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N-Acetyl cysteine is an early but also a late preventive agent against carbon tetrachloride-induced liver necrosis
Toxicology ELSEVIER SCIENCE IRELAND
letters
Toxicology Letters 71 (1994) 87-95
WAcetyl cysteine is an early but also a late preventive agent against carbon tetrachloride-induced liver necrosis E.G. Valles, CR.
de Castro,
J.A. Castro*
Cenrro de Investigaciones Toxicokigicas (CEITOX) CITEFA/CONICET (1603) Villa Martelli, Buenos Aires, Argentina
Zufiiategui
4380,
(Received 24 May 1993; revision received 8 July 1993; accepted 8 July 1993)
Abstract
N-Acetyl cysteine (NAC) treatment 30 min before or 6 or 10 h after carbon tetrachloride (CC&) administration significantly prevented the liver necrosis produced by the hepatotoxin at 24 h. NAC pretreatment was able to partially decrease the covalent binding of Ccl, reactive metabolites at 1 and 3 h of poisoning and, to a small extent, the concentration of Ccl, reaching the liver at 3 h. NAC also diminished partially the Ccl,-promoted increases in lipid peroxidation at 3 h, but had an enhancing effect of its own of small intensity. Results suggest that early and late protective effects of NAC might be attributable to its prior conversion to cysteine and glutathione. Key words: N-Acetyl cysteine; Liver damage; Carbon tetrachloride; Lipid peroxidation; Selfresponse injury; Covalent binding; Reactive metabolites
1. Introduction
IV-Acetyl cysteine (NAC) is an amino acid analog that has found wide clinical applications either because of its cytoprotective properties or as antidote in a variety of intoxications [1,2]. Its use has been backed by a large number of detailed experi-
mental studies either in animals or in in vitro systems [1,2]. In the case of carbon tetrachloride (CC&), NAC has been effectively employed in cases of human poisoning without previous detailed mechanistical studies in animals [1,3-51. * Corresponding author, Tel.: 54-I-761~0031/0081
(ext. 239); Fax: 54-I-761-591
0378-4274/94/%07.00 0 1994 Elsevier Science Ireland SSDI 0378-4274(93)03023-M
Ltd. All rights reserved
l/3063.
88
In this work, we attempt to provide NAC in cases of human CCL, poisoning relative protective efficiency of NAC previously developed in our laboratory
E.G. V&es et al. / Taxied.
Lett. 31 (1994) 83-9s
rational experimental support to the use of and to develop terms of comparison for the against Ccl4 intoxication with treatments or reported by others in literature [6].
2. Materials and methods 2.1. Chemicals “CC14 (spec. act., 2.8 m~~mmol) was purchased from New England Nuclear. NAC was purchased from Sigma Chemical Co. All other chemicals were of the best available quality. 2.2. Animals and treatments Sprague-Dawley male rats (220-270 g) were used in the experiments. Food was withdrawn 12-14 h before CC14 intoxication, but the animals had free access to water. Temperature in the animal room was 22 f 2°C and the relative humidity was between 45 and 65%. Lights in the animal room were on from 06:OOto 18:OOh. Five to 8 animals per group were used as stated for each experiment. NAC was given p.o. in water solution at the dose of 2000 mg/kg (400 mgml solution) 30 min before, 6 or 10 h after CC14. Control rats received the equivalent amount of water. ‘VC14 was given i.p. as 5% CC& (v/v) in olive oil (30 x lo6 dp~ml) at a dose of 5 ml of the solution/kg. Ccl4 (5 ml/kg) was given i-p. as a 20% (v/v) solution in olive oil. Control rats received onfy olive oil i.p. The animals were killed by decapitation at different times after Ccl4 administration; livers were rapidly removed and processed. Whenever blood samples were taken, animals were kept under light ether anesthesia, and blood was obtained with heparinized syringes from the inferior vena cava. 2.3. Enzymatic and chemical determinations Isolation of the microsomal fraction was performed as previously reported [7]. The in vivo incorporation of radioactivity from 14CC14to microsomal lipids was determined according to procedures described previously 171. Results are given in dpm per mg of microsomal lipid. Lipids were extracted according to the procedure reported by Castro et al. [7]. The 14CCf4concentrations in liver were estimated by a procedure described by Recknagel and Litteria [8] up to the microdiffusion step, and then the t4CC14collected in the toluene in the center well of the cell was transferred to a scintillation counting vial and counted. Results were corrected for quenching by the channels ratio method. Lipid peroxidation in vivo was measured by conjugated diene ultraviolet absorption of the microsomal lipid extracts [9]. The results are expressed as the absorbance at 243 nm x 1000 for a solution having 1 mg of microsomal lipid/ml. NADPHlinked isocitric acid dehydrogenase (ICD) activity in plasma was determined according to Sterkel et al. [lo]. Activity is given in units: 1 unit being the amount of the enzyme producing 1 nmol NADPH/ml plasma/h at 25°C. 2.4. Histological techniques After removal of the livers, small portions of the left and central lobes were
89
E.G. Valles et al. / Toxicol. Lett. 71 (1994) 87-95
