Glutathione synthesis and export in experimental liver cirrhosis induced by thioacetamide: Relations to ultrastructural changes

Exp. Pathol. 1999; 36: 113-122 VEB Gustav Fischer Vcrlag .lena

Institute of Phannacology and Toxicologyt), and Institute of Pathological Biochemistry') of the Friedrich Schiller University lena, G.D.R.

Glutathione synthesis and export in experimental liver cirrhosis induced by thioacetamide: Relations to ultrastructural changes

By M. KRETZSCHMAR 1), H. FRANKE c), T. ZIMMERMANN'), R. DARGEL 2) and W. KLiNGER 1) With 3 figures

Address for correspondence: Dr. med. M. KRETZSCHMAR, Institute of Pharmacology and Toxicology of the Friedrich Schiller University, LbbderstraBe I, lena, DDR - 6900 Key words: liver cirrhosis; glutathione system; thioacetamide; hepatocyte; ultrastructure

Summary Micro- and macronodular experimental liver cirrhosis was induced in female rats by administration of 0.03 % thioacetamide (TAA) in drinking water for 3 or 6 months, respectively. The glutathione (GSH) status (content, synthesis, export) and ultrastructural changes of liver were investigated 14 d after withdrawal of T AA. The hepatic level of GSH was increased after 6 months T AA treatment. The levels of oxidized glutathione (GSSG) were not changed after 3 months or 6 months TAA administration. The GSH synthesis was not disturbed in the cirrhotic livers; only the ratio between the 2 synthesizing enzymes was changed in macronodular liver cirrhosis. The plasma GSH content was reduced in both cases, independent of the stage of liver cirrhosis. The electron microscopic studies on cirrhotic rat livers revealed a series of characteristic structural changes, such as disorganization and total lack of the microvilli border, appearance of basement membrane-like deposits within the narrowed space of Disse, disappearance of the highly porous endothelial cell lining and partly an intensively detoriated blood supply within the pseudolobules. It is suggested that all these changes may contribute to a disturbance of the GSH export from the hepatocytes into the blood. It is very likely, however, that the alterations of the sinusoidal cell surface play the most important role. We conclude from our data: I. The GSH/GSSG redox potential is shifted in favour of the reduced form in this cirrhosis model. This shift seems to be connected with later stages of cirrhogenesis. 2. A GSH export disturbance is responsible for the decreased plasma GSH level in liver cirrhosis.

Introduction The biological importance of intracellular glutathione (GSH) for many physiological processes and in the protection of cells from oxidative stress is well established (for review see 13). 8

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Extracellular (plasma) GSH is an important scavenger of reactive oxygen species produced by activated granulocytes. Studies in rats and other species have elucidated the central role of liver in the complex inter-organ regulation of GSH metabolism. Liver exports GSH produced in hepatic cytosol at a rapid rate mainly into plasma. A smaller part is exported into bile. The liver is able to generate cysteine from methionine, and therefore the capacity of the liver to synthesize GSH is not limited by the availability of dietary cysteine. This fact is important for supplying extrahepatic tissues with GSH or cysteine (11). However, in spite of the clinical importance of liver cirrhosis, only little information is available on GSH concentration, synthesis and export in experimental liver cirrhosis and in the human disease. Recently BVRGVNDER and LAvTERBVRG (4) reported the first data about production ofGSH in healthy humans and patients with cirrhosis by an analysis of plasma GSH kinetics. They demonstrated that liver cirrhosis in man is associated with decreased GSH plasma concentration. The estimated export of GSH into the circulation was significantly decreased. The authors concluded from these data that GSH synthesis in cirrhotic liver is decreased. The thioacetamide (TAA)-induced cirrhosis-like liver lesion in rats was found to be similar to major morphological and biochemical features of the human disease (3, 15). Therefore we used this model to investigate the relations between morphological changes in TAA-induced chronic liver injury and glutathione status (content, synthesis, export) and to compare these findings with different stages of experimental cirrhosis. GSH is exported into the circulation through the sinusoidal membrane by a carrier transport mechanism (2). Therefore, we analysed by electron microscopy mainly the ultrastructure of the perisinusoidal space, i.e. the sinusoidal pole of the liver parenchymal cells (LPC), the space of Disse, the sinusoidal endothelium and the cytotopical relation of the LPC to the microcirculation (6).

