Hepatic ATP content and hyperammonemia induced by CCl4 in rats

Tozicology, 51 (1988)111--117 Elsevier ScientificPublishers Ireland Ltd.

HEPATIC ATP CONTENT AND HYPERAMMONEMIA INDUCED BY CCI, I N R A T S

HIRO-AKIYAMAMOT0 and NARUMI SUGIHARA Department of The First Hygienic Chemistry. Faculty of PAarmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyam~ Hiroshima 7"29-0"2(Japan) (Received August 31st. 1987) (Accepted January 5th. 1988)

SUMMARY An investigation of the mechanism of development of hyperammonemia observed in CCl,-induced hepatic encephalopathy was performed in rats. CCI, (1.0 ml/kg 3 times per week for over 10 weeks) caused a severe hyperammohernia and depletion of hepatic ATP contents in only those rats with hepatic encephalopathy. However, CCI~ (1.0 ml/kg 3 times per week for 7 weeks) did not cause hepatic encephalopathy and did not change in blood ammonia levels. Administration of 2,4-dinitrophenol (2,4-DNP) in these CC14-treated rats caused hepatic encephalopathy within 30 min after injection and then the increase of 140 ~g/dl in blood ammonia levels and the decrease of 80% in hepatic ATP contents were observed. However, the administration of 2,4DNP in CCl~-untreated rats did not cause hepatic encephalopathy within 30 min after injection although the increase of 70 ~g/dl in blood ammonia levels and the decrease of 80% in hepatic A T P contents were observed. Hepatic activities of carbamylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), important enzymes of the urea cycle, were significantly inhibited by 85% and 600/0 respectively, in rats treated with CCI 4 plus 2,4DNP. However, in rats treated with 2,4-DNP and without CCI,, the hepatic activities of CPS and ASS were inhibited only 25% and 0°/0, respectively. These findings suggest that the severe hyperammonemia, which may be produced by the decrease of hepatic ATP content and the inhibition of CPS and ASS, may play an important role in induction of hepatic encephalopathy. K e y words: Ammonia; Hepatic encephalopathy; ATP; Urea cycle enzymes;

2,4-Dinitophenol; CC14 Address all correspondence and reprint requests to: Hiro-aki Yamamoto, Ph.D., Department of the First Hygienic Chemistry. Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University. Fukuyama. Hiroshima 7294)2,Japan. 0300-4~X/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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INTRODUCTION It has been known that hepatic coma is one of the chief causes of primary hepatic disease. It is very important to elucidate the mechanism of development of hepatic encephalopathy, since the death rate in these patients is high. Knowledge of the mechanism may result in appropriate therapy. In our previous report [1], it was shown that CCI, (1.0 ml/kg 3 times per week for over 10 weeks) caused hepatic encephalopathy in 80% of the treated rats. Accompanying the hepatic encephalopathy were, hematemesis, abdominal dropsy and hyperammonemia, conditions observed in hepatic coma patients. Furthermore, the blood ammonia levels were tremendously changed in only those rats with hepatic encephalopathy [1]. Therefore, it is of interest to investigate the mechanism of hyperammonemia observed in hepatic encephalopathy induced by CC14. In this experiment, we studied the relationship between blood ammonia levels and the hepatic ATP, a substrate of CPS and ASS enzymes, contents in rats treated with 2,4-DNP, which blocks ATP synthesis as an uncouplar of oxidative phosphorylation in mitochondria. MATERIALS AND METHODS Male Wistar rats (150--180 g) were purchased from Ishikawa Pref. Animals Laboratories (Ishikawa, Japan). The animals were housed in a cage in a temperature- and light-controlled (12 hr dark/light) room and were given diet of commercial chow and water ad libitum. Rats received an intraperitoneal injection of CCI 4 (1 ml/kg 3 times per week for 7 weeks). These treated rats were further injected i.p. with 2,4-DNP (30 mg/kg in 0.2 ml of dimethyl sulfoxide) and killed 20--30 rain after administration.

