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Protective effect of colchicine on acute liver damage induced by carbon tetrachloride
.lournalofHepatology. 1988: 6:337-342
337
Elsevier HEP 00415
Protective effect of colchicine on acute liver damage induced by carbon tetrachloride
Marisabel Mourelle, C. Villalon and J.L. Amezcua Department of Pharmacology and Toxicology, Centro de lnvestigaci6n y de Estudios Avanzados del lnstituto Polit~cnico Nacional, Mexico City (Mexico)
(Received 20 July 1987) (Accepted 26 January 1988)
Summary Pretreatment of rats with colchicine (10 ktg/day) for 7 days protected against CCl4-induced acute liver damage. CCI 4 intoxication was demonstrated histologically and by increased serum activities of glutamic-pyruvic transaminase, alkaline phosphatase and y-glutamyi transpeptidase. Furthermore, an increase in liver lipid peroxidation and a decrease in plasma membrane y-glutamyl transpeptidase activity were found. Colchicine increased the LD50 of CCI 4 2.5-fold and prevented the release of intracellular enzymes, as well as the decrease in y-glutamyl transpeptidase activity in the plasma membrane. It also completely prevented the lipid peroxidation produced by CCI4 and limited the extent of the histological changes. Our results suggest that the protective effect of coichicine may be mediated through its action on an early toxic event, because treatment of the animals with colchicine produced a significant decrease in CCl4-induced lipid peroxidation.
Introduction Carbon tetrachloride (CCI4) intoxication in the rat is an experimental model used widely to study necrosis and steatosis of the liver [1-3]. It is well established that, after the 'in vivo' administration of CCI 4, the earliest morphological alterations appear within 15 min and involve the endoplasmic reticulum of hepatocytes [1-3]. These changes are followed within the first hour by structural alterations of the Golgi ap-
paratus, plasma membrane and mitochondria [4,5]. These alterations might be related to the peroxidation of polyunsaturated fatty acids included in the structure of cellular membranes, as free radicals arising from CCl 4 metabolism initiate lipoperoxidation processes [6,7] and also inactivate calcium pump activity [8]. All these alterations eventually lead to liver cell death which is accompanied by the release into the blood of intrahepatic enzymes. Previous studies have demonstrated that colchi-
This work was partially supported by Grant PCCBBNA 020846 from Consejo Nacional de Ciencia y Tecnologfa, M6xico. Correspondence: Marisabel Mourelle, Ph.D., Department of Pharmacology and Toxicology, Centro de lnvestigaci6n y de Estudios Avanzados del Instituto Polit6cnico Nacional, Apartado Postal 14-740, 07000 M6xico, D.F., M6xico. 0168-8278/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
338 cine partially prevents the production of liver cirrhosis in rats when given simultaneously with CC14, and ameliorates the disease in animals that are already cirrhotic [9,10]. In chronic studies we have shown that improvement of liver function by colchicine is probably due in part to its action on hepatocyte plasma membrane, modifying several enzymes which regulate normal hepatocyte function [9,10]. However, until now, most studies on colchicine hepatoprotection have been done in models of chronic liver damage and no information seems to be available on the early effects of colchicine against acute liver toxicity induced by CC14. In view of this, we wanted to explore the capability of colchicine to prevent the early pathological changes in liver plasma membrane functions and to determine whether colchicine hepatoprotection is reflected in a change of the C C I 4 LDs0.