immediately fixed in Bouin’s solution, embedded in paraffin and stained with hematoxylin-eosin. The specimens were coded to avoid bias and were evaluated histologically by three independent observers. To quantify the morphological changes, liver sections were graded for necrotic changes using an arbitrary scale: + = slight (about 20-30% necrosis); ++ = moderate (about 50% necrosis); +++ = marked (about 75% necrosis) and ++++ = very intense (about 90-100% necrosis). Results reported as representative for each experimental condition were the means of observations made by all observers for a given experimental condition. 2.5. Statistics A decision tree for selecting the hypothesis testing statistical procedures described by Gad and Weil [l l] was applied to the results of every experiment. Homogeneity of variance was established using either the Barlett homogeneity of variance test or the F-test for comparisons involving three or more groups or two groups, respectively. According to significance of homogeneity of variance, one of the following tests were applied: Student’s r-test or the Cochran t-test. Parametric analysis of variance tests were used for comparisons involving three or more groups of data [l 11. 3. Results 3.1. Effect of NAC in vivo on microsomal lipid peroxidation (LP) produced by Ccl, at different times after administration NAC administered alone produced a minor but statistically significant enhancement of LP in microsomal lipids at 1 h but not at 3 h (Table 1). However, NAC values at 3 h exhibited a tendency to be enhanced, considering that the mean value
Table 1 CCl&duced Treatmenta
Control CCI, NAC NAC + Ccl,
lipid peroxidation
of microsomal
lipids in rats previously
Lipid peroxidation
(x f S.D.)b,C
lh
% of control
3h
100 265 137 241
212 540 337 421
147 389 201 354
f + f f
22 46dqf 43 26d,f
treated
with NAC
% of control zt * + f
60 134e.f 136 87e,’
100 255 158 198
‘Sprague-Dawley male rats (260-280 g) that had not been fed for 12-14 h were injected i.p. with CCI, as a 20% solution (v/v) in olive oil at a dose of 5 ml/kg 30 min after NAC administration. NAC was given p.o. in water solution at a dose of 2000 mg/kg. Control received the equivalent amount of olive oil and water. Five animals per group were used in this experiment. bThe lipid peroxidation value is expressed as absorbance at 243 nm x 1000 for a solution having 1 mg of microsomal lipid/ml. ‘The P value for the overall effect of the NAC on the CC&-induced lipid peroxidation obtained by analysis of variance was P < 0.05. dCCI, vs. Control at I h; NAC + CC& vs. Control at 1 h and NAC + Ccl, vs. NAC at 1 h: P < 0.001 Student’s t-test (N, + N2 - 2). eCCI, vs. Control at 3 h; NAC + Ccl, vs. Control at 3 h: P < 0.01, Student’s t-test (N, + N, - 2). ‘Ccl, vs. CCI, + NAC at 1 and 3 h: P > 0.05, Student’s r-test (N, + N, - 2).
Table 2 Covalent binding of “CC14-reactive metabolites of rats previously treated with NAC
to hepatic
Treatment”
14CC14 concentration in liver (dpmig liver + SD.)
Covalent binding 14C from Ccl, Lipid f SD.
lh %CI, “CCl,
of
microsomal
(dpm/mg)
lipids and “CC14 concentrations
R x IO1 (zt S.D.)b
Lipid
+ NAC
331 z!z 56 254 f 46d
92408 76352
+ 20 105 f 11 596’
3.73 f 0.99 3.38 f 0.72’
14CC14 + NAC
412 f 39 368 + 35’
65877 45208
f f
6.53 f 1.55 8.62 f 2.52’
3h
“cc14
14323 10984d
“Sprague Dawley male rats (220-240 g) that had not been fed for 12-14 h were injected i.p. with 14CC14 as 5% Ccl, (v/v) solution in olive oil (30 x IO6 dpm/ml) at a dose of 5 ml solution/kg, 30 min after NAC. NAC was administered p.0. in water solution at the dose of 2000 mgikg, 30 min before “Ccl,. Controls received the equivalent amount of water. The animals were sacrificed either I or 3 h after 14CC14 administration, and their livers were processed for microsomal lipid extraction and counting (see Materials and methods for details). Eight animals per group were used. bR is the ratio of irreversible binding to lipids to 14CC14 concentations in liver. ‘P > 0.05, Student’s r-test (N, + N, - 2). dP < 0.01, Student’s t-test (N, + N, - 2). eP > 0.05, Student’s r-test (N, + Nz - 2). ‘P < 0.05, Student’s r-test (N, + N? - 2).
observed was 58% higher than that in the control group (Table 1). NAC administration to CC&-poisoned animals, in contrast, significantly decreased the intensity of the Ccl,-promoted LP process at both 1 and 3 h of poisoning, as evidenced by the two-way analysis of variance of results (Table 1). Notwithstanding, comparison of LP values between groups Ccl, and NAC + CC& using t-test, indicated that results in one group were not significantly different from those in the other at either 1 or 3 h of poisoning (Table 1). 3.2. Effect of pretreatment with NAC on the covalent binding (CB) of “CC14 to hepatic microsomal lipids and 14CC14 concentrati0n.s in liver NAC administration slightly, but significantly, reduced the intensity of the CB of CC& reactive metabolites to microsomal lipids at 1 or 3 h (Table 2) and the “CC14 concentrations reaching the liver at 3 h, but not at 1 h (Table 2). However, the intrinsic ability of the liver microsomal fraction to activate CC14 (as defined by the ability to activate Ccl4 to reactive metabolites that bind covalently to microsomal lipids per unit of concentration) R was not significantly modified by NAC treatment at 1 h and slightly, but significantly, increased at 3 h (Table 2). 3.3. Effect of NAC administration on Ccl,-induced liver necrosis The extent of CC14-induced liver necrosis was evidenced by histological
examina-
91
E.G. Vales et al. / Toxicol. Lett. 71 (1994) 87-95 Table 3 Effect of NAC on CC&-induced Treatment” 30 min before Control ccl, NAC Ccl, + NAC 6 h after Control CCI, NAC CCI, + NAC 10 h after Control CCI, NAC Ccl, + NAC
hepatic
necrosis
ICDb* (units f S.D.) 303 f 37 88200 f 39800 4875 f 1153
24 h after administration
of CC14 to rats
Degree of histologically
observed
necrosis
++++ -
4500 f 2001d
215 f 58 100160 f 46151 3112 i 918 9373
l
7609=
203 86760 3124 27780
f f f zt
26 21092 823 16491f
++++ +
++++ ++
“Ccl4 and NAC were administered at the doses indicated in Table 1. NAC was given 30 min before, 6 or IO h after the hepatotoxin. The animals were sacrificed 24 h after Ccl, administration. Eight animals per group were used in these experiments. bIsocitric acid dehydrogenase: 1 unit of enzyme is the amount required to form 1 nmol NADPH/ml plasma/h at 25°C. ‘The P value of the overall effect of NAC on the Ccl,-induced increase in ICD, obtained by two-way analysis of variance, was P < 0.05 at 30 min, 6 and IO h. dCCI, + NAC vs. Ccl,: P < 0.001, Student’s t-test (Nt + N, - 2). ‘CC14 + NAC vs. CCld P < 0.02, Student’s t-test (N, + N, - 2). ‘Ccl, + NAC vs. Ccl,: P < 0.01, Student’s t-test (N, + N, - 2).