Materials and Methods Animals Female virgin Uje: WIST rats, 4 months of age at the beginning of the experiments, were used in this study. They were kept under conventional conditions. Tap water and food (pelleted diet R 13, VEB Versuchstierproduktion Schonwalde, G.D.R.) were given ad libitum if not stated otherwise.

TM-treatment Animals of the experimental groups received tap water containing 300 mill TAA (Merck Darmstadt, F.R.G.) beginning from the 4th up to the 6th or the 9th month oflife. After 2 additional weeks under the same conditions, but without TAA administration (to exclude acute toxic effects of TAA) the animals were used in the experiments. Age matched animals receiving tap water were used as controls.

Tissue and blood sampling At 7.00 a.m. the rats were sacrificed under ether anaesthesia by decapitation. Arterial blood from the carotides was collected with a small pipet, put immediately into plastic tubes with reaction medium (see below) and stored on ice. Thereafter the abdomen was opened and the liver was rapidly removed. Liver samples for electron microscopic studies were processed as described below. Samples for biochemical investigations were immediately placed in buffer (see below) on ice.

Biochemical investigations Liver tissue glutathione concentration Liver samples were homogenized with II volumes of 200 mM sodium phosphate -5 mM EDTA buffer (PH 8.0) and 4 volumes of25 % metaphosphoric acid. The deproteinized mixture was cleared by centrifugation (12,000 g, 30 min, O°C) in a cooling centrifuge (K 24, VEB MLW Engelsdorf, G.D.R.). Liver GSH was determined by modified ELLMAN'S method (6): 0.2 ml supernatant were added 114

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Table 1. Levels of reduced (GSH) and oxidized (GSSG) glutathione in thioacetamide (TAA)induced micro- (3 months) or macronodular (6 months treatment) experimental liver cirrhosis. Arithmetic means ± SEM are given (n == 6). * P :S 0.05 vs controls. Treatment

GSSG

GSH/GSSG

Age (months)

GSH

Controls TAA 3 months

6

8.59 ±0.08

0.48 ±0.01

17.8±1.0

6

8.65 ±0.14

0.51 ± 0.05

17.2±1.0

Controls TAA 6 months

9

6.44±0.26

0.36 ± 0.02

17.7±1.2

9

7.66±0.62*

0.36 ± 0.04

21.6 ± 1.8*

([!mol!g liver w.w.)

Table 2. Glutathione synthesis [y-glutamyl-cysteinyl-synthetase (GCS) and GSH-synthetase (GSHS)] in cirrhotic rat liver produced by TAA-treatment. For data and symbols see table I. Treatment

Age (months)

Total Pi releated

GCS

GSHS

(nmoles P/mg protein X min) Controls TAA 3 months

6

33.08 ± 2.16

21.48 ± 2.40

11.60 ± 1.27

6

36.64±4.77

28.54 ± 3.98

8.10±1.06

Controls TAA 6 months

9

36.07 ± 2.48

25.92±2.30

10.15 ± 1.39

9

34.06 ± 1.66

19.24± 1.37*

16.78± 1.14*

Table 3. Total (GSH + GSSG) glutathione (tGSH) levels in arterial plasma and ratio between plasma and liver GSH content from rats with thioacetamide (TAA)-induced liver cirrhosis of different grades. For data and symbols see table I. Treatment

Age (months)

Plasma tGSH ([!mol!l)

