, and SAM administration prevented glycogen depletion (p<0.05), although the content of gly- cogen did not reach control values in the bile-duct-lig- ated+SAM group (Fig. 5, upper panel).

Lipid peroxidation levels (expressed as malondialdelyde content) increased significantly in bile-duct-ligated rats, but SAM treatment completely prevented this effect (Fig. 5 lower panel).

Fig. 6 shows the Na+/K+-ATPase activity in plasma membranes derived from erythrocytes (upper panel) and from hepatocytes (lower panel). Biliary obstruction caused a significant decrease in Na+/K+-ATPase activity

S-ADENOSYL-L-METHIONINE AND LIVER DAMAGE 97

. ' . / " "": ; " 5 ; " " ~ " ~ ' ~ " ' ~ , ~ " ~ " . . . . • . . . . : - f ~ " ~

- - ' -~" - : : ",*<,;:: '7 , ' , :~ ," : e ~ / . . . . - , - , - , . / -:

• - ~ " - - " ~ ;~• ' ' - " " " " ' ~ " : ' ' . - t ~ ' .; , 2"- , i % - )

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Fig. 1. Liver sections from control rats A (sham operated), bile-duct-ligated rats (BDL) B, BDL rats that receive S-adenosyM-methionine (SAM) (20 mg/kg i.m. 3 times a day) C, and sham-operated rats receiving SAM (SAM control) D. CV: Central vein. Arrows show collagen. Trichromic staining. 125X.

in both cell types and SAM treatment completely pre- served this ATPase activity in erythrocytes and partially in hepatocytes (/7<0.05). It is worth noting that SAM ad- ministration alone produced an increase in erythrocyte Na÷/K÷-ATPase activity.

Plasma membrane CaZ+-ATPase activity derived from erythrocytes (Fig. 7, upper panel) and from hepatocytes (Fig. 7, lower panel) decreased sharply in bile-duct-ligated rats. In this case SAM treatment completely preserved ATPase activity. Furthermore, SAM administration p e r

se increased erythrocyte Ca>-ATPase activity.

Discussion

Electron microscopy examination showed enlarged ca- naliculi after bile-duct ligation, probably due to an in- crease in the intrahepatic bile pressure, as reported by Metz et al. (22). Mitochondria showed a loss of cristae

and internal structure, which is similar to changes that occur in human extrahepatic cholestasis (23).

The changes in mitochondria structure in this study support results by Koyama et al. (24), who found that various parameters of liver mitochondrial respiratory function, such as respiratory control ratio, O~ consump- tion and ATP synthesis decreased in biliary obstruction.

It is important to emphasize that SAM-treatment im- proved the morphology and ultrastructure of livers as well as the biochemical markers of liver damage, indicating an important hepatoprotective effect of this methyl donor•

Lipid peroxidation has been associated with several models of liver injury (25-29). In fact, we observed an important increase in lipoperoxidation levels after bile- duct ligation which was prevented by SAM. Since SAM metabolism is related to reduced glutathione, at least part of the beneficial effect o f SAM might occur by the inhi- bition of lipoperoxidative processes. However, we pre-

98 P. MURIEL et al.

Fig. 2. Electron micrograph of livers from: control (sham-operated) rat .4,; bile-duct-ligated (BDL) rat B; a sham-operated rat that received SAM C; a liver derived from a BDL rat that received SAM; D shows collagen fibers (CF) from a BDL rat. Filled arrows show the bile canaliculi (BC); arrows heads, mitochondria; small arrows, rough endoplasmic reticulum; open arrows, tight junctions; N, means nucleus.

viously observed that lipid peroxidation occurs before

liver damage and that some antioxidants prevent lipoper-

oxidation but not biliary obstruction-induced liver dam-

age (unpublished observations), thus suggesting that lipo-

peroxidation is not a mechanism for liver injury in

experimental biliary obstruction. This indicates that, in

biliary obstruction, iipoperoxidation is independent of,

and may be even a consequence rather than a cause of,

250 A

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Fig. 3. Total (panel A), conjugated (panel B), and unconjugated (panel C) bilirubins determined in serum from sham-operated (SIIAM), bile-duct-ligated (BDL), BDL+S-adenosyl- l -methionine (SAM) and SAM-treated rats. Each bar represents the mean value of experiments performed in duplicate assays with samples from 10 an ima l s_SEM. *Means different from the control group (SHAM), p<0.05. • Means different from the bile-duct-ligated group (BDL), p<0.05.