tion and also by determination of ICD levels in plasma (Table 3). NAC given alone only produced at 24 h a minor degree of hydropic degeneration of the hepatocytes and trabecular disorganization. No necrosis was observed in hepatocytes of animals treated with NAC alone. There was, however, a significant increase in ICD activity in this group of animals in relation to that found in controls (Table 3). Administration of CC& to animals produced an intense centrolobular necrosis of the liver (++++) at 24 h (Fig. 1) and a marked increase in ICD activity in relation to controls (Table III). Treatment with NAC 30 min before or 6 or 10 h after Ccl, significantly decreased the extent of the histologically evident necrosis produced by the hepatotoxin at 24 h (Fig. 2) or the levels of ICD observed in the CClrpoisoned animals (Table 3). 4. Discussion
In full agreement with previous observations in human beings poisoned with CC& and treated with NAC [1,3-51, we reproduced the preventive actions of this
92
E.G. Valles et al. / Toxicol. Lett. 71 (1994) 87-95
Fig. I. Liver section of a rat 24 h after administration of Ccl,. x 120.
Note the centrilobular
necrosis.
H & E
N-acetylated derivative of cysteine against the effects of the hepatotoxin in rats. Protection afforded by NAC was better when administered 30 min before than when given 6 or 10 h after CCL,. This suggested that NAC was able to exert beneficial actions at the early stages of poisoning (before 6 h), but also at the late steps of intox-
Fig. 2. Liver section of a rat 24 h after admmlstration administration of Ccl,. The centrilobular
of Ccl,. This rat was treated with NAC 6 h after zones are well preserved. H & E x 120.
E.G. Valles et al. / Toxicol. ht.
93
71 (1994) 87-95
ication (after 6 h). According to present views on the mechanism of CCL hepatotoxicity, the early part of the process is dominated by factors such as Ccl, absorption, Ccl4 activation to * CC& and CC1302 ’ free radicals, the covalent binding of ’ Ccl3 to cellular components, and the H abstraction from PUFA by the CC130z ’ and the ’ Ccl,, to initiate LP [ 12-141. Later stages of Ccl4 poisoning involve perturbation of Ca2+ homeostasis and the effect of it on degradative processes mediated by proteases, phospholipases and potentially by other enzymes such as endonucleases [6,14-171. The thiol status of proteins was also found to be critical at this late stage and fundamentally preserved by GSH [18,19]. The NAC protective effects on the early stage of CC& poisoning observed under our 30 min prior to CC& administration experiment might be due to the contribution of several factors, including a decrease in the CB, decreases in the levels of CCL, reaching the liver, and partial blockade of CC&-promoted lipid peroxidation. The analysis of NAC effects on CC&-promoted LP are complex and are not fully understood with the presently available information. However, our experiments indicate that NAC could be in part a chain-breaking antioxidant, as previously defined by Halliwell and Gutteridge [20]. In effect, NAC-treated CCL,-poisoned animals had a significant decrease in CCL,-promoted LP, as evidenced by the conjugated diene hyperconjugation technique. This procedure is able to reveal modulations of the initiation stage of LP involved in chain-breaking antioxidants effects ]201. NAC administration alone also sparked a moderate LP process in our experiments, which might explain the minor cell injury evidenced in hepatocytes from NAC-treated animals. This is not unusual behavior for a thiol-containing compound as is NAC. In effect, many compounds having SH groups have exhibited both LPstimulating or -inhibiting properties, according to their concentration or other conditions [21-231. Additional beneficial effects of NAC in the LP process occurring after the initiation stage are likely to occur because it is known that NAC is able to destroy several reactive oxygen species produced during LP [24,25]. Concerning NAC protective effects at the late stage of Ccl4 poisoning, they might be related to the ability of NAC to generate the GSH precursor amino acid cysteine [25]. Both cysteine itself [26] and GSH or precursors [27,28] were found to be late protective agents against CCla-induced liver necrosis. Irrespective of the full understanding of the mechanism of NAC preventive effects, the mere fact that it is protective when given at late stages of the process gives sufficient rational support for its use in cases of humans poisoned with CCL,. If beneficial effects of NAC only operated at early stages of poisoning, the usefulness of its employment would be limited. 5. Acknowledgement This work was partially supported by Grant DK 13195-20 from the National Institutes of Health. USA. 6. References I
Zambon Group (1992) Biomedical Documentation Morel1 Arti GraIiche, Osnago, Italy, pp. 7-106.
Centre.
N-Acetylcysteine.
Antidotal
Properties.
94
E.G. Vulles ct al / Touical.