Materials and Methods
Treatments of animals Male Wistar rats weighing 200-250 g and fed a Purina Chow diet ad libitum were used in these experiments. Animals were divided into four groups: group 1 received CC14 (0.5 ml/100 g) dissolved in mineral oil (1:1, v/v). In group 2, the same dose of CC14 dissolved in water was given 24 h after the last dose of a 7 day regime of colchicine (10 llg/animal/day). Animals of group 3 received mineral oil instead of CCI~ and those of group 4 were given mineral oil and colchicine. All the treatments were administered by means of an intragastric tube. Since water was the vehicle of colchicine we felt that there was no need for vehicle administration by gavage in those groups not receiving colchicine. These groups were in turn divided into subgroups of 10 rats each to be sacrificed at 3, 24, 48, 72 and 96 h after the administration of CC14 or mineral oil. The subgroups sacrificed at 3 h were used for the measurement of lipoperoxidation in liver homogenates. Lipoperoxidation was quantified by measuring malondialdehyde production by the thiobarbituric acid method [11]. One additional group of rats which had
M. MOURELLE et al. received coichicine as stated above were sacrificed 24 h after the last dose of colchicine to measure cytochrome P-450 in the livers according to Omura and Sato [12]. The results were compared with those of rats receiving no treatment at all. In the other subgroups blood was collected by heart puncture under light diethyl ether anesthesia and serum was obtained by centrifugation. Livers were quickly removed and were used to prepare homogenates, postmitochondrial fractions (15 000 x g supernatants), and plasma membranes according to Neville's method [13]. Small liver sections fixed in formalin (10%) were used for hematoxylin-eosin and Sudan staining. The serum activity of alanine aminotransferase (ALT) was determined according to Reitman and Frankel [14]. Alkaline phosphatase (ALP) activity was measured in serum using the method of Berger and Rudolph [15] and ~,-glutamyl transpeptidase (GGTP) by the method of Glossmann and Neville [16] in both serum and liver plasma membranes. Protein contents were determined according to Bradford [17] using bovine serum albumin as standard. Additional rats without pretreatment or treated with colchicine as mentioned before were used to determine the oral LDso of CCI~. For this purpose, subgroups of 10 rats received different doses of CC14 from 0.1 to 1 ml/100 g. For statistical analysis Student's t-tests for unpaired samples were performed and a difference was considered significant when P < 0.01. The LDs0 values of C C I 4 w e r e calculated according to the method of Litchfield and Wilcoxon [18].
Results
Histological examination (Fig. 1) showed that the administration of CC14 produced an intense centrolobular necrosis with polymorphonuclear infiltration and edema. Hepatocytes in the portal zones were enlarged and showed intense vacuolation. The normal architecture was lost. The liver sections of animals pretreated with colchicine showed a less marked effect of CC14: there were only small focal areas of cen-
COLCHICINE IN CCI4-INDUCED ACUTE LIVER D A M A G E
~AI
339
I1~" .
Fig. 1. Liver sections from control (a) and colchicine-treated animals (b) and sections from animals treated with CCI 4 alone (c) and CCI 4 + colchicine (d). (Hematoxylin plus eosin staining; final magnification x 125.)
trolobular necrosis and the cells of the periportal zones were well preserved. Neither mineral oil nor colchicine by themselves produced any histological change and no diarrhoea was observed in those animals treated with 10/~g/day of colchicine for 7 days. CC14 administration produced sharp changes in the activity of serum ALP, A L T and G G T P as well as in the plasma liver membrane activity of G G T P (Figs. 2-5). These changes were maximal at 24 h and then all the activities tended to return to the control values although at different rates. As can be seen in Figs. 2-5, the recovery of serum and liver plasma membrane G G T P activity was virtually complete at 48 h whereas serum A L T transferase and serum ALP remained significantly different from the control values. Malondialdehyde production was enhanced in the liver homogenates of CCl4-treated rats (Fig. 6). Colchicine pretreatment prevented most of the
CCl4-induced changes. As can be seen, in Figs. 3, 4 and 6 colchicine completely prevented the changes in serum activities of ALP and G G T P as well as those in the liver plasma membrane activity of GGTP. A smaller degree of protection (although still significant) was shown in the A L T activity measured in serum (Fig. 2). Quite remarkably, colchicine completely prevented the increase in malondialdehyde production induced by CCI4 in the liver homogenates. Colchicine treatment prevented polymorphonuclear infiltration associated with CCI 4 treatment; therefore, the possibility that polymorphonuclearderived radicals also mediate the lipid peroxidation observed following CC14 treatment cannot be excluded. Colchicine produced an increase in the oral LDs0 of C C l 4 from 3.9 g/kg in the control to 9.7 g/kg in the pretreated group.
340
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Fig. 2. Time course of the effect of GGl 4 and eolehicine on serum A L T activity. A , oil; A , oil + colchicine; 0 , CCI4; ©, CGI~ + colchieine. Each point represents the mean _+ S.E. of ]0 independent observations performed in different rats. Values marked with an asterisk (*) were significantly different from controls given oil, P < 0.001.