Determination of A TP A part of rat liver was immediately frozen in liquid nitrogen and homogenized in ice,~old 0.6 N HCIO,. After centrifugation, ATP in the supernatant was assayed with a Boehringer-Mannheim ATP-test kit acording to the method of Bficher [2].

Preparation of liver mitochondria, microsomes and cytosol Rats were killed by decapitation and the liver was homogenized in ice,cold 0.25 M sucrose buffer (pH 6.8) containing 5 mM Hepes, 1 mM dithiothreitol and 1 mM EGTA. This 10°/0 homogenate was centrifuged at 1000 g for 5 min. The supernatant was centrifuged at 7800 g for 10 rain to give a mitoehondria pellet. The supernatant was further centrifuged at 105 000 g for 60 min to yield a cytosol fraction and microsome pellets. Pellets were washed with 20 mM Hepes buffer (pH 6.8) by centrifugation at 7800 or 105 000 g. These pellets were homogenized with 20 mM Hepes buffer (pH 6.8) for use in the experiments described below.

De termination of Mg~*-ATPase activity Mg2*-ATPase activity was measured by the colorimetric determination of 112

phosphate hydrolyzed from A T P [3]. To measure mitochondrial Mg2"-ATPase activity, aliquots containing 2 8 0 - 6 2 0 ~g protein were incubated at 37 °C for 10 min in I ml of 20 mM Hepes buffer (pH 6.8) containing 5 mM MgCI~, 1 mM EGTA and I mM ATP. After incubation the reaction was stopped by the addition of 1 ml of 10°/0 (w/v) trichloroacetic acid (TCA). To measure microsoreal Mg~÷-ATPase activity, aliquots containing 3 6 0 - 4 9 0 ~g protein were incubated as described for the determination of mitochondrial ATPase activity except that 1/~g oligomycin was added.

Determination of CPS activity Mitochondria containing 7 5 - 1 6 0 ~g protein was incubated at 37°C for 10 min in 1 ml of 50 mM Hepes buffer (pH 8.0) containing 5 mM N-acetylglutamate, 15 mM MgCI 2, 10 mM NH4C1, 50 mM NaHCO 8, 5 mM ornithine and 10 mM ATP. After incubation, the reaction was stopped by the addition of 1 ml of 10°/o TCA. After centrifugation, the amount of citrulline produced in the supernatant was determined by the diacetylmonooxime method [4]. Determination of A S S activity Cytosol fraction (approx. 600/~g protein) was incubated at 37 °C for 10 rain in 1 ml of 50 mM Tris--HCl buffer (pH 7.5) containing 5 mM MgCI 2, 5 mM aspartate, 0.5 mM citrulline and 1 mM ATP. After incubation, the reaction was stopped by the addition of I ml 10% TCA. After centrifugation, the amount of citrulline used by the reaction was determined by the diacetylmonooxime method [5]. Determination of ammonia Blood was taken from the jugular vein of rats just before and 20--30 min after 2,4-DNP injection and the ammonia concentration was immediately determined by the method of Okuda [6]. Other methods Protein was determined by the phenol method [7]. All data were compared by an analysis of variance. When the analysis indicated that a significant difference existed, the means of selected groups were compared by Student's t-test. RESULTS

Effect of 2,4-DNP on ammonia levels in blood Blood ammonia levels of just before and 20-- 30 rain after 2,4-DNP treatment were determined in rats treated with or without CCI4. 2,4-DNP increased in the blood ammonia levels to 70/~g/dl as compared to that of just before treatment of 2,4-DNP in CCl~-untreated rats but did not cause hepatic encephalopathy 30 rain after treatment. However, in rats treated with CCI4, 2,4-DNP increased to 140 ~g/dl in blood ammonia levels as compared to that of just before the treatment (Fig. 1) and caused hepatic encephalopathy (loss of righting reflex) within 20--30 rain after the treatment. 113

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Fig. 1. Effects of 2,4-DNP on ammon/a concentration of blood in rats. Ammonia concentration of blood was determined as described in Methods. The ordinate represents amount of ammonia increase (~Jdl) in blood as compared to that of control (DMS). Values represents the mean ± S~E.M., • = 6. Asterisks ind/eate significant increase from control (*P < 0.05, **P < 0.001).