Lert. 71
i IYY41 87-Y>
Flanagan, R.J. and Meredith, T.J. (1991) Use of N-Acetylcysteine in clinical toxicology. Am. J. Med. 91 (Suppl. 3C), 13lS-139s. Ruprah, M., Mant, T.G. and Flanagan, R.J. (1985) Acute carbon tetrachloride poisoning in 19 patients: implications for diagnosis and treatment. Lancet I 1 1027-1029. 4
6
8 9
14 15
16 17
18 19 20 21
22 23 24
25
Mathieson, P.W., Williams. G. and MC Sweeney, J.E. (1985) Survival after massive Ingestion of carbon tetrachloride treated by intravenous infusion of acetylcysteine. Hum. Toxicol. 4. 627-631. Davis, M. (1986) Protective agents for acetaminophen overdose. Semin. Liver Dis. 6, 138-233. Castro, J.A. (1990) Prevention ofchemically induced liver injury. In: R.S. Goldstein, W. Hewitt and J.B. Hook (Eds.), Toxic Interactions, Academic Press. New York. pp. 233-257. Castro, J.A. and Diaz Gbmez, M.l. (1972) Studies on the irreversible binding of “C-CC14 to microsomal lipids in rats under varying experimental conditions. Toxicol. Appl. Pharmacol. 23. 541-552. Recknagel, R. and Litteria, M. (1960) Biochemical changes in carbon tetrachloride fatty liver. Concentration of carbon tetrachloride in liver and blood. Am. J. Pathol. 36, 521-531. Klaassen, C. and Plaa. G.L. (1969) Comparison of the biochemical alterations elicited in livers from rats treated with carbon tetrachloride. chloroform, 1,1,2-trichloroethane and I. I, I-trichloroethane. Biochem. Pharmacol. 18. 2019-2027. Sterkel, R., Spencer, S.. Wolfson, S. and Williams-Ashman. H. (1958) Serum isocitric dehydrogenase activity with particular reference to liver desease. J. Lab. Clin. Med. 52. 176-180. Gad, S.C. and Weil, C.S. (1982) Statistics for toxicologist. In: A.W. Hayes (Ed.), Principles and Methods of Toxicology. Raven Press, New York, pp. 273-320. Castro, J.A. (1984) Mechanistical studies and prevention of free radical cell inJury. In Proceedings IX International Congress of Pharmacology, MacMillan Co., London, Vol. 2, pp. 243-270. Slater, T.F. (1982) Activation of carbon tetrachloride: chemical principles and biological significance. In: D.C. McBrien and T.F. Slater (Eds.), Free Radicals. Lipid Peroxidation and Cancer. Academic Press. New York. pp. 243-270. Recknagel. R.O., Glende, E.A.. Dolak. J.A. and Waller. R.L. (1989) Mechanisms of carbon tetrachloride toxicity. Pharmacol. Ther. 43, 139-154. de Ferreyra. E.C., Villarruel. M.C.. Bernacchi, A.S., de Fenos, O.M. and Castro, J.A. (1992) Prevention of carbon tetrachloride-induced liver necrosis by the chelator Alizarin sodium sulfonate. Exp. Mol. Pathol. 56, 197-207. Villarruel, M.C., Fernandez. G., de Fenos, O.M. and Castro. J.A. (1990) Modulation of the course of CC],-induced liver injury by the anticalmodulin drug thioridaaine. Toxicol. Lett. 51. 13-21. de Ferreyra. E.C., Villarruel, M.C.. Bernacchi. AS., Fernandez. G., de Fenos. O.M. and Castro. J.A. (1989) Late preventive effects against carbon tetrachloride-induced liver necrosis of the calcium chelating agent Calcion. Arch. Toxicol. 63, 450-455. Reed. D.J. (1990) Gluthathione: toxicological implications. Annu. Rev. Pharmacol. Toxicol. 30. 603-63 I. Brigelius, R. (1985) Mixed disulfides: biological functions and increase in oxidative stress. In: H. Sies (Ed.). Oxidative Stress, Academic Press, New York, pp. 243-272. Halliwell, B. and Gutteridge, J.M.C. (1990) Free Radicals in Biology and Medicine. 2nd edn. Oxford University Press, Oxford, pp. 234-260. Kamutaki, T., Sugita, 0.. Ozawa, N. and Kitagawa. H. (1977) Stabilization and induction of a lipid peroxidation inhibitor present in the soluble fraction of rat liver homogenates. Toxicol. Appl. Pharmacol. 40. 283-289. Haenen, G.R., Vermeulen, N.P.. Timmerman, H. and Blast, A. (1989) Effects of thiols on lipid peroxidation in rat liver microsomes. Chem.-Biol. Interact. 71. 201-212. Zimmerman. W.F. and Keys, S. (1991) Effects of antioxidants dithiothreitol and vitamin E on phospholipid metabolism in isolated rod outer segments. Exp. Eye Res. 52, 607-612. Aruoma. 0.1.. Halliwell. B., Hoey, B.M. and Butler, J. (1989) The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical. superoxide and hypoclorous acid. Free Rad. Biol. Med. 6, 593-597. Moldeus, P.. Cotgreave, I.A. and Bergreen, M. (1986) Lung protection by a thiol-containing antioxidant: N-acetylcysteine. Respiration 50 (Suppl. I), 31-42.
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E.G. Valles et al. / Toxicol. Lett. 71 (1994) 87-95
26
de Ferreyra, E.C., Castro, J.A., Diaz Gomez, M.I., D’Acosta, O.M. (1974) Prevention and treatment of carbon tetrachloride about its mechanism. Toxicol. Appl. Pharmacol. 27, 558-568.
27
de Toranzo, E.G.D., de Ferreyra, E.C., de Fenos, O.M. and Castro, J.A. (1983) Prevention of carbon tetrachloride-induced liver necrosis by several amino acids. Br. J. Exp. Pathol. 64, 166-171. Gorla, N., de Ferreyra, E.C., Villarruel, M.C., de Fenos, O.M. and Castro, J.A. (1983) Studies on the mechanism of gluthathione prevention of carbon tetrachloride-induced liver injury. Br. J. Exp. Pathol. 64, 388-395.