I
48
after
72
I
96
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Fig. 4. Time course of the effect of CCI 4 and colchicine on serum G G T P activity. A , oil; A , oil + colchicine; 0 , CCI4; ©, CCI 4 + colchicine. Each point represents the m e a n + S.E. of 10 independent observations performed in different rats. Value marked with an asterisk (*) was significantly different from controls given oil, P < 0.001.
Table 1 shows that colchicine treatment at 10/~g/ day for 7 days produced a small but yet significant (20%) decrease in cytochrome P-450.
Discussion
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I
1
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96
(hs)
Fig. 3. Time course of the effect of CCI 4 and colchicine on serum A L P activity. A , oil: &, oil + colchicine; O, CCI~: @, CCla + colchicine. Each point represents the mean + S.E. of 10 independent observations performed in different rats. Values marked with an asterisk (*) were significantly different from controls given oil, P < 0,001.
O-
I
I
I
l
0
24
48
72
Time
after
CCI4 (hs)
Fig. 5. Time course of the effect of CCla and colchicine on G G T P activity determined in isolated plasma m e m b r a n e s . A , oil; A oil + colchicine: O. CCI4; ©, CCI 4 + colchicine. Each point represents the mean value of 10 independent determinations. Value marked with an asterisk (*) was significantly different from controls given oil, P < 0.001.
C O L C H I C I N E IN C C I . c l N D U C E D A C U T E L I V E R D A M A G E
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treatment. Bars represent the mean + S.E. of 10 independent observations performed in different rats. Value marked with an asterisk (*) was significantly different from controls given oil, P < 0.001. a g a i n s t C C l 4 hepatotoxicity [19]. A drug interfering with C C I 4 action on the liver could act at different levels: on absorption, distribution, metabolism, covalent binding to lipids and proteins, lipid peroxidation, or it could act at later stages in a sequence leading to necrosis, or even through quite different and
TABLE 1 E F F E C T OF C O L C H I C I N E T R E A T M E N T ON H E P A T I C C Y T O C H R O M E P-450 C O N T E N T
Determinations were made 24 h after the last dose of a 7 day regime of colchicine, 10 pg/rat, given orally. Results are the means _ S.E. of 10 independent experiments performed in different rats. Treatment
Cytochrome P-450 (nmol/mg protein)
None Colchicine
200.0 + 18.0 155.2 + 10.0"
* Significantly different from control values. P < 0.001.
341 unknown mechanisms. Although the detailed mechanism by which CCI 4 produces hepatotoxicity is still a subject for debate, free radical formation, presumably arising during the CCI 4 activation step by the microsomal enzymes, appears to be a prerequisite step for various toxic effects in the liver [1]. The results presented in this study show that the administration of colchicine to rats effectively protected against CCl4-induced liver damage. The protection appeared to occur at the activation step of CC14 by microsomal enzymes, since the administration of colchicine modified the CCl4-induced lipid peroxidation processes and cytochrome P-450 content. Our results also suggest that protection observed in the above studies cannot be attributed to a delay in absorption of CCI4 by colchicine as demonstrated by the time course experiments. Since diarrhoea is a common side effect of colchicine [20], we deliberately avoided incurring it by fractioning colchicine administration over a period of 7 days before giving CCI 4. However, the slow accumulation of colchicine in the body [20] provided enough colchicine in the liver to produce the expected effects. The dose of CC14 was intentionally high so as to guarantee that any protection offered by colchicine could not be attributed to some minor changes in CC14 absorption or metabolism. In fact, cytochrome P-450 is reduced (about 20%) by our schedule of colchicine administration. However, we feel that such an effect on the metabolic machinery in charge of CCI 4 bioactivation is unlikely to account for the extensive protection provided by colchicine. We suggest that some other actions, perhaps at the level of the propagation of lipid peroxidation or a membrane-stabilizing effect, cannot be ruled out as a part of the hepatoprotective mechanisms of colchicine in CC14 acute intoxication. We have shown that colchicine also prevents the early changes in a plasma membrane-associated enzyme. Plasma membrane functions are also modified quite early in rats treated acutely with CC14. A change of the lipid composition of the plasma membrane has been shown to occur in the early stages of CCl4-induced hepatotoxicity [21]. This, in turn, has been suggested as an explanation for the changes in the activities of the lipid-embedded
342
M. MOURELLE et al.
e n z y m e s of the plasma m e m b r a n e
[9]. It is w o r t h
which
act
through
enhanced
lipid
peroxidation
noting that the m e m b r a n e d e f e c t lasts l o n g e r t h a n the
[22,23]. T h e c o m p l e x actions of colchicine in the liver
activated lipid p e r o x i d a t i o n ,
have yet to be e l u c i d a t e d .