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Fig. 2. Effects of 2,4-DNP on hepatic A T P contents in rats. ATP contents were determined in rats liver as described in Methods. Values represent the mean ± S . E ~ . , n = 8. Asterisks ind/eate significantly different from control (ep < 0.01, , , p < 0.001).

114

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Fig. 3. Effects of 2,4-DNP on Mg"-ATPase activity of hepatic mitechondria or microsomes in rats. Mg"-ATPase activity of mitochondria and mim'osomes in liver from rats. Microsomes and mitochondria were prepared from whole liver homogenate in rats (see Methods}. Values represents the mean ± S.E.M., n = 6. Asterisks indicate significantly different from control ( * P < 0 . 0 5 , * * / ' < 0.01}.

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Fig. 4. Effects of 2,4-DNP on activities of CPS and A S S in liver from rats. Ut'~ and ASS activities were assayed as described in Methods. The ordinate represents the % of inhibition of CPS or ASS activity as compared to control (CPS activity; 1.03 ± 0.05 ~mol citrulllnehng protein, ASS activity; 1.14 ± 0.15/~nol c/trull/ne/mg protein). Values represent the mean ± S.E.M., n = 10. Asterisks indicate significantly different from control {'P < 0.05, **P < 0.01).

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Effects of ~ 4-DNP on A TP content in liver Hepatic ATP content in rats treated with CCI~ for 7 weeks was significantly decreased by 38% from that of control. However, the ATP content was elicited a tremendous decrease (80°/0) by 2,4-DNP treatment (Fig. 2). Effects of CCl, and ~ - D N P on Mg~*-ATPase activity of mitochondria and microsomes in liver Hepatic activity of Mg2°-ATPase from rats treated with CCI, (Fig. 3) was increased by 24% and 169% in mitochondria and microsomes, respectively. However, 2,4-DNP did not change the hepatic activity of Mg2+-ATPase in mitoehondria and microsomes from rats treated with and without CCI~. Effects of ~ - D N P on activity of CPS or A S S Hepatic activities of CPS and ASS, important enzymes of the urea cycle, were only inhibited 25% and 00/0 by 2,4-DNP in CCl,-untreated rats, respectively. However, in rat treated with CCI4, 2,4-DNP elicited an inhibition of 85% or 600/0 in hepatic activity of CPS or ASS, respectively, although CCI~ alone inhibited by 30% only in both activities (Fig. 4). DISCUSSION In a previous report we showed that CCl~ (1.0 ml/kg 3 times per week for over 10 weeks) caused hepatic encephalopathy in 800/0 of the treated rats [1]. Accompanying the hepatic encephalopathy were, loss of righting reflex, hematemesis, abdominal dropsy and hyperammonemia, conditions observed in hepatic coma patients. All rats demonstrating hepatic encephalopathy displayed a high ammonia level in blood. This previous finding suggested that the increase in blood ammonia levels may play an important role in development of hepatic encephalopathy induced by CCI4. Furthermore, hepatic ATP content was significantly decreased by 60°/0 in rats with hepatic encephalopathy and CPS and ASS activities were dependent on ATP concentration in vitro [1]. To elucidate the mechanism of hyperammonemia observed in hepatic encephalopathy induced by CCI4 in rats, in this experiment, we investigated the relationship between the hepatic ATP contents and the blood ammonia levels in rats treated with 2,4-DNP, which blocks ATP synthesis as an uncouplar of oxidative phosphorylation. 2,4-DNP elicited a decrease of 80% in hepatic ATP contents and an increase of 70 ~g/dl in blood ammonia levels but did not cause hepatic encephalopathy in CCl4-untreated rats. In addition, 2,4-DNP did not change ASS activity in liver. On the other hand, in rats treated with CCI~, 2,4-DNP elicited a decrease of 800/0 in hepatic ATP contents, an increase of 140 ~g/dl in blood ammonia levels and an inhibition of 600/0 in hepatic ASS activity and caused hepatic encephalopathy. These results indicate that a depletion of hepatic ATP content contributes in a little increase of blood ammomia levels in rats but do not contribute directly in a tremendous increase of blood ammonia levels observed in hepatic encephalopathy. Hepatic activities of CPS and ASS were inhibited by 85% and 60% by 2,4116