28
N., de Castro, hepatotoxicity
CR. and de Fenos, by cysteine. Studies
letters
Toxicology Letters 71 (1994) 87-95
WAcetyl cysteine is an early but also a late preventive agent against carbon tetrachloride-induced liver necrosis E.G. Valles, CR.
de Castro,
J.A. Castro*
Cenrro de Investigaciones Toxicokigicas (CEITOX) CITEFA/CONICET (1603) Villa Martelli, Buenos Aires, Argentina
Zufiiategui
4380,
(Received 24 May 1993; revision received 8 July 1993; accepted 8 July 1993)
Abstract
N-Acetyl cysteine (NAC) treatment 30 min before or 6 or 10 h after carbon tetrachloride (CC&) administration significantly prevented the liver necrosis produced by the hepatotoxin at 24 h. NAC pretreatment was able to partially decrease the covalent binding of Ccl, reactive metabolites at 1 and 3 h of poisoning and, to a small extent, the concentration of Ccl, reaching the liver at 3 h. NAC also diminished partially the Ccl,-promoted increases in lipid peroxidation at 3 h, but had an enhancing effect of its own of small intensity. Results suggest that early and late protective effects of NAC might be attributable to its prior conversion to cysteine and glutathione. Key words: N-Acetyl cysteine; Liver damage; Carbon tetrachloride; Lipid peroxidation; Selfresponse injury; Covalent binding; Reactive metabolites
1. Introduction
IV-Acetyl cysteine (NAC) is an amino acid analog that has found wide clinical applications either because of its cytoprotective properties or as antidote in a variety of intoxications [1,2]. Its use has been backed by a large number of detailed experi-
mental studies either in animals or in in vitro systems [1,2]. In the case of carbon tetrachloride (CC&), NAC has been effectively employed in cases of human poisoning without previous detailed mechanistical studies in animals [1,3-51. * Corresponding author, Tel.: 54-I-761~0031/0081
(ext. 239); Fax: 54-I-761-591
0378-4274/94/%07.00 0 1994 Elsevier Science Ireland SSDI 0378-4274(93)03023-M
Ltd. All rights reserved
l/3063.
88
In this work, we attempt to provide NAC in cases of human CCL, poisoning relative protective efficiency of NAC previously developed in our laboratory
E.G. V&es et al. / Taxied.
Lett. 31 (1994) 83-9s
rational experimental support to the use of and to develop terms of comparison for the against Ccl4 intoxication with treatments or reported by others in literature [6].
2. Materials and methods 2.1. Chemicals “CC14 (spec. act., 2.8 m~~mmol) was purchased from New England Nuclear. NAC was purchased from Sigma Chemical Co. All other chemicals were of the best available quality. 2.2. Animals and treatments Sprague-Dawley male rats (220-270 g) were used in the experiments. Food was withdrawn 12-14 h before CC14 intoxication, but the animals had free access to water. Temperature in the animal room was 22 f 2°C and the relative humidity was between 45 and 65%. Lights in the animal room were on from 06:OOto 18:OOh. Five to 8 animals per group were used as stated for each experiment. NAC was given p.o. in water solution at the dose of 2000 mg/kg (400 mgml solution) 30 min before, 6 or 10 h after CC14. Control rats received the equivalent amount of water. ‘VC14 was given i.p. as 5% CC& (v/v) in olive oil (30 x lo6 dp~ml) at a dose of 5 ml of the solution/kg. Ccl4 (5 ml/kg) was given i-p. as a 20% (v/v) solution in olive oil. Control rats received onfy olive oil i.p. The animals were killed by decapitation at different times after Ccl4 administration; livers were rapidly removed and processed. Whenever blood samples were taken, animals were kept under light ether anesthesia, and blood was obtained with heparinized syringes from the inferior vena cava. 2.3. Enzymatic and chemical determinations Isolation of the microsomal fraction was performed as previously reported [7]. The in vivo incorporation of radioactivity from 14CC14to microsomal lipids was determined according to procedures described previously 171. Results are given in dpm per mg of microsomal lipid. Lipids were extracted according to the procedure reported by Castro et al. [7]. The 14CCf4concentrations in liver were estimated by a procedure described by Recknagel and Litteria [8] up to the microdiffusion step, and then the t4CC14collected in the toluene in the center well of the cell was transferred to a scintillation counting vial and counted. Results were corrected for quenching by the channels ratio method. Lipid peroxidation in vivo was measured by conjugated diene ultraviolet absorption of the microsomal lipid extracts [9]. The results are expressed as the absorbance at 243 nm x 1000 for a solution having 1 mg of microsomal lipid/ml. NADPHlinked isocitric acid dehydrogenase (ICD) activity in plasma was determined according to Sterkel et al. [lo]. Activity is given in units: 1 unit being the amount of the enzyme producing 1 nmol NADPH/ml plasma/h at 25°C. 2.4. Histological techniques After removal of the livers, small portions of the left and central lobes were
89
E.G. Valles et al. / Toxicol. Lett. 71 (1994) 87-95
immediately fixed in Bouin’s solution, embedded in paraffin and stained with hematoxylin-eosin. The specimens were coded to avoid bias and were evaluated histologically by three independent observers. To quantify the morphological changes, liver sections were graded for necrotic changes using an arbitrary scale: + = slight (about 20-30% necrosis); ++ = moderate (about 50% necrosis); +++ = marked (about 75% necrosis) and ++++ = very intense (about 90-100% necrosis). Results reported as representative for each experimental condition were the means of observations made by all observers for a given experimental condition. 2.5. Statistics A decision tree for selecting the hypothesis testing statistical procedures described by Gad and Weil [l l] was applied to the results of every experiment. Homogeneity of variance was established using either the Barlett homogeneity of variance test or the F-test for comparisons involving three or more groups or two groups, respectively. According to significance of homogeneity of variance, one of the following tests were applied: Student’s r-test or the Cochran t-test. Parametric analysis of variance tests were used for comparisons involving three or more groups of data [l 11. 3. Results 3.1. Effect of NAC in vivo on microsomal lipid peroxidation (LP) produced by Ccl, at different times after administration NAC administered alone produced a minor but statistically significant enhancement of LP in microsomal lipids at 1 h but not at 3 h (Table 1). However, NAC values at 3 h exhibited a tendency to be enhanced, considering that the mean value