thus suggesting that,
once the lipid p e r o x i d a t i o n is initiated, a m o r e stable change, p r o b a b l y in the lipid c o m p o s i t i o n of the m e m b r a n e , r e m a i n s r e s p o n s i b l e for the a l t e r a t i o n of
Acknowledgements
the plasma m e m b r a n e functions. T h e s e results suggest that colchicine h e p a t o p r o t e c t i o n is not c o n f i n e d
T h e a u t h o r s w a n t to express their g r a t i t u d e to Miss
to the p r e v e n t i o n or reversal of c h r o n i c liver d a m a g e
C o n c e p c i 6 n A v a l o s and M a r i s e l a Vidal P~rez for the
but also m o d i f i e s s o m e f u n c t i o n s of the h e p a t o c y t e in
e x c e l l e n t secretarial assistance and Mr. A l f r e d o Pa-
the early stages of r e s p o n s e
dilla for p r e p a r i n g the figures.
to c h e m i c a l injuries
References 1 Recknagel RO, Glende EA, Ugazio G, Koch RR. Srinivasan S. New data in support of the lipoperoxidation theory for carbon tetrachloridc liver injury. In: Eliakim M, Eshchar J, Zimmerman H J, eds. International Symposium on Hepatotoxicity, New York: Academic Press. 1974: 7-17. 2 Dinman BD, Hamdi EA, Fox CD, Frajola WJ. CCI4 toxicity. III: Hepato-structural and enzymatic changes. Arch Environ Health 1967: 7: 630. 3 Cagen SZ, Klaasen CD. Carbon tetrachloride-induced hepatotoxicity: studies in developing rats and protection by zinc. Fed Proc 1980; 39: 3124-3128. 4 Bassi M. Electron microscopy of rat liver after carbon tetrachloride poisoning. Exp Cell Res 1960; 20: 313-323. 5 Reynolds ES. Liver parenchymal cell injury. I. Initial alterations of the cell following poisoning with carbon tetrachloride. J Cell Biol 1963; 19: 139-157. 6 Recknagel RO, Glende EA. Carbon tetrachloride hepatotoxicity: an example of lethal cleavage. CRC Crit Rev Toxicol 1973; 2: 263. 7 Binni A, Botti B, Calligaro A, Congiu L, Tomasi A, Vannini V. Morphological changes and free radical rate in early hepatic injury from carbon tetrachloride and monobromotrichloromethane. J Submicrosc Cytol 1978; 10: 215. 8 Moore L, Davenport R, Landon EJ. Calcium uptake of a rat liver microsomal subcellular fraction in response to 'in vivo' administration, of carbon tetrachloride. J Biol Chem 1976: 251: 1197-1201. 9 Mourelle M, Rojkind M, Rubalcava B. Colchicine improves the alterations in the liver adenylate cyclase system of cirrhotic rats. Toxicology 1981; 21: 213. 10 Yahuaca P, Amaya A, Rojkind M, Mourelle M. Cryptic adenosine triphosphatase activities in plasma membranes of CCI4-cirrhotic rats. Its modulation by changes in cholesterol/phospholipid ratios, Lab Invest 1985; 53: 541. 11 Buege JA, Aust SD. Microsomal lipid peroxidation.