164-+11 41"

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S-ADENOSYL-L-METHIONINE AND LIVER DAMAGE 99

78,3 +- 9.8 t

R 100"t 4.9

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< 60

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20.

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Fig. 4. Serum activities of alkaline phosphatase (upper panel) and 7" glutamyl transpeptidase (lower panel) determined in serum from sham-operated (SHAM), bile-duct-ligated (BDL), BDL+S-adenos- yl-l-methionine (SAM) and SAM-treated rats. Each bar represents the mean value of experiments performed in duplicate assays with samples from l0 animals_+SEM. *Means different from the control group (SHAM), p<0.05. • Means different from the bile-duct-lig- ated group (BDL), p<0.05.

cell death. Treatment with SAM could prevent liver dam-

age by another mechanism and thus lipid peroxidat ion as

a consequence. However, the present data do not conclus-

ively show that SAM does not act through a mechanism

of glutathione enhancement, since butionine sulfoximine

(an inhibitor of glutathione synthesis) has been shown to

abrogate the beneficial effects of N-acetylcysteine and

SAM in paracetamol- induced hepatotoxici ty (30,31).

ATPases which are located in the plasma membrane

of all animal cells maintain the characteristic intracellular

concentration of ions and determine the t ransmembrane

electrochemical gradient (32). The electrochemical sodium

gradient in the hepatocyte provides the driving force for

100 P. MURIEL et al.

¢...)

3-

.>=_ "6

==

0

200-

2~±0.18

T

0, t6 + 0.02 ' k

1.4 +-OA2 " K - I

2.8 + 0.05

155 ± 1.5

8 5 . 6 ± 1 . 0

SHAM BOL

95.4 +- 2.0 I

87.6+ 1.9

BDL SAM +

SAM

Fig. 5. Glycogen content (upper panel) and lipid peroxidation (lower panel) expressed as the content of malondialdehyde determined in livers from sham-operated (SHAM), bile-duct-ligated (BDL), BDl.+S-adenosyl-l-methionine (SAM) and SAM-treated rats. Each bar represents the mean value of experiments performed in duplicate assays with samples from I0 animals-SEM. *Means different from the control group (SHAM), p<0.05. • Means different from the bile- duct-ligated group (BDL), p<0.05.

Na+-coupled transport of a variety of solutes (33), includ- ing certain amino acids (34-36) and bile acids (37,38). In addition, intracellular Ca 2+ is essential to regulate a large number of liver enzymatic activities, such as those partici- pating in gluconeogenesis and glycogenolysis (39). It is also well known that red cells can extrude Ca 2+ ions across plasma membranes against a large gradient by an ATP-fuelled pumping mechanism. It has been demon- strated that the mechanical and permeability properties of erythrocyte membranes depend upon the intracellular level of Ca 2+ and ATP (40). In addition, Na+/K+-ATPase is an integral part of the red cell membrane (41). This

,.i: .° ,

Na*I K *-ATPase 3.03"-.0.17

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2.08-+0.20

O . k

lO-

5 -

9.31 ± 0.61

T

SHAM

9.30-+0.65

BDL BDL SAM +

SAM

Fig. 6. Effect of bile duct ligation (BDL) and S-adenosyl-l-methion- ine (SAM) on erythrocyte (upper panel) and hepatocyte (lower panel) plasma membrane Na-/K+-ATPase activity. Each bar repre- sents the mean value of experiments performed in duplicate assays with samples from 10 animals+_SEM. *Means different from the control group (SHAM), p<0.05. • Means different from the bile- duct-ligated group (BDL), p<0.05.

6-

= 4 5-:

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Co z+- ATPase 6.12- + 0.~6

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I

SHAM

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BOL

3.G5 + 0,32 al,00¢0.t?

il BDL SAM

÷

SAM

Fig. 7. Effect of bile duct ligation (BDL) and S-adenosyl-l-methion- ine (SAM) on erythrocyte (upper panel) and hepatocyte (lower panel) plasma membranes Ca2+-ATPase activity. Each bar repre- sents the mean value of experiments performed in duplicate assays with samples from 10 animals+_SEM. *Means different from the control group (SHAM), p<0.05. • Means different from the bile- duct-ligated group (BDL), p<0.05.