Table 1 CCl&duced Treatmenta
Control CCI, NAC NAC + Ccl,
lipid peroxidation
of microsomal
lipids in rats previously
Lipid peroxidation
(x f S.D.)b,C
lh
% of control
3h
100 265 137 241
212 540 337 421
147 389 201 354
f + f f
22 46dqf 43 26d,f
treated
with NAC
% of control zt * + f
60 134e.f 136 87e,’
100 255 158 198
‘Sprague-Dawley male rats (260-280 g) that had not been fed for 12-14 h were injected i.p. with CCI, as a 20% solution (v/v) in olive oil at a dose of 5 ml/kg 30 min after NAC administration. NAC was given p.o. in water solution at a dose of 2000 mg/kg. Control received the equivalent amount of olive oil and water. Five animals per group were used in this experiment. bThe lipid peroxidation value is expressed as absorbance at 243 nm x 1000 for a solution having 1 mg of microsomal lipid/ml. ‘The P value for the overall effect of the NAC on the CC&-induced lipid peroxidation obtained by analysis of variance was P < 0.05. dCCI, vs. Control at I h; NAC + CC& vs. Control at 1 h and NAC + Ccl, vs. NAC at 1 h: P < 0.001 Student’s t-test (N, + N2 - 2). eCCI, vs. Control at 3 h; NAC + Ccl, vs. Control at 3 h: P < 0.01, Student’s t-test (N, + N, - 2). ‘Ccl, vs. CCI, + NAC at 1 and 3 h: P > 0.05, Student’s r-test (N, + N, - 2).
Table 2 Covalent binding of “CC14-reactive metabolites of rats previously treated with NAC
to hepatic
Treatment”
14CC14 concentration in liver (dpmig liver + SD.)
Covalent binding 14C from Ccl, Lipid f SD.
lh %CI, “CCl,
of
microsomal
(dpm/mg)
lipids and “CC14 concentrations
R x IO1 (zt S.D.)b
Lipid
+ NAC
331 z!z 56 254 f 46d
92408 76352
+ 20 105 f 11 596’
3.73 f 0.99 3.38 f 0.72’
14CC14 + NAC
412 f 39 368 + 35’
65877 45208
f f
6.53 f 1.55 8.62 f 2.52’
3h
“cc14
14323 10984d
“Sprague Dawley male rats (220-240 g) that had not been fed for 12-14 h were injected i.p. with 14CC14 as 5% Ccl, (v/v) solution in olive oil (30 x IO6 dpm/ml) at a dose of 5 ml solution/kg, 30 min after NAC. NAC was administered p.0. in water solution at the dose of 2000 mgikg, 30 min before “Ccl,. Controls received the equivalent amount of water. The animals were sacrificed either I or 3 h after 14CC14 administration, and their livers were processed for microsomal lipid extraction and counting (see Materials and methods for details). Eight animals per group were used. bR is the ratio of irreversible binding to lipids to 14CC14 concentations in liver. ‘P > 0.05, Student’s r-test (N, + N, - 2). dP < 0.01, Student’s t-test (N, + N, - 2). eP > 0.05, Student’s r-test (N, + Nz - 2). ‘P < 0.05, Student’s r-test (N, + N? - 2).
observed was 58% higher than that in the control group (Table 1). NAC administration to CC&-poisoned animals, in contrast, significantly decreased the intensity of the Ccl,-promoted LP process at both 1 and 3 h of poisoning, as evidenced by the two-way analysis of variance of results (Table 1). Notwithstanding, comparison of LP values between groups Ccl, and NAC + CC& using t-test, indicated that results in one group were not significantly different from those in the other at either 1 or 3 h of poisoning (Table 1). 3.2. Effect of pretreatment with NAC on the covalent binding (CB) of “CC14 to hepatic microsomal lipids and 14CC14 concentrati0n.s in liver NAC administration slightly, but significantly, reduced the intensity of the CB of CC& reactive metabolites to microsomal lipids at 1 or 3 h (Table 2) and the “CC14 concentrations reaching the liver at 3 h, but not at 1 h (Table 2). However, the intrinsic ability of the liver microsomal fraction to activate CC14 (as defined by the ability to activate Ccl4 to reactive metabolites that bind covalently to microsomal lipids per unit of concentration) R was not significantly modified by NAC treatment at 1 h and slightly, but significantly, increased at 3 h (Table 2). 3.3. Effect of NAC administration on Ccl,-induced liver necrosis The extent of CC14-induced liver necrosis was evidenced by histological
examina-
91
E.G. Vales et al. / Toxicol. Lett. 71 (1994) 87-95 Table 3 Effect of NAC on CC&-induced Treatment” 30 min before Control ccl, NAC Ccl, + NAC 6 h after Control CCI, NAC CCI, + NAC 10 h after Control CCI, NAC Ccl, + NAC
hepatic
necrosis
ICDb* (units f S.D.) 303 f 37 88200 f 39800 4875 f 1153
24 h after administration
of CC14 to rats
Degree of histologically
observed
necrosis
++++ -
4500 f 2001d
215 f 58 100160 f 46151 3112 i 918 9373
l
7609=
203 86760 3124 27780
f f f zt
26 21092 823 16491f
++++ +
++++ ++
“Ccl4 and NAC were administered at the doses indicated in Table 1. NAC was given 30 min before, 6 or IO h after the hepatotoxin. The animals were sacrificed 24 h after Ccl, administration. Eight animals per group were used in these experiments. bIsocitric acid dehydrogenase: 1 unit of enzyme is the amount required to form 1 nmol NADPH/ml plasma/h at 25°C. ‘The P value of the overall effect of NAC on the Ccl,-induced increase in ICD, obtained by two-way analysis of variance, was P < 0.05 at 30 min, 6 and IO h. dCCI, + NAC vs. Ccl,: P < 0.001, Student’s t-test (Nt + N, - 2). ‘CC14 + NAC vs. CCld P < 0.02, Student’s t-test (N, + N, - 2). ‘Ccl, + NAC vs. Ccl,: P < 0.01, Student’s t-test (N, + N, - 2).