Methods Enzymol 1978; 53: 302. 12 Omura T, Sato R. Carbon monoxide-binding pigment of liver microsomes. Evidence for its hemoprotein nature. J Biol Chem 1964; 239: 2370-2378. 13 Neville D. Isolation of an organ specific protein antigen from cell-surface of rat liver. Biochim Biophys Acta 1968; 154: 540-552. 14 Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; 28: 56-63. 15 Berger L, Rudolph GN. Alkaline and acid phosphatase. In: Meites E, ed. Standard Methods of Clinical Chemistry. New York: Academic Press. 1963; 56. 16 Glossmann M, Neville D. y-Glutamyltransferase in kidney brush border membranes. FEBS Lett 1972; 19: 340. 17 Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248. 18 Litchfield JT, Wilcoxon F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 1949; 962 99-113. 19 Ferreyra EC, De Fenos OM, Bernacchi AS, De Castro CR, Castro JA. Treatment of carbon te'trachloride induced liver necrosis with chemical compounds. Toxicol Appl Pharmacol 1977; 42: 513-521. 20 Wallace SL, Omokoku B, Ertel NH. Colchicine plasma levels: implications as to pharmacology and mechanism of action. Am J Med 1970; 48: 443-448. 21 Martinez-Calva I, Campos-Apaes E, Rosales Vega E, Mourelle M. Vitamin E improves membrane lipid alterations induced by CCIa intoxication. J Appl Toxicol 1984; 4: 270-272. 22 Comporti M. Biology of disease. Lab Invest 1985; 53: 599-623. 23 Recknagel RO, Ghoshal AK. Lipoperoxidation as a vector in carbon tetrachloride hepatotoxicity. Lab Invest 1966; 15: 132-148.
337
Elsevier HEP 00415
Protective effect of colchicine on acute liver damage induced by carbon tetrachloride
Marisabel Mourelle, C. Villalon and J.L. Amezcua Department of Pharmacology and Toxicology, Centro de lnvestigaci6n y de Estudios Avanzados del lnstituto Polit~cnico Nacional, Mexico City (Mexico)
(Received 20 July 1987) (Accepted 26 January 1988)
Summary Pretreatment of rats with colchicine (10 ktg/day) for 7 days protected against CCl4-induced acute liver damage. CCI 4 intoxication was demonstrated histologically and by increased serum activities of glutamic-pyruvic transaminase, alkaline phosphatase and y-glutamyi transpeptidase. Furthermore, an increase in liver lipid peroxidation and a decrease in plasma membrane y-glutamyl transpeptidase activity were found. Colchicine increased the LD50 of CCI 4 2.5-fold and prevented the release of intracellular enzymes, as well as the decrease in y-glutamyl transpeptidase activity in the plasma membrane. It also completely prevented the lipid peroxidation produced by CCI4 and limited the extent of the histological changes. Our results suggest that the protective effect of coichicine may be mediated through its action on an early toxic event, because treatment of the animals with colchicine produced a significant decrease in CCl4-induced lipid peroxidation.
Introduction Carbon tetrachloride (CCI4) intoxication in the rat is an experimental model used widely to study necrosis and steatosis of the liver [1-3]. It is well established that, after the 'in vivo' administration of CCI 4, the earliest morphological alterations appear within 15 min and involve the endoplasmic reticulum of hepatocytes [1-3]. These changes are followed within the first hour by structural alterations of the Golgi ap-
paratus, plasma membrane and mitochondria [4,5]. These alterations might be related to the peroxidation of polyunsaturated fatty acids included in the structure of cellular membranes, as free radicals arising from CCl 4 metabolism initiate lipoperoxidation processes [6,7] and also inactivate calcium pump activity [8]. All these alterations eventually lead to liver cell death which is accompanied by the release into the blood of intrahepatic enzymes. Previous studies have demonstrated that colchi-
This work was partially supported by Grant PCCBBNA 020846 from Consejo Nacional de Ciencia y Tecnologfa, M6xico. Correspondence: Marisabel Mourelle, Ph.D., Department of Pharmacology and Toxicology, Centro de lnvestigaci6n y de Estudios Avanzados del Instituto Polit6cnico Nacional, Apartado Postal 14-740, 07000 M6xico, D.F., M6xico. 0168-8278/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
338 cine partially prevents the production of liver cirrhosis in rats when given simultaneously with CC14, and ameliorates the disease in animals that are already cirrhotic [9,10]. In chronic studies we have shown that improvement of liver function by colchicine is probably due in part to its action on hepatocyte plasma membrane, modifying several enzymes which regulate normal hepatocyte function [9,10]. However, until now, most studies on colchicine hepatoprotection have been done in models of chronic liver damage and no information seems to be available on the early effects of colchicine against acute liver toxicity induced by CC14. In view of this, we wanted to explore the capability of colchicine to prevent the early pathological changes in liver plasma membrane functions and to determine whether colchicine hepatoprotection is reflected in a change of the C C I 4 LDs0.