Ratio plasma contentl Ii ver content

Controls TAA 3 months

6

20.52 ± 1.06

2.15 ± 0.18

6

13.47± 1.51*

1.39±0.14*

Controls TAA 6 months

9

20.09 ± 0.42

2.80 ± 0.20

9

14.71 ± 1.38*

l.76±0.15*

to 1.6 ml buffer (as described above) and 0.2 milO mM 5,5' -dithiobis(2-nitrobenzoic acid) (Serva Heidelberg, F.R.G.). The absorbance was read at 412 nm on a spectrophotometer SPEKOL 11 (VEB Carl Zeiss JENA, G.D. R.) against blanks. For standard a GSH (Serva) solution was prepared immediately before use. 8*

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Fig.I. Control rat liver showing the sinusoidal cell pole of two liver parenchymal cells, (N) cell nucleus. Note the numerous microvilli projecting into the space of Disse and the slender, highly porous projections of the sinusoidal endothelium (J'). (be) bile capillary, (S) sinusoid, (rf) reticulin fibr ils, (EC) endothelial cell. X 12,500.

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Fig.2. Macronodular rat liver , assembl y of some liver parenchymal cells (LPC) localized remote from the sinusoids. Note the structural depolarization of the hepatocytes. (F) fibrocyte , (NC) necrotic cell, (N) cell nucleus. X 7 ,500.

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Oxidized glutathione (OSSO) was determined with 0.5 ml supernatant as described by HISSIN and HILF (10). Results were expressed in I-lmol/g liver (w.w.). OSH synthesis activity in rat liver cytoplasm Liver samples were homogenized with 2 volumes of 100 mM Tris-HCl - 1 mM EDTA buffer (PH 7. 1). Cytoplasm was prepared by a so-called rapid method: At first homogenate was centrifuged at 9,000 g (O°C) for 20 min; 1 ml of supernatant was mixed with 2.5 ml 25 mM magnesium chloride and then centrifuged under the conditions described above. Cytoplasm was dialysed in Visking dialysis tubings type 8/32 (Serva) against buffer for 12 h at 4 0c. y-glutamylcysteinyl synthetase (OCS) activity was determined by a modified method ofT ATAISHi et al. (20). A mixture containing 100 nmoles glutamic acid (Reanal Budapest, Hungaria), 100 nmoles L-cysteine (Reanal), 100 nmoles ATP-Na2 (Reanal), 100 nmoles magnesium chloride, and about 1 mg protein in buffer (as described above), final volume 1 ml, was incubated for 1 hat 37°C. The reaction was finished by adding 1 mIlO % trichloroacetic acid. The reaction in ATP-dependent and the release of inorganic phosphate (Pi) are in proportion to enzyme activity. Pi was measured in the deproteinized supernatant: 0.2 Ji supernatant were diluted with 1.4 ml sodium acetate buffer (PH 4.09),0.5 ml freshly prepared 1 % ammonium molybdate/1 % ascorbic acid mixture (I + 1) added, and incubated at 37°C for 15 min. After standing at room temperature for 30 min the absorbance was read at 700 nm against blanks. Potassium phosphate solution was used as a standard. OSH synthetase (OSHS) activity was determined at the same way; the incubation mixture contained 100 nmoles glycine (VEB LCA Feinchemikalien Sebnitz, O.D.R.) supplementary. Enzyme activity was calculated from the difference of total OSH synthesis and OCS activity. The results were expressed in nmoles Pi released/mg protein per minute. Total glutathione (tOSH) content of plasma tOSH (OSH + OSSO) content of arterial plasma was determined as described by ADAMS and LAuTERBURG (I). Protein content of cytoplasm Protein determination was carried out with the Biuret-reaction using bovine serum albumine as a standard (10). Statistics Experimental groups comprised 6 animals. The data are given as arithmetic means ± SEM. The statistical significance of differences between the sets of experimental data was assessed using Wilcoxon's U-test, p ::; 0.05.

Morphological investigations The evaluation of the stages of experimentally induced liver cirrhosis was performed by macroscopic inspection of the surface of the Ii ver lobes and by examination of ultrathin sections. For electron microscopy, in each case from 3 rats (controls, micro- and macronodular livers) tissue samples were taken, prefixed in buffered 3 % glutaraldehyde for 30 min and postfixed in 2 % buffered osmium tetroxide for 3 h. The tissue samples were embedded in Micropal (Ferak, Berlin! West). Details of the technical processing of the liver samples have been described elsewhere (9).