tion and also by determination of ICD levels in plasma (Table 3). NAC given alone only produced at 24 h a minor degree of hydropic degeneration of the hepatocytes and trabecular disorganization. No necrosis was observed in hepatocytes of animals treated with NAC alone. There was, however, a significant increase in ICD activity in this group of animals in relation to that found in controls (Table 3). Administration of CC& to animals produced an intense centrolobular necrosis of the liver (++++) at 24 h (Fig. 1) and a marked increase in ICD activity in relation to controls (Table III). Treatment with NAC 30 min before or 6 or 10 h after Ccl, significantly decreased the extent of the histologically evident necrosis produced by the hepatotoxin at 24 h (Fig. 2) or the levels of ICD observed in the CClrpoisoned animals (Table 3). 4. Discussion
In full agreement with previous observations in human beings poisoned with CC& and treated with NAC [1,3-51, we reproduced the preventive actions of this
92
E.G. Valles et al. / Toxicol. Lett. 71 (1994) 87-95
Fig. I. Liver section of a rat 24 h after administration of Ccl,. x 120.
Note the centrilobular
necrosis.
H & E
N-acetylated derivative of cysteine against the effects of the hepatotoxin in rats. Protection afforded by NAC was better when administered 30 min before than when given 6 or 10 h after CCL,. This suggested that NAC was able to exert beneficial actions at the early stages of poisoning (before 6 h), but also at the late steps of intox-
Fig. 2. Liver section of a rat 24 h after admmlstration administration of Ccl,. The centrilobular
of Ccl,. This rat was treated with NAC 6 h after zones are well preserved. H & E x 120.
E.G. Valles et al. / Toxicol. ht.
93
71 (1994) 87-95
ication (after 6 h). According to present views on the mechanism of CCL hepatotoxicity, the early part of the process is dominated by factors such as Ccl, absorption, Ccl4 activation to * CC& and CC1302 ’ free radicals, the covalent binding of ’ Ccl3 to cellular components, and the H abstraction from PUFA by the CC130z ’ and the ’ Ccl,, to initiate LP [ 12-141. Later stages of Ccl4 poisoning involve perturbation of Ca2+ homeostasis and the effect of it on degradative processes mediated by proteases, phospholipases and potentially by other enzymes such as endonucleases [6,14-171. The thiol status of proteins was also found to be critical at this late stage and fundamentally preserved by GSH [18,19]. The NAC protective effects on the early stage of CC& poisoning observed under our 30 min prior to CC& administration experiment might be due to the contribution of several factors, including a decrease in the CB, decreases in the levels of CCL, reaching the liver, and partial blockade of CC&-promoted lipid peroxidation. The analysis of NAC effects on CC&-promoted LP are complex and are not fully understood with the presently available information. However, our experiments indicate that NAC could be in part a chain-breaking antioxidant, as previously defined by Halliwell and Gutteridge [20]. In effect, NAC-treated CCL,-poisoned animals had a significant decrease in CCL,-promoted LP, as evidenced by the conjugated diene hyperconjugation technique. This procedure is able to reveal modulations of the initiation stage of LP involved in chain-breaking antioxidants effects ]201. NAC administration alone also sparked a moderate LP process in our experiments, which might explain the minor cell injury evidenced in hepatocytes from NAC-treated animals. This is not unusual behavior for a thiol-containing compound as is NAC. In effect, many compounds having SH groups have exhibited both LPstimulating or -inhibiting properties, according to their concentration or other conditions [21-231. Additional beneficial effects of NAC in the LP process occurring after the initiation stage are likely to occur because it is known that NAC is able to destroy several reactive oxygen species produced during LP [24,25]. Concerning NAC protective effects at the late stage of Ccl4 poisoning, they might be related to the ability of NAC to generate the GSH precursor amino acid cysteine [25]. Both cysteine itself [26] and GSH or precursors [27,28] were found to be late protective agents against CCla-induced liver necrosis. Irrespective of the full understanding of the mechanism of NAC preventive effects, the mere fact that it is protective when given at late stages of the process gives sufficient rational support for its use in cases of humans poisoned with CCL,. If beneficial effects of NAC only operated at early stages of poisoning, the usefulness of its employment would be limited. 5. Acknowledgement This work was partially supported by Grant DK 13195-20 from the National Institutes of Health. USA. 6. References I
Zambon Group (1992) Biomedical Documentation Morel1 Arti GraIiche, Osnago, Italy, pp. 7-106.
Centre.
N-Acetylcysteine.