Materials and Methods
Treatments of animals Male Wistar rats weighing 200-250 g and fed a Purina Chow diet ad libitum were used in these experiments. Animals were divided into four groups: group 1 received CC14 (0.5 ml/100 g) dissolved in mineral oil (1:1, v/v). In group 2, the same dose of CC14 dissolved in water was given 24 h after the last dose of a 7 day regime of colchicine (10 llg/animal/day). Animals of group 3 received mineral oil instead of CCI~ and those of group 4 were given mineral oil and colchicine. All the treatments were administered by means of an intragastric tube. Since water was the vehicle of colchicine we felt that there was no need for vehicle administration by gavage in those groups not receiving colchicine. These groups were in turn divided into subgroups of 10 rats each to be sacrificed at 3, 24, 48, 72 and 96 h after the administration of CC14 or mineral oil. The subgroups sacrificed at 3 h were used for the measurement of lipoperoxidation in liver homogenates. Lipoperoxidation was quantified by measuring malondialdehyde production by the thiobarbituric acid method [11]. One additional group of rats which had
M. MOURELLE et al. received coichicine as stated above were sacrificed 24 h after the last dose of colchicine to measure cytochrome P-450 in the livers according to Omura and Sato [12]. The results were compared with those of rats receiving no treatment at all. In the other subgroups blood was collected by heart puncture under light diethyl ether anesthesia and serum was obtained by centrifugation. Livers were quickly removed and were used to prepare homogenates, postmitochondrial fractions (15 000 x g supernatants), and plasma membranes according to Neville's method [13]. Small liver sections fixed in formalin (10%) were used for hematoxylin-eosin and Sudan staining. The serum activity of alanine aminotransferase (ALT) was determined according to Reitman and Frankel [14]. Alkaline phosphatase (ALP) activity was measured in serum using the method of Berger and Rudolph [15] and ~,-glutamyl transpeptidase (GGTP) by the method of Glossmann and Neville [16] in both serum and liver plasma membranes. Protein contents were determined according to Bradford [17] using bovine serum albumin as standard. Additional rats without pretreatment or treated with colchicine as mentioned before were used to determine the oral LDso of CCI~. For this purpose, subgroups of 10 rats received different doses of CC14 from 0.1 to 1 ml/100 g. For statistical analysis Student's t-tests for unpaired samples were performed and a difference was considered significant when P < 0.01. The LDs0 values of C C I 4 w e r e calculated according to the method of Litchfield and Wilcoxon [18].
Results
Histological examination (Fig. 1) showed that the administration of CC14 produced an intense centrolobular necrosis with polymorphonuclear infiltration and edema. Hepatocytes in the portal zones were enlarged and showed intense vacuolation. The normal architecture was lost. The liver sections of animals pretreated with colchicine showed a less marked effect of CC14: there were only small focal areas of cen-
COLCHICINE IN CCI4-INDUCED ACUTE LIVER D A M A G E
~AI
339
I1~" .
Fig. 1. Liver sections from control (a) and colchicine-treated animals (b) and sections from animals treated with CCI 4 alone (c) and CCI 4 + colchicine (d). (Hematoxylin plus eosin staining; final magnification x 125.)
trolobular necrosis and the cells of the periportal zones were well preserved. Neither mineral oil nor colchicine by themselves produced any histological change and no diarrhoea was observed in those animals treated with 10/~g/day of colchicine for 7 days. CC14 administration produced sharp changes in the activity of serum ALP, A L T and G G T P as well as in the plasma liver membrane activity of G G T P (Figs. 2-5). These changes were maximal at 24 h and then all the activities tended to return to the control values although at different rates. As can be seen in Figs. 2-5, the recovery of serum and liver plasma membrane G G T P activity was virtually complete at 48 h whereas serum A L T transferase and serum ALP remained significantly different from the control values. Malondialdehyde production was enhanced in the liver homogenates of CCl4-treated rats (Fig. 6). Colchicine pretreatment prevented most of the
CCl4-induced changes. As can be seen, in Figs. 3, 4 and 6 colchicine completely prevented the changes in serum activities of ALP and G G T P as well as those in the liver plasma membrane activity of GGTP. A smaller degree of protection (although still significant) was shown in the A L T activity measured in serum (Fig. 2). Quite remarkably, colchicine completely prevented the increase in malondialdehyde production induced by CCI4 in the liver homogenates. Colchicine treatment prevented polymorphonuclear infiltration associated with CCI 4 treatment; therefore, the possibility that polymorphonuclearderived radicals also mediate the lipid peroxidation observed following CC14 treatment cannot be excluded. Colchicine produced an increase in the oral LDs0 of C C l 4 from 3.9 g/kg in the control to 9.7 g/kg in the pretreated group.