DNP plus CCI~, respectively (Fig. 4). Since ASS is a rate limiting enzyme in the urea cycle causality, the decreased ASS activity may play an important role in the increase of blood ammonia levels. Furthermore, since we have found that ASS activity is inhibited by depletion of hepatic ATP contents [1], both decreases of ASS activity and ATP contents in liver may cause an increase of blood ammonia levels. These findings suggest that hyperammonemia, which is produced by the decrease in hepatic ATP content and by the inhibition of ASS activity, may play an important role in induction of hepatic encephalopathy. James et al. [8] have found that hyperammonemia contributes to encephalopathy indirectly, by raising the brain concentration of neutral amino acids which alter neurotransmitter metabolism, rather than directly, by toxic effects on neuronal metabolism. Therefore, it is suggested that hyperammonemia produced by CCI, administration may contribute to encephalopathy by raising the brain concentration of neutral amino acids [9,10]. However, since a number of reports have suggested that organic amines [11,12], short chain fatty acids [13] or mercaptans [13] produced by hepatic coma may also have a role in hepatic encephalopathy, it could be important to further investigate the relationship between blood ammonia levels and the other factors involved in hepatic encephalopathy. REFERENCES H.-A. Yamamoto and N. Sugihara, Blood ammonia levels and hepatic encephalophathy induced by CCI, in rats. Toxicol. Appl. Pharmacol. 91 (1987) 461. 2 T. Bflcher, 0 b e r ein phosphatubertragendes garungsferment. Biochim. Biophys. Acta, 1 (1947) 292. 3 A.P. Dawson and D.V. Fulton, Some properties of the CaZ'-stimulated ATPase of a rat liver microsomal fraction. Biochem. J., 210 (1983) 405. 4 M. Mori and P.P. Cohen, Preparation of crystalline carbamyl phosphate synthetase I from frog liver. J. Biol. Chem., 25 (1978) 8337. 5 S. Takada, T. Saheki, Y. Igarashi and T. Katsunuma, Studies on rat liver argininosuccinate synthetase inhibition by various amino acids. J. Biochem., 85 (1979) 1309. 6 H. Okuda and S. Fujita, Direct colorimetric determination of blood ammonia. Salshin Igaku, 21 (1966) 622. 7 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with folin phenol reagent. J. Biol. Chem., 193 (1951) 262. 8 J.H. James, V. Ziparo, B. Jeppsson and J.E. Fischer, Hyperammonemia, plasma amino acid in balance and blood-brain amino acid transport: A unified theory of portal systemic encephalopathy. Lancet, 13 (1979) 772. 9 J.E. Fischer, J.M. Funovis, A. Aguirre, J.H. James, J.M. Keane, R.I.C. Wesdorp, N. Yoshimura and T. Westman, The role of plasma amino acids in hepatic encephalopathy. Surgery, 78 (1975) 276. 10 A. Ishikawa, H. Ishiyama, T. Enomoto0 A. Ozaki, K. Fukao, T. Okamura, Y. Iwasaki and H.-H.-A. Yamamoto, Hepatic coma and amino acids in the nerve endings of the central nervous system. Life Sci., 37 (1985) 2129. 11 J.E. Fischer and R.J. Baldessarini, False neurotransmitters and hepatic failure. Lancet, 2 (1971) 75. 12 A. Smith, Rossi-FaneUi, V. Ziparo, J.H. James, B. Perelle and J.E. Fischer, Alterations in plasma and CSF amino acids, amines and metabolites in hepatic coma. Ann. Surg., 137 (1978) 343. 13 L. Zieve, W.M. Doizaki and F.J. Zieve, Synergism between mercaptans and ammonia or fatty acids in the production of coma: a possible role for mercaptans in the pathogenesis of hepatic coma. J. Lab. Clin. Med., 83 (1974) 16. 1

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