Antidotal
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i IYY41 87-Y>
Flanagan, R.J. and Meredith, T.J. (1991) Use of N-Acetylcysteine in clinical toxicology. Am. J. Med. 91 (Suppl. 3C), 13lS-139s. Ruprah, M., Mant, T.G. and Flanagan, R.J. (1985) Acute carbon tetrachloride poisoning in 19 patients: implications for diagnosis and treatment. Lancet I 1 1027-1029. 4
6
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18 19 20 21
22 23 24
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Mathieson, P.W., Williams. G. and MC Sweeney, J.E. (1985) Survival after massive Ingestion of carbon tetrachloride treated by intravenous infusion of acetylcysteine. Hum. Toxicol. 4. 627-631. Davis, M. (1986) Protective agents for acetaminophen overdose. Semin. Liver Dis. 6, 138-233. Castro, J.A. (1990) Prevention ofchemically induced liver injury. In: R.S. Goldstein, W. Hewitt and J.B. Hook (Eds.), Toxic Interactions, Academic Press. New York. pp. 233-257. Castro, J.A. and Diaz Gbmez, M.l. (1972) Studies on the irreversible binding of “C-CC14 to microsomal lipids in rats under varying experimental conditions. Toxicol. Appl. Pharmacol. 23. 541-552. Recknagel, R. and Litteria, M. (1960) Biochemical changes in carbon tetrachloride fatty liver. Concentration of carbon tetrachloride in liver and blood. Am. J. Pathol. 36, 521-531. Klaassen, C. and Plaa. G.L. (1969) Comparison of the biochemical alterations elicited in livers from rats treated with carbon tetrachloride. chloroform, 1,1,2-trichloroethane and I. I, I-trichloroethane. Biochem. Pharmacol. 18. 2019-2027. Sterkel, R., Spencer, S.. Wolfson, S. and Williams-Ashman. H. (1958) Serum isocitric dehydrogenase activity with particular reference to liver desease. J. Lab. Clin. Med. 52. 176-180. Gad, S.C. and Weil, C.S. (1982) Statistics for toxicologist. In: A.W. Hayes (Ed.), Principles and Methods of Toxicology. Raven Press, New York, pp. 273-320. Castro, J.A. (1984) Mechanistical studies and prevention of free radical cell inJury. In Proceedings IX International Congress of Pharmacology, MacMillan Co., London, Vol. 2, pp. 243-270. Slater, T.F. (1982) Activation of carbon tetrachloride: chemical principles and biological significance. In: D.C. McBrien and T.F. Slater (Eds.), Free Radicals. Lipid Peroxidation and Cancer. Academic Press. New York. pp. 243-270. Recknagel. R.O., Glende, E.A.. Dolak. J.A. and Waller. R.L. (1989) Mechanisms of carbon tetrachloride toxicity. Pharmacol. Ther. 43, 139-154. de Ferreyra. E.C., Villarruel. M.C.. Bernacchi, A.S., de Fenos, O.M. and Castro, J.A. (1992) Prevention of carbon tetrachloride-induced liver necrosis by the chelator Alizarin sodium sulfonate. Exp. Mol. Pathol. 56, 197-207. Villarruel, M.C., Fernandez. G., de Fenos, O.M. and Castro. J.A. (1990) Modulation of the course of CC],-induced liver injury by the anticalmodulin drug thioridaaine. Toxicol. Lett. 51. 13-21. de Ferreyra. E.C., Villarruel, M.C.. Bernacchi. AS., Fernandez. G., de Fenos. O.M. and Castro. J.A. (1989) Late preventive effects against carbon tetrachloride-induced liver necrosis of the calcium chelating agent Calcion. Arch. Toxicol. 63, 450-455. Reed. D.J. (1990) Gluthathione: toxicological implications. Annu. Rev. Pharmacol. Toxicol. 30. 603-63 I. Brigelius, R. (1985) Mixed disulfides: biological functions and increase in oxidative stress. In: H. Sies (Ed.). Oxidative Stress, Academic Press, New York, pp. 243-272. Halliwell, B. and Gutteridge, J.M.C. (1990) Free Radicals in Biology and Medicine. 2nd edn. Oxford University Press, Oxford, pp. 234-260. Kamutaki, T., Sugita, 0.. Ozawa, N. and Kitagawa. H. (1977) Stabilization and induction of a lipid peroxidation inhibitor present in the soluble fraction of rat liver homogenates. Toxicol. Appl. Pharmacol. 40. 283-289. Haenen, G.R., Vermeulen, N.P.. Timmerman, H. and Blast, A. (1989) Effects of thiols on lipid peroxidation in rat liver microsomes. Chem.-Biol. Interact. 71. 201-212. Zimmerman. W.F. and Keys, S. (1991) Effects of antioxidants dithiothreitol and vitamin E on phospholipid metabolism in isolated rod outer segments. Exp. Eye Res. 52, 607-612. Aruoma. 0.1.. Halliwell. B., Hoey, B.M. and Butler, J. (1989) The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical. superoxide and hypoclorous acid. Free Rad. Biol. Med. 6, 593-597. Moldeus, P.. Cotgreave, I.A. and Bergreen, M. (1986) Lung protection by a thiol-containing antioxidant: N-acetylcysteine. Respiration 50 (Suppl. I), 31-42.
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26
de Ferreyra, E.C., Castro, J.A., Diaz Gomez, M.I., D’Acosta, O.M. (1974) Prevention and treatment of carbon tetrachloride about its mechanism. Toxicol. Appl. Pharmacol. 27, 558-568.
27
de Toranzo, E.G.D., de Ferreyra, E.C., de Fenos, O.M. and Castro, J.A. (1983) Prevention of carbon tetrachloride-induced liver necrosis by several amino acids. Br. J. Exp. Pathol. 64, 166-171. Gorla, N., de Ferreyra, E.C., Villarruel, M.C., de Fenos, O.M. and Castro, J.A. (1983) Studies on the mechanism of gluthathione prevention of carbon tetrachloride-induced liver injury. Br. J. Exp. Pathol. 64, 388-395.
28
N., de Castro, hepatotoxicity
CR. and de Fenos, by cysteine. Studies