340
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I
0
I
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Time
_
I
I
I
I
0
24
48
72
Time
after
CCI4
I
96 (hs)
Fig. 2. Time course of the effect of GGl 4 and eolehicine on serum A L T activity. A , oil; A , oil + colchicine; 0 , CCI4; ©, CGI~ + colchieine. Each point represents the mean _+ S.E. of ]0 independent observations performed in different rats. Values marked with an asterisk (*) were significantly different from controls given oil, P < 0.001.
I
48
after
72
I
96
CCI 4 (hs)
Fig. 4. Time course of the effect of CCI 4 and colchicine on serum G G T P activity. A , oil; A , oil + colchicine; 0 , CCI4; ©, CCI 4 + colchicine. Each point represents the m e a n + S.E. of 10 independent observations performed in different rats. Value marked with an asterisk (*) was significantly different from controls given oil, P < 0.001.
Table 1 shows that colchicine treatment at 10/~g/ day for 7 days produced a small but yet significant (20%) decrease in cytochrome P-450.
Discussion
41'
250-
A variety of drugs have been found to protect
c
E ..u
200-
800 -
-6 E
.=/
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i . ~oo-
~'
50-
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f.
400-
./ tl-
I
I
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1
48 after
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I
1
72
96
(hs)
Fig. 3. Time course of the effect of CCI 4 and colchicine on serum A L P activity. A , oil: &, oil + colchicine; O, CCI~: @, CCla + colchicine. Each point represents the mean + S.E. of 10 independent observations performed in different rats. Values marked with an asterisk (*) were significantly different from controls given oil, P < 0,001.
O-
I
I
I
l
0
24
48
72
Time
after
CCI4 (hs)
Fig. 5. Time course of the effect of CCla and colchicine on G G T P activity determined in isolated plasma m e m b r a n e s . A , oil; A oil + colchicine: O. CCI4; ©, CCI 4 + colchicine. Each point represents the mean value of 10 independent determinations. Value marked with an asterisk (*) was significantly different from controls given oil, P < 0.001.
C O L C H I C I N E IN C C I . c l N D U C E D A C U T E L I V E R D A M A G E
3.0
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o
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0 Fig. 6. Effect of CCla and colchicine on e n d o g e n o u s lipoperoxidation. Malondialdehyde ( M D A ) was measured 3 h after CCla
treatment. Bars represent the mean + S.E. of 10 independent observations performed in different rats. Value marked with an asterisk (*) was significantly different from controls given oil, P < 0.001. a g a i n s t C C l 4 hepatotoxicity [19]. A drug interfering with C C I 4 action on the liver could act at different levels: on absorption, distribution, metabolism, covalent binding to lipids and proteins, lipid peroxidation, or it could act at later stages in a sequence leading to necrosis, or even through quite different and
TABLE 1 E F F E C T OF C O L C H I C I N E T R E A T M E N T ON H E P A T I C C Y T O C H R O M E P-450 C O N T E N T
Determinations were made 24 h after the last dose of a 7 day regime of colchicine, 10 pg/rat, given orally. Results are the means _ S.E. of 10 independent experiments performed in different rats. Treatment
Cytochrome P-450 (nmol/mg protein)
None Colchicine
200.0 + 18.0 155.2 + 10.0"
* Significantly different from control values. P < 0.001.