Fig.3. a) Macronodular rat liver showing a sinusoid (S) bordered by continuous, non-porous endothelial cell projections (cp). Note the narrowed space of Disse containing amorphous deposits. (E) erythrocyte, (ly) lymphocyte, (PC) liver parenchymal cell, X 9,000. b) Higher magnified area of the peri sinusoidal space of fig. 3 a in order to demonstrate the nearly homogeneous deposits ()") in the space of Disse. X 14,000. 118

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Results The TAA administration for 3 months resulted in liver cirrhosis of the micronodular type in all treated rats; the administration for 6 months predominantly resulted in liver cirrhosis of the macronodular type.

Biochemical investigations The results of the biochemical investigations are presented in tables 1-3. The hepatic GSH- and GSSG levels were in normal range afterTAA-administration over 3 months. An increased GSH level was observed in livers from animals treated with TAA over 6 months, but the GSSG content was unchanged in comparison with the controls. The total GSH synthesis activity was unchanged after 3 months as well as after 6 months T AAtreatment. After the longer period of T AA-administration the ratio between the 2 synthesizing enzyme activities was changed. We observed a decrease ofGCS and, on the other hand, an increase of GSHS activities. The plasma tGSH content was reduced in both cases. The extent of this slope is independent of the macroscopic grade of the cirrhosis. According to data of STOHS and LA WSON (19) we observed lower hepatic GSH levels in the older animals. Electron microscopy Controls: The characteristic ultrastructural appearance of control LPC, their sinusoidal cell pole, the adjacent space ofDisse and the sinusoidal cell lining is to be seen in fig. 1. The cell surface which faces the sinusoids is well studded with numerous microvilli. They project into the space of Disse which is electron-lucent and contains only some VLDL particles or occassionally some bundles of reticulin fibers. The long slender projections of the sinus endothelial cells are fenestrated, i.e. they form sieve plates, additionally, they are perforated by gaps of 1- 3 [lm in diameter with the consequence that the space ofDisse communicates well with the microcirculatory system. The liver parenchyma is arranged in cell plates which in sections appeared to be one or two cells in thickness, therefore at least one or two sell poles of each LPC is in close contact with the blood circulation. Cirrhotic livers: Liver cirrhosis leads to a series of characteristic morphologic changes which may cause an intensive disturbance of the transport of certain metabolites from the LPC into the blood circulation. The livers of micro- or macronodulartype reveal a diffusely nodular parenchyma. Within the pseudolobules the LPC are not arranged in single-cell plates, but in groups being 4 and more cells thick. In this way many of the LPC are localized very remote from the sinusoids. These hepatocytes have lost their microvilli , and obviously in dependence on the blood supply the pseudolobules consists either predominantly ofLPC of atrophic (fig. 2) or hypertrophic appearance (not shown). Generally, in macronodular livers the structural alterations within the perisinusoidal region are much stronger than in micronodular ones. It is almost a constant finding that the sinusoidal lining cells show a capillarization, that means, the typical fenestration of the long cell projections has disappeared (fig. 3 a, b).Remarkable is furthermore the common diminution of the space of Disse, an alteration, which is regularly combined with a complete loss of the microvilli border. Beside this, the narrowed space of Disse is filled over long distances with an amorphous homogenous material of moderate electron density (fig. 3 b) and locally increased amounts of reticulin fibrils occur (not shown).

Discussion The main topic of our study was to elucidate the GSH status (content, synthesis, export) in an animal liver cirrhosis model and to compare it with the ultrastructural changes at different stages of experimental cirrhosis. The TAA-induced liver cirrhosis is assessed as a suitable animal model to reproduce morphological and biochemical features of the human disease (3, 15). The GSH content in cirrhotic liver is found to be increased after 6 months TAA-administration, which has not been reported before. This increase indicates that the GSH/GSSG redox potential is shifted in favour of the reduced form. This agrees with the unchanged GSSG levels after T AA administration observed in our study. The assumption is also supported by the results of CERDAN et al. (5).