S-ADENOSYL-L-METHIONINE AND LIVER DAMAGE 101

pump can mediate a number of ouabain-sensitive trans- port processes, each with characteristic cation and sub- strate requirements (41). Moreover, our previous studies (42) showed that erythrocytes may be good indicators of hepatocyte function, and the present results confirm those findings since erythrocyte alterations produced by CCI4 resembled their counterpart on hepatocytes. However, it should be pointed out that a SAM effect in non CCI4- treated rats was observed in erythrocyte but not in he- patocyte ATPases. The reasons for these results are at present unknown.

Although the beneficial effect of SAM on ATPase ac- tivities might be due to alterations in the regulatory mech- anisms of each enzyme, the normalization of membrane lipid composition and fluidity is probably responsible for that improvement (43). Several factors such as the lipid/ protein ratio, amount of cholesterol, cholesterol/phospho- lipids ratio, and degree of saturation of fatty acyl chains among others, can modify the fluidity of the membrane (44).

Strittmater and co-workers (45) incubated normal erythrocyte membranes in the presence of SAM and dem- onstrated increased Ca -'+ stimulated ATP hydrolysis, in- creased 45Ca -~+ efflux from erythrocyte ghosts and syn- thesis of phosphatidyl-N-monomethylethanolamine. Membrane fluidity is increased by phospholipid methylation, which may be the mechanism for ATPase stimulation. Our results in vivo support those of Strittmat- er in vitro, who found increased ATPase activity in the groups receiving SAM. In fact, increases in phosphatidyl- N-monomethylethanolamine content and membrane flu- idity mediated by SAM (45) could explain the increase in erythrocyte ATPase activity above control values.

However, the quantity of membrane lipids which be- come methylated is relatively small (46, 47). For example, Panagia et al. (47) found that about 3 pmol of methyl groups per mg protein became incorporated into sarco- lemma lipids at 10/.tM S-adenosyl-l-methionine. Since the phospholipid content of sarcolemma is about 2/.tmol/mg (47), only about 1 in 106 phospholipids is affected, and thus an extremely sensitive interaction would be required. Other hypotheses are also possible: incubation of membranes with S-adenosyl-l-methionine also causes protein to be- come methylated (48). SAM might stimulate membrane ATPases by methylating them rather than phosphatidyle- thanolamine. In fact, carboxymethylation of proteins has also been reported to modify enzyme activities (49). How- ever, since both methylation of phosphatidylethanolamine and proteins are stimulated by SAM (50), it is not clear which of these methylation reactions is responsible for mo- dulating liver plasma membrane ATPase activity.

Hirata & Axelrod (51) suggested that enzymatic

methylation of phosphatidylethanolamine in erythrocyte membranes of rats increased the lipid fluidity of the mem- branes. Subsequent studies in other plasma membranes, however, have failed to support the contention that phos- pholipid methylation modulates membrane fluidity (52). More recently the same group of workers (53) demon- strated that transmethylation of membrane phospho- lipids, but not membrane non-polar lipids or proteins, in- fluenced the fluidity of rat colonic basolateral membranes, which in turn modified Na+/K+-ATPase activity. How- ever, more experiments are required to determine this re- action in liver plasma membrane ATPases.

The mechanism by which SAM protects liver and mem- brane function is not easily understood, since SAM is the methyl donor in most transmethylation reactions and is utilized by methyltransferases in the formation of many biological compounds.

The exogenous SAM supply is probably responsible for restoring transmethylation reactions lost in liver damage, because the synthesis of this metabolite is markedly re- duced in this disease (4).

Acknowledgements

The authors express their gratitude to Ms Concepci6n Avalos for secretarial assistance and Mr. Alfredo Padilla for preparing the figures. The authors also acknowledge the technical support provided by Personnel of the UME, CINVESTAV, specially the assistance of Lourdes Rojas, BSc. This work was supported in part by Grant 0589-N9110 from CONACYT, Mrxico.

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