341 unknown mechanisms. Although the detailed mechanism by which CCI 4 produces hepatotoxicity is still a subject for debate, free radical formation, presumably arising during the CCI 4 activation step by the microsomal enzymes, appears to be a prerequisite step for various toxic effects in the liver [1]. The results presented in this study show that the administration of colchicine to rats effectively protected against CCl4-induced liver damage. The protection appeared to occur at the activation step of CC14 by microsomal enzymes, since the administration of colchicine modified the CCl4-induced lipid peroxidation processes and cytochrome P-450 content. Our results also suggest that protection observed in the above studies cannot be attributed to a delay in absorption of CCI4 by colchicine as demonstrated by the time course experiments. Since diarrhoea is a common side effect of colchicine [20], we deliberately avoided incurring it by fractioning colchicine administration over a period of 7 days before giving CCI 4. However, the slow accumulation of colchicine in the body [20] provided enough colchicine in the liver to produce the expected effects. The dose of CC14 was intentionally high so as to guarantee that any protection offered by colchicine could not be attributed to some minor changes in CC14 absorption or metabolism. In fact, cytochrome P-450 is reduced (about 20%) by our schedule of colchicine administration. However, we feel that such an effect on the metabolic machinery in charge of CCI 4 bioactivation is unlikely to account for the extensive protection provided by colchicine. We suggest that some other actions, perhaps at the level of the propagation of lipid peroxidation or a membrane-stabilizing effect, cannot be ruled out as a part of the hepatoprotective mechanisms of colchicine in CC14 acute intoxication. We have shown that colchicine also prevents the early changes in a plasma membrane-associated enzyme. Plasma membrane functions are also modified quite early in rats treated acutely with CC14. A change of the lipid composition of the plasma membrane has been shown to occur in the early stages of CCl4-induced hepatotoxicity [21]. This, in turn, has been suggested as an explanation for the changes in the activities of the lipid-embedded
342
M. MOURELLE et al.
e n z y m e s of the plasma m e m b r a n e
[9]. It is w o r t h
which
act
through
enhanced
lipid
peroxidation
noting that the m e m b r a n e d e f e c t lasts l o n g e r t h a n the
[22,23]. T h e c o m p l e x actions of colchicine in the liver
activated lipid p e r o x i d a t i o n ,
have yet to be e l u c i d a t e d .
thus suggesting that,
once the lipid p e r o x i d a t i o n is initiated, a m o r e stable change, p r o b a b l y in the lipid c o m p o s i t i o n of the m e m b r a n e , r e m a i n s r e s p o n s i b l e for the a l t e r a t i o n of
Acknowledgements
the plasma m e m b r a n e functions. T h e s e results suggest that colchicine h e p a t o p r o t e c t i o n is not c o n f i n e d
T h e a u t h o r s w a n t to express their g r a t i t u d e to Miss
to the p r e v e n t i o n or reversal of c h r o n i c liver d a m a g e
C o n c e p c i 6 n A v a l o s and M a r i s e l a Vidal P~rez for the
but also m o d i f i e s s o m e f u n c t i o n s of the h e p a t o c y t e in
e x c e l l e n t secretarial assistance and Mr. A l f r e d o Pa-
the early stages of r e s p o n s e
dilla for p r e p a r i n g the figures.
to c h e m i c a l injuries
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Methods Enzymol 1978; 53: 302. 12 Omura T, Sato R. Carbon monoxide-binding pigment of liver microsomes. Evidence for its hemoprotein nature. J Biol Chem 1964; 239: 2370-2378. 13 Neville D. Isolation of an organ specific protein antigen from cell-surface of rat liver. Biochim Biophys Acta 1968; 154: 540-552. 14 Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; 28: 56-63. 15 Berger L, Rudolph GN. Alkaline and acid phosphatase. In: Meites E, ed. Standard Methods of Clinical Chemistry. New York: Academic Press. 1963; 56. 16 Glossmann M, Neville D. y-Glutamyltransferase in kidney brush border membranes. FEBS Lett 1972; 19: 340. 17 Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248. 18 Litchfield JT, Wilcoxon F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 1949; 962 99-113. 19 Ferreyra EC, De Fenos OM, Bernacchi AS, De Castro CR, Castro JA. Treatment of carbon te'trachloride induced liver necrosis with chemical compounds. Toxicol Appl Pharmacol 1977; 42: 513-521. 20 Wallace SL, Omokoku B, Ertel NH. Colchicine plasma levels: implications as to pharmacology and mechanism of action. Am J Med 1970; 48: 443-448. 21 Martinez-Calva I, Campos-Apaes E, Rosales Vega E, Mourelle M. Vitamin E improves membrane lipid alterations induced by CCIa intoxication. J Appl Toxicol 1984; 4: 270-272. 22 Comporti M. Biology of disease. Lab Invest 1985; 53: 599-623. 23 Recknagel RO, Ghoshal AK. Lipoperoxidation as a vector in carbon tetrachloride hepatotoxicity. Lab Invest 1966; 15: 132-148.