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The authors studied the activities of hepatic enzyme of the pentose phosphate shunt as well as other cytosolic NADP-linked enzymes in vivo during administration of TAA. They found an increased capacity for the generation of NADPH. The pentose phosphate pathway is the main source of N AD PH for the GSSG-redox -cyc Ie (and other N ADPH -dependent enzyme systems, e. g. cytochrome PA50). Therefore we assume that chronic T AA-treatment increases the activity of GSSG-reductase, the key enzyme of the GSSG-redox-cycle. Whether the shift in GSH/GSSG redox potential is connected with the hepatic carcinogenic effect of prolonged TAA-administration (14, 17) or whether it is a general phenomenon in cirrhogenesis cannot be decided at this time. The shift seems to be connected with the later stages of cirrhogenesis; the GSH levels after 3 months of T AA-administration were unchanged in our study. In correspondence with investigations on patients with cirrhosis (4) we could show a decreased tGSH plasma level. These authors described a reduced GSH export from liver into plasma in cirrhotic patients. Since all patients had disturbed hepatic functions the authors suggested a decrease in hepatic synthesis of GSH. In the rat the efflux of GSH into blood in the physiological range is proportional to the intracellular GSH concentration ( 15). Therefore plasma GSH concentration reflects the intrahepatic level of GSH. This ratio is disturbed in this animal cirrhosis model, most pronounced in the macronodular form. The intrahepatic GSH level was unchanged (after 3 months T AA) or increased (after 6 months TAA). To elucidate this difference we measured the GSH synthesis in these two stages of T AA-induced liver cirrhosis. Surprisingly the GSH synthesis rate was normal in comparison with control animals. Comparable data have not been reported before. Various hepatic functions were decreased in the T AA-induced experimental cirrhosis in the same way as in human liver (8,21). Further investigations are necessary to e1arify this contradiction. The changed ratio between the two GSH synthesizing enzyme activities is notable. In connection with the other data this result points to a general change-over of the complex GSH metabolism in the cirrhotic liver. The results demonstrate that the lowered plasma tGSH levels were not caused by a disturbance ofGSH synthesis in contradiction to the assumption of BURG UNDER and LAUTERBURG (4). Therefore we hypothesized that depression of plasma tGSH content could be caused by a GSH-export disturbance. GSH is exported into plasma through the sinusoidal membrane ofhepatocytes by a carrier transport mechanism (2). We investigated the ultrastructural changes in T AA-induced liver cirrhosis to elucidate the assumed GSH-export disturbance and focused our interest on hepatic sinusoids. Our ultrastructural studies demonstrate that liver cirrhosis in rats is combined with a series of characteristic structural alterations. These are the disappearance of the microvilli border, the appearance of the basement membrane-like deposits in the narrowed space of Disse, the disappearance of the highly porous endothelial cell wall and the extensive disorganization of the microvasculatory system. All these changes may contribute to an effective inhibition of the GSH export from LPC into the blood. Similar ultrastructural changes can be observed in human liver cirrhosis (16). Therefore we assume that GSH export disturbance is the cause of decreased GSH plasma concentration detected in patients with cirrhosis, too. With respect to the fact that the GSH export takes place by a carrier transport via the sinusoidal plasma membrane of the LPC (2) and that toxically induced lesions of the microvilli may be accompanied by alterations of the molecular organization of the plasma membrane leading to changes in the function of those membrane proteins which act as enzymes and which arc concerned with the transmembrane transport (6, 18), the alterations of the microvilli border appear to be of greatest importance among all morphologic changes in the cirrhotic livers here described.

Acknowledgements Mrs.

Valuable technical assistance was provided by Mrs. HANNA ZUBER for manuscript preparation.

THEA SIEMER

and Mrs.

HEIDRUN GUDER.

We are grateful to

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