Toxicology Letters, 16 (1983) 63-67 Elsevier Biomedical Press

63

MODULATION OF GALACTOSAMINE-INDUCED LIVER INJURY BY

SOME AMINO ACIDS OR TRITON WR1339

(Hepatotoxicity; potentiation; protection)

ELIDA C. DE FERREYRA, OLGA M. DE FENOS and JOSE A. CASTRO

Centro de Investigaciones Toxicoldgicas (CEITOX), CITEFA/CONICET, Zufriategui y Varela, 1603

Villa Martelli, Pcia. de Buenos Aires (Argentina)

(Received September 9th, 1982)

(Revision received and accepted October 12th, 1982)

SUMMARY

Galactosamine administration to rats (600 mg/kg, i.p.) produces a severe liver necrosis at 24 h.

Pretreatment with cystine, tryptophan or methionine (600 mg/kg, p.o., 30 min before the hepatotoxin)

significantly prevents galactosamine-induced liver necrosis. Glutamic or aspartic acids, however, were

not protective under a similar dosage regimen. The prior administration of Triton WR1339 (1 g/kg, i.v.,

30 min before galactosamine) significantly enhanced the liver response to galactosamine-induced liver

injury, whereas the administration of tyrosine resulted in death of the animals.

INTRODUCTION

We previously reported that aspartic acid, cystine, methionine and tyrosine prevented CC4-induced liver injury [I]. The protective effects were not due to interference with the formation from CC& of reactive metabolites or to effects on CCld-induced lipid peroxidation, but to amino acid-induced changes in the response of the liver cell to injury [I].

Similarly, prior administration of Triton WR1339 significantly enhances liver injury by Ccl*, thioacetamide or dimethylnitrosamine [2-51 by increasing the cell susceptibility to injury and not because of effects on activation or covalent binding or lipid peroxidation [4, 51.

If such non-specific effects were involved in either the protective effects of some amino acids or in the enhancing effects of Triton WR1339, then the possibility exists that a similar process operates with other hepatotoxins.

Abbreviations: GAL, galactosamine hydrochloride; ICD, isocitric acid dehydrogenase.

0378-4274/83/0000-0000ooo/$03.00 @ Elsevier Biomedical Press

64

In this work, we tested the possibility for modifying effects of amino acids or Triton WR1339, on galactosamine-induced liver injury.

MATERIALS AND METHODS

D( +) galactosamine hydrochloride (GAL), Triton WR1339 (Tyloxapol, a sodium alkylaryl polyether) and the amino acids were purchased from Sigma Chemical Company.

Sprague-Dawley male rats (250-290 g) were used throughout these experiments. The rats were fasted 12-14 h before administration of GAL. GAL was given i.p. at a dose of 600 mg/kg dissolved in 0.9% NaCl solution and neutralized with sodium hydroxide to pH 7.0. Amino acids were given p.o. at a dose of 600 mg/kg dissolved in water adjusted to pH 1 with HCl 30 min before the hepatotoxin. Controls received water or 0.9% saline solution. Triton WR1339 dissolved in 0.9% NaCl solution (200 mg/ml) was given i.v. at a dose of 1 g/kg 30 min before GAL administration. Controls received 0.9% NaCl solution. The animals were killed 24 h after GAL administration. The livers were rapidly removed and processed. When blood samples were taken, the animals were kept under light ether anesthesia and blood was collected from the vena cava into a syringe rinsed with heparin solution. ICD activity in plasma was measured according to Sterkel et al. [6], one unit of enzyme is that required to form 1 nmol NADPH per ml of plasma per h at 25°C. Histological techniques, methods of evaluation of damage and of statistical evaluation of the data were as previously reported [7].

RESULTS

Effect of the prior administration of some amino acids or Triton WR1339 on GAL- induced increases in ICD activity in plasma

GAL administration significantly increased ICD levels in plasma at 24 h, while the effect was reduced significantly following prior treatment with cystine, tryptophan or methionine (Table I). GAL administration to tyrosine-pretreated animals resulted in increased toxicity and all animals died. Pretreatment with Triton WR1339 significantly increased ICD levels resulting from GAL administration (Table I).

Histological observations in livers from animals receiving GAL and pretreatment with either some amino acids or Triton WR1339

Treatment with the amino acids tested or Triton WR1339 alone did not modify the normal architecture of the liver at 24 h. Administration of GAL produced at 24 h a marked (+ + +) centrilobular necrosis. Tryptophan markedly reduced (+)

TABLE I

EFFECT OF THE PRIOR ADMINISTRATION OF SOME AMINO ACIDS OR TRITON WR1339 ON GAL-INDUCED INCREASES IN ICD ACTIVITY IN PLASMA

Treatmenta ICD Degree of histologically observable necrosis

Control GAL GAL + cystine GAL + tryptophan GAL + glutamic acid

Control GAL GAL + aspartic acid GAL + methionine

Control Cystine Tryptophan Aspartic acid Glutamic acid Methionine

Control GAL Triton WR1339 Triton WR1339 + GAL

80 k 40 10450 + 5670 3 900 * 2 930b

200 * 85b 9 955 * 12 100

110 * 50 16415 + 11 850 13415 f 8 940 2 475 + 1 610b

90 * 70 150 2 45 100 + 30 115 & 27 95 f 46

120 + 30

160 + 40 7 050 + 3 870b

185 & 55 145 000 & 28 585b.c

---

+++ ++ + +++

---

+++ +++ ++

--- ---

--- ++

++++

aRats (250-290 g) were fasted 12-14 h before administration of GAL (600 g/kg, i.p.). Amino acids (600 mg/kg, p.o.) or Triton WR1339 (1 g/kg, i.v.) were given 30 min before GAL. Controls received the equivalent amount of 0.9% NaCl solution or water. Seven animals per group were used in these experiments. bP < 0.05. ‘P < 0.001 for the difference between GAL and GAL + Triton WR1339.

the extent of necrosis produced by GAL at 24 h when given 30 min before the hepatotoxin; cystine and methionine also diminished (+ +) the alteration produced by GAL. Glutamic and aspartic acids failed to prevent the necrosis produced by GAL, when given 30 min before the hepatotoxin, and the animals were killed after 24 h. In contrast, Triton WR1339 markedly increased the extent of necrosis produced by GAL at 24 h ( + + + +).

DISCUSSION

Two (cystine and methionine) of the four amino acids previously reported to be effective against CCL-induced liver injury (aspartic acid, cystine, methionine and tyrosine) [I] were also able partially to prevent GAL liver necrosis. A further two

66

amino acids (glutamic acid and tryptophan), that in our studies with CCL gave borderline effects [l] were, in the case of GAL, either non-protective (glutamic acid), or exacerbated the toxic effect leading to death of the animals (tyrosine).

Prior administration of Triton WR1339 increased liver susceptibility to GAL as observed previously with CCL, thioacetamide or dimethylnitrosamine [2-51, These facts on the one hand point to similarities in behaviour of both hepatotoxins, but on the other hand provide evidence that they are not completely alike in some aspects critical to the pathogenesis of the necrotic process.

This should not be surprising in light of the completely different mechanisms and course of the deleterious processes of these necrogenic compounds. CC4 is believed to cause liver damage through a process of covalent binding of reactive metabolites to macromolecules or by promoting lipid peroxidation [8, 91; thioacetamide because it is activated to a sulfoxide and sulfone interacting with proteins [lo, 1 l] and dimethylnitrosamine because it is transformed to a carbonium ion [12] or to other active compounds arising during reductive metabolism [13, 141. All these processes have their origins in the liver endoplasmic reticulum: GAL, in contrast, is believed to act by depletion of uridine nucleotides and by causing an early change in the plasma membrane [ 15, 161.

Concerning the nature of the processes shared in part by all these hepatotoxins and the relevance to cell injury, our past and present findings [l, 4, 51 suggest that a balance between protein synthesis and protein degradation might be somehow involved.

ACKNOWLEDGEMENTS

This work was supported by Grant AM 1319513 from the National Institutes of Health (USA).

REFERENCES

1 E.G.D. de Toranzo, E.C. de Ferreyra, O.M. de Fenos and J.A. Castro, Prevention of carbon

tetrachloride-induced liver necrosis by several amino acids, Meeting of the Argentinian Society of

Toxicology, Rosario, Argentina, August, 1981.

2 H. Iturriaga, I. Posalaki and E. Rubin, Aggravation of hepatic necrosis by lysosomal injury, Exp.

Mol. Pathol., 10 (1969) 231-239.

3 H. Iturriaga, S. Vaisman, M.E. Pino and P. Pereda, Lysosomal injury and hepatic necrosis. Effects

of Triton WR 1339 on liver cells in rats, Exp. Mol. Pathol., 14 (1971) 350-361.

4 E.C. de Ferreyra, E.G.D. de Toranzo, O.M. de Fenos and J.A. Castro, Studies on the mechanism

of enhancement of carbon tetrachloride hepatotoxicity by Triton WR 1339, Br. J. Exp. Pathol., 62

(1981) 512-518.

5 M.I. Diaz Gomez, H.M. Godoy, A. Marzi, E.C. de Ferreyra, O.M. de Fenos and J.A. Castro,

Enhancement of dimethylnitrosamine acute effects in rat liver by prior treatment with Triton WR

1339, J. Natl. Cancer Inst., 67 (1981) 1089-1092.

6 R. Sterkel, S. Spencer, S. Wolfson and H. Williams-Ashman, Serum isocitric dehydrogenase activity

with particular reference to liver disease, J. Lab. Med., 52 (1958) 176-184.

67

7 E.C. de Ferreyra, O.M. de Fenos, AS. Bernacchi, C.R. de Castro and J.A. Castro, Treatment of carbon tetrachloride-induced liver necrosis with chemical compounds, Toxicol. Appl. Pharmacol., 42 (1977) 513-521.

8 R. Recknagel, Carbon tetrachloride hepatotoxicity, Pharmacol. Rev., 19 (1967) 145-208. 9 A.S. Bernacchi, C.R. de Castro, E.G.D. de Toranzo, A. Marzi, E.C. de Ferreyra, O.M. de Fenos

and J.A. Castro, Pyrazole prevention of carbon tetrachloride-induced ultrastructural changes in rat liver, Br. J. Exp. Pathol., 61 (1981) 505-511.

10 A.L. Hunter, M.A. Holscher and R.A. Neal, Thioacetamide-induced hepatic necrosis, I. Involvement of the mixed function oxidase enzyme system, J. Pharmacol. Exp. Ther., 200 (1977) 439 (abstract).

11 E.C. de Ferreyra, O.M. de Fenos and J.A. Castro, Effect of different chemicals on thioacetamide induced liver necrosis, Toxicology, 16 (1980) 205-214.

12 P.N. Magee and J.A. Barnes, Carcinogenic nitroso compounds, Adv. Cancer Res., 10 (1967) 163-246.

13 H.M. Godoy, M.I. Diaz G6mez and J.A. Castro, Mechanism of dimethylnitrosamine metabolism and activation in rats, J. Natl. Cancer Inst., 61 (1978) 1285-1289.

14 H.M. Godoy, M.I. Diaz G6mez and J.A. Castro, Relationship between dimethylnitrosamine metabolism or activation and its ability to induce liver necrosis in rats, J. Natl. Cancer Inst., 64 (1980) 533-538.

15 J.L. Farber and S. El Mofty, The biochemical pathology of liver cell necrosis, Am. J. Pathol., 81 (1975) 237-249.

16 W. Reuter, C. Bauer, W. Bachmann and R. Lesch, The galactosamine refractory regenerating rat liver, in R. Lesch and W. Reuter (Eds.), Liver Regeneration After Experimental Injury, Stratton Intercontinental Medical Book Corp., New York, 1975, pp. 